We utilized the Hypertension Pathotypes cohort, a multinational, multi-decade study of the genetic underpinnings of hypertension in both hypertensives and normotensives. LSD-1 and β2AR are represented by tagging SNPs rs587168 and rs1042713, respectively. All participants consumed a low sodium (<20 mmol/day) then a liberal sodium diet (> 200 mmol/day) for 7 days. The outcome of interest was the change in ΔSBP between the two diets.  Statistical comparison of ΔSBP were performed with an interaction term for each pair of SNPs coded as “risk” or “non-risk” based on prior published analyses, and then with models adjusting for age, gender, BMI, and known diagnosis of hypertension individually and in a full model. The analysis was in Caucasian (n=468) and African–descent (n=73) subjects who had phenotype and genotype data.

In those of African-descent with and without hypertension, β2AR gene variance has no independent effect on ΔSBP (p = 0.8). LSD-1 has a minimally significant effect on ΔSBP (beta 3.7, p = 0.123 in an unadjusted model). However, the interaction between the two SNPs was highly significant (beta_interaction -25.1, p <0.001), and remained so even after adjusting for age, gender, BMI, and diagnosis of hypertension (beta -24.1; p <0.001). Of note, of these classic predictors, only female gender was a significant confounder. Unadjusted ΔSBP with no risk alleles was 6.0 mm Hg; 19.3 and 20.8 for having a single risk genotype (β2AR risk and LSD-1, respectively); and 8.8 mm Hg for having both. Similar analysis for interaction in the Caucasian cohort was not significant, with a p-value of 0.3.

Thus, LSD-1 and β2 adrenergic receptor risk genotypes interact in a not only a non-additive, but potentially protective, manner in populations of African-descent but not Caucasians. That the presence of two risk alleles was associated with less salt-sensitivity than having either risk allele alone may explain in part blood pressure heterogeneity within a population. Future work will explore the interaction of genes via a network model.

It is well known that obese patients are highly complicated with hypertension. Recently, “undetermined” adipocyte-derived factor(s) have been recognized as one of the etiologies of obesity-related hypertension independent of angiotensin (A) II. In the present study, we aim to identify adipocyte-derived factor(s) that stimulate aldosterone synthase gene (CYP11B2) expression/aldosterone secretion that may induce obesity-related hypertension.

yMethodsz

We first differentiated mouse fibroblast 3T3-L1 cells into adipocytes, and collected their supernatants. The supernatants were then incubated with human adrenocortical carcinoma-derived H295R cells, and their ability to stimulate CYP11B2 mRNA expression was determined by qRT-PCR using RNA extracted from H295R cells. Thereafter, the supernatants were fractionated by ultrafiltration to obtain active fractions that stimulate CYP11B2 expression, and proteins included in the fractions were analyzed by LC-MS/MS.

yResultsz

The supernatants obtained from 3T3-L1 adipocytes were shown to stimulate CYP11B2 expression, whose activity was abolished by heating at 96°C for 5 min. AII could not be observed in the supernatants when determined by LC-MS/MS. Fractionation by molecular weight using ultrafiltration demonstrated that the ability to stimulate CYP11B2 expression was detected in fractions between 50 and 100 kDa. The proteins included in the fractions were analyzed by LC-MS/MS, and several candidates were obtained. We are now trying to identify adipocyte-derived factor(s) that can stimulate CYP11B2 expression from them.

yConclusionz

We here have demonstrated the existence of “undetermined” adipocyte-derived factor(s) that have the ability of stimulate CYP11B2 expression. The factor(s) were heat sensitive, different from AII, and their molecular weight was between 50 and 100 kDa. The factor(s) may therefore be one of the causes of hypertension and arteriosclerosis through the increase of aldosterone secretion in obese patients. The structural and functional elucidation of the factor(s) will give us clues for the future innovation of drugs against obesity-related hypertension.

Methods: Wistar rats (~250g) were submitted to ovariectomy (OVX) and on the following day they were treated with estradiol cypionate (10µg/rat, sc, OVX+E2) or vehicle (corn oil, 0.1mL/rat, sc) for eight days. On the eighth day, the rats received an icv (lateral ventricle) injection of ERK1/2 inhibitor (U0126, 1mM/2µL/rat) or p38MAPK inhibitor (SB203580, 50µM/2µL/rat) or PKC inhibitor (Chelerythrine, 1mM/ 2µL/rat) or vehicle (DMSO 5%, 2µL/rat) and 20 min after they were injected with angiotensin II (ANGII, 25ng/2μL/rat) or vehicle (0.9% saline, 2μL/rat). After five min of ANGII injection the animals were decapitated for blood collection for OT and AVP analysis by specific radioimmunoassays. Data were analyzed using ANOVA three-way, followed by Newman-Keuls post-test and the level of significance was set at 5%.

Results: The pretreatment with estradiol attenuated both OT (ANGII x E2 interaction: F=11.0, df=1, p<0.05, n=9) and AVP secretion (ANGII x E2 interaction: F=8.2, df=1, p<0.05, n=9) induced by ANGII in OVX rats. PKC and p38MAPK inhibition blocked OT secretion (ANGII x inhibitor interaction: F=5.2, df=1, p<0.05, n=17; F=7.4, df=1, p<0.05, n=13, respectively). On the other hand, ERK1/2 inhibition attenuated AVP (ANGII x inhibitor interaction: F=6.6, df=1, p<0.05, n=13) induced by ANGII in OVX rats. PKC, p38MAPK and ERK1/2 inhibitors did not affect the ANGII-induced OT and AVP secretion in OVX+E2 rats.

Conclusions: The present results show for the first time the physiological relevance of PKC, p38MAPK and ERK1/2 signaling pathways in the neurohypophysial hormone release induced by ANGII in female rats. These data suggest that ANGII evokes dual signaling pathways, ERK1/2 for AVP secretion and PKC/p38MAPK for OT secretion.

To study the effect of DEN in treatment-naive patients and patients switched from other osteoporosis therapies in a real-world clinic setting, we conducted a retrospective chart review of all patients treated with DEN at an osteoporosis referral centre in Vancouver, Canada.  The study group consisted of all males and females treated with DEN 60 mg SC every six months for at least one year, and in whom baseline and follow-up BMD data were available.  Annual BMD measurements at either hip or spine were performed at the site with the lowest T-score.  Patients were either treatment-naive when starting DEN, or switched from one of four medications: alendronate (ALE), risedronate (RIS), zoledronic acid (ZOL), or teriparatide (TER).  Data analysis was conducted using ANOVA with Bonferroni correction for multiple comparisons.

758 consecutive patients were included: 310 followed at the hip and 448 followed at the spine.  Baseline characteristics were similar between hip and spine follow-up groups.  Treatment-naive and all prior-treatment groups increased BMD on DEN.  Responder analysis showed that 52% and 87% of patients followed at the hip and spine respectively had increased BMD by more than 3% by the end of follow-up at 4 years.  Treatment-naive patients showed a greater increase in hip BMD on DEN compared to patients switching from ZOL after 1 year (p=0.006).  This difference persisted at 3 years (p=0.005).  Treatment-naive patients followed with spine BMD also showed a greater response to DEN compared to those switching from ZOL after 1 year (p=0.007).  Patients switched from ALE, RIS, and TER showed similar increases in hip or spine BMD compared to treatment-naive patients initiating DEN therapy.

Consistent with reported data from clinical trials, DEN increased BMD at hip and spine both in treatment-naive and in patients switching from prior osteoporosis therapy.  The observed increase in BMD was greater than reported in controlled trials (1,3).  Lower adherence to preceding therapy and the use of generic bisphosphonates in clinic patients may account for some of this difference.  There is also greater variability in clinic patient characteristics compared to clinical trial participants.  Our results will inform clinicians regarding anticipated improvements in BMD when transitioning to denosumab.

   The effects of modified-release calcifediol (MRC) on elevated plasma iPTH and serum markers of bone turnover were examined in two identical, randomized, double-blind, placebo-controlled trials conducted in patients with SHPT (>85 pg/mL), stage 3 or 4 CKD and low serum 25D (10-30 ng/mL).  The trials randomized a total of 429 subjects from 77 US sites, stratified by stage, 2:1 to receive oral MRC or placebo for 26 weeks.  MRC dosing started at 30 μg/d at bedtime and increased to 60 μg/d after 12 weeks if plasma iPTH remained above 70 pg/mL.  Of the 429 subjects, 356 (83%) completed treatment and were grouped according to their 25D levels (0-20, >20-40, >40-60, >60-80 or >80 ng/mL) at the end of treatment (EOT).  Mean EOT plasma iPTH, serum total 1,25-dihydroxyvitamin D (1,25D), and serum calcium (Ca), phosphorus (P) and bone formation/resorption markers, as well as urine Ca and P, were compared among the five groups.    

   Baseline demographics were similar for the five groups with mean age of 63.2-68.7 years, mean serum 25D of 16.5-21.5 ng/mL, mean plasma iPTH of 134.8-156.5 pg/mL and mean eGFR of 30.1-32.3 mL/min/1.73m².  More than 95% of subjects treated with MRC achieved serum 25D levels of  greater than 30 ng/mL at EOT.  Mean serum 1,25D progressively rose with increasing serum 25D regardless of CKD stage.  Similarly, and independent of CKD stage, mean plasma iPTH, serum collagen type 1 C-telopeptide and serum procollagen type 1 N-terminal propeptide progressively decreased with increasing 25D.   Serum bone-specific alkaline phosphatase decreased with serum 25D increasing up to >40-60 ng/mL.  No increases in mean serum Ca or P, or urine Ca or P were apparent with increasing serum 25D levels. Treatment-emergent adverse events did not associate with serum 25D levels and appeared to be higher at lower 25D levels.

   In conclusion, MRC increased serum 25D and 1,25D, and reduced plasma iPTH and other serum bone markers in patients with stage 3 or 4 CKD, SHPT and VDI with no effect on serum Ca or P, or urine Ca or P.  Decreases in both plasma iPTH and bone markers were maximal at serum 25D levels above 60 ng/mL.  These findings indicate that serum 25D levels recommended for non-CKD patients are insufficient to control SHPT in stage 3 or 4 CKD and that higher, more effective levels (>60 ng/mL) can be safely achieved with daily MRC.

Methods: This was a 12-month, multicenter, randomized, double-blind, double-dummy study in 643 postmenopausal women aged ≥ 55 years who had received oral BP for ≥ 2 years and had a bone mineral density (BMD) T-score ≤ –2.5 at the lumbar spine, total hip, or femoral neck. Subjects were randomized 1:1 to DMAb 60 mg subcutaneously (SC) every 6 months (Q6M) + placebo (IV once) or ZOL 5 mg IV once + placebo (SC Q6M) for 12 months, and received daily calcium (≥ 1000 mg) and vitamin D (≥ 800 IU). A subset of 117 subjects (61 DMAb, 56 ZOL) was enrolled to have serum iPTH and serum albumin-adjusted Ca evaluated at baseline, day 10 (Ca only), and months 1, 3, 6, 6+10 days (Ca only), 7, 9, and 12.

Results: Significant median percentage increases from baseline in serum iPTH were observed at months 1 (33.9%), 3 (19.7%), and 9 (10.3%) with DMAb but only at month 1 with ZOL (15.1%; all p<0.05). Median percentage changes from baseline in serum iPTH were significantly greater at months 3 and 9 with DMAb (19.7% and 10.3%, respectively) vs ZOL (–5.6% and –8.9%, respectively) (p<0.05). There were no significant percentage changes from baseline in serum albumin-adjusted Ca in either group (except at month 12 in the ZOL group [+1.0%, p<0.01]), and no difference was observed between treatment groups at all visits.

Conclusion: In postmenopausal women with osteoporosis previously treated with oral BP, DMAb treatment was associated with greater transient increases in serum iPTH vs ZOL and greater gains in BMD at all measured skeletal sites. These data help support the hypothesis that the unique mechanism of action of DMAb is due to its direct effect to fully and rapidly inhibit osteoclastic activity at cortical and trabecular bone and its indirect effect to increase serum iPTH.

Experimental Design: Adult C57BL6 male mice were fed a normal diet (ND) or HFD and received twice-daily IP injection of nicotine (0.75 mg/kg BW) or saline for 16 weeks. An additional group nicotine-treated mice on a HFD received twice daily IP injections of 1 mg/kg BW of mecamylamine, a nonselective nicotinic acetylcholine receptor antagonist, for 16 weeks.

Results:HFD alone led to ventricular lipid accumulation compared with mice fed with ND with or without nicotine. Nicotine treatment effectively prevented such HFD-induced ventricular lipid accumulation. A very low incidence of CM apoptosis, expressed as the percentage of TUNEL-positive nuclei per total (apoptotic plus non-apoptotic) nuclei was noted in mice fed ND with or without nicotine or HFD alone. However, combined with a HFD, nicotine led to a significant (P<0.05) increase in CM apoptosis. Mecamylamine treatment fully prevented nicotine and HFD-induced CM apoptosis. Induction of CM apoptosis was associated with increased oxidative stress and activation of caspase 2-mediated intrinsic pathway signaling coupled with inactivation of AMP-activated protein kinase (AMPK). Furthermore, nicotine treatment significantly (P<0.05) blunted the inhibitory effects of HFD on fibroblast growth factor 21 and silent information regulator 1.

Conclusion: We conclude that nicotine when combined with a HFD triggers CM apoptosis though generation of oxidative stress and inactivation of AMPK together with activation of the caspase 2-mediated intrinsic apoptotic signaling. The clinical implication of this study is that smiking plus a HFD may lead to heart failure and other forms of cardiomyopathy.

 Methods: We retrospectively reviewed medical records of eight patients who have used U-500R via CSII. Two patients were excluded from the statistical analysis because of the unavailability of a follow-up hemoglobin A1c (HbA1c) in one patient and discontinuation of CSII in the second patient due to contact dermatitis.

Results: All patients had type II diabetes with an average weight of 113.25 Kg and daily insulin dose of 2.75 U/Kg at baseline. After initiating the U-500R via CSII, the mean HbA1c decreased from 9.07% to 8.4%. The reduction in HbA1c was associated with a significant reduction in the average total daily dose of insulin from 303.33 U/day to 234 U/day (P= 0.02).

Conclusion: The use of U-500R via CSII appears to be an effective treatment option in achieving better glycemic control with less insulin in type II diabetics with severe insulin resistance. The ability to infuse precise amounts of U-500R at a continuous basal rate with boluses makes the U-500R via CSII a suitable option for type II diabetics who have not achieved their glycemic goals. Prospective studies to assess long-term effectiveness, side effects and potential cost savings with the use of U-500R via CSII are necessary.

A registry was established to assess long-term effectiveness and safety of TU in a urological setting in comparison to an untreated hypogonadal control group.

Material and Methods:

Observational, prospective, cumulative registry study in 656 men (age: 60.72 ± 7.15 years) with total testosterone (T) levels below 12.1 nmol/L and symptoms of hypogonadism. 360 men received parenteral TU 1000 mg/12 weeks following an initial 6-week interval for up to 10 years. 296 men had opted against TTh and served as controls. Median follow-up in both groups was 7 years. Measurements were taken at least twice a year, and 8-year data were analysed. Mean changes over time between the two groups were compared by means of a mixed effects model for repeated measures with a random effect for intercept and fixed effects for time, group and their interaction. Changes were adjusted for age, weight, waist circumference, blood pressure, and lipids to account for baseline differences between the two groups.

Results:

In the T group, 113 men (31.4%) had type 2 diabetes mellitus (T2DM), in the control group, 114 (38.5%).

Across all patients, fasting glucose (mmol/L) decreased from 5.65 ± 0.7 to 5.23 ± 0.05 (p<0.0001) in the T group and remained stable from 5.57 ± 0.36 to 5.56 ± 0.34 (NS) in the controls. The model-adjusted estimated difference between groups at 8 years was -0.61 mmol/L (p<0.0001).

HbA_1c (%) decreased from 6.87 ± 1.42 to 5.59 ± 0.44 in the T group and increased from 6.09 ± 1.22 to 6.38 ± 1.44 in the controls. The difference between groups at 8 years was -1.83% (p<0.0001 for all).

The triglyceride:HDL ratio, a surrogate marker for insulin resistance, decreased from 5.5 ± 1.99 to 2.65 ± 0.67 (p<0.0001) in the T group and from 6.48 ± 3.61 to 5.76 ± 3.56 (p=0.0102) in the controls. The difference between groups at 8 years was -1.24 mmol/L (p<0.0001 for all).

The TyG index, another surrogate marker for insulin resistance, decreased from 4.13 ± 0.1 to 3.95 ± 0.02 in the T group and increased from 4.1 ± 0.09 to 4.13 ± 0.09 in the controls. The difference between groups at 8 years was -0.24 (p<0.0001 for all).

Medication adherence in the testosterone group was 100 per cent as injections were administered in the office.

There were two deaths in the T group and 21 deaths in the control group. No patient dropped out.

Conclusions:

Long-term TTh with TU in an unselected cohort of hypogonadal men resulted in improvements in glycaemic control, whereas there was a worsening except for fasting glucose in untreated controls. Long-term TU was well tolerated and excellent adherence suggested a high level of patient satisfaction.

The number of patients using U-500 regular insulin has significantly increased in recent years.  These patients are severely insulin resistant requiring high doses of insulin to achieve glycemic control.  However, it has been observed that a patient’s insulin requirements may dramatically decrease upon admission to the hospital. The medical literature is sparse on this issue, with no formal published guidelines. Therefore, we sought to systematically investigate this phenomenon in our institution.

Methods

We performed a retrospective chart review of patients with U-500 insulin outpatient regimens who were admitted to the San Antonio Military Medical Center over a five-year period. Each patient’s outpatient total daily dose (TDD) of insulin was compared to the average inpatient TDD. The outpatient estimated average glucose (eAG) was calculated from the HgbA1c and compared to the average inpatient glucose level.

Results

We collected data on 27 patients with a total of 62 separate admissions. The average age was 64.4 years with a mean body mass index of 38.9 kg/m² and eAG of 203mg/dl [74 – 109] (HgbA1c of 8.7% [0 – 5.6]). All patients were converted from U-500 to various U-100 insulin regimens upon admission. The average TDD of insulin received in the hospital was lower than their outpatient TDD, 91 units vs. 337 units (p < 0.001) or 27% of their outpatient insulin dose. Overall, 89% of patients received ≤ 50% of their outpatient TDD while in the hospital. The average inpatient glucose was slightly higher than the outpatient eAG, 234 mg/dl vs. 203 mg/dl (p < 0.003).

Discussion

Patients using U-500 insulin routinely have their TDD of insulin reduced upon admission in our institution. Possible factors for decreased insulin requirements include controlled hospital diet, NPO status, and strict adherence to insulin injections by nursing staff. U-500 insulin dosing is prone to errors in the hospital setting, so conversion to U-100 insulin with a dose reduction is a preferred option. Despite a significant reduction in insulin TDD, these patients had clinically similar glucose levels. Therefore, we propose that it is reasonable to convert U-500 patients to U-100 insulin on admission with starting doses between 25 to 50% of their home TDD. Patients’ glucose should be closely monitored and insulin titrated as needed to maintain adequate glucose control.

Conclusion

Patients taking U-500 insulin as an outpatient should be converted to a U-100 basal-bolus regimen at 25 to 50% of their home TDD upon hospital admission. Further prospective trial data is needed to best evaluate the ideal approach to this situation.

Methods:  In a retrospective cohort study, we reviewed the records of patients with T1DM treated at the University of Vermont Medical Center Endocrinology Clinic. We grouped patients as those using CSII, those using CSII + CGM, and those using MDI. We examined age, sex, duration of diabetes, distance from clinic, HbA1c within 12 months, and presence of diabetic complications.  Results:  We reviewed 314 patient records (39% of the clinic’s T1DM population ). 74% of patients reviewed (n=232) used CSII ± CGM.  Groups did not differ in age or sex. Patients using CSII had increased duration of DM compared to patients using MDI or CSII + CGM (F(2,309)=6.967, p=0.0011). Patients who used CSII ± CGM tended to live further away (ß=0.01 per mile from clinic, SE=0.01, p=0.04).  HbA1c was significantly higher in the MDI group (8.53% ± 2.13) versus CSII with and without CGM (adjusted mean=7.34%, t=-4.602, p=0.001; adjusted mean=7.79%, t=3.327, p=0.003, respectively). Duration of disease was significantly associated with cardiovascular disease, retinopathy, peripheral neuropathy, and renal disease.  CSII ± CGM was not associated with decreased incidence of diabetic complications.  

Conclusions:  Contrary to our expectations, patients with CSII ± CGM lived further from clinic and did not have lower occurrences of comorbidities and complications, despite having lower HbA1c levels.  Patients living far away may either be selected by their provider or request CGM ± CSII for better glycemic control to decrease frequency of visits to the endocrinologist. Patients may begin CGM ± CSII too late in the disease course to avert complications, thus having multiple comorbidities and complications despite improved glycemic control as measured by HbA1c.

Methods:Seven transgender men (18-39 years old) naïve to cross-sex hormone therapy and voice therapy were recruited from an outpatient academic endocrinology practice. Each subject underwent prospective voice testing at baseline and following 3, 6 and 9 months after therapy with intramuscular testosterone enanthate or cypionate injections (50-100 mg every 2 weeks).

Results:All subjects showed a dramatic increase in serum testosterone levels at 3, 6 and 9 months consistent with medication use. Baseline fundamental frequencies during a reading task ranged from 134-183 Hz; four voices were consistent with female norms and three were in the “gender neutral” or high end of typical male range. The decline in fundamental frequency over 9 months ranged from 15-74 Hz with an average of 44 Hz. The average decline was 19 Hz between 0-3 months, 19 Hz between 3-6 months and 8 Hz between 6-9 months. Six out of seven subjects had a significant decline within the first 3 months and four out of seven subjects had the largest decline during this period. At 9 months six out of seven subjects had a male range voice and one out of seven had a “gender neutral” voice.

Conclusions:Transgender men show a heterogeneity of baseline fundamental frequencies as well as a heterogeneity of responses to testosterone therapy. Most transgender men can expect significant and meaningful changes in voice within six months of starting testosterone therapy, but the rate and degree of change over the first nine months will vary by individual.

Methods: Prospective observational study, including 29 GID individuals (20 FtoM/9 MtoF), attended in the Gender Identity Disorder Unit (UTIG) of Hospital Clinic from July 2012 to November 2013, who accepted to participate. CHT in FtoM consisted of intramuscular testosterone undecanoate (1000 mg every 2-3 months) except for one who received transdermal testosterone (50mg/day). All MtoF received estradiol valerate 2-4mg/day; cyproterone acetate (25-50mg/day) was associated in some of them. Contraindications for CHT were ruled out; subjects had not previously received hormonal treatment, and had no history of cardiovascular disease or HIV. Anthropometric, hormonal, metabolic and coagulation parameters were assessed at baseline, and at 6 and 12 months of CHT. A substudy in FtoM included body composition by DEXA, endothelial dysfunction by flow-mediated dilation (FMD) and Intima-media thickness (IMT) by carotid ultrasound at baseline, 6 and 12 months.

Results: At baseline, FtoM had higher percentage of total fat mass, LH, estradiol and SHBG than MtoF, while MtoF presented higher HOMA, hemoglobin, hematocrit and testosterone than FtoM. Changes after 6 and 12 moths of CHT: FtoM -Anthropometry: increased BMI (p: 0.001). Hormonal: decreased LH (p: 0.008), SHBG (p<0.001) and increased testosterone (p<0.001). Metabolism: increased total cholesterol (p: 0.043), LDL (p: 0.019) and triglycerides (p: 0.001), and decreased HDL (p: 0.035); increased homocysteine (p: 0.003) and leucocytes (p:<0.001). Coagulation: increased hemoglobin (p<0.001) and hematocrit (p<0.001) and decreased platelets (p: 0.011). MtoF– Anthropometry: increased BMI (p: 0.015) and decreased waist to hip ratio (p: 0.050). Hormonal: decreased testosterone (p: 0.003) and increased FSH (p: 0.035), SHBG (p: 0.002) and prolactin (p: 0.016). Metabolism: decreased triglycerides (p: 0.019) and increased HDL (p: 0.019). Coagulation: decreased hemoglobin (p: 0.023), hematocrit (p: 0.019), platelets (p: 0.021) and ICAMs (p: 0,014). Substudy of body composition in FtoM: decreased total fat mass (p: 0.050), increased total lean mass (p: 0.007) and decreased ginecoid fat distribution (p: 0.008);  no changes in FMD or IMT.

Conclusion: CHT in FtoM has negative effects that could increase the cardiovascular risk, whereas beneficial changes were observed in MtoF.

Hypothesis: Gastric bypass surgery will result in sustained weight loss over time with an increased likelihood of weight normalization compared to published rates for medical therapy.

Methods: We carried out retrospective chart review of patients undergoing gastric bypass surgery (GB) at the University of Michigan between January 1, 2008 and November 1, 2010 who also followed up in the post bariatric surgery clinic (n=219). We evaluated the sustainability of weight loss and the likelihood of achieving a normal weight (BMI<25 kg/m2) 5 years after gastric bypass surgery.

Results: There were 183 females (age: 43±11 years, BMI 47.0±7.9 kg/m2) and 36 males (age: 48±9 years, BMI 46.9±7.6 kg/m2). Out of the female patients, 123 had data at 2 years and had lost a mean of 72.7±25.7% of their excess weight. Four and 5-year follow up data are available on 112 and 105 patients, and mean % excess weight lost from baseline was 61.6±24.5 and 59.2±25.8 % respectively. For males, 2, 4 and 5-year data was available on 22, 22 and 18 patients, and mean % excess weight lost from baseline was 55.2±41.3, 39.6±64.9, and 29.7±72.5% respectively. The difference between the two sexes did not attain statistical significance (p=0.06, 0.07, 0,12 respectively), but males displayed greater variability in the amount of weight loss compared to females. The number of female patients achieving a BMI of 25 kg/m2 or better during the 5 years of observation was 31 (16.9 %) while only 5 of these patients reached normal weight by year 2 and sustained it for more than 3 years.  There were only 2 males (5.5%) who reached a BMI of 25 during the 5-year follow-up. From the females, only 13 patients out of 105 who returned for follow up regained more than half of the weight they lost while none regained all the weight they lost. In males, 4 of the 18 patients returning regained more than half of the weight they lost and 2 experienced regain of all weight lost.

Conclusions: Our data adds to the growing body of evidence that GB provides sustainable weight loss in patients with morbid obesity who return for follow-up. Although the percentage of patients who reached and sustained normal weight is greater than what is reported with medical weight loss, this rate was still low, underscoring the challenge of weight normalization in patients with a very high initial body mass index. Further studies are required to determine preoperative markers that can predict long-term success with bariatric surgery and whether weight normalization can be an attainable goal for specific subgroups of patients.

 Objective: Assess the efficacy and safety of orally administered IN DDAVP for the management of infants with neurogenic DI.

 Design and methods: Retrospective review of clinical and laboratory data of fifteen infants (mean age: 4.5 months) with neurogenic DI treated at a tertiary care center. Treatment was with diluted IN DDAVP formulation (10 mcg/mL) administered orally via a tuberculin syringe to the buccal mucosa.

 Results: After initial DDAVP titration over 2-3 days, IN DDAVP doses ranged from 1 μg to 5 μg twice daily given orally. Mean sodium concentration at DI diagnosis was 159±6.6 mmol/L (range, 151-178) and improved to 142±3.5 mmol/L (range, 137-147) with the orally administered IN DDAVP. Normal serum sodium concentrations were achieved without major fluctuations. Serum sodium was then maintained in the outpatient setting at a mean of 146±5.2 mmol/L (mean duration of follow-up: 4 months).

 Conclusions: Orally administered IN formulation of DDAVP provides a practical and safe treatment alternative for neurogenic DI in infancy. Our experience was most notable for avoidance of severe hypo- and hypernatremia during DDAVP titration and ongoing outpatient management of DI. The possibility for smaller dosage increments and ease of administration make IN DDAVP administered orally preferable over other DDAVP treatment options in infants.

Objective: To evaluate the utility of molecular analysis to improve CAH diagnosis in our NBS program.

Material and Methods: N17OHP were measured by IFMA assays (Autodelfia-Perkin Elmer) and cutoffs adjusted for birth-weight. Confirmatory tests included serum 17OHP, androstenedione, testosterone and cortisol analyses. DNA were extracted from 70 newborns with increased neonatal (N) and serum 17OHP levels, between 1999-2014. CYP21A2 genotypes were determined by allele-specific PCR and MLPA techniques; entire gene sequencing was performed when necessary.

Results: Among all newborns with positive tests, 40 (57%) presented genotypes predicting classical forms (21 males); 33/40 (83%) presented the SW form. Regarding patients carrying the I2 splice as the less severe affected allele (22), 16 (73%) presented slightly or moderate hyponatremia in the first days of life; in the 6 remaining, the clinical form distinction was not possible, due to the precocious treatment introduction.  All patients carrying the p.I172N mutation, in the less affected allele, had the SV form. Genotyping identified mutations in 100% of classical alleles. Among the 30 asymptomatic newborns (15 males) with persistently increased serum 17OHP levels, genotyping predicted NC form in 15 (21.5%) and the other 15 with non-affected genotypes (including 6 heterozygotes) were discharged. Mean N17OHP in classical patients (confirmed by genotyping) was 327 (±163) ng/mL, whereas in the others, including FPR and nonclassical newborns, was 137 (±128) ng/mL. In affected newborns, mutations derived from gene conversion events were found in 89% of the alleles; the most frequent were I2 splice (35%), p.Q318X (23%) and p.R356W (19% of alleles). Mutations not derived from pseudogene were found in 11% of the alleles: gene founder effect was observed with the p.G424S, p.R408C and IVS2-2A>G mutations.

Conclusion: molecular testing was a useful supplemental tool especially in identifying false-positive results in CAH-NBS, preventing unnecessary follow-up of cases with inconclusive hormonal tests. We observed a good genotype/phenotype correlation being helpful to predict the clinical forms in asymptomatic affected newborns. Additionally, molecular diagnosis in our population should take into account mutations not derived from pseudogene.

Subjects and Methods: Ninety-five children with increasing weight (male:67) aged median 10.0 (5.0 to 14.5) were involved in this study. Mean percent obesity was +43.6%(+12.8~+113.8%,<20%:5,20~30%:13,30~50%:49,50%<:28). Serum P IIIP levels and liver function were measured in all subjects. Abdominal CT or ultrasonography were performed in 78 cases.

Results: 24% of the patients had elevated AST/ALT/γGTP levels. 39 out of 78 are diagnosed to have fatty liver/mild fatty changes. Serum PIIIP levels were ranged from 0.65 to 3.0 U/ml (reference range: 0.3~0.8). There was no significant difference in P IIIP levels between patients with or without fatty liver diagnosed by imaging (1.17±0.30 (mean±SD) and 1.18±0.45 U/ml, respectively). However, there was significant difference of P IIIP levels in two groups with or without obesity (0.83±0.18 and 1.16±0.37 U/ml, respectively, ; P = 0.01). There was significant positive correlation between P IIIP levels with subcutaneous fat amount.

Discussion: P IIIP levels were significantly higher in patients with obesity. P IIIP levels did not significantly differ in patients with/without fatty liver change assessed by CT or ultrasonography. It may be due to the lower sensitivity of images, or P IIIP levels reflecting fibrotic changes of liver rather than fatty changes and no complete concordance of these two conditions. 

Conclusion: Serum P IIIP levels were elevated with progression of obesity, indicating that liver fibrosis with fatty changes can occur and progress in children and adolescents. Serum P IIIP measurement may be a useful tool to detect early pathologic changes of liver.

There is increasing concern about the endocrine disrupting chemicals and their involvement in obesity, diabetes, fertility and cancer. However, much baseline data is missing and many of these chemicals are lipophilic and sequester in fat; therefore as the prevalence of obesity is increasing in most populations this study was performed.  The aim of this study was to provide baseline data on the concentrations of chlorinated and brominated dioxins and related compounds as well as polybrominated diphenyl ethers to assess whether concentrations of these compounds are higher in obese than control subjects.

Materials and Methods

Patients undergoing Roux-en-y gastric bypass surgery for weight loss and control patients who were undergoing abdominal surgery for non-bariatric reasons were recruited with informed consent for the study.  Anthropometric parameters were measured at the day of surgery.  During surgery, visceral and subcutaneous adipose tissue biopsies, liver biopsy and blood samples were taken.

Results

Patients undergoing bariatric surgery were younger on average than control patients (47.9 (12.7) vs. 68.5 (14.2) years) and on average had higher BMI (47.1 (10.8) vs. 25.3 (4.9) kg/m²). 

Tissue concentrations were measured in samples of visceral and subcutaneous fat and in liver biopsies. Since the bariatric and control surgery groups were significantly different in age, had overlapping body mass index (BMI) ranges, and due to the known age-dependent patterns of tissue concentrations for chlorinated toxic equivalency (TEQ), the data from the two groups were combined for further analysis. Brominated TEQ concentrations were relatively low compared to chlorinated TEQ, constituting less than 5% of adipose tissue TEQ and less than 10% of liver TEQ.  The most frequently detected PBDD/F compounds were 2,3,7,8-tetrabromodibenzodioxin, 2,3,7,8-tetrabromodibenzofuran, and 2,3,4,7,8-pentabromodibenzofuran. The PBDE compounds presented here are those that were consistently detected in the samples.  Of these, BDE 153 was present at the highest concentrations, followed by BDE 47.

Multivariate linear regressions showed chlorinated TEQ in visceral fat was significantly positively associated with both age and BMI.  In contrast, brominated TEQ compounds showed no significant association with any of the factors considered.  BDE 47 was borderline significantly negatively associated with age, while BDE 153 showed a borderline significant negative relationship to BMI.  Gender was not a significant factor for any analyte. 

Conclusion

Subcutaneous fat concentrations were highly correlated with visceral fat concentrations for all analytes. This confirms that concentrations of these compounds in fat depots in the body appear to be generally in equilibrium, an observation previously made for chlorinated TEQ compounds but not previously demonstrated in humans for PBDD/Fs and PBDEs.

Timed-pregnant SD rats were provided ad lib access to TCC supplemented diet (0.1% w/w) starting at gestational day (GD) 4 until postnatal day (PND) 16 after birth. Fecal samples were collected from dams during gestation and lactation. Cecum content was collected from neonates during lactation only. A group of age-matched unexposed dams and their neonates served as controls. The V4 region of the 16S rRNA region was sequenced via the MiSeq platform.  Sequences were analyzed with the Phyloseq and Vegan packages in R.

Exposure to TCC during gestation and lactation led to perturbations in microbial community structure across time in both dams and neonates. Phylogenetic diversity was significantly reduced among exposed dams from GD 11 until sacrifice and neonates at PNDs 12 and 16. Weighted Unifrac analysis of microbiota from TCC exposed dams revealed significant dysbiosis of the gut microbiota by GD 18, a trend that continued to 16 days after delivery when the samples were last collected. Using the same metric among neonates, in both control and TCC exposed animals, an initial stochastic pattern emerges at PND 3. At PND 6, an overall restructuring occurs where both control and TCC exposed communities converge. By PND 12, communities start separating based on exposure status and become significantly different at PND 16.

Our results both demonstrate the impact of TCC exposure on gut microbial structure during gestation and lactation, as well as provide insight into the neonatal bacterial colonization process. The ability of TCC to drive microbial dysbiosis warrants future investigation to determine the clinical health outcomes resulting from non-prescription antimicrobial use during sensitive exposure windows.

These data are consistent with known literature and support the validity of our new mouse models in studying the ProkR2 signaling system and physiological functions. A valid animal model that expresses Cre-recombinase in ProkR2 cells will allow for further elucidation of the GnRH system including the differential roles of ProkR2 on the neurodevelopment and migration of GnRH neurons as well as the neuroendocrine effects of ProkR2 signaling in the hypothalamo-pituitary-gonadal axis.

It has been reported that treatment with metformin is associated with smaller tumor size, higher complete remission rate and longer progression free survival in diabetic patients with differentiated thyroid cancer (DTC). Furthermore, metformin inhibits DTC cell line growth in vitro via down-regulation of mTOR signaling pathway.  Thus, we hypothesized that metformin will affect disease specific survival by decreasing thyroid cancer growth and progression in a metastatic mouse model of thyroid cancer. 

Methods

We used NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice that had tail vein injection of 160,000 human DTC cells (FTC-133) transfected with a linearized pGL4.51[luc2/CMV/Neo] vector. When lung engraftment was documented with quantification of luminescence, the mice were randomly assigned into 2 groups:

- control group (C), treated with placebo (water via gastric gavage), n = 11 animals (4 males, 7 females)

- metformin (MF) group - treated with 12.5 mg MF in 250 ul of water via gavage, n=10 mice (4 males, 6 females)

We performed weekly imaging studies with Xenogen IVIS.

The mice were followed until they reached endpoints for euthanasia established by our local Animal Advisory Committee. The disease-specific survival time was calculated from the day of starting the experiment until death.  The lung and liver tissue containing metastatic lesions were removed, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and then paraffin embedded. Representative H-E stained slides containing the cross section of the lung and liver tissue underwent pathologic evaluation. Pulmonary metastatic tumor burden was expressed as the percentage of tumor divided by percentage of total lung tissue evaluated.

Results

Control mice had more rapid tumor growth as compared with MF treated mice.  Weekly bioluminescence ratios were established by normalizing to baseline: week 1 - C 3.88 vs M 1.78, p=0.1; week 2 - C 9.2 vs M 3.13, p=0.056; week 3 - C 71.4 vs M 22.9, p=0.14; week 4 - C 8080 vs M 167, p=0.1. Disease-specific survival was significantly longer in MF group compared with C group (median 36 days vs 29 days, p=0.002, respectively). Pathology evaluation of H-E stained slides revealed that all mice which died before the endpoint of the experiment had tumor involving 70-90% of normal lung tissue.  The experiment was finished at 36 days when remaining mice from control group met euthanasia criteria. The pathology evaluation of the tissues obtained at day 36 of the experiment revealed that the median tumor burden was 90% of lung volume in C group vs 60% in MF group, p=0.01.

Conclusions

Our study suggests that metformin treatment increases disease specific survival by delaying progression of thyroid cancer metastases in vivo. 

Our aim was to prospectively study subjects with RTHβ during pregnancy, prenatally determine the fetus’ genotype (affected or normal) and evaluate whether treatment of the mother with antithyroid drugs has an impact on fetal development and early postnatal course, including thyroid function tests (TFTs) at birth. 

Thirteen RTHβ females were prospectively studied in fourteen pregnancies. Genetic testing of the fetus was performed by Sanger sequencing of fetal samples either from chorionic villi or amniotic fluid. The decision to treat was based on maternal TFTs and the fetus’ genotype. No antithyroid treatment was started in females carrying affected fetuses, whereas in case of unaffected fetus, the target was a free T₄ not higher than 20% of the upper limit of normal. Treatment was adjusted by monthly monitoring of maternal TFTs.

The RTHβ females harbored nine different THRB gene mutations: A317T (3 subjects), R320C and M334R (2 subjects each), L450H, E460K, V349M, P453T, R429Q and M310L. Two females had a history of miscarriages and three had older affected children. The genotype distribution was 8 unaffected and 4 affected full term deliveries. Two stillbirths were reported, both involving affected fetuses, one of which due to placental insufficiency. No perinatal and postnatal complications were documented besides one case of fetal distress attributed to pneumonia in an affected infant born to an A317T mother. The mean birth weight (±standard deviation) was 3.181 (±0.33) kg; no significant difference was noted between unaffected and affected infants (3.09±0.39 vs 3.33±0.22 kg). Mean TSH levels at birth were 3.26±1.26 uU/ml and were not different between unaffected and affected infants (4.06±0.69 vs 3.06±1.25 uU/mL).

This study emphasizes the role of prenatal diagnosis in guiding the management of RTHβ subjects in pregnancy. Maintaining T₄ levels not higher than 20% of the upper limit of normal in case of unaffected fetuses prevented low birth weight and postnatal TSH suppression, expected based on previous data. Study of additional pregnant subjects with RTΗβ will crystallize our knowledge on maternal-fetal interaction in RTHβ and help optimize clinical care of both the mother and the fetus.

1)  Duparc C et al., J Clin Endocrinol Metab. 2015 100(4):E550-60.

Objective: To evaluate the hypotheses that dietary sodium intake variability and insufficiently suppressed PRA can result in inaccurate interpretation of the ARR for PA screening.

Methods: 241 untreated stage I hypertensives with ARR > 20, underwent one week of high sodium diet (HS) such that 24h urine sodium was >200 mmol/d, and subsequently one week of low sodium diet (LS), such that 24h urine sodium was <50 mmol/d. All ARR measurements were taken in the morning after overnight supine rest. Subjects were considered to have a “positive screen” for PA if they had ARR > 20 in addition to PRA ≤ 1.0 ng/mL/h and serum aldosterone ≥ 6 ng/dL. PA was considered to be “confirmed” if positive screens also had a 24h urine aldosterone of ≥12 mcg/d on HS.

Results: 33% (79/241) of the population met criteria for a positive screen for PA; the remaining 67% had a negative screen for PA despite having ARR>20 due to a combination of low aldosterone (< 6 ng/dL) and a highly suppressed PRA. When the 79 subjects with a positive screen for PA underwent LS diet, 56% (44/79) no longer met criteria for a positive screen. This subset of “false negative” screens for PA on LS were characterized as having: higher PRA on LS (2.03 ± 1.9 vs. 0.44 ± 0.3 ng/mL/h; P<0.001) and on HS (0.25 ± 0.13 vs. 0.16 ± 0.1 ng/mL/h; P=0.001), Caucasian race predominance (90 vs. 62%; P=0.002), but no difference in serum aldosterone levels, when compared to subjects whose screen remained positive on LS. Multivariable logistic regression showed that odds for false-negative PA screening on LS were associated with a PRA > 0.3 ng/mL/h on HS (OR=3.7 [1.1–12.5]) and Caucasian race (OR = 8.3 [CI 2.1–32.2]). Most notably, among the 48/79 subjects who had confirmed PA, 52% (25/48) no longer met criteria for a positive screen for PA when placed on LS.

Conclusions: Among individuals with untreated stage I hypertension, we observed that substantial inaccuracies in PA screening can occur if dietary sodium balance and absolute values of PRA and aldosterone are not considered. Our findings suggest that case-detection for PA could be markedly improved by: 1) ensuring a high dietary sodium intake when measuring ARR; and 2) interpreting the ARR in the context of a sufficiently suppressed PRA.

First, we analysed mitochondrial function in vivo in the different cell types of the adult mouse SVZ. Following stereotaxic injection of a mitochondrial membrane potential dye, JC-1, into the lateral ventricle of the brain, we observed greater mitochondrial activity in DCX+ neuroblasts than in EGFR+ proliferative progenitor cells and NG2+ oligodendrocyte precursors. Furthermore, preliminary data suggest that a short-term hypothyroidism also affects mitochondrial activity in the SVZ. Second, we studied the expression pattern of the activated form of the DRP1 protein (pDRP1^S616). It has been shown that THs can induce the translocation of DRP1 to the mitochondria (5), where it can mediate mitochondrial fission, and in turn impact mitochondrial respiration. We showed that pDRP1^S616 is preferentially present in the cytosol of cells differentiating toward a neuronal fate in the adult SVZ.

Taken together, the results show that THs direct NSCs differentiation toward a neuronal rather than an oligodendroglial phenotype in association with metabolic modulation. We hypothesize that it is through their impact on mitochondrial activity that THs govern NSCs commitment. Future work addressing the interplay between THs and mitochondrial metabolism underlying this cell fate decision will involve studying the impact of modulating DRP1 in vivo and in vitro in different thyroid contexts. This work should provide new insights on molecular and cellular mechanisms governing adult NSCs fate in control conditions and in response to changing energy metabolism.

Methods: To determine the effect of maternal diet on hypothalamic gene expression (n = 5) of Pomc, Mc4r (melanocortin 4 receptor; αMSH receptor), and Npy in female offspring exposed to maternal VitD deficiency, we used qRTPCR. To determine if exposure to maternal VitD deficiency affected hypothalamic responsiveness to excitatory peptide, αMSH, we administered MTII (αMSH analog) via an intraperitoneal injection to female offspring exposed to control (n=7) or maternal VitD deficient diets (n=5) and collected serial blood samples to measure serum LH levels.

 Results: Compared to controls, female offspring exposed to maternal VitD deficiency exhibited attenuated LH release in response to MTII (13.69 vs 7.03 ng/ml; p< 0.005). Reduced responsiveness to MTII was not associated with gross changes in hypothalamic, Mc4r, mRNA expression. Female offspring exposed to maternal VitD deficiency had a 0.5-fold reduction in hypothalamic Pomc (p< 0.05) and a 3-fold increase in Npy mRNA expression (p< 0.05).

Conclusion: Maternal VitD deficiency disrupts expression of hypothalamic Pomc and Npy mRNA and responsiveness to MTII. These findings suggest that in utero VitD signaling may act as a metabolic cue that is important for the programming of the reproductive neuroendocrine axis of adult offspring.  Moreover, these data suggest maternal VitD deficiency may program female reproductive axis dysfunction through adverse effects on the balance of neuropeptides that transmit information about energy homeostasis.

By comparing age- and exposure-matched mice that exhibited anorexia-like behavior from those that did not, we are beginning to identify neuroanatomical and molecular correlates of subsequent risk of anorexic behavior. We found that levels of arginine vasopressin (AVP) in the serum were more than 2-fold higher in mice that previously exhibited anorexic behavior, similar to observations in AN patients (1). Moreover, we found that exposure of Val66Met carriers to social isolation stress was associated with increased AVP levels in the serum before the onset of aphagic behavior. We then explored whether increased risk of anorexic behavior was also correlated with changes in other components of the BDNF and AVP signaling pathways in the brain. We identified marked increases in the expression of genes encoding the AVP receptor, Avpr1a, and the BDNF p75^NTR receptor (Ngfr) in the amygdala, while expression in the hypothalamus, prefrontal cortex, hippocampus and pituitary were not affected. The idea that AVPR1A signaling is predictive of and contributes to severe dietary restriction is supported by pharmacological experiments in animals (2, 3) and observations that a variant in the AVPR1A gene that is associated with increased risk of severe dietary restriction in humans is also associated with increased amygdala reactivity (4). This study raises the possibility that developmental impacts on the AVP system could be exploited to develop novel therapeutic compounds to prevent and/or treat AN.

Methods: This multi-center, active-controlled study enrolled women with PMO who had taken an oral bisphosphonate for ≥ 3 years prior to screening and alendronate (70 mg weekly or equivalent) in the year prior to screening; had a bone mineral density (BMD) T-score ≤ –2.5 at the total hip (TH), lumbar spine (LS), or femoral neck (FN); and had a history of nonvertebral fracture after age 50 or vertebral fracture. Subjects received daily calcium and vitamin D and were randomized to receive subcutaneous romosozumab 210 mg once monthly or TPTD 20 μg once daily. The primary endpoint was percent change from baseline in BMD by DXA at the TH through month 12 (the average at months 6 and 12). Secondary endpoints included percent change from baseline at months 6 and 12 in BMD by DXA at the TH, LS, and FN; hip integral and cortical BMD by quantitative computed tomography (QCT); and estimated hip strength by finite element analysis. Imaging assessments were done blinded to treatment.

Results: A total of 436 women were randomized to receive romosozumab (N = 218) or TPTD (N = 218) with a mean age of 72 years. Baseline mean TH, LS, and FN T-scores were –2.2, –2.9, and –2.5, respectively. Through 12 months, the mean (95% CI) percent change from baseline in TH BMD by DXA was 2.6% (2.2, 3.0) with romosozumab and –0.6% (–1.0, –0.2) with TPTD (p < 0.0001 between groups). TH BMD changes at months 6 and 12 were significantly larger with romosozumab than with TPTD (p < 0.0001): month 6, 2.3% (1.9, 2.7) vs –0.8% (–1.2, –0.4); month 12, 2.9% (2.5, 3.4) vs –0.5% (–0.9, 0), respectively. Romosozumab also resulted in significantly larger BMD gains at the LS at months 6 and 12 vs TPTD (p < 0.0001): month 6, 7.2% (6.6, 7.8) vs 3.5% (2.9, 4.0); month 12, 9.8% (9.0, 10.5) vs 5.4% (4.7, 6.1), respectively. QCT assessments of the hip demonstrated significantly greater gains in integral and cortical BMD with romosozumab vs TPTD at months 6 and 12 (p < 0.0001). Estimated hip strength gains were also significantly larger with romosozumab at both time points (p < 0.0001) and declined from baseline in TPTD-treated subjects at month 6. The subject incidences of treatment emergent adverse events and adverse events of interest were generally balanced between treatment groups.

Conclusions: In subjects transitioning from bisphosphonate therapy, romosozumab was well-tolerated and was associated with significant BMD gains at both the hip and spine compared with TPTD. BMD gains in the cortical compartment contributed to the greater treatment effect of romosozumab at the hip. Estimated hip strength improved with romosozumab over 12 months but decreased early with TPTD. A global phase 3 program evaluating romosozumab for the treatment of PMO is ongoing.

Objective: To investigate associations between stimulant use and bone mass in pediatric subjects.

Design: Cross-sectional analysis of data from the 2005–2010 National Health and Nutrition Examination Study (NHANES).

Participants: 6489 NHANES participants ages 8–20 years (mean 13.58 + 3.58).

Outcomes: Total femur, femoral neck and lumbar spine bone mineral content (BMC) and density (BMD) assessed via dual-energy X-ray absorptiometry (DXA).

Results: 159 of 6489 subjects used stimulants. Stimulant use was an independent predictor of bone mass after multivariable adjustment for age, gender, height and weight Z score, socioeconomic status, physical activity, cotinine level and race/ethnicity. Lumbar spine BMC was 5.1% lower among stimulant users versus non-users (mean difference 0.704 g, SE ± 0.02 g, p = 0.005). Lumbar spine BMD was 3.9% lower (mean difference 0.037 g/cm2, SE ± 0.001 g/cm2, p = 0.002). Femoral neck BMC was similarly negatively associated with stimulant use while BMD approached significance. Compared to nonusers, femoral neck BMC was 5.3% lower among stimulant users (mean difference 0.242 g, SE ± 0.0093 g, p = 0.009) and BMD was 3.7% lower (mean difference 0.034 g/cm2, SE ± 0.0157 g/cm2, p = 0.08). Subjects treated with stimulants for more than 6 months had lower lumbar spine BMD and BMC than those using for less than six months and non-users.

Conclusion: In this NHANES study, pediatric subjects treated with stimulants had lower DXA measurements of the lumbar spine and femoral neck compared to non-users. As stimulant medications are first-line pharmacotherapies for ADHD, their potential effects on pediatric bone health need to be clarified. These findings support the need for future prospective studies to examine the effects of stimulant use on bone mass in this population.

Aim: To investigate the prevalence and risk factors for low BMD in an established TS cohort.

Method: A retrospective, cross-sectional study of TS patients who underwent DXA scanning at Monash Health, Victoria, Australia from 1998 to 2015. Cases were identified through the hospital Adult TS clinic and Bone Density Department databases. Data from baseline DXA included areal bone mineral density (aBMD) and Z-scores. To adjust for smaller bone size in TS patients, bone mineral apparent density (BMAD) of the lumbar spine (2) and femoral neck (3) was calculated. Low bone mass in young pre-menopausal women was defined as a Z-score < -2.0 (4). Medical history, including ERT and growth hormone therapy (GHT), was collected from medical records. Statistical analysis included descriptive statistics and multivariate regression analysis.

Results: Seventy-nine TS patients were included, and fractures occurred in 30.4% of cases. The median age of TS diagnosis was 11 (range 0-65) years. Primary amenorrhoea was common (81.8%), and the median age of commencing estrogen was delayed at 16 (range 11-46) years. Non-continuous ERT use was reported in 37.9% and GHT was reported in 53%. The median (range) age, height and body mass index was 29 (14-66) years, 148.5 (127.8 – 170.0) cm, and 25.6 (12.4 – 49.5) kg/m², respectively. The most common osteoporotic risk factor was gonadal failure (89.4%), followed by vitamin D deficiency (40%). Low bone mass in the spine and femoral neck occurred in 26.4% and 7.7%, respectively. Multivariate regression analysis demonstrated that spine and hip aBMD, and BMAD, were inversely associated with age of commencing ERT or years of estrogen deficiency (adjusting for age at DXA and BMI). After adjusting for confounding, GHT was associated with neither aBMD nor BMAD.

Conclusion: Low spinal bone mass is common in TS. Both the delay in commencing estrogen and years of estrogen deficiency are important independent risk factors for reductions in spine and hip bone mass. Avoiding estrogen deficiency is important in optimizing bone health in TS. This depends on early diagnosis of TS to allow age-appropriate pubertal induction and to maximize ERT compliance. Transitional multidisciplinary TS clinics have a role in the early initiation of ERT and in monitoring bone health in TS.

Methods:VLBW adults (135 subjects) and controls (69 subjects), mean age 27.2 yr, were assessed (demographics, clinical and anthropometrics)  over a 2 day period at one centre (Christchurch, New Zealand) during which metabolic studies were performed. Fasting overnight blood samples were drawn for assay of NTproBNP and NTproCNP along with other VRF including cholesterol/ HDL cholesterol ratio, triglycerides, and insulin-glucose ratio. Spearman coefficients were used to determine univariate associations.

Results: VLBW adults were significantly shorter (males 4.1 cm, females 3.9 cm) than controls (p<0.05 and <0.01 respectively). Systolic blood pressure was significantly increased in males (122±1.5 vs 116±2.1 mmHg, p<0.04) but not in females. Body Mass Index (26.4±0.5, VLBW; 27.7±0.6 controls) did not differ. Compared to controls, plasma NTproCNP was higher in male (23.4±0.4 vs 17.2±0.5 pmol/L, p<0.005) and female (18.7±0.4 vs 17.2±0.5 pmol/L, p<0.05) VLBW subjects whereas NTproBNP and other VRF did not differ between the two groups. Across all participants (n=204), NTproCNP was significantly associated with systolic (r=0.33) and diastolic blood pressure (r=0.19), chol/HDL ratio (r=0.21), triglycerides (r=0.24) and insulin resistance (r=0.19). Unexpectedly, associations were all significantly inverse with NTproBNP (range of r values -0.21 to -0.38). Across all subjects, the ratio of NTproCNP to NTproBNP was correlated with a composite index of vascular risk (r=0.46), stronger than the correlation of either NTproCNP (r=0.29) or NTproBNP alone (r=-0.39).

Conclusion: Alone among VRF, and as early as the 3^rd decade, plasma NTproCNP is significantly raised in both males and females born with VLBW. The differential association of NTproCNP (positive) and NTproBNP (inverse) with VRF in this young adult population likely reflects an adaptive response to vascular stress (increasing CNP) and genetic polymorphisms raising BNP (2) and conferring reduced risk of future metabolic (3) and vascular complications (4). The ratio of NTproCNP to NTproBNP could be a novel index of metabolic vascular health in young adults.

Methods: We studied 362 healthy men (mean age 33 years) who were enrolled into one of 3 cohorts: 1) placebo GnRH agonist + placebo testosterone gel (controls, n=35); 2) GnRH agonist (goserelin acetate 3.6 mg monthly) + randomization to placebo gel or testosterone gel 1.25 g, 2.5 g, 5 g, or 10 g daily (n=167), and 3) GnRH agonist (goserelin acetate 3.6 mg monthly) + aromatase inhibitor (anastrazole 1 mg daily) + randomization to placebo gel or testosterone gel 1.25 g, 2.5 g, 5 g, or 10 g daily (n=160). At 12 weeks, we analyzed serum BNP, serum total testosterone, and serum estradiol levels.

Results: As expected, mean levels of serum total testosterone and estradiol differed between testosterone dosage groups (p<0.0001), with higher levels in higher dosage groups. The range of estradiol levels was much narrower in the cohort that received an aromatase inhibitor compared to the cohorts that did not receive an aromatase inhibitor. BNP levels were negatively associated with serum total testosterone levels in the cohort that received an aromatase inhibitor (r = -0.21, p<0.009), in the cohorts that did not receive an aromatase inhibitor (r = -0.14, p<0.05), and in all cohorts combined (r = -0.17, p=0.001); differences remained significant after adjusting for age and BMI. In contrast, BNP levels were not associated with estradiol levels. In multivariable models including serum total testosterone, estradiol, age and BMI, BNP levels were associated negatively with serum total testosterone levels (partial r = -0.19, p=0.0005) but were not associated with estradiol levels.

Conclusions: In men randomized to receive varying doses of testosterone supplementation, there is an inverse association between circulating testosterone and BNP levels.  In contrast, there is no clear association between estradiol and BNP, within the range of estradiol levels seen in men. Further investigation is needed to elucidate the mechanisms underlying the sex-specific differences in NP levels, a topic that has potentially important implications for sex-related disparities in hypertension and other cardiovascular disorders.

Methods: We evaluated data from 711 participants of the Princeton Lipid Research Cohort with information regarding levels of insulin, adiponectin, and MetS severity during the time period 1998-2003 (mean age 39.5 years), 595 of whom with data regarding MetS severity in childhood from 1973-1976 (mean age 12.9 years), and 417 with disease status from 2010-2014 (mean age 50.9 years).  We used linear regression to assess relationships between childhood MetS severity Z-score and adult insulin and adiponectin levels; we used Pearson correlation to assess relationships between current levels. Finally, we used logistic regression to assess individual and independent relationships between insulin, adiponectin and MetS severity Z-score on future T2DM and CVD.   

Results: Childhood MetS severity Z-score exhibited positive association with adult levels of insulin (p<0.01) and inverse association with adult levels of adiponectin (p<0.01). Current MetS severity Z-score was linked to fasting insulin (Pearson’s r=0.618, p<0.0001) and adiponectin (r=-0.467, p<0.0001). In individual analyses, higher levels of insulin and MetS severity Z-score as adults (mean age 39.5 years) were related to future odds of incident T2DM and CVD over the next 11.2 years (all p<0.001), while lower levels of adiponectin were only related to odds of future T2DM (p<0.0001). In a model that included insulin, adiponectin and MetS severity Z-score, adiponectin was not linked to either future diabetes or CVD; both insulin (p<0.05) and MetS severity Z-score (p<0.01) were related to risk of future T2DM, while only MetS Z-score was related to future CVD (p<0.001).

Conclusion: The severity of MetS exhibits tight links with adiponectin and fasting insulin, including between childhood and adulthood.  Both fasting insulin and MetS severity Z-score were related to future T2DM independent of adiponectin; of these 3 measures, only MetS severity Z-score was an independent predictor of future CVD.  These data suggest potential clinical utility in assessing MetS severity as a marker of risk.

Methods: Patients with partial and generalized LD participating in IRB-approved studies at the National Institutes of Health underwent onetime non-contrast cardiac CT for coronary calcium score (Agatston) followed by a standard cardiac multidimensional CT angiogram (CCTA). Patients with LD (n=19; 14 male and 5 female) were compared with age-, sex-, and BMI-matched controls.

From CCTA, the following scores were calculated to quantify atherosclerosis- 1) segment-involvement score (SIS), 2) segment-stenosis score (SSS). The coronary circulation was divided into 16 segments. The SIS (on a scale of 0-16, with 16 the most involved) was calculated based on the number of coronary artery segments involved with any atherosclerotic plaque. Each of the involved coronary artery segments were graded based on the degree of stenoses (from 0-3). The SSS was derived by addition of stenosis score of each individual coronary artery segment and thus ranged from 0-48. Previous studies have shown that subjects with SIS and SSS greater than 5 have higher mortality compared to those with scores below 5.

Results: Age (33.8±13.0 vs. 33.6 ±12.6 yrs) and BMI (24.2±5.2 and 26.8±5.4 kg/m2) were not significantly different between LD and control groups. In the LD group, 36.8% had generalized LD and the remainder had partial LD. As expected, more patients with LD were diabetic (89.4% vs. 4.7%, p<0.001), hypertensive (52.6% vs. 15.7%, p=0.03), and had higher triglycerides (1322 ± 2935 vs. 104± 70 mg/dL, p= 0.0001). Despite these classical CV risk factors, only 57.8% of subjects with LD and 42.1% of controls had SIS > 5 (p=0.35). Similarly, the proportion of subjects with SSS >5 was not different between the two groups (21% vs. 15.7%). Coronary calcium scores also did not differ significantly between the two groups though the number of subjects with calcium scores greater than 0 tended to be higher in LD vs controls (63.1% vs. 21%, p=0.07).

Conclusion: Contrary to our hypothesis, these findings suggest that despite the presence of insulin resistance and other CV risk factors, young patients with LD do not have increased coronary atherosclerotic burden.

Material and Methods: We conducted a double-blinded, randomized, placebo controlled crossover study involving 22 adult subjects with type 1 diabetes who used an insulin pump or multiple daily injections, had a self-reported average frequency of hypoglycemia <60 mg/dl of at least twice a week, and reported inconsistent or absent symptoms with blood glucose <50 mg/dl. Participants administered their own insulin as usual while receiving either glucagon or placebo (filled from coded vial blinding devices) for 24 hours at a time from an automated bionic pancreas system. During the 2-week study, days were randomized to glucagon or placebo in blocks of two (7 days of each). The primary outcome was area over the curve <60 mg/dl (AOC<60), a measure of total hypoglycemia exposure, on glucagon vs. placebo days. Secondary outcomes included AOC<60 during the nighttime (11:00 PM – 7:00 AM), time <60 mg/dl, incidents of symptomatic hypoglycemia, mean CGM glucose, mean glucagon dosing on glucagon days, and daily self-reported nausea on a visual analog scale.

Results: The AOC <60 mg/dl was reduced by 75% on glucagon vs. placebo days (851 ± 748 vs. 3,414 ± 2,242 mg/dl·min, p<0.001). There was a 91% reduction in AOC<60 at night on glucagon vs. placebo days (117 ± 204 vs. 1,309 ± 1,476 mg/dl·min, p<0.0001). Subjects spent 74% less time with BG <60 mg/dL on glucagon vs. placebo days (1.2 ± 0.8% versus 4.7 ± 3.6%; p<0.0001). There were half as many symptomatic hypoglycemia episodes on glucagon vs. placebo days (0.6 ± 0.4 versus 1.2 ± 0.8 incidents per day; p<0.0001). Blinding was effective, with subjects correctly guessing their daily assignment to glucagon or placebo in a daily survey on 42% of days (similar to chance). There was no difference in mean CGM glucose on glucagon vs. placebo days (153 ± 28 vs. 152 ± 27 mg/dl, p=0.6). The mean total daily dose of glucagon was 0.48 mg on glucagon days. There was a trend for more self-reported nausea on glucagon vs. placebo days, but this did not reach statistical significance (1.1 ± 0.6 vs. 0.4 ± 0.7 cm on a 10 cm scale, p=0.05). There was no unexpected or severe adverse events on either glucagon or placebo days.

Conclusions: Automated glucagon administration effectively reduced hypoglycemia in patients with type 1 diabetes, and was well tolerated.

Results: In 2012 Dartmouth-Hitchcock Medical Center developed a collaborative program for total pancreatectomy with islet auto-transplant (TP-IAT), initially using off-site islet isolation at Massachusetts General Hospital and then transitioned to on-site intra-operative islet isolation. We have successfully treated 30 patients thus far, and have had encouraging results from a diabetes perspective. Before the surgery, 11% of the patients already had diabetes requiring treatment (metformin and/or insulin), 15% had pre-diabetes, and 74% had normal A1c <5.6%. The average A1c was 5.7+1.1% (+SD) with a range of 4.6-8.4%. All patients had normal pre-operative stimulated c-peptide (3.2+ 2.1 ng/ml, normal range 1.1-4.4 ng/ml), suggestive of preserved endogenous insulin production. Average islet equivalent (IEq) yields was 4,683 IEq/Kg (range 843-10,214); 10% of patients had low yields (< 2,500), 57% had moderate yields (2,500-5,000), and 33% had high yields (>5,000 IEq/Kg). Three months after the surgery, 40% of patients required no insulin with near normal mean A1c 6.3% while 83% had A1c at target (<7%). Six months post-operatively, the average A1c was 6.8% while 71% of patients had A1c at target. After 12 months, the average A1c had risen to 7.5%, but 62% of the patients still had glycemic control at target, and 11 of 21 (52%) of patients required no insulin at all. Since most of the patients maintained reasonable islet graft function with normal c-peptide levels, we typically started treatment with oral agents (e.g. metformin and then DPP-4 inhibitor) without the need of insulin for patients who subsequently had suboptimal diabetic control.

Conclusion: TP-IAT techniques, either using off-site or on-site intra-operative islet isolation, resulted in a lower rate of insulin-dependent diabetes, and should be seen as a viable treatment option for patients with intractable chronic pancreatitis. Furthermore, patients generally experienced much less pain and returned to work with a better quality of life. Therefore, islet auto-transplantation either on-site or off-site islet isolation can be used to avert the burden of complicated and costly diabetic care for carefully selected patients requiring total or sub-total pancreatectomy.

Objective: To search whether high protein B consisting of whey protein has a greater impact on WL, overall PPG, S and HbA1c than B with different protein sources.

Methods: 48 T2D, 22 males, BMI 32.1 0.9 kg/m², aged 58.9 4.5 y, were randomized to 3 isocaloric (1500 kcal) WL diets during 12 weeks. The 3 diets had breakfast (660 kcal), lunch (567 kcal) and dinner (276 kcal) with the same composition at lunch (% of carb:prot:fat 20:45:25%) and dinner (13:40:47%), but differed in the B composition and protein content.

The 3 meals were: 1) A high-carbohydrate B Diet (CBd), n=15, with 13 g protein at B: (65:15:20%), i.e., ready-to-eat cereals. 2) A high-protein B Diet (PBd), n=16, with 36 g protein in B: (40:40:20%), i.e., eggs, tuna and cheese. 3) A whey B Diet (WBd), n=17, with 36 g protein in B, (40:40:20%), i.e., whey protein shake. Additionally, on 3 separate days between day 10 and 15 after diet initiation, all participants underwent 3 all day CBd, PBd and WBd meal tests, to assess PPG, insulin, intact GLP-1 and S after B, lunch and D.

Results: After 12 weeks, WL was -3.5 ± 0.3 kg (-3.8%) in CBd, -6.1 ± 0.3 kg (- 6.8 %) in PBd and the greater WL was found in WBd, -7.6 ± 0.3 kg (- 8.4%)  (p<0.0001). Compared to CBd, the % of WL in PBd was higher by 44 %, and in WBd was greater by 55% (p<0.0001).

The reduction of HbA1c was lower in CBd by -0.36 ± 0.04%, in PBd was -0.6 ± 0.04% and the greater reduction was found in WBd, 0.89 ± 0.05% (p<0.0001). The % of change for HbA1c was 4.6 % in CBd, 7.7% in PBd and the greatest reduction changes were in WBd by 11.5% (p<0.0001). Compared to CBd the % of the reduction of HbA1c was greater by 41 % in PBd, and by 64 % in WBd (p<0.0001).

Overall AUC for PPG was 95522 ± 565 mg/dl*min in CBd, 84065 ± 299 mg/dl*min in PBd and 77452 ± 292 mg/dl*min in WBd (p<0.0001). Compared to CBd the AUC for overall PPG was 12 % lower in PBd and 19 % lower in WBd (p<0.0001). The AUC for overall Insulin was 27% higher in PBd and 38% higher in WBd (p<0.0001). The AUC for overall intact GLP-1 increased more than in CBd, by 32% in PBd and by 41% in WBd (p<0.0001) and AUC for overall S was enhanced by 24% in PBd and by 30% in WBd compared to CBd (p<0.0001).

Conclusions: This study demonstrates that increasing protein content from 13 g to 36 g at breakfast has a significant impact on WL, overall S, HbA1c and overall PPG. However, for the same protein content, whey protein vs other protein sources, yields additional benefits on WL, overall S, reduction of PPG and HbA1c. Whey protein should be considered an important adjuvant in the management of T2D

Methods/design: We present preliminary results of an ongoing randomized controlled trial (RCT) designed to comprehensively examine the effect of a lifestyle intervention strategy (behavioral diet therapy for weight loss and exercise training) in older (age: 65-85 yrs.) overweight/obese (BMI ≥ 27 kg/m²) adults with T2D. Main outcomes for this RCT included changes in: a) glycemic metabolic control (HbA1c), b) body weight and body composition (lean body mass, fat mass and visceral fat mass using DXA), c) physical function (Physical Performance Test [PPT], aerobic capacity [VO_2peak]), and d) bone mineral density (BMD) and bone quality (trabecular bone score, TBS).  Subjects were randomized to intensive lifestyle intervention (LI group) or healthy-lifestyle control (HL group) for 6 months.

Results: To date, 17 subjects (age: 70.2±3.8 yrs., BMI: 35.3±6.0 kg/m²_, HbA1c: 7.3±1.2%) have been enrolled and 13 subjects (76.5%) have completed the interventions. There were no significant differences in age and baseline BMI and HbA1c between the LI (n=10) and HL (n=7) groups. After 6 months, HbA1c significantly improved in the LI group compared with HL (-0.7±0.2 vs 0.2±0.4%; p<0.001). Body weight tended to decrease in the LI relative to HL (-8.1±3.2 vs. -1.9±7.7 kg; p=0.08) associated with significant decreases in fat mass (-4.6±6.6 vs. -2.1±6.6 kg, p=0.02) and visceral fat mass (-0.2±0.2 vs 0.0±0.1 kg; p=0.04) but relative preservation of lean body mass (0.3±3.4 vs. 0.9±5.1 kg; p=0.80). In addition, PPT (3.3±1.6 vs. 1.2±6.5) and VO_2peak (2.7±1.1 vs. 0.4±1.5) also significantly improved in LI compared to HL. Finally, although there were no significant changes in BMD, TBS significantly improved in the LI group but not in the HL group (0.09±0.0 vs -0.02; p=0.04).  

Conclusion: These preliminary findings suggest that an intensive lifestyle intervention is not only feasible but importantly confers beneficial effects on glucose control, body composition, and physical function in older adults with T2D.  Further, they provide evidence that bone quality may improve independent of changes in BMD in response to lifestyle intervention. Long-term studies involving a larger sample are needed to follow up on these encouraging results and examine underlying mechanisms.

In this randomized, nonblinded, single-center trial, to evaluate the effectiveness on glycemic control, diabetes remission, security and weight control we evaluated three treatment arms: clinical, gastric bypass and sleeve with ileal transposition (sleeve-IT); in 42 obese class I patients with uncontrolled type 2 diabetes.

Patients’ average age (±SD) was 51±7 years old, and 62% were women. The average glycated hemoglobin level was 9.3±1.9%. Glycemic control was defined as glycated hemoglobin level of 6.5% or less, that was achieved in 8% (1 of 12 patients) in clinical group versus 46% (6 of 13 patients) in the bypass group and 100% (12 of 12 patients) in the sleeve-TI group (p= 0,002 sleeve vs bypass). Diabetes remission was defined as glycemic control without medication and was achieved in 30% (4 of 13 patients) in the bypass group versus 75% (9 of 12 patients) in sleeve-TI group (p=0,02 sleeve vs bypass). Glycemic control improved in all three groups, with an average glycated hemoglobin level of 8±1.3% in the clinical group, 6.9±1.0% in the bypass group and 5,6±0,6% in the sleeve-TI group (p=0,01 sleeve vs bypass). Weight loss was greater in the bypass and sleeve-TI (−22.5± 8,2kg and −18±6,4 kg, respectively) than in the clinical group (−4,7±5,1 kg) (p=0.003 for bypass and p=0,017 for sleeve-TI versus clinical group). Drugs to lower glucose, lipid, and blood-pressure levels decreased after both surgical groups. Four patients experienced serious adverse event. There were no deaths or life-threatening complications.

Although it has been recommended in 2010, by International Diabetes Federation, surgery in diabetic patients with BMI between 30-35 when clinical treatment is not successful, few studies have been published. This study showed that the sleeve-TI surgery has better results in glycemic control than the clinical and bypass treatment. The protocol follow-up will continue for 24 to 36 months to assess the maintenance of the results (NCT01857076).

Forty-six subjects with T1D (mean age [± SD]: 44.0 ± 10.4 years) received a single dose (0.1, 0.2 or 0.4 U/kg) of faster aspart or IAsp in a randomized, double-blind, crossover treatment sequence. PK/PD properties of faster aspart and IAsp were evaluated in an automated euglycemic glucose clamp setting (ClampArt, blood glucose [BG] target 5.5 mmol/L [100 mg/dL]; duration 12 h post-dose).

Across all three doses, onset of appearance with faster aspart occurred approximately twice as fast (mean [95% CI] and relative differences: 0.1 U/kg, –4.7 min [–6.0; –3.4], 47%; 0.2 U/kg, –4.7 min [–5.4; –4.0], 54%; 0.4 U/kg, –4.1 min [–5.3; –3.0], 56%) as with IAsp. Time to 50% maximum exposure (C_max) was observed significantly earlier (8–12 min; 24–32%) across all doses with faster aspart, compared with IAsp. Insulin exposure (AUC_{IAsp, 0–30 min}) was higher in the first 30 min with faster aspart (treatment ratio [95% CI]: 0.1 U/kg, 1.54 [1.29; 1.83]; 0.2 U/kg, 2.09 [1.83; 2.38]; 0.4 U/kg, 2.20 [1.85; 2.62]), compared with IAsp. For both faster aspart and IAsp, total exposure (AUC_{IAsp,0–12 h}) and C_maxincreased with increasing dose levels.

Onset of action (time to first lowering of BG by 0.3 mmol/L [5 mg/dL] from baseline) occurred up to 26% faster with faster aspart (mean [95% CI] and relative differences: 0.1 U/kg, -5.0 min [–10.0; –0.0], 20%; 0.2 U/kg, –5.8 min [–8.6; –3.1], 26%; 0.4 U/kg, –5.6 min [–8.8; –2.4], 26%) than with IAsp. Likewise, time to 50% maximum glucose infusion rate occurred significantly earlier (9–12 min; 22–25%) with faster aspart than with IAsp across all doses. The early glucose-lowering effect for faster aspart was up to twofold greater within the first 30 min (treatment ratio [95% CI]: 0.1 U/kg, 1.48 [1.00; 2.41]; 0.2 U/kg, 1.92 [1.51; 2.58]; 0.4 U/kg, 2.13 [1.60; 3.06]), compared with IAsp. Total and maximum glucose-lowering effects were similar between faster aspart and IAsp. Both treatments were well tolerated and had no safety issues; no injection-site reactions were observed.

Faster aspart showed a faster onset of exposure and a greater initial absorption than IAsp, which was associated with a faster onset and greater early action, compared with IAsp across all three doses. Hence, the fast absorption properties of faster aspart are preserved across a clinically relevant dose range.

Objective: To assess the clinical characteristics and long-term outcomes in young men with suspected PAIS.

Methods: Through the I-DSD Registry, information on the initial presentation and current clinical status was gathered on young men diagnosed as having PAIS (n=52) who presented before the age of 16 yrs and who had undergone genetic analysis of AR.

Results: The median age at presentation and at the time of the study was 1 month (1 day-16 years) and 22 yrs (16-52), respectively. Of the cohort, 29 (56%) had 20 different AR mutations reported. Median external masculinisation score (EMS) at time of diagnosis for AR mut+ve and –ve cases was 7 and 6, respectively (p=0.83). Median current EMS was 9 and 10, in the two respective groups (p=0.09). Testosterone therapy was reported in 16 AR mut+ve (53%) and 6 (26%) AR mut-ve cases. Thirty-one (60%) men required at least one surgical procedure, with AR mut+ve men being more likely to require multiple surgeries for hypospadias repair (p=0.004). All AR mut+ve men had gynaecomastia compared to 9% of the AR mut–ve men. Of the 6 men who had mastectomy, 5 (83%) were AR mut+ve.

Conclusions: Boys with PAIS with an identifiable mutation in AR are at increased risk of poorer medical and surgical outcomes in adulthood. Routine genetic analysis of AR in boys with XY DSD will guide prognosis and personalised management.

Rafael Loch Batista, MD; Marlene Inacio, PhD, Sorahia Domenice MD, PhD; Flávia Siqueira Cunha, MD; Nathalia Lisboa, MD; Vinicius Nahime Brito MD, PhD; Elaine Maria Frade Costa, MD, PhD, Berenice Bilharinho de Mendonça, MD, PhD.

Several psychosexual issues are relevant and rarely explored in patients with disorders of sex development (DSD). There are few reports on the psychosexual outcomes of 46,XY DSD and most of the studies are in 46,XX DSD due to CAH. Patients with 46,XY DSD exhibit a variety of phenotypes at birth ranging from typical female genitalia to undervirilized male external genitalia. Changing from female to male social sex is common, especially in 5α-RD2  and in 17β-HSD3 deficiencies. We evaluated the psychosexual outcomes in 140 patients with 46,XY DSD with different etiologies (testosterone synthesis defects (n=33), 5α-RD2 deficiency (n=30), androgen resistance (n=34), defects in gonadal differentiation (n=33) and with indeterminate etiology (n=10) applying a specific questionnaires on sexual issues. Among these patients, 27 (23.9%) changed the social sex in adulthood (24 female to male and 3 male to female). The psychosexual outcomes were compared between the groups that changed social sex vs the group that kept the social sex in adulthood and between the male and female final social sex in adulthood. In male social sex, we did not observe differences between the group that changed with group that kept the male social sex (p >0.05). In male and female social sex, comparing patients who changed social sex versus the ones who kept the assigned sex, we observed differences on the frequency of orgasm (p=0.015), masturbation (0.02), satisfactory intercourse (<0.001) and sexual life satisfaction (0.016). All parameters were better in the male sex. There was better satisfaction with sexual life in males (73%) compared to females (51%), although these rates are lower than that observed in non-DSD control population (1, 2). We conclude that psychosexual outcomes in male and female 46,XY DSD patients in adulthood are adequate but the patients with male social sex shows better parameters.

References: (1) Moreira ED Jr, Abdo CH, Torres EB, et al. Urology 2001 Oct;58(4):583-8. (2) Abdo CH, Oliveira WM Jr, Moreira ED Jr et al. Int J Impot Res. 2004 Apr;16(2):160-6.

Nothing to Disclose: RLB, MI, SD, FSC, NL, VNB, EMFC, BBM

Objectives: To address these concerns, we recently formed a multi-disciplinary Gender Dysphoria Team with endocrinology, mental health, pharmacy, speech therapy, and vocational rehab providers at the Salt Lake City Veterans Affairs Medical Center (SLC VAMC).  Providers meet with patients individually and bi-monthly as a team to discuss patients and treatment plans. We defined baseline characteristics of mental health diagnoses in our population.

Methods: We examined SLC VAMC electronic medical records between January 1, 2014 and October 1, 2015 for encounters using the ICD-9 code for Gender Identity Disorder in Adolescents or Adults. Charts with the code were then examined for ICD-9 codes for tobacco use disorder, posttraumatic stress disorder (PTSD), depression, and anxiety-related conditions. Other ICD-9 codes for mental health diagnoses and suicide attempt/ideation requiring crisis contact were noted.

Results: Thirty-nine patients were found with the GID ICD-9 code. Of those, 31 (79.5%) identified as male-to-female, 8 (20.1%) female-to-male; 19 (48.7%) had a history of suicide attempt/ideation. PTSD was the most commonly identified mental health diagnosis (18/39 (46.1%)), depression second (16 (41.0%)), followed by tobacco use disorder (13 (33.3%)), other (substance abuse, bipolar disorder, schizotypal disorder) (9 (23.1%)), and anxiety (6 (15.4%)). When further examined, four (10.2%) patients were identified as having no mental health co-morbidities, 17 (42.6%) carried one mental health diagnosis and 18 (46.2%) carried 2 or more diagnoses (maximum number documented in one patient: 4 (10.2%)).

Conclusions: Of patients with GID at the SLC VAMC, 90% have mental health co-morbidities, nearly 50% having two or more diagnoses. Nearly 50% required intervention for suicide attempt/ideation. We have formed the multi-disciplinary Gender Dysphoria Team in an effort to reduce the impact of these diagnoses on the management of GID, however, further research into the optimization of management for this population is still needed.

Objectives: To investigate effects of CSHT on BMD during the first year of treatment in male-to-female (MtFs) and female-to-male transgender patients (FtMs). 

Methods: This study is a prospective observational study and part of ENIGI (European Network for Investigation of Gender Incongruence). 205 adult patients who completed one year of CSHT were included. In 107 FtMs and 98 MtFs a dual-energy X-ray absorptiometry was performed to measure lumbar spine BMD before and after a year CSHT. FtMs received intramuscular testosterone undecanoate (1000mg/12 weeks), testosterone gel (50mg/day) or testosterone esters intramuscular (250mg/2 weeks). MtFs were treated with oral estradiol valerate (2-4mg/day) or an estradiol patch (200ug/week). Most MtFs received cyproteronacetate (50mg/day) simultaneously.

Results: At baseline the mean lumbar spine BMD of FtMs was 1.02 g/cm² (SD±0.13) and after one year 1.03 g/cm² (SD±0.13), reflecting a mean increase of 1.12% (95%CI 0.32 – 1.92%). In MtFs at baseline and after one year, the lumbar spine BMD was 0.98 g/cm² (SD±0.14) and 1.01 g/cm²(SD±0.14), respectively, reflecting a mean increase of 3.71% (95%CI 2.82 - 4.60%).

Conclusion: After one year CSHT the spine BMD increased in both groups, but more in MtFs. This confirms the role of estrogen on bone in biological males.

Supported by FAPESP grant#2014/10965-6 and NIH/NCI award R01 CA172220.

We retrieved anthropometric data of 33.537 subjects from the CrescNet® data base and included those with measurements of at least two time points.

Overweight and obesity were present in 10% and 9% of the adolescents (15-18 years), respectively. Changes from normal weight to overweight/obesity in childhood were considered and probability of being overweight/obese in adolescence given a certain BMI-SDS in childhood was calculated.

Retrospectively seen, 53% of the children, who were obese in adolescence were normal weight until the age of 4 years. By the age of 5 years, however, half of them were overweight/obese. Prospectively, 60% of children who are overweight already at the age of 4 years remained overweight/obese in adolescence. The probability of being overweight/obese in adolescence in those who are already obese at the age of 4 was 79%. In a subgroup of 26.344 children we considered the influence of BMI acceleration in different age periods. Dynamically, children in the age of 2 to 5 years, who had a rise in BMI-SDS by 0.2-2 during 1 year had a higher risk of being overweight/obese in adolescence than older children with the same BMI acceleration.

In conclusion, overweight/obesity in adolescence is already determined in early childhood. Periods of increased BMI acceleration in early childhood should be recognised for an optimized strategy in obesity prevention.

Methods: The study sample included patients age ≥18 years old with BMI ≥ 30 kg/m² (first observed BMI measurement was defined as index BMI) who had no medical conditions associated with unintentional WL and had ≥4 BMI measurements per year for ≥5 years.  Patients were categorized into 4 weight groups based on their amount of weight change during a WL period of two quarters following the index BMI: stable weight= within <5% of index BMI; modest WL= ≥5 to <10% of index BMI lost; moderate WL= ≥10 to <15% of index BMI lost; and high WL= ≥15% of index BMI lost.  Patterns of weight change were also assessed following the WL period for 8 consecutive quarters (i.e., the weight maintenance [WM] period).  Subgroup analyses were conducted among patients with class II obesity (index BMI≥35), pre-diabetes, and type 2 diabetes (based on diagnoses and lab values).

Results: Of 194,490 patients, 177,743 patients were included in the main analysis as they were classified in one of the 4 weight groups of interest during the WL period: 151,236 (85.1%) patients were in the stable weight group, 16,559 (9.3%) were in the modest WL group, 4,017 (2.3%) were in the moderate WL group, and 5,931 (3.3%) were in the high WL group.  Though 7.2% of the modest WL group continued to lose weight in the first quarter of the WM period, this dropped to 2.0% by the eighth quarter; in the moderate WL group these proportions were 13.3% and 4.1%; and 18.8% and 11.1% for the high WL group.  A decreasing trend in the overall proportion of patients who maintained their weight throughout the WM period was also observed.  In the modest, moderate, and high WL groups 40.0%, 35.9%, and 18.6%, of patients, respectively, regained ≥50% of lost weight during the WM period.  Cyclers were defined as patients who did not consistently lose, maintain, or gain weight in each quarter of the WM period.  The high WL group had the lowest proportion of cyclers with 58.3%, while 71.5% of the modest WL group and 74.1% of the moderate WL group were cyclers.  This trend was similar in the subgroups analyzed, though among patients with class II obesity those with high initial WL were more likely to continue to lose weight during the WM period compared to the overall sample and other subgroups.      

Conclusion: Weight cycling and regain was commonly observed among patients.  Patients who lost more weight during the WL period (overall and in subgroups) were more likely to keep the weight off and continue losing weight.

Objective: To identify novel biomarkers in obese adolescents, assessed at the time of study enrollment, that predict successful response to a 4-month lifestyle modification intervention using non-targeted proteomics and metabolomics.

Methods: The primary outcomes of this behavioral weight loss trial are published.(1) Outcome measures included 4- and 12-month changes in body mass index (BMI), proteomics (1126 proteins, SOMAScan, SomaLogic, Inc), metabolomics (417 metabolites, Metabolon, Inc). Normalized protein and metabolite levels were compared between weight loss response groups using rank product (RP) testing (2) to obtain p values and false discovery rates (FDRs) through a permutation procedure. Proteins/metabolites were also mapped to curated sets for pathway analysis.

Results: Participants (n=40) were 74% female, 38% African-American, 14.9 years old (±1.2 SD), with baseline BMI Z-score of 2.31 (±0.29 SD). On average, BMI decreased by 7.2±1.3% after 4 months, and decreased by 5.5±2.2% after 12 months. Subjects were divided into 4 groups based on their weight loss responses: “Group 1” (n=13), strong 4- and 12-month responses, “Group 2” (n=10), strong 4-month response, but some weight regain by 12 months, “Group 3” (n=15), modest but consistent response over 4 and 12 months, and “Group 4” (n=9), poor response at both 4 and 12 months. We prioritized comparisons between “Group 1” (treatment-responsive) and “Group 4” (treatment-resistant) participants.

The most apparent group-specific differences were levels of immunomodulatory and neutrophil associated proteins that were higher in treatment-resistant participants (p<0.001, FDR <1%, for both). In addition, fibroblast growth factor 19 (FGF19), an insulin-independent modulator of hepatic protein and glycogen synthesis, was also significantly higher in treatment-resistant subjects (p=0.0001, FDR=1%). Finally, levels of proteins related to metabolism of amino acids (Reactome R-HSA-7121) were different between treatment-resistant versus treatment-responsive obesity (p=0.0016, FDR=3%), and levels multiple individual protein and amino acid derivatives were also nominally different, e.g., gamma-glutamylmethionine (higher in treatment-resistant group, p=0.0048, FDR=25%).

Conclusions: We speculate that increased activity of immunomodulatory and neutrophil associated pathways is related to treatment-resistant obesity in adolescents. In the future, leveraging biomarker profiles may facilitate individualized, rational treatment formulations for pediatric obesity. Investigation of the above signals in a separate validation cohort is ongoing.

 Methods: Two lean cohorts (BMI 5^th to ≤75%ile; one from low-income primary care clinic, one from longitudinal epidemiologic study) and two severely obese cohorts (BMI≥99%ile; one from low-income primary care clinic, one from obesity referral clinic) were selected based on BMI between ages 2-6.  Growth data were abstracted from birth through age 6 for each. In addition, sex, race, and health insurance type were collected. Repeated measures mixed modeling and logistic regression were used to distinguish growth characteristics of the lean and obese cohorts.

 Results: 783 lean (647 low-income, 136 population-based) and 480 severely obese (365 low income, 115 referral clinic) participants were included. Sex distribution of patients was similar among all groups. Both the low-income populations were predominantly African-American at 79% and 62%, respectively, while the population-based and referral clinic had a significantly lower proportion of African-Americans, at 24% and 23%, respectively. Clinical obesity onset occurred at median 2.00 [IQR: 1.24, 3.11] years of age in the low income obese, and at 1.36 [0.61, 3.02] years in the referral obese group. However, BMI differed significantly between severely obese and lean cohorts by 4 months of age (p<0.0001) while WHO weight-for-age percentile differed by 2 months (p<0.0001), prior to obesity onset. BMI high-specificity (95%) thresholds accurately differentiated severely obese from lean groups at 6, 12, and 18 months of age (51%, 52% and 95% sensitivity, respectively).

 Conclusions:  BMI trajectories in children who become severely obese by age 6 differ from children who remain normal weight as early as 4-6 months of age, approximately 12-18 months prior to the median onset of clinical obesity. Infant BMI at 6, 12, or 18 months above 85^th percentile on WHO growth chart is highly specific for identifying children at risk for early onset severe obesity and should prompt close monitoring.

Using case matching to control for differences in age, sex, body mass index (BMI) and estimated glomerular filtration rate (eGFR), fasting PP was significantly higher in individuals with T2DM than either NGT (T2DM n = 105, PP median 35.0 pmol/L (interquartile range 21.7-62.2); NGT n = 105, PP 25.2 pmol/L (12.8-31.1); p < 0.0001) or IFG/IGT (T2DM n = 84, PP 51.6 pmol/L (21.6-62.8); IFG/IGT n = 84; PP 23.4 (15.8-46.7); p = 0.002), while PP did not differ significantly between individuals with IFG/IGT  and those with NGT.  This difference was particularly interesting given that the clinical distinction between T2DM and IFG/IGT is predicated on the greatly increased vascular disease risk in T2DM. 

Among individuals with T2DM and with adequate retinal screening within 1 year of the study date (n=236), we found that PP was significantly higher in those with background retinopathy (66.1 pmol/L (38.5-102.3), p < 0.01) or moderate non-proliferative or proliferative retinopathy (61.0 pmol/L (40.29-91.05) p < 0.05) than individuals without retinopathy (35.58 pmol/L (21.68-64.77)).   In common with other peptide hormones, fasting PP is increased in renal impairment due to reduced filtration and metabolism.  However, PP was significantly elevated in participants with T2DM who showed evidence of persistent microalbuminuria (50.3 pmol/L (28.4-75.0) cf. 35.0 pmol/L (21.7-65.0), p = 0.046) when those with eGFR < 60 mL/min/1.73m² were excluded. We used a logistic regression model to further investigate the association of elevated PP with diabetic retinopathy.  Log-transformed PP remained a significant predictor of retinopathy when age, sex, duration of diabetes, HbA1c, systolic blood pressure, eGFR and log-transformed serum triglycerides were included as covariates (p = 0.039, OR =  3.672 per 10-fold increase in PP).  

Measuring visceral adiposity is expensive and difficult to do accurately in a clinical context.  We speculate that PP not only acts as a measure of visceral adiposity but may also reflect its contribution to vascular risk.  Given that retinopathy is associated with, and usually precedes, other microvascular and macrovascular complications of T2DM, the association we describe raises the possibility that fasting PP measurement may be of use in vascular risk stratification.

Materials and methods: data of 473,983 newborns (NBs) were retrospectively evaluated. N17OHP was measured by IFMA (AutoDelfia) and cutoffs (99^th and 99.8^th percentiles) adjusted according to birthweight (BW1:<1500g; BW2:1500-2000g; BW3:2001-2500g; BW4: >2500g), and to age at sample collection (before or after 72hs of life). For confirmatory tests, serum 17OHP analyses were performed by RIA and LC-MS/MS and 21-deoxicortisol (21DF), Δ4 and cortisol levels by LC-MS/MS. Asymptomatic NBs with persistently increased serum 17OHP levels had entire CYP21A2 sequenced in peripheral DNA samples.

Results: the recall rate was 0.05% (n=221) using the P99^th of N17OHP levels and decreased to 0.03% (n=149) using the P99.8^th; additionally, PPV increased from 11% (P99^th) to 17% (P99.8^th). N17OHP cutoffs in samples collected before 72hs of life were significantly lower than those collected after. Since in our state since most samples are collected earlier, different cutoffs according to BW and age at sample collection were determined. Twenty-six NBs were diagnosed (22 SW, 4 SV, 12 males), confirmed by sequencing. N17OHP levels ranged from 53-494 ng/mL (serum equivalence) in SW form and from 36-53 ng/mL in SV form. Serum confirmatory tests were performed in 149 NBs. In affected NBs, serum 17OHP levels (LC-MS/MS) ranged from 56-668 ng/mL in SW and from 54-117 ng/mL in SV form. FPR persisted using 17OHP measurements in 70% of samples by RIA and 13% by LC-MS/MS. PPV of LC-MS/MS methodology was significantly higher than RIA (52 vs.27%). Serum 21DF and steroid ratios (17OHP/cortisol; 17OHP+Δ4/cortisol; 17OHP+21DF/cortisol) presented similar FPR and PPV values in comparison to 17OHP by LC-MS/MS. Serum 21DF and steroid ratio values overlapped between affected NBs and those with FPR. Among asymptomatic NBs with persistently increased serum 17OHP levels, molecular analysis identified 2 with NC form.

Conclusions: N17OHP levels adjusted to P99.8^th and to sample collection time improve the CAH-NBS by reducing the FPR rate without missing the diagnosis of classical forms. Although serum 17OHP by RIA is widely used as confirmatory test in our country, the use of 17OHP by LC- MS/MS significantly reduced the FPR. The 21DF and steroid ratio measurements did not provide higher accuracy than serum 17OHP by LC-MS/MS. Molecular analysis could be restricted for asymptomatic NBs with persistently increased 17OHP levels.

Treatment protocol: HC-treatment in children with CAH is applied 8 hourly  with a 1^st dose (D1) in the morning (6-8h) , 2^nd dose (D2) in the early afternoon (14-16h) and 3^rd dose(D3) late at night (22-24h). Therapy monitoring used saliva-17-OHP profiles collected at home directly prior to the HC medication (5 - 10fold concentrations of age-specific reference range used as target range). In case of out of range salivary 17-OHP profiles the HC dose prior to the sampling point was lowered or increased. Patients are followed clinically (and by saliva profile) every 3 months, blood-sampling takes place once a year at the clinic appointment. In a sub-cohort of children participating in the pharmacokinetic part of the TAIN-treatment trial blood sampling was timed exactly prior the morning dose of HC.

Methods: We describe the dosing scheme after individualized adaptation by 17OHP saliva profiles of 31 children aged 1-11 years (females n=15, males n=16) in a longitudinal period of 2 years. Growth indicators, dosing and treatment adaptation frequency at the different medication times as well as ACTH and androgen levels in randomly or timed blood sampling were used as outcome parameters.  8 infants started saliva sampling within the study period.

Results: The resulting dosing scheme based on our “pre-dose-at-home-saliva-sampling” is different from published recommendations (40 – 20 – 40%).  Dose adaptation frequency was 59/248 visits (30% D1, 29% D2, 41% D3). Single dose adaptation occurred in 65%, two-dose adaptation in 20% and three-dose adaptation in 15%.   Mean total dose at T0 (10,77 mg/sqm/d) / T-12 month (10,88 mg/sqm/d) / T-24 month (11,03 mg/sqm/d)  was declining during the study period. Blood androgen levels were different between randomly and timed sampling. Height SDS and weight SDS remained stable in saliva controlled patients.

Conclusions: Dose adaptation by saliva sampling combined with 8hourly dosing scheme results in a rise of the late night dose and lowering the afternoon doses. Total HC doses are within the recommended ranges for HC replacement-doses in CAH-children. Regularly saliva profiling is easy to perform and leads to an individualized treatment based on a more frequent dose-adaptation. Saliva sampling was well tolerated and without stress for the children. Random and timed blood androgen levels are of limited value for therapy optimization.

PATIENTS AND METHODS:  This randomized controlled study included 30 peripubertal boys (age range 9.1-14.2 yrs) with idiopathic short stature (ISS) (3). Sixteen boys were treated with letrozole (2.5mg/d) for 2 yrs and 14 received placebo. Boys were followed up for 3 yrs, hormonal and MKRN3 levels were obtained with 6 mo intervals for 2 yrs, and analyzed using summary measures.

RESULTS: The boys showed an age-dependent decline in serum MKRN3 levels (mean regression coefficient -6.6±7.2 pg/mL per year, P < 0.001), with no difference between the two groups. Importantly, MKRN3 levels declined before Tanner genital stage 2, but not thereafter (-29.3±27.5 vs -5.6±15.1 pg/mL per year) (P < 0.05). During Tanner genital stage 1, the rate of MKRN3 change correlated negatively with the rate of increases in testosterone (r= -0.4, n=28, P < 0.05), LH (r= -0.5, n=26, P < 0.01) and inhibin B (r= -0.44, n=26, P < 0.05) levels.

CONCLUSIONS: In boys, peripheral MKRN3 levels decrease prior to clinical onset of puberty in an inverse association with circulating markers of HPG axis activity. Inhibition of estrogen biosynthesis has no effect on circulating MKRN3 levels.

We have studied 61 normal/overweight girls (Tanner I-V) via q 10 min sampling that included the 1900 to 2300 h time block—a period of wakefulness in all cases. LH pulse count for this 4 h time block was determined. Sex steroids were sampled q 60 min. Simple and partial Spearman rank correlations were used to assess relationships between LH pulse count while awake and sex steroid levels. Since both P and T may influence pulse frequency, and since both may change across puberty, we assessed the correlation between daytime LH pulse count and the molar ratio between P and free T (P-to-T molar ratio, with each hormone in pmol/L). Subjects were also analyzed in groups defined by daytime LH pulse counts: 0 (“none,” n=10), 1-2 (“some,” n=13), and ≥3 (“more,” n=38).

Among the sex steroids assessed, daytime LH pulse count correlated best with free T (R=0.68, p<0.0001), versus estradiol (R=0.40, p=0.001) and P (R=0.34, p=0.007). Daytime LH pulse count also exhibited a significant negative correlation with P-to-T molar ratio (R=-0.67, p<0.0001). When correcting for bone age (BA) or Tanner stage (TS), most correlations lost statistical significance—except for free T corrected for TS (R=0.40, p=0.002), P corrected for BA (R=-0.47, p=0.01), and P-to-T molar ratio corrected for either BA (R=-0.45, p=0.003) or TS (R=-0.49, p=0.01). Girls with no daytime LH pulses (“none”) tended to have lower free T levels (2.7 ± 0.5 pmol/L [mean ± SEM]) compared to “some” (19.5 ± 8.0) and “more” (22.8 ± 3.6) groups (p=0.057 and p<0.0001, respectively). Likewise, girls in the “none” group had higher P-to-T molar ratios (291 ± 49 pmol/L / pmol/L) compared to “some” (138 ± 24) and “more” (90 ± 12) groups (p=0.02 and p=0.003, respectively).

These results indicate that both increasing free T and decreasing P-to-T molar ratio correlate with daytime LH pulse counts, even when corrected for pubertal status. These data are consistent with the notion that rising T levels across puberty antagonize the ability of P to restrain daytime GnRH secretion, thus allowing a gradual increase in daytime LH frequency.

Objective:  Longitudinally evaluate gonadal function and fertility potential in a pediatric and young adult population after RIC HSCT.

Methods:  Children and young adults ≥ 1 year after a single RIC HSCT regimen were followed in our prospective cohort study to evaluate late effects of HSCT on the endocrine system and fertility potential. Subjects were evaluated for pubertal development and hormonal status, and semen analysis was obtained for specifically consented males.  All patients received RIC regimen prior to HSCT, with the majority receiving Fludarabine and Melphalan +/- Campath, and none had total body irradiation. Five of 14 patients (36%) had received prior chemotherapy for malignancy.  The median age at time of HSCT was 13.6 years (range 3.4-24.3). The average age at time of semen analysis following HSCT was 20.3 years (range 15.6-25.2), with a median time from HSCT of 5.6 years (range 1.9-10.0).

Results:  Preliminary results were obtained from 14 male subjects >11 years of age after RIC HSCT.  Of those, 11 (79%) were pubertal.  Of the 9 pubertal subjects with available laboratory testing, only one (11%) had abnormally elevated gonadotropins (LH and FSH).  Similarly, only one (11%) had an abnormally low testosterone level.  The remainder had normal testing for Tanner stage.  Interestingly, 4 of 9 (44%) had abnormally low inhibin B levels, suggestive of reduced fertility and Sertoli cell dysfunction.  Semen analysis was abnormal in all 8 subjects tested: azoospermia was diagnosed in 88% (7/8) and oligoteratospermia in 12% (1/8).  

Conclusion:  Our analysis suggests that, despite its potential benefits, RIC HSCT may be associated with a high risk of impaired spermatogenesis and infertility, similar to the risk associated with traditional, myeloablative HSCT. However, gonadotropins and testosterone appear to be normal in most young male subjects after RIC HSCT. Therefore, normal pubertal development does not ensure fertility potential following RIC HSCT.  Whereas our data suggest inhibin B levels can be useful for screening those at risk for infertility, semen analysis is a more reliable, definitive measure.  Risk of infertility should be included in counseling about RIC HSCT, and fertility preservation should be discussed and offered to these patients prior to HSCT if possible. Additional studies to confirm these data in a larger cohort are underway.

Objective: We aimed to share data on the largest cohort of patients with the IGSF1 deficiency syndrome to date and formulate recommendations for clinical management.

Methods: We collected clinical and biochemical characteristics of 69 male patients (35 children, 34 adults) and 56 female carriers of an IGSF1 mutation (3 children, 53 adults) from 30 unrelated families according to a standardized clinical protocol. At evaluation, 89% of boys were treated with levothyroxine, as were 44% of adult males, and 5% of females.

Results: Several additional symptoms were identified in male patients. Thyroid gland volume was small in 74%, birth weight was high in 25%, and head circumference was large in 20%. Late adrenarche was observed in patients with prolactin deficiency, and adult DHEA levels were decreased in 40%. In general, the timing of pubertal testicular growth was normal or even advanced, in contrast to a late rise in pubertal testosterone levels. Hypocortisolism was documented in 6 of 28 evaluated newborns, although cortisol levels were normal later on. Male patients' waist circumference was increased in 60%, but blood lipids were normal. Female carriers showed low FT₄ and low-normal FT₄ in 18% and 60%, respectively, delayed age at menarche in 31%, mild prolactin deficiency in 22%, increased waist circumference in 57%, and a negative correlation between FT₄ concentrations and metabolic parameters. Based on the large number of patients identified in only three years, IGSF1 deficiency represents the most common genetic cause of relatively isolated central hypothyroidism. 

Conclusion: IGSF1 deficiency syndrome represents the most common genetic cause of central hypothyroidism and is associated with multiple other characteristics. The results allow the formulation of recommendations for mutational analysis, endocrine work-up, and long-term care.

To address the question of whether BPA acts directly, we utilized an ex vivo culture method of the fetal mammary gland (3). In this method, the direct action of estrogen and estrogen-mimics can be tested during E14 to 19, a critical window of exposure. This method allows for the direct observation of development as it occurs.

Mammary buds of CD1 mice were dissected at E14 and cultured for 5 days. The explants were exposed to BPA or 17β-estradiol (E2). Morphometric analysis was performed on the explant whole-mounts and markers of epithelial and mesenchymal development were detected by immunofluorescence.

We show that BPA exerts a direct effect on the fetal mammary gland. Exposure of the explants to 10^-9M BPA significantly increased ductal growth (89711 ± 6836 µm²; p=0.028; n=20) while 10 ^-6M BPA decreased it (33518 ± 3761 µm²; p=0.006; n=9) compared to controls (67615 ± 8806 µm²; n=15). Epithelial growth was significantly diminished in mammary buds exposed to 10^-9 (48337 ± 5200 µm²; p=0.005; n=16), 10^-11 (49441 ± 4604 µm²; p=0.000; n=39) and 10^-13M (54414 ± 6404 µm²; p=0.031; n=15) E2 compared to controls (76802 ± 6305 µm²; n=43); lower doses had no effect.

Our findings show that fetal mammary gland development is altered by direct action of estrogenic compounds. BPA shows a non-monotonic dose response curve whereby low dose increases ductal development and high doses inhibit it. Moreover, increased ductal development was observed at doses comparable to those producing similar effects in vivo (2). In contrast, estradiol resulted in a monotonic inhibition of ductal growth. Finally, in addition to its usefulness to the understanding of the hormonal regulation of mammary gland development, this ex vivo culture system could serve as a bioassay to test the numerous new chemicals that are synthesized each year and released into the environment without proper assessment of hormonal action on critical targets like the mammary gland.

 We find that exposure of the developing liver to TBT, or treatment of liver cells in vitro, alters the expression of key genes involved in energy homeostasis, including Shp and Acox.  Altered expression of these genes correlates with increased H3K4me3, indicating that TBT-induced changes in transcription are associated with altered histone methyltransferase (HMT) activity.  To test the hypothesis that TBT engagement of PPARγ/RXRα is directly responsible for altered HMT recruitment/activity, we have developed a DNA pulldown assay employing recombinant PPARγ and RXRα proteins incubated with rat liver FAO cell nuclear extract (NE), DMSO vehicle or TBT, and a biotinylated Shp gene promoter containing an evolutionarily conserved PPAR/RXR binding site. After pulldown with streptavidin beads, bound CoR proteins were detected by mass spectrometry (MS). From the MS data, we identified proteins involved in chromatin remodeling, tethering/looping, and histone modifications (histone acetyltransferases (HATs) and HMTs) that were enriched at least 2-fold with TBT. Similar CoR recruitments were seen using a biotinylated 3xPPRE template derived from the rat Acox promoter.

Importantly, TBT recruited p300, DPY30, a core component of SET/MLL HMT complexes, and SRC-2 to PPREs in an RXR-dependent manner. Consistent with this finding, the RXR inhibitor HX531 reduced TBT-induced recruitment of these three CoRs from HeLa cell NE to 3xPPREs. Combined these data suggest that specific CoRs are recruited to target genes by TBT binding to RXR, and point to a model where TBT binding to the PPAR/RXR heterodimer recruits distinct histone “modifying” CoRs to “write” histone marks that will persist to reprogram TBT-target genes in the adult liver to a metabolic setpoint favoring obesity.

* LST and CEF contributed equally to this work.

Background: The long-term legacy of pediatric critical illness comprises a severe and unexplained attention deficit.¹ Phthalates, which may be neurotoxic, are used to soften plastic indwelling medical devices and can leach into the circulation.^{2, 3}

Hypothesis: We hypothesized that in children treated in the pediatric intensive care unit (PICU), circulating phthalates leaching from indwelling medical devices contribute to their long-term attention deficit.

Methods: Circulating plasma concentrations of di(2-ethylhexyl)phthalate (DEHP) metabolites were quantified in 100 healthy children and 449 children who had been treated in PICU and were neurocognitively tested 4 years later. In a development patient cohort (N=228), a multivariable bootstrap study identified stable thresholds of exposure to circulating DEHP metabolites above which there was an independent association with worse neurocognitive outcome. Subsequently, in a second patient cohort (N=221), the observed independent associations were validated.

Results: Plasma concentrations of DEHP metabolites, that were virtually undetectable [0.029(0.027-0.031) µmol/l] in healthy children, were 4.41(3.76-5.06) µmol/l in critically ill children upon PICU admission (P<0.001). Plasma DEHP metabolite concentrations decreased rapidly but remained 18-times elevated until PICU discharge (P<0.001). After adjusting for baseline risk factors and duration of PICU stay, and further for PICU complications and treatments, exceeding the potentially harmful threshold for exposure to circulating DEHP metabolites was independently associated with the attention deficit (all P≤0.008) and impaired motor coordination (all P≤0.02). The association with the attention deficit was confirmed in the validation cohort (all P≤0.01). This phthalate exposure effect explained half of the attention deficit in post-PICU patients.

Conclusions and Relevance: Exposure to phthalates leaching from indwelling medical devices used for intensive medical care in children was independently and robustly associated with their long-term attention deficit. Development of alternative plasticizers for this application may be indicated.

Exogenous administration of FSH and LH rescued anovulation and lead to subsequent pregnancy in approximately one-third of the ERKdko animals. However, litter sizes from ERKdko mice were significantly smaller than controls (2.4±0.29 vs. 9±1 pups, n=7, n=3, respectively, p<0.01). Furthermore, 15/17 pups from ERKdko mothers recovered were stillborn, and the two live pups died within hours of birth. No ERKdko pups were alive by postnatal day 3 (0% vs. 85.7± 11.7% survival in control litters, n=7, n=3, respectively, p<0.01). Gestation length was significantly longer in ERKdko animals (23.9±0.55 days vs. 20.25±0.25 days, n=5 and n=4 respectively, p<0.01), and parturition lasted significantly longer (2.25±0.42 days vs. 1 day in control animals, n=4 each, p<0.05).

To dissect the mechanisms underlying this profound reproductive phenotype, we started to examine the placentas for potential GnRHR expression during gestation. We performed qRT-PCR on C57/Blk6 mouse implantation sites/placentas from embryonic day (e)5.5 (n=4), e10.5 (n=4), e12.5 (n=3), and e18.5 (n=1) and compared them to pituitary GnRHR mRNA expression levels. Strikingly, we found that placental GnRHR mRNA levels were comparable to those in the pituitary at e5.5 and e10.5, and 9-fold greater in the placenta at e18.5. These studies suggest that GnRHR-mediated Cre expression in the placenta may effectively inactivate the floxed ERK1/2 target genes in this tissue. We speculate that loss of ERK signaling in the placenta of ERKdko animals causes alterations of the maternal/fetal interface leading to disruption of initiation and/or progression of parturition, resulting in fetal mortality. Further, these studies reveal a potentially important model of placental-specific Cre recombinase.

To test the hypothesis that Sirt1 level or activity is altered with OVX, 9-week-old C57BL/6 female mice (n=10/group) were subjected to OVX or SHAM operation, and were left untreated for 6 weeks. OVX and SHAM mice were sacrificed 1 and 6 weeks post operation. The remaining OVX mice were treated for 6 weeks with either sc daily 10mg/kg 17-β estradiol (E2), SRT3025, a Sirt1 activator, administered by gavage kindly provided by Sirtris/GSK, at 100 mg/kg/day once daily or a vehicle. SHAM mice were left untreated. Mice were kept on standard chow diet, weighed weekly, and serum was collected 1,6,12 weeks post operation. Upon sacrifice uterine weight was determined to ensure a successful OVX, livers and brains were collected. In vitro experiments were conducted in a model cell line.

OVX induced a significant weight gain of 27% and a 61% decrease in uterine weight over the 12 week period. Treatment with SRT3025 blunted OVX-induced weight gain (1.9% vs 4.6% in SRT3025-treated and untreated OVX mice, respectively), whereas E2-treated mice had a weight gain of 3.9%. Uterine weight was similar in OVX untreated and OVX SRT3025-treated mice (36.3±21.04 mg and 45.7±22.0 mg, respectively vs. 97.6±19.5 mg in E2-treated mice), suggesting no uterine estrogen-like effect. Strikingly, a dramatic decrease of 40% in Sirt1 protein level was observed 6 weeks post operation in liver and brain obtained from OVX compared to SHAM mice. Liver Sirt1 mRNA expression was significantly elevated by 1.8 fold in OVX compared to SHAM mice. To elucidate underlying mechanisms, C3H10T1/2 cells were exposed to 10-100nM E2, 1-10uM fulvestrant or serum derived from OVX and SHAM mice 1 week post operation. While no effect in Sirt1 protein level was detected in E2- and fulvestrant-treated cells, serum derived from OVX mice induced a marked decrease in Sirt1 expression, suggesting that OVX-related humoral factors influence Sirt1 expression.

In conclusion, reduced Sirt1 is a possible contributor to OVX-induced weight gain. Reduced Sirt1 appears to result from indirect effects of estrogen withdrawal. Pharmacologic activation of Sirt1 blunted OVX-associated weight gain without inducing an undesired effect of increased uterine weight. Future studies are needed to evaluate the effects of Sirt1 activation on menopause-related metabolic derangements and co-morbidities.

To test this hypothesis, we analyzed the skeletal muscle of TRα1PV mice 4 days after cardiotoxin-induced muscle injury and compared with wild type (WT) animals. We injected TRα-knockdown C2C12 myoblasts into injured skeletal muscle and compared the in vivo behavior, 4 days after transplantation, with control C2C12 myoblasts. The cells were labeled with DiI to facilitate in vivo tracking.   

We found significant muscle inflammation in TRα1PV mice, 4 days after muscle injury, compared with WT mice.  The total number of PAX7-positive SCs in skeletal muscle of TRα1PV mice was significantly lower than control, both before and 4 days after muscle injury, indicating SC pool exhaustion. PAX7/Ki67 double staining after muscle injury showed significantly lower SC proliferation in TRα1PV mice. Ki67 was co-expressed in about 50% of the PAX7-expressing SCs in the control muscle, but less than 5% in the TRα1PV muscle. The mice injected with control-SCs showed efficient engraftment and migration of the donor cells in close proximity to the muscle fiber. Mice injected with TRα-knockdown SCs showed few donor cells localized in the sub laminar space.

Our data indicate that TRα plays an important role in maintenance of the SC niche. Impaired skeletal muscle regeneration in TRα1PV mice may be explained by insufficient SC activation and proliferation, and progressive loss of the satellite cell pool. Regulation of SC proliferation provide a therapeutic target  to enhance skeletal muscle regeneration.

Tumorigenic clone of PTC cells (BHP10-3SCp) were implanted in nude mice, following recombinant human TSH (rhTSH) or saline for 3 weeks (n=10/each). Tumors of rhTSH group were bigger than saline group from day15 to day 20 (1733.4±793.5 vs 1148.8±471.1mm³, p<0.05). At day 20, 3 of 10 mice showed tumor bleeding in rhTSH group, while saline group showed none. Immunofluorescent staining of CD31 showed higher density of vasculature in rhTSH and saline group. Intravascular injection of FITC-labeled nanoparticles showed dilated tortious vascularity in rhTSH group compared to saline group. Moreover, tumors of rhTSH group showed higher density of F4/80-positive macrophages than that of saline group. To evaluate the mechanistic insight of the TSH actions on tumor angiogenesis, TSH were treated to BHP10-3SCp cells and vascular endothelial growth factor (VEGF) expressions were evaluated. mRNA expressions were up-regulated by 3-fold and 9-fold at 48 and 72 hrs, respectively. Conditioned medium (CM) of TSH-treated BHP10-3SCp cells showed increased VEGF concentrations than that of control-CM. Next, TSH-CMs were treated into microvascular endothelial cells (HMVEC) or macrophages (THP-1). TSH-CM treated group showed enhanced HMVEC migration and tube formation potentials than control-CM group, and these effects were attenuated with treatment of Bevacizumab, a humanized anti-VEGF antibody. Finally, TSH-CM also increased migration potentials in THP-1 cells.

In conclusion, TSH supported PTC tumor growth by enhancing tumor angiogenesis and macrophage recruitment into tumor microenvironment and its action on PTC tumor angiogenesis was partly mediated by VEGF, which may be a potential therapeutic target in TSH-dependent PTC progressions.

Methods This open-label, randomized, controlled trial was performed in Ningyang County, Shandong Province, China. Mild SCH patients who were diagnosed with twice thyroid function tests were randomly assigned to receive L-thyroxine replacement therapy (25 μg once daily initially, adjusted according to the reevaluation results until thyroid function returned normal) or no treatment for 15 months. The primary outcome was the change in serum total cholesterol (TC) concentrations analyzed in the per-protocol population. Subgroup analyses were also performed in subjects with different thyrotropin or TC concentrations at baseline. This study is registered with ClinicalTrials.gov, number NCT01848171.

Results Between July and December 2013, 378 subjects were enrolled and 369 subjects were included in the final analysis (210 in the intervention group and 159 in the control group). With the L-thyroxine replacement therapy, serum TC levels were decreased by 0.41 mmol/L (p<0.001) in the intervention group, while the decline was 0.17 mmol/L (p=0.019) in the control group. The decrease was more obvious in the intervention group than the control group (p=0.012). Subgroup analyses showed that in the subjects with different thyrotropin concentrations at baseline, L-thyroxine resulted in similar decreases of TC levels in each subgroup (all declines were approximately 0.41 mmol/L, p<0.001). In addition, L-thyroxine replacement therapy could also benefit the subjects with different TC concentrations at baseline. Even in the subjects with absolutely normal TC levels (less than 5.18 mmol/L), serum TC concentrations were kept unchanged in the intervention group (p=0.936), while they were increased by 0.35 mmol/Lin the control group (p=0.004). Variation trend for serum low-density lipoprotein cholesterol levels was similar with that for serum TC levels.

Conclusion Mild SCH patients could benefit from L-thyroxine replacement therapy on serum lipid profiles. Our study might provide reliable and important evidence for evidence-based medicine and help clinicians offer effective treatment advices to the mild SCH patients.

Methods:We examined associations between TSH (mIU/L), free T4 (pmol/L), and total T3 (nmol/L) and incidence of hip fracture, atrial fibrillation, dementia, coronary heart disease (CHD), heart failure, and total mortality in 360 US community-dwelling men and women aged 65 years and over who were enrolled in the Cardiovascular Health Study and taking levothyroxine. Cox proportional hazard models were used to examine the relationship between each thyroid test and incident events; TSH was log-transformed. All models were adjusted for age, sex, race, and outcome specific covariates.

Results:Mean age was 74.8 years and 79% were women. Only 51% of these levothyroxine users were euthyroid; 13% had overt hyperthyroidism, 16% had subclinical hyperthyroidism, and 20% had an elevated TSH.  There were 28 cases of incident hip fracture. Higher TSH levels were associated with a lower risk of hip fracture (HR 0.84; 95% CI 0.71-0.98, p=0.03 for lnTSH) and, concordantly, higher free T4 levels were associated with a higher risk of hip fracture (HR 2.63, 95% CI 1.26-5.50, p=0.01). There were 31 cases of incident dementia and 57 cases of incident CHD. Higher free T4 levels were associated with a higher risk of dementia (HR 2.57, 95% CI 1.18-5.56), p=0.02) and a lower risk of CHD (HR 0.42, 95% CI 0.19-0.93, p=0.03). There were 57 cases of incident atrial fibrillation and 70 cases of incident heart failure. There were no associations between any thyroid function test and atrial fibrillation or heart failure. There were 122 deaths. Higher total T3 levels were associated with lower risk of mortality (HR 0.35, 95% CI 0.19-0.64, p=0.001), consistent with nonthyroidal illness syndrome.

Conclusions: Higher free T4 concentrations are associated with a higher risk of hip fracture and dementia, and a lower risk of CHD, in older levothyroxine users. Clinical trials are needed to evaluate the optimal target concentrations for thyroid hormone replacement in older people.

Design and Method: We compared the transcriptome of zona glomerulosa (ZG), zona fasciculata (ZF), and APAs in 13 human adrenals, finding 28 genes >5-fold over-expressed in ZG vs ZF¹. Expression of neurofilament medium (NEFM), a regulator of dopamine receptors, was 14.8-fold higher (p= 9.16E^-12) in ZG than ZF, and 4-fold down-regulated in ZF-like APAs carrying KCNJ5 mutations in comparison to small ZG-like APAs (p=-4.35E^-03). NEFM protein expression and subcellular localisation were evaluated by immunohistochemistry of human adrenals, and immunofluorescence microscopy in H295R and HEK293 cells. Aldosterone production and secretion and the dopamine receptors gene expression were investigated by silencing NEFM in adrenocarcinoma H295R cells. Aldosterone response to dopamine receptor 2 antagonist metoclopramide (10^-7M) was measured in H295R cells transfected with mutant DelI157 KCNJ5 constructs.

Results: ZG selective expression of NEFM was confirmed by qPCR at 200-fold (cf. ZF). Immunohistochemistry showed ZG selectivity for NEFM staining, which was cytoplasmic and peri-membranous in normal ZG and small ZG-like APAs but absent in the normal ZF and ZF-like APAs. NEFM fluorescence staining in H295R was not filamentous (as in neurons and in HEK293) but present in nuclei and cytoplasm. Silencing of NEFM in H295R cells caused significant down-regulation of CYP11B2 and NR4A2by 40% and 70%, respectively (p= 0.01 and 0.05), and 40% increase  of aldosterone secretion after 72 hrs (from 205 to 280 pM/ug protein, p= 0.004). Moreover, dopamine receptor 1 and 2 were down-regulated by 45% and 70%, respectively (p= 0.007 and 0.02).

Aldosterone increase in response to dopamine receptor 2 blockade with metoclopramide was blunted in H295R cells transfected with KCNJ5 mutant construct whereas it increased by 200% in controls (p= 0.0159).

Conclusions: We postulate that NEFM plays a role in regulating basal aldosterone production and its response to dopamine. Down-regulation of NEFM in ZF-like APAs could explain the low expression levels of dopamine receptor 2 observed in some APAs, and the variable responses of aldosterone secretion to metoclopramide.²

MethodsFFive Wister rats were fed with high, normal and low salt diet for 8 weeks. Plasma and urinary aldosterone, PRA were measured by RIA. The gene expression of CYP11B2 was measured by real time quantitative PCR in the adrenal glands. Isolated DNAs from rat adrenal glands were treated with bisulfite and amplified using primers specific for the human CYP11B2 promoter regions.

ResultsFPlasma and urinary aldosterone and PRA were significantly decreased by high salt diet compared with normal or low salt diet (p<0.05). High salt diet significantly decreased mRNA levels of CYP11B2 gene and increased metylation ratio of this gene (p<0.05).

ConclusionsFHigh salt diet may influence the methylation ratio of CYP11B2 gene and regulate aldosterone biosynthesis.

Method: in a within-patient design, we compared baseline and post-metoclopramide values of lateralization index (LI) and relative aldosterone secretion index (RAI) of  each adrenal gland in 103 consecutive patients undergoing AVS, using a conclusive diagnosis of aldosterone-producing adenoma (APA) as gold standard.

Results: Metoclopramide increased aldosterone in inferior vena cava (IVC) and in adrenal vein blood of both APA and the contralateral side. Post-metoclopramide LI provided an accurate identification of APA (p < 0.001), but did not improve the diagnostic accuracy over baseline LI, because of a similar increase of RAI of both sides (p<0.005). The baseline non-dominant RAI  showed low accuracy for the identification of a contralateral APA. By contrast, the post metoclopramide RAI values raised consistently >1.00 on the APA side, and bilaterally in all but one (non-dominat RAI = 0.91) non-APA patients; at variance, it failed to do so in the side contralateral to the tumor in 46% of the APA patients.

Conclusions: albeit increasing aldosterone secretion, metoclopramide did not improve the assessment of lateralization. However, a post-metoclopramide RAI < 0.91 offered 46% sensitivity for identifying an APA contralaterally and 100% specificity. Thus post metoclopramide AVS can allow a diagnosis in many cases which would have been non-diagnosed because of non bilaterally-selective AVS studies.

Objective: We evaluated diagnostic discrepancy in subtyping of PA by different criteria based on the PA guideline of both Endocrine Society (ENDO) and Japan Endocrine Society (JES).

Methods: We evaluated 105 consecutive PA patients undergoing AVS from 2011 to 2014. The selectivity index (SI) was PCC_adrenal/PCC_IVC, AVS was considered bilaterally successful when the SI on both sides was >3 without ACTH stimulation, and >10 with ACTH stimulation, respectively. Lateralization index (LI) and contralateral index (CI) was calculated as PAC_dominant/PCC_dominant/PAC_non-dominant/PCC_non-dominant and PAC_nondominant/PCC_nondominant /PAC_IVC/PCC_IVC.  We used several criteria for unilateral PA as follows: 1^st criteria was LI>2 and 2^nd criteria was PAC_dominant>14000 pg/mL and 3^rd criteria was LI >2.6 and 4^th criteria was LI >4 after ACTH stimulation.

Results: Seventy-five out of 105(71%) cases were successful in baseline AVS and ACTH stimulated AVS at the same time. In baseline AVS, the prevalence of unilateral PA in 1^st criteria were 38/75 (51%). Moreover, in ACTH stimulated AVS, the unilateral PA prevalence in 2^nd, 3^rd and 4^th criteria were 28/75 (37%), 15/75 (20%) and 6/75 (8%), respectively. In every criteria, diagnostic discrepancy between baseline and ACTH stimulated AVS were observed in approximately 16-45% cases. In most of cases demonstrating diagnostic discrepancy, diagnoses were changed from unilateral to bilateral PA by ACTH injection. Furthermore, ACTH stimulation significantly decreased LI in all unilateral PA diagnosed on 1^st criteria (p<0.05).

Conclusion: The criteria changed the diagnosis of PA subtype. Especially, ACTH stimulation increased the prevalence of bilateral PA. Further studies are necessary to clarify which AVS procedure is more accurate.

Abbreviations; PCC: plasma cortisol concertation, PAC: plasma aldosterone concertation, IVC: inferior vena cava

Objectives: To investigate the accuracy of adrenal CT in subtype diagnosis and to develop a prediction score for bilateral subtype in PA patients with no adrenal tumor on CT.

Methods: WAVES-J database of PA patients underwent AVS between 2006 and 2013 at nine referral centers in Japan were studied. Total 393 patients who had undergone successful AVS with cosyntropin stimulation were included in this study. Concordant rate between the adrenal CT and AVS was evaluated. Variables for bilateral subtype were identified by multivariate logistic regression analysis and a prediction score was constructed. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic ability of the score.

Results: Forty two % (165/393) of the patients showed adrenal tumor(s) (a unilateral tumor in 40%, bilateral tumors in 2%), while no tumor was detected in the rest of the patients. The subtype diagnosis by CT was concordant with that by AVS in 68% (269/393): 38% (68/156) of patients with a unilateral tumor, 56% (5/9) of patients with bilateral tumors and 89% (204/228) of patients without a tumor were concordant with the subtype diagnosis by the AVS. Multivariate logistic regression analysis in patients without a tumor revealed that female gender (β=1.476, p=0.005), plasma aldosterone concentration (pg/ml) to plasma renin activity ratio ≤550 (β=1.115, p=0.023) and serum potassium ≥3.8 mEq/l (β=1.970, p=0.001) were the independent predictors of bilateral subtype. The prediction score for bilateral subtype based on 3 variables, which each one point was attributed to, showed that a score of 3 points had 40% sensitivity and 96% specificity, and that of 2 points had 82% sensitivity and 63% specificity in ROC curve analysis.

Conclusions: This study demonstrated that concordant rate of the subtype diagnosis by adrenal CT imaging and AVS in PA patients were high in those without adrenal tumor but not in those with adrenal tumors. Our prediction score could be useful in selecting patients with bilateral lesions who don’t need to undergo AVS, especially if no tumor was detected on CT.

Objective: Aim of the study was to investigate aldosterone concentration and its gradient in the adrenal vein of non-PA hypertensive patients.

Design, Setting and Participants: This retrospective study was conducted as a multi-center collaborative study involving nine referral centers in Japan. We studied the results of cosyntropin-stimulated AVS in 41 hypertensive patients who showed negative results in 2 confirmatory tests, captopril challenge test and saline infusion test. AVS was conducted by the decision of respective attending physicians based on various clinical settings.

Main outcome measures: Plasma aldosterone concentration (PAC), aldosterone to cortisol (A/C) ratio, its higher to lower ratio (lateralized index: LI) in the adrenal vein with cosyntropin stimulation were measured.

Results: Median PAC in the adrenal vein was 25737 pg/ml (range: 5154-69920) in the higher side and 13347 pg/ml (range: 1866-36190) in the lower side (P<0.001). There was a significant gradient in A/C ratio between the higher and lower sides [27.2 pg/ml/µg/dl (5.4-66.0) vs 17.3 pg/ml/µg/dl (4.0-59.0); P<0.001] with LI ranging from 1.01 to 3.87. The aldosterone lateralization gradient was between 1 to 2 in 33 patients and 2 to 4 in 8 patients. None of the patients showed LI ≥4.

Conclusions: The present study demonstrated that PAC in the adrenal veins showed significant variation and lateralization gradient even in non-PA hypertensive patients. AVS aldosterone lateralization gradients between 2 and 4 should be interpreted with caution in patients with PA because these gradients can be found in patients without PA.

Objective: This study aimed to determine the selectivity of the new imaging agents ¹⁸F-CDP2230 (PET) and ¹²³I-CDP2440 (single photon emission computed tomography: SPECT) for CYP11B2 over CYP11B1 and determine whether the biodistribution profiles are favorable for imaging CYP11B2.

Methods: The half maximal inhibitory concentrations (IC₅₀) of CDP2230 and CDP2440 for the enzymatic activities of CYP11B2 and CYP11B1 were determined using cells with stable expression of either enzyme. In vitro autoradiography (ARG) of human adrenal sections with APA was performed to confirm the specific binding ability of the new imaging agents to CYP11B2-expressing regions. Biodistribution studies were performed in rats.

Results: The selectivity of CDP2230 and CDP2440 for CYP11B2 over CYP11B1 was higher than that of metomidate analogues. In vitro ARG revealed that the binding of the new imaging agents to CYP11B2-expressing regions in the adrenal gland were more specific than that of the metomidate analogue. Moreover, the biodistribution study showed that the new imaging agents accumulated in adrenal glands with low background uptake.

Conclusions: Our study revealed high selectivity of the new imaging agents for CYP11B2 over CYP11B1 with favorable biodistribution for imaging CYP11B2. ¹⁸F-CDP2230 and ¹²³I-CDP2440 are promising imaging agents for the subtype diagnosis of PA.

Methods: We developed a novel quick cortisol assay (QCA) that uses immunochromatography and gold nanoparticles, and that can be performed either semi-quantitatively or quantitatively (United States Patent No. 8927219, Japan Patent No.5590651). The assay was evaluated during non-ACTH stimulated AVS in two prospective randomized, controlled studies. In a single-center study, PA patients were assigned to undergo AVS incorporating the semi-quantitative QCA (n=30), quantitative QCA (n=30), or without the QCA (n=30); and the rates of successful AVS were determined. The multicenter study was performed at seven medical centers, including Kanazawa University Hospital(K)(Kanazawa, Japan), Houju Memorial Hospital(H)(Nomi, Japan), Sanda Municipal Hospital(S)(Sanda, Japan), Akashi Medical Center(A)(Akashi, Japan), Ishikawa Prefectural Hospital(I)(Kanazawa, Japan), Saiseikai Ishikawa Hospital(SI)(Kanazawa, Japan), and Takaoka Municipal Hospital(T)(Takaoka, Japan) from March 2012 to March 2015. In a multicenter study, the success rates of AVS performed with (n=148) or without the semi-quantitative QCA (n=145) were determined.

Results: Cortisol concentrations were measured during AVS within 6 min in the radiology suite, without additional technical assistance, and excellently correlated with a conventional reference assay (r=0·997, p<0·001). In the single-center study, there was no difference in the AVS success rates using the semi-quantitative (93%) and quantitative QCAs (93%); however, both rates were significantly higher than the rate of AVS without QCA (63%; p<0·001). The success rate of AVS performed in the multicenter study was 94% for the semi-quantitative QCA, which was significantly higher than the rate for the control patients (56%; p<0·001).

Conclusions: Our novel QCA was rapidly and easily performed at the point-of-care, and improved the AVS success rate.

Aim: To identify, evaluate and compare the expression levels of miRNAs with potential binding sites on the 3'-UTR of the human HSD11B2 mRNA, in urinary exosomes and peripheral leukocytes  (PBMC).

Subjects and Methods: Subjects were classified in two groups according their serum F/E ratio: high F/E ratios (> percentil 90) and subjects with normal F/E ratio. Total RNA was isolated from PBMC (n=94) and in a subset spot-urinary exosomes (n=20). To identify exosomes we used electronic microscopy and aquaporin 2 (AQP2) expression. Bioinformatic analyses were performed by MirWalk 2.0, which identified potential miRNAs binding to HSD11B2 RNA. Expression of HSD11B2, miRNAs, AQP2 and RNU6 (housekeeping gene) were evaluated by qRT-PCR. The results were expressed as relative units (RU). Comparisons were performed by Kruskal-Wallis and Dunn analyses. 

Results: Bioinformatic analyses identified 12 miRNAs that potentially bind to the 3'UTR region of HSD11B2. We successfully amplified 4 miRNA in leukocytes (miR-101, 488, 615 and 1205) and 2 miRNA in exosomes by qRT-PCR, being miR-488 and miR-615 expressed in both samples. AQP2 expression was only expressed in urinary exosomes and not in PBMC. In the total group, the expression in PBMC of miR-1205 (8.9 [4.7 - 19.0] RU) was higher than expression of miR-101 (0.3 [0.1- 1.3] RU; p <0.05), miR-615 (2.1 [0.7- 5.1] RU; p <0.05) and miR-488 (0.4 [0.1- 1.3] RU; p <0.05). In the total group, we observed an increased expression of miR-615 and miR-488 in exosomes than PBMC (241.2 [27.6-4143] vs 2.1 [0.7-5.1] UR; p <0.0001) and (630.9 [91.2-1080] vs 0.4 [0.1-1.3] RU; p 0.0015), respectively. In urinary exosomes, the expression of miR-488 was about 6 times higher in subjects with high F/E ratios (n = 10) vs normal F/E ratios (n = 10) (1025 [915.1-1134] vs 176.4 [6.1 to 346 7] RU; p <0.0001), and the expression of HSD11B2 tended to be lower in the group subject to high F/E vs normal F/E (1150 [376-1968] vs 4730 [1312-8108] RU; p = 0.1). We found no differences in expression of miRNA in leukocytes.

Conclusion: We identified a high expression of miR-488 and miR-615 that could modify the expression of HSD11B2 when evaluated in urinary exosomes from subjects with high F/E ratio. We provide new evidence regarding the usefulness of exosomes and their content of miRNA as regulators and potential biomarkers of 11βHSD2 activity.

Methods and Materials: Angiotensin (A) II and RXR pan-agonist PA024 were used as reagents. We added various concentrations of PA024 on H295R cells derived from human adrenocortical carcinoma. We thereafter analyzed CYP11B2 mRNA expression by quantitative real-time PCR, CYP11B2 promoter activity by luciferase assay, aldosterone secretion by enzyme immunoassay, and intracellular Ca²⁺ level by Calcium Kit-Fluo 4, respectively.

Results: PA024 dose-dependently suppressed AII-induced CYP11B2 mRNA expression and CYP11B2 promoter activity. Additionally, high-dose PA024 significantly decreased aldosterone secretion. The suppression of CYP11B2 promoter activity by PA024 was observed in the deletion mutants analyses using the region from -1521 (full length) to -135 including NBRE-1, Ad4, and Ad5 elements. Therefore, transcription factors binding to these elements may be involved in the suppression. PA024 also suppressed mRNA expression of transcription factors Nurr1 and NGFIB in a dose-dependent manner. Since PA024-mediated suppression of CYP11B2 promoter activity was rescued by Nurr1 overexpression, decrease of Nurr1 expression may contribute to the suppression. On the other hand, PA024 did not affect on intracellular Ca²⁺ concentrations, cellular proliferation, and apoptosis. PA024 also inhibited mRNA expression of StAR, HSD3β2, and CYP21A2. Therefore, PA024-mediated suppression of these enzymes (and protein) may also be involved in the inhibition of aldosterone synthesis pathway.

Conclusions: These data indicate that RXR pan-agonist PA024 may possibly be a candidate of novel anti-hypertensive/anti-arteriosclerotic drugs via the suppression of aldosterone synthesis/secretion. We are currently investigating its in vivo effect using transgenic Tsukuba hypertensive mice (hRN8-12 x hAG2-5) which demonstrate AII-mediated CYP11B2 overexpression.

Setting and Design: This is a retrospective analysis of 64 patients who were diagnosed with PA and underwent AVS. Of these, 35 and 23 patients were diagnosed with UHA and BHA, respectively, including 25 patients pathologically confirmed as having APA. Their plasma adrenocortical hormone levels at 0:00, 6:00, 12:00, and 18:00 and 24-h urinary aldosterone level under the condition of 6 g daily dietary salt intake were measured.

Results: Serum sodium; PAC at 0:00, 6:00, 12:00 and 18:00; PFC at 12:00 and 18:00; and 24-h urinary aldosterone levels were statistically significantly higher in those with UHA than those with BHA. In contrast, the UHA group had lower levels of serum potassium and ACTH at 0:00. The area under the ROC curve for PAC at 0:00, 6:00, 12:00, and 18:00 and 24-h urinary aldosterone to discriminate UHA and BHA was 0.840 [95% confidence interval (CI); 0.73−0.95], 0.936 (95% CI; 0.87−1.00), 0.882 (95% CI; 0.79−0.98), 0.824 (95% CI; 0.71−0.94), 0.873 (95% CI; 0.78−0.97), respectively.

Conclusions: We firstly demonstrated the diagnostic value of PAC in different blood collecting times and 24-h urinary aldosterone level to discriminate between UHA and BHA. This test can be performed when we screen PA, and it is far less expensive than AVS. Thus, by omitting unnecessary AVS procedures, we can reduce the associated medical costs.

Methods: Seventy one hypertensive patients (male:female=33:38) were diagnosed as PA by confirmatory tests including captopril challenge, furosemide-upright posture, oral salt loading and rapid ACTH tests, and subtype diagnosis was performed by AVS (unilateral (UNI):bilateral  (BIL)=27:44).  The cut-off value for UNI was determined by ROC analysis and compared usefulness of confirmatory tests for subtype diagnosis.

Results: Blood pressure, pulse rate and BMI were comparable between UNI and BIL.  UNI PA patients present with younger age, lower serum potassium, higher plasma and urine aldosterone excretion.  Among 4 confirmatory test, rapid ACTH test was shown to be most useful to predict subtype diagnosis.  For UNI diagnosis, the cut-off value of peak aldosterone was 39.8ng/dL (83% sensitivity, 75% specificity, 59% positive predictive value, 91% negative predictive value).  The cut-off value of peak aldosterone/cortisol ratio was 1.63 (83% sensitivity, 71% specificity, 56% positive predictive value, 91% negative predictive value).

Conclusions: Rapid ACTH test may be an useful test for PA subtype diagnosis with a cut-off value of peak plasma aldosterone 39.8ng/dL or a cut-off value of peak plasma aldosterone/cortisol ratio 1.63.  When peak aldosterone or aldosterone/cortisol ratio is lower than cut-off values in rapid ACTH test, AVS can be skipped due to high probability of BIL PA.

Methods:One hundred and twenty-three patients with primary aldosteronism (PA) and one hundred and thirty-six patients with essential hypertension were consecutively recruited from Zhongshan hospital, Fudan university, 2012-2015. Statistical analysis was conducted with the Medcalc software.

Results:The results suggested that patients with primary aldosteronism had similar eGFR (P>0.05), higher PAC (P<0.001), lower PRA (P<0.001), and lower serum potassium levels (P<0.001), than patients with essential hypertension. eGFR was correlated inversely with PRA, and positively with calculated ARR, both in the patients with primary aldosteronism or essential hypertension. Primary aldosteronism patients from the lowest tertile of ARR (<17.8 pg/dl per ng/ml.h) or highest tertile of PRA (>1.4 ng/ml.h) were associated with higher BP, plasma potassium, and albuminuria and lower eGFR than other patients (P<0.01). In the whole study population, the optimal cut- off point according to Youden’s J statistic was an ARR of 25.2 pg/ml per ng/ml.h (AUC=0.845, 95% Confidence interval: 0.730 to 0.908, P<0.001); in subjects with eGFR higher than 90 ml/min/1.73m², the optimal cut- off point was an ARR of 42.0 pg/dl per ng/ml.h (AUC=0.891, 95% Confidence interval: 0.789 to 0.972, P<0.001); in subjects with eGFR lower than 90 ml/min/1.73m², the optimal cut- off point was an ARR of 19.3 (AUC=0.820, 95%CI: 0.714 to 0.911, P<0.001).

Conclusions: Unsuppressed renin and lower ARR levels were associated with more severe renal damage and decreased eGFR in patients with primary aldosteronism. Diagnostic criteria of ARR by stratified eGFR may be an optimal strategy for the screening of primary aldosteronism.

Magnesium (Mg) is an essential cofactor for many metabolic reactions, which homeostasis is maintained by bone, intestine and kidney. Primary aldosteronism (PA) can cause Mg deficiency because aldosterone inhibits reabsorption of urinary Mg in kidney. Moreover, hypomagnesemia may increase urinary potassium excretion and impair hypocalcemic-induced parathyroid hormone secretion.

Objectives

This study aimed to evaluate serum Mg level (S-Mg) and urinary Mg excretion (U-Mg) in PA, and analyze the associations among Mg, plasma aldosterone concentration (PAC) and other variables.

Material & Method

We performed the retrospective study for 332 PA patients, who were diagnosed with PA by a couple of confirmatory tests, captopril challenge test and adrenal vein sampling at our center from April 2007 to September 2015, and measured S-Mg. Hypokalemia was actively corrected by prescribing potassium replacement.

Results

The mean age of the participants was 53.2 ± 11.0 (mean ± SD) years and the percentage of male was 46.7%. The baseline parameters at the diagnosis were as follows: plasma renin activity 0.20 ±0.18 ng/ml/hr; PAC 27.7 ± 24.4 ng/dl; urinary aldosterone excretion (U-Aldo) 16.9 ± 13.6 μg/day; aldosterone-to-renin ratio 210.0 ± 246.9; S-Mg 2.22 ± 0.18 mg/dl; U-Mg 0.09 ± 0.04 g/day; fractional excretion of Mg (FEMg) 3.8 ± 2.0%; serum potassium (S-K) 4.08 ± 0.55 mM; urinary potassium excretion (U-K) 59.0 ± 30.7 mmol/day; estimated glomerular filtration rate 77.4 ± 16.2 ml/min/173m². Using Spearman’s correlation, S-Mg was positively correlated with S-K (r_s = 0.204, p = 0.0002). Among the participants (n = 128), who were measured U-Mg, U-Mg was positively correlated with U-K (r_s = 0.336, p = 0.0001), PAC (r_s = 0.251, p = 0.0042) and U-Aldo (r_s = 0.348, p = 0.0001). Furthermore, FEMg in unilateral PA (n = 67) was significantly higher than that in bilateral PA (p = 0.024) as well as PAC (p < 0.001). In multiple regression analysis, PAC was an independent prognostic factor for U-Mg (β = 0.021, p = 0.02), and U-Mg was also an independent prognostic factor for U-K (β = 131.7, p = 0.019) after adjustment for PAC.

Conclusion

Our study implied that high level of aldosterone may increase U-Mg as well as U-K.

All tumors with known mutations contained aldosterone at higher levels than the negative controls. The aldosterone content showed a small but not significant decline with length of time in the freezer (r²= 0.036, p=0.23). No difference in aldosterone content was observed between APAs with G151R (n=13) and L168R KCNJ5 mutations (n=11). ATP1A1/ATP2B3 mutated tumours contained a significantly higher amount of aldosterone compared to KCNJ5 mutated tumours, 4.3 vs 1.3 pmol/mg (p=0.0041). In the multinodular adrenals, a higher level of aldosterone was observed in mutated nodules. In adrenals with low CYP11B2 expression low aldosterone content was observed. Importantly, of the tumours classified as non-mutated, 6/13 (46%) did not contain aldosterone, suggesting a non-functional nodule. In conclusion, this method demonstrated the differences between nodules and different mutations in terms of aldosterone release, implying a heterogeneous secretion pattern and may represent an easy and inexpensive way of diagnosing aldosterone production.

Endocrine Society Guidelines from 2008 recommend confirmatory testing when primary aldosteronism (PA) is suspected, with subsequent adrenal vein sampling (AVS), followed by adrenalectomy in unilateral, and medical treatment in bilateral disease. Our aim was to describe clinical characteristics, diagnostic procedures and long-term outcomes for PA patients in Western Norway.

Material and methods

Data from all suspected PA patients investigated at Haukeland University Hospital in the period 1998-2012 were retrospectively evaluated for inclusion. Patients with verified PA after saline infusion testing (SIT) or who otherwise were considered highly likely of having PA, (elevated aldosterone/renin-ratio, hypokalemia and/or radiological evidence of adenoma or hyperplasia), were included. Clinical, biochemical, radiology findings, and AVS-reports were retrieved. All patients still alive by August 2014 were invited to a clinical follow-up visit.

Results

One hundred and eight patients were included. The median age at PA-diagnosis was 54 years (range 30-85); the median duration of hypertension was 10 years (range 0-30). Eighty-five percent (85%) were hypokalemic. Eighty-two (76%) had PA confirmed by SIT. AVS was performed in 95 (88%) patients, of whom 50 (53%) had unilateral, and 20 (21%) bilateral disease; the remaining did not have representative AVS-results. For patients with representative AVS, the proportion bilateral disease increased from 17% (5/29) before 2008, to 37% (15/41) from 2008 onwards.           

Altogether, 68/108 (63%) were adrenalectomized, of whom 48 (71%) had AVS-confirmed unilateral disease. The remaining were operated based on radiological findings alone. Forty patients were medically treated.

Follow-up visits were performed for 72/108 (67%); median 72 months (range 27-186) after treatment. Blood pressure reductions were similar in adrenalectomized (n= 52) and medically treated (n=20) patients (26/8 vs. 20/16 mm Hg, p=ns). Among the adrenalectomized (n=52), eleven (21%) were cured of HT, thirty-four (65%) had improved, and seven (13%) showed no improvement of HT. Nine of the cured patients had available pathology reports, all showing adenomas. Among the patients not cured, 63% had adenomas and 37% hyperplasia. Using logistic regression analysis, only female sex significantly predicted cure of hypertension(p= 0.029).

Conclusion

AVS was performed in a high proportion of our patients, of whom the majority had unilateral disease and hypokalemia, indicating that patients with bilateral disease and milder PA may still be underdiagnosed. The cure rate of HT after adrenalectomy was low and associated with female sex.

Objective: To investigate the effect of chronic GC exposure on AT inflammation by quantifying macrophage infiltration in AT biopsies from patients with active CD compared to BMI-matched controls.

Methods:  Immunohistochemistry on subcutaneous abdominal AT biopsies from 6 active CD patients were compared to 9 overweight BMI-matched controls. To qualitatively assess for varying architectural phenotypes within the AT, antibodies for vimentin, an intermediary filament that stains mesenchymal cells, and CD31, an endothelial marker, were used. Macrophage infiltration was evaluated by the presence of pan-macrophage CD68 and activated macrophage CD163-positive cells. 

Results:  The 6 enrolled CD patients (6 female) had a median age of 30.5 years (range 19-53), median BMI of 34.5 kg/m² (range 29.6-61.8), and median waist circumference (WC) of 103.4 cm (range 98.4-157.5).  The 9 control patients (5 female) had a median age of 27 years (range 24-41), median BMI of 31 kg/m² (range 21.8-37.5), and median WC of 89.1 cm (range 77.5-105.4).  There were no differences in mean age (p=0.72) or BMI (p=0.06) between CD patients and controls, but CD patients had a higher mean WC vs. controls (p=0.02). Analysis of AT immunohistochemistry revealed a qualitative increase in the expression of Vimentin and CD31 in AT from CD patients vs. matched controls.  100% of AT samples evaluated from CD patients (N=5) exhibited a focal to diffuse increase in CD68+ macrophages, with CLS (i.e. aggregates of CD68+ macrophages surrounding adipocytes), compared to 1 out of 9 (11%) control AT samples with focal CD68+ cells and no CLS. 

Conclusions:  Our preliminary qualitative data demonstrate that, compared to BMI-matched controls, AT from patients with CD are characterized by an influx of CD68+ macrophages and a possible increase in vimentin and vascular networks. These findings suggest that chronic excess GC exposure has a paradoxical pro-inflammatory morphologic impact on AT. Additional studies are ongoing to quantify and further phenotype AT macrophage content in patients with CD.

To evaluate the influence of long-term vs. short-term cortisol replacement therapy on the adrenomedullary system and cognitive functions.

Fourteen patients with primary or secondary AI were divided into two groups, depending on the duration of disease and HC replacement therapy (less or at least 15 years). All subjects underwent standardized neurocognitive testing; in addition, cortisol and catecholamine levels as well as physiological parameters and quality of life (QoL) were assessed.

Patients with HC replacement therapy >15 years (n=7) received significantly higher equivalent glucocorticoid (GC) doses than those with a shorter lasting therapy (n=7; p=0.048). Neuropsychological tests, QoL, physiological parameters and cortisol levels did not differ significantly between both groups. While epinephrine concentrations in plasma were subnormal in both groups, norepinephrine levels were in the normal range but significantly lower in patients on short-term HC replacement therapy (p= 0.025). 

We showed that duration of cortisol replacement therapy may have an impact on catecholamine release, but does not seem to have an impact on cognitive functions and QoL.

To study the effects of physiologic alterations in cortisol mileu on mood changes during late pregnancy, we prospectively evaluated 75 healthy pregnant subjects after 36 weeks of gestation and at 3-4 weeks of postpartum period. During each visit blood samples were taken to determine the levels of total cortisol (TC) and cortisol binding globulin (CBG) at 8 a.m. Free cortisol (FC) was calculated using Coolen's equation (3) and free cortisol index (FCI) was defined as serum TC/CBG. Concurrently, status of depression, anxiety and stress were graded using Beck Depression Inventory (BDI),  Beck Anxiety Inventory (BAI) and Percieved Stress Scale (PSS). Additional demographic data and support mechanisms of each subject were also questionnaired.

The mean age of the participants was 28.5±4.9 years and the median week of gestation was 39 [IQR: 38-40]. After gestation TC levels decreased from 130.4 [IQR: 101.1-167.5] to 53 [IQR: 41.7-73.1] nmol/l, FC levels from 22 [IQR: 14.2-38.3] to 9.9 [IQR: 6.4-15.2] nmol/l and FCI from 28.9 [IQR: 21.6-40.1] to 25.5 [IQR: 16.9-36.8] nmol/mg (p<0.001 for all). At late pregnancy and early postpartum period the median score on BDI was 9 [IQR: 6-14.5] and 7 [IQR: 3-11.5] (p=0.006), on BAI was 15 [IQR: 9-19] and 6 [IQR: 2-12] (p<0.001) and on PSS was 23 [IQR: 19-28.5] and 22 [IQR: 17-26] (p=0.02), respectively. Both at late pregnancy and postpartum period neither TC levels nor FC measures were correlated with the concurrent scores on BDI, BAI and PSS. However higher FC levels during late pregnancy were associated with lower scores on stress and depression at early postpartum period, albeit the latter was not statistically significant (r= -0.2, p=0.03 and r= -0.2, p=0.06). Additionally, as FCI increased during late pregnancy both the scores on stress and depression decreased during early postpartum period (r= -0.4, p=0.02 and r= -0.3, p=0.01).

We speculate that physiologic hypercortisolism during late pregnancy may not add a burden on psychology of pregnant cases. Moreover increased cortisol levels during  latest terms of pregnancy may cause indirect and ultimate alterations, having protective effects on mood of female cases during postpartum period.

Methods: Patients admitted to the NIH Clinical Center for the evaluation and treatment of active CS are eligible for study. During an inpatient admission, baseline REE is measured by indirect calorimetry (TrueOne 2400) after waking, at rest, and prior to receiving any medications. Baseline body composition is measured by DXA and blood is drawn in the morning after a 12 hour fast. These measurements are repeated at inpatient admissions at 6 and 12 months after successful surgery. Data were compared between two intervals by paired t-test or Wilcoxon signed rank test and are presented as mean ± SEM.

Results: 29 patients have enrolled in this study, of which 11 have completed a 6 month follow-up visit after surgical treatment. These 11 patients are 82% female (n=9) and 37± 4 years of age. All 11 subjects had ACTH dependent CS (11 pituitary, 1 bronchial carcinoid). REE adjusted for LBM significantly increased 32 ± 6% 6 months after surgery (pre: 27 ± 2 kCal/d/kg, post: 34 ± 1 kCal/d/kg, p<0.001). Unadjusted REE also significantly increased (pre: 1284 ± 98 kCal/d, post: 1644 ± 89, p<0.001). LBM did not significantly change (pre: 49 ± 4 kg, post: 49 ± 3, p=0.905), but BMI (pre: 37 ± 4 kg/m2, post: 34 ± 3, p=0.012) and percent body fat (pre: 46 ± 3%, post: 42 ± 3, p=0.025) decreased. Patients lost an average of 9 ± 3 kg, or 7.1 ± 2.5%, of body weight (p=0.022). Excluding one patient on levothyroxine, TSH (pre: 1.2 ± 0.2, post: 2.9 ± 0.4, p<0.001), total T3 (pre: 78 ± 5, post: 130 ± 5, p<0.001), and free T4 (pre: 0.9 ± 0.1, post: 1.1 ± 0.0, p=0.038) increased. We await additional data on the 12 month visits.

Discussion: Weight loss observed in the 6 months after successful surgery in CS patients is associated with higher REE and higher circulating levels of TSH and thyroid hormones. This suggests that decreases in REE associated with lower thyroid hormone levels may contribute to weight gain in CS prior to surgery. The effect of cortisol excess on binding proteins and deiodinase activity is being examined. It is unclear if this effect will persist at one year after cure of CS or whether the expected decrease in REE with further weight loss will be observed.

Methods: We retrospectively studied pts with CS admitted to our protocols at the NIH Clinical Center from 1/2012 to 10/2015. Ectopic ACTH syndrome (EAS) or Cushing’s disease (CD) was diagnosed based on pathology. Pts were considered to have DM based on a HbA1c ≥6.5%, a fasting plasma glucose (FPG) ≥125 mg/dl, or if they were on oral hypoglycemic medications (meds) or insulin therapy. After surgery, pts were inpatients for 7-15 days; hydrocortisone (HC) (10-12mg/m²) was started after remission was confirmed. Usually insulin was used for glycemic control until discharge. Pts returned every 3-6 months (mo) for f/u and HC dose was adjusted based on weight and recovery of adrenal function. Resolution of DM was defined as HbA1c<6.5% off all meds.

Results: 50 pts achieved remission after tumor resection. At baseline (BL), 15 (10 female) had a diagnosis of DM (13=CD, 2=EAS); 10 pts took oral antidiabetic meds and 5 took insulin 0.3-1.3units/kg/d. BL HbA1c was 6.3-9.4%. Evaluable pre and post-operative insulin usage data was available for 1^st week after surgery in 11 pts: 2 pts previously on oral meds did not need insulin, 9 had a decrease in daily insulin dose, with 5 requiring none by day 7.

Of the 15 pts, at discharge, 4 required no DM meds, 4 had a decrease in insulin or med dosage, and 5 had no change to DM regimen. Despite overall decreased insulin use after surgery, 2 pts were discharged on higher med doses than BL due to uncontrolled DM on admission (HbA1c: 9.4, 7.6%).

8/15 pts were seen for 3-mo f/u. Of these, 4 were on no DM meds, insulin dose decreased in 2, while 2 pts had no change in regimen. HbA1c decreased in 7 and was unchanged in one. FPG decreased in all. By 6 mo, 6/8 took no meds, one was on a decreased regimen from BL, and one had no change. HbA1c was <6.5% (5-6.2%) in all. 7/8 came back for 12 mo f/u. Of these, 6 were on no meds or on decreased regimen with HbA1c and FPG in non-diabetic range. HbA1c (6.6%) increased in one pt due to over-replacement with HC but normalized by 18 mo.

2/3 pts first seen at 6 mo f/u were on no DM meds with HbA1c and FPG in non-diabetic range, the third pt had a decrease in DM regimen with decreasing HbA1c (6.8%) and FPG levels. One pt only seen at 9 mo had a normal HbA1c and FPG off all meds. 3 pts have not yet had a f/u after recent surgery. Overall, DM resolved in 10/12 pts with f/u by 3-18 mo. 

Conclusion: Insulin sensitivity and diabetic regimen can change rapidly, as early as the 1^st week, after surgical remission of CS. We recommend close monitoring of blood glucose levels (especially in the 1^st month), pt education and long-term f/u of these pts for optimal management of DM post-remission of CS.

Methods - We collected scalp hair samples in 66 TS patients, and 216 sex-matched adult controls from the large population-based LifeLines cohort study. Subjects who used systemic and/or topical on scalp corticosteroids were excluded from the analysis. The proximal 3 cm of the hair samples, corresponding to cortisol exposure in the preceding three months, were processed before cortisol was extracted in methanol and further purified using solid phase extraction. HCC were subsequently quantified using LC-MS/MS. Anthropometry, fasting biochemical metabolic parameters were collected, and psychological stress was assessed using the Perceived Stress Scale (PSS) questionnaire.

Results - HCC could be quantified in 55 TS patients and in 186 adult female controls (median age: 31.0 and 41.0 years, respectively). TS patients had significantly higher long-term cortisol levels than controls (mean [95% CI]: 3.38 [2.61-4.37] vs. 2.33 [2.17-2.51], P<0.001, adjusted for age). In a stratified analysis, increased HCC appeared to be most pronounced in TS patients with isochromosome-Xq variant, but there was no significant difference between different karyotypes. In TS patients, HCC were positively associated with total cholesterol levels (standardized β=0.318, P=0.049, adjusted for age and BMI), but not with blood pressure, BMI, fasting triglycerides, LDL- and HDL-cholesterol, or fasting glucose. PSS-scores in TS patients were relatively high (mean (SD): 24.8 (±8.6)) and tended to increase with HCC, albeit not statistically significant.

Conclusions - Compared to sex-matched population controls, Turner syndrome patients are chronically exposed to higher systemic cortisol levels, which is associated with increased total cholesterol levels, and potentially contributes to an elevated cardiovascular disease risk.

To better understand this pathophysiological mechanism, we evaluated several key coagulation elements in patients with CS before and after intervention and in a control group.

This was a prospective observational study, including 85 consecutive patients, aged ≥18 years, that were admitted to the National Institutes of Health for evaluation of suspected CS. Coagulation profile was taken before and 6-12 months following surgery (either transsphenoidal surgery or adrenalectomy). The patients were divided into three groups: CD group (n=22) including patients with pathologically proven CD; AdCS group (n=21) including patients that underwent adrenalectomy due to confirmed autonomous cortisol secretion from the adrenal(s); and a control group (n=42) including patients that were screened for CS, and were found to be normocortisolemic.

Before intervention, patients with CD had higher mean UFC levels and PM plasma cortisol levels compared with the AdCS group, and both had higher levels compared with the control group (p<0.001 each). At baseline, patients with CS had higher levels of FVIII (p=0.04) and vWF (p=0.02) compared with controls, as well as higher PC (p=0.03) and AT-III (p=0.02) levels. Both PC and AT-III decreased significantly after intervention (p values of 0.02 and 0.006, respectively). Patient's with CD had higher levels of vWF (p=0.03) and AT-III (p=0.01) before surgery than patients with AdCS. Moreover, patients with CD had decrease in AT-III (p=0.016) and PC levels (p=0.032) after intervention, whereas patients with AdCS did not show similar dynamics. UFC levels correlated with AT-III (r=0.5, p<0.001), PC (r=0.4, p=0.008), PS (r=0.5, p<0.001), vWF (r=0.3, p=0.01), FVIII (r=0.5, p=0.001), and aPTT (r= -0.5, p=0.001). Decrease in UFC levels following intervention correlated positively with the dynamics of FVIII (r=0.8, p<0.001), and PC (r=0.6, p=0.02).

In conclusion, our data shows that patients with CD experienced significant decreases in PC and AT-III after intervention, a finding that was not present among patients with AdCS, and might be explained by higher cortisol levels in CD patients. This study also confirmed prior findings that patients with CS have an increase in endogenous procoagulants and anticoagulants, as well as in antifibrinolytics.

Methods: 30 patients and 27 subjects without clinical or laboratory evidence of EHC were enrolled. We identified Cushing’s disease in 12, overt adrenal Cushing’s syndrome in 9 and subclinical Cushing’s syndrome in 9 subjects. All subjects underwent a full ophthalmological examination including best corrected visual acuity, slit-lamp assessment of lens opacities using the LOCSIII, slit-lamp gonioscopy, intraocular pressure measurement, fundus examination and diurnal IOP evalaution at 8:00 a.m., 12:00 p.m. and 4:00 p.m.

Results: Briefly, results of this study revealed no significant differences between the groups. We also did not observe a clear relationship between the prevalence of ophthalmological disturbances and the severity of the EHC (subclinical Cushing’s vs. overt Cushing’s syndrome).

Discussion: We attribute these results to relatively low potency of hypercortisolism in endogenous Cushing syndrome when compared to exogenous Cushing syndrome. Depending on the duration of exposure, total daily dose and the glucocorticoid type, glucocorticoid drugs are usually associated with more rapid development of cushingoid features. Additionally, there might be a referral bias for our patients when compared to the general population. Patients in this study have been evaluated in a tertiary health care unit by endocrinologists. This might be associated with early diagnosis, definitive treatment and the reduction of hypercortisolemia related co-morbidities.

Subjects and methods: We collected scalp hair samples from the posterior vertex in 295 adult participants of LifeLines (median age: 42 yrs, 75% females), a large prospective population based cohort study in the north of the Netherlands (www.LifeLines.nl). Approximately 20 mg of the proximal 3 cm of hair was weighed, and washed using isopropanol. We extracted steroids in methanol, followed by solid phase extraction. HairE and HairF were quantified on a Waters Xevo TQ-S LC-MS/MS system. In all subjects, we performed anthropometry, and collected the long-term difficulties inventory (LDI), list of threatening experiences (LTE) and a questionnaire about hair characteristics and corticosteroid use. Fasting metabolic values were available in 97% of participants.

Results: HairE and HairF could be quantified in 98 and 90% of samples, respectively. In multiple linear regression, both HairE and HairF increased with age, male sex, black hair color, and occurrences of perspiration on the scalp, and decreased with hair washing frequency (P<0.05). Only HairE was increased with brown hair color and fasting glucose (P<0.05). HairE was lower in users of systemic corticosteroids (4.1 vs. 8.5 pg/mg hair, P<0.001), and in individuals who only used local corticosteroids (7.1 vs. 8.5, P=0.028). Life events (LTE) were associated with higher HairF (simple standardized β: 0.131, P=0.039), but this association may be mediated by the frequency of perspiration on the scalp (adjusted β: 0.068, P=0.306). In this subpopulation of the LifeLines cohort, hair glucocorticoids were not independently associated with BMI, waist circumference, blood pressure or fasting lipids.

Conclusion: HairE can be a useful marker to detect mild adrenal suppression due to corticosteroid use in the general population, even when only inhaled, nasal or topical corticosteroids are used. These findings suggest that commonly used local corticosteroids induce systemic effects.

Objective: To investigate whether micro- and macrovascular health is impaired after long-term remission of CS,  in patients with no or adequately treated co-morbidities compared to healthy control subjects 

Design and setting: Cross-sectional case–control study in two tertiary referral centers.

Patients and main outcome measures: 63 patients (remission of CS for ≥ 4 years) and 63 well matched controls were compared. In group A (58 patients and 58 controls) serum biomarkers associated with endothelial dysfunction, intima media thickness, pulse wave velocity and pulse wave analysis were studied. In group B (14 patients and 14 controls) endothelium dependent and independent vasodilation was studied in both conduit arteries (flow mediated dilation of brachial artery) and forearm skeletal muscle resistance arteries (vasodilator response to intra-arterial acetylcholine, sodium-nitroprusside and  N^G-monomethyl-L-arginine using venous occlusion plethysmography).

Results There were no significant differences between the outcome measures of vascular health of patients and controls  in Group A and B.

Conclusion: In conclusion, vascular health of patients after long-term remission of Cushing’s syndrome seems to be comparable to that of healthy gender-, age and BMI matched controls, provided that the patients have no, or adequately controlled co-morbidities. Therefore, the effects of hypercortisolism per se on the vasculature may be reversible, which accentuates the need for stringent treatment of metabolic co-morbidities in these patients.

Objective: To investigate physical fitness level, as measured by peak oxygen uptake (VO_{2 peak}) during a maximal exercise stress test and  to explore its relation with energy expenditure (EE), in patients in long-term remission of CS and matched controls.

Patients and methods: Patients in long-term (>4 years) remission of CS, who did not use any medication, except for thyroid hormone substitution,  were eligible. For each patient a sex-, estrogen status-, age-, BMI-, smoking-, ethnicity-, and physical activity level (measured via the METS-score) matched control subject was recruited from the general population. Physical fitness level was assessed using a maximal exercise stress test on a bicycle ergometer with an incremental exercise protocol. Daily EE was assed using an activity monitor (Sensewear Pro, worn for one week).

Main outcome measures: Peak oxygen uptake (VO_2peak) during a maximal exercise stress test.

Results:  The patients in  long-term remission of CS (n=17) had a significantly lower peak oxygen uptake and maximal workload than the  matched controls (both P<0.05). Combined with a higher breath-by-breath minute ventilation (V_E)/VO₂ and a lower ventilatory threshold VO₂in patients these results prompt the consideration of mitochondrial myopathy.  No significant differences were found in respiratory exchange ratio, peak heart rate and blood lactate-levels (all P>0.05).  Daily EE did not differ between the two groups.

Conclusions:  Patients in long-term remission of CS have a lower physical fitness level with indications of limitation in muscle respiratory chain function, despite similar daily EE, compared to well-matched healthy controls. The lower physical fitness level in patients is a  promising potential target for future (early and late) rehabilitation programs.

Objective: To examine the association between OXT and mitochondrial oxidative phosphorylation (OXPHOS) (5) in peripheral energy-requiring tissues using a two-pronged approach, bioinformatics and in vitro measurements.

Methods: Bioinformatics. NCBI/GEO gene expression profiles were queried using the locally developed Awsomics toolbox. A curated collection of 35 datasets from experiments including pharmacologic or genetic disruption of the mitochondrial respiratory chain (RC) was used to identify conditions in which OXT was differentially expressed, using ANOVA with Bonferroni correction. Tissue culture. The effects of physiologic OXT (10uM x 24 hours) (6) on mitochondrial parameters was assessed using Fluorescence-Activated Cell Sorting Analysis (9) in liver-like hepatoma (HepG2) and muscle-like rhabdomyosarcoma (RD) cells under different nutrient conditions (5 mM versus 25 mM glucose).

Results: Bioinformatics. In 7/35 datasets, OXT showed differential expression. OXT was decreased 39% in skeletal muscle from humans with a primary mitochondrial RC diseases, and also decreased in 2 skin fibroblast cell lines from humans with mitochondrial RC diseases (specifically, coenzyme Q10 deficiency and mitochondrial ATP synthase deficiency), 60% and 57%, respectively. In contrast, in human neuroblastoma cells treated for ~1 week with rotenone (a mitochondrial RC complex I inhibitor), liver from mice with liver-specific Pdss2 deletion and associated coenzyme Q9 deficiency, and in cochlea from mice with mtDNA mutations, a marked increase in OXT expression was observed (54%, 224%, and 145%, respectively). Tissue Culture. In RD muscle-like cells, OXT produced a 45% ± 24% (SEM) increase in mitochondrial content in 5 mM glucose; in 25 mM glucose, 24% ± 41% (SEM). In HepG2 liver-like cells, OXT produced a 40% ± 25% (SEM) increase in mitochondrial content in 5 mM glucose; in 25 mM glucose, 8% ± 7% (SEM). Accounting for content, mitochondrial oxidant burden was decreased in RD cells (72% and 75% in 5 and 25 mM glucose, respectively) and also in HepG2 cells (83% and 84% in 5 and 25 mM glucose, respectively).

Conclusions: OXT expression is often altered in response to OXPHOS disruption in peripheral tissues; as in previous studies (7), the direction of effect is tissue-specific and may be compensatory. Indeed, short-term treatment of energy-modulating cell types (liver, muscle) in vitro with OXT increased mitochondrial content, which could be the basis of increased energy expenditure. Follow-up mechanistic studies are ongoing.