Endogenous Humanin Regulates Beta Cell Viability and Glucose-Stimulated Insulin Secretion in Mouse Beta Cells
Presentation Number: SUN 605
Date of Presentation: April 2nd, 2017
Manal Habib*1, Akari Utsunomiya2, Bo-Chul Shin3, Hiromi Nakamura1, Vladislava Paharkova1, Nicole Ng1, Julian Whitelegge4, Sangeeta Dhawan5, Kuk-Wha Lee6 and Sherin U Devaskar1
1Mattel Children's Hospital, UCLA, Los Angeles, CA, 2Hiroshima University Hospital, 3Mattel Children's Hospital, UCLA, 4The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute, David Geffen School of Medicine,UCLA, 5David Geffen School of Medicine, UCLA, Los Angeles, CA, 6Florida Hospital, Orlando, FL
Humanin (HN) is a short polypeptide transcribed from an open reading frame within a region of mtDNA. The human homologue exhibits cytoprotective properties against Alzheimer’s disease-, myocardial infarction-, and diabetes-associated cellular insults. We have previously reported that HN improves survival of beta cells in response to cytokine-induced cell death and delays onset of diabetes in the NOD mouse. We also previously demonstrated that HN and insulin co-localize in rat INS-1 cells, and that knockdown of HN abrogates glucose-stimulated insulin secretion (GSIS) in the mouse insulin secreting cell line, MIN6.
To this end in MIN6 cells, we hypothesized that: 1) Glycemia regulates mouse HN (mHN) expression, and 2) mHN knockdown will decrease cell viability, increase apoptosis and impair oxidative phosphorylation.
To test these hypothesis, we first examined the glucose-dependent effect of HN expression. MIN6 cells were incubated in glucose-containing media at 0, 5, 16, and 32 mM in a time course experiment. mHN was assessed by Western blots and immunostaining, both of which revealed a dose- and time-dependent increase in mHN expression. We next examined the biological role of endogenous HN by knocking down (KD) HN. mHN expression in MIN6 cells was effectively knocked down with siRNA targeting the nuclear sequence, which allowed measurement of beta cell viability by the MTT assay, and apoptosis by the caspase 3/7 assay. Under hyperglycemic conditions, upon mHN KD, there was a 50% decrease in beta cell viability, and 40% increase in the rate of apoptosis. To determine the mechanism by which mHN regulates glucose stimulated insulin secretion, under mHN KD, we measured ATP in a time-dependent manner and observed a ~20% decrease (p<0.05) which led us to undertake the Seahorse assessment of oxidative phosphorylation and glycolysis, GLUT2 translocation by immunostaining and assessment of glucose-stimulated insulin secretion in the presence of calcium channel blockers. These experiments uncovered the mechanisms responsible for mHN's regulation of GSIS.
In summary, our preliminary data demonstrate that HN is a novel binding partner of insulin and is essential for GSIS. KD of HN results in impaired beta cell function and viability. This is the first description of a physiologic role for endogenous HN in a biologic process, and presents additional avenues for discovery in beta cell pathophysiology, perhaps paving the way towards amelioration of type I diabetes.
Nothing to Disclose: MH, AU, BCS, HN, VP, NN, JW, SD, KWL, SUD