Immunohistochemical Localization of [D-Leu-4]-OB3, a Synthetic Peptide Leptin Mimetic, in the Arcuate Nucleus of the Mouse Hypothalamus Following Oral Delivery in Dodecyl Maltoside
Presentation Number: LBSat-43
Date of Presentation: April 2nd, 2016
Brian M Anderson, Lauren Jacobson and Patricia Grasso*
Albany Medical College, Albany, NY
[D-Leu-4]-OB3, a synthetic peptide leptin mimetic, regulates energy balance and glucose homeostasis in genetically obese insulin-resistant ob/ob and db/db mice, and in streptozotocin-induced hyperglycemic non-obese Swiss Webster mice. We have recently shown that [D-Leu-4]-OB3, in a manner similar to that of leptin, activates STAT3 via phosphorylation of ERK1/2 and PI-3K, suggesting that these signals may ultimately result in peptide effects on transcriptional and translational events associated with energy balance and glycemic regulation. In the present study, we describe the biodistribution of [D-Leu-4]-OB3 binding in the hypothalamus of normal male Swiss Webster mice, C57BL/6J wild-type mice, and genetically obese leptin-deficient ob/ob mice (N = 6 per strain). Six-week old mice were given [D-Leu-4]-OB3 (40 mg/kg) orally by gavage in 0.3% dodecyl maltoside (trade name Intravail®). 50 minutes following treatment, the mice were deeply anesthetized with pentobarbital. Whole body fixation was achieved by paraformaldehyde perfusion through the left ventricle. The brains were removed, post-fixed in paraformaldehyde, and cryoprotected in sucrose. Free-floating coronal sections were cut at 25-microns and stored in sucrose at -20 C until processed for imaging by immunofluorescence microscopy. For imaging, the sections were incubated with a rabbit polyclonal antibody to [D-Leu-4]-OB3 (previously validated for ELISA) at a dilution of 1:500, followed by incubation with Cy3-labeled goat anti-rabbit secondary antibody at 1:400. The sections were then mounted on charged glass slides and examined. In all three strains of mice, dense staining was concentrated along the wall and at the base of the third ventricle at the level of the arcuate nucleus. This area is known to contain first order leptin receptor-containing anorexigenic proopiomelanocortin (POMC)/cocaine- and amphetamine-regulated transcript (CART)- expressing neurons as well as orexigenic neuropeptide Y (NPY)/Agouti-related protein (AgRP)-expressing neurons. Contrary to what has been reported for leptin binding, however, no [D-Leu-4]-OB3 binding was noted in any other region of the hypothalamus at the time points examined. These results are highly significant in that they represent the first visual evidence of [D-Leu-4]-OB3 crossing the blood-brain barrier in an area of the hypothalamus known to regulate energy balance. Together with our previously reported signaling data, these findings are consistent with a central mechanism of action for [D-Leu-4]-OB3 involving the activation of hypothalamic leptin receptors.
Nothing to Disclose: BMA, LJ, PG