Hansen M. Hsiung

Absence of Cocaine- and Amphetamine-Regulated Transcript Results in Obesity in Mice Fed a High Caloric Diet

Cart (cocaine- and amphetamine-regulated transcript) was first identified to be a major brain mRNA up-regulated by cocaine and amphetamine. The CART protein has been established as a satiety factor closely associated with the action of leptin. To assess CART’s role as an anorexigenic signal, we have generated CART-deficient mice by gene targeting. On a high fat diet, CARTdeficient and female heterozygous mice, but not male heterozygous mice, showed statistically significant increases in weekly food consumption, body weight, and fat mass compared with their wild-type littermates. Furthermore, CART-deficient and female heterozygous mice were significantly heavier when fed a high fat diet than on a regular chow diet at 17 wk of age and at the 14th wk of the feeding studies. However, wild-type or male heterozygous mice showed no weight variations attributable to caloric contents of the diet at that age. Contrary to the obese phenotypes shown in MC4R-, proopiomelanocortin-, or leptindeficient mice, our results showed that CART deficiency predisposed mice to become obese on a calorically dense diet. The results also show that CART may not be a major anorectic signal compared with proopiomelanocortin or leptin in the regulation of energy homeostasis. (Endocrinology 142: 4394–4400, 2001)

Structure-Activity Relationships of β -MSH Derived Melanocortin-4 Receptor Peptide Agonists

The recent emergence of obesity as a major health threat in the industrialized world has intensified the search for novel and effective pharmacologic treatment. The proopiomelanocortin (POMC)-melanocortin 4 receptor (MC4R) axis has been shown to regulate food intake and energy homeostasis and is considered among the most promising antiobesity targets. Our initial efforts in this area have focused on affinity and selectivity directed optimization of the native beta-MSH(5-22) sequence and resulted in the discovery of a potent MC4R agonist: Ac-Tyr-Arg-[Cys-Glu-His-D-Phe-Arg-Trp-Cys]-NH(2) (10). Subcutaneous administration of this peptide produced an excellent in vivo efficacy in reducing food intake and increasing fat metabolism. Additionally, suppression of food intake was observed in wild type but not in MC4R deficient mice, suggesting that the effects observed in the wild type mice were mediated through MC4R signaling. Subsequent optimization efforts led to the identification of a novel series of disulfide constrained hexapeptides as exemplified by Ac-[hCys-His-D-Phe-Arg-Trp-Cys]-NH(2) (100). These cyclic hexapeptides showed a further improved potency in binding MC4R and an enhanced selectivity over MC1R. At a dose of 0.07 mg/kg analog 102 reduced food intake by 38% and increased fat utilization by 58% in rats. These cyclic peptides provide novel and enhanced reagents for the elucidation of melanocortin receptors biology and may find applications in the treatment of obesity and related metabolic disorders.

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor.

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His1, 125I-Tyr10,Nle27]hGHRH(1-32)-NH2 increased linearly with protein concentration (10-45 micrograms protein/ tube). Binding reached equilibrium after 90 min at 30 degrees C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (Kd = 1.04 +/- 0.19 nM, Bmax = 3.9 +/- 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC50) for porcine GHRH (2.8 +/- 0.51 nM), rat GHRH (3.1 +/- 0.69 nM), [N-Ac-Tyr1, D-Arg2]hGHRH(3-29)-NH2 (3.9 +/- 0.58 nM), and [D-Thr7]GHRH(1-29)-NH2 (189.7 +/- 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.

Potent peptide agonists for human melanocortin 3 and 4 receptors derived from enzymatic cleavages of human β-MSH(5–22) by dipeptidyl peptidase I and dipeptidyl peptidase IV

Human beta-MSH(1-22) was first isolated from human pituitary as a 22-amino acid (aa) peptide derived from a precursor protein, pro-opiomelanocortin (POMC). However, Bertagna et al. demonstrated that a shorter human beta-MSH(5-22), (DEGPYRMEHFRWGSPPKD), is a true endogenous peptide produced in human hypothalamus. In this report, we demonstrated that in vitro enzymatic cleavage of native human beta-MSH(5-22) with two ubiquitous dipeptidyl peptidases (DPP), DPP-I and DPP-IV, generated two potent MC3/4R peptide analogues, beta-MSH(7-22) (GPYRMEHFRWGSPPKD) and beta-MSH(9-22) (YRMEHFRWGSPPKD). In fact, the MC4R binding affinity and functional potency of beta-MSH(7-22) (Ki=4.6 nM, EC50=0.6 nM) and beta-MSH(9-22) (Ki=5.7 nM, EC50=0.6 nM) are almost an order of magnitude greater than those of their parent peptide, beta-MSH(5-22) (MC4R, Ki=23 nM, EC50= 3nM). Furthermore, the DPP-I/DPP-IV cleaved peptide, beta-MSH(9-22), when administered intracerebroventricularly (ICV) at a dose of 3 nmol/rat, potently induced an acute negative energy balance in a diet-induced obese rat model, while its parent molecule, beta-MSH(5-22), administered at the same dose did not have any effect. These data suggest that DPP-I and DPP-IV may play a role in converting the endogenous beta-MSH(5-22) to more potent peptides that regulate energy homeostasis in the hypothalamus.