FGF21 Is an Insulin-Dependent Postprandial Hormone in Adult Humans.

Context: Fibroblast growth factor 21 (FGF21) secretion has been shown to respond directly to carbohydrate consumption, with glucose, fructose, and sucrose all reported to increase plasma levels of FGF21 in rodents and humans. However, carbohydrate consumption also results in secretion of insulin. Objective: The aim of this study was to examine the combined and independent effects of hyperglycemia and hyperinsulinemia on total and bioactive FGF21 in the postprandial period in humans, and determine whether this effect is attenuated in conditions of altered insulin secretion and action. Methods: Circulating glucose, insulin, total and bioactive FGF21, and fibroblast activation protein were measured in adults with and without type 2 diabetes (T2D) following an oral glucose tolerance test (OGTT), and under a series of insulin and glucose clamp conditions and following high-fat diet in healthy adults. Results: Circulating total and bioactive FGF21 levels responded acutely to OGTT, and their ratio was attenuated in T2D patients with reduced postprandial insulin response. The clamp studies revealed that insulin but not glucose accounts for the postprandial rise in FGF21. Finally, there was an attenuated rise in FGF21 in response to a high-fat dietary intervention that is known to alter insulin-stimulated substrate utilization in metabolically active tissues. Conclusions: Insulin rather than glucose per se increases total and bioactive FGF21 in the postprandial period in adult humans. Understanding the impact of T2D on bioactive FGF21 will have a significant effect upon the efficacy of therapeutic agents designed to target the FGF21 pathway.

Antibody-Mediated Targeting of the FGFR1c Isoform Increases Glucose Uptake in White and Brown Adipose Tissue in Male Mice.

The increased prevalence of obesity and its cardiometabolic implications demonstrates the imperative to identify novel therapeutic targets able to effect meaningful metabolic changes in this population. Antibody-mediated targeting of fibroblast growth factor receptor 1c isoform (FGFR1c) has been shown to ameliorate hyperglycemia and protect from diet- and genetically-induced obesity in rodents and nonhuman primates. However, it is currently unknown which tissue(s) contribute to this glucose-lowering effect. Thus, to elucidate this effect, we treated euglycemic mice with H7, a monoclonal antibody that selectively targets FGFR1c, and used whole-body positron emission computed tomography with a glucose tracer (18F-fluorodeoxyglucose). Treatment with H7 increased basal glucose uptake in white adipose tissue (WAT), brown adipose tissue (BAT), the brain, and liver but reduced it in the quadriceps muscles. Consequentially, blood glucose was significantly reduced in response to treatment. Under insulin-stimulated conditions, the effects of H7 were maintained in WAT, BAT, liver, and muscle. Treatment with H7 decreased triglyceride (TG) content and increased adipose TG lipase content in white adipose tissue, while increasing activation of acetyl coenzyme A carboxylase, suggesting futile cycling of TGs, albeit favoring net hydrolysis. We demonstrated, in vitro, this is a direct effect of treatment in adipose tissue, as basal cellular respiration and glucose uptake were increased in response to treatment. Taken together, these data suggest that antibody-mediated targeting of FGFR1c exerts its powerful glucose-lowering efficacy primarily due to increased glucose uptake in adipose tissue.

Discrete Aspects of FGF21 In Vivo Pharmacology Do Not Require UCP1.

A primary target of the pleiotropic metabolic hormone FGF21 is adipose tissue, where it initiates a gene expression program to enhance energy expenditure, an effect presumed to be centered on augmented UCP1 expression and activity. In UCP1 null (UCP1KO) mice, we show that the effect of FGF21 to increase the metabolic rate is abolished. However, in contrast to prior expectations, we found that increased UCP1-dependent thermogenesis is only partially required to achieve the beneficial effects of FGF21 treatment. In UCP1KO mice, there appears to be an underlying reduction in food intake following FGF21 administration, facilitating weight loss equal to that observed in wild-type animals. Furthermore, we show that UCP1-dependent thermogenesis is not required for FGF21 to improve glycemic control or to reduce circulating cholesterol or free fatty acids. These data indicate that several important metabolic endpoints of FGF21 are UCP1 independent; however, the contribution of UCP1-dependent thermogenesis to other discrete aspects of FGF21 biology requires further study.

A novel and selective beta-melanocyte-stimulating hormone-derived peptide agonist for melanocortin 4 receptor potently decreased food intake and body weight gain in diet-induced obese rats.

alphaMSH has generally been accepted as the endogenous ligand for melanocortin 4 receptor (MC4R), which plays a major role in energy homeostasis. Targeting MC4R to develop antiobesity agents, many investigators have performed a structure-activity relationship (SAR) studies based on alphaMSH structure. In this report, we performed a SAR study using human betaMSH (5 - 22) (DEGPYRMEHFRWGSPPKD, peptide 1) as a lead sequence to develop potent and selective agonists for MC4R and MC3R. The SAR study was begun with a truncation of N terminus of betaMSH (5 - 22) together with acetylation of the N terminus and amidation of the C terminus of the peptide. Introduction of a cyclic disulfide constrain and replacement of L-Phe with D-Phe afforded a super potent agonist (peptide 5). Furthermore truncation at the C terminus generated a small and potent MC4R and MC3R agonist (Ac-YRcyclo[CEHdFRWC]amide, peptide 6), which exhibited no MC5R and greatly reduced MC1R activity. Molecular modeling of Ac-YRcyclo[CEHdFRWC]amide (peptide 6) revealed that Arg2 in the peptide formed a salt bridge with Glu4. Subcutaneous or intracerebroventricular administration of peptide 6 in rats showed potent in vivo efficacy as evidenced by its effects in reducing energy balance, increasing fat use, and decreasing weight gain in both acute and chronic rat metabolic studies. Furthermore, the antiobesity effect by peptide 6 was manifested only in wild-type but not MC4R-deficient mice, indicating that antiobesity effects of the peptide were attributed largely through MC4R but not MC3R agonist activity of the peptide.

Potent peptide agonists for human melanocortin 3 and 4 receptors derived from enzymatic cleavages of human beta-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.

Discovery of a beta-MSH-derived MC-4R selective agonist.

A series of novel, disulfide-constrained human beta-melanocyte stimulating hormone (beta-MSH)-derived peptides were optimized for in vitro melanocortin-4 receptor (MC-4R) binding affinity, agonist efficacy, and selectivity. The most promising of these, analogue 18, was further studied in vivo using chronic rat food intake and body weight models.

Central H3R activation by thioperamide does not affect energy balance.

The central histamine 3 receptor (H3R) is a presynaptic autoreceptor that regulates neuronal release and synthesis of histamine, and is thought to play a key role in controlling numerous central nervous system (CNS)-mediated parameters, including energy homeostasis. Thioperamide, the prototypical selective H3R antagonist, was used to examine the role that H3R plays in regulating energy balance in vivo. Thioperamide was administered either intraperitoneally or orally to rats and the pharmacokinetic parameters were examined along with central H3R binding and histaminergic system activation. Food intake and metabolic parameters of either route of thioperamide administration were likewise examined. In a dose-dependent manner, both the intraperitoneal and oral route of administration resulted in similar ex vivo binding curves and tele-methylhistamine dose-response curves despite the route of administration. However, only intraperitoneal administration of 30 mg/kg thioperamide resulted in a significant decrease in 24-h food intake (60% lower than control) and respiratory quotient (RQ), while the oral route of delivery did not. Moreover, the decrease in RQ with the 30 mg/kg ip administration also decreased energy expenditure (EE) thus resulting in an unchanged energy balance. The decrease in food intake and EE was coupled with a conditioned taste aversion with the 30-mg/kg ip administration. These data indicate that the activation of the central H3R system by thioperamide does not play a direct role in decreasing food intake or altering energy homeostasis.

Vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor 2 deficiency in mice results in growth retardation and increased basal metabolic rate.

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are two closely related peptides that bind two homologous G protein-coupled receptors, VIP/PACAP receptor 1 (VPAC1R) and VIP/PACAP receptor II (VPAC2R), with equally high affinity. Recent reports suggest that VPAC2R plays a role in circadian rhythm and T cell functions. To further elucidate the functional activities of VPAC2R, we generated VPAC2R-deficient mice by deleting exons VIII-X of the VPAC2R gene. The VPAC2R-deficient mice showed retarded growth and had reduced serum IGF-I levels compared with gender-matched, wild-type siblings. The mutant mice appeared healthy and fertile at a young adult age. However, older male mutant mice exhibited diffuse seminiferous tubular degeneration with hypospermia and reduced fertility rate. The mutant mice appeared to have an increase in insulin sensitivity. VPAC2R-deficient mice had increased lean mass and decreased fat mass with reduced serum leptin levels. Indirect calorimetry experiments showed that the respiratory quotient values immediately following the transition into the dark cycle were significantly higher in male knockout mice for about 4 h. Additionally, male and female VPAC2R-deficient mice presented an increased basal metabolic rate (23% and 10%, respectively) compared with their wild-type siblings. Our results suggest that VPAC2R plays an important role in growth, basal energy expenditure, and male reproductive functions.