Antiobesity action of peripheral exenatide (exendin-4) in rodents: effects on food intake, body weight, metabolic status and side-effect measures.

BACKGROUND: Exenatide (exendin-4) is an incretin mimetic currently marketed as an antidiabetic agent for patients with type 2 diabetes. In preclinical models, a reduction in body weight has also been shown in low-fat-fed, leptin receptor-deficient rodents. OBJECTIVE: To more closely model the polygenic and environmental state of human obesity, we characterized the effect of exenatide on food intake and body weight in high-fat-fed, normal (those with an intact leptin signaling system) rodents. As glucagon-like peptide-1 receptor agonism has been found to elicit behaviors associated with visceral illness in rodents, we also examined the effect of peripheral exenatide on kaolin consumption and locomotor activity. METHODS AND RESULTS: High-fat-fed C57BL/6 mice and Sprague-Dawley rats were treated with exenatide (3, 10 and 30 microg/kg/day) for 4 weeks via subcutaneously implanted osmotic pumps. Food intake and body weight were assessed weekly. At 4 weeks, body composition and plasma metabolic profiles were measured. Kaolin consumption and locomotor activity were measured in fasted Sprague-Dawley rats following a single intraperitoneal injection of exenatide (0.1-10 microg/kg). Exenatide treatment in mice and rats dose-dependently decreased food intake and body weight; significant reductions in body weight gain were observed throughout treatment at 10 and 30 microg/kg/day (P<0.05). Decreased body weight gain was associated with a significant decrease in fat mass (P<0.05) with sparing of lean tissue. Plasma cholesterol, triglycerides and insulin were also significantly reduced (P<0.05). Exenatide at 10 microg/kg significantly reduced food intake (P<0.05) but failed to induce kaolin intake. In general, locomotor activity was reduced at doses of exenatide that decreased food intake, although a slightly higher dose was required to produce significant changes in activity. CONCLUSION: Systemic exenatide reduces body weight gain in normal, high-fat-fed rodents, a model that parallels human genetic variation and food consumption patterns, and may play a role in metabolic pathways mediating food intake.

Effects of PYY[3-36] in rodent models of diabetes and obesity.

BACKGROUND: Peptide YY (PYY) is a 36 amino-acid peptide secreted from ileal L cells following meals. The cleaved subpeptide PYY[3-36] is biologically active and may constitute the majority of circulating PYY-like immunoreactivity. The peptide family that includes PYY, pancreatic peptide and neuropeptide Y is noted for its orexigenic effect following intracerebroventricular administration. OBJECTIVE: To investigate the effects of peripheral (intraperitoneal and chronic subcutaneous) infusions of PYY[3-36] on food intake, body weight and glycemic indices. DESIGN/RESULTS: Food intake was measured in normal mice and in several rodent models of obesity and type II diabetes. In marked contrast to the reported central orexigenic effects, in the present study, PYY[3-36] acutely inhibited food intake by up to 45%, with an ED(50) of 12.5 microg/kg in fasted female NIH/Swiss mice. A 4-week infusion reduced weight gain in female ob/ob mice, without affecting the cumulative food intake. In diet-induced obese male mice, PYY[3-36] infusion reduced cumulative food intake, weight gain and epididymal fat weight (as a fraction of carcass) with similar ED(50)'s (466, 297 and 201 microg/kg/day, respectively) and prevented a diet-induced increase in HbA1c. Infusion at 100 microg/kg/day for 8 weeks in male fa/fa rats reduced the weight gain (288+/-11 vs 326+/-12 g in saline-infused controls; P<0.05), similar to effects in a pair-fed group. In female ob/ob and db/db mice, there was no acute effect of PYY[3-36] on plasma glucose concentrations. In male diabetic fatty Zucker rats, PYY[3-36] infused for 4 weeks reduced HbA1c and fructosamine (ED(50)'s 30 and 44 microg/kg/day). CONCLUSION: Peripheral PYY[3-36] administration reduced the food intake, body weight gain and glycemic indices in diverse rodent models of metabolic disease of both sexes. These findings justify further exploration of the potential physiologic and therapeutic roles of PYY[3-36].

Comparison of the in vitro and in vivo pharmacology of adrenomedullin, calcitonin gene-related peptide and amylin in rats.

Adrenomedullin has been reported to be structurally similar to a group of peptides that includes amylin, calcitonin and calcitonin gene-related peptide (CGRP). Human and rat adrenomedullin displaced [125I]CGRP from membranes of SK-N-MC cells (CGRP receptors) with affinities intermediate between those of rat amylin and rat CGRP alpha (Ki values 0.12 +/- 0.06, 0.017 +/- 0.007, 3.83 +/- 1.14 and 0.007 +/- 0.001 nM, respectively). In contrast Ki values for displacement of [125I]rat amylin from accumbens membranes (amylin receptors), and [125I]salmon calcitonin from T47D cells (calcitonin receptors) were lower than with rat amylin or rat CGRP alpha in these preparations (51 +/- 5, 34 +/- 2, 0.024 +/- 0.002, 0.31 +/- 0.07 nM, respectively, at amylin receptors; 33 +/- 5, 69 +/- 29, 2.7 +/- 1.5 and 13 +/- 3 nM, respectively, at calcitonin receptors). In anesthetized rats, the hypotensive potency of adrenomedullin was between that of amylin and CGRP alpha. In contrast, for amylin or calcitonin agonist actions (inhibition of [14C]glycogen formation in soleus muscle, hyperlactemia, hypocalcemia and inhibition of gastric emptying), human adrenomedullin was without measurable effect. Thus, in its binding behaviour and in its biological actions, adrenomedullin appeared to behave as a potent CGRP agonist, but as a poor amylin or calcitonin agonist.

Molecular cloning and functional expression of a third isoform of the human calcitonin receptor and partial characterization of the calcitonin receptor gene.

We have cloned and expressed two isoforms of the human calcitonin (hCT) receptor. Primers designed from the published sequence of a CT receptor cloned from an ovarian small cell carcinoma line were used for the polymerase chain reaction amplification of related products from human breast carcinoma MCF-7 cells. Two complementary DNAs were isolated. One clone lacks a 16-amino acid insert in the first intracellular loop and is virtually identical to the receptor recently cloned from the T47D human breast carcinoma cell line. The second clone is another splice variant lacking both the 16-amino acid insert in the first intracellular domain as well as the first 47 amino acids of the amino-terminus extracellular domain. COS-7 cells transfected with either receptor isoform bound [125I]salmon CT with high affinity and responded to hCT with increases in cAMP. Tissue distribution studies revealed the truncated extracellular domain 1 isoform transcripts in human skeletal muscle, kidney, brain, and lung. Analysis of a hCT receptor genomic clone demonstrated an exon/intron organization similar to that of the porcine CT receptor gene, except for a distinct exon coding for the alternatively spliced insert in the first intracellular domain.

Differential antagonism of amylin's metabolic and vascular actions with amylin receptor antagonists.

High affinity amylin binding sites are present in the rat nucleus accumbens. These sites bind [125I]amylin with an affinity of 27 pM and have high affinity for salmon calcitonin (sCT) and moderately high affinity for calcitonin gene related peptide (CGRP). N-terminally truncated peptides were tested for their ability to compete for [125I]amylin binding to these sites and to antagonize the metabolic and vascular actions of amylin. CGRP(8-37), sCT(8-32), and ac-[Asn30,Tyr32]sCT(8-32) (AC187) inhibited [125I]amylin binding to rat nucleus accumbens. Order of potency at inhibiting amylin binding (AC187 > sCT(8-32) > CGRP(8-37)) differed from the order of potency at inhibiting [125I]CGRP binding to SK-N-MC neuroblastoma cells (CGRP(8-37) > AC187 > sCT(8-32)) . AC187 was the most potent antagonist of amylin's effects on isolated rat soleus muscle glycogen metabolism, and it was more effective than either sCT(8-32) or CGRP(8-37) at reducing amylin-stimulated hyperlactemia in rats. In contrast, CGRP(8-37) was the most potent peptide at antagonizing amylin-induced hypotension in rats. Amylin's hypotensive actions appear to be mediated by a weak action at CGRP receptors, while its metabolic actions are mediated by receptors with a distinct antagonist profile. AC187 is a potent antagonist of amylin binding sites in nucleus accumbens and of amylin's metabolic actions.

Regulation of muscle glycogen metabolism by CGRP and amylin: CGRP receptors not involved.

The aim of the present study was to determine whether amylin and calcitonin gene-related peptide (CGRP) act through shared or distinct receptors to inhibit insulin-stimulated incorporation of [14C]-glucose into glycogen. Rat amylin was 3 fold more potent than either rat alpha CGRP or rat beta CGRP at reducing glycogen synthesis from [14C]-glucose in insulin-treated rat soleus muscle. This action was blocked by peptide antagonists, with the rank order of potency being AC187 > salmon calcitonin8-32 (sCT8-32) > h-alpha CGRP8-37 for antagonism of either amylin or CGRP. The antagonist potency order correlated with affinity for amylin receptors measured in rat nucleus accumbens but not CGRP receptors measured in rat L6 muscle cells. Inhibition of glucose incorporation into glycogen by amylin and CGRP appears to be mediated by shared receptors that have the pharmacological characteristics of amylin receptors, and are distinct from previously described CGRP receptors.

Molecular physiology of amylin.

Amylin is a 37-amino acid peptide first isolated, purified, and characterized from the amyloid deposits in the pancrease of type 2 diabetics. It is synthesized and secreted primarily from pancreatic beta cells along with insulin. The ability of amylin to potently reduce insulin-stimulated incorporation of glucose into glycogen in skeletal muscle requires both an intact 2Cys-7Cys disulfide bond and a COOH-terminal amide. Amylin has structural and functional relationships to two other messenger proteins, calcitonin and CGRP. Amylin has relatively potent calcitonin-like activity on bone metabolism and weaker CGRP-like activity on the vasculature. CGRP is a slightly weaker agonist than amylin for metabolic responses. Although rat calcitonins are weak, teleost fish calcitonins are very potent agonists for amylin's metabolic effects. This group of peptides appears to act on a family of related G protein-coupled receptors; several variant calcitonin receptors have recently been cloned and expressed. These receptors appear to be coupled to adenylyl cyclase in many instances; recent evidence supports the view that amylin's effects on skeletal muscle occur, at least in large part, through activation of the cAMP pathway.

Salmon calcitonin binding and stimulation of cyclic AMP generation in rat skeletal muscle.

Salmon calcitonin potently competes for amylin binding sites in rat brain and has amylin-like actions upon glucose metabolism in rat muscle. We report here that [125I]-salmon calcitonin binds to rat hindlimb muscle membranes with high affinity (Kd = 0.47 pM). Binding was inhibited by rat amylin (Ki = 2 nM), rat alpha CGRP (Ki = 8 nM), rat beta CGRP (Ki = 11 nM), and rat calcitonin (Ki = 64 nM). Binding was maximal when measured in a hypotonic NaHepes buffer, and was significantly reduced in affinity when salts of Mg++, Ca++, Na+ or K+ were present. Incubation of rat hindlimb muscle membranes with salmon calcitonin at concentrations of 10 pM and above stimulated cyclic AMP generation. These results describe a skeletal muscle binding site which may mediate some of the actions of exogenous salmon calcitonin and of endogenous amylin and related peptides upon skeletal muscle fuel metabolism.

Molecular cloning of two receptors from rat brain with high affinity for salmon calcitonin.

Two receptors with high affinity for salmon calcitonin were cloned from the nucleus accumbens region of rat brain. The deduced 479 amino acid sequence of cDNA clone L2175-D20 (designated C1a receptor) is 78% and 66% identical with those reported for human and porcine calcitonin receptors, respectively. Clone U3237-A2 codes for a receptor (designated C1b) that is identical to C1a except for a 37 amino acid insert in the second extracellular domain. COS-7 cells transfected with either transcript bound [125I]salmon calcitonin with high affinity (Kd = 8 pM for C1a; Kd = 48 pM for C1b) and responded to salmon calcitonin with increases in cAMP. Tissue distribution studies revealed C1a transcript in rat brain, skeletal muscle, kidney and lung, whereas C1b was predominantly found in brain.

Co-secretion of amylin and insulin from cultured islet beta-cells: modulation by nutrient secretagogues, islet hormones and hypoglycemic agents.

Amylin is a pancreatic islet beta-cell peptide hormone which modulates carbohydrate metabolism in skeletal muscle and liver, and could contribute to impaired insulin sensitivity in Type II diabetes. Here we report the first description of amylin secretion from isolated beta-cells. We measured amylin secretion from HIT T15 beta-cells exposed to glucose, arginine, glucagon, somatostatin, tolbutamide, glyburide, or metformin. With the exception of glucagon at concentrations above 1 microM, all compounds induced parallel, dose-dependent changes in secretion of amylin and insulin. We conclude that: 1) insulin and amylin are co-secreted from islet beta-cells; (2) nutrient secretagogues and peptide modulators exert direct effects on beta-cells to alter amylin and insulin secretion; (3) most modulators of islet beta-cell secretion alter amylin and insulin in parallel, but differential secretion can occur; and (4) HIT cell line is a useful model in which to study amylin metabolism.