An Albumin-Exendin-4 Conjugate Engages Central and Peripheral Circuits Regulating Murine Energy and Glucose Homeostasis

Background & Aims: Glucagon-like peptide-1 (GLP-1) regulates glucose homeostasis through multiple mechanisms including direct actions on the endocrine pancreas and indirect activation of central nervous system circuits regulating gastric emptying, satiety, and body weight. Because native GLP-1 is rapidly degraded, there is considerable interest in development of more potent GLP-1 receptor (GLP-1R) agonists with sustained activity; however, the extent to which much larger GLP-1R agonists will mimic some or all of the actions of smaller peptides remains uncertain. Methods: We studied the actions of CJC-1134-PC, a recombinant human serum albumin-exendin-4 conjugated protein, at the GLP-1R using heterologous cells expressing the GLP-1R in vitro and both wild-type and Glp1r−/− mice in vivo. Results: CJC-1134-PC activated GLP-1R-dependent signaling in baby hamster kidney-GLP-1R cells and acutely lowered blood glucose in wild-type but not in Glp1r−/− mice. Moreover, acute administration of CJC-1134-PC rapidly activated c-Fos expression in multiple regions of the central nervous system, acutely inhibited gastric emptying, and produced sustained inhibition of food intake in a GLP-1R-dependent manner. Furthermore, chronic daily treatment of high-fat diet-fed wild-type mice with CJC-1134-PC for 4 weeks led to improved glucose tolerance, increased levels of glucose-stimulated insulin, decreased HbA1c, and weight loss associated with decreased hepatic triglyceride content. Conclusions: These findings illustrate that a high-molecular-weight exendin-4-albumin conjugate retains the ability to mimic a full spectrum of GLP-1R-dependent actions, including activation of central nervous system circuits regulating gastric emptying, food intake, and body weight. Background & Aims: Glucagon-like peptide-1 (GLP-1) regulates glucose homeostasis through multiple mechanisms including direct actions on the endocrine pancreas and indirect activation of central nervous system circuits regulating gastric emptying, satiety, and body weight. Because native GLP-1 is rapidly degraded, there is considerable interest in development of more potent GLP-1 receptor (GLP-1R) agonists with sustained activity; however, the extent to which much larger GLP-1R agonists will mimic some or all of the actions of smaller peptides remains uncertain. Methods: We studied the actions of CJC-1134-PC, a recombinant human serum albumin-exendin-4 conjugated protein, at the GLP-1R using heterologous cells expressing the GLP-1R in vitro and both wild-type and Glp1r−/− mice in vivo. Results: CJC-1134-PC activated GLP-1R-dependent signaling in baby hamster kidney-GLP-1R cells and acutely lowered blood glucose in wild-type but not in Glp1r−/− mice. Moreover, acute administration of CJC-1134-PC rapidly activated c-Fos expression in multiple regions of the central nervous system, acutely inhibited gastric emptying, and produced sustained inhibition of food intake in a GLP-1R-dependent manner. Furthermore, chronic daily treatment of high-fat diet-fed wild-type mice with CJC-1134-PC for 4 weeks led to improved glucose tolerance, increased levels of glucose-stimulated insulin, decreased HbA1c, and weight loss associated with decreased hepatic triglyceride content. Conclusions: These findings illustrate that a high-molecular-weight exendin-4-albumin conjugate retains the ability to mimic a full spectrum of GLP-1R-dependent actions, including activation of central nervous system circuits regulating gastric emptying, food intake, and body weight. Glucagon-like peptide-1 (GLP-1) is a naturally occurring peptide hormone that is released from intestinal L cells in response to nutrient ingestion. GLP-1 lowers blood glucose levels in both preclinical studies and in human subjects with type 2 diabetes mellitus (T2DM) through multiple distinct actions including stimulation of glucose-dependent insulin secretion, suppression of glucagon secretion, and inhibition of gastric emptying.1Deacon C.F. Therapeutic strategies based on glucagon-like peptide 1.Diabetes. 2004; 53: 2181-2189Crossref PubMed Scopus (282) Google Scholar, 2Drucker D.J. The biology of incretin hormones.Cell Metab. 2006; 3: 153-165Abstract Full Text Full Text PDF PubMed Scopus (1678) Google Scholar GLP-1 also promotes satiety, suppresses energy intake, and causes weight loss in healthy, obese, and diabetic humans. In preclinical studies, GLP-1 increases β-cell mass via induction of β-cell proliferation and neogenesis and inhibition of β-cell apoptosis.3Xu G. Stoffers D.A. Habener J.F. et al.Exendin-4 stimulates both β-cell replication and neogenesis, resulting in increased β-cell mass and improved glucose tolerance in diabetic rats.Diabetes. 1999; 48: 2270-2276Crossref PubMed Scopus (1081) Google Scholar, 4Stoffers D.A. Kieffer T.J. Hussain M.A. et al.Insulinotropic glucagon-like peptide-1 agonists stimulate expression of homeodomain protein IDX-1 and increase β-cell mass in mouse pancreas.Diabetes. 2000; 49: 741-748Crossref PubMed Scopus (515) Google Scholar, 5Li Y. Hansotia T. Yusta B. et al.Glucagon-like peptide-1 receptor signaling modulates β-cell apoptosis.J Biol Chem. 2003; 278: 471-478Crossref PubMed Scopus (525) Google Scholar The combined properties of GLP-1 to reduce glycemia and promote weight loss, in association with its potential to restore or sustain β-cell mass and function, have sparked considerable interest in the use of GLP-1 as a therapeutic agent for the treatment of T2DM.6Drucker D.J. Nauck M.A. The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes.Lancet. 2006; 368: 1696-1705Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar A major limitation to the therapeutic use of the native GLP-1 molecule is its very short half-life in the circulation, which has been attributed to both rapid cleavage by the ubiquitous proteolytic enzyme dipeptidyl peptidase-4 (DPP-4) and renal clearance. Consequently, GLP-1 must be injected repeatedly or infused continuously to sustain clinical efficacy in vivo. Thus, pharmaceutic strategies based on sustained GLP-1 receptor (GLP-1R) activation have been focused on the development of long-acting DPP-4-resistant GLP-1R agonists such as exenatide and liraglutide. Exenatide is a synthetic version of the lizard salivary gland-derived peptide exendin-4 (Ex-4) that is resistant to DPP-4.7Eng J. Kleinman W.A. Singh L. et al.Isolation and characterization of exendin 4, an exendin 3 analogue from Heloderma suspectum venom.J Biol Chem. 1992; 267: 7402-7405Abstract Full Text PDF PubMed Google Scholar Preclinical and clinical studies demonstrate that exenatide is a long-acting GLP-1R agonist,8Young A.A. Gedulin B.R. Bhavsar S. et al.Glucose-lowering and insulin-sensitizing actions of exendin-4: studies in obese diabetic (ob/ob, db/db) mice, diabetic fatty Zucker rats, and diabetic rhesus monkeys (Macaca mulatta).Diabetes. 1999; 48: 1026-1034Crossref PubMed Scopus (388) Google Scholar, 9Kolterman O.G. Buse J.B. Fineman M.S. et al.Synthetic exendin-4 (exenatide) significantly reduces postprandial and fasting plasma glucose in subjects with type 2 diabetes.J Clin Endocrinol Metab. 2003; 88: 3082-3089Crossref PubMed Scopus (520) Google Scholar and it has been approved for clinical use in the United States and Europe. Liraglutide is a DPP-4-resistant fatty-acylated GLP-1 peptide analog that binds noncovalently to serum albumin following subcutaneous administration, thereby reducing its renal clearance and extending its pharmacokinetic profile.10Agerso H. Jensen L.B. Elbrond B. et al.The pharmacokinetics, pharmacodynamics, safety and tolerability of NN2211, a new long-acting GLP-1 derivative, in healthy men.Diabetologia. 2002; 45: 195-202Crossref PubMed Scopus (479) Google Scholar Liraglutide exhibits more potent and persistent glucose-lowering effects in diabetic patients compared with native GLP-1 and is currently undergoing evaluation in phase III clinical trials in patients with T2DM. Although exenatide and liraglutide effectively lower blood glucose levels in T2DM patients, the requirement for twice or once daily administration of these agents, respectively, has fostered ongoing efforts to generate GLP-1R agonists with more sustained effectiveness. One such putative agent is CJC-1134-PC, a conjugate consisting of Ex-4 that has been covalently bonded, ex vivo, to recombinant human serum albumin (HSA) via a chemical linker (see Supplementary Figure 1 online at www.gastrojournal.org). Preliminary results from clinical trials in diabetic humans indicate that CJC-1134-PC has a half-life of approximately 8 days, making it suitable for once weekly dosing.11Wang M, Kipnes MS, Matheson S, et al. Safety and pharmacodynamics of CJC-1134-PC, a novel GLP-1 receptor agonist, in patients with type 2 diabetes mellitus: a randomized, placebo-controlled, double-blind, dose-escalation study (Poster 498). Propanel Abstracts of the 67th Scientific Sessions of the American Diabetes Association, June 22–26, 2007, Chicago, IL, p A133.Google Scholar Nevertheless, in short-term studies over 4 weeks, CJC-1134-PC produced only modest effects on body weight; hence, it remains uncertain whether this high-molecular-weight GLP-1R agonist is capable of engaging central nervous system (CNS) circuits regulating appetite and body weight. To determine whether the considerably larger Ex-4-albumin hybrid CJC-1134-PC retains the identical spectrum of biologic actions exhibited by the much smaller GLP-1R peptide agonist Ex-4, we examined the effects of CJC-1134-PC on GLP-1R-dependent actions in vitro as well as in short- and long-term studies in mice. Cell culture medium was purchased from HyClone (Logan, UT). Serum, G418, and antibiotics were from Invitrogen-Gibco (Burlington, ON). Forskolin and 3-isobutyl-1-methylxantine (IBMX) were obtained from Sigma Chemical Co (St Louis, MO). HSA and CJC-1134-PC were provided by Conjuchem (Montreal, QC). Ex-4, glucose-dependent insulinotropic peptide, exendin (9–39), and cholecystokinin-8 were purchased from California Peptide Research Inc (Napa, CA). Wild-type C57BL/6 mice (4 or 7 weeks old) were from Charles River Laboratories (Montreal, QC). Glp1r−/− mice on the CD-1 genetic background have been described.12Scrocchi L.A. Brown T.J. MacLusky N. et al.Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide receptor gene.Nat Med. 1996; 2: 1254-1258Crossref PubMed Scopus (660) Google Scholar Glp1r−/− mice backcrossed onto the C57BL/6 genetic background for 6 generations13Hansotia T. Maida A. Flock G. et al.Extrapancreatic incretin receptors modulate glucose homeostasis, body weight, and energy expenditure.J Clin Invest. 2007; 117: 143-152Crossref PubMed Scopus (221) Google Scholar were used in the current studies. All mice were maintained under a 12-hour light/dark cycle (lights on at 7 am and off at 7 pm) with free access to food and water, except where noted. All experiments were conducted in accordance with protocols and guidelines approved by the University Health Network Animal Care Committee. For acute studies, 8- to 10-week-old wild-type or Glp1r−/− male mice were used. For chronic studies, 4-week-old wild-type male mice were fed either standard rodent chow or a high-fat diet (HFD; 45% kcal from fat; Research Diets Inc, New Brunswick, NJ) for 4 weeks then maintained on their respective diets for an additional 4 weeks. Mice on the standard rodent chow diet were given twice daily intraperitoneal (IP) injections of phosphate-buffered saline (PBS); HFD mice were randomized to receive (1) twice daily IP injections of PBS, (2) twice daily IP injections of Ex-4 (24 nmol/kg), (3) once daily IP injections of HSA (100 nmol/kg), or (4) once daily IP injections of CJC-1134-PC (100 nmol/kg). Twice daily injections were at ∼8 am and 6 pm. For mice receiving once daily injections, drugs were administered in the evening, but mice were also given IP injections of PBS in the morning to ensure that all mice were exposed to identical experimental conditions. Blood glucose levels in whole blood were measured using a Glucometer Elite blood glucose meter (Bayer, Toronto, ON). Plasma insulin levels were measured using a Rat/Mouse Insulin ELISA kit (LINCO Research, St Charles, MO), and pancreatic insulin levels were determined using a Rat insulin RIA kit (LINCO Research). HbA1c levels were measured using a DCA 2000+ Analyzer (Bayer). Plasma glucagon and leptin were measured using a Mouse Endocrine LINCOplex kit (LINCO Research). Serum monocyte chemoattractant protein-1 (MCP-1), total plasminogen activator inhibitor-1 (PAI-1), and resistin levels were measured using a Mouse Adipokine LINCOplex kit (LINCO Research). Hepatic and/or serum levels of total cholesterol and triglycerides were obtained using specific colorimetric assays (Wako Cholesterol E and Wako L-Type TG-H; Wako Chemicals, Richmond, VA). Baby hamster kidney fibroblast cells stably transfected with the rat GLP-1R5Li Y. Hansotia T. Yusta B. et al.Glucagon-like peptide-1 receptor signaling modulates β-cell apoptosis.J Biol Chem. 2003; 278: 471-478Crossref PubMed Scopus (525) Google Scholar were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Cells were incubated with 1 μmol/L exendin (9–39) or medium alone for 5 minutes at 37°C, followed by an additional 10-minute incubation in the presence of increasing concentrations of Ex-4 or CJC-1134-PC. All reactions were carried out in triplicate and terminated by the addition of ice-cold absolute ethanol. cAMP concentration was measured in ethanol extracts using a cAMP RIA kit (Biomedical Technologies, Stoughton, MA). Oral glucose tolerance tests (OGTT) and intraperitoneal (IP) glucose tolerance tests (IPGTT) were performed after an overnight fast of 16–18 hours after administration of glucose (1.5 mg/g body weight). For acute studies, CJC-1134-PC or HSA was given via IP injection 1 hour prior to glucose administration, whereas glucose-dependent insulinotropic peptide was injected 10 minutes before glucose administration. For chronic studies, no drugs were administered prior to the glucose tolerance tests. Mice were fasted overnight for 16–18 hours, weighed, and then given IP injections of PBS or 10 nmol/kg of Ex-4, HSA, or CJC-1134-PC. The mice were then placed into individual cages containing preweighed rodent chow with free access to water. Food was removed from the cages at 2, 4, 8, and 24 hours following treatment, and food intake (g/g body weight) was calculated. The rate of gastric emptying was determined as described14Asakawa A. Inui A. Yuzuriha H. et al.Characterization of the effects of pancreatic polypeptide in the regulation of energy balance.Gastroenterology. 2003; 124: 1325-1336Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar 4 hours after peptide injections. The gastric emptying rate was calculated using the following equation: gastric emptying rate (%) = [1 − (stomach content wet weight/food intake)] × 100. The number of c-Fos immunopositive neurons was quantified in regions of the mouse CNS as described.15Elias C.F. Kelly J.F. Lee C.E. et al.Chemical characterization of leptin-activated neurons in the rat brain.J Comp Neurol. 2000; 423: 261-281Crossref PubMed Scopus (321) Google Scholar, 16Yamamoto H. Kishi T. Lee C.E. et al.Glucagon-like peptide-1-responsive catecholamine neurons in the area postrema link peripheral glucagon-like peptide-1 with central autonomic control sites.J Neurosci. 2003; 23: 2939-2946PubMed Google Scholar Sections were processed for immunocytochemical detection of Fos using an avidin-biotin-immunoperoxidase method (Vectastain ABC Elite Kit; Vector Laboratories, Burlingame, CA) as described.17Yamamoto H. Lee C.E. Marcus J.N. et al.Glucagon-like peptide-1 receptor stimulation increases blood pressure and heart rate and activates autonomic regulatory neurons.J Clin Invest. 2002; 110: 43-52Crossref PubMed Scopus (453) Google Scholar The Fos antibody (Sigma-Aldrich, Oakville, ON) was used at a dilution of 1:50,000. Sections corresponding to CNS regions at the level of the area postrema, nucleus of the solitary tract, and paraventricular nucleus of the hypothalamus were defined according to Franklin and Paxinos.18Franklin K. Paxinos G. The mouse brain in sterotaxic coordinates. Academic Press, Burlington, MA1997Google Scholar Total body fat and lean mass were measured using a mouse whole-body magnetic resonance analyzer (Echo Medical Systems, Houston, TX). Pancreatic sections were immunostained for insulin as described.13Hansotia T. Maida A. Flock G. et al.Extrapancreatic incretin receptors modulate glucose homeostasis, body weight, and energy expenditure.J Clin Invest. 2007; 117: 143-152Crossref PubMed Scopus (221) Google Scholar Morphometric measurements of insulin-positive area and total pancreatic area were determined using ImageScope software (Aperio Technologies Inc, Vista, CA), and β-cell mass was calculated using the following equation: (insulin positive area/total pancreatic area) × pancreas weight. For each pancreas sample, the β-cell mass was averaged from 2 different slides that were obtained from the same pancreas sectioned at 2 different levels. For histologic examination of hepatic lipids, the liver was removed, embedded in Tissue-Tek OCT Compound (Sakura Finetek, Torrence, CA), frozen rapidly in a dry ice/ethanol bath, and stored at −80°C. Frozen tissue was cut into 8-μm sections and stained with Oil-red O using standard protocols. For hepatic lipid determinations, hepatic lipids were extracted as described.19Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J Biol Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar RNA was extracted from pancreas samples using TRI reagent (Sigma Chemical Co) and first-strand complementary DNA (cDNA) generated using standard protocols. Primers and polymerase chain reaction (PCR) conditions for the selected genes were those recommended by Applied Biosystems (Foster City, CA). Relative messenger RNA (mRNA) levels were quantified using the 2-ΔΔCt method.20Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔC(T)) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (120953) Google Scholar All data are presented as means ± SE. Statistical significance was determined by 1- or 2-way ANOVA and Bonferroni post hoc test using Prism version 4.02 software (GraphPad Software, San Diego, CA). A P value < .05 was considered to be statistically significant. To determine whether covalent attachment of a large molecule like HSA impaired the ability of the Ex-4 moiety within CJC-1134-PC to activate the GLP-1R, we compared the potency of CJC-1134-PC vs Ex-4 in vitro. Both Ex-4 and CJC-1134-PC increased cAMP levels in baby hamster kidney-GLP-1R cells in a dose- and GLP-1R-dependent manner (see Supplementary Figure 2 online at www.gastrojournal.org) with half maximal effective concentration values for CJC-1134-PC and Ex-4 (3.47 nmol/L for CJC-1134-PC and 2.62 nmol/L for Ex-4). We next examined whether CJC-1134-PC can reproduce the repertoire of actions associated with the smaller Ex-4 peptide. CJC-1134-PC significantly reduced glucose excursion after oral and IP glucose challenge in a dose-dependent manner in wild-type mice (Figure 1A and C) but not in Glp1r−/− mice (Figure 1E and F) with significant increases in insulin/glucose ratios at doses of 10 and 100 nmol/kg (Figure 1B and D). In contrast, glucose-dependent insulinotropic peptide potently decreased glucose excursion in Glp1r−/− mice (Figure 1E). CJC-1134-PC significantly reduced food intake in wild-type mice (Figure 2), and the effect of CJC-1134-PC and Ex-4 to decrease feeding lasted for at least 24 hours (Figure 2A). Moreover, at 4–8 hours and 8–24 hours following injection, the reduction in food intake was significantly greater with CJC-1134-PC compared with Ex-4 (Figure 2). The anorectic action of CJC-1134-PC was not simply a nonspecific aversive response because it required a functional GLP-1 receptor (Figure 2A). The mechanisms through which GLP-1 regulates gastric emptying remain incompletely understood and may require interaction with neural pathways.21Imeryuz N. Yegen B.C. Bozkurt A. et al.Glucagon-like peptide-1 inhibits gastric emptying via vagal afferent-mediated central mechanisms.Am J Physiol. 1997; 273: G920-G927PubMed Google Scholar, 22Nagell C.F. Wettergren A. Orskov C. et al.Inhibitory effect of GLP-1 on gastric motility persists after vagal deafferentation in pigs.Scand J Gastroenterol. 2006; 41: 667-672Crossref PubMed Scopus (37) Google Scholar Both Ex-4 and CJC-1134-PC significantly diminished the gastric emptying rate in wild-type mice (Figure 2B) but not in Glp1r−/− mice (Figure 2C). In contrast, cholecystokinin-8, which inhibits gastric emptying through a distinct mechanism, significantly reduced the gastric emptying rate in Glp1r−/− mice (Figure 2C). To determine whether GLP-1R agonists communicate with the CNS to inhibit feeding and gastric emptying, we first compared the effects of peripheral administration of Ex-4 vs CJC-1134-PC on c-Fos positivity, a marker of neuronal activity (Figure 3). Ex-4 and CJC-1134-PC produced a similar pattern of c-Fos expression in the mouse CNS that included the area postrema, nucleus of the solitary tract, and paraventricular nucleus of the hypothalamus (Figure 3A, B, and C, respectively), regions associated with the control of feeding and gut motility. Moreover, the relative level of c-Fos activation following IP CJC-1134-PC was comparable with that of Ex-4 (Figure 3A–C). Conversely, IP injection of Ex-4 or CJC-1134-PC failed to increase c-Fos expression in Glp1r−/− mice (Figure 3A–C, bar graphs). Immunohistochemical examination of the mouse CNS following IP administration of HSA did not identify any HSA-immunopositive staining (data not shown). Because larger molecules like CJC-1134-PC are unlikely to readily cross the normal blood-brain barrier, these data, taken together with the results of previous experiments,23Baggio L.L. Huang Q. Brown T.J. et al.A recombinant human glucagon-like peptide (GLP)-1-albumin protein (albugon) mimics peptidergic activation of GLP-1 receptor-dependent pathways coupled with satiety, gastrointestinal motility, and glucose homeostasis.Diabetes. 2004; 53: 2492-2500Crossref PubMed Scopus (311) Google Scholar suggest that GLP-1R agonists do not require direct access to the CNS to modify feeding behavior and gastric motility. Prolonged administration of GLP-1 or Ex-4 reduces blood glucose levels and promotes weight loss in individuals with T2DM.6Drucker D.J. Nauck M.A. The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes.Lancet. 2006; 368: 1696-1705Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar, 24Zander M. Madsbad S. Madsen J.L. et al.Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and β-cell function in type 2 diabetes: a parallel-group study.Lancet. 2002; 359: 824-830Abstract Full Text Full Text PDF PubMed Scopus (1161) Google Scholar, 25DeFronzo R.A. Ratner R.E. Han J. et al.Effects of exenatide (exendin-4) on glycemic control and weight over 30 weeks in metformin-treated patients with type 2 diabetes.Diabetes Care. 2005; 28: 1092-1100Crossref PubMed Scopus (1331) Google Scholar Accordingly, mice were rendered obese and glucose intolerant and then treated with once or twice daily IP injection of CJC-1134-PC or Ex-4, respectively, for 4 weeks (see Supplementary Figure 3 online at www.gastrojournal.org). Nonfasting blood glucose levels in HFD-fed CJC-1134-PC- and Ex-4-treated mice were lower compared with control mice (Figure 4A). Mice on the HFD gained more weight compared with mice maintained on the normal chow diet during the first 4 weeks (Figure 4B). However, HFD-fed mice treated with CJC-1134-PC or Ex-4 lost weight, and, after 4 weeks of treatment, mice treated with CJC-1134-PC had body weights similar to normal chow-fed PBS-treated mice (Figure 4B). Serum HbA1c levels were reduced in HFD-fed mice treated with CJC-1134-PC but not Ex-4 (Figure 4C). Chronic administration of Ex-4 or CJC-1134-PC in HFD-fed mice produced significant reductions in glucose excursions following glucose challenge (Figure 5A, B, D, and E), and CJC-1134-PC but not Ex-4 increased insulin-to-glucose ratios measured following glucose administration (Figure 5C and F). Chronic treatment with CJC-1134-PC or Ex-4 had no effect on plasma glucagon levels measured in these same samples (data not shown). Magnetic resonance imaging (MRI) data demonstrated that Ex-4 and CJC-1134-PC reduced total and percent body fat mass and increased percent lean mass relative to HFD-fed control treated mice (Figure 6A and B). Furthermore, body weight and epididymal fat pad weights were significantly decreased in CJC-1134-PC- and Ex-4-treated HFD-fed mice and were comparable with those observed in PBS-treated mice maintained on a normal chow diet (Figure 6C and data not shown).Figure 6CJC-1134-PC decreases body weight and fat mass in HFD-fed wild-type mice. MRI determinations of (A) fat mass and (B) lean mass, expressed as total (grams; [g]) or normalized to body weight (%), in high-fat diet (HFD)-fed wild-type mice after 2 weeks of treatment with PBS, exendin-4 (Ex-4), human serum albumin (HSA), or CJC-1134-PC or in mice maintained on normal chow and treated with PBS (RC PBS) for 2 weeks. (C) Body weight in HFD-fed mice after 4 weeks of treatment with PBS, exendin-4 (Ex-4), human serum albumin (HSA), or CJC-1134-PC or in mice maintained on normal chow and treated with PBS (RC PBS). Data are expressed as means ± SE; n = 7–8 mice/group. ##P < .01, ###P < .001 for Ex-4- vs PBS-treated mice. **P < .01, ***P < .001 for CJC-1134-PC- vs HSA-treated mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CJC-1134-PC or Ex-4 had no effect on serum MCP-1 or PAI-1 levels in HFD-fed mice (Figure 7A and B), whereas treatment with Ex-4, but not CJC-1134-PC, led to significant reductions in serum resistin (Figure 7C). Serum leptin levels were significantly lower in both CJC-1134-PC- and Ex-4-treated mice (Figure 7D). Serum cholesterol levels were reduced in HFD-fed mice following administration of CJC-1134-PC but not Ex-4 (Figure 8A). Surprisingly, chronic treatment with both CJC-1134-PC and Ex-4 resulted in significant elevations in serum triglyceride levels (Figure 8B). Although hepatic cholesterol levels were unchanged (data not shown), total hepatic triglyceride content was reduced following treatment with CJC-1134-PC or Ex-4 in HFD-fed mice (Figure 8C). Oil-Red-O staining (see Supplementary Figure 4 online at www.gastrojournal.org) further illustrated the reduced hepatic lipid content in CJC-1134-PC- and Ex-4-treated mice. The decrease in hepatic triglyceride, in association with increased plasma triglyceride levels, raised the possibility that mice treated with CJC-1134-PC or Ex-4 fail to accumulate triglyceride in the liver, perhaps leading to increased circulating levels of triglycerides. Hence, we examined the expression of hepatic genes whose products are important for lipid absorption, transport, metabolism, or synthesis. Real-time quantitative PCR demonstrated that treatment with CJC-1134-PC or Ex-4 was associated with significantly reduced levels of mRNA transcripts for fatty acid binding proteins 1 and 2 (Fabp1 and Fabp2) and CD36, respectively (Figure 8D–F). Conversely, CJC-1134-PC or Ex-4 administration did not alter the mRNA levels of apolipoprotein B, hepatic lipase, peroxisomal proliferator-activated receptor α, or sterol regulatory element binding protein-1 (Srebf1) (data not shown). Ex-4, but not CJC-1134-PC, increased insulin mRNA levels compared with saline-treated mice (see Supplementary Figure 5A online at www.gastrojournal.org). In contrast, pancreas insulin content was significantly reduced in CJC-1134-PC-treated mice relative to levels in mice treated with HSA but was not significantly different in Ex-4- vs control-treated mice (see Supplementary Figure 5B online at www.gastrojournal.org). Although both CJC-1134-PC and Ex-4 increased pancreas weights in HFD-fed mice (see Supplementary Figure 5C online at www.gastrojournal.org), no significant increases in β-cell mass were observed following chronic treatment with either agent (see Supplementary Figure 5D online at www.gastrojournal.org). GLP-1R agonists also increase the expression of genes that contribute to β-cell glucose sensing, proliferation, neogenesis, or cytoprotection.2Drucker D.J. The biology of incretin hormones.Cell Metab. 2006; 3: 153-165Abstract Full Text Full Text PDF PubMed Scopus (1678) Google Scholar, 26Wang Y.H. Egan J.M. Raygada M. et al.Glucagon-like peptide-1 affects gene transcription and messenger ribonucleic acid stability of components of the insulin secretory system in RIN 1046-38 cells.Endocrinology. 1995; 136: 4910-4917Crossref PubMed Google Scholar, 27Drucker D.J. Glucagon-like peptides: regulators of cell proliferation, differentiation, and apoptosis.Mol Endocrinol. 2003; 17: 161-171Crossref PubMed Scopus (419) Google Scholar mRNA levels of Gck, Glp1r, and Pdx1 were increased in pancreatic RNA from both CJC-1134-PC- and/or Ex-4-treated mice (see Supplementary Figure 5E–G online at www.gastrojournal.org). Surprisingly however, Ex-4, but not CJC-1134-PC, markedly increased levels of mRNA transcripts for Irs2 (see Supplementary Figure 5H online at www.gastrojournal.org). Although there i