ABSTRACT
OBJECTIVE: Glucose-dependent insulinotropic polypeptide (GIP) and Glucagon-like peptide-1 (GLP-1) are incretin hormones that exert overlapping yet distinct actions on islet ß-cells. We recently observed that GIP, but not GLP-1, upregulated islet expression of Transcription Factor 7 (TCF7), a gene expressed in immune cells and associated with the risk of developing type 1 diabetes. TCF7 has also been associated with glucose homeostasis control in the liver. Herein we studied the relative metabolic importance of TCF7 expression in hepatocytes vs. islet ß-cells in mice. METHODS: Tcf7 expression was selectively inactivated in adult mouse hepatocytes using adenoviral Cre expression and targeted in ß-cells using two different lines of insulin promoter-Cre mice. Glucose homeostasis, plasma insulin and triglyceride responses, islet histology, hepatic and islet gene expression, and body weight gain were evaluated in mice fed regular chow or high fat diets. Tcf7 expression within pancreatic islets and immune cells was evaluated using published single cell RNA-seq (scRNA-seq) data, and in islet RNA from immunodeficient Rag2-/-Il2rg-/- mice. RESULTS: Reduction of hepatocyte Tcf7 expression did not impair glucose homeostasis, lipid tolerance or hepatic gene expression profiles linked to control of metabolic or immune pathways. Similarly, oral and intraperitoneal glucose tolerance, plasma insulin responses, islet histology, body weight gain, and insulin tolerance were not different in mice with targeted recombination of Tcf7 in insulin-positive ß-cells. Surprisingly, islet Tcf7 mRNA transcripts were not reduced in total islet RNA containing endocrine and associated non-endocrine cell types from Tcf7ßcell-/- mice, despite Cre-mediated recombination of islet genomic DNA. Furthermore, glucose tolerance was normal in whole body Tcf7-/- mice. Analysis of scRNA-seq datasets localized pancreatic Tcf7 expression to islet progenitors during development, and immune cells, but not within differentiated islet ß-cells or endocrine lineages within mature islets. Moreover, the expression of Tcf7 was extremely low in islet RNA from Rag2-/-Il2rg-/- mice and, consistent with expression within immune cells, Tcf7 was highly correlated with levels of Cd3g mRNA transcripts in RNA from wild type mouse islets. CONCLUSIONS: These findings demonstrate that Tcf7 expression is not a critical determinant of glucose homeostasis in mice. Moreover, the detection of Tcf7 expression within islet mRNA is attributable to the expression of Tcf7 RNA in islet-associated murine immune cells, and not in islet ß-cells.
Subject(s)
Blood Glucose/metabolism , Hepatocyte Nuclear Factor 1-alpha/metabolism , Hepatocytes/metabolism , Homeostasis/genetics , Insulin-Secreting Cells/metabolism , Non-alcoholic Fatty Liver Disease/metabolism , Animals , Cells, Cultured , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Diet, High-Fat/adverse effects , Disease Models, Animal , Female , Gene Expression , Hepatocyte Nuclear Factor 1-alpha/genetics , Insulin/blood , Insulin/genetics , Interleukin Receptor Common gamma Subunit/genetics , Interleukin Receptor Common gamma Subunit/metabolism , Lymphocytes/metabolism , Male , Mice , Mice, Knockout , Non-alcoholic Fatty Liver Disease/genetics , Triglycerides/blood , Weight Gain/geneticsABSTRACT
OBJECTIVE: Glucagon-like peptide-1 is a nutrient-sensitive hormone secreted from enteroendocrine L cells within the small and large bowel. Although GLP-1 levels rise rapidly in response to food ingestion, the greatest density of L cells is localized to the distal small bowel and colon. Here, we assessed the importance of the distal gut in the acute L cell response to diverse secretagogues. METHODS: Circulating levels of glucose and plasma GLP-1 were measured in response to the administration of L cell secretagogues in wild-type mice and in mice with (1) genetic reduction of Gcg expression throughout the small bowel and large bowel (GcgGut-/-) and (2) selective reduction of Gcg expression in the distal gut (GcgDistalGut-/-). RESULTS: The acute GLP-1 response to olive oil or arginine administration was markedly diminished in GcgGut-/- but preserved in GcgDistalGut-/- mice. In contrast, the increase in plasma GLP-1 levels following the administration of the GPR119 agonist AR231453, or the melanocortin-4 receptor (MC4R) agonist LY2112688, was markedly diminished in the GcgDistalGut-/- mice. The GLP-1 response to LPS was also markedly attenuated in the GcgGut-/- mice and remained submaximal in the GcgDistalGut-/- mice. Doses of metformin sufficient to lower glucose and increase GLP-1 levels in the GcgGut+/+ mice retained their glucoregulatory activity, yet they failed to increase GLP-1 levels in the GcgGut-/- mice. Surprisingly, the actions of metformin to increase plasma GLP-1 levels were substantially attenuated in the GcgDistalGut-/- mice. CONCLUSION: These findings further establish the importance of the proximal gut for the acute response to nutrient-related GLP-1 secretagogues. In contrast, we identify essential contributions of the distal gut to (i) the rapid induction of circulating GLP-1 levels in response to pharmacological selective agonism of G-protein-coupled receptors, (ii) the increased GLP-1 levels following the activation of Toll-Like Receptors with LPS, and iii) the acute GLP-1 response to metformin. Collectively, these results reveal that distal gut Gcg + endocrine cells are rapid responders to structurally and functionally diverse GLP-1 secretagogues.
Subject(s)
Enteroendocrine Cells/metabolism , Glucagon-Like Peptide 1/metabolism , Glucagon/metabolism , Animals , Blood Glucose/analysis , Colon/metabolism , Colon/physiology , Enteroendocrine Cells/pathology , Female , Gene Expression/genetics , Gene Expression Regulation/genetics , Glucagon/genetics , Glucagon-Like Peptide 1/blood , Glucagon-Like Peptide 1/genetics , Glucagon-Like Peptide-2 Receptor/metabolism , Glucose/metabolism , Insulin/metabolism , Intestine, Small/metabolism , Intestine, Small/physiology , Male , Metformin/pharmacology , Mice , Mice, Knockout , Proglucagon/metabolismABSTRACT
OBJECTIVE: Fibroblast Activation Protein (FAP), an enzyme structurally related to dipeptidyl peptidase-4 (DPP-4), has garnered interest as a potential metabolic drug target due to its ability to cleave and inactivate FGF-21 as well as other peptide substrates. Here we investigated the metabolic importance of FAP for control of body weight and glucose homeostasis in regular chow-fed and high fat diet-fed mice. METHODS: FAP enzyme activity was transiently attenuated using a highly-specific inhibitor CPD60 and permanently ablated by genetic inactivation of the mouse Fap gene. We also assessed the FAP-dependence of CPD60 and talabostat (Val-boroPro), a chemical inhibitor reportedly targeting both FAP and dipeptidyl peptidase-4 RESULTS: CPD60 robustly inhibited plasma FAP activity with no effect on DPP-4 activity. Fap gene disruption was confirmed by assessment of genomic DNA, and loss of FAP enzyme activity in plasma and tissues. CPD60 did not improve lipid tolerance but modestly improved acute oral and intraperitoneal glucose tolerance in a FAP-dependent manner. Genetic inactivation of Fap did not improve glucose or lipid tolerance nor confer resistance to weight gain in male or female Fap-/- mice fed regular chow or high-fat diets. Moreover, talabostat markedly improved glucose homeostasis in a FAP- and FGF-21-independent, DPP-4 dependent manner. CONCLUSION: Although pharmacological FAP inhibition improves glucose tolerance, the absence of a metabolic phenotype in Fap-/-mice suggest that endogenous FAP is dispensable for the regulation of murine glucose homeostasis and body weight. These findings highlight the importance of characterizing the specificity and actions of FAP inhibitors in different species and raise important questions about the feasibility of mouse models for targeting FAP as a treatment for diabetes and related metabolic disorders.
Subject(s)
Dipeptidyl Peptidase 4/metabolism , Gelatinases/metabolism , Glucose/metabolism , Membrane Proteins/metabolism , Serine Endopeptidases/metabolism , Animals , Blood Glucose/metabolism , Body Weight/physiology , Diabetes Mellitus/drug therapy , Diet, High-Fat , Dipeptidyl Peptidase 4/blood , Dipeptidyl-Peptidase IV Inhibitors/administration & dosage , Endopeptidases , Female , Fibroblast Growth Factors/metabolism , Gelatinases/physiology , Glucagon-Like Peptide 1/blood , Homeostasis/physiology , Insulin/metabolism , Male , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Serine Endopeptidases/physiology , Weight GainABSTRACT
OBJECTIVE: Pro-opiomelanocortin (POMC)-derived peptides act on neurons expressing the Melanocortin 4 receptor (MC4R) to reduce body weight. Setmelanotide is a highly potent MC4R agonist that leads to weight loss in diet-induced obese animals and in obese individuals with complete POMC deficiency. While POMC deficiency is very rare, 1-5% of severely obese individuals harbor heterozygous mutations in MC4R. We sought to assess the efficacy of Setmelanotide in human MC4R deficiency. METHODS: We studied the effects of Setmelanotide on mutant MC4Rs in cells and the weight loss response to Setmelanotide administration in rodent studies and a human clinical trial. We annotated the functional status of 369 published MC4R variants. RESULTS: In cells, we showed that Setmelanotide is significantly more potent at MC4R than the endogenous ligand alpha-melanocyte stimulating hormone and can disproportionally rescue signaling by a subset of severely impaired MC4R mutants. Wild-type rodents appear more sensitive to Setmelanotide when compared to MC4R heterozygous deficient mice, while MC4R knockout mice fail to respond. In a 28-day Phase 1b clinical trial, Setmelanotide led to weight loss in obese MC4R variant carriers. Patients with POMC defects upstream of MC4R show significantly more weight loss with Setmelanotide than MC4R deficient patients or obese controls. CONCLUSIONS: Setmelanotide led to weight loss in obese people with MC4R deficiency; however, further studies are justified to establish whether Setmelanotide can elicit clinically meaningful weight loss in a subset of the MC4R deficient obese population.
Subject(s)
Receptor, Melanocortin, Type 4/agonists , Receptor, Melanocortin, Type 4/deficiency , alpha-MSH/analogs & derivatives , Adrenal Insufficiency/drug therapy , Adrenal Insufficiency/metabolism , Adult , Amino Acid Sequence , Animals , Female , HEK293 Cells , Humans , Male , Mice , Mice, Knockout , Middle Aged , Obesity/drug therapy , Obesity/metabolism , Pro-Opiomelanocortin/deficiency , Pro-Opiomelanocortin/metabolism , Receptor, Melanocortin, Type 4/genetics , Receptor, Melanocortin, Type 4/metabolism , alpha-MSH/pharmacologyABSTRACT
GPR119 was originally identified as an orphan ß-cell receptor; however, subsequent studies demonstrated that GPR119 also regulates ß-cell function indirectly through incretin hormone secretion. We assessed the importance of GPR119 for ß-cell function in Gpr119-/- mice and in newly generated Gpr119ßcell-/- mice. Gpr119-/- mice displayed normal body weight and glucose tolerance on a regular chow (RC) diet. After high-fat feeding, Gpr119-/- mice exhibited reduced fat mass, decreased levels of circulating adipokines, improved insulin sensitivity, and better glucose tolerance. Unexpectedly, oral and intraperitoneal glucose tolerance and the insulin response to glycemic challenge were not perturbed in Gpr119ßcell-/- mice on RC and high-fat diets. Moreover, islets from Gpr119-/- and Gpr119ßcell-/- mice exhibited normal insulin responses to glucose and ß-cell secretagogues. Furthermore, the selective GPR119 agonist AR231453 failed to directly enhance insulin secretion from perifused islets. In contrast, AR231453 increased plasma glucagon-like peptide 1 (GLP-1) and insulin levels and improved glucose tolerance in wild-type and Gpr119ßcell-/- mice. These findings demonstrate that ß-cell GPR119 expression is dispensable for the physiological control of insulin secretion and the pharmacological response to GPR119 agonism, findings that may inform the lack of robust efficacy in clinical programs assessing GPR119 agonists for the therapy of type 2 diabetes.
Subject(s)
Blood Glucose/metabolism , Diabetes Mellitus, Experimental/metabolism , Glucagon-Like Peptide 1/metabolism , Insulin Resistance , Insulin-Secreting Cells/metabolism , Receptors, G-Protein-Coupled/genetics , Adipokines/metabolism , Animals , Apoptosis , Diet, High-Fat , Gene Expression Profiling , Glucagon/metabolism , Glucagon-Like Peptide 1/drug effects , Glucose/metabolism , Glucose Tolerance Test , Incretins/metabolism , Insulin/metabolism , Insulin Secretion , Male , Mice, Knockout , Oxadiazoles/pharmacology , Pyrimidines/pharmacology , Receptors, G-Protein-Coupled/agonistsABSTRACT
CONTEXT: Activation of the melanocortin-4 receptor (MC4R) with the synthetic agonist RM-493 decreases body weight and increases energy expenditure (EE) in nonhuman primates. The effects of MC4R agonists on EE in humans have not been examined to date. OBJECTIVE, DESIGN, AND SETTING: In a randomized, double-blind, placebo-controlled, crossover study, we examined the effects of the MC4R agonist RM-493 on resting energy expenditure (REE) in obese subjects in an inpatient setting. STUDY PARTICIPANTS AND METHODS: Twelve healthy adults (6 men and 6 women) with body mass index of 35.7 ± 2.9 kg/m(2) (mean ± SD) received RM-493 (1 mg/24 h) or placebo by continuous subcutaneous infusion over 72 hours, followed immediately by crossover to the alternate treatment. All subjects received a weight-maintenance diet (50% carbohydrate, 30% fat, and 20% protein) and performed 30 minutes of standardized exercise daily. Continuous EE was measured on the third treatment day in a room calorimeter, and REE in the fasting state was defined as the mean of 2 30-minute resting periods. RESULTS: RM-493 increased REE vs placebo by 6.4% (95% confidence interval, 0.68-13.02%), on average by 111 kcal/24 h (95% confidence interval, 15-207 kcal, P = .03). Total daily EE trended higher, whereas the thermic effect of a test meal and exercise EE did not differ significantly. The 23-hour nonexercise respiratory quotient was lower during RM-493 treatment (0.833 ± 0.021 vs 0.848 ± 0.022, P = .02). No adverse effect on heart rate or blood pressure was observed. CONCLUSIONS: Short-term administration of the MC4R agonist RM-493 increases REE and shifts substrate oxidation to fat in obese individuals.
Subject(s)
Anti-Obesity Agents/administration & dosage , Energy Metabolism/drug effects , Obesity/metabolism , Receptor, Melanocortin, Type 4/agonists , alpha-MSH/analogs & derivatives , Adult , Combined Modality Therapy , Cross-Over Studies , Double-Blind Method , Female , Humans , Male , Middle Aged , Obesity/therapy , Rest , Weight Reduction Programs , Young Adult , alpha-MSH/administration & dosageABSTRACT
The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.
Subject(s)
GTP-Binding Protein alpha Subunits, Gs , Neurons/metabolism , Paraventricular Hypothalamic Nucleus/cytology , Potassium Channels, Inwardly Rectifying/metabolism , Receptor, Melanocortin, Type 4/metabolism , Action Potentials , Agouti-Related Protein/metabolism , Animals , Eating/genetics , Energy Metabolism , Female , HEK293 Cells , Homeostasis/genetics , Humans , Ligands , Male , Melanocortins/metabolism , Mice , Receptor, Melanocortin, Type 4/genetics , Signal Transduction/genetics , alpha-MSH/metabolismABSTRACT
The melanocortin-4 receptor (MC4R) is expressed in the brainstem and vagal afferent nerves and regulates a number of aspects of gastrointestinal function. Here we show that the receptor is also diffusely expressed in cells of the gastrointestinal system, from stomach to descending colon. Furthermore, MC4R is the second most highly enriched GPCR in peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) expressing enteroendocrine L cells. When vectorial ion transport is measured across mouse or human intestinal mucosa, administration of α-MSH induces a MC4R-specific PYY-dependent antisecretory response consistent with a role for the MC4R in paracrine inhibition of electrolyte secretion. Finally, MC4R-dependent acute PYY and GLP-1 release from L cells can be stimulated in vivo by intraperitoneal (i.p.) administration of melanocortin peptides to mice. This suggests physiological significance for MC4R in L cells and indicates a previously unrecognized peripheral role for the MC4R, complementing vagal and central receptor functions.
Subject(s)
Enteroendocrine Cells/metabolism , Glucagon-Like Peptide 1/metabolism , Peptide YY/metabolism , Receptor, Melanocortin, Type 4/metabolism , Acids, Heterocyclic/pharmacology , Animals , Colon/cytology , Colon/drug effects , Colon/metabolism , Enteroendocrine Cells/drug effects , Humans , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oxadiazoles/pharmacology , Peptides, Cyclic/pharmacology , Receptor, Melanocortin, Type 4/agonistsABSTRACT
Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson-Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson-Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson-Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3' or 5' of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.
Subject(s)
Nucleotides/chemistry , Nucleotides/genetics , RNA Stability/genetics , RNA/chemistry , RNA/genetics , Base Pairing/genetics , Inverted Repeat Sequences/genetics , Models, Theoretical , Nucleic Acid Conformation , ThermodynamicsABSTRACT
Haploinsufficiency of the melanocortin-4 receptor (MC4R) results in melanocortin obesity syndrome, the most common monogenic cause of severe early onset obesity in humans. The syndrome, which produces measurable hyperphagia, has focused attention on the role of MC4R in feeding behavior and macronutrient intake. Studies show that inhibition of MC4R signaling can acutely increase the consumption of high-fat foods. The current study examines the chronic feeding preferences of mice with deletion of one or both alleles of the MC4R to model the human syndrome. Using two-choice diet paradigms with high-fat or high-carbohydrate foods alongside normal chow, we show, paradoxically, that deletion of one allele has no effect, whereas deletion of both alleles of the MC4R actually decreases preference for palatable high-fat and high-sucrose foods, compared with wild-type mice. Nonetheless, we observed hyperphagic behavior from increased consumption of the low-fat standard chow when either heterozygous or homozygous mutant animals were presented with dietary variety. Thus, decreased MC4R signaling in melanocortin obesity syndrome consistently yields hyperphagia irrespective of the foods provided, but the hyperphagia appears driven by variety and/or novelty, rather than by a preference for high-fat or high-carbohydrate foodstuffs.
Subject(s)
Eating/genetics , Food Preferences/physiology , Hyperphagia/genetics , Obesity/etiology , Receptor, Melanocortin, Type 4/genetics , Animals , Body Weight , Diet, High-Fat , Dietary Carbohydrates , Eating/physiology , Gene Deletion , Hyperphagia/complications , Male , Mice , Mice, Knockout , Receptor, Melanocortin, Type 4/physiologyABSTRACT
Defective melanocortin signaling causes hyperphagic obesity in humans and the melanocortin-4 receptor knockout mouse (MC4R(-/-)). The human disease most commonly presents, however, as haploinsufficiency of the MC4R. This study validates the MC4R(+/-) mouse as a model of the human disease in that, like the MC4R(-/-), the MC4R(+/-) mouse also exhibits a sustained hyperphagic response to dietary fat. Furthermore, both saturated and monounsaturated fats elicit this response. N-acylphosphatidylethanolamine (NAPE) is a signaling lipid induced after several hours of high-fat feeding, that, if dysregulated, might explain the feeding behavior in melanocortin obesity syndrome. Remarkably, however, MC4R(-/-) mice produce elevated levels of NAPE and are fully responsive to the anorexigenic activity of NAPE and oleoylethanolamide. Interestingly, additional differences in N-acylethanolamine (NAE) biochemistry were seen in MC4R(-/-) animals, including reduced plasma NAE levels and elevated hypothalamic levels of fatty acid amide hydrolase expression. Thus, while reduced expression of NAPE or NAE does not explain the high-fat hyperphagia in the melanocortin obesity syndrome, alterations in this family of signaling lipids are evident. Analysis of the microstructure of feeding behavior in response to dietary fat in the MC4R(-/-) and MC4R(+/-) mice indicates that the high-fat hyperphagia involves defective satiation and an increased rate of food intake, suggesting defective satiety signaling and enhanced reward value of dietary fat.