RESUMO
The melanocortin system regulates metabolic homeostasis and inflammation. Melanocortin agonists have contradictorily been reported to both increase and decrease metabolic rate and body temperature. We find two distinct physiologic responses occurring at similar doses. Intraperitoneal administration of the nonselective melanocortin agonist MTII causes a melanocortin-4 receptor (Mc4r)-mediated hypermetabolism/hyperthermia. This is preceded by a profound, transient hypometabolism/hypothermia that is preserved in mice lacking any one of Mc1r, Mc3r, Mc4r, or Mc5r. Three other melanocortin agonists also caused hypothermia, which is actively achieved via seeking a cool environment, vasodilation, and inhibition of brown adipose tissue thermogenesis. These results suggest that the hypometabolic/hypothermic effect of MTII is not due to a failure of thermoregulation. The hypometabolism/hypothermia was prevented by dopamine antagonists, and MTII selectively activated arcuate nucleus dopaminergic neurons, suggesting that these neurons may contribute to the hypometabolism/hypothermia. We propose that the hypometabolism/hypothermia is a regulated response, potentially beneficial during extreme physiologic stress.
Assuntos
Temperatura Corporal/efeitos dos fármacos , Receptores de Melanocortina/agonistas , alfa-MSH/análogos & derivados , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Antagonistas de Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Tipo 1 de Melanocortina/agonistas , Receptor Tipo 1 de Melanocortina/genética , Receptor Tipo 1 de Melanocortina/metabolismo , Receptor Tipo 3 de Melanocortina/agonistas , Receptor Tipo 3 de Melanocortina/genética , Receptor Tipo 3 de Melanocortina/metabolismo , Receptor Tipo 4 de Melanocortina/agonistas , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo , Receptores de Melanocortina/genética , Receptores de Melanocortina/metabolismo , alfa-MSH/farmacologiaRESUMO
The chemical uncoupler 2,4-dinitrophenol (DNP) was an effective and widely used weight loss drug in the early 1930s. However, the physiology of DNP has not been studied in detail because toxicity, including hyperthermia and death, reduced interest in the clinical use of chemical uncouplers. To investigate DNP action, mice fed a high fat diet and housed at 30 °C (to minimize facultative thermogenesis) were treated with 800 mg/liter DNP in drinking water. DNP treatment increased energy expenditure by â¼ 17%, but did not change food intake. DNP-treated mice weighed 26% less than controls after 2 months of treatment due to decreased fat mass, without a change in lean mass. DNP improved glucose tolerance and reduced hepatic steatosis without observed toxicity. DNP treatment also reduced circulating T3 and T4 levels, Ucp1 expression, and brown adipose tissue activity, demonstrating that DNP-mediated heat generation substituted for brown adipose tissue thermogenesis. At 22 °C, a typical vivarium temperature that is below thermoneutrality, DNP treatment had no effect on body weight, adiposity, or glucose homeostasis. Thus, environmental temperature should be considered when assessing an anti-obesity drug in mice, particularly agents acting on energy expenditure. Furthermore, the beneficial effects of DNP suggest that chemical uncouplers deserve further investigation for the treatment of obesity and its comorbidities.
Assuntos
2,4-Dinitrofenol/farmacologia , Adiposidade/efeitos dos fármacos , Regulação da Temperatura Corporal/efeitos dos fármacos , Dieta/efeitos adversos , Metabolismo Energético/efeitos dos fármacos , Obesidade/tratamento farmacológico , Desacopladores/farmacologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Animais , Ingestão de Alimentos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Canais Iônicos/biossíntese , Camundongos , Proteínas Mitocondriais/biossíntese , Obesidade/induzido quimicamente , Obesidade/metabolismo , Obesidade/patologia , Proteína Desacopladora 1RESUMO
Increased hepatic glucose production is a key pathophysiological feature of type 2 diabetes. Like all other cell types, hepatocytes express many G protein-coupled receptors (GPCRs) that are linked to different functional classes of heterotrimeric G proteins. The important physiological functions mediated by G(s)-coupled hepatic glucagon receptors are well-documented. In contrast, little is known about the in vivo physiological roles of hepatocyte GPCRs that are linked to G proteins of the G(q) family. To address this issue, we established a transgenic mouse line (Hep-Rq mice) that expressed a G(q)-linked designer receptor (Rq) in a hepatocyte-selective fashion. Importantly, Rq could no longer bind endogenous ligands but could be selectively activated by a synthetic drug, clozapine-N-oxide. Clozapine-N-oxide treatment of Hep-Rq mice enabled us to determine the metabolic consequences caused by selective activation of a G(q)-coupled GPCR in hepatocytes in vivo. We found that acute Rq activation in vivo led to pronounced increases in blood glucose levels, resulting from increased rates of glycogen breakdown and gluconeogenesis. We also demonstrated that the expression of the V(1b) vasopressin receptor, a G(q)-coupled receptor expressed by hepatocytes, was drastically increased in livers of ob/ob mice, a mouse model of diabetes. Strikingly, treatment of ob/ob mice with a selective V(1b) receptor antagonist led to reduced glucose excursions in a pyruvate challenge test. Taken together, these findings underscore the importance of G(q)-coupled receptors in regulating hepatic glucose fluxes and suggest novel receptor targets for the treatment of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/induzido quimicamente , Ativadores de Enzimas/efeitos adversos , Quinases de Receptores Acoplados a Proteína G/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicogenólise/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Células Cultivadas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Ativadores de Enzimas/farmacologia , Feminino , Quinases de Receptores Acoplados a Proteína G/química , Quinases de Receptores Acoplados a Proteína G/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Hipoglicemiantes/uso terapêutico , Masculino , Camundongos , Camundongos Obesos , Camundongos Transgênicos , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/química , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Organismos Livres de Patógenos EspecíficosRESUMO
Spinophilin (SPL), a multidomain scaffolding protein known to modulate the activity of different G-protein-coupled receptors, regulates various central nervous system (CNS) functions. However, little is known about the role of SPL expressed in peripheral cell types including pancreatic ß cells. In this study, we examined the ability of SPL to modulate the activity of ß-cell M(3) muscarinic acetylcholine receptors (M3Rs), which play an important role in facilitating insulin release and maintaining normal blood glucose levels. We demonstrated, by using both in vitro and in vivo approaches (mouse insulinoma cells and SPL-deficient mice), that SPL is a potent negative regulator of M3R-mediated signaling and insulin release. Additional biochemical and biophysical studies, including the use of bioluminescence resonance energy transfer technology, suggested that SPL is able to recruit regulator of G-protein signaling 4 (RGS4) to the M3R signaling complex in an agonist-dependent fashion. Since RGS4 is a member of the RGS family of proteins that act to reduce the lifetime of activated G proteins, these findings support the concept that the inhibitory effects of SPL on M3R activity are mediated by RGS4. These data suggest that SPL or other G-protein-coupled receptor-associated proteins may serve as novel targets for drug therapy aimed at improving ß-cell function for the treatment of type 2 diabetes.
Assuntos
Resistência à Insulina/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptor Muscarínico M3/metabolismo , Animais , Western Blotting , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Resistência à Insulina/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Lipodystrophies are characterized by a loss of white adipose tissue, which causes ectopic lipid deposition, peripheral insulin resistance, reduced adipokine levels, and increased food intake (hyperphagia). The growth factor myostatin (MSTN) negatively regulates skeletal muscle growth, and mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. MSTN inhibition may therefore be efficacious in ameliorating diabetes. To test this hypothesis, we inhibited MSTN signaling in a diabetic model of generalized lipodystrophy to analyze its effects on glucose metabolism separate from effects on adipose mass. A-ZIP/F1 lipodystrophic mice were crossed to mice expressing a dominant-negative MSTN receptor (activin receptor type IIB) in muscle. MSTN inhibition in A-ZIP/F1 mice reduced blood glucose, serum insulin, triglyceride levels, and the rate of triglyceride synthesis, and improved insulin sensitivity. Unexpectedly, hyperphagia was normalized by MSTN inhibition in muscle. Blood glucose and hyperphagia were reduced in double mutants independent of the adipokine leptin. These results show that the effect of MSTN inhibition on insulin sensitivity is not secondary to an effect on adipose mass and that MSTN inhibition may be an effective treatment for diabetes. These results further suggest that muscle may play a heretofore unappreciated role in regulating food intake.
Assuntos
Diabetes Mellitus/genética , Hiperfagia/genética , Resistência à Insulina/genética , Lipodistrofia/complicações , Músculo Esquelético/metabolismo , Miostatina/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Glucose/farmacologia , Hiperfagia/etiologia , Hiperfagia/metabolismo , Insulina/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/genética , Lipodistrofia/genética , Lipodistrofia/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/efeitos dos fármacos , Miostatina/metabolismoRESUMO
OBJECTIVE: Obesity, insulin resistance, and type 2 diabetes form a tightly correlated cluster of metabolic disorders in which adipose is one of the first affected tissues. The role of hypoxia and hypoxia-inducible factor 1 (HIF1) in the development of high-fat diet (HFD)-induced obesity and insulin resistance was investigated using animal models. RESEARCH DESIGN AND METHODS: Mice with adipocyte-specific targeted disruption of the genes encoding the HIF1 obligatory subunits Hif1α or Arnt (Hif1ß) were generated using an aP2-Cre transgene with the Cre/LoxP system. The mice were fed an HFD for 12 weeks and their metabolic phenotypes were determined. Gene expression patterns in adipose tissues were also determined by microarray and quantitative PCR. RESULTS: On an HFD, adipocyte-specific ARNT knockout mice and adipocyte-specific HIF1α knockout mice exhibit similar metabolic phenotypes, including reduced fat formation, protection from HFD-induced obesity, and insulin resistance compared with similarly fed wild-type controls. The cumulative food intake remained similar; however, the metabolic efficiency was lower in adipocyte-specific HIF1α knockout mice. Moreover, indirect calorimetry revealed respiratory exchange ratios were reduced in adipocyte-specific HIF1α knockout mice. Hyperinsulinemic-euglycemic clamp studies demonstrated that targeted disruption of HIF1α in adipocytes enhanced whole-body insulin sensitivity. The improvement of insulin resistance is associated with decreased expression of Socs3 and induction of adiponectin. CONCLUSIONS: Inhibition of HIF1 in adipose tissue ameliorates obesity and insulin resistance. This study reveals that HIF1 could provide a novel potential therapeutic target for obesity and type 2 diabetes.
Assuntos
Adipócitos/metabolismo , Adiposidade/fisiologia , Gorduras na Dieta/efeitos adversos , Regulação da Expressão Gênica/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Resistência à Insulina , Tecido Adiposo Marrom , Tecido Adiposo Branco , Envelhecimento , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Gorduras na Dieta/administração & dosagem , Técnica Clamp de Glucose , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Fígado/metabolismo , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Consumo de OxigênioRESUMO
OBJECTIVE: The role of adenosine (ADO) in the regulation of glucose homeostasis is not clear. In the current study, we used A1-ADO receptor (A1AR)-deficient mice to investigate the role of ADO/A1AR signaling for glucose homeostasis. RESEARCH DESIGN AND METHODS: After weaning, A1AR(-/-) and wild-type mice received either a standard diet (12 kcal% fat) or high-fat diet (HFD; 45 kcal% fat). Body weight, fasting plasma glucose, plasma insulin, and intraperitoneal glucose tolerance tests were performed in 8-week-old mice and again after 12-20 weeks of subsequent observation. Body composition was quantified by magnetic resonance imaging and epididymal fat-pad weights. Glucose metabolism was investigated by hyperinsulinemic-euglycemic clamp studies. To describe pathophysiological mechanisms, adipokines and Akt phosphorylation were measured. RESULTS: A1AR(-/-) mice were significantly heavier than wild-type mice because of an increased fat mass. Fasting plasma glucose and insulin were significantly higher in A1AR(-/-) mice after weaning and remained higher in adulthood. An intraperitoneal glucose challenge disclosed a significantly slower glucose clearance in A1AR(-/-) mice. An HFD enhanced this phenotype in A1AR(-/-) mice and unmasked a dysfunctional insulin secretory mechanism. Insulin sensitivity was significantly impaired in A1AR(-/-) mice on the standard diet shortly after weaning. Clamp studies detected a significant decrease of net glucose uptake in A1AR(-/-) mice and a reduced glucose uptake in muscle and white adipose tissue. Effects were not triggered by leptin deficiency but involved a decreased Akt phosphorylation. CONCLUSIONS: ADO/A1AR signaling contributes importantly to insulin-controlled glucose homeostasis and insulin sensitivity in C57BL/6 mice and is involved in the metabolic regulation of adipose tissue.
Assuntos
Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Receptor A1 de Adenosina/genética , Receptor A1 de Adenosina/metabolismo , Transdução de Sinais/fisiologia , Adiposidade , Animais , Glicemia/metabolismo , Peso Corporal , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Ingestão de Alimentos , Feminino , Insulina/sangue , Insulina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Atividade Motora , Transdução de Sinais/genéticaRESUMO
Adipose-specific inactivation of both AP-1 and CCAAT-enhancer-binding protein (C/EBP) families of B-ZIP transcription factors in transgenic mice causes severe lipoatrophy. To evaluate whether inactivation of only C/EBP members was critical for lipoatrophy, A-C/EBP, a dominant-negative protein that specifically inhibits the DNA binding of the C/EBP members, was expressed in adipose tissue. For the first 2 weeks after birth, aP2-A-C/EBP mice had no white adipose tissue (WAT), drastically reduced brown adipose tissue (BAT), and exhibited marked hepatic steatosis, hyperinsulinemia, and hyperlipidemia. However, WAT appeared during the third week, coinciding with significantly improved metabolic functioning. In adults, BAT remained reduced, causing cold intolerance. At 30 weeks, the aP2-A-C/EBP mice had only 35% reduced WAT, with clear morphological signs of lipodystrophy in subcutaneous fat. Circulating leptin and adiponectin levels were less than the wild-type levels, and these mice exhibited impaired triglyceride clearance. Insulin resistance, glucose intolerance, and reduced free fatty acid release in response to ß3-adrenergic agonist suggest improper functioning of the residual WAT. Gene expression analysis of inguinal WAT identified reduced mRNA levels of several enzymes involved in fatty acid synthesis and glucose metabolism that are known C/EBPα transcriptional targets. There were increased levels for genes involved in inflammation and muscle differentiation. However, when dermal fibroblasts from aP2-A-C/EBP mice were differentiated into adipocytes in tissue culture, muscle markers were elevated more than the inflammatory markers. These results demonstrate that the C/EBP family is essential for adipose tissue development during the early postnatal period, the regulation of glucose and lipid homeostasis in adults, and the suppression of the muscle lineage.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Lipodistrofia/etiologia , Lipodistrofia/metabolismo , Proteínas/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Western Blotting , Composição Corporal/genética , Composição Corporal/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Calorimetria Indireta , Células Cultivadas , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Proteínas de Ligação a Ácido Graxo/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Lipodistrofia/genética , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Proteínas/genética , Triglicerídeos/metabolismoRESUMO
Previous studies have shown that ß-cell M(3) muscarinic acetylcholine receptors (M3Rs) play a key role in maintaining blood glucose homeostasis by enhancing glucose-dependent insulin release. In this study, we tested the hypothesis that long-term, persistent activation of ß-cell M3Rs can improve glucose tolerance and ameliorate the metabolic deficits associated with the consumption of a high-fat diet. To achieve the selective and persistent activation of ß-cell M3Rs in vivo, we generated transgenic mice that expressed the Q490L mutant M3R in their pancreatic ß-cells (ß-M3-Q490L Tg mice). The Q490L point mutation is known to render the M3R constitutively active. The metabolic phenotypes of the transgenic mice were examined in several in vitro and in vivo metabolic tests. In the presence of 15 mm glucose and the absence of M3R ligands, isolated perifused islets prepared from ß-M3-Q490L Tg mice released considerably more insulin than wild-type control islets. This effect could be completely blocked by incubation of the transgenic islets with atropine (10 µm), an inverse muscarinic agonist, indicating that the Q490L mutant M3R exhibited ligand-independent signaling (constitutive activity) in mouse ß-cells. In vivo studies showed that ß-M3-Q490L Tg mice displayed greatly improved glucose tolerance and increased serum insulin levels as well as resistance to diet-induced glucose intolerance and hyperglycemia. These results suggest that chronic activation of ß-cell M3Rs may represent a useful approach to boost insulin output in the long-term treatment of type 2 diabetes.
Assuntos
Glicemia/metabolismo , Células Secretoras de Insulina/metabolismo , Receptor Muscarínico M3/metabolismo , Análise de Variância , Animais , Atropina/farmacologia , Glicemia/efeitos dos fármacos , Genótipo , Homeostase/efeitos dos fármacos , Insulina/sangue , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Antagonistas Muscarínicos/farmacologia , Fenótipo , Receptor Muscarínico M3/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Glucose homeostasis in mammals is mainly regulated by insulin signaling. It was previously shown that SIRT6 mutant mice die before 4 weeks of age, displaying profound abnormalities, including low insulin, hypoglycemia, and premature aging. To investigate mechanisms underlying the pleiotropic phenotypes associated with SIRT6 deficiency, we generated mice carrying targeted disruption of SIRT6. We found that 60% of SIRT6(-/-) animals had very low levels of blood glucose and died shortly after weaning. The remaining animals, which have relatively higher concentrations of glucose, survived the early post-weaning lethality, but most died within one year of age. Significantly, feeding the mice with glucose-containing water increased blood glucose and rescued 83% of mutant mice, suggesting that the hypoglycemia is a major cause for the lethality. We showed that SIRT6 deficiency results in more abundant membrane association of glucose transporters 1 and 4, which enhances glucose uptake. We further demonstrated that SIRT6 negatively regulates AKT phosphorylation at Ser-473 and Thr-308 through inhibition of multiple upstream molecules, including insulin receptor, IRS1, and IRS2. The absence of SIRT6, consequently, enhances insulin signaling and activation of AKT, leading to hypoglycemia. These data uncover an essential role of SIRT6 in modulating glucose metabolism through mediating insulin sensitivity.
Assuntos
Glucose/farmacocinética , Hipoglicemia/etiologia , Insulina/farmacologia , Sirtuínas/fisiologia , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Feminino , Imunofluorescência , Genes Letais , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Hipoglicemia/metabolismo , Hipoglicemia/patologia , Técnicas Imunoenzimáticas , Resistência à Insulina , Masculino , Camundongos , Camundongos Knockout , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Distribuição TecidualRESUMO
Therapeutic strategies that augment insulin release from pancreatic beta-cells are considered beneficial in the treatment of type 2 diabetes. We previously demonstrated that activation of beta-cell M(3) muscarinic receptors (M3Rs) greatly promotes glucose-stimulated insulin secretion (GSIS), suggesting that strategies aimed at enhancing signaling through beta-cell M3Rs may become therapeutically useful. M3R activation leads to the stimulation of G proteins of the G(q) family, which are under the inhibitory control of proteins known as regulators of G protein signaling (RGS proteins). At present, it remains unknown whether RGS proteins play a role in regulating insulin release. To address this issue, we initially demonstrated that MIN6 insulinoma cells express functional M3Rs and that RGS4 was by far the most abundant RGS protein expressed by these cells. Strikingly, siRNA-mediated knockdown of RGS4 expression in MIN6 cells greatly enhanced M3R-mediated augmentation of GSIS and calcium release. We obtained similar findings using pancreatic islets prepared from RGS4-deficient mice. Interestingly, RGS4 deficiency had little effect on insulin release caused by activation of other beta-cell GPCRs. Finally, treatment of mutant mice selectively lacking RGS4 in pancreatic beta-cells with a muscarinic agonist (bethanechol) led to significantly increased plasma insulin and reduced blood glucose levels, as compared to control littermates. Studies with beta-cell-specific M3R knockout mice showed that these responses were mediated by beta-cell M3Rs. These findings indicate that RGS4 is a potent negative regulator of M3R function in pancreatic beta-cells, suggesting that RGS4 may represent a potential target to promote insulin release for therapeutic purposes.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas RGS/metabolismo , Receptor Muscarínico M3/metabolismo , Análise de Variância , Animais , Betanecol , Glicemia/metabolismo , Western Blotting , Células COS , Cálcio/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Eletroporação , Imunoprecipitação , Secreção de Insulina , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: An increase in the rate of hepatic glucose production is the major determinant of fasting hyperglycemia in type 2 diabetes. A better understanding of the signaling pathways and molecules that regulate hepatic glucose metabolism is therefore of great clinical importance. Recent studies suggest that an increase in vagal outflow to the liver leads to decreased hepatic glucose production and reduced blood glucose levels. Since acetylcholine (ACh) is the major neurotransmitter of the vagus nerve and exerts its parasympathetic actions via activation of muscarinic ACh receptors (mAChRs), we examined the potential metabolic relevance of hepatocyte mAChRs. RESEARCH DESIGN AND METHODS: We initially demonstrated that the M(3) mAChR is the only mAChR subtype expressed by mouse liver/hepatocytes. To assess the physiological role of this receptor subtype in regulating hepatic glucose fluxes and glucose homeostasis in vivo, we used gene targeting and transgenic techniques to generate mutant mice lacking or overexpressing M(3) receptors in hepatocytes only. RESULTS: Strikingly, detailed in vivo phenotyping studies failed to reveal any significant metabolic differences between the M(3) receptor mutant mice and their control littermates, independent of whether the mice were fed regular or a high-fat diet. Moreover, the expression levels of genes for various key transcription factors, signaling molecules, and enzymes regulating hepatic glucose fluxes were not significantly altered in the M(3) receptor mutant mice. CONCLUSIONS: This rather surprising finding suggests that the pronounced metabolic effects mediated by activation of hepatic vagal nerves are mediated by noncholinergic signaling pathways.
Assuntos
Glucose/metabolismo , Hepatócitos/metabolismo , Células de Kupffer/metabolismo , Receptor Muscarínico M3/metabolismo , Animais , Glicemia/metabolismo , Western Blotting , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Receptor Muscarínico M3/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para CimaRESUMO
BACKGROUND: Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. RESULTS: We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgf beta 1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. CONCLUSION: AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.
Assuntos
Dependovirus/genética , Diabetes Mellitus Tipo 1/terapia , Terapia Genética/métodos , Vetores Genéticos/genética , Ilhotas Pancreáticas/virologia , Transdução Genética/métodos , Animais , Linhagem Celular , Dependovirus/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/cirurgia , Feminino , Vetores Genéticos/metabolismo , Humanos , Técnicas In Vitro , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos , Ratos WistarRESUMO
BACKGROUND: Does diet-induced obesity persist after an obesigenic diet is removed? We investigated this question by providing male C57BL/6 mice with free access to two different obesigenic diets followed by a switch to chow to determine if obesity was reversible. METHODOLOGY/PRINCIPAL FINDINGS: Male C57BL/6 mice were randomly assigned to five weight-matched groups: 1) C group that continuously received a chow diet; 2) HF group on a 60% high fat diet; 3) EN group on the high fat diet plus liquid Ensure; 4) HF-C group switched from high fat to chow after 7 weeks; 5) EN-C group switched from high fat plus Ensure to chow after 7 weeks. All food intake was ad libitum. Body weight was increased after 7 weeks on both obesigenic diets (44.6+/-0.65, 39.8+/-0.63, and 28.6+/-0.63 g for EN, HF, and C groups, respectively) and resulted in elevated concentrations of serum insulin, glucose, and leptin and lower serum triglycerides. Development of obesity in HF and EN mice was caused by increased energy intake and a relative decrease of average energy output along with decreased ambulatory activity. After the switch to chow, the HF-C and EN-C groups lost weight but subsequently maintained a state of persistent obesity in comparison to the C group (34.8+/-1.2, 34.1+/-1.2 vs. 30.8+/-0.8 g respectively; P<0.05) with a 40-50% increase of body fat. All serum hormones and metabolites returned to control levels with the exception of a trend for increased leptin. The HF-C and EN-C groups had an average energy output in line with the C group and the persistent obesity was maintained despite a non-significant increase of energy intake of less than 1 kcal/d at the end of the study. CONCLUSION: Our results illustrate the importance of considering the history of energy imbalance in determining body weight and that a persistent elevation of body weight after removal of obesigenic diets can result from very small increases of energy intake.
Assuntos
Dieta/efeitos adversos , Obesidade/etiologia , Tecido Adiposo/patologia , Animais , Glicemia/metabolismo , Distribuição da Gordura Corporal , Peso Corporal , Modelos Animais de Doenças , Ingestão de Energia , Metabolismo Energético , Glicerol/sangue , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora , Obesidade/patologia , Obesidade/fisiopatologia , Fatores de TempoRESUMO
Adipose tissue grows by two mechanisms: hyperplasia (cell number increase) and hypertrophy (cell size increase). Genetics and diet affect the relative contributions of these two mechanisms to the growth of adipose tissue in obesity. In this study, the size distributions of epididymal adipose cells from two mouse strains, obesity-resistant FVB/N and obesity-prone C57BL/6, were measured after 2, 4, and 12 weeks under regular and high-fat feeding conditions. The total cell number in the epididymal fat pad was estimated from the fat pad mass and the normalized cell-size distribution. The cell number and volume-weighted mean cell size increase as a function of fat pad mass. To address adipose tissue growth precisely, we developed a mathematical model describing the evolution of the adipose cell-size distributions as a function of the increasing fat pad mass, instead of the increasing chronological time. Our model describes the recruitment of new adipose cells and their subsequent development in different strains, and with different diet regimens, with common mechanisms, but with diet- and genetics-dependent model parameters. Compared to the FVB/N strain, the C57BL/6 strain has greater recruitment of small adipose cells. Hyperplasia is enhanced by high-fat diet in a strain-dependent way, suggesting a synergistic interaction between genetics and diet. Moreover, high-fat feeding increases the rate of adipose cell size growth, independent of strain, reflecting the increase in calories requiring storage. Additionally, high-fat diet leads to a dramatic spreading of the size distribution of adipose cells in both strains; this implies an increase in size fluctuations of adipose cells through lipid turnover.
Assuntos
Adipócitos/patologia , Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/patologia , Gorduras na Dieta/metabolismo , Modelos Biológicos , Obesidade/patologia , Obesidade/fisiopatologia , Animais , Crescimento Celular , Proliferação de Células , Tamanho Celular , Simulação por Computador , Hiperplasia/patologia , Hiperplasia/fisiopatologia , Hipertrofia/patologia , Hipertrofia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Myostatin (Mstn) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Mstn(-/-) mice have a dramatic increase in muscle mass, reduction in fat mass, and resistance to diet-induced and genetic obesity. To determine how Mstn deletion causes reduced adiposity and resistance to obesity, we analyzed substrate utilization and insulin sensitivity in Mstn(-/-) mice fed a standard chow. Despite reduced lipid oxidation in skeletal muscle, Mstn(-/-) mice had no change in the rate of whole body lipid oxidation. In contrast, Mstn(-/-) mice had increased glucose utilization and insulin sensitivity as measured by indirect calorimetry, glucose and insulin tolerance tests, and hyperinsulinemic-euglycemic clamp. To determine whether these metabolic effects were due primarily to the loss of myostatin signaling in muscle or adipose tissue, we compared two transgenic mouse lines carrying a dominant negative activin IIB receptor expressed specifically in adipocytes or skeletal muscle. We found that inhibition of myostatin signaling in adipose tissue had no effect on body composition, weight gain, or glucose and insulin tolerance in mice fed a standard diet or a high-fat diet. In contrast, inhibition of myostatin signaling in skeletal muscle, like Mstn deletion, resulted in increased lean mass, decreased fat mass, improved glucose metabolism on standard and high-fat diets, and resistance to diet-induced obesity. Our results demonstrate that Mstn(-/-) mice have an increase in insulin sensitivity and glucose uptake, and that the reduction in adipose tissue mass in Mstn(-/-) mice is an indirect result of metabolic changes in skeletal muscle. These data suggest that increasing muscle mass by administration of myostatin antagonists may be a promising therapeutic target for treating patients with obesity or diabetes.
Assuntos
Tecido Adiposo/metabolismo , Resistência à Insulina/fisiologia , Insulina/metabolismo , Músculo Esquelético/metabolismo , Miostatina/antagonistas & inibidores , Animais , Dieta , Gorduras na Dieta , Glucose/metabolismo , Técnica Clamp de Glucose , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/anatomia & histologia , Miostatina/metabolismo , Oxirredução , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The ubiquitously expressed G protein alpha-subunit G(s)alpha is required for receptor-stimulated intracellular cAMP responses and is an important regulator of energy and glucose metabolism. We have generated skeletal muscle-specific G(s)alpha-knockout (KO) mice (MGsKO) by mating G(s)alpha-floxed mice with muscle creatine kinase-cre transgenic mice. MGsKO mice had normal body weight and composition, and their serum glucose, insulin, free fatty acid, and triglyceride levels were similar to that of controls. However, MGsKO mice were glucose intolerant despite the fact that insulin sensitivity and glucose-stimulated insulin secretion were normal, suggesting an insulin-independent mechanism. Isolated muscles from MGsKO mice had increased basal glucose uptake and normal responses to a stimulator of AMP-activated protein kinase (AMPK), which indicates that AMPK and its downstream pathways are intact. Compared with control mice, MGsKO mice had reduced muscle mass with decreased cross-sectional area and force production. In addition, adult MGsKO mice showed an increased proportion of type I (slow-twitch, oxidative) fibers based on kinetic properties and myosin heavy chain isoforms, despite the fact that these muscles had reduced expression of peroxisome proliferator-activated receptor coactivator protein-1alpha (PGC-1alpha) and reduced mitochondrial content and oxidative capacity. Therefore G(s)alpha deficiency led to fast-to-slow fiber-type switching, which appeared to be dissociated from the expected change in oxidative capacity. MGsKO mice are a valuable model for future studies of the role of G(s)alpha signaling pathways in skeletal muscle adaptation and their effects on whole body metabolism.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/deficiência , Intolerância à Glucose/metabolismo , Resistência à Insulina , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Glicemia/metabolismo , Cromograninas , Metabolismo Energético , Ácidos Graxos/metabolismo , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Intolerância à Glucose/genética , Intolerância à Glucose/patologia , Intolerância à Glucose/fisiopatologia , Insulina/sangue , Masculino , Camundongos , Camundongos Knockout , Contração Muscular , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Lenta/patologia , Força Muscular , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/genética , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Transdução de Sinais , Fatores de Tempo , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
Most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity. The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation. Here, we tested the hypothesis that the absence of M3 receptors in mice might protect against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. In all cases, the lack of M3 receptors greatly ameliorated impairments in glucose homeostasis and insulin sensitivity but had less robust effects on overall adiposity. Under all experimental conditions tested, M3 receptor-deficient mice showed a significant elevation in basal and total energy expenditure, most likely due to enhanced central sympathetic outflow and increased rate of fatty-acid oxidation. These findings suggest that the M3 receptor may represent a potential pharmacologic target for the treatment of obesity and associated metabolic disorders.
Assuntos
Corpos Aórticos/metabolismo , Doenças Metabólicas/etiologia , Obesidade , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/fisiologia , Animais , Epinefrina/urina , Canais Iônicos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Norepinefrina/urina , Receptor Muscarínico M3/deficiência , Proteína Desacopladora 3RESUMO
The complex imprinted Gnas locus encodes several gene products including G(s)alpha, the ubiquitously expressed G protein alpha-subunit required for receptor-stimulated cAMP generation, and the neuroendocrine-specific G(s)alpha isoform XLalphas. XLalphas is only expressed from the paternal allele, whereas G(s)alpha is biallelically expressed in most tissues. XLalphas knock-out mice (Gnasxl(m+/p-)) have poor suckling and perinatal lethality, implicating XLalphas as critical for postnatal feeding. We have now examined the metabolic phenotype of adult Gnasxl(m+/p-) mice. Gnasxl(m+/p-) mice had reduced fat mass and lipid accumulation in adipose tissue, with increased food intake and metabolic rates. Gene expression profiling was consistent with increased lipid metabolism in adipose tissue. These changes likely result from increased sympathetic nervous system activity rather than adipose cell-autonomous effects, as we found that XLalphas is not normally expressed in adult adipose tissue, and Gnasxl(m+/p-) mice had increased urinary norepinephrine levels but not increased metabolic responsiveness to a beta3-adrenergic agonist. Gnasxl(m+/p-) mice were hypolipidemic and had increased glucose tolerance and insulin sensitivity. The similar metabolic profile observed in some prior paternal Gnas knock-out models results from XLalphas deficiency (or deficiency of the related alternative truncated protein XLN1). XLalphas (or XLN1) is a negative regulator of sympathetic nervous system activity in mice.
Assuntos
Metabolismo Energético , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Glucose/química , Glucose/metabolismo , Tecido Adiposo/metabolismo , Animais , Composição Corporal , Catecolaminas/metabolismo , Cromograninas , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Lipídeos/química , Camundongos , Camundongos Knockout , Modelos Genéticos , Sistema Nervoso Simpático/patologia , Triglicerídeos/metabolismoRESUMO
The antiobesity and antidiabetic effects of the beta3-adrenergic agonists were investigated on nonobese type 2 diabetic MKR mice after injection with a beta3-adrenergic agonist, CL-316243. An intact response to acute CL-316243 treatment was observed in MKR mice. Chronic intraperitoneal CL-316243 treatment of MKR mice reduced blood glucose and serum insulin levels. Hyperinsulinemic euglycemic clamps exhibited improvement of the whole body insulin sensitivity and glucose homeostasis concurrently with enhanced insulin action in liver and adipose tissue. Treating MKR mice with CL-316243 significantly lowered serum and hepatic lipid levels, in part due to increased whole body triglyceride clearance and fatty acid oxidation in adipocytes. A significant reduction in total body fat content and epididymal fat weight was observed along with enhanced metabolic rate in both wild-type and MKR mice after treatment. These data demonstrate that beta3-adrenergic activation improves the diabetic state of nonobese diabetic MKR mice by potentiation of free fatty acid oxidation by adipose tissue, suggesting a potential therapeutic role for beta3-adrenergic agonists in nonobese diabetic subjects.