RESUMO
Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic alpha cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr(-/-)). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr(-/-) mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to alpha cell, and to a lesser extent, delta cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr(-/-) mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and alpha and delta cell numbers. Furthermore, the lean phenotype of Gcgr(-/-) mice suggests glucagon action may be involved in the regulation of whole body composition.
Assuntos
Glicemia/metabolismo , Glucagon/sangue , Ilhotas Pancreáticas/patologia , Receptores de Glucagon/genética , Receptores de Glucagon/fisiologia , Animais , Peso Corporal , Calorimetria , Divisão Celular , AMP Cíclico/metabolismo , Epididimo/metabolismo , Epinefrina/farmacologia , Glucose/metabolismo , Hormônios/metabolismo , Hiperplasia , Imuno-Histoquímica , Insulina/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Fenótipo , Fatores de TempoRESUMO
Glucose transporter (GLUT) 4 is the insulin responsive glucose transporter in adipose tissue, skeletal muscle, and heart. Insulin elicits increased glucose uptake by recruiting GLUT4 from a specialized intracellular storage site to the cell surface. Expression of various proteins that colocalize with GLUT4 and/or are involved in insulin-stimulated GLUT4 translocation was examined in adipocytes as well as skeletal and cardiac muscles from GLUT4 null mice. Our data demonstrate that expression of insulin-regulated aminopeptidase (IRAP) is divergently regulated in GLUT4 null tissues, e.g., upregulated 1.6-fold in GLUT4 null adipocytes and downregulated in GLUT4 null skeletal muscle (40%) and heart (60%). IRAP exhibited abnormal subcellular distribution and impaired insulin-stimulated translocation in GLUT4-deficient tissues. We propose the compartment containing IRAP and proteins normally associated with GLUT4 vesicle traffics constitutively to the cell surface in GLUT4 null adipocytes and skeletal muscle.
Assuntos
Adipócitos/metabolismo , Aminopeptidases/metabolismo , Membrana Celular/enzimologia , Proteínas de Transporte de Monossacarídeos/deficiência , Proteínas Musculares , Proteínas Adaptadoras de Transporte Vesicular , Adipócitos/química , Adipócitos/efeitos dos fármacos , Aminopeptidases/análise , Animais , Compartimento Celular/efeitos dos fármacos , Compartimento Celular/fisiologia , Membrana Celular/química , Separação Celular , Cruzamentos Genéticos , Cistinil Aminopeptidase , Transportador de Glucose Tipo 4 , Insulina/farmacologia , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Proteínas de Transporte de Monossacarídeos/análise , Proteínas de Transporte de Monossacarídeos/genética , Músculo Esquelético/química , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Proteínas Qa-SNARE , Proteínas R-SNARE , Receptores da Transferrina/análise , Receptores da Transferrina/metabolismo , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Proteínas rab4 de Ligação ao GTP/metabolismoRESUMO
Conjugated linoleic acid (CLA) isomers have a number of beneficial health effects, as shown in biomedical studies with animal models. Previously, we reported that a mixture of CLA isomers improved glucose tolerance in ZDF rats and activated peroxisome proliferator-activated receptor (PPAR)-gamma response elements in vitro. Here, our aim was to elucidate the effect(s) of specific CLA isomers on whole-body glucose tolerance, insulin action in skeletal muscle, and expression of genes important in glucose and lipid metabolism. ZDF rats were fed either a control diet (CON), one of two CLA supplemented diets (1.5% CLA) containing differing isoforms of CLA (47% c9,t11; 47.9% c10,t12, 50:50; or 91% c9,t11, c9,t11 isomers), or were pair-fed CON diet to match the intake of 50:50. The 50:50 diet reduced adiposity and improved glucose tolerance compared with all other ZDF treatments. Insulin-stimulated glucose transport and glycogen synthase activity in skeletal muscle were improved with 50:50 compared with all other treatments. Neither phosphatidlyinositol 3-kinase activity nor Akt activity in muscle was affected by treatment. Uncoupling protein 2 in muscle and adipose tissue was upregulated by c9,t11 and 50:50 compared with ZDF controls. PPAR-gamma mRNA was downregulated in liver of c9,t11 and pair-fed ZDF rats. Thus, the improved glucose tolerance in 50:50 rats is attributable to, at least in part, improved insulin action in muscle, and CLA effects cannot be explained simply by reduced food intake.
Assuntos
Glicemia/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Insulina/fisiologia , Ácidos Linoleicos/farmacologia , Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Músculo Esquelético/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas/genética , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Glicemia/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Suplementos Nutricionais , Ingestão de Energia/efeitos dos fármacos , Ácidos Graxos não Esterificados/sangue , Comportamento Alimentar/efeitos dos fármacos , Teste de Tolerância a Glucose , Insulina/sangue , Canais Iônicos , Isomerismo , Leptina/sangue , Ácidos Linoleicos/administração & dosagem , Masculino , Músculo Esquelético/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/genética , Ratos , Ratos Zucker , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Transcrição Gênica/efeitos dos fármacos , Triglicerídeos/sangue , Desacopladores/metabolismo , Proteína Desacopladora 2RESUMO
To understand the long-term metabolic and functional consequences of increased GLUT4 content, intracellular substrate utilization was investigated in isolated muscles of transgenic mice overexpressing GLUT4 selectively in fast-twitch skeletal muscles. Rates of glycolysis, glycogen synthesis, glucose oxidation, and free fatty acid (FFA) oxidation as well as glycogen content were assessed in isolated EDL (fast-twitch) and soleus (slow-twitch) muscles from female and male MLC-GLUT4 transgenic and control mice. In male MLC-GLUT4 EDL, increased glucose influx predominantly led to increased glycolysis. In contrast, in female MLC-GLUT4 EDL increased glycogen synthesis was observed. In both sexes, GLUT4 overexpression resulted in decreased exogenous FFA oxidation rates. The decreased rate of FFA oxidation in male MLC-GLUT4 EDL was associated with increased lipid content in liver, but not in muscle or at the whole body level. To determine how changes in substrate metabolism and insulin action may influence energy balance in an environment that encouraged physical activity, we measured voluntary training activity, body weight, and food consumption of MLC-GLUT4 and control mice in cages equipped with training wheels. We observed a small decrease in body weight of MLC-GLUT4 mice that was paradoxically accompanied by a 45% increase in food consumption. The results were explained by a marked fourfold increase in voluntary wheel exercise. The changes in substrate metabolism and physical activity in MLC-GLUT4 mice were not associated with dramatic changes in skeletal muscle morphology. Collectively, results of this study demonstrate the feasibility of altering muscle substrate utilization by overexpression of GLUT4. The results also suggest that as a potential treatment for type II diabetes mellitus, increased skeletal muscle GLUT4 expression may provide benefits in addition to improvement of insulin action.
Assuntos
Glicólise/fisiologia , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas Musculares , Músculo Esquelético/metabolismo , Animais , Transporte Biológico , Peso Corporal/fisiologia , Ingestão de Alimentos/fisiologia , Ácidos Graxos não Esterificados/metabolismo , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 4 , Glicogênio/biossíntese , Glicogênio/metabolismo , Glicólise/efeitos dos fármacos , Insulina/farmacologia , Fígado/metabolismo , Masculino , Camundongos , Proteínas de Transporte de Monossacarídeos/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiologia , Ácido Oleico/metabolismo , Tamanho do Órgão , Oxirredução , Condicionamento Físico Animal/fisiologia , Caracteres Sexuais , Distribuição Tecidual , Triglicerídeos/metabolismoRESUMO
To investigate whether GLUT4 is required for exercise/hypoxia-induced glucose uptake, we assessed glucose uptake under hypoxia and normoxia in extensor digitorum longus (EDL) and soleus muscles from GLUT4-deficient mice. In EDL and soleus from wild type control mice, hypoxia increased 2-deoxyglucose uptake 2-3-fold. Conversely, hypoxia did not alter 2-deoxyglucose uptake in either EDL or soleus from either male or female GLUT4-null mice. Next we introduced the fast-twitch skeletal muscle-specific MLC-GLUT4 transgene into GLUT4-null mice to determine whether changes in the metabolic milieu accounted for the lack of hypoxia-mediated glucose transport. Transgenic complementation of GLUT4 in EDL was sufficient to restore hypoxia-mediated glucose uptake. Soleus muscles from MLC-GLUT4-null mice were transgene-negative, and hypoxia-stimulated 2-deoxyglucose uptake was not restored. Although ablation of GLUT4 in EDL did not affect normoxic glycogen levels, restoration of GLUT4 to EDL led to an increase in glycogen under hypoxic conditions. Male GLUT4-null soleus displayed reduced normoxic glycogen stores, but female null soleus contained significantly more glycogen under normoxia and hypoxia. Reduced normoxic levels of ATP and phosphocreatine were measured in male GLUT4-null soleus but not in EDL. However, transgenic complementation of GLUT4 prevented the decrease in hypoxic ATP and phosphocreatine levels noted in male GLUT4-null and control EDL. In conclusion, we have demonstrated that GLUT4 plays an essential role in the regulation of muscle glucose uptake in response to hypoxia. Because hypoxia is a useful model for exercise, our results suggest that stimulation of glucose transport in response to exercise in skeletal muscle is totally dependent upon GLUT4. Furthermore, the compensatory glucose transport system that exists in GLUT4-null soleus muscle is not sensitive to hypoxia/muscle contraction.
Assuntos
Desoxiglucose/metabolismo , Teste de Complementação Genética , Hipóxia/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas Musculares , Músculo Esquelético/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Transportador de Glucose Tipo 4 , Ácido Láctico/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Cadeias Leves de Miosina/genética , Fosfocreatina/metabolismoRESUMO
The expression and signal transduction of the glucagon receptor (GR) have been studied in betaTC3 cells. Northern blot and RT-PCR analysis indicated the expression of the GR gene in betaTC3 cells. One-5 nM glucagon stimulated a 2.5-fold increase in the IP(S) production. At glucagon concentrations higher than 5 nM, the production of IP(S) was blunted but not abolished. The accumulation of intracellular cAMP was observed following the stimulation with 5 nM of glucagon. A maximal 4.5-fold increase in cAMP was observed using 250 nM glucagon and higher. Comparative studies using a glucagon anatogonist, des-His1[Glu]9glucagon, showed no effect on intracellular cAMP and IPs in betaTC3 cells. Our data shows that the GR gene is expressed in betaTC3 cells. The GR in betaTC3 cells transmits its intracellular signal by causing the accumulation of both IP(S) and cAMP.
Assuntos
Receptores de Glucagon/biossíntese , Transdução de Sinais , Adenilil Ciclases/metabolismo , Northern Blotting , Linhagem Celular , AMP Cíclico/metabolismo , Expressão Gênica , Glucagon/antagonistas & inibidores , Glucagon/farmacologia , Fosfatos de Inositol/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Receptores de Glucagon/genética , Receptores de Glucagon/fisiologia , Fosfolipases Tipo C/metabolismoRESUMO
Because the insulin-responsive glucose transporter, GLUT4, is expressed in renal vascular and glomerular cells, we determined the effects of experimental diabetes mellitus on GLUT4 expression and glucose uptake by these tissues. Quantitative reverse-transcription polymerase chain reaction studies of microdissected afferent microvessels and renal glomeruli showed that, after 1 wk of diabetes, GLUT4 mRNA was decreased to 26 and 34% of control values, respectively. GLUT4 immunoblots of renal glomerular and microvessel samples showed that GLUT4 polypeptide was decreased to 51% of control values. These results were confirmed by indirect immunofluorescence, which showed decreased GLUT4 expression in glomerular cells and in vascular smooth muscle cells of the afferent microvasculature of diabetic animals. Uptake of the glucose analogue, 2-deoxyglucose, was also depressed in microvessels of diabetic rats to 57% of control values, supporting the conclusion that fewer total glucose transporters were available for glucose uptake into diabetic renal glomerular and microvascular cells. Thus both GLUT4 expression and glucose uptake by glomerular and microvascular cells are decreased in diabetic animals. These results have led us to suggest a mechanism by which decreased renal GLUT4 expression could contribute to glomerular hyperfiltration and hypertension seen in early diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Expressão Gênica , Glomérulos Renais/metabolismo , Rim/irrigação sanguínea , Microcirculação/metabolismo , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas Musculares , Artéria Renal/metabolismo , Animais , Sequência de Bases , Transporte Biológico , Primers do DNA , Desoxiglucose/metabolismo , Imunofluorescência , Transportador de Glucose Tipo 4 , Immunoblotting , Insulina/farmacologia , Masculino , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/análise , Proteínas de Transporte de Monossacarídeos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Valores de Referência , Circulação RenalRESUMO
Polyclonal antibodies to the amino- or carboxyl-terminated peptide sequences of the GLUT4 transporter protein were used in immunoelectron microscopic studies to demonstrate the location and insulin-induced translocation of GLUT4 in intact isolated rat adipocytes. Labeling of untreated adipocytes with the amino-terminal antibody revealed 95% of GLUT4 was intracellular, associated with plasma membrane invaginations or vesicles contiguous with or within 75 nm of the cell membrane. Insulin treatment increased plasma membrane labeling approximately 13-fold, to 52% of the total transporters, and decreased intracellular labeling proportionately. In contrast, labeling of untreated adipocytes with the carboxyl-terminal antibody or with a monoclonal antibody (1F8) that binds to the carboxyl terminus of GLUT4 detected fewer transporters, only approximately 40% of which were intracellular. In insulin-treated cells, plasma membrane labeling increased approximately 20-fold, but the total number of labeled transporters also increased approximately 13-fold. The number of intracellular transporters was not changed. The insulin-induced increase in plasma membrane labeling was reversible. Thus, the vast majority of GLUT4 transporters in untreated adipocytes are intracellular in invaginations or vesicles attached or close to the plasma membrane. Insulin treatment causes translocation of transporters to the plasma membrane, which involves flow of transporters from invaginations to the cell surface and possible fusion of subplasma membrane vesicles with the plasma membrane. Differences in the labeling of intracellular transporters by peptide antibodies suggested the carboxyl-terminal epitope of intracellular transporters was masked. The unmasking of the carboxyl terminus during translocation to the plasma membrane may be part of the mechanism by which insulin stimulates glucose transport in rat adipocytes.
Assuntos
Tecido Adiposo/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/ultraestrutura , Sequência de Aminoácidos , Animais , Anticorpos , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Epitopos/análise , Masculino , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/imunologia , Proteínas de Transporte de Monossacarídeos/ultraestrutura , Peptídeos/síntese química , Peptídeos/imunologia , Ratos , Ratos EndogâmicosRESUMO
Rat adipose cells treated with Staphylococcus aureus alpha-toxin are permeable and retain their ability to respond to insulin after hormone treatment. The GLUT 4 glucose transporter isoform, specific to fat and muscle cells, is translocated normally from low density microsomes to the plasma membrane in permeabilized cells. Addition of guanosine 5'-O-(3-thiotriphosphate), guanylyl imidodiphosphate, or guanylyl beta, gamma-methylenediphosphate to permeabilized adipocytes induces an insulin-like translocation of GLUT 4 to the plasma membrane; GTP or adenosine 5'-(beta, gamma-imino)triphosphate has no effect. No translocation of GLUT 4 is observed when GTP analogs are added to intact adipocytes. These results suggest the involvement of a GTP-binding protein in insulin-triggered recruitment of GLUT 4 to the cell surface.
Assuntos
Tecido Adiposo/metabolismo , Membrana Celular/metabolismo , Guanosina Trifosfato/farmacologia , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/farmacologia , Compartimento Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanilil Imidodifosfato/farmacologia , Proteínas Hemolisinas/farmacologia , Técnicas Imunológicas , Técnicas In Vitro , Dados de Sequência Molecular , Ratos , Saponinas/farmacologiaRESUMO
At least two genetically distinct glucose transporters (GTs) coexist in adipose cells, one cloned from human hepatoma cells and rat brain (HepG2/brain) and another from rat skeletal muscle, heart, and adipose cells (adipose cell/muscle). Here we demonstrate differential regulation of these two GTs in adipose cells of diabetic and insulin-treated diabetic rats and compare changes in the expression of each GT with marked alterations in insulin-stimulated glucose transport activity. Adipose cell/muscle GTs detected by immunoblotting with the monoclonal antiserum 1F8 (James, D. E., R. Brown, J. Navarro, and P. F. Pilch. 1988. Nature (Lond.). 333:183-185), which reacts with the protein product of the newly cloned adipose cell/muscle GT cDNA, decrease 87% with diabetes and increase to 8.5-fold diabetic levels with insulin treatment. These changes concur qualitatively with previous detection of GTs by cytochalasin B binding and with insulin-stimulated 3-O-methylglucose transport. Northern blotting reveals that the adipose/muscle GT mRNA decreases 50% with diabetes and increases to 6.8-fold control (13-fold diabetic) levels with insulin treatment. In contrast, GTs detected with antisera to the carboxyl terminus of the HepG2 GT or to the human erythrocyte GT show no significant change with diabetes or insulin treatment. The HepG2/brain GT mRNA is unchanged with diabetes and increases threefold with insulin treatment. These results suggest that (a) altered expression of the adipose cell/muscle GT forms the molecular basis for the dysregulated glucose transport response to insulin characteristic of diabetes, (b) the expression of two types of GTs in rat adipose cells is regulated independently, and (c) alterations in mRNA levels are only part of the mechanism for in vivo regulation of the expression of either GT species.