Tissue-specific Ablation of the GLUT4 Glucose Transporter or the Insulin Receptor Challenges Assumptions about Insulin Action and Glucose Homeostasis
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.r300019200
ISSN1083-351X
AutoresYasuhiko Minokoshi, C. Ronald Kahn, Barbara B. Kahn,
Tópico(s)Adipose Tissue and Metabolism
ResumoThe prevalence of type 2 diabetes mellitus is growing worldwide. Most forms of type 2 diabetes are polygenic with complex inheritance patterns strongly influenced by environmental factors. The specific gene defects are unknown, but they affect both insulin action and insulin secretion. Glucose homeostasis is maintained by the fine orchestration of insulin secretion and insulin action to promote glucose transport into muscle and adipocytes and to inhibit hepatic glucose output. Resistance to these effects of insulin is a classic pathogenic feature of obesity and type 2 diabetes. Insulin action on lipid metabolism also has important effects on glucose homeostasis. Recent studies using tissue-specific gene targeting of the GLUT4 glucose transporter or the insulin receptor in mice reveal intercommunication among insulin target tissues which can modify the impact of genetic defects in individual tissues. These studies provide new concepts regarding the importance of adipose tissue versus muscle in whole-body insulin sensitivity and the role of proximal versus distal components of the insulin action cascade in the pathogenesis of obesity and type 2 diabetes. The insulin receptor (IR) 1The abbreviations used are: IR, insulin receptor; GLUT, glucose transporter; G4KO, GLUT4 knock-out; IRKO, insulin receptor knock-out; IGF, insulin-like growth factor; GTG, gold thioglucose.1The abbreviations used are: IR, insulin receptor; GLUT, glucose transporter; G4KO, GLUT4 knock-out; IRKO, insulin receptor knock-out; IGF, insulin-like growth factor; GTG, gold thioglucose. is present in virtually all mammalian cells, and insulin binding results in activation of several phosphorylation-dephosphorylation cascades (1Saltiel A.R. Kahn C.R. Nature. 2001; 414: 799-806Crossref PubMed Scopus (3813) Google Scholar). The phosphoinositide 3-kinase cascade is necessary, albeit insufficient, for stimulation of glucose transport, and the mitogen-activated protein kinase cascade underlies the mitogenic effects of insulin. These insulin signaling cascades have pleiotrophic metabolic and growth-promoting effects (see Fig. 1 in Supplemental Material). Insulin stimulation of glucose transport in muscle and adipose cells is essential for maintenance of glucose homeostasis. This is mediated by sodium-independent, facilitated-diffusion glucose transporters (GLUTs). In tissues with insulin-sensitive glucose transport (i.e. skeletal and cardiac muscle and adipose cells), GLUT4 is the predominant glucose transporter and GLUT1 plays a minor role. The large stimulatory effect of insulin in these tissues results from the translocation of GLUT4-containing vesicles from intracellular storage sites to the plasma membrane where they dock and fuse with the membrane (2Bryant N. Govers R. James D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 267-277Crossref PubMed Scopus (907) Google Scholar, 3Khan A. Pessin J.E. Diabetologia. 2002; 45: 1475-1483Crossref PubMed Scopus (305) Google Scholar), markedly augmenting glucose transport into the cell. GLUT4 is present primarily in white and brown adipocytes, skeletal muscle, and cardiac muscle, with expression in discrete areas of other tissues (e.g. brain and kidney) that are not traditionally thought to play a major role in glucose homeostasis (4Shepherd P.R. Abel E.D. Kahn B.B. LeRoith D.R. Olefsky J.M. Taylor S.I. Diabetes Mellitus: A Fundamental and Clinical Text. J. P. Lippincott Co., Philadelphia2000: 627-641Google Scholar). In insulin-resistant states including obesity and type 2 diabetes, GLUT4 expression is reduced in adipocytes but not in skeletal muscle (4Shepherd P.R. Abel E.D. Kahn B.B. LeRoith D.R. Olefsky J.M. Taylor S.I. Diabetes Mellitus: A Fundamental and Clinical Text. J. P. Lippincott Co., Philadelphia2000: 627-641Google Scholar, 5Shepherd P.R. Kahn B.B. N. Engl. J. Med. 1999; 341: 248-257Crossref PubMed Scopus (1043) Google Scholar). This down-regulation in adipocytes was not thought to be important because skeletal muscle accounts for up to 85% of glucose disposal following a glucose infusion (6DeFronzo R. Jacot E. Jequier E. Maeder E. Wahren J. Felber J. Diabetes. 1981; 30: 1000-1007Crossref PubMed Scopus (1368) Google Scholar) and at least 50% following glucose ingestion (7Basu A. Basu R. Shah P. Vella A. Johnson C.M. Jensen M. Nair K.S. Schwenk W.F. Rizza R.A. Diabetes. 2001; 50: 1351-1362Crossref PubMed Scopus (130) Google Scholar), whereas adipose tissue accounts for much less. Glucose transport is the rate-controlling step in skeletal-muscle glucose metabolism in normal and type 2 diabetic subjects (8Cline G.W. Petersen K.F. Krssak M. Shen J. Hundal R.S. Trajanoski Z. Inzucchi S. Dresner A. Rothman D.L. Shulman G.I. N. Engl. J. Med. 1999; 341: 240-246Crossref PubMed Scopus (510) Google Scholar). Impaired glucose uptake in skeletal muscle is present even in non-diabetic relatives of type 2 diabetic subjects and is a risk factor for developing diabetes (9Kahn B.B. Rossetti L. Nat. Genet. 1998; 20: 223-225Crossref PubMed Scopus (31) Google Scholar, 10DeFronzo R.A. Diabetes Rev. 1997; 5: 177-269Google Scholar). Defects in GLUT4 trafficking or function in skeletal muscle are thought to be most important in the development of insulin resistance. To understand the role of IR and GLUT4 in glucose homeostasis, mice were engineered to destroy the function of these genes. Genetic ablation of IR in all tissues results in lethality at 4–5 days after birth due to severe diabetic ketoacidosis (11Accili D. Drago J. Lee E.J. Johnson M.D. Cool M.H. Salvatore P. Asico L.D. Jose P.A. Taylor S.I. Westphal H. Nat. Genet. 1996; 12: 106-109Crossref PubMed Scopus (472) Google Scholar). When GLUT4 is “knocked out” of all tissues (GLUT4-null), mice are growth-retarded, with markedly reduced fat mass, cardiomegaly, and shortened lifespan but no diabetes (12Katz E. Stenbit A. Hatton K. DePinho R. Charron J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (392) Google Scholar). In contrast, at least 50% of heterozygous GLUT4-null mice developed diabetes by 6 months of age (13Stenbit A.E. Tsao T.S. Li J. Burcelin R. Geenen D.L. Factor S.M. Houseknecht K. Katz E.B. Charron M.J. Nat. Med. 1997; 3: 1096-1101Crossref PubMed Scopus (273) Google Scholar). Hence, to distinguish the role of the insulin receptor and GLUT4 in adipose tissue and muscle in glucose homeostasis, diabetes, and adiposity, tissue-specific knock-out mice were made using Cre/loxP gene targeting (14Gu H. Zou Y. Rajewsky K. Cell. 1993; 73: 1155-1164Abstract Full Text PDF PubMed Scopus (810) Google Scholar). These mice challenge long held concepts about the control of glucose homeostasis (Fig. 1). Muscle-specific GLUT4 knock-out mice (muscle-G4KO) were made by breeding mice carrying the GLUT4 gene with exon 10 flanked by loxP sites to mice carrying a transgene encoding the Cre recombinase enzyme under the control of the muscle creatine kinase promoter/enhancer (15Zisman A. Peroni O.D. Abel E.D. Michael M.D. Mauvais-Jarvis F. Lowell B.B. Wojtaszewski J.F. Hirshman M.F. Virkamaki A. Goodyear L.J. Kahn C.R. Kahn B.B. Nat. Med. 2000; 6: 924-928Crossref PubMed Scopus (552) Google Scholar). GLUT4 protein levels were reduced about 95% in all skeletal muscles and heart. GLUT1 protein levels were normal in skeletal muscle but were increased by 1.5–2-fold in hearts of muscle-G4KO mice. An even greater induction of cardiac GLUT1 expression was seen in GLUT4-null mice (12Katz E. Stenbit A. Hatton K. DePinho R. Charron J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (392) Google Scholar) and in mice with cardiac-specific GLUT4 knock-out (cardiac-G4KO) (16Abel E.D. Kaulbach H.C. Tian R. Hopkins J. Duffy J. Doetschman T. Minnemann T. Boers M.-E. Hadro E. Oberste-Berghaus C. Quist W. Lowell B.B. Ingwall J.S. Kahn B.B. J. Clin. Invest. 1999; 104: 1703-1714Crossref PubMed Scopus (260) Google Scholar). In contrast to GLUT4-null mice (12Katz E. Stenbit A. Hatton K. DePinho R. Charron J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (392) Google Scholar), muscle-G4KO mice have normal body weight and fat pad weight at least until 6 months of age. Skeletal muscle mass is also normal. Heart weight is increased, consistent with GLUT4-null mice (12Katz E. Stenbit A. Hatton K. DePinho R. Charron J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (392) Google Scholar) and cardiac-G4KO mice (16Abel E.D. Kaulbach H.C. Tian R. Hopkins J. Duffy J. Doetschman T. Minnemann T. Boers M.-E. Hadro E. Oberste-Berghaus C. Quist W. Lowell B.B. Ingwall J.S. Kahn B.B. J. Clin. Invest. 1999; 104: 1703-1714Crossref PubMed Scopus (260) Google Scholar). Lifespan is normal in muscle-G4KO mice in contrast to the shortened lifespan in GLUT4-null mice. Ex vivo, basal, insulin-stimulated, and contraction-stimulated glucose transport are markedly reduced in both slow twitch, oxidative and fast twitch, glycolytic muscles of muscle-G4KO mice. These mice have hyperglycemia, glucose intolerance, and insulin resistance as early as 8 weeks of age, which persists for up to 1 year of age (15Zisman A. Peroni O.D. Abel E.D. Michael M.D. Mauvais-Jarvis F. Lowell B.B. Wojtaszewski J.F. Hirshman M.F. Virkamaki A. Goodyear L.J. Kahn C.R. Kahn B.B. Nat. Med. 2000; 6: 924-928Crossref PubMed Scopus (552) Google Scholar). A subset of mice have frank diabetes with severe insulin resistance. Muscle-G4KO mice do not develop dyslipidemia or other aspects of the metabolic syndrome, in contrast to mice with muscle-specific insulin receptor knock-out (muscle-IRKO, also MIRKO (17Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar)) (see below). Insulin-stimulated glucose transport in muscle in vivo is markedly reduced in muscle-G4KO mice (18Kim J.K. Zisman A. Fillmore J.J. Peroni O.D. Kotani K. Perret P. Zong H. Dong J. Kahn C.R. Kahn B.B. Shulman G.I. J. Clin. Invest. 2001; 108: 153-160Crossref PubMed Scopus (149) Google Scholar). Surprisingly, insulin-stimulated glucose transport in adipose tissue and suppression of hepatic glucose production by insulin are also impaired. The effects in adipose tissue and liver appear to be at least partly due to glucose toxicity because phloridzin treatment, which normalizes blood glucose in diabetic animals by increasing glucose excretion by the kidney, normalized insulin action in adipose tissue and liver of muscle-G4KO mice (18Kim J.K. Zisman A. Fillmore J.J. Peroni O.D. Kotani K. Perret P. Zong H. Dong J. Kahn C.R. Kahn B.B. Shulman G.I. J. Clin. Invest. 2001; 108: 153-160Crossref PubMed Scopus (149) Google Scholar). In muscle-G4KO and GLUT4-null mice, glucose uptake by the liver may partially compensate for impaired glucose uptake in muscle and other tissues (12Katz E. Stenbit A. Hatton K. DePinho R. Charron J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (392) Google Scholar, 15Zisman A. Peroni O.D. Abel E.D. Michael M.D. Mauvais-Jarvis F. Lowell B.B. Wojtaszewski J.F. Hirshman M.F. Virkamaki A. Goodyear L.J. Kahn C.R. Kahn B.B. Nat. Med. 2000; 6: 924-928Crossref PubMed Scopus (552) Google Scholar). Why Is the Defect in Glucose Homeostasis in Muscle-G4KO Mice More Severe than in the Homozygous, Whole-body GLUT4-null Mice?—This may reflect differences in the developmental stage at which GLUT4 is deleted or in genetic background. Alternatively, the absence of GLUT4 from discrete regions of the brain, kidney, and other tissues may modify the phenotype in GLUT4-null mice. Additionally, the negative nutritional balance in GLUT4-null mice may prevent the emergence of diabetic features. Heterozygous GLUT4+/–-null mice become more diabetic than GLUT4-null mice (13Stenbit A.E. Tsao T.S. Li J. Burcelin R. Geenen D.L. Factor S.M. Houseknecht K. Katz E.B. Charron M.J. Nat. Med. 1997; 3: 1096-1101Crossref PubMed Scopus (273) Google Scholar). Moreover, diabetes is averted when GLUT4 expression is restored specifically in muscle of GLUT4+/– mice (19Tsao T.-S. Stenbit A.E. Factor S.M. Chen W. Rossetti L. Charron M.J. Diabetes. 1999; 48: 775-782Crossref PubMed Scopus (47) Google Scholar), confirming the importance of GLUT4 in muscle to maintain glucose homeostasis. Transgenic overexpression of GLUT1 in skeletal muscle also increases basal glucose uptake and muscle glycogen synthesis but insulin-mediated glucose uptake is impaired (20Gulve E. Ren J. Marshall B. Gao J. Hansen P. Holloszy J. Mueckler M. J. Biol. Chem. 1994; 269: 18366-18370Abstract Full Text PDF PubMed Google Scholar, 21Marshall B.A. Mueckler M.M. Am. J. Physiol. 1994; 267: E738-E744Crossref PubMed Google Scholar). Effects of GLUT4 Ablation on the Heart—Muscle-G4KO, cardiac-G4KO, and GLUT4-null mice all develop cardiac hypertrophy. GLUT4-null homozygous (22Weiss R.G. Chatham J.C. Georgakopolous D. Charron M.J. Wallimann T. Kay L. Walzel B. Wang Y. Kass D.A. Gerstenblith G. Chacko V.P. FASEB J. 2002; 16: 613-615Crossref PubMed Scopus (42) Google Scholar) and heterozygous (13Stenbit A.E. Tsao T.S. Li J. Burcelin R. Geenen D.L. Factor S.M. Houseknecht K. Katz E.B. Charron M.J. Nat. Med. 1997; 3: 1096-1101Crossref PubMed Scopus (273) Google Scholar) mice show cardiac dysfunction under normal conditions. In contrast, cardiac-G4KO mice have normal contractile function under basal conditions (16Abel E.D. Kaulbach H.C. Tian R. Hopkins J. Duffy J. Doetschman T. Minnemann T. Boers M.-E. Hadro E. Oberste-Berghaus C. Quist W. Lowell B.B. Ingwall J.S. Kahn B.B. J. Clin. Invest. 1999; 104: 1703-1714Crossref PubMed Scopus (260) Google Scholar) but decreased recovery after hypoxia (23Tian R. Abel E.D. Circulation. 2001; 103: 2961-2966Crossref PubMed Scopus (178) Google Scholar). The increase in GLUT1 expression in the heart of these models clearly cannot compensate for the absence of GLUT4 suggesting that GLUT4 is essential for normal cardiac function at least under some conditions. Furthermore, cardiac-G4KO mice are normal metabolically, thus eliminating the possibility that hyperinsulinemia or other metabolic abnormalities play a major etiologic role in the cardiac hypertrophy. In GLUT4-null mice, however, metabolic abnormalities including alterations in cardiac substrate availability may contribute to impaired cardiac function. Muscle-IRKO (also MIRKO (17Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar)), created by breeding mice with exon 4 of the insulin receptor flanked by loxP sites to the muscle creatine kinase-Cre mice, exhibit a muscle-specific >95% reduction in insulin receptor content and early insulin signaling events (17Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar). Insulin-stimulated glucose uptake and activation of glycogen synthase in muscle in vivo and in vitro are severely impaired. Despite this, muscle glycogen content is relatively normal or only mildly decreased. Unexpectedly, muscle-IRKO mice show no alteration in glucose tolerance in the awake state, and they maintain euglycemia for at least the first 11 months of life (17Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar). Moreover, plasma insulin concentrations and the blood glucose lowering effect of exogenous insulin are normal. These data sharply contrast with the insulin resistance and glucose intolerance in muscle-G4KO (above) and adipose-G4KO mice (below). Euglycemic clamp studies reveal insulin resistance in muscle-IRKO mice (24Kim J.K. Michael M.D. Previs S.F. Peroni O.D. Mauvais-Jarvis F. Neschen S. Kahn B.B. Kahn C.R. Shulman G.I. J. Clin. Invest. 2000; 105: 1791-1797Crossref PubMed Scopus (262) Google Scholar), but this is not evident under ambient conditions (17Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar). In contrast to muscle-G4KO mice, muscle-IRKO mice have features of “the metabolic syndrome” including increased fat mass, serum triglycerides, and serum free fatty acids (Table I) (17Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar). Glucose uptake in adipose tissue in vivo is increased 3-fold in muscle-IRKO mice whereas glucose uptake in adipocytes is normal in vitro (24Kim J.K. Michael M.D. Previs S.F. Peroni O.D. Mauvais-Jarvis F. Neschen S. Kahn B.B. Kahn C.R. Shulman G.I. J. Clin. Invest. 2000; 105: 1791-1797Crossref PubMed Scopus (262) Google Scholar). This in vivo repartitioning of glucose from muscle to adipose tissue suggests that impairment of insulin signaling in muscle, but not impairment of glucose uptake per se, can lead to altered adipose insulin sensitivity, increased adiposity, and abnormal plasma lipid profiles. This phenomenon illustrates that tissue “cross-talk” occurs when insulin action is impaired in one insulin target tissue. Interestingly, muscle-IRKO mice and cardiac-specific IRKO mice (25Belke D. Betuing S. Tuttle M. Graveleau C. Young M. Pham M. Zhang D. Cooksey R. McClain D. Litwin S. Taegtmeyer H. Severson D. Kahn C.R. Abel E.D. J. Clin. Invest. 2002; 109: 629-639Crossref PubMed Scopus (334) Google Scholar) have decreased cardiac size in contrast to the cardiac hypertrophy when GLUT4 is absent from the heart.Table IComparison of muscle-G4KO, muscle-IRKO, adipose-G4KO, and adipose-IRKO miceMuscle-G4KOMuscle-IRKOAdipose-G4KOAdipose-IRKOGrowthNormalNormalNormalNormalAdiposityNormalIncreasedNormalDecreasedMuscle massNormalDecreasedNormalNormalHeart massHypertrophyDecreasedNormalNormalMetablic parametersGlucoseIncreasedNormalNormal/increasedNormal/lower (after GTG)LactateDecreasedNormalNormal/decreasedNormalFFANormalIncreasedNormalNormalTGNormalIncreasedNormalDecreasedInsulinIncreasedNormal/slight increaseIncreasedDecreased (fasting)Leptin (per adipose mass)NormalN/DNormalIncreasedAdiponectinNormalN/DNormalIncreasedTNF-αN/DN/DNormalN/DGlucose metabolismWhole-bodyGlucose toleranceImpairedNormal (awake)aWhen anesthetized, glucose tolerance was slightly impaired (26).ImpairedNo aging- or obesity-induced glucose intoleranceInsulin sensitivityImpairedNormal (ITT)/decreased (clamp)ImpairedNo aging- or obesity-induced insulin resistanceIndividual tissuesGlucose uptake Muscle (insulin)DecreasedDecreasedDecreased (in vivo)/normal (in vitro)N/D Muscle (contraction)DecreasedNormalN/DN/D Adipose tissue (insulin)Decreased (clamp)Normal (in vitro)/increased (clamp)DecreasedDecreasedInsulin-induced suppression of hepatic glucose productionDecreasedNormalDecreasedN/Da When anesthetized, glucose tolerance was slightly impaired (26Wojtaszewski J.F. Higaki Y. Hirshman M.F. Michael M.D. Dufresne S.D. Kahn C.R. Goodyear L.J. J. Clin. Invest. 1999; 104: 1257-1264Crossref PubMed Scopus (176) Google Scholar). Open table in a new tab The data suggest that insulin signaling through its cognate receptor in muscle is not essential for the maintenance of glucose disposal in mice (17Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar). The glucose intolerance in muscle-G4KO mice (15Zisman A. Peroni O.D. Abel E.D. Michael M.D. Mauvais-Jarvis F. Lowell B.B. Wojtaszewski J.F. Hirshman M.F. Virkamaki A. Goodyear L.J. Kahn C.R. Kahn B.B. Nat. Med. 2000; 6: 924-928Crossref PubMed Scopus (552) Google Scholar), but not in muscle-IRKO mice, suggests that insulin-independent glucose uptake contributes importantly to the maintenance of glucose homeostasis. Unlike mice with muscle-G4KO, mice with muscle-IRKO retain normal basal and contraction-stimulated glucose transport (26Wojtaszewski J.F. Higaki Y. Hirshman M.F. Michael M.D. Dufresne S.D. Kahn C.R. Goodyear L.J. J. Clin. Invest. 1999; 104: 1257-1264Crossref PubMed Scopus (176) Google Scholar). Indeed, muscle-IRKO mice display normal exercise-stimulated glucose uptake and a normal synergistic action of exercise plus insulin on muscle glucose uptake. In contrast, contraction-induced glucose uptake is severely impaired in muscle-G4KO mice (15Zisman A. Peroni O.D. Abel E.D. Michael M.D. Mauvais-Jarvis F. Lowell B.B. Wojtaszewski J.F. Hirshman M.F. Virkamaki A. Goodyear L.J. Kahn C.R. Kahn B.B. Nat. Med. 2000; 6: 924-928Crossref PubMed Scopus (552) Google Scholar). Therefore, contraction of postural muscles and other forms of physical activity that elicit non-insulin-dependent glucose uptake could contribute to the maintenance of glucose tolerance in muscle-IRKO mice. In addition, signaling through the insulin-like growth factor-1 (IGF-1) receptor in muscle may become important when IR is absent. Interference with both IR and IGF-1 receptor signaling with a dominant-negative IGF-1 receptor expressed in muscle leads to insulin resistance and type 2 diabetes (27Fernandez A.M. Kim J.K. Yakar S. Dupont J. Hernandez-Sanchez C. Castle A.L. Filmore J. Shulman G.I. Le Roith D. Genes Dev. 2001; 15: 1926-1934Crossref PubMed Scopus (297) Google Scholar), and deletion of the IGF-1 gene leads to severe muscle insulin resistance (28Yakar S. Liu J.L. Fernandez A.M. Wu Y. Schally A.V. Frystyk J. Chernausek S.D. Mejia W. Le Roith D. Diabetes. 2001; 50: 1110-1118Crossref PubMed Scopus (297) Google Scholar). Adipose-G4KO mice were made by crossing mice carrying the “floxed” GLUT4 allele with transgenic mice expressing Cre recombinase driven by the adipose-specific fatty acid binding protein (aP2) promoter/enhancer (29Abel E.D. Peroni O.D. Kim J.K. Kim Y.-B. Boss O. Hadro E. Minnemann T. Shulman G.I. Kahn B.B. Nature. 2001; 409: 729-733Crossref PubMed Scopus (928) Google Scholar). GLUT4 protein levels were reduced by 70–99% in both brown and white adipose tissue whereas GLUT4 levels were preserved in skeletal muscle and heart. In adipocytes from adipose-G4KO mice, basal glucose uptake tends to be reduced, and insulin-stimulated glucose uptake is markedly blunted. Unlike the global GLUT4-null mice (12Katz E. Stenbit A. Hatton K. DePinho R. Charron J. Nature. 1995; 377: 151-155Crossref PubMed Scopus (392) Google Scholar), reduction of GLUT4 selectively in adipose tissue does not result in growth retardation or decreased adipose mass or adipocyte size (29Abel E.D. Peroni O.D. Kim J.K. Kim Y.-B. Boss O. Hadro E. Minnemann T. Shulman G.I. Kahn B.B. Nature. 2001; 409: 729-733Crossref PubMed Scopus (928) Google Scholar). The latter suggests that the small amount of glucose transported by GLUT1 in adipocytes may be adequate for the formation of glycerol 3-phosphate required for triglyceride synthesis. Heart weight is also normal in adipose-G4KO mice in contrast to the cardiomegaly observed in GLUT4-null mice and in cardiac-specific (16Abel E.D. Kaulbach H.C. Tian R. Hopkins J. Duffy J. Doetschman T. Minnemann T. Boers M.-E. Hadro E. Oberste-Berghaus C. Quist W. Lowell B.B. Ingwall J.S. Kahn B.B. J. Clin. Invest. 1999; 104: 1703-1714Crossref PubMed Scopus (260) Google Scholar) and muscle-specific (15Zisman A. Peroni O.D. Abel E.D. Michael M.D. Mauvais-Jarvis F. Lowell B.B. Wojtaszewski J.F. Hirshman M.F. Virkamaki A. Goodyear L.J. Kahn C.R. Kahn B.B. Nat. Med. 2000; 6: 924-928Crossref PubMed Scopus (552) Google Scholar) G4KO mice. Although white adipose tissue accounts for less than 10% of whole-body glucose uptake (30James D. Burleigh K. Kraegen E. Diabetes. 1985; 34: 1049-1054Crossref PubMed Google Scholar), adipose-G4KO mice have insulin resistance and glucose intolerance, and a subset of these mice develops extreme insulin resistance and diabetes. The range in severity of the phenotype is expected when studying outbred strains with a mixture of genetic modifiers. Hyperinsulinemic/euglycemic clamp studies reveal an ∼50% reduction in whole-body glucose uptake in adipose-G4KO mice. As expected, insulin-stimulated 2-deoxyglucose uptake into white and brown adipose in vivo is markedly reduced. However, unexpectedly, glucose uptake into skeletal muscle in vivo is also impaired despite preserved GLUT4 expression in muscle. As basal and insulin-stimulated glucose uptake are normal in muscle from these mice ex vivo, it appears that this defect is secondary to the in vivo milieu. Furthermore, in addition to the insulin resistance in muscle, the insulin-induced suppression of hepatic glucose production is impaired in adipose-G4KO mice. Insulin-stimulated activation of phosphoinositide 3-kinase is impaired in muscle and liver of adipose-G4KO mice in vivo (29Abel E.D. Peroni O.D. Kim J.K. Kim Y.-B. Boss O. Hadro E. Minnemann T. Shulman G.I. Kahn B.B. Nature. 2001; 409: 729-733Crossref PubMed Scopus (928) Google Scholar) and could contribute to the defective insulin responses in these tissues. These data suggest that reduction of insulin-stimulated glucose transport in adipocytes secondarily induces insulin resistance in other insulin target tissues. Adipocytes can indirectly regulate insulin action and substrate metabolism in muscle and liver and insulin secretion from pancreatic β-cells by altered release of free fatty acids, leptin, tumor necrosis factor-α, resistin, and adiponectin (31Kahn B.B. Flier J.S. J. Clin. Invest. 2000; 106: 473-481Crossref PubMed Scopus (2390) Google Scholar, 32Frühbeck G. Gómez-Ambrosi J. Muruzábal F.J. Burrell M.A. Am. J. Physiol. 2001; 280: E827-E847Crossref PubMed Google Scholar). However, serum levels of these molecules are not altered in adipose-G4KO mice (Table I) (29Abel E.D. Peroni O.D. Kim J.K. Kim Y.-B. Boss O. Hadro E. Minnemann T. Shulman G.I. Kahn B.B. Nature. 2001; 409: 729-733Crossref PubMed Scopus (928) Google Scholar). 2B. B. Kahn, unpublished results. Insulin resistance also occurs by increasing lipid depots in muscle and liver, but these were not increased in adipose-G4KO mice. Thus, the insulin resistance in muscle and liver of adipose-G4KO mice is likely to be caused by altered secretion of an as yet unidentified adipocyte-derived molecule that affects insulin action in other tissues (Fig. 1). Additional evidence for a “systemic impact” of adipose-GLUT4 is the fact that adipose-specific overexpression of GLUT4 results in enhanced glucose disposal and insulin sensitivity in vivo (33Shepherd P.R. Gnudi L. Tozzo E. Yang H. Leach F. Kahn B.B. J. Biol. Chem. 1993; 268: 22243-22246Abstract Full Text PDF PubMed Google Scholar). Adipocyte-specific insulin receptor knock-out (adipose-IRKO, also FIRKO (34Blüher M. Michael M. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar)) mice were generated by breeding insulin receptor-floxed mice (17Brüning J.C. Michael M.D. Winnay J.N. Hayashi T. Horsch D. Accili D. Goodyear L.J. Kahn C.R. Mol. Cell. 1998; 2: 559-569Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar) with transgenic mice that express Cre recombinase driven by the aP2 promoter/enhancer. Whereas basal glucose uptake in adipocytes from adipose-IRKO mice is unchanged, insulin-stimulated glucose uptake is reduced by ∼90% (34Blüher M. Michael M. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). Insulin also failed to stimulate glucose metabolism or suppress lipolysis in these adipocytes. Growth is normal in adipose-IRKO mice up to 4 weeks of age. By 8 weeks, however, these mice gained less weight than controls. In contrast to adipose-G4KO mice that have normal adipose mass, adipose-IRKO mice have reduced white adipose and brown adipose tissue mass and whole-body triglyceride content (34Blüher M. Michael M. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). Blood glucose concentrations were not altered in adipose-IRKO mice at 2–8 months, but fasting plasma insulin concentrations were lower suggesting increased insulin sensitivity (Table I). Serum triglyceride levels were also reduced. Glucose and insulin tolerance tests were normal at 2 and 10 months of age, whereas control mice showed age-related glucose intolerance and insulin resistance. Thus, adipose-IRKO mice are protected against age-related glucose intolerance as well as obesity. Serum adiponectin concentration was increased in adipose-IRKO mice. Furthermore, plasma leptin levels expressed per mg of fat pad were ∼3-fold elevated, and the linear relationship between leptin levels and body weight seen in normal mice was lost. Leptin protein levels were normal in adipose tissue of adipose-IRKO mice. Thus, the absence of IR in adipose tissue alters leptin and adiponectin synthesis, secretion, or clearance. Adipose-IRKO Mice Are Resistant to Obesity—Mice were injected with gold thioglucose (GTG), which ablates glucose-sensing neurons in the ventromedial hypothalamus. GTG treatment increased food intake in both adipose-IRKO and control mice (34Blüher M. Michael M. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar), and as previously observed, control mice treated with GTG became obese. Despite hyperphagia, adipose-IRKO mice did not develop obesity, diabetes, or steatosis in contrast to GTG-injected controls. Thus, adipose-IRKO protects against GTG-induced, as well as age-related, obesity and obesity-related diabetes, and insulin resistance. The protection from obesity could be explained by the lack of the lipogenic and anti-lipolytic effects of insulin in adipocytes. However, it is also likely that energy expenditure is enhanced, because adipose IRKO mice are lean despite hyperphagia. The reduced adipose mass in adipose-IRKO mice is not due to fewer adipocytes. Histology showed polarization of adipocytes into small and large in contrast to a normal distribution in control mice (34Blüher M. Michael M. Peroni O.D. Ueki K. Carter N. Kahn B.B. Kahn C.R. Dev. Cell. 2002; 3: 25-38Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). IR expression in both large and small adipocytes of adipose-IRKO mice is reduced by 85–99%, indicating that the heterogeneity is not due to differences in efficiency of gene recombination. However, expression levels of some proteins, e.g. fatty-acid synthase and the adipogenic transcription factors SREBP-1 and C/EBPα, are different in small and large adipocytes, and this may contribute to the heterogeneity. In contrast to adipose-IRKO mice, brown adipocyte-specific insulin receptor knock-out (BAT-IRKO) mice exhibit an age-dependent loss of brown adipose tissue that is associated with impaired glucose tolerance without insulin resistance (35Guerra C. Navarro P. Valverde A.M. Arribas M. Bruning J. Kozak L.P. Kahn C.R. Benito M. J. Clin. Invest. 2001; 108: 1205-1213Crossref PubMed Google Scholar). This appears to be primarily due to loss of β-cell mass and defective insulin secretion. Thus, absence of IR in white adipose tissue has a protective effect over the impaired glucose homeostasis resulting from the absence of IR in brown adipose tissue alone. In addition, brown adipose tissue may affect pancreatic β-cell mass and function. Adipose-IRKO Mice Have Enhanced Longevity—Surprisingly, mean lifespan in adipose-IRKO mice is increased by 134 days (18%) (36Blüher M. Kahn B.B. Kahn C.R. Science. 2003; 299: 572-574Crossref PubMed Scopus (1047) Google Scholar) in contrast to the shortened lifespan in GLUT4-null mice. Calorie restriction in many species is associated with increased longevity, but it has not been clear whether this is due to decreased food intake or the resultant leanness. Because adipose-IRKO mice do not eat less, the effect of calorie restriction on longevity is likely to be due to reduced adipose mass per se. Combined with data showing that decreased signaling through an insulin-like pathway increases longevity in Caenorhabditis elegans and Drosophila melanogaster (37Guarente L. Kenyon C. Nature. 2000; 408: 255-262Crossref PubMed Scopus (1091) Google Scholar), these new data imply that selective reduction of insulin signaling in certain metabolically important organs such as adipose tissue could result in extended longevity (36Blüher M. Kahn B.B. Kahn C.R. Science. 2003; 299: 572-574Crossref PubMed Scopus (1047) Google Scholar). Tissue-conditional knock-out mice advance our understanding of the mechanisms underlying insulin resistance. Marked reduction of GLUT4 in muscle or adipose tissue causes insulin resistance in other insulin target tissues secondarily and increases the risk for diabetes. Hence, there is cross-talk among insulin target tissues. This may have implications for the genetics of type 2 diabetes because a single genetic defect in one insulin target tissue can result in insulin resistance in other tissues, leading to type 2 diabetes. This may explain, at least in part, the surprising finding that reduction of GLUT4 in adipose tissue causes a similar degree of insulin resistance as reduction of GLUT4 in muscle. In contrast, ablation of IR in adipose tissue or muscle has only a subtle direct impact on glucose homeostasis, possibly due to compensation by IGF-1 receptor signaling and by insulin-independent stimulation of glucose transport by contraction. However, IR in both muscle and adipose tissue is critical for the regulation of adiposity.
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