Adipose tissue regulates insulin sensitivity: role of adipogenesis, de novo lipogenesis and novel lipids.

Obesity, the major cause of the current global epidemic of type 2 diabetes (T2D), induces insulin resistance in peripheral insulin target tissues. Several mechanisms have been identified related to cross-talk between adipose tissue, skeletal muscle and liver. These mechanisms involve both increased free fatty acid release and altered secretion of adipokines from adipose tissue. A major determinant of metabolic health is the ability of subcutaneous adipose tissue (SAT) to store excess fat rather than allowing it to accumulate in ectopic depots including liver (i.e. in nonalcoholic fatty liver disease), muscle and heart, or in epicardial/pericardial and visceral fat depots which promote the metabolic complications of obesity. The ability to recruit and differentiate precursor cells into adipose cells (adipogenesis) in SAT is under genetic regulation and is reduced in high-risk individuals who have first-degree relatives with T2D. Early recruitment of new adipose cells is dependent on the cross-talk between canonical WNT and BMP4 signalling; WNT enhances their undifferentiated and proliferative state whereas BMP4 induces their commitment to the adipogenic lineage. Dysregulation of these signalling pathways is associated with impaired adipogenesis and impaired ability to respond to the need to store excess lipids in SAT. This leads to hypertrophic, dysfunctional and insulin-resistant adipose cells with a reduced content of GLUT4, the major insulin-regulated glucose transporter, which in turn reduces adipose tissue glucose uptake and de novo lipogenesis. We recently identified that reduced GLUT4 and lipogenesis in adipocytes impairs the synthesis of a novel family of lipids secreted by adipose tissue (and potentially other tissues), branched fatty acid esters of hydroxy fatty acids (FAHFAs). FAHFAs have beneficial metabolic effects, including enhancing insulin-stimulated glucose transport and glucose-stimulated GLP1 and insulin secretion, as well as powerful anti-inflammatory effects. FAHFA levels are reduced in subcutaneous adipose tissue in insulin-resistant individuals, and this novel family of lipids may become of future therapeutic use.

Targeted disruption of the glucose transporter 4 selectively in muscle causes insulin resistance and glucose intolerance.

The prevalence of type 2 diabetes mellitus is growing worldwide. By the year 2020, 250 million people will be afflicted. Most forms of type 2 diabetes are polygenic with complex inheritance patterns, and penetrance is strongly influenced by environmental factors. The specific genes involved are not yet known, but impaired glucose uptake in skeletal muscle is an early, genetically determined defect that is present in non-diabetic relatives of diabetic subjects. The rate-limiting step in muscle glucose use is the transmembrane transport of glucose mediated by glucose transporter (GLUT) 4 (ref. 4), which is expressed mainly in skeletal muscle, heart and adipose tissue. GLUT4 mediates glucose transport stimulated by insulin and contraction/exercise. The importance of GLUT4 and glucose uptake in muscle, however, was challenged by two recent observations. Whereas heterozygous GLUT4 knockout mice show moderate glucose intolerance, homozygous whole-body GLUT4 knockout (GLUT4-null) mice have only mild perturbations in glucose homeostasis and have growth retardation, depletion of fat stores, cardiac hypertrophy and failure, and a shortened life span. Moreover, muscle-specific inactivation of the insulin receptor results in minimal, if any, change in glucose tolerance. To determine the importance of glucose uptake into muscle for glucose homeostasis, we disrupted GLUT4 selectively in mouse muscles. A profound reduction in basal glucose transport and near-absence of stimulation by insulin or contraction resulted. These mice showed severe insulin resistance and glucose intolerance from an early age. Thus, GLUT4-mediated glucose transport in muscle is essential to the maintenance of normal glucose homeostasis.

Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.

Adiponectin (Ad) is a hormone secreted by adipocytes that regulates energy homeostasis and glucose and lipid metabolism. However, the signaling pathways that mediate the metabolic effects of Ad remain poorly identified. Here we show that phosphorylation and activation of the 5'-AMP-activated protein kinase (AMPK) are stimulated with globular and full-length Ad in skeletal muscle and only with full-length Ad in the liver. In parallel with its activation of AMPK, Ad stimulates phosphorylation of acetyl coenzyme A carboxylase (ACC), fatty-acid oxidation, glucose uptake and lactate production in myocytes, phosphorylation of ACC and reduction of molecules involved in gluconeogenesis in the liver, and reduction of glucose levels in vivo. Blocking AMPK activation by dominant-negative mutant inhibits each of these effects, indicating that stimulation of glucose utilization and fatty-acid oxidation by Ad occurs through activation of AMPK. Our data may provide a novel paradigm that an adipocyte-derived antidiabetic hormone, Ad, activates AMPK, thereby directly regulating glucose metabolism and insulin sensitivity in vitro and in vivo.

High circulating levels of RBP4 and mRNA levels of aP2, PGC-1alpha and UCP-2 predict improvement in insulin sensitivity following pioglitazone treatment of drug-naïve type 2 diabetic subjects.

CONTEXT: High levels of circulating retinol-binding protein 4 (RBP4) and baseline expression of adipogenic genes correlate with subsequent improvement in insulin sensitivity following Thiazolidinedione (TZD) treatment. OBJECTIVE: The aim was to identify baseline characteristics and early changes related to TZD treatment that could predict a good treatment response. DESIGN: Subjects were examined with oral glucose tolerance test, intravenous glucose tolerance test, hyperinsulinaemic euglycaemic clamp, body composition and standard blood sampling at baseline and after 4 and 12 weeks treatment. Subcutaneous adipose tissue biopsies were taken from the abdominal region at baseline, after 3 days and 4 weeks treatment to examine the gene expression profile. SETTING: Research laboratory in a University hospital. PARTICIPANTS: Ten newly diagnosed and previously untreated type 2 diabetic subjects were treated with pioglitazone for 3 months. MAIN OUTCOME MEASURES: Baseline characteristics and early changes related to TZD treatment that could predict the response after 3 months. RESULTS: Pioglitazone improved insulin sensitivity after 4 weeks combined with lower glucose and insulin levels without any change in BMI. It was accompanied by lower circulating resistin and plasminogen activator inhibitor-1 levels rapidly increased levels of circulating total and high molecular weight adiponectin as well as adiponectin and adipocyte fatty acid-binding protein (aP2) mRNA expression in the adipose tissue. High levels of circulating RBP4 at baseline and adipose tissue expression of aP2, proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1alpha) and uncoupling protein 2 (UCP-2) predicted a good treatment response measured as improvement in insulin-stimulated whole-body glucose uptake after 3 months. CONCLUSIONS: Circulating levels of RBP4 as an index of insulin sensitivity and mRNA levels of adipogenic genes correlate with the subsequent improvement in insulin sensitivity following TZD treatment.

Divergent mechanisms for the insulin resistant and hyperresponsive glucose transport in adipose cells from fasted and refed rats. Alterations in both glucose transporter number and intrinsic activity.

The effects of fasting and refeeding on the glucose transport response to insulin in isolated rat adipose cells have been examined using 3-O-methylglucose transport in intact cells and cytochalasin B binding and Western blotting in subcellular membrane fractions. After a 72-h fast, basal glucose transport activity decreases slightly and insulin-stimulated activity decreases greater than 85%. Following 48 h of fasting, insulin-stimulated glucose transport activity is diminished from 3.9 +/- 0.5 to 1.3 +/- 0.3 fmol/cell per min (mean +/- SEM). Similarly, the concentrations of glucose transporters are reduced with fasting in both the plasma membranes from insulin-stimulated cells from 38 +/- 5 to 18 +/- 3 pmol/mg of membrane protein and the low density microsomes from basal cells from 68 +/- 8 to 34 +/- 9 pmol/mg of membrane protein. Ad lib. refeeding for 6 d after a 48-h fast results in up to twofold greater maximally insulin-stimulated glucose transport activity compared with the control level (7.1 +/- 0.4 vs. 4.5 +/- 0.2 fmol/cell per min), before returning to baseline at 10 d. However, the corresponding concentration of glucose transporters in the plasma membranes is restored only to the control level (45 +/- 5 vs. 50 +/- 5 pmol/mg of membrane protein). Although the concentration of glucose transporters in the low density microsomes of basal cells remains decreased, the total number is restored to the control level due to an increase in low density microsomal protein. Thus, the insulin-resistant glucose transport in adipose cells from fasted rats can be explained by a decreased translocation of glucose transporters to the plasma membrane due to a depleted intracellular pool. In contrast, the insulin hyperresponsive glucose transport observed with refeeding appears to result from (a) a restored translocation of glucose transporters to the plasma membrane from a repleted intracellular pool and (b) enhanced plasma membrane glucose transporter intrinsic activity.

Mechanism for enhanced glucose transport response to insulin in adipose cells from chronically hyperinsulinemic rats. Increased translocation of glucose transporters from an enlarged intracellular pool.

The mechanism for the increased glucose transport response to insulin in adipose cells from chronically hyperinsulinemic rats was examined. Rats were infused with insulin s.c. for 2 wk. Isolated adipose cells were incubated with and without insulin, 3-O-methylglucose transport was measured, and glucose transporters in subcellular membrane fractions were assessed by cytochalasin B binding. Adipose cells from insulin-treated rats showed no change in basal but a 55% increase in insulin-stimulated glucose transport activity compared with those from control rats (7.1 +/- 0.8 vs. 4.6 +/- 0.5 fmol/cell per min, mean +/- SEM) and a corresponding increase in the concentration of glucose transporters in the plasma membranes (44 +/- 5 vs. 32 +/- 6 pmol/mg of membrane protein). In the low-density microsomes, glucose transporter concentrations in both basal and insulin-stimulated states were the same, but the total numbers were greater in cells from the insulin-treated rats because of a 39% increase in low-density microsomal protein. Therefore, chronic experimental hyperinsulinemia in the rat enhances the stimulatory action of insulin on glucose transport in the adipose cell by increasing the concentration of glucose transporters in the plasma membranes. This results from an enlarged intracellular pool due to increased intracellular protein and enhanced glucose transporter translocation in response to insulin.

Divergent regulation of the Glut 1 and Glut 4 glucose transporters in isolated adipocytes from Zucker rats.

We have studied the relationship between glucose uptake rate and Glut 1 and Glut 4 protein and mRNA levels per fat cell in lean (FA/FA) and obese (fa/fa) Zucker rats at 5, 10, and 20 wk of age, and after induction of acute diabetes with streptozotocin. 5 wk obese rats exhibit insulin hyperresponsive glucose uptake, whereas 20 wk obese rats show insulin resistant glucose uptake. The relative abundance of Glut 1 and Glut 4 mRNA and protein per equal amount of total RNA and total membrane protein, respectively, is lower in adipocytes from obese rats. However, at all ages the enlargement of fat cells from obese rats is accompanied by a severalfold increase in total RNA and total membrane protein per cell. Thus, on a cellular basis, mRNA and protein levels of Glut 4 increases in young obese rats and gradually declines as a function of age. Basal glucose uptake is increased severalfold in fat cells from obese rats, and in parallel Glut 1 expression per cell in obese rats is two- to threefold increased over lean rats at all ages. Acute diabetes in 20 wk obese rats causes a profound downregulation of glucose uptake and a concomitant reduction of both Glut 1 and Glut 4 protein levels. Thus, changes in Glut 4 expression are a major cause of alteration in insulin-stimulated glucose uptake of adipocytes during evolution of obesity and diabetes in Zucker rats.

Regulation of glucose transporter-specific mRNA levels in rat adipose cells with fasting and refeeding. Implications for in vivo control of glucose transporter number.

Fasting in the rat is associated with a rapid and progressive decrease in insulin-stimulated glucose transport activity in adipose cells, which is not only restored to normal, but increased transiently to supranormal levels by refeeding. The mechanisms for these changes in glucose transport activity appear to involve alterations in both glucose transporter number and intrinsic activity (glucose turnover number). In this study, we use the human hepatoma Hep G2 glucose transporter complementary DNA clone to examine the molecular basis for these alterations. Extractable RNA per adipose cell is decreased 35% with 3 d of fasting and increased to 182% of control with 6 d of refeeding after 2 d of fasting. This parallels changes in adipose cell intracellular water, so that total RNA/water space remains relatively constant. When the changes in total RNA/cell are taken into account, Northern and slot blot analyses with quantitative densitometry reveal a 36% decrease in specific glucose transporter mRNA level in cells from the fasted rats. The mRNA level in cells from the fasted/refed rats is restored to normal. These observations correlate closely with previous measurements of glucose transporter number in adipose cell membrane fractions using cytochalasin B binding and Western blotting. The levels of specific mRNAs for tubulin and actin on a per cell basis show similar but more dramatic changes and mRNAs encoding several differentiation-dependent adipose cell proteins are also significantly affected. Thus, the levels of mRNA for multiple adipose cell genes are affected by fasting and refeeding. In particular, this demonstrates that in vivo metabolic alterations can influence the level of a glucose transporter mRNA in adipose cells. This may have implications for the regulation of glucose transporter number and glucose transport activity.

Decreased in vivo glucose uptake but normal expression of GLUT1 and GLUT4 in skeletal muscle of diabetic rats.

This study was designed to determine whether altered glucose transporter expression is essential for the in vivo insulin-resistant glucose uptake characteristic of streptozocin-induced diabetes. Immunofluorescence in rat skeletal muscle colocalizes GLUT4 with dystrophin, both intrinsic to muscle fibers. In contrast, GLUT1 is extrinsic to muscle fibers, probably in perineurial sheath. Immunoblotting shows that levels of GLUT1 and GLUT4 protein per DNA in hindlimb muscle are unaltered from control levels at 7 d of diabetes but decrease to approximately 20% of control at 14 d of diabetes. This decrease is prevented by insulin treatment. In adipose cells of 7 d diabetic rats, GLUT4 levels are depressed. Thus, GLUT4 undergoes tissue-specific regulation in response to diabetes. GLUT4 and GLUT1 mRNA levels in muscle are decreased 62-70% at both 7 and 14 d of diabetes and are restored by insulin treatment. At 7 d of diabetes, when GLUT4 protein levels in muscle are unaltered, in vivo insulin-stimulated glucose uptake measured by euglycemic clamp is 54% of control. This reflects impairment in both glycogen synthesis and glycolysis and the substrate common to these two pathways, glucose-6-phosphate, is decreased approximately 30% in muscle of diabetic rats. These findings suggest a defect early in the pathway of glucose utilization, probably at the step of glucose transport. Because GLUT1 and GLUT4 levels are unaltered at 7 d of diabetes, reduced glucose uptake in muscle probably reflects impaired glucose transporter translocation or intrinsic activity. Later, at 14 d of diabetes, GLUT1 and GLUT4 protein levels are reduced, suggesting that sequential defects may contribute to the insulin-resistant glucose transport characteristic of diabetes.

Normalization of blood glucose in diabetic rats with phlorizin treatment reverses insulin-resistant glucose transport in adipose cells without restoring glucose transporter gene expression.

Evidence is emerging for a direct role of glucose, independent of changes in insulin, in the regulation of cellular glucose transport and glucose utilization in vivo. In this study we investigate potential cellular and molecular mechanisms for this regulatory effect of glucose by determining how normalization of glycemia without insulin therapy in diabetic rats influences 3-O-methylglucose transport and the expression and translocation of two genetically distinct species of glucose transporters (GTs) in adipose cells. These results are compared with alterations in glucose disposal in vivo measured by euglycemic clamp. In rats rendered diabetic by 90% pancreatectomy, insulin-stimulated glucose transport in adipose cells is decreased 50% in parallel with reduced insulin-mediated glucose disposal in vivo. Levels of adipose/muscle GTs measured by immunoblotting are decreased in adipose cell subcellular membrane fractions, as are the corresponding mRNA levels assessed by Northern blotting of total adipose cell RNA. Normalization of blood glucose in diabetic rats with phlorizin, which impairs renal tubular glucose reabsorption and thus enhances glucose excretion, restores insulin-stimulated glucose transport in adipose cells and insulin-mediated glucose disposal in vivo. Importantly, levels of the adipose/muscle GT protein remain 43% reduced in the low-density microsomes in the basal state and 46% reduced in the plasma membranes in the insulin-stimulated state. Adipose/muscle GT mRNA levels remain approximately 50% depressed. Levels of the HepG2/brain GT protein and mRNA are unaltered by diabetes or phlorizin treatment. Thus, changes in ambient glucose independent of changes in ambient insulin can regulate the glucose transport response to insulin in isolated adipose cells and changes in responsiveness parallel alterations in glucose uptake in vivo. Since this effect can occur without alteration in the expression of the two species of glucose transporters present in adipose cells or in their translocation to the plasma membrane in response to insulin, it may result from changes in GT functional activity.

Differential regulation of two glucose transporters in adipose cells from diabetic and insulin-treated diabetic rats.

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.

Expression of ob mRNA and its encoded protein in rodents. Impact of nutrition and obesity.

The mutant gene responsible for obesity in the ob/ob mouse was recently identified by positional cloning (Zhang Y., R. Proenca, M. Maffel, M. Barone, L. Leopold, and J.M. Friedman. 1994. Nature (Lond.) 372:425). The encoded protein and to represent and "adipostat" signal reflecting the state of energy stores. We confirm that the adipocyte is the source of ob mRNA and that the predicted 16-kD ob protein is present in rodent serum as detected by Western blot. To evaluate the hypothesis that it might represent an adipostat, we assessed serum levels of ob protein and expression of ob mRNA in adipose cells and tissue of rodents in response to a variety of perturbations which effect body fat mass. Both ob protein and ob mRNA expression are markedly increased in obesity. The levels of ob protein are approximately 5-10-fold elevated in serum of db/db mice, in mice with hypothalamic lesions caused by neonatal administration of monosodium glutamate (MSG), and in mice with toxigene induced brown fat ablation, (UCP-DTA). Very parallel changes are observed in adipocyte ob mRNA expression in these models and in ob/ob mice. As predicted however, no serum ob protein could be detected in the ob/ob mice. By contrast to obesity, starvation of normal rats and mice for 1-3 d markedly suppresses ob mRNA abundance, and this is reversed with refeeding. Similarly, ob protein concentration in normal mice falls to undetectable levels with starvation. In the ob/ob, UCP-DTA and MSG models, overexpression of ob mRNA is reversed by caloric restriction. These data support the hypothesis that expression of ob mRNA and protein are regulated as a function of energy stores, and that ob serves as a circulating feedback signal to sites involved in regulation of energy homeostasis.

Normal insulin-dependent activation of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 diabetes.

To determine whether the serine/threonine kinase Akt (also known as protein kinase B) is activated in vivo by insulin administration in humans, and whether impaired activation of Akt could play a role in insulin resistance, we measured the activity and phosphorylation of Akt isoforms in skeletal muscle from 3 groups of subjects: lean, obese nondiabetic, and obese type 2 diabetic. Vastus lateralis biopsies were taken in the basal (overnight fast) and insulin-stimulated (euglycemic clamp) states. Insulin-stimulated glucose disposal was reduced 31% in obese subjects and 63% in diabetic subjects, compared with lean subjects. Glycogen synthase (GS) activity in the basal state was reduced 28% in obese subjects and 49% in diabetic subjects, compared with lean subjects. Insulin-stimulated GS activity was reduced 30% in diabetic subjects. Insulin treatment activated the insulin receptor substrate-1-associated (IRS-1-associated) phosphoinositide 3-kinase (PI 3-kinase) 6.1-fold in lean, 3.7-fold in obese, and 2.4-fold in diabetic subjects. Insulin also stimulated IRS-2-associated PI 3-kinase activity 2.2-fold in lean subjects, but only 1.4-fold in diabetic subjects. Basal activity of Akt1/Akt2 (Akt1/2) and Akt3 was similar in all groups. Insulin increased Akt1/2 activity 1.7- to 2. 0-fold, and tended to activate Akt3, in all groups. Insulin-stimulated phosphorylation of Akt1/2 was normal in obese and diabetic subjects. In lean subjects only, insulin-stimulated Akt1/2 activity correlated with glucose disposal rate. Thus, insulin activation of Akt isoforms is normal in muscle of obese nondiabetic and obese diabetic subjects, despite decreases of approximately 50% and 39% in IRS-1- and IRS-2-associated PI 3-kinase activity, respectively, in obese diabetic subjects. It is therefore unlikely that Akt plays a major role in the resistance to insulin action on glucose disposal or GS activation that is observed in muscle of obese type 2 diabetic subjects.

Redistribution of substrates to adipose tissue promotes obesity in mice with selective insulin resistance in muscle.

Obesity and insulin resistance in skeletal muscle are two major factors in the pathogenesis of type 2 diabetes. Mice with muscle-specific inactivation of the insulin receptor gene (MIRKO) are normoglycemic but have increased fat mass. To identify the potential mechanism for this important association, we examined insulin action in specific tissues of MIRKO and control mice under hyperinsulinemic-euglycemic conditions. We found that insulin-stimulated muscle glucose transport and glycogen synthesis were decreased by about 80% in MIRKO mice, whereas insulin-stimulated fat glucose transport was increased threefold in MIRKO mice. These data demonstrate that selective insulin resistance in muscle promotes redistribution of substrates to adipose tissue thereby contributing to increased adiposity and development of the prediabetic syndrome.

Glucose toxicity and the development of diabetes in mice with muscle-specific inactivation of GLUT4.

Using cre/loxP gene targeting, transgenic mice with muscle-specific inactivation of the GLUT4 gene (muscle GLUT4 KO) were generated and shown to develop a diabetes phenotype. To determine the mechanism, we examined insulin-stimulated glucose uptake and metabolism during hyperinsulinemic-euglycemic clamp in control and muscle GLUT4 KO mice before and after development of diabetes. Insulin-stimulated whole body glucose uptake was decreased by 55% in muscle GLUT4 KO mice, an effect that could be attributed to a 92% decrease in insulin-stimulated muscle glucose uptake. Surprisingly, insulin's ability to stimulate adipose tissue glucose uptake and suppress hepatic glucose production was significantly impaired in muscle GLUT4 KO mice. To address whether these latter changes were caused by glucose toxicity, we treated muscle GLUT4 KO mice with phloridzin to prevent hyperglycemia and found that insulin-stimulated whole body and skeletal muscle glucose uptake were decreased substantially, whereas insulin-stimulated glucose uptake in adipose tissue and suppression of hepatic glucose production were normal after phloridzin treatment. In conclusion, these findings demonstrate that a primary defect in muscle glucose transport can lead to secondary defects in insulin action in adipose tissue and liver due to glucose toxicity. These secondary defects contribute to insulin resistance and to the development of diabetes.

Cardiac hypertrophy with preserved contractile function after selective deletion of GLUT4 from the heart.

Glucose enters the heart via GLUT1 and GLUT4 glucose transporters. GLUT4-deficient mice develop striking cardiac hypertrophy and die prematurely. Whether their cardiac changes are caused primarily by GLUT4 deficiency in cardiomyocytes or by metabolic changes resulting from the absence of GLUT4 in skeletal muscle and adipose tissue is unclear. To determine the role of GLUT4 in the heart we used cre-loxP recombination to generate G4H(-/-) mice in which GLUT4 expression is abolished in the heart but is present in skeletal muscle and adipose tissue. Life span and serum concentrations of insulin, glucose, FFAs, lactate, and beta-hydroxybutyrate were normal. Basal cardiac glucose transport and GLUT1 expression were both increased approximately 3-fold in G4H(-/-) mice, but insulin-stimulated glucose uptake was abolished. G4H(-/-) mice develop modest cardiac hypertrophy associated with increased myocyte size and induction of atrial natriuretic and brain natriuretic peptide gene expression in the ventricles. Myocardial fibrosis did not occur. Basal and isoproterenol-stimulated isovolumic contractile performance was preserved. Thus, selective ablation of GLUT4 in the heart initiates a series of events that results in compensated cardiac hypertrophy.

Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes.

Phosphatidylinositol 3-kinase (PI3K) activation is necessary for many insulin-induced metabolic and mitogenic responses. However, it is unclear whether PI3K activation is sufficient for any of these effects. To address this question we increased PI3K activity in differentiated 3T3-L1 adipocytes by adenovirus-mediated expression of both the inter-SH2 region of the regulatory p85 subunit of PI3K (iSH2) and the catalytic p110 alpha subunit (p110). Coexpression resulted in PI3K activity that exceeded insulin-stimulated activity by two- to fivefold in cytosol, total membranes, and the low density microsome (LDM) fraction, the site of greatest insulin stimulation. While insulin increased glucose transport 15-fold, coexpression of iSH2-p110 increased transport (5.2-) +/- 0.7-fold with a parallel increase in GLUT4 translocation to the plasma membrane. Constitutive activation of PI3K had no effect on maximally insulin-stimulated glucose transport. Neither basal nor insulin-stimulated activity of glycogen synthase or mitogen-activated protein kinase was altered by iSH2-p110 coexpression. DNA synthesis was increased twofold by insulin in control 3T3-L1 adipocytes transduced with beta-galactosidase-encoding recombinant adenovirus, while iSH2-p110 coexpression increased DNA synthesis fivefold. These data indicate that (i) increased PI3K activity is sufficient to activate some but not all metabolic responses to insulin, (ii) activation of PI3K to levels exceeding the effect of insulin in adipocyte LDM results in only a partial stimulation of glucose transport, and (iii) increased PI3K activity in the absence of growth factor or oncoprotein stimulation is a potent stimulus of DNA synthesis.

Regulation of early events in integrin signaling by protein tyrosine phosphatase SHP-2.

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in growth factor and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS-1)/SIRP-1alpha, a transmembrane glycoprotein with adhesion molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol. 16:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon plating of murine fibroblasts onto specific extracellular matrices. Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl phosphorylation is catalyzed by Src family protein tyrosine kinases (PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon 3(-/-) and wild-type cell lines to being plated on fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl phosphorylation of several focal adhesion proteins, MAPK activation, and the ability to spread on fibronectin were defective in SHP-2 mutant fibroblasts but were restored upon SHP-2 expression. Our data suggest a positive-feedback model in which, upon integrin engagement, basal levels of c-Src activity catalyze the tyrosyl phosphorylation of SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps by further activating Src PTKs, SHP-2 transduces positive signals for downstream events such as MAPK activation and cell shape changes.

Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice.

Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.