Altered hepatic and muscle substrate utilization provoked by GLUT4 ablation.

Studies were conducted to explore altered substrate utilization and metabolism in GLUT4 null mice. Liver fatty acid synthase mRNA and fatty acid synthesis rates were dramatically increased in GLUT4 null mice compared with control mice and were supported by increased rates of the pentose phosphate pathway oxidative phase and sterol regulatory binding protein mRNA expression. Increased GLUT2 protein content, glucokinase mRNA, and glucose-6-phosphate in GLUT4 null mice may provide substrate for the enhanced fatty acid synthesis. Increased fatty acid synthesis, however, did not lead to hepatic triglyceride accumulation in GLUT4 null mice because of increased hepatic triglyceride secretion rates. GLUT4 null mice rapidly cleared orally administered olive oil, had reduced serum triglyceride concentrations in the fed and the fasted state, and increased skeletal muscle lipoprotein lipase when compared with controls. Oleate oxidation rates were increased in GLUT4 null skeletal muscle in association with mitochondrial hyperplasia/hypertrophy. This study demonstrated that GLUT4 null mice had increased hepatic glucose uptake and conversion into triglyceride for subsequent use by muscle. The ability of GLUT4 null mice to alter hepatic carbohydrate and lipid metabolism to provide proper nutrients for peripheral tissues may explain (in part) their ability to resist diabetes when fed a normal diet.

Metabolic adaptations in skeletal muscle overexpressing GLUT4: effects on muscle and physical activity.

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.

Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice.

Adipocyte complement-related protein (30 kDa) (Acrp30), a secreted protein of unknown function, is exclusively expressed in differentiated adipocytes; its mRNA is decreased in obese humans and mice. Here we describe novel pharmacological properties of the protease-generated globular head domain of Acrp30 (gAcrp30). Acute treatment of mice with gAcrp30 significantly decreased the elevated levels of plasma free fatty acids caused either by administration of a high fat test meal or by i.v. injection of Intralipid. This effect of gAcrp30 was caused, at least in part, by an acute increase in fatty acid oxidation by muscle. As a result, daily administration of a very low dose of gAcrp30 to mice consuming a high-fat/sucrose diet caused profound and sustainable weight reduction without affecting food intake. Thus, gAcrp30 is a novel pharmacological compound that controls energy homeostasis and exerts its effect primarily at the peripheral level.

Preservation of glucose metabolism in hypertrophic GLUT4-null hearts.

GLUT4-null mice lacking the insulin-sensitive glucose transporter are not diabetic but do exhibit abnormalities in glucose and lipid metabolism. The most striking morphological consequence of ablating GLUT4 is cardiac hypertrophy. GLUT4-null hearts display characteristics of hypertrophy caused by hypertension. However, GLUT4-null mice have normal blood pressure and maintain a normal cardiac contractile protein profile. Unexpectedly, although they lack the predominant glucose transporter in the heart, GLUT4-null hearts transport glucose and synthesize glycogen at normal levels, but gene expression of rate-limiting enzymes involved in fatty acid oxidation is decreased. The GLUT4-null heart represents a unique model of hypertrophy that may be used to study the consequences of altered substrate utilization in normal and pathophysiological conditions.

Amelioration of insulin resistance but not hyperinsulinemia in obese mice overexpressing GLUT4 selectively in skeletal muscle.

The effects of gold-thioglucose (GTG) treatment were examined in mice overexpressing GLUT4 selectively in skeletal muscle (MLC-GLUT4 mice) and in age-matched controls. Groups of MLC-GLUT4 and control mice were injected with GTG or saline at 5 weeks of age. At 12 weeks following the injections, GTG-treated control mice exhibited a 35% increase in body weight versus saline-treated controls. Similarly, a 30% increase in body weight was observed in GTG-treated MLC-GLUT4 mice compared with saline-treated MLC-GLUT4 mice 12 weeks after the injections. In saline-treated lean MLC-GLUT4 and control mice, intraperitoneal injection of insulin decreased blood glucose in 1 hour by 63% and 38%, respectively. Insulin also decreased blood glucose by 40% in GTG-treated obese MLC-GLUT4 mice after 1 hour. However, insulin did not reduce blood glucose levels in GTG-treated obese control mice. The ability of insulin to clear blood glucose in GTG-treated obese MLC-GLUT4 mice is associated with increased skeletal muscle GLUT4 content and white adipose tissue (WAT) GLUT4 content as compared with GTG-treated obese controls. However, fasting blood glucose levels in GTG-treated obese MLC-GLUT4 and control mice were elevated by approximately 30% compared with saline-treated groups. Lastly, although GTG-treated obese MLC-GLUT4 mice exhibited improved glucose clearance in response to insulin, they nevertheless remained as hyperinsulinemic as GTG-treated obese control mice. These results suggest that genetic overexpression of GLUT4 in skeletal muscle may ameliorate the development of insulin resistance associated with obesity but cannot restore normal glucose and insulin levels.

In vitro analysis of the glucose-transport system in GLUT4-null skeletal muscle.

We have characterized the glucose-transport system in soleus muscle from female GLUT4-null mice to determine whether GLUT1, 3 or 5 account for insulin-stimulated glucose-transport activity. Insulin increased 2-deoxyglucose uptake 2.8- and 2.1-fold in soleus muscle from wild-type and GLUT4-null mice, respectively. Cytochalasin B, an inhibitor of GLUT1- and GLUT4-mediated glucose transport, inhibited insulin-stimulated 2-deoxyglucose uptake by >95% in wild-type and GLUT4-null soleus muscle. Addition of 35 mM fructose to the incubation media was without effect on insulin-stimulated 3-O-methylglucose transport activity in soleus muscle from either genotype, whereas 35 mM glucose inhibited insulin-stimulated (20 nM) 3-O-methylglucose transport by 65% in wild-type and 99% in GLUT4-null mice. We utilized the 2-N-4-1-(1-azi-2,2,2-triflu oroethyl)benzoyl-1, 3-bis(D-mannose-4-yloxy)-2-propylamine (ATB-BMPA) exofacial photolabel to determine if increased cell-surface GLUT1 or GLUT4 content accounted for insulin-stimulated glucose transport in GLUT4-null muscle. In wild-type soleus muscle, cell-surface GLUT4 content was increased by 2.8-fold under insulin-stimulated conditions and this increase corresponded to the increase in 2-deoxyglucose uptake. No detectable cell-surface GLUT4 was observed in soleus muscle from female GLUT4-null mice under either basal or insulin-stimulated conditions. Basal cell-surface GLUT1 content was similar between wild-type and GLUT4-null mice, with no further increase noted in either genotype with insulin exposure. Neither GLUT3 nor GLUT5 appeared to account for insulin-stimulated glucose-transport activity in wild-type or GLUT4-null muscle. In conclusion, insulin-stimulated glucose-transport activity in female GLUT4-null soleus muscle is mediated by a facilitative transport process that is glucose- and cytochalasin B-inhibitable, but which is not labelled strongly by ATB-BMPA.

Prevention of insulin resistance and diabetes in mice heterozygous for GLUT4 ablation by transgenic complementation of GLUT4 in skeletal muscle.

Impaired skeletal muscle glucose utilization under insulin action is a major defect in the etiology of type 2 diabetes. This is underscored by a new mouse model of type 2 diabetes generated by genetic disruption of one allele of glucose transporter 4 (GLUT4+/-), the insulin-responsive glucose transporter in muscle and adipose tissue. Male GLUT4+/- mice exhibited decreased GLUT4 expression and glucose uptake in muscle that accompanied impaired whole-body glucose utilization, hyperinsulinemia, hyperglycemia, and heart histopathology. To determine whether development of the diabetic phenotype in GLUT4+/- mice can be forestalled by preventing the onset of impaired muscle GLUT4 expression and glucose utilization, standard genetic crossing was performed to introduce a fast-twitch muscle-specific GLUT4 transgene--the myosin light chain (MLC) promoter-driven transgene MLC-GLUT4--into GLUT4+/- mice (MLC-GLUT4+/- mice). GLUT4 expression and 2-deoxyglucose uptake levels were normalized in fast-twitch muscles of MLC-GLUT4+/- mice. In contrast to GLUT4+/- mice, MLC-GLUT4+/- mice exhibited normal whole-body glucose utilization. In addition, development of hyperinsulinemia and hyperglycemia observed in GLUT4+/- mice was prevented in MLC-GLUT4+/- mice. The occurrence of diabetic heart histopathology in MLC-GLUT4+/- mice was reduced to control levels. Based on these results, we propose that the onset of a diabetic phenotype in GLUT4+/- mice can be avoided by preventing decreases in muscle GLUT4 expression and glucose uptake.

Restoration of hypoxia-stimulated glucose uptake in GLUT4-deficient muscles by muscle-specific GLUT4 transgenic complementation.

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.

GLUT4 heterozygous knockout mice develop muscle insulin resistance and diabetes.

GLUT4, the insulin-responsive glucose transporter, plays an important role in postprandial glucose disposal. Altered GLUT4 activity is suggested to be one of the factors responsible for decreased glucose uptake in muscle and adipose tissue in obesity and diabetes. To assess the effect of GLUT4 expression on whole-body glucose homeostasis, we disrupted the murine GLUT4 gene by homologous recombination. Male mice heterozygous for the mutation (GLUT4 +/-) exhibited a decrease in GLUT4 expression in adipose tissue and skeletal muscle. This decrease in GLUT4 expression did not result in obesity but led to increased serum glucose and insulin, reduced muscle glucose uptake, hypertension, and diabetic histopathologies in the heart and liver similar to those of humans with non-insulin-dependent diabetes mellitus (NIDDM). The male GLUT4 +/- mice represent a good model for studying the development of NIDDM without the complications associated with obesity.

Muscle-specific transgenic complementation of GLUT4-deficient mice. Effects on glucose but not lipid metabolism.

We have taken the approach of introducing the muscle-specific myosin light chain (MLC)-GLUT4 transgene into the GLUT4-null background to assess the relative role of muscle and adipose tissue GLUT4 in the etiology of the GLUT4-null phenotype. The resulting MLC-GLUT4-null mice express GLUT4 predominantly in the fast-twitch extensor digitorum longus (EDL) muscle. GLUT4 is nearly absent in female white adipose tissue (WAT) and slow-twitch soleus muscle of both sexes of MLC-GLUT4-null mice. GLUT4 content in male MLC-GLUT4-null WAT is 20% of that in control mice. In transgenically complemented EDL muscle, 2-deoxyglucose (2-DOG) uptake was restored to normal (male) or above normal (female) levels. In contrast, 2-DOG uptake in slow-twitch soleus muscle of MLC-GLUT4-null mice was not normalized. With the normalization of glucose uptake in fast-twitch skeletal muscle, whole body insulin action was restored in MLC-GLUT4-null mice, as shown by the results of the insulin tolerance test. These results demonstrate that skeletal muscle GLUT4 is a major regulator of skeletal muscle and whole body glucose metabolism. Despite normal skeletal muscle glucose uptake and insulin action, the MLC-GLUT4-null mice exhibited decreased adipose tissue deposits, adipocyte size, and fed plasma FFA levels that are characteristic of GLUT4-null mice. Together these results indicate that the defects in skeletal muscle and whole body glucose metabolism and adipose tissue in GLUT4-null mice arise independently.

The metabolic consequences of altered glucose transporter expression in transgenic mice.

Glucose transporters are a family of membrane proteins which mediate glucose uptake across the cell membrane. The facilitative glucose transporter proteins are products of unique genes and are expressed in a tissue-specific manner. They are very similar structurally, containing 12 putative membrane spanning domains. Functionally they vary in their affinity for glucose and sensitivity to hormones such as insulin. Glucose homeostasis depends mainly on controlled changes in glucose transport in insulin-responsive tissues such as skeletal muscle and adipose cells where both glucose transporter 1 and glucose transporter 4 are expressed. Glucose transporter 4 is the major glucose transporter in these tissues and translocates from an intracellular vesicle to the cell membrane in response to insulin. Alterations of the level of expression of these glucose transporters should result in changes in insulin sensitivity and modification of whole-body metabolism. To test these hypotheses transgenic mouse models have been generated which overexpress glucose transporters in specific tissues or in the whole body. Glucose transporter 1 and glucose transporter 4 have been overexpressed specifically in skeletal muscle and glucose transporter 4 specifically in adipose tissue. Mice have also been made which overexpress glucose transporter 4 in the whole body. Using homologous recombination technology to disrupt the glucose transporter 4 gene, a "knockout" mouse has been created which expresses no glucose transporter 4. The metabolic consequences of these genetic manipulations on the level of expression of glucose transporters in the mouse are reviewed. The future applications of transgenic mouse technology in creating models which mimic human diseases are also discussed.

Diverse effects of Glut 4 ablation on glucose uptake and glycogen synthesis in red and white skeletal muscle.

The ability of muscles from Glut 4-null mice to take up and metabolize glucose has been studied in the isolated white EDL and red soleus muscles. In EDL muscles from male or female Glut 4-null mice, basal deoxyglucose uptake was lower than in control muscles and was not stimulated by insulin. In parallel, glycogen synthesis and content were decreased. Soleus muscles from male Glut 4-null mice took up twice more deoxyglucose in the absence of insulin than control muscles, but did not respond to insulin. In females, soleus deoxyglucose uptake measured in the absence of hormone was similar in Glut 4-null mice and in control mice. This uptake was stimulated twofold in Glut 4-null mice and threefold in control mice. Basal glycogen synthesis was increased by 4- and 2.2-fold in male and female null mice, respectively, compared to controls, and insulin had no or small (20% stimulation over basal) effect. These results indicate that while EDL muscles behaved as expected, soleus muscles were able to take up a large amount of glucose in the absence (males) or the presence of insulin (females). Whether this is due to a change in Glut 1 intrinsic activity or targeting and/or to the appearance of another glucose transporter remains to be determined.

Regulation of hexokinase II gene expression by glucose flux in skeletal muscle.

UNLABELLED: The in vivo studies of transcriptional regulation by glucose, in general, have yielded ambiguous interpretations due to the closed loop relationship between insulin and glucose. Insulin cannot be held as a constant since elevated glucose levels will elicit a corresponding rise in insulin and current animal models of insulinopenia are associated with a plethora of counter-regulatory hormone responses. One potential solution to increase intracellular glucose flux without a further increase in insulin was achieved by transgenic overexpression of the insulin-sensitive glucose transporter, GLUT4, in specific skeletal muscles (previously described in Tsao, T.-S., Burcelin, R., Katz, E. B., Huang, L., and Charron, M. J. (1996) Diabetes 45, 28-36). Using these MLC-GLUT4 transgenic mice as a model, we investigated the effects of increased glucose flux on hexokinase II (HK II) gene expression in skeletal muscle. Under conditions where blood glucose levels were normal and insulin levels decreased by 36%, HK II mRNA level was reduced in non-GLUT4-overexpressing tissues (i.e. heart and adipose tissue) of 2-4-month-old male MLC-GLUT4 transgenic mice. This reduction in HK II mRNA was prevented in skeletal muscle, where overexpression of GLUT4 caused a 2.5-fold increase in basal and insulin-stimulated glucose uptake. The levels of HK II mRNA in heart, muscle, and adipose tissue are paralleled by HK II enzymatic activity. IN CONCLUSION: 1) due to relative mild insulinopenia, HK II expression is decreased in non-GLUT4-overexpressing tissues of MLC-GLUT4 mice compared to age/sex-matched controls, and 2) GLUT4-mediated increase in cellular glucose flux can prevent the decrease in HK II expression (in GLUT4-overexpressing tissues) as a result of relative mild insulinopenia. Indeed, during the process of aging, the return of circulating insulin levels of MLC-GLUT4 mice to normal levels is associated with the normalization of HK II expression in all tissues of MLC-GLUT4 and age/sex-matched control mice. We propose that: 1) glucose flux has an amplifying effect on the ability of insulin to stimulate skeletal muscle HK II gene expression and 2) insulin-dependent glucose flux may be a potential mechanism by which HK II gene expression is regulated by sensitivity to insulin.

Enhanced insulin action due to targeted GLUT4 overexpression exclusively in muscle.

Dysregulation of GLUT4, the insulin-responsive glucose transporter, is associated with insulin resistance in skeletal muscle. Although skeletal muscle is the major target of insulin action, muscle GLUT4 has not been linked causally to whole-body insulin sensitivity and regulation of glucose homeostasis. To address this, we generated a line of transgenic mice that overexpresses GLUT4 in skeletal muscle. We demonstrate that restricted overexpression of GLUT4 in fast-twitch skeletal muscles of myosin light chain (MLC)-GLUT4 transgenic mice induces a 2.5-fold increase in insulin-stimulated 2-deoxyglucose uptake in transgene-overexpressing cells. Consequently, glycogen content is increased in the fast-twitch skeletal muscles under insulin action (5.75 +/- 1.02 vs. 3.24 +/- 0.26 mg/g). This indicates that insulin-stimulated glucose transport is partly rate-limiting for glycogen synthesis. At the whole-body level, insulin-stimulated glucose turnover is increased 2.5-fold in unconscious MLC-GLUT4 mice. Plasma glucose and insulin levels in MLC-GLUT4 mice are altered as a result of increased insulin action. In 2- to 3-month-old MLC-GLUT4 mice, fasting insulin levels are decreased (0.43 +/- 0.05 vs. 0.74 +/- 0.10 microgram/l), whereas normal fasting glycemia is maintained. Conversely, 7- to 9-month-old MLC-GLUT4 mice exhibit decreased fasting glycemia (5.75 +/- 0.73 vs. 8.11 +/- 0.57 mmol/l) with normal insulin levels. Fasting plasma lactate levels are elevated in both age groups (50-100%). Additionally lipid metabolism is affected by skeletal muscle GLUT4 overexpression. This is indicated by changes in plasma free fatty acid and beta-hydroxybutyrate levels. These studies underscore the importance of GLUT4 in the regulation of glucose homeostasis and its interaction with lipid metabolism.

Amino acids enhance renal tubular absorption of the los-molecular-weight proteins insulin and growth hormone.

The effect of amino acids(AA) on the tubular absorption of low-molecular-weight (LMW) proteins was studied in isolated rat kidneys. Kidneys were perfused with an albumin-electrolyte solution that contained insulin or human growth hormone (hGH) and, unless otherwise stated, the following L-amino acids: glycine, isoleucine, serine, alanine, methionine, proline, arginine, and aspartic acid. In kidneys perfused without AA, fractional urinary insulin clearance (FCi) averaged 7.4 +/- 1.54%, whereas in the presence of multiple AA the FCi was significantly lower (0.68 +/- 0.2%, P less than 0.01). Addition of glycine or alpha-aminoisobutyric acid (AIB) alone also reduced the FCi significantly (1.79 +/- 0.66 and 1.59 +/-1.06%, respectively). By contrast, perfusion with the other AA individually did not alter the FCi. The fractional urinary hGH clearance was also significantly lower in kidneys perfused with multiple AA (0.94 +/- 0.47%) than in those perfused without AA (9.07 +/- 1.2%). We conclude that tubular absorption of filtered insulin and hGH is enhanced by the presence of AA. The mechanism is unclear, but enhancement of insulin absorption can be produced by glycine and AIB alone. This raises the possibility of a link between the absorption of insulin and the glycine and AIB shared transport system, but excludes a primary metabolic effect because AIB is nonmetabolizable.