-AMP-activated protein kinase (AMPK) is a meta-bolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific ␥3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK ␥3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse ␥3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK ␥3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK ␥3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK ␥3 isoform as a therapeutic target for prevention and treatment of insulin resistance.AMPK 1 is a heterotrimeric serine/threonine protein kinase composed of a catalytic ␣ subunit and non-catalytic ␤ and ␥ subunits (1, 2). The mammalian genome contains seven AMPK genes encoding two ␣, two ␤, and three ␥ isoforms. AMPK signaling is elicited by cellular stresses that deplete ATP (and consequently elevate AMP) by either inhibiting ATP production (e.g. hypoxia) or accelerating ATP consumption (e.g. muscle contraction). AMPK is activated allosterically by AMP and through phosphorylation of Thr 172 in the ␣ subunit by an upstream AMPK kinase, the tumor-suppressor protein kinase LKB1 (3, 4). AMPK is likely to be important for diverse functions in many cell types, but particular interest has been focused on elucidating the role of AMPK in the regulation of lipid and carbohydrate metabolism in skeletal muscle (5-10). AMPK activity has been correlated with an increase in glucose uptake and fatty acid oxidation and an inhibition of glycogen synthase activity and fatty acid synthesis. Exercise, as well as skeletal muscle contractions in vitro, leads to AMPK activation. Pharmacological activation of AMPK also can be achieved using 5-aminoimidazole-4-carboxamide-1-␤-D-ribonucleoside (AICAR). Once taken up by the cell, AICAR is phosphorylated to 5-aminoimidazole-4-carboxamide riboside monophosphate (ZMP) and mimics effects of AMP on AMPK (1, 2). AMPK function is closely related to glycogen storage. AMPK phosphorylates glycogen synthase in vitro (11) and co-immunoprecipitates with glycogen synthase and glycogen phosphorylase from skeletal muscle (12). Mutations of the ␥3 or ␥2 subunit, respectively, affect glycogen storage in pigs (13, 14) or glycogen storage associated with cardiac abnormalities in humans (15). The recent identification of a glycogen-binding domain in the AMPK ␤1 subunit provides a molecular relationship between AMPK and glycogen (16,17). The formation of heterotrimers appears to be...

We characterized metabolic and mitogenic signaling pathways in isolated skeletal muscle from well-matched t y p e 2 diabetic and control subjects. Time course studies of the insulin receptor, insulin receptor substrate ( I R S )-1/2, and phosphatidylinositol (PI) 3-k i n a s e revealed that signal transduction through this pathway was engaged between 4 and 40 min. Insulin-stimulated ( 0 . 6 -6 0 nmol/l) tyrosine phosphorylation of the insulin receptor -subunit, mitogen-activated protein (MAP) kinase phosphorylation, and glycogen synthase activity were not altered in type 2 diabetic subjects. In contrast, insulin-stimulated tyrosine phosphorylation of IRS-1 and anti-phosphotyrosine-associated PI 3-kinase activity were reduced 40-55% in type 2 diabetic subjects at high insulin concentrations (2.4 and 60 nmol/l, respectively). Impaired glucose transport activity was noted at all insulin concentrations (0.6-60 nmol/l). Aberrant protein expression cannot account for these insulin-signaling defects because expression of insulin receptor, IRS-1, IRS-2, MAP kinase, or glycogen synthase was similar between type 2 diabetic and control subjects. In skeletal muscle from type 2 diabetic subjects, IRS-1 phosphorylation, PI 3-kinase activity, and glucose transport activity were impaired, whereas insulin receptor tyrosine phosphorylation, MAP kinase phosphorylation, and glycogen synthase activity were normal. Impaired insulin signal transduction in skeletal muscle from type 2 diabetic patients may partly account for reduced insulin-stimulated glucose transport; however, additional defects are likely to play a role. D i a b e t e s 4 9 :2 8 4-292, 2000 S keletal muscle is a primary site of insulin-stimulated glucose disposal, which accounts for 70-80% of postprandial glucose disposal (1). In vivo studies reveal that insulin resistance in skeletal muscle is one of the first measurable defects associated with type 2 diabetes (2,3). The molecular basis for the development of whole-body insulin resistance remains unclear, although decreased insulin-stimulated glucose transport activity has been observed in isolated skeletal muscle from lean and obese people with type 2 diabetes (4-8). Because glucose transport is an early step in peripheral glucose utilization, a defect in glucose transport most likely plays a major role in the pathogenesis of peripheral insulin resistance (9). Thus, an understanding of the mechanisms that control glucose transport into insulin-sensitive tissues is essential to develop strategies for reestablishing normal glucose homeostasis in people with type 2 diabetes.Insulin-stimulated glucose transport is achieved by translocation of the major insulin-responsive glucose transp o r t e r, GLUT4, from an intracellular vesicle storage site to the plasma membrane and transverse tubules (10-12). Reduced glucose transport activity in skeletal muscle from people with type 2 diabetes may be a consequence of impaired insulin signal transduction (13,14) and/or alterations in the traffic and translocation of GLUT4 to the...

Aims/hypothesis: We used oestrogen receptor-α (ERα) knockout (ERKO) and receptor-β (ERβ) knockout (BERKO) mice to investigate the mechanism(s) behind the effects of oestrogens on glucose homeostasis. Methods: Endogenous glucose production (EGP) was measured in ERKO mice using a euglycaemic-hyperinsulinaemic clamp. Insulin secretion was determined from isolated islets. In isolated muscles, glucose uptake was assayed by using radiolabelled isotopes. Genome-wide expression profiles were analysed by high-density oligonucleotide microarray assay, and the expression of the genes encoding stearoyl-CoA desaturase 1 and the Leptin receptor (Scd1 and Lepr, respectively) was confirmed by RT-PCR. Results: ERKO mice had higher fasting blood glucose, plasma insulin levels and IGT. The plasma leptin level was increased, while the adiponectin concentration was decreased in ERKO mice. Levels of both glucose-and arginine-induced insulin secretion from isolated islets were similar in ERKO and wild-type mice. The euglycaemichyperinsulinaemic clamp revealed that suppression of EGP by increased insulin levels was blunted in ERKO mice, which suggests a pronounced hepatic insulin resistance. Microarray analysis revealed that in ERKO mice, the genes involved in hepatic lipid biosynthesis were upregulated, while genes involved in lipid transport were downregulated. Notably, hepatic Lepr expression was decreased in ERKO mice. In vitro studies showed a modest decrease in insulin-mediated glucose uptake in soleus and extensor digitorum longus (EDL) muscles of ERKO mice. BERKO mice demonstrated normal glucose tolerance and insulin release. Conclusions/interpretation: We conclude that oestrogens, acting via ERα, regulate glucose homeostasis mainly by modulating hepatic insulin sensitivity, which can be due to the upregulation of lipogenic genes via the suppression of Lepr expression.

Conjugated linoleic acid (CLA) isomers have a number of beneficial health effects, as shown in biomedical studies with animal models. Previously, we reported that a mixture of CLA isomers improved glucose tolerance in ZDF rats and activated peroxisome proliferatoractivated receptor (PPAR)-␥ response elements in vitro. Here, our aim was to elucidate the effect(s) of specific CLA isomers on whole-body glucose tolerance, insulin action in skeletal muscle, and expression of genes important in glucose and lipid metabolism. ZDF rats were fed either a control diet (CON), one of two CLA supplemented diets (1.5% CLA) containing differing isoforms of CLA (47% c9,t11; 47.9% c10,t12, 50:50; or 91% c9,t11, c9,t11 isomers), or were pair-fed CON diet to match the intake of 50:50. The 50:50 diet reduced adiposity and improved glucose tolerance compared with all other ZDF treatments. Insulin-stimulated glucose transport and glycogen synthase activity in skeletal muscle were improved with 50:50 compared with all other treatments. Neither phosphatidlyinositol 3-kinase activity nor Akt activity in muscle was affected by treatment. Uncoupling protein 2 in muscle and adipose tissue was upregulated by c9,t11 and 50:50 compared with ZDF controls. PPAR-␥ mRNA was downregulated in liver of c9,t11 and pair-fed ZDF rats. Thus, the improved glucose tolerance in 50:50 rats is attributable to, at least in part, improved insulin action in muscle, and CLA effects cannot be explained simply by reduced food intake.

Level of physical activity is linked to improved glucose homeostasis. We determined whether exercise alters the expression and͞or activity of proteins involved in insulin-signal transduction in skeletal muscle. Wistar rats swam 6 h per day for 1 or 5 days. Epitrochlearis muscles were excised 16 h after the last exercise bout, and were incubated with or without insulin (120 nM). Insulin-stimulated glucose transport increased 30% and 50% after 1 and 5 days of exercise, respectively. Glycogen content increased 2-and 4-fold after 1 and 5 days of exercise, with no change in glycogen synthase expression. Protein expression of the glucose transporter GLUT4 and the insulin receptor increased 2-fold after 1 day, with no further change after 5 days of exercise. Insulinstimulated receptor tyrosine phosphorylation increased 2-fold after 5 days of exercise. Insulin-stimulated tyrosine phosphorylation of insulin-receptor substrate (IRS) 1 and associated phosphatidylinositol (PI) 3-kinase activity increased 2.5-and 3.5-fold after 1 and 5 days of exercise, despite reduced (50%) IRS-1 protein content after 5 days of exercise. After 1 day of exercise, IRS-2 protein expression increased 2.6-fold and basal and insulin-stimulated IRS-2 associated PI 3-kinase activity increased 2.8-fold and 9-fold, respectively. In contrast to IRS-1, IRS-2 expression and associated PI 3-kinase activity normalized to sedentary levels after 5 days of exercise. Insulin-stimulated Akt phosphorylation increased 5-fold after 5 days of exercise. In conclusion, increased insulin-stimulated glucose transport after exercise is not limited to increased GLUT4 expression. Exercise leads to increased expression and function of several proteins involved in insulin-signal transduction. Furthermore, the differential response of IRS-1 and IRS-2 to exercise suggests that these molecules have specialized, rather than redundant, roles in insulin signaling in skeletal muscle.

AMP-activated protein kinase (AMPK) activation by AICAR (5-amino-imidazole carboxamide riboside) is correlated with increased glucose transport in rodent skeletal muscle via an insulin-independent pathway. We determined in vitro effects of insulin and/or AICAR exposure on glucose transport and cell-surface GLUT4 content in skeletal muscle from nondiabetic men and men with type 2 diabetes. AICAR increased glucose transport in a dose-dependent manner in healthy subjects. Insulin and AICAR increased glucose transport and cell-surface GLUT4 content to a similar extent in control subjects. In contrast, insulin-and AICAR-stimulated responses on glucose transport and cell-surface GLUT4 content were impaired in subjects with type 2 diabetes. Importantly, exposure of type 2 diabetic skeletal muscle to a combination of insulin and AICAR increased glucose transport and cell-surface GLUT4 content to levels achieved in control subjects. AICAR increased AMPK and acetyl-CoA carboxylase phosphorylation to a similar extent in skeletal muscle from subjects with type 2 diabetes and nondiabetic subjects. Our studies highlight the potential importance of AMPK-dependent pathways in the regulation of GLUT4 and glucose transport activity in insulin-resistant skeletal muscle. Activation of AMPK is an attractive strategy to enhance glucose transport through increased cell surface GLUT4 content in insulin-resistant skeletal muscle. Diabetes 52:1066 -1072, 2003 S keletal muscle is the principal site of glucose uptake under insulin-stimulated conditions, accounting for ϳ75% of glucose disposal after glucose infusion (1,2). Insulin-mediated glucose uptake and utilization is markedly impaired in patients with type 2 diabetes (2-4). Although the primary cause for type 2 diabetes is still unknown, insulin resistance is an early defect in the pathogenesis of this metabolic disorder, as it is observed already in normal glucose-tolerant relatives of patients with type 2 diabetes (5-7). In vivo studies using a nuclear magnetic resonance technique provide evidence that the major rate-limiting impairment in the regulation of insulin-stimulated glycogen synthesis in skeletal muscle from patients with type 2 diabetes is localized at the level of glucose transport (8). Moreover, in vitro studies have directly shown that insulin-stimulated glucose transport is markedly reduced in isolated skeletal muscle from patients with type 2 diabetes (4,9,10) and insulin-resistant morbidly obese (BMI ϳ50 kg/m 2 ) subjects with or without type 2 diabetes (11), as well as in women with gestational diabetes (12). Thus, reduced skeletal muscle glucose transport may be among the early factors accounting for increased risk of developing type 2 diabetes.In skeletal muscle, glucose transport can be activated by distinct and separate pathways mediated by insulin, insulin-mimicking agents, and insulin-like growth factors, as well as by muscle contraction (exercise), cellular stress, and hypoxia (13). PI 3-kinase is involved in insulin-, but not contraction-stimulated, glucose...

Cell surface GLUT4 levels in skeletal muscle from nine t y p e 2 diabetic subjects and nine healthy control subjects have been assessed by a new technique that involves the use of a biotinylated photo-affinity label. A profound impairment in GLUT4 translocation to the skeletal muscle cell surface in response to insulin was observed in type 2 diabetic patients. Levels of insulinstimulated cell surface GLUT4 above basal in type 2 diabetic patients were only ~10% of those observed in healthy subjects. The magnitude of the defect in GLUT4 translocation in type 2 diabetic patients was greater than that observed for glucose transport activity, which was ~50% of that in healthy subjects. Reduced GLUT4 translocation is therefore a major contributor to the impaired glucose transport activity in skeletal muscle from type 2 diabetic subjects. When a marked impairment in GLUT4 translocation occurs, the contribution of other transporters to transport activity becomes apparent. In response to hypoxia, marked reductions in skeletal muscle cell surface GLUT4 levels were also observed in type 2 diabetic patients. Therefore, a defect in a common late stage in signal transduction and/or a direct impairment in the GLUT4 translocation process accounts for reduced glucose transport in t y p e 2 diabetic patients. D i a b e t e s 4 9 :6 4 7-654, 2000

Type II (non-insulin-dependent) diabetes mellitus is one of the major causes of disability and death, these being due to the complications accompanying the disease, [1]. Today, the therapeutic tools commonly utilised to treat Type II diabetes mellitus include diet, exercise, anti-diabetic drugs and insulin treatment. These therapies have been successful in keeping metabolic control within an acceptable range. However, the management of Type II diabetes mellitus has failed with respect to the prevention of the disease Diabetologia (2002) AbstractAims/hypothesis. The 5'AMP-activated protein kinase is an important mediator of muscle contractioninduced glucose transport and a target for pharmacological treatment of Type II (non-insulin-dependent) diabetes mellitus. The 5'AMP-activated protein kinase can be activated by 5-aminoimidazole-4-carboxamide ribonucleoside. We hypothesised that 5-aminoimidazole-4-carboxamide ribonucleoside treatment could restore glucose homeostasis in ob/ob mice. Methods. Lean and ob/ob mice were given 5-aminoimidazole-4-carboxamide ribonucleoside (1 mg´g body wt ±1´d ay ±1 s.c) or 0.9 % NaCl (vehicle) for 1±7 days. Results. Short-term 5-aminoimidazole-4-carboxamide ribonucleoside treatment normalised glucose concentrations in ob/ob mice within 1 h, with effects persisting over 4 h. After 1 week of daily injections, 5-aminoimidazole-4-carboxamide ribonucleoside treatment corrected hyperglycaemia, improved glucose tolerance, and increased GLUT4 and hexokinase II protein expression in skeletal muscle, but had deleterious effects on plasma non-esterified fatty acids and triglycerides. Treatment with 5-aminoimidazole-4-carboxamide ribonucleoside increased liver glycogen in fasted and fed ob/ob mice and muscle glycogen in fasted, but not fed ob/ob and lean mice. Defects in insulin-stimulated phosphatidylinositol 3-kinase and glucose transport in skeletal muscle from ob/ob mice were not corrected by 5-aminoimidazole-4-carboxamide ribonucleoside treatment. While ex vivo insulin-stimulated glucose transport was reduced in isolated muscle from ob/ob mice, the 5-aminoimidazole-4-carboxamide ribonucleoside stimulated response was normal. Conclusion/interpretation. The 5-aminoimidazole-4-carboxamide ribonucleoside mediated improvements in glucose homeostasis in ob/ob mice can be explained by effects in skeletal muscle and liver. Due to the apparently deleterious effects of 5-aminoimidazole-4-carboxamide ribonucleoside on the blood lipid profile, strategies to develop tissue-specific and pathway-specific activators of 5'AMP-activated protein kinase should be considered in order to improve glucose homeostasis. [Diabetologia (2002) 45: 56±65] Keywords Glucose transport, glycogen, lipids, insulin signalling, glucose tolerance, obesity, GLUT4, hexokinase II, glycogen synthase, myocyte enhancer factor 2. Corresponding author: J. R. Zierath, Department of Physiology and Pharmacology, Integrative Physiology Karolinska Institutet, von Eulers väg 4, II, Stockholm, Sweden, e-mail: Juleen. Zierath@fyfa.ki.se...