Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats

Activation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofurano-side (AICAR) increases glucose transport in skeletal muscle via an insulin-independent pathway. To examine the effects of AMPK activation on skeletal muscle glucose transport activity and whole-body carbohydrate and lipid metabolism in an insulin-resistant rat model, awake obese Zuckerfa/fa rats (n = 26) and their lean (n = 23) littermates were infused for 90 min with AICAR, insulin, or saline. The insulin infusion rate (4 mU.kg(-1).min(-1)) was selected to match the glucose requirements during AICAR (bolus, 100 mg/kg; constant, 10 mg.kg(-1).min(-1)) isoglycemic clamps in the lean rats. The effects of these identical AICAR and insulin infusion rates were then examined in the obese Zucker rats. AICAR infusion increased muscle AMPK activity more than fivefold (P < 0.01 vs. control and insulin) in both lean and obese rats. Plasma triglycerides, fatty acid concentrations, and glycerol turnover, as assessed by [2-13C]glycerol, were all decreased in both lean and obese rats infused with AICAR (P < 0.05 vs. basal), whereas insulin had no effect on these parameters in the obese rats. Endogenous glucose production rates, measured by [U-13C]glucose, were suppressed by >50% during AICAR and insulin infusions in both lean and obese rats (P < 0.05 vs. basal). In lean rats, rates of whole-body glucose disposal increased by more than two-fold (P < 0.05 vs. basal) during both AICAR and insulin infusion; [3H]2-deoxy-D-glucose transport activity increased to a similar extent, by >2.2-fold (both P < 0.05 vs. control), in both soleus and red gastrocnemius muscles of lean rats infused with either AICAR or insulin. In the obese Zucker rats, neither AICAR nor insulin stimulated whole-body glucose disposal or soleus muscle glucose transport activity. However, AICAR increased glucose transport activity by approximately 2.4-fold (P < 0.05 vs. control) in the red gastrocnemius from obese rats, whereas insulin had no effect. In summary, acute infusion of AICAR in an insulin-resistant rat model activates skeletal muscle AMPK and increases glucose transport activity in red gastrocnemius muscle while suppressing endogenous glucose production and lipolysis. Because type 2 diabetes is characterized by diminished rates of insulin-stimulated glucose uptake as well as increased basal rates of endogenous glucose production and lipolysis, these results suggest that AICAR-related compounds may represent a new class of antidiabetic agents.     

Activation of glucose transport by AMP-activated protein kinase via stimulation of nitric oxide synthase

Glucose transport in skeletal muscle is stimulated by two distinct stimuli, insulin and exercise. The mechanism by which exercise stimulates glucose transport is not known, although it is distinct from the insulin-mediated pathway. Recently, it has been shown that AMP-activated protein kinase (AMPK) is activated by exercise in skeletal muscle, whereas pharmacological activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAR) leads to increased glucose transport. It has been postulated, therefore, that AMPK may be the link between exercise and glucose transport. To address this, we have examined the signaling pathway involved in the stimulation of glucose uptake after activation of AMPK. Here we show that activation of AMPK by AICAR in rat muscle and mouse H-2Kb muscle cells activates glucose transport approximately twofold. AMPK in H-2Kb cells is also activated by hyperosmotic stress and the mitochondrial uncoupling agent, dinitrophenol, both of which lead to increased glucose transport. In contrast, insulin, which activates glucose transport two- to-threefold in both rat muscle and H-2Kb cells, has no effect on AMPK activity. A previous study has shown that AMPK phosphorylates and activates endothelial nitric oxide synthase (NOS). We show here that NOS activity in H-2Kb cells is activated after stimulation of AMPK by AICAR. Treatment of H-2Kb cells or rat muscle with NOS inhibitors completely blocks the increase in glucose transport after activation of AMPK. In addition, an inhibitor of guanylate cyclase also blocks activation of glucose transport by AICAR in H-2Kb cells. These results indicate that activation of AMPK in muscle cells stimulates glucose transport by activation of NOS coupled to downstream signaling components, including cyclic GMP.     

AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 beta-d-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (alpha1beta1gamma1 and alpha2beta2gamma1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (alpha2 AMPK knockout [KO], alpha2 AMPK kinase dead [KD], and gamma3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing alpha2 and gamma3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in alpha2 AMPK KO and KD but not gamma3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.     

Rosiglitazone treatment enhances acute AMP-activated protein kinase-mediated muscle and adipose tissue glucose uptake in high-fat-fed rats

AMP-activated protein kinase (AMPK) has been implicated in the insulin-sensitizing actions of thiazolidinediones (TZDs), but it is not known whether TZD treatment can enhance tissue glucose uptake in response to AMPK activation. The present study investigated the influence of the TZD rosiglitazone on glucose turnover induced by intravenous infusion of the AMPK activator 5-aminoimidazole 4-carboxamide riboside (AICAR) under euglycemic and iso-insulinemic conditions in insulin-resistant high-fat-fed rats. We found that rosiglitazone treatment significantly enhanced AICAR-stimulated whole-body glucose disposal by 27% in high-fat-fed rats, and a 44% greater glucose infusion rate (both P < 0.01 vs. vehicle control rats) was required to maintain euglycemia. Along with this, both AICAR-stimulated glucose uptake and glucose incorporation into glycogen in muscle and adipose tissue were enhanced (P < 0.05). The enhanced glucose uptake and glycogen synthesis in muscle were associated with increased activity of total AMPK and the AMPKalpha2 subunit. In comparison, these effects were not apparent in rats fed standard rodent diet. Thus, our findings suggest that in addition to ameliorating insulin resistance, TZDs may enhance AMPK-stimulated glucose clearance into peripheral tissues in insulin-resistant states.     

AMP-activated protein kinase activation by AICAR increases both muscle fatty acid and glucose uptake in white muscle of insulin-resistant rats in vivo

Insulin-stimulated glucose uptake is increased in white but not red muscle of insulin-resistant high-fat-fed (HF) rats after administration of the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). To investigate whether a lesser AICAR effect on glucose uptake in red muscle was offset by a greater effect on fatty acid (FA) uptake, we examined acute effects of AICAR on muscle glucose and FA fluxes in HF rats. HF rats received AICAR (250 mg/kg) subcutaneously. At 30 min, a mixture of either (3)H-(R)-2-bromopalmitate/(14)C-palmitate or (3)H-2-deoxyglucose/(14)C-glucose was administered intravenously to assess muscle FA and glucose uptake. AICAR decreased plasma levels of glucose (approximately 25%), insulin (approximately 60%), and FAs (approximately 30%) at various times over the next 46 min (P < 0.05 vs. controls). In white muscle, AICAR increased both FA (2.4-fold) and glucose uptake (4.9-fold), associated with increased glycogen synthesis (6-fold). These effects were not observed in red muscle. We conclude that both glucose and FA fluxes are enhanced by AICAR more in white versus red muscle, consistent with the relative degree of activation of AMPK. Therefore, a lesser effect of AICAR to alleviate muscle insulin resistance in red versus white muscle is not explained by a relatively greater effect on FA uptake in the red muscle.     

Ca2+/calmodulin-dependent protein kinase kinase-alpha regulates skeletal muscle glucose uptake independent of AMP-activated protein kinase and Akt activation

Studies in nonmuscle cells have demonstrated that Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) are upstream regulators of AMP-activated protein kinase (AMPK) and Akt. In skeletal muscle, activation of AMPK and Akt has been implicated in the regulation of glucose uptake. The objective of this study was to determine whether CaMKKalpha regulates skeletal muscle glucose uptake, and whether it is dependent on AMPK and/or Akt activation. Expression vectors containing constitutively active CaMKKalpha (caCaMKKalpha) or empty vector were transfected into mouse muscles by in vivo electroporation. After 2 weeks, caCaMKKalpha was robustly expressed and increased CaMKI (Thr(177/180)) phosphorylation, a known CaMKK substrate. In muscles from wild-type mice, caCaMKKalpha increased in vivo [(3)H]-2-deoxyglucose uptake 2.5-fold and AMPKalpha1 and -alpha2 activities 2.5-fold. However, in muscles from AMPKalpha2 inactive mice (AMPKalpha2i), caCaMKKalpha did not increase AMPKalpha1 or -alpha2 activities, but it did increase glucose uptake 2.5-fold, demonstrating that caCaMKKalpha stimulates glucose uptake independent of AMPK. Akt (Thr(308)) phosphorylation was not altered by CaMKKalpha, and caCaMKKalpha plus insulin stimulation did not increase the insulin-induced phosphorylation of Akt (Thr(308)). These results suggest that caCaMKKalpha stimulates glucose uptake via insulin-independent signaling mechanisms. To assess the role of CaMKK in contraction-stimulated glucose uptake, isolated muscles were treated with or without the CaMKK inhibitor STO-609 and then electrically stimulated to contract. Contraction increased glucose uptake 3.5-fold in muscles from both wild-type and AMPKalpha2i mice, but STO-609 significantly decreased glucose uptake (approximately 24%) only in AMPKalpha2i mice. Collectively, these results implicate CaMKKalpha in the regulation of skeletal muscle glucose uptake independent of AMPK and Akt activation.     

AICAR administration causes an apparent enhancement of muscle and liver insulin action in insulin-resistant high-fat-fed rats

Exercise improves insulin sensitivity. As AMP-activated protein kinase (AMPK) plays an important role in muscle metabolism during exercise, we investigated the effects of the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) on insulin action in insulin-resistant high-fat-fed (HF) rats. Rats received a subcutaneous injection of 250 mg/kg AICAR (HF-AIC) or saline (HF-Con). The next day, euglycemic-hyperinsulinemic clamp studies were performed. Glucose infusion rate during the clamp was enhanced (50%) in HF-AIC compared with HF-Con rats. Insulin-stimulated glucose uptake was improved in white but not in red quadriceps, whereas glycogen synthesis was improved in both red and white quadriceps of HF-AIC rats. HF-AIC rats also showed increased insulin suppressibility of hepatic glucose output (HGO). AICAR-induced responses in both liver and muscle were accompanied by reduced malonyl-CoA content. Clamp HGO correlated closely with hepatic triglyceride content (r = 0.67, P < 0.01). Thus, a single dose of AICAR leads to an apparent enhancement in whole-body, muscle, and liver insulin action in HF rats that extends beyond the expected time of AMPK activation. Whether altered tissue lipid metabolism mediates AICAR effects on insulin action remains to be determined. Follow-up studies suggest that at least some of the post-AICAR insulin-enhancing effects also occur in normal rats. Independent of this, the results suggest that pharmacological activation of AMPK may have potential in treating insulin-resistant states and type 2 diabetes.     

Caffeine-induced impairment of insulin action but not insulin signaling in human skeletal muscle is reduced by exercise

We investigated the effects of caffeine ingestion on skeletal muscle glucose uptake, glycogen synthase (GS) activity, and insulin signaling intermediates during a 100-min euglycemic-hyperinsulinemic (100 microU/ml) clamp. On two occasions, seven men performed 1-h one-legged knee extensor exercise at 3 h before the clamp. Caffeine (5 mg/kg) or placebo was administered in a randomized, double-blind fashion 1 h before the clamp. During the clamp, whole-body glucose disposal was reduced (P < 0.05) in caffeine (37.5 +/- 3.1 micromol x min(-1) x kg(-1)) vs. placebo (54.1 +/- 2.9 micromol x min(-1) x kg(-1)). In accordance, the total area under the curve over 100 min (AUC(0--100 min)) for insulin-stimulated glucose uptake in caffeine was reduced (P < 0.05) by approximately 50% in rested and exercised muscle. Caffeine also reduced (P < 0.05) GS activity before and during insulin infusion in both legs. Exercise increased insulin sensitivity of leg glucose uptake in both caffeine and placebo. Insulin increased insulin receptor tyrosine kinase (IRTK), insulin receptor substrate 1-associated phosphatidylinositol (PI) 3-kinase activities, and Ser(473) phosphorylation of protein kinase B (PKB)/Akt significantly but similarly in rested and exercised legs. Furthermore, insulin significantly decreased glycogen synthase kinase-3alpha (GSK-3alpha) activity equally in both legs. Caffeine did not alter insulin signaling in either leg. Plasma epinephrine and muscle cAMP concentrations were increased in caffeine. We conclude that 1) caffeine impairs insulin-stimulated glucose uptake and GS activity in rested and exercised human skeletal muscle; 2) caffeine-induced impairment of insulin-stimulated muscle glucose uptake and downregulation of GS activity are not accompanied by alterations in IRTK, PI 3-kinase, PKB/Akt, or GSK-3alpha but may be associated with increases in epinephrine and intramuscular cAMP concentrations; and 3) exercise reduces the detrimental effects of caffeine on insulin action in muscle.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and metformin inhibit hepatic glucose phosphorylation by an AMP-activated protein kinase-independent effect on glucokinase translocation

AMP-activated protein kinase (AMPK) controls glucose uptake and glycolysis in muscle. Little is known about its role in liver glucose uptake, which is controlled by glucokinase. We report here that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), metformin, and oligomycin activated AMPK and inhibited glucose phosphorylation and glycolysis in rat hepatocytes. In vitro experiments demonstrated that this inhibition was not due to direct phosphorylation of glucokinase or its regulatory protein by AMPK. By contrast, AMPK phosphorylated liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase without affecting activity. Inhibitors of the endothelial nitric oxide synthase, stress kinases, and phosphatidylinositol 3-kinase pathways did not counteract the effects of AICAR, metformin, or oligomycin, suggesting that these signaling pathways were not involved. Interestingly, the inhibitory effect on glucose phosphorylation of these well-known AMPK activators persisted in primary cultured hepatocytes from newly engineered mice lacking both liver alpha1 and alpha2 AMPK catalytic subunits, demonstrating that this effect was clearly not mediated by AMPK. Finally, AICAR, metformin, and oligomycin were found to inhibit the glucose-induced translocation of glucokinase from the nucleus to the cytosol by a mechanism that could be related to the decrease in intracellular ATP concentrations observed in these conditions.     

Exercise increases nuclear AMPK alpha2 in human skeletal muscle

An acute bout of exercise increases skeletal muscle glucose uptake, improves glucose homeostasis and insulin sensitivity, and enhances muscle oxidative capacity. Recent studies have shown an association between these adaptations and the energy-sensing 5' AMP-activated protein kinase (AMPK), the activity of which is increased in response to exercise. Activation of AMPK has been associated with enhanced expression of key metabolic proteins such as GLUT-4, hexokinase II (HKII), and mitochondrial enzymes, similar to exercise. It has been hypothesized that AMPK might regulate gene and protein expression through direct interaction with the nucleus. The purpose of this study was to determine if nuclear AMPK alpha(2) content in human skeletal muscle was increased by exercise. Following 60 min of cycling at 72 +/- 1% of VO(2peak) in six male volunteers (20.6 +/- 2.1 years; 72.9 +/- 2.1 kg; VO(2peak) = 3.62 +/- 0.18 l/min), nuclear AMPK alpha(2) content was increased 1.9 +/- 0.4-fold (P = 0.024). There was no change in whole-cell AMPK alpha(2) content or AMPK alpha(2) mRNA abundance. These results suggest that nuclear translocation of AMPK might mediate the effects of exercise on skeletal muscle gene and protein expression.     

The alpha2-5'AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading

The 5'AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-beta-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). In muscle of mice with targeted deletion of the alpha2-AMPK gene, phosphorylation of GS site 2 was decreased under basal conditions and unchanged by AICAR treatment. In contrast, in alpha1-AMPK knockout mice, the response to AICAR was normal. Fuel surplus (glucose loading) decreased AMPK activation by AICAR, but the phosphorylation of the downstream targets acetyl-CoA carboxylase-beta and GS was normal. Fractionation studies suggest that this suppression of AMPK activation was not a direct consequence of AMPK association with membranes or glycogen, because AMPK was phosphorylated to a greater extent in response to AICAR in the membrane/glycogen fraction than in the cytosolic fraction. Thus, the downstream action of AMPK in response to AICAR was unaffected by glucose loading, whereas the action of the kinase upstream of AMPK, as judged by AMPK phosphorylation, was decreased. The fact that alpha2-AMPK is a GS kinase that inactivates GS while simultaneously activating glucose transport suggests that a balanced view on the suitability for AMPK as an antidiabetic drug target should be taken.     

AMP kinase-induced skeletal muscle glucose but not long-chain fatty acid uptake is dependent on nitric oxide

The purpose of this study was to examine the effects of AMP kinase (AMPK) activation on in vivo glucose and long-chain fatty acid (LCFA) uptake in skeletal muscle and to examine the nitric oxide (NO) dependence of any putative effects. Catheters were chronically implanted in the carotid artery and jugular vein of male Sprague-Dawley rats. After 4 days of recovery, rats were given either water or water containing 1 mg/ml nitro-l-arginine methylester (l-NAME) for 2.5 days. After an overnight fast, rats underwent one of five protocols: saline, 5-aminoimidazole-4-carboxamide-1-B-d-ribofuranoside (AICAR) (10 mg. kg(-1). min(-1)), l-NAME, AICAR + l-NAME, or AICAR + Intralipid (20%, 0.02 ml. kg(-1). min(-1)). Glucose was clamped at approximately 6.5 mmol/l in all groups, and an intravenous bolus of 2-deoxy[(3)H]glucose and [(125)I]-15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid was administered to obtain indexes of glucose (K(g)) and LCFA (K(f)) uptake and clearance. At 150 min, soleus, gastrocnemius, and superficial vastus lateralis were excised for tracer determination. Both K(g) and K(f) increased with AICAR in all muscles studied. K(g) decreased with increasing muscle composition of type 1 slow-twitch fibers, whereas K(f) increased. In addition, AICAR-induced increases in K(g) but not K(f) were abolished by l-NAME in the majority of muscles examined. This shows that the mechanisms by which AMPK stimulates glucose and LCFA uptake are distinct.     

5-amino-imidazole carboxamide riboside increases glucose transport and cell-surface GLUT4 content in skeletal muscle from subjects with type 2 diabetes

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.     

Effect of exercise intensity on skeletal muscle AMPK signaling in humans

The effect of exercise intensity on skeletal muscle AMP-activated protein kinase (AMPK) signaling and substrate metabolism was examined in eight men cycling for 20 min at each of three sequential intensities: low (40 +/- 2% VO(2) peak), medium (59 +/- 1% VO(2) peak), and high (79 +/- 1% VO(2) peak). Muscle free AMP/ATP ratio only increased at the two higher exercise intensities (P < 0.05). AMPK alpha 1 (1.5-fold) and AMPK alpha 2 (5-fold) activities increased from low to medium intensity, with AMPK alpha 2 activity increasing further from medium to high intensity. The upstream AMPK kinase activity was substantial at rest and only increased 50% with exercise, indicating that, initially, signaling through AMPK did not require AMPK kinase posttranslational modification. Acetyl-CoA carboxylase (ACC)-beta phosphorylation was sensitive to exercise, increasing threefold from rest to low intensity, whereas neuronal NO synthase (nNOS) micro phosphorylation was only observed at the higher exercise intensities. Glucose disappearance (tracer) did not increase from rest to low intensity, but increased sequentially from low to medium to high intensity. Calculated fat oxidation increased from rest to low intensity in parallel with ACC beta phosphorylation, then declined during high intensity. These results indicate that ACC beta phosphorylation is especially sensitive to exercise and tightly coupled to AMPK signaling and that AMPK activation does not depend on AMPK kinase activation during exercise.     

Effect of AICAR treatment on glycogen metabolism in skeletal muscle

AMP-activated protein kinase (AMPK) is proposed to stimulate fat and carbohydrate catabolism to maintain cellular energy status. Recent studies demonstrate that pharmacologic activation of AMPK and mutations in the enzyme are associated with elevated muscle glycogen content in vivo. Our purpose was to determine the mechanism for increased muscle glycogen associated with AMPK activity in vivo. AMPK activity and glycogen metabolism were studied in red and white gastrocnemius muscles from rats treated with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) in vivo, and also in muscles incubated with AICAR in vitro. In vivo AICAR treatment reduced blood glucose and increased blood lactate compared with basal values. AICAR increased muscle alpha2 AMPK activity, glycogen, and glucose-6-phosphate concentrations. Glycogen synthase activity was increased in the red gastrocnemius but was decreased in the white gastrocnemius. Glycogen phosphorylase activity increased in both muscles, with an inhibition initially observed in the red gastrocnemius. In vitro incubation with AICAR activated alpha2 AMPK but had no effect on either glycogen synthase or glycogen phosphorylase. These results suggest that AICAR treatment does not promote glycogen accumulation in skeletal muscle in vivo by altering glycogen synthase and glycogen phosphorylase. Rather, the increased glycogen is due to the well-known effects of AICAR to increase glucose uptake.     

The Mammalian target of rapamycin pathway regulates nutrient-sensitive glucose uptake in man

The nutrient-sensitive kinase mammalian target of rapamycin (mTOR) and its downstream target S6 kinase (S6K) are involved in amino acid-induced insulin resistance. Whether the mTOR/S6K pathway directly modulates glucose metabolism in humans is unknown. We studied 11 healthy men (29 years old, BMI 23 kg/m(2)) twice in random order after oral administration of 6 mg rapamycin, a specific mTOR inhibitor, or placebo. An amino acid mixture was infused to activate mTOR, and somatostatin-insulin-glucose clamps created conditions of low peripheral hyperinsulinemia (approximately 100 pmol/l, 0-180 min) and prandial-like peripheral hyperinsulinemia (approximately 450 pmol/l, 180-360 min). Glucose turnover was assessed using d-[6,6-(2)H(2)]glucose infusion (n = 8). Skeletal muscle biopsies were performed at baseline and during prandial-like peripheral hyperinsulinemia (n = 3). At low peripheral hyperinsulinemia, whole-body glucose uptake was not affected by rapamycin. During prandial-like peripheral hyperinsulinemia, rapamycin increased glucose uptake compared with placebo by 17% (R(d 300-360 min), 75 +/- 5 vs. 64 +/- 5 micromol x kg(-1) x min(-1), P = 0.0008). Rapamycin affected endogenous glucose production neither at baseline nor during low or prandial-like peripheral hyperinsulinemia. Combined hyperaminoacidemia and prandial-like hyperinsulinemia increased S6K phosphorylation and inhibitory insulin receptor substrate-1 (IRS-1) phosphorylation at Ser312 and Ser636 in the placebo group. Rapamycin partially inhibited this increase in mTOR-mediated S6K phosphorylation and IRS-1 Ser312 and Ser636 phosphorylation. In conclusion, rapamycin stimulates insulin-mediated glucose uptake in man under conditions known to activate the mTOR/S6K pathway.     

5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle.

It has previously been reported that exercise causes an increase in glucose uptake in skeletal muscle and also an increase in 5' AMP-activated protein kinase (AMPK) activity. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA-riboside), an analog of adenosine, is taken up into cells and phosphorylated to form AICA-riboside monophosphate (ZMP), which can also activate AMPK. This study was designed to determine whether the increase in glucose uptake observed with AMPK activation by AICA-riboside is due to GLUT4 translocation from an intracellular location to the plasma membranes, similar to that seen in response to contraction. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine erythrocytes, 8 mmol/l glucose, and +/-2 mmol/AICA-riboside or +/-60 nmol/l insulin. Perfusion medium containing AICA-riboside was found to significantly increase AMPK activity, glucose uptake, and GLUT4 translocation in skeletal muscle above basal levels. Insulin-perfused muscles showed significant increases in glucose uptake and GLUT4 translocation, but AMPK activation was not significantly changed from basal levels. These results provide evidence that the increased glucose uptake observed with AMPK activation by AICA-riboside in perfused rat hindlimb muscles is due to an increase in the translocation of GLUT4 to surface membranes.     

5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes

Incubation of skeletal muscle with 5-aminoimidazole-4carboxamide ribonucleoside (AICAR), a compound that activates 5'-AMP-activated protein kinase (AMPK), has been demonstrated to stimulate glucose transport and GLUT4 translocation to the plasma membrane. In this study, we characterized the AMPK cascade in 3T3-L1 adipocytes and the response of glucose transport to incubation with AICAR. Both isoforms of the catalytic alpha-subunit of AMPK are expressed in 3T3-L1 adipocytes, in which AICAR stimulated AMPK activity in a time- and dose-dependent fashion. AICAR stimulated 2-deoxy-D-glucose transport twofold and reduced insulin-stimulated uptake to 62% of the control transport rate dose-dependently, closely correlating with the activation of AMPK. AICAR also inhibited insulin-stimulated GLUT4 translocation, assessed using the plasma membrane lawn assay. The effects of AICAR on insulin-stimulated glucose transport are not mediated by either adenosine receptors or nitric oxide synthase and are mediated downstream of phosphatidylinositol 3'-kinase stimulation. We propose that in contrast to skeletal muscle, in which AMPK stimulation promotes glucose transport to provide ATP as a fuel, AMPK stimulation inhibits insulin-stimulated glucose transport in adipocytes, inhibiting triacylglycerol synthesis, to conserve ATP under conditions of cellular stress. Investigation of the mode of action of AICAR and AMPK may, therefore, give insight into the mechanism of insulin action.     

Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes

Metformin is an effective hypoglycemic drug that lowers blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in skeletal muscle; however, the molecular site of metformin action is not well understood. AMP-activated protein kinase (AMPK) activity increases in response to depletion of cellular energy stores, and this enzyme has been implicated in the stimulation of glucose uptake into skeletal muscle and the inhibition of liver gluconeogenesis. We recently reported that AMPK is activated by metformin in cultured rat hepatocytes, mediating the inhibitory effects of the drug on hepatic glucose production. In the present study, we evaluated whether therapeutic doses of metformin increase AMPK activity in vivo in subjects with type 2 diabetes. Metformin treatment for 10 weeks significantly increased AMPK alpha2 activity in the skeletal muscle, and this was associated with increased phosphorylation of AMPK on Thr172 and decreased acetyl-CoA carboxylase-2 activity. The increase in AMPK alpha2 activity was likely due to a change in muscle energy status because ATP and phosphocreatine concentrations were lower after metformin treatment. Metformin-induced increases in AMPK activity were associated with higher rates of glucose disposal and muscle glycogen concentrations. These findings suggest that the metabolic effects of metformin in subjects with type 2 diabetes may be mediated by the activation of AMPK alpha2.     

Distinct signals regulate AS160 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle

Insulin and contraction increase GLUT4 translocation in skeletal muscle via distinct signaling mechanisms. Akt substrate of 160 kDa (AS160) mediates insulin-stimulated GLUT4 translocation in L6 myotubes, presumably through activation of Akt. Using in vivo, in vitro, and in situ methods, insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR all increased AS160 phosphorylation in mouse skeletal muscle. Insulin-stimulated AS160 phosphorylation was fully blunted by wortmannin in vitro and in Akt2 knockout (KO) mice in vivo. In contrast, contraction-stimulated AS160 phosphorylation was only partially decreased by wortmannin and unaffected in Akt2 KO mice, suggesting additional regulatory mechanisms. To determine if AMPK mediates AS160 signaling, we used AMPK alpha2-inactive (alpha2i) transgenic mice. AICAR-stimulated AS160 phosphorylation was fully inhibited, whereas contraction-stimulated AS160 phosphorylation was partially reduced in the AMPK alpha2i transgenic mice. Combined AMPK alpha2 and Akt inhibition by wortmannin treatment of AMPK alpha2 transgenic mice did not fully ablate contraction-stimulated AS160 phosphorylation. Maximal insulin, together with either AICAR or contraction, increased AS160 phosphorylation in an additive manner. In conclusion, AS160 may be a point of convergence linking insulin, contraction, and AICAR signaling. While Akt and AMPK alpha2 activities are essential for AS160 phosphorylation by insulin and AICAR, respectively, neither kinase is indispensable for the entire effects of contraction on AS160 phosphorylation.