Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle

Insulin, contraction, and the nitric oxide (NO) donor, sodium nitroprusside (SNP), all increase glucose transport in skeletal muscle. Some reports suggest that NO is a critical mediator of insulin- and/or contraction-stimulated transport. To determine if the mechanism leading to NO-stimulated glucose uptake is similar to the insulin- or contraction-dependent signaling pathways, isolated soleus and extensor digitorum longus (EDL) muscles from rats were treated with various combinations of SNP (maximum 10 mmol/l), insulin (maximum 50 mU/ml), electrical stimulation to produce contractions (maximum 10 min), wortmannin (100 nmol/l), and/or the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) (0.1 mmol/l). The combinations of SNP plus insulin and SNP plus contraction both had fully additive effects on 2-deoxyglucose uptake. Wortmannin completely inhibited insulin-stimulated glucose transport and only slightly inhibited SNP-stimulated 2-deoxyglucose uptake, whereas L-NMMA did not inhibit contraction-stimulated 2-deoxyglucose uptake. SNP significantly increased the activity of the alpha1 catalytic subunit of 5'AMP-activated protein kinase (AMPK), a signaling molecule that has been implicated in mediating glucose transport in fuel-depleted cells. Addition of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (1 mg/ml) to the drinking water of rats for 2 days failed to affect the increase in muscle 2-deoxyglucose uptake in response to treadmill exercise. These data suggest that NO stimulates glucose uptake through a mechanism that is distinct from both the insulin and contraction signaling pathways.     

Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism

5'AMP-activated protein kinase (AMPK) can be activated in response to cellular fuel depletion and leads to switching off ATP-consuming pathways and switching on ATP-regenerating pathways in many cell types. We have hypothesized that AMPK is a central mediator of insulin-independent glucose transport, which enables fuel-depleted muscle cells to take up glucose for ATP regeneration under conditions of metabolic stress. To test this hypothesis, rat epitrochlearis muscles were isolated and incubated in vitro under several conditions that evoke metabolic stress accompanied by intracellular fuel depletion. Rates of glucose transport in the isolated muscles were increased by all of these conditions, including contraction (5-fold above basal), hypoxia (8-fold), 2,4-dinotrophenol (11-fold), rotenone (7-fold), and hyperosmolarity (8-fold). All of these stimuli simultaneously increased both alpha1 and alpha2 isoform-specific AMPK activity. There was close correlation between alpha1 (r2 = 0.72) and alpha2 (r2 = 0.67) AMPK activities and the rate of glucose transport, irrespective of the metabolic stress used, all of which compromised muscle fuel status as judged by ATP, phosphocreatine, and glycogen content. 5-Aminoimidazole-4-carboxamide ribonucleoside, a pharmacological AMPK activator that is metabolized to an AMP-mimetic ZMP, also increased both glucose transport and AMPK activity but did not change fuel status. Insulin stimulated glucose transport by 6.5-fold above basal but did not affect AMPK activity. These results suggest that the activation of AMPK may be a common mechanism leading to insulin-independent glucose transport in skeletal muscle under conditions of metabolic stress.     

Dissociation of AMP-activated protein kinase activation and glucose transport in contracting slow-twitch muscle

5'AMP-activated protein kinase (AMPK) has been suggested to be a key regulatory protein in exercise signaling of muscle glucose transport. To test this hypothesis, we investigated whether muscle glycogen levels affect AMPK activation and glucose transport stimulation similarly during contractions. Rats were preconditioned by a combination of swimming exercise and diet to obtain a glycogen-supercompensated group (high muscle glycogen content [HG]) with approximately 3-fold higher muscle glycogen levels than a glycogen-depleted group (low muscle glycogen content [LG]). In perfused fast-twitch muscles, contractions induced significant increases in AMPK activity and glucose transport and decreases in acetyl-CoA carboxylase (ACC) activity in both HG and LG groups. Contraction-induced glucose transport was nearly 2-fold (P < 0.05) and AMPK activation was 3-fold (P < 0.05) higher in the LG group compared with the HG group, whereas ACC deactivation was not different between groups. Thus, there was a significant positive correlation between AMPK activity and glucose transport in contracting fast-twitch muscles (r = 0.80, P < 0.01). However, in slow-twitch muscles with HG, glucose transport was increased 6-fold (P < 0.05) during contractions, whereas AMPK activity did not increase. In contracting slow-twitch muscles with LG, the increase in AMPK activity (315%) and the decrease in ACC activity (54 vs. 34% at 0.2 mmol/l citrate, LG vs. HG) was higher (P < 0.05) compared with HG muscles, whereas the increase in glucose transport was identical in HG and LG. In conclusion, in slow-twitch muscles, high glycogen levels inhibit contraction-induced AMPK activation without affecting glucose transport. This observation suggests that AMPK activation is not an essential signaling step in glucose transport stimulation in skeletal muscle.     

Glycogen-dependent effects of 5-aminoimidazole-4-carboxamide (AICA)-riboside on AMP-activated protein kinase and glycogen synthase activities in rat skeletal muscle.

5'-AMP-activated protein kinase (AMPK) functions as a metabolic switch in mammalian cells and can be artificially activated by 5-aminoimidazole-4-carboxamide (AICA)-riboside. AMPK activation during muscle contraction is dependent on muscle glycogen concentrations, but whether glycogen also modifies the activation of AMPK and its possible downstream effectors (glycogen synthase and glucose transport) by AICA-riboside in resting muscle is not known. Thus, we have altered muscle glycogen levels in rats by a combination of swimming exercise and diet and investigated the effects of AICA-riboside in the perfused rat hindlimb muscle. Two groups of rats, one with super-compensated muscle glycogen content (approximately 200-300% of normal; high glycogen [HG]) and one with moderately lowered muscle glycogen content (approximately 80% of normal; low glycogen [LG]), were generated. In both groups, the degree of activation of the alpha2 isoform of AMPK by AICA-riboside depended on muscle type (white gastrocnemius > red gastrocnemius > soleus). Basal and AICA-riboside-induced alpha2-AMPK activity were markedly lowered in the HG group (approximately 50%) compared with the LG group. Muscle 2-deoxyglucose uptake was also increased and glycogen synthase activity decreased by AICA-riboside. Especially in white gastrocnemius, these effects, as well as the absolute activity levels of AMPK-alpha2, were markedly reduced in the HG group compared with the LG group. The inactivation of glycogen synthase by AICA-riboside was accompanied by decreased gel mobility and was eliminated by protein phosphatase treatment. We conclude that acute AICA-riboside treatment leads to phosphorylation and deactivation of glycogen synthase in skeletal muscle. Although the data do not exclude a role of other kinases/phosphatases, they suggest that glycogen synthase may be a target for AMPK in vivo. Both basal and AICA-riboside-induced AMPK-alpha2 and glycogen synthase activities, as well as glucose transport, are depressed when the glycogen stores are plentiful. Because the glycogen level did not affect adenine nucleotide concentrations, our data suggest that glycogen may directly affect the activation state of AMPK in skeletal muscle.     

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.     

Isoform-specific regulation of 5' AMP-activated protein kinase in skeletal muscle from obese Zucker (fa/fa) rats in response to contraction

Glucose transport can be activated in skeletal muscle in response to insulin via activation of phosphoinositide (PI) 3-kinase and in response to contractions or hypoxia, presumably via activation of 5' AMP-activated protein kinase (AMPK). We determined the effects of insulin and muscle contraction/hypoxia on PI 3-kinase, AMPK, and glucose transport activity in epitrochlearis skeletal muscle from insulin-resistant Zucker (fa/ fa) rats. Insulin-stimulated glucose transport in isolated skeletal muscle was reduced 47% in obese versus lean rats, with a parallel 42% reduction in tyrosine-associated PI 3-kinase activity. Contraction and hypoxia elicited normal responses for glucose transport in skeletal muscle from insulin-resistant obese rats. Isoform-specific AMPK activity was measured in skeletal muscle in response to insulin, contraction, or hypoxia. Contraction increased AMPKalpha1 activity 2.3-fold in lean rats, whereas no effect was noted in obese rats. Hypoxia increased AMPKalpha1 activity to a similar extent (more than sixfold) in lean and obese rats. Regardless of genotype, contraction, and hypoxia, each increased AMPKalpha2 activity more than fivefold, whereas insulin did not alter either AMPKalpha1 or -alpha2 activity in skeletal muscle. In conclusion, obesity-related insulin resistance is associated with an isoform-specific impairment in AMPKalpha1 in response to contraction. However, this impairment does not appear to affect contraction-stimulated glucose transport. Activation of AMPKalpha2 in response to muscle contraction/ exercise is associated with a parallel and normal increase in glucose transport in insulin-resistant skeletal muscle.     

Downregulation of AMPK Accompanies Leucine- and Glucose-Induced Increases in Protein Synthesis and Insulin Resistance in Rat Skeletal Muscle

OBJECTIVE

Branched-chain amino acids, such as leucine and glucose, stimulate protein synthesis and increase the phosphorylation and activity of the mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase (p70S6K). We examined in skeletal muscle whether the effects of leucine and glucose on these parameters and on insulin resistance are mediated by the fuel-sensing enzyme AMP-activated protein kinase (AMPK).

RESEARCH DESIGN AND METHODS

Rat extensor digitorum longus (EDL) muscle was incubated with different concentrations of leucine and glucose with or without AMPK activators. Muscle obtained from glucose-infused rats was also used as a model.

RESULTS

In the EDL, incubation with 100 or 200 μmol/l leucine versus no added leucine suppressed the activity of the α2 isoform of AMPK by 50 and 70%, respectively, and caused concentration-dependent increases in protein synthesis and mTOR and p70S6K phosphorylation. Very similar changes were observed in EDL incubated with 5.5 or 25 mmol/l versus no added glucose and in muscle of rats infused with glucose in vivo. Incubation of the EDL with the higher concentrations of both leucine and glucose also caused insulin resistance, reflected by a decrease in insulin-stimulated Akt phosphorylation. Coincubation with the AMPK activators AICAR and α-lipoic acid substantially prevented all of those changes and increased the phosphorylation of specific sites of mTOR inhibitors raptor and tuberous sclerosis complex 2 (TSC2). In contrast, decreases in AMPK activity induced by leucine and glucose were not associated with a decrease in raptor or TSC2 phosphorylation.

CONCLUSIONS

The results indicate that both leucine and glucose modulate protein synthesis and mTOR/p70S6 and insulin signaling in skeletal muscle by a common mechanism. They also suggest that the effects of both molecules are associated with a decrease in AMPK activity and that AMPK activation prevents them.AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that has classically been defined in terms of its role in restoring ATP levels in energy-depleted cells. In skeletal muscle, AMPK is typically activated by such factors as glucose deprivation and contraction (exercise) (1,2). The activated AMPK in turn enhances processes that generate ATP, such as fatty acid oxidation and glucose transport, and downregulates others that consume ATP and can be diminished temporarily without jeopardizing the cell (e.g., protein and lipid synthesis). Much less studied is the notion that a decrease in AMPK below baseline values may also be a physiologically or pathophysiologically relevant event. In keeping with such a possibility, decreased AMPK activity has been observed in tissues of many obese insulin-resistant rodents (3) and in liver (4,5) and adipose tissue (6) of rats starved for 48 h when they are refed. One consequence of decreased AMPK activity could be increases in mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K) signaling and protein synthesis because both are decreased by AMPK activation (7).In the present study, we assessed whether fuel-induced increases in protein synthesis, mTOR/p70S6K signaling, and insulin resistance in skeletal muscle are mediated by decreases in AMPK activity. Toward this end, rat extensor digitorum longus (EDL) muscles were incubated for different time periods with various concentrations of leucine or glucose and the above parameters were assessed. The results indicate that elevated concentrations of leucine and glucose decrease AMPK activity, increase protein synthesis and mTOR/p70S6 phosphorylation, and cause insulin resistance and that activation of AMPK by pharmacological agents prevents these events from occurring. Finally, the data suggest that the decrease in AMPK activity caused by both leucine and glucose is not mediated by changes in the AMP-to-ATP ratio but is associated with an increase in the lactate/pyruvate ratio.     

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.     

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.     

Acyl-CoA inhibition of hexokinase in rat and human skeletal muscle is a potential mechanism of lipid-induced insulin resistance

There are strong correlations between impaired insulin-stimulated glucose metabolism and increased intramuscular lipid pools; however, the mechanism by which lipids interact with glucose metabolism is not completely understood. Long-chain acyl CoAs have been reported to allosterically inhibit liver glucokinase (hexokinase IV). The aim of the present study was to determine whether long-chain acyl CoAs inhibit hexokinase in rat and human skeletal muscle. At subsaturating glucose concentrations, 10 micromol/l of the three major long-chain acyl-CoA species in skeletal muscle, palmitoyl CoA (16:0), oleoyl CoA (18:1, n = 9), and linoleoyl CoA (18:2, n = 6), reduced hexokinase activity of rat skeletal muscle to 61 +/- 3, 66 +/- 7, and 57 +/- 5% of control activity (P < 0.005), respectively. The inhibition was concentration-dependent (P < 0.005) with 5 pmol/l producing near maximal inhibition. Human skeletal muscle hexokinase was also inhibited by long-chain acyl CoAs (5 pmol/l palmitoyl CoA decreased activity to 75 +/- 6% of control activity, P < 0.005). Inhibition of hexokinase in rat and human muscle by long-chain acyl CoAs was additive to the inhibition of hexokinase by glucose-6-phosphate (an allosteric inhibitor of hexokinase). This inhibition of skeletal muscle hexokinase by long-chain acyl CoA suggests that increases in intramuscular lipid metabolites could interact directly with insulin-mediated glucose metabolism in vivo by decreasing the rate of glucose phosphorylation and decreasing glucose-6-phosphate concentrations.     

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.     

Ablation of AMP-activated protein kinase alpha2 activity exacerbates insulin resistance induced by high-fat feeding of mice

OBJECTIVE—We determined whether muscle AMP-activated protein kinase (AMPK) has a role in the development of insulin resistance.RESEARCH DESIGN AND METHODS—Muscle-specific transgenic mice expressing an inactive form of the AMPK α2 catalytic subunit (α2i TG) and their wild-type littermates were fed either a high-fat (60% kcal fat) or a control (10% kcal fat) diet for 30 weeks.RESULTS—Compared with wild-type mice, glucose tolerance in α2i TG mice was slightly impaired on the control diet and significantly impaired on the high-fat diet. To determine whether the whole-body glucose intolerance was associated with impaired insulin sensitivity in skeletal muscle, glucose transport in response to submaximal insulin (450 μU/ml) was measured in isolated soleus muscles. On the control diet, insulin-stimulated glucose transport was reduced by ∼50% in α2i TG mice compared with wild-type mice. High-fat feeding partially decreased insulin-stimulated glucose transport in wild-type mice, while high-fat feeding resulted in a full blunting of insulin-stimulated glucose transport in the α2i TG mice. High-fat feeding in α2i TG mice was accompanied by decreased expression of insulin signaling proteins in gastrocnemius muscle.CONCLUSIONS—The lack of skeletal muscle AMPK α2 activity exacerbates the development of glucose intolerance and insulin resistance caused by high-fat feeding and supports the thesis that AMPK α2 is an important target for the prevention/amelioration of skeletal muscle insulin resistance through lifestyle (exercise) and pharmacologic (e.g., metformin) treatments.The development of insulin resistance in skeletal muscle precedes the onset of type 2 diabetes by decades (1). Although the underlying mechanism is not fully understood, in recent years there has been growing interest in AMP-activated protein kinase (AMPK) as a potential target to attenuate skeletal muscle insulin resistance. As examples, two well-known therapies for type 2 diabetes, physical exercise and metformin, both activate AMPK in skeletal muscle (2). Despite this emphasis on AMPK, there is little direct evidence showing that AMPK is in fact critical in the development of skeletal muscle insulin resistance.AMPK is an energy-sensing enzyme that is activated by acute increases in the cellular AMP-to-ATP ratio. In skeletal muscle, AMPK activity is increased by stimuli such as exercise, hypoxia, ischemia, and osmotic stress, all of which reduced cellular energy level (2). When intracellular ATP decreases, AMPK acts to switch off ATP-consuming pathways, such as glycogen, fatty acid, and protein synthesis pathways, and acts to switch on alternative pathways for ATP regeneration, such as glucose transport, glycolysis, and fatty acid oxidation. AMPK may also play a role in enhancing insulin sensitivity and/or responsiveness for glucose transport (3–6) in skeletal muscle.AMPK is a heterotrimeric serine/threonine kinase that consists of a catalytic α-subunit and regulatory β- and γ-subunits (7–11). In skeletal muscle, α2 (12,13) is the major catalytic isoform expressed. To examine a possible role of AMPK in the development of insulin resistance, muscle-specific transgenic mice expressing an inactive form of the AMPK α2 catalytic subunit isoform (α2i TG mice) (14) and their wild-type littermates were subjected to a high-fat diet. Here, we show that ablation of muscle AMPK α2 activity worsens the glucose intolerance induced by high-fat feeding. This exacerbated glucose intolerance is associated with a marked decrease in muscle glucose transport in response to insulin, measured in vitro in isolated muscles. These results demonstrate that AMPK α2 activity is an important factor contributing to insulin action on glucose transport in skeletal muscle. Therefore, activation of muscle AMPK α2 by exercise and/or pharmacological stimulation can be a significant strategy to improve insulin resistance in patients with type 2 diabetes.     

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.     

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.     

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.     

AMP-activated protein kinase: a new beta-cell glucose sensor?: Regulation by amino acids and calcium ions

Stimulation of AMP-activated protein kinase (AMPK) in skeletal muscle and liver is seen as an exciting prospect for the treatment of type 2 diabetes. However, we have recently demonstrated that changes in AMPK activity accompany the exposure of pancreatic islet beta-cells to elevated glucose concentrations and may be involved in the activation of insulin secretion. Here, we discuss this hypothesis and explore the potential role of changes in AMPK activity in the actions of other secretagogues. Amino acids decreased AMPK activity in MIN6 beta-cells with an order of potency for inhibition: arg=leu < gln= leu + glu < glucose, which was closely correlated with the stimulation of insulin release (r2=0.76). By contrast, increases in intracellular Ca2+ concentration provoked by cell depolarization with KCl activated AMPK in the face of increased free intracellular ATP concentrations. Elevation of intracellular cAMP levels with isobutylmethylxanthine or forskolin had no effect on AMPK activity. We conclude that metabolizable amino acids regulate AMPK in the beta-cell via increases in the cytosolic ATP/AMP ratio and via phosphorylation by the upstream kinase LKB1. Intracellular Ca2+ ions may activate AMPK by calmodulin kinase 1 kinase-mediated phosphorylation. The latter may act as a novel feedback mechanism to inhibit excessive insulin secretion under some circumstances.     

Skeletal muscle AMP-activated protein kinase phosphorylation parallels metabolic phenotype in leptin transgenic mice under dietary modification

Leptin augments glucose and lipid metabolism independent of its effect on satiety. Administration of leptin in rodents increases skeletal muscle beta-oxidation by activating AMP-activated protein kinase (AMPK). We previously reported that, as hyperleptinemic as obese human subjects, transgenic skinny mice overexpressing leptin in liver (LepTg) exhibit enhanced insulin sensitivity and lipid clearance. To assess skeletal muscle AMPK activity in leptin-sensitive and -insensitive states, we examined phosphorylation of AMPK and its target, acetyl CoA carboxylase (ACC), in muscles from LepTg under dietary modification. Here we show that phosphorylation of AMPK and ACC are chronically augmented in LepTg soleus muscle, with a concomitant increase in the AMP-to-ATP ratio and a significant decrease in tissue triglyceride content. Despite preexisting hyperleptinemia, high-fat diet (HFD)-fed LepTg develop obesity, insulin-resistance, and hyperlipidemia. In parallel, elevated soleus AMPK and ACC phosphorylation in regular diet-fed LepTg is attenuated, and tissue triglyceride content is increased in those given HFD. Of note, substitution of HFD with regular diet causes a robust recovery of soleus AMPK and ACC phosphorylation in LepTg, with a higher rate of body weight reduction and a regain of insulin sensitivity. In conclusion, soleus AMPK and ACC phosphorylation in LepTg changes in parallel with its insulin sensitivity under dietary modification, suggesting a close association between skeletal muscle AMPK activity and sensitivity to leptin.     

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.     

Impaired energy metabolism in skeletal muscle during phosphate depletion

The effects of phosphate depletion (PD) of 4, 8, and 12 weeks on skeletal muscle energy metabolism were studied in rats fed a phosphate deficiency diet and compared with rats pairfed with a normal phosphate diet. Skeletal muscle biopsy specimens were examined for energy production, transport, and utilization. The results show that already by 4 weeks of PD, the concentration of inorganic phosphorus of the skeletal muscle was significantly reduced and remained low thereafter. There was significant (P less than 0.01) and direct correlation between the cellular inorganic phosphorus and that of serum phosphorus. Adenine nucleotides, ATP, ADP, AMP, and creatine phosphate levels did not change. Mitochondrial respiration and oxidative phosphorylation were impaired by PD. Total cellular mitochondrial and myofibrillar creatine phosphokinase activities were significantly reduced at 4 weeks of PD and fell further at 8 and 12 weeks. There was a significant (P less than 0.01) and direct correlation between the activity of total extractable creatine phosphokinase and both serum and cellular levels of inorganic phosphorus. These data show that chronic PD is associated with a decrease in energy production, transfer, and utilization by skeletal muscle and provides information on the molecular events responsible for the myopathy of PD.     

Ca2+ and AMPK both mediate stimulation of glucose transport by muscle contractions

It is now generally accepted that activation of AMP-activated protein kinase (AMPK) is involved in the stimulation of glucose transport by muscle contractions. However, earlier studies provided evidence that increases in cytosolic Ca(2+) mediate the effect of muscle contractions on glucose transport. The purpose of this study was to test the hypothesis that both the increase in cytosolic Ca(2+) and the activation of AMPK are involved in the stimulation of glucose transport by muscle contractions. Caffeine causes release of Ca(2+) from the sarcoplasmic reticulum. Incubation of rat epitrochlearis muscles with a concentration of caffeine that raises cytosolic Ca(2+) to levels too low to cause contraction resulted in an approximate threefold increase in glucose transport. Caffeine treatment also resulted in increased phosphorylation of calmodulin-dependent protein kinase (CAMK)-II in epitrochlearis muscle. The stimulation of glucose transport by caffeine was blocked by the Ca(2+)-CAMK inhibitors KN62 and KN93. Activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) also resulted in an approximate threefold increase in glucose transport in the epitrochlearis. The increases in glucose transport induced by AICAR and caffeine were additive, and their combined effect was not significantly different from that induced by maximally effective contractile activity. KN62 and KN93 caused an approximately 50% inhibition of the stimulation of glucose transport by contractile activity. Our results provide evidence that both Ca(2+) and AMPK are involved in the stimulation of glucose transport by muscle contractions. They also suggest that the stimulation of glucose transport by Ca(2+) involves activation of CAMK.