Inhibition of insulin signaling and glycogen synthesis by phorbol dibutyrate in rat skeletal muscle

2001 ◽  
Vol 281 (1) ◽  
pp. E8-E15 ◽  
Author(s):  
Yenshou Lin ◽  
Samar I. Itani ◽  
Theodore G. Kurowski ◽  
David J. Dean ◽  
Zhijun Luo ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Insulin Signaling ◽  
Phorbol Esters ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Insulin Receptor Tyrosine Kinase ◽  
Pkc Isoforms ◽  
Glucose Incorporation

Numerous studies have shown a correlation between changes in protein kinase C (PKC) distribution and/or activity and insulin resistance in skeletal muscle. To investigate which PKC isoforms might be involved and how they affect insulin action and signaling, studies were carried out in rat soleus muscle incubated with phorbol esters. Muscles preincubated for 1 h with 1 μM phorbol 12,13-dibutyrate (PDBu) showed an impaired ability of insulin to stimulate glucose incorporation into glycogen and a translocation of PKC-α, -βI, -θ, and -ε, and probably -βII, from the cytosol to membranes. Preincubation with 1 μM PDBu decreased activation of the insulin receptor tyrosine kinase by insulin and to an even greater extent the phosphorylation of Akt/protein kinase B and glycogen synthase kinase-3. However, it failed to diminish the activation of phosphatidylinositol 3′-kinase by insulin. Despite these changes in signaling, the stimulation by insulin of glucose transport (2-deoxyglucose uptake) and glucose incorporation into lipid and oxidation to CO2 was unaffected. The results indicate that preincubation of skeletal muscle with phorbol ester leads to a translocation of multiple conventional and novel PKC isoforms and to an impairment of several, but not all, events in the insulin-signaling cascade. They also demonstrate that these changes are associated with an inhibition of insulin-stimulated glycogen synthesis but that, at the concentration of PDBu used here, glucose transport, its incorporation into lipid, and its oxidation to CO2 are unaffected.

Cellular depletion of atypical PKCλ is associated with enhanced insulin sensitivity and glucose uptake in L6 rat skeletal muscle cells

2010 ◽  
Vol 299 (3) ◽  
pp. E402-E412 ◽  
Author(s):  
Clare Stretton ◽  
Ashleigh Evans ◽  
Harinder S. Hundal
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Insulin Receptor ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Dominant Negative Mutant

Atypical protein kinase C (aPKC) isoforms (λ and ζ) have been implicated in the control of insulin-stimulated glucose uptake in adipose and skeletal muscle, but their precise role in this process remains unclear, especially in light of accumulating evidence showing that, in response to numerous stimuli, including insulin and lipids such as ceramide, activation of aPKCs acts to negatively regulate key insulin-signaling molecules, such as insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB)/cAMP-dependent PKC (Akt). In this study, we have depleted PKCλ in L6 skeletal muscle cells using RNA interference and assessed the effect this has upon insulin action. Muscle cells did not express detectable amounts of PKCζ. Depletion of PKCλ (>95%) had no significant effect on the expression of proteins participating in insulin signaling [i.e., insulin receptor, IRS-1, phosphatidylinositol 3-kinase (PI 3-kinase), PKB, or phosphate and tensin homolog deleted on chromosome 10] or those involved in glucose transport [Akt substrate of 160 kDa, glucose transporter (GLUT)1, or GLUT4]. However, PKCλ-depleted muscle cells exhibited greater activation of PKB/Akt and phosphorylation of its downstream target glycogen synthase kinase 3, in the basal state and displayed greater responsiveness to submaximal doses of insulin with respect to p85-PI 3-kinase/IRS-1 association and PKB activation. The increase in basal and insulin-induced signaling resulted in an associated enhancement of basal and insulin-stimulated glucose transport, both of which were inhibited by the PI 3-kinase inhibitor wortmannin. Additionally, like RNAi-mediated depletion of PKCλ, overexpression of a dominant-negative mutant of PKCζ induced a similar insulin-sensitizing effect on PKB activation. Our findings indicate that aPKCs are likely to play an important role in restraining proximal insulin signaling events but appear dispensable with respect to insulin-stimulated glucose uptake in cultured L6 muscle cells.

Role of PDK1 in insulin-signaling pathway for glucose metabolism in 3T3-L1 adipocytes

2002 ◽  
Vol 282 (6) ◽  
pp. E1385-E1394 ◽  
Author(s):  
Tetsuya Yamada ◽  
Hideki Katagiri ◽  
Tomoichiro Asano ◽  
Masatoshi Tsuru ◽  
Kouichi Inukai ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Metabolism ◽  
Signaling Pathway ◽  
Glucose Transport ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Insulin Signaling Pathway ◽  
Ph Domain

To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the insulin-signaling pathway for glucose metabolism, wild-type (wt), the kinase-dead (kd), or the plecstrin homology (PH) domain deletion (ΔPH) mutant of PDK1 was expressed using an adenovirus gene transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were found in both membrane and cytosol fractions, whereas ΔPH-PDK1, which exhibited PDK1 activity similar to that of wt-PDK1, was detected exclusively in the cytosol fraction. Insulin dose dependently activated protein kinase B (PKB) but did not change atypical protein kinase C (aPKC) activity in control cells. aPKC activity was not affected by expression of wt-, kd-, or ΔPH-PDK1 in either the presence or the absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced activation of PKB as well as insulin-induced phosphorylation of glycogen synthase kinase (GSK)3α/β, a direct downstream target of PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither ΔPH-PDK1 nor kd-PDK1 expression affected PKB activity, GSK3 phosphorylation, or glycogen synthesis. Thus membrane localization of PDK1 via its PH domain is essential for insulin signaling through the PDK1-PKB-GSK3α/β pathway. Glucose transport activity was unaffected by expression of wt-PDK1, kd-PDK1, or ΔPH-PDK1 in either the presence or the absence of insulin. These findings suggest the presence of a signaling pathway for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.

Oxidative stress-induced insulin resistance in rat skeletal muscle: role of glycogen synthase kinase-3

2008 ◽  
Vol 294 (3) ◽  
pp. E615-E621 ◽  
Author(s):  
Betsy B. Dokken ◽  
Vitoon Saengsirisuwan ◽  
John S. Kim ◽  
Mary K. Teachey ◽  
Erik J. Henriksen
Keyword(s):  
Oxidative Stress ◽  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Transport ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Oxidant Stress ◽  
Glycogen Synthesis ◽  
Active Form ◽  
Gsk 3Β

Oxidative stress can contribute to the multifactorial etiology of whole body and skeletal muscle insulin resistance. No investigation has directly assessed the effect of an in vitro oxidant stress on insulin action in intact mammalian skeletal muscle. Therefore, the purpose of the present study was to characterize the molecular actions of a low-grade oxidant stress (H2O2) on insulin signaling and glucose transport in isolated skeletal muscle of lean Zucker rats. Soleus strips were incubated in 8 mM glucose for 2 h in the absence or presence of 100 mU/ml glucose oxidase, which produces H2O2 at ∼90 μM. By itself, H2O2 significantly ( P < 0.05) activated basal glucose transport activity, net glycogen synthesis, and glycogen synthase activity and increased phosphorylation of insulin receptor (Tyr), Akt (Ser473), and GSK-3β (Ser9). In contrast, this oxidant stress significantly inhibited the expected insulin-mediated enhancements in glucose transport, glycogen synthesis, and these signaling factors and allowed GSK-3β to retain a more active form. In the presence of CT-98014, a selective GSK-3 inhibitor, the ability of insulin to stimulate glucose transport and glycogen synthesis during exposure to this oxidant stress was enhanced by 20% and 39% ( P < 0.05), respectively, and insulin stimulation of the phosphorylation of insulin receptor, Akt, and GSK-3 was significantly increased by 36–58% ( P < 0.05). These results indicate that an oxidant stress can directly and rapidly induce substantial insulin resistance of skeletal muscle insulin signaling, glucose transport, and glycogen synthesis. Moreover, a small, but significant, portion of this oxidative stress-induced insulin resistance is associated with a reduced insulin-mediated suppression of the active form of GSK-3β.

Insulin-Sensitive Phospholipid Signaling Systems and Glucose Transport. Update II

2001 ◽  
Vol 226 (4) ◽  
pp. 283-295 ◽  
Author(s):  
Robert V. Farese
Keyword(s):  
Protein Kinase ◽  
Glucose Metabolism ◽  
Glucose Transport ◽  
Intracellular Signaling ◽  
Glucose Transporter ◽  
De Novo ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Cell Types ◽  
Biologically Active

Insulin provokes rapid changes in phospholipid metabolism and thereby generates biologically active lipids that serve as intracellular signaling factors that regulate glucose transport and glycogen synthesis. These changes include: (i) activation of phosphatidylinositol 3-kinase (PI3K) and production of PIP3; (ii) PIP3-dependent activation of atypical protein kinase Cs (PKCs); (iii) PIP3-dependent activation of PKB; (iv) PI3K-dependent activation of phospholipase D and hydrolysis of phosphatidyicholine with subsequent increases in phosphatidic acid (PA) and diacyiglycerol (DAG); (v) PI3K-independent activation of glycerol-3-phosphate acylytansferase and increases in de novo synthesis of PA and DAG; and (vi) activation of DAG-sensitive PKCs. Recent findings suggest that atypical PKCs and PKB serve as important positive regulators of insulin-stimulated glucose metabolism, whereas mechanisms that result in the activation of DAG-sensitive PKCs serve mainly as negative regulators of insulin signaling through PI3K. Atypical PKCs and PKB are rapidly activated by insulin in adipocytes, liver, skeletal muscles, and other cell types by a mechanism requiring PI3K and its downstream effector, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), which, in conjunction with PIP3, phosphorylates critical threonine residues in the activation loops of atypical PKCs and PKB. PIP3 also promotes increases in autophosphorylation and allosteric activation of atypical PKCs. Atypical PKCs and perhaps PKB appear to be required for insulin-induced translocation of the GLUT 4 glucose transporter to the plasma membrane and subsequent glucose transport. PKB also appears to be the major regulator of glycogen synthase. Together, atypical PKCs and PKB serve as a potent, integrated PI3K/PDK-1-directed signaling system that is used by insulin to regulate glucose metabolism.

Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle

2005 ◽  
Vol 288 (6) ◽  
pp. E1188-E1194 ◽  
Author(s):  
Betsy B. Dokken ◽  
Julie A. Sloniger ◽  
Erik J. Henriksen
Keyword(s):  
Skeletal Muscle ◽  
Tyrosine Phosphorylation ◽  
Glucose Transport ◽  
Insulin Signaling ◽  
Insulin Action ◽  
Glycogen Synthase ◽  
Serine Phosphorylation ◽  
Zucker Rats ◽  
Type I ◽  
Insulin Resistant

Glycogen synthase kinase-3 (GSK3) has been implicated in the multifactorial etiology of skeletal muscle insulin resistance in animal models and in human type 2 diabetic subjects. However, the potential molecular mechanisms involved are not yet fully understood. Therefore, we determined if selective GSK3 inhibition in vitro leads to an improvement in insulin action on glucose transport activity in isolated skeletal muscle of insulin-resistant, prediabetic obese Zucker rats and if these effects of GSK3 inhibition are associated with enhanced insulin signaling. Type I soleus and type IIb epitrochlearis muscles from female obese Zucker rats were incubated in the absence or presence of a selective, small organic GSK3 inhibitor (1 μM CT118637, Ki < 10 nM for GSK3α and GSK3β). Maximal insulin stimulation (5 mU/ml) of glucose transport activity, glycogen synthase activity, and selected insulin-signaling factors [tyrosine phosphorylation of insulin receptor (IR) and IRS-1, IRS-1 associated with p85 subunit of phosphatidylinositol 3-kinase, and serine phosphorylation of Akt and GSK3] were assessed. GSK3 inhibition enhanced ( P <0.05) basal glycogen synthase activity and insulin-stimulated glucose transport in obese epitrochlearis (81 and 24%) and soleus (108 and 20%) muscles. GSK3 inhibition did not modify insulin-stimulated tyrosine phosphorylation of IR β-subunit in either muscle type. However, in obese soleus, GSK3 inhibition enhanced (all P < 0.05) insulin-stimulated IRS-1 tyrosine phosphorylation (45%), IRS-1-associated p85 (72%), Akt1/2 serine phosphorylation (30%), and GSK3β serine phosphorylation (39%). Substantially smaller GSK3 inhibitor-mediated enhancements of insulin action on these insulin signaling factors were observed in obese epitrochlearis. These results indicate that selective GSK3 inhibition enhances insulin action in insulin-resistant skeletal muscle of the prediabetic obese Zucker rat, at least in part by relieving the deleterious effects of GSK3 action on post-IR insulin signaling. These effects of GSK3 inhibition on insulin action are greater in type I muscle than in type IIb muscle from these insulin-resistant animals.

Polymyxin B selectively inhibits insulin effects on transport in isolated muscle

1987 ◽  
Vol 252 (2) ◽  
pp. E248-E254
Author(s):  
T. Gremeaux ◽  
J. F. Tanti ◽  
E. Van Obberghen ◽  
Y. Le Marchand-Brustel
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Glycogen Synthase ◽  
Polymyxin B ◽  
Acid Uptake ◽  
Insulin Effect ◽  
Insulin Receptor Tyrosine Kinase ◽  
Free System

Polymyxin B (PMB), a cyclic decapeptide antibiotic, inhibits the hypoglycemic effect of insulin in vivo. To elucidate the mechanism of PMB action, we have studied its effect in vitro on insulin-stimulated pathways in the mouse skeletal muscle. PMB, added to the incubation mixture, specifically inhibited insulin-stimulated 2-deoxyglucose transport and alpha-aminoisobutyric acid uptake in the isolated soleus muscle but did not affect the basal rates of transport (measured in the absence of insulin). PMB did not alter insulin binding and hexokinase activity. PMB effect was observed at all deoxyglucose concentrations tested, and PMB was also able to inhibit vanadate-stimulated glucose transport. By contrast, insulin activation of glycogen synthase was not prevented by PMB. Basal and maximally insulin-stimulated insulin receptor tyrosine kinase activity, tested in a cell-free system, was similar for both autophosphorylation and phosphorylation of exogenous substrates in the absence or in the presence of PMB. Furthermore, the insulin sensitivity of the kinase was increased in the presence of PMB. Our results suggest that the anti-insulin effect of PMB observed in vivo is due to an inhibition of insulin-stimulated glucose transport in the skeletal muscle perhaps through a specific blockade of the insulin-induced translocation of the glucose carriers.

Control of glycogen synthesis is shared between glucose transport and glycogen synthase in skeletal muscle fibers

2000 ◽  
Vol 278 (2) ◽  
pp. E234-E243 ◽  
Author(s):  
Iñaki Azpiazu ◽  
Jill Manchester ◽  
Alexander V. Skurat ◽  
Peter J. Roach ◽  
John C. Lawrence
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Muscle Fibers ◽  
Fiber Types ◽  
Oxidative Potential ◽  
Glycogen Accumulation ◽  
Tibialis Muscle ◽  
Fast Twitch

The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [14C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.

scholarly journals Overexpression of SH2-Containing Inositol Phosphatase 2 Results in Negative Regulation of Insulin-Induced Metabolic Actions in 3T3-L1 Adipocytes via Its 5′-Phosphatase Catalytic Activity

2001 ◽  
Vol 21 (5) ◽  
pp. 1633-1646 ◽  
Author(s):  
Tsutomu Wada ◽  
Toshiyasu Sasaoka ◽  
Makoto Funaki ◽  
Hiroyuki Hori ◽  
Shihou Murakami ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Phosphatase Activity ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Dominant Negative ◽  
Inositol Phosphatase

ABSTRACT Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5′-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5′-phosphatase-defective SHIP2 (ΔIP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor β subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or ΔIP-SHIP2. Because WT-SHIP2 possesses the 5′-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of ΔIP-SHIP2, indicating that ΔIP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase Cλ in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase Cλ, whereas these activations were increased by expression of ΔIP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of ΔIP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3β and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of ΔIP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5′-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.

Glucose infusion causes insulin resistance in skeletal muscle of rats without changes in Akt and AS160 phosphorylation

2007 ◽  
Vol 293 (5) ◽  
pp. E1358-E1364 ◽  
Author(s):  
Andrew J. Hoy ◽  
Clinton R. Bruce ◽  
Anna Cederberg ◽  
Nigel Turner ◽  
David E. James ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glucose Infusion ◽  
Metabolic Effects ◽  
Synthesis Rate ◽  
Muscle Sample

Hyperglycemia is a defining feature of Type 1 and 2 diabetes. Hyperglycemia also causes insulin resistance, and our group (Kraegen EW, Saha AK, Preston E, Wilks D, Hoy AJ, Cooney GJ, Ruderman NB. Am J Physiol Endocrinol Metab Endocrinol Metab 290: E471–E479, 2006) has recently demonstrated that hyperglycemia generated by glucose infusion results in insulin resistance after 5 h but not after 3 h. The aim of this study was to investigate possible mechanism(s) by which glucose infusion causes insulin resistance in skeletal muscle and in particular to examine whether this was associated with changes in insulin signaling. Hyperglycemia (∼10 mM) was produced in cannulated male Wistar rats for up to 5 h. The glucose infusion rate required to maintain this hyperglycemia progressively lessened over 5 h (by 25%, P < 0.0001 at 5 h) without any alteration in plasma insulin levels consistent with the development of insulin resistance. Muscle glucose uptake in vivo (44%; P < 0.05) and glycogen synthesis rate (52%; P < 0.001) were reduced after 5 h compared with after 3 h of infusion. Despite these changes, there was no decrease in the phosphorylation state of multiple insulin signaling intermediates [insulin receptor, Akt, AS160 (Akt substrate of 160 kDa), glycogen synthase kinase-3β] over the same time course. In isolated soleus strips taken from control or 1- or 5-h glucose-infused animals, insulin-stimulated 2-deoxyglucose transport was similar, but glycogen synthesis was significantly reduced in the 5-h muscle sample (68% vs. 1-h sample; P < 0.001). These results suggest that the reduced muscle glucose uptake in rats after 5 h of acute hyperglycemia is due more to the metabolic effects of excess glycogen storage than to a defect in insulin signaling or glucose transport.

scholarly journals Effect of acute activation of 5′-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle

2007 ◽  
Vol 102 (3) ◽  
pp. 1007-1013 ◽  
Author(s):  
Licht Miyamoto ◽  
Taro Toyoda ◽  
Tatsuya Hayashi ◽  
Shin Yonemitsu ◽  
Masako Nakano ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Muscle Glycogen ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycolytic Flux ◽  
Ampk Activation ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Isolated Rat

5′-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the α1- and α2-isoforms of AMPK, with a corresponding increase in the rate of 3- O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3- O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.

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