AMP-activated protein kinase activation prevents denervation-induced decline in gastrocnemius GLUT-4.

This study was designed to determine whether the reductions in GLUT-4 seen in 3-day-denervated muscles can be prevented through chemical activation of 5'-AMP-activated protein kinase (AMPK). Muscle AMPK can be chemically activated in rats using subcutaneous injections with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). In this study, the tibial nerve was sectioned on one side; the other was sham operated but without nerve section. Acute injections of AICAR resulted in significantly increased AMPK activity in denervated gastrocnemius but not soleus muscles. Acetyl-CoA carboxylase activity, a reporter of AMPK activation, declined in both gastrocnemius and soleus in both denervated and contralateral muscles. Three days after denervation, GLUT-4 levels were significantly decreased by approximately 40% in gastrocnemius muscles and by approximately 30% in soleus muscles. When rats were injected with AICAR (1 mg/g body wt) for 3 days, the decline in GLUT-4 levels was prevented in denervated gastrocnemius muscles but not in denervated soleus muscles. The extent of denervation-induced muscle atrophy was similar in AICAR-treated vs. saline-treated rats. These studies provide evidence that some effects of denervation may be prevented by chemical activation of the appropriate signaling pathways.     

Regulation of nutrient uptake by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that is a key regulator of energy balance at both the cellular and whole-body level. AMPK acts to stimulate ATP production and reduce ATP consumption when cellular ATP levels fall, thereby normalizing energy balance. Given the central role of AMPK in cellular carbohydrate and lipid metabolism, AMPK activation has been proposed to be a therapeutic target for conditions associated with dysfunctional nutrient metabolism including obesity, type 2 diabetes, hepatic steatosis, cardiovascular diseases and cancer. One way by which increased ATP production can be achieved is by increasing the supply of nutrient substrates. In the 1990s, AMPK activation was demonstrated to stimulate glucose uptake in striated muscle, thereby improving substrate supply for ATP production. Subsequently AMPK activation was postulated to underlie the increase in glucose uptake that occurs during muscle contraction. More recently, however, several lines of evidence have demonstrated that AMPK activation is unlikely to be required for contraction-mediated glucose uptake. Furthermore, despite the importance of AMPK in cellular and whole-body metabolism, far fewer studies have investigated either the role of AMPK in glucose uptake by non-muscle tissues or whether AMPK regulates the uptake of fatty acids. In the present review, we discuss the role of AMPK in nutrient uptake by tissues, focusing on glucose uptake out with muscle and fatty acid uptake.     

Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle.

AMP-activated protein kinase (AMPK) is emerging as an important energy-sensing/signaling system in skeletal muscle. This kinase is activated allosterically by 5'-AMP and inhibited allosterically by creatine phosphate. Phosphorylation of AMPK by an upstream kinase, AMPK kinase (also activated allosterically by 5'-AMP), results in activation. It is activated in both rat and human muscle in response to muscle contraction, the extent of activation depending on work rate and muscle glycogen concentration. AMPK can also be activated chemically in resting muscle with 5-aminoimidazole-4-carboxamide-riboside, which enters the muscle and is phosphorylated to form ZMP, a nucleotide that mimics the effect of 5'-AMP. Once activated, AMPK is hypothesized to phosphorylate proteins involved in triggering fatty acid oxidation and glucose uptake. Evidence is also accumulating for a role of AMPK in inducing some of the adaptations to endurance training, including the increase in muscle GLUT-4, hexokinase, uncoupling protein 3, and some of the mitochondrial oxidative enzymes. It thus appears that AMPK has the capability of monitoring intramuscular energy charge and then acutely stimulating fat oxidation and glucose uptake to counteract the increased rates of ATP utilization during muscle contraction. In addition, this system may have the capability of enhancing capacity for ATP production when the muscle is exposed to endurance training.     

Regulation of AMP-activated protein kinase and acetyl-CoA carboxylase phosphorylation by palmitate in skeletal muscle cells

The purpose of this study was to investigate the effects of long-chain fatty acids (LCFAs) on AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation and beta-oxidation in skeletal muscle. L6 rat skeletal muscle cells were exposed to various concentrations of palmitate (1-800 microM). Subsequently, ACC and AMPK phosphorylation and fatty acid oxidation were measured. A 2-fold increase in both AMPK and ACC phosphorylation was observed in the presence of palmitate concentrations as low as 10 microM, which was also accompanied by a significant increase in fatty acid oxidation. The effect of palmitate on AMPK and ACC phosphorylation was dose-dependent, reaching maximum increases of 3.5- and 4.5-fold, respectively. Interestingly, ACC phosphorylation was coupled with AMPK activation at palmitate concentrations ranging from 10 to 100 microM; however, at concentrations >200 microM, ACC phosphorylation and fatty acid oxidation remained high even after AMPK phosphorylation was completely prevented by the use of a selective AMPK inhibitor. This indicates that LCFAs regulate ACC activity by AMPK-dependent and -independent mechanisms, based on their abundance in skeletal muscle cells. Here, we provide novel evidence that the AMPK/ACC pathway may operate as a mechanism to sense and respond to the lipid energy charge of skeletal muscle cells.     

Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A

Winder, W. W., H. A. Wilson, D. G. Hardie, B. B. Rasmussen,C. A. Hutber, G. B. Call, R. D. Clayton, L. M. Conley, S. Yoon, and B. Zhou. Phosphorylation of rat muscle acetyl-CoA carboxylase byAMP-activated protein kinase and protein kinase A. J. Appl. Physiol. 82(1): 219-225, 1997This studywas designed to compare functional effects of phosphorylation of muscleacetyl-CoA carboxylase (ACC) by adenosine 3,5-cyclicmonophosphate-dependent protein kinase (PKA) and by AMP-activatedprotein kinase (AMPK). Muscle ACC (272 kDa) was phosphorylated and thensubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresisfollowed by autoradiography. Functional effects of phosphorylation weredetermined by measuring ACC activity at different concentrations ofeach of the substrates and of citrate, an activator of the enzyme. Themaximal velocity(V_max) and theMichaelis constants(K_m) for ATP,acetyl-CoA, and bicarbonate were unaffected by phosphorylation by PKA.Phosphorylation by AMPK increased theK_m for ATP andacetyl-CoA. Sequential phosphorylation by PKA and AMPK, first withoutlabel and second with label, appeared to reduce the extent of label incorporation, regardless of the order. The activation constant (K_a) forcitrate activation was increased to the same extent by AMPKphosphorylation, regardless of previous or subsequent phosphorylation by PKA. Thus muscle ACC can be phosphorylated by PKA but with noapparent functional effects on the enzyme. AMPK appears to be the moreimportant regulator of muscle ACC.

     

AMP-activated protein kinase kinase activity and phosphorylation of AMP-activated protein kinase in contracting muscle of sedentary and endurance-trained rats

This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) in muscles from nontrained and endurance-trained rats. Rats were trained 5 days/wk, 2 h/day for 8 wk at a final intensity of 32 m/min up a 15% grade with 30-s sprints at 53 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in trained and nontrained rats for 5 min at frequencies of 0.4/s and 1/s. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately twofold in response to training. Phosphorylation of AMP-activated protein kinase (AMPK) determined by Western blot and AMPK activity of immunoprecipitates (both isoforms) was increased at both stimulation rates in both trained and nontrained muscles. AMPKK activity was 73% lower in resuspended polyethylene glycol precipitates of muscle extracts from the trained compared with nontrained rats. AMPKK activity did not increase in either trained or nontrained muscle in response to electrical stimulation, even though phospho-AMPK did increase. These results suggest that AMPKK is activated during electrical stimulation of both trained and nontrained muscle by mechanisms other than covalent modification.     

AMP-activated protein kinase regulates the expression of monocarboxylate transporter 4 in skeletal muscle

AimsThe aim of this study was to determine the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, on monocarboxylate transporter 4 (MCT4) expression in rat skeletal muscle and a prototypic embryonal rhabdomyosarcoma cell line (RD cells).Main methodsWe examined the alteration in Glucose transporter 4 (GLUT4) and MCT4 mRNA levels by quantitative real-time PCR. Alteration in GLUT4 and MCT4 protein levels was examined by Western blotting.Key findingsIn an in vivo study, AICAR increased MCT4 mRNA and protein levels in a fiber-type specific manner. In an in vitro study, AICAR increased MCT4 mRNA and protein levels. Moreover, AICAR-induced MCT4 expression was blocked by Compound C, an AMPK inhibitor.SignificanceIn this study, we found that AMPK activation induced expression of MCT4 in RD cells and rat skeletal muscle in a fiber-type specific manner. These results indicate the possible involvement of an AMPK-mediated pathway associated with MCT4 expression in skeletal muscle.     

Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase β (CaMKKβ) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease in basal AMPK activity and a decrease of AMPK activity in the presence of the AMPK activator phenformin. We also demonstrate that stimulation of 3T3L1 adipocytes with intracellular [Ca(2+) ]-raising agents results in an activation of the AMPK pathway. The inhibition of CaMKK isoforms, particularly CaMKKβ, by the inhibitor STO-609 or by siRNAs, blocked Ca(2+) -, but not phenformin-, AICAR-, or forskolin-induced activation of AMPK, indicating that CaMKK activated AMPK in response to Ca(2+) . Collectively, we show that LKB1 is required to maintain normal AMPK-signaling in non-stimulated adipocytes and in the presence of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes.     

Regulation of cGMP-dependent protein kinase expression by Rho and Kruppel-like transcription factor-4

Type I cGMP-dependent protein kinase (PKG I) plays a major role in vascular homeostasis by mediating smooth muscle relaxation in response to nitric oxide, but little is known about the regulation of PKG I expression in smooth muscle cells. We found opposing effects of RhoA and Rac1 on cellular PKG I expression: (i) cell density-dependent changes in PKG I expression varied directly with Rac1 activity and inversely with RhoA activity; (ii) RhoA activation by calpeptin suppressed PKG I, whereas RhoA down-regulation by small interfering RNA increased PKG I expression; and (iii) PKG I promoter activity was suppressed in cells expressing active RhoA or Rho-kinase but was enhanced in cells expressing active Rac1 or a dominant negative RhoA. Sp1 consensus sequences in the PKG I promoter were required for Rho regulation and bound nuclear proteins in a cell density-dependent manner, including the Krüppel-like factor 4 (KLF4). KLF4 was identified as a major trans-acting factor at two proximal Sp1 sites; active RhoA suppressed KLF4 DNA binding and trans-activation potential on the PKG I promoter. Experiments with actin-binding agents suggested that RhoA could regulate KLF4 via its ability to induce actin polymerization. Regulation of PKG I expression by RhoA may explain decreased PKG I levels in vascular smooth muscle cells found in some models of hypertension and vascular injury.     

AMP-activated protein kinase regulates beta-catenin transcription via histone deacetylase 5

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; it is inhibited under obese conditions and is activated by exercise and by many anti-diabetic drugs. Emerging evidence also suggests that AMPK regulates cell differentiation, but the underlying mechanisms are unclear. We hypothesized that AMPK regulates cell differentiation via altering β-catenin expression, which involves phosphorylation of class IIa histone deacetylase 5 (HDAC5). In both C3H10T1/2 cells and mouse embryonic fibroblasts (MEFs), AMPK activity was positively correlated with β-catenin content. Chemical inhibition of HDAC5 increased β-catenin mRNA expression. HDAC5 overexpression reduced and HDAC5 knockdown increased H3K9 acetylation and cellular β-catenin content. HDAC5 formed a complex with myocyte enhancer factor-2 to down-regulate β-catenin mRNA expression. AMPK phosphorylated HDAC5, which promoted HDAC5 exportation from the nucleus; mutation of two phosphorylation sites in HDAC5, Ser-259 and -498, abolished the regulatory role of AMPK on β-catenin expression. In conclusion, AMPK promotes β-catenin expression through phosphorylation of HDAC5, which reduces HDAC5 interaction with the β-catenin promoter via myocyte enhancer factor-2. Thus, the data indicate that AMPK regulates cell differentiation and development via cross-talk with the wingless and Int (Wnt)/β-catenin signaling pathway.     

Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle.

Muscle contraction causes an increase in activity of 5'-AMP-activated protein kinase (AMPK). This study was designed to determine whether chronic chemical activation of AMPK will increase mitochondrial enzymes, GLUT-4, and hexokinase in different types of skeletal muscle of resting rats. In acute studies, rats were subcutaneously injected with either 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mg/g body wt) in 0.9% NaCl or with 0.9% NaCl alone and were then anesthetized for collection and freezing of tissues. AMPK activity increased in the superficial, white region of the quadriceps and in soleus muscles but not in the deep, red region of the quadriceps muscle. Acetyl-CoA carboxylase (ACC) activity, a target for AMPK, decreased in all three muscle types in response to AICAR injection but was lowest in the white quadriceps. In rats given daily, 1 mg/g body wt, subcutaneous injections of AICAR for 4 wk, activities of citrate synthase, succinate dehydrogenase, and malate dehydrogenase were increased in white quadriceps and soleus but not in red quadriceps. Cytochrome c and delta-aminolevulinic acid synthase levels were increased in white, but not red, quadriceps. Carnitine palmitoyl-transferase and hydroxy-acyl-CoA dehydrogenase were not significantly increased. Hexokinase was markedly increased in all three muscles, and GLUT-4 was increased in red and white quadriceps. These results suggest that chronic AMPK activation may mediate the effects of muscle contraction on some, but not all, biochemical adaptations of muscle to endurance exercise training.     

Regulation of Na+-coupled glucose carrier SGLT1 by AMP-activated protein kinase

Abstract

AMP-activated protein kinase (AMPK), a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilization. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na⁺-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active ^γR70QAMPK (α1β1γ1(R70Q)) alone, the addition of glucose to the extracellular bath generated a current (I_g), which was half maximal (K_M) at ≈ 650 μM glucose concentration. Coexpression of ^γR70QAMPK did not affect K_M but significantly enhanced the maximal current (≈ 1.7 fold). Coexpression of wild type AMPK or the kinase dead ^αK45RAMPK mutant (α1(K45R)β1γ1) did not appreciably affect I_g. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 μM) enhanced the SGLT1 protein abundance in the cell membrane of Caco2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na⁺-coupled glucose transport.     

Regulation of AMP-activated protein kinase by a pseudosubstrate sequence on the gamma subunit

The AMP-activated protein kinase (AMPK) system monitors cellular energy status by sensing AMP and ATP, and is a key regulator of energy balance at the cellular and whole-body levels. AMPK exists as heterotrimeric alphabetagamma complexes, and the gamma subunits contain two tandem domains that bind the regulatory nucleotides. There is a sequence in the first of these domains that is conserved in gamma subunit homologues in all eukaryotes, and which resembles the sequence around sites phosphorylated on target proteins of AMPK, except that it has a non-phosphorylatable residue in place of serine. We propose that in the absence of AMP this pseudosubstrate sequence binds to the active site groove on the alpha subunit, preventing phosphorylation by the upstream kinase, LKB1, and access to downstream targets. Binding of AMP causes a conformational change that prevents this interaction and relieves the inhibition. We present several lines of evidence supporting this hypothesis.     

Chronic activation of 5'-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle.

This study was designed to determine whether chronic chemical activation of AMP-activated protein kinase (AMPK) would increase glucose transporter GLUT-4 and hexokinase in muscles similarly to periodic elevation of AMPK that accompanies endurance exercise training. The adenosine analog, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), has previously been shown to be taken up by cells and phosphorylated to form a compound (5-aminoimidazole-4-carboxamide ribonucleotide) that mimics the effect of AMP on AMPK. A single injection of AICAR resulted in a marked increase in AMPK in epitrochlearis and gastrocnemius/plantaris muscles 60 min later. When rats were injected with AICAR (1 mg/g body wt) for 5 days in succession and were killed 1 day after the last injection, GLUT-4 was increased by 100% in epitrochlearis muscle and by 60% in gastrocnemius muscle in response to AICAR. Hexokinase was also increased approximately 2. 5-fold in the gastrocnemius/plantaris. Gastrocnemius glycogen content was twofold higher in AICAR-treated rats than in controls. Chronic chemical activation of AMPK, therefore, results in increases in GLUT-4 protein, hexokinase activity, and glycogen, similarly to those induced by endurance training.