AMP-activated Protein Kinase Substrate Research Articles

The Outcome of Renal Ischemia-Reperfusion Injury Is Unchanged in AMPK-β1 Deficient Mice

AimActivation of the master energy-regulator AMP-activated protein kinase (AMPK) in the heart reduces the severity of ischemia-reperfusion injury (IRI) but the role of AMPK in renal IRI is not known. The aim of this study was to determine whether activation of AMPK by acute renal ischemia influences the severity of renal IRI.MethodsAMPK expression and activation and the severity of renal IRI was studied in mice lacking the AMPK β1 subunit and compared to wild type (WT) mice.ResultsBasal expression of activated AMPK, phosphorylayed at αThr172, was markedly reduced by 96% in AMPK-β1−/− mice. Acute renal ischaemia caused a 3.2-fold increase in α1-AMPK activity and a 2.5-fold increase in α2-AMPK activity (P<0.001) that was associated with an increase in AMPK phosphorylation of the AMPK-α subunit at Thr172 and Ser485, and increased inhibitory phosphorylation of the AMPK substrate acetyl-CoA carboxylase. After acute renal ischemia AMPK activity was reduced by 66% in AMPK-β1−/− mice compared with WT. There was no difference, however, in the severity of renal IRI at 24-hours between AMPK-β1−/− and WT mice, as measured by serum urea and creatinine and histological injury score. In the heart, macrophage migration inhibitory factor (MIF) released during IRI contributes to AMPK activation and protects from injury. In the kidney, however, no difference in AMPK activation by acute ischemia was observed between MIF−/− and WT mice. Compared with the heart, expression of the MIF receptor CD74 was found to be reduced in the kidney.ConclusionThe failure of AMPK activation to influence the outcome of IRI in the kidney contrasts with what is reported in the heart. This difference might be due to a lack of effect of MIF on AMPK activation and lower CD74 expression in the kidney.

A Surface Plasmon Resonance-Based Two-Dimensional Screen for Protein Kinase Substrates Identifies Fumarase as AMPK Target

This study describes a novel in vitro two-dimensional screening approach for kinase substrates that combines a biophysical interaction assay based on surface plasmon resonance and a standard in vitro phosphorylation assay. The interaction assay was optimized with appropriate prepurification procedures on a Biacore instrument. It selects for substrates interacting with a specific kinase, and can thus identify substrates that are preferentially phosphorylated e.g. by a specific kinase isoform. This approach was applied to isoforms of the heterotrimeric AMP-activated protein kinase (AMPK). AMPK is an emerging central cellular signaling hub in energy homeostasis and proliferation, but its signaling network is still incompletely understood. Using soluble rat liver proteins and full-length AMPK α2-β2-γ1 complex, several putative AMPK substrates were identified by mass spectrometry. One of them, fumarate hydratase (fumarase), was confirmed as an in vitro AMPK target which preferentially interacted with and was phosphorylated by the AMPKα2 isoform as shown by yeast-two-hybrid and in vitro phosphorylation assays. AMPK-mediated phosphorylation of fumarate hydratase led to significant activation of enzymatic activity in vitro and in vivo, suggesting that it is a bona fide AMPK substrate. This may have different physiological consequences, since the enzyme has a dual localization in the mitochondrial matrix and the cytosol. [AK and CP contributed equally to this work]

Chemical Genetic Screen for AMPKα2 Substrates Uncovers a Network of Proteins Involved in Mitosis

The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK invivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.

IRE1-Dependent Activation of AMPK in Response to Nitric Oxide

While there can be detrimental consequences of nitric oxide production at pathological concentrations, eukaryotic cells have evolved protective mechanisms to defend themselves against this damage. The unfolded-protein response (UPR), activated by misfolded proteins and oxidative stress, is one adaptive mechanism that is employed to protect cells from stress. Nitric oxide is a potent activator of AMP-activated protein kinase (AMPK), and AMPK participates in the cellular defense against nitric oxide-mediated damage in pancreatic β-cells. In this study, the mechanism of AMPK activation by nitric oxide was explored. The known AMPK kinases LKB1, CaMKK, and TAK1 are not required for the activation of AMPK by nitric oxide. Instead, this activation is dependent on the endoplasmic reticulum (ER) stress-activated protein IRE1. Nitric oxide-induced AMPK phosphorylation and subsequent signaling to AMPK substrates, including Raptor, acetyl coenzyme A carboxylase, and PGC-1α, is attenuated in IRE1α-deficient cells. The endoribonuclease activity of IRE1 appears to be required for AMPK activation in response to nitric oxide. In addition to nitric oxide, stimulation of IRE1 endoribonuclease activity with the flavonol quercetin leads to IRE1-dependent AMPK activation. These findings indicate that the RNase activity of IRE1 participates in AMPK activation and subsequent signaling through multiple AMPK-dependent pathways in response to nitrosative stress.

Coptidis Rhizoma Water Extract Stimulates 5′-AMP-Activated Protein Kinase in Rat Skeletal Muscle

Aim Coptidis Rhizoma (CR), the dried rhizomes of Asian herbs (including Coptis chinensis Franch), has been used to treat diabetes mellitus for thousands of years. We explored the possibility that CR acts directly on skeletal muscle, the major organ responsible for glucose homeostasis, and activates 5′-AMP-activated protein kinase (AMPK), a signaling intermediary leading to metabolic enhancement of skeletal muscle. Methods Isolated rat epitrochlearis and soleus muscles were incubated in a buffer containing a CR water extract (CE), and activation of AMPK and related events were examined. Results In response to CE treatment, phosphorylation of Thr 172 at the catalytic α subunit of AMPK, an essential step for full kinase activation, increased in both muscles. Phosphorylation of Ser 79 of acetyl CoA carboxylase (ACC), an endogenous substrate of AMPK, increased concomitantly. Analysis of isoform-specific AMPK activity revealed that CE activated both the α1 and α2 isoforms of the catalytic subunit. Importantly, the maximal effect of CE on AMPK phosphorylation was significantly greater than that of berberine (BBR), indicating that the action of CE is not totally ascribed to BBR. Conclusion We propose that CE is an acute activator of AMPK in both fast- and slow-twitch skeletal muscles.

AMP-activated Protein Kinase (AMPK) Activation and Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Induce Ca2+-independent Deposition of Tight Junction Components at the Plasma Membrane

Extracellular Ca(2+) is essential for the development of stable epithelial tight junctions. We find that in the absence of extracellular Ca(2+), AMP-activated protein kinase (AMPK) activation and glycogen synthase kinase (GSK)-3β inhibition independently induce the localization of epithelial tight junction components to the plasma membrane. The Ca(2+)-independent deposition of junctional proteins induced by AMPK activation and GSK-3β inhibition is independent of E-cadherin. Furthermore, the nectin-afadin system is required for the deposition of tight junction components induced by AMPK activation, but it is not required for that induced by GSK-3β inhibition. Phosphorylation studies demonstrate that afadin is a substrate for AMPK. These data demonstrate that two kinases involved in regulating cell growth and metabolism act through distinct pathways to influence the deposition of the components of epithelial tight junctions.

Abstract 4071: Sestrins modulate the expression of the metabolic stress sensor AMPK in breast cancer cells

Abstract Introduction: Sestrins (SESN) are stress response mediators that accumulate in cells exposed to genotoxic stresses, such as ultraviolet and ionizing radiation (IR). The three mammalian SESN (1-3) were recently shown to modulate signalling events. SESN1/2 are suggested to interact with and increasing the activity of the metabolic stress sensor AMP-activated protein kinase (AMPK). SESN1/2 have been postulated to act as scaffolding proteins that can bind targets of the AMPK metabolic pathway including AMPKα and TSC2. However, the direct mechanism in which SESN1/2 increase AMPK activity is still being elucidated. Furthermore, the ability of SESNs to modulate the expression of AMPK subunits in untreated or IR treated cells has not been examined. Methods: Immunoprecipitation of the AMPK subunits (α1-2, β1-2, γ1-3) with subunit-specific antibodies was preformed to determine the function AMPK heterotrimer complexes in MCF7 breast cancer cells. Overexpression of full length SESN2 cDNA was achieved in MCF7 cells using tetracycline inducible promoter system. SESN1/2 siRNA was introduced into cells using HiPerFect transfection reagent and the Qiagen protocol. Protein and mRNA levels were measured with western blotting and RT-PRC, respectively. Cells were treated with 0 – 8 Gy IR, and cell survival was measured using clonogenic assays. Results: Through immunoprecipitation we identified that the most prominent AMPK functional complex in MCF7 cells is the α1β1γ1 heterotrimer. SESN2 overexpression increases both mRNA and protein expression levels of AMPKα1 and AMPKβ1, and was co-immunoprecipitated with the AMPKβ1 subunit. Similarly, increasing SENS2 expression increased the phosphorylation levels of AMPKα (Thr172) and AMPKβ1 (Ser108), as well as phosphorylation of the AMPK substrate ACC. Conversely, siRNA against SESN2 decreased both AMPKα1 and AMPKβ1 mRNA and protein levels, while SESN1 siRNA blocked basal and IR-induced phosphorylation of AMPKα (Thr172). In addition, SESN2 overexpression sensitized MCF7 cells to the cytotoxic effects of IR. Conclusions: To date our work suggests that upregulation of SESNs in breast cancer cells modulates AMPK by stabilizing the AMPK (α1β1γ1) heterotrimer and by enhancing the subunit expression of AMPK. We observed that SESN2 was shown to directly interact with the AMPKβ1 subunit and also enhance its Ser108 phosphorylation, an additional marker of AMPK activity. Furthermore, silencing SESN1 expression blocks basal IR-induced AMPKα (Thr172) phosphorylation, while SESN2 overexpression acts as a radiation sensitizer in MCF7 cells. Taken together, this data indicates that SESNs not only regulate AMPK activity, but also play a role in stabilizing this enzymes basal and stress-induced expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2011-4071

Autophagy: Regulation by Energy Sensing

Autophagy is inhibited by the mTOR signaling pathway, which is stimulated by increased amino acid levels. When cellular energy production is compromised, AMP-activated protein kinase is activated, mTOR is inhibited and autophagy is stimulated. Two recent studies have shed light on the molecular mechanism by which AMPK controls autophagic flux.

An active part of Artemisia sacrorum Ledeb. inhibits adipogenesis via the AMPK signaling pathway in 3T3-L1 adipocytes

Artemisia sacrorum Ledeb. (Compositae) (ASL) has long been used in Oriental folk medicine to treat diverse hepatic diseases. In this study, we investigated the effect of ASL on adipocyte differentiation in 3T3-L1 cells. ASL significantly suppressed 3T3-L1 differentiation in a concentration-dependent manner. A significant increase of AMP-activated protein kinase (AMPK) was observed when the cells were treated with ASL. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase, a substrate of AMPK. These effects were abolished by pre-treatment with the AMPK inhibitor, compound C. In addition, ASL down-regulated the adipogenesis-related gene expression of the sterol regulatory element-binding protein 1c (SREBP1c) and its target genes, such as fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1) and glycerol-3-phosphate acyltransferase (GPAT) in a concentration-dependent manner. These effects were abolished by pre-treatment with compound C. ASL significantly reduced the gene expression of the peroxisome proliferator-activated receptor γ (PPARγ) and of the CCAAT/enhancer binding protein-α (C/EBPα), two key transcription factors in adipogenesis. Meanwhile, adipocyte fatty acid binding protein (aP2) gene expression was also reduced in a concentration-dependent manner. These findings indicated that ASL exerts anti-adipogenic activity via AMPK activation and may act to prevent obesity.

Novel candidate substrates of AMP-activated protein kinase identified in red blood cell lysates

AMPK is a metabolic stress-sensing kinase with important functions for red blood cell (RBC) survival. By using a proteomic approach, we identified putative AMPK targets in hemoglobin-depleted lysates of RBC, including metabolic enzymes, cytoskeletal proteins and enzymes involved in the oxidative stress response. These data tie in with the phenotypic observations of AMPKα1-deficient RBC and provide reference for future studies.

AMP-activated Protein Kinase Antagonizes Pro-apoptotic Extracellular Signal-regulated Kinase Activation by Inducing Dual-specificity Protein Phosphatases in Response to Glucose Deprivation in HCT116 Carcinoma

Mitogen-activated protein kinase (MAPK) pathways are involved in the regulation of cellular responses, including cell proliferation, differentiation, cell growth, and apoptosis. Because these responses are tightly related to cellular energy level, AMP-activated protein kinase (AMPK), which plays an essential role in energy homeostasis, has emerged as another key regulator. In the present study, we demonstrate a novel signal network between AMPK and MAPK in HCT116 human colon carcinoma. Glucose deprivation activated AMPK and three MAPK subfamilies, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK. Under these conditions, inhibition of endogenous AMPK by expressing a dominant-negative form significantly potentiated ERK activation, indicating that glucose deprivation-induced AMPK is specifically antagonizing ERK activity in HCT116 cells. Moreover, we provide novel evidence that AMPK activity is critical for p53-dependent expression of dual-specificity phosphatase (DUSP) 1 & 2, which are negative regulators of ERK. Notably, ERK exhibits pro-apoptotic effects in HCT116 cells under glucose deprivation. Collectively, our data suggest that AMPK protects HCT116 cancer cells from glucose deprivation, in part, via inducing DUSPs, which suppresses pro-apoptotic ERK, further implying that a signal network between AMPK and ERK is a critical regulatory point in coupling the energy status of the cell to the regulation of cell survival.

Oligomycin-induced Bioenergetic Adaptation in Cancer Cells with Heterogeneous Bioenergetic Organization

Cancer cells constantly adapt to oxidative phosphorylation (OXPHOS) suppression resulting from hypoxia or mitochondria defects. Under the OXPHOS suppression, AMP-activated protein kinase (AMPK) regulates global metabolism adjustments, but its activation has been found to be transient. Whether cells can maintain cellular ATP homeostasis and survive beyond the transient AMPK activation is not known. Here, we study the bioenergetic adaptation to the OXPHOS inhibitor oligomycin in a group of cancer cells. We found that oligomycin at 100 ng/ml completely inhibits OXPHOS activity in 1 h and induces various levels of glycolysis gains by 6 h, from which we calculate the bioenergetic organizations of cancer cells. In glycolysis-dominant cells, oligomycin does not induce much energy stress as measured by glycolysis acceleration, ATP imbalance, AMPK activation, AMPK substrate acetyl-CoA carboxylase phosphorylation at Ser(79), and cell growth inhibition. In OXPHOS-dependent LKB1 wild type cells, oligomycin induces 5-8% ATP drops and transient AMPK activation during the initial 1-2 h. After AMPK activation is completed, oligomycin-induced increase of acetyl-CoA carboxylase phosphorylation at Ser(79) is still detected, and cellular ATP is back at preoligomycin treatment levels by sustained elevation of glycolysis. Cell growth, however, is inhibited without an increase in cell death and alteration in cell cycle distribution. In OXPHOS-dependent LKB1-null cells, no AMPK activation by oligomycin is detected, yet cells still show a similar adaptation. We also demonstrate that the adaptation to oligomycin does not invoke activation of hypoxia-induced factor. Our data suggest that cancer cells may grow and survive persistent OXPHOS suppression through an as yet unidentified regulatory mechanism.

Korean red ginseng attenuates hepatic lipid accumulation via AMPK activation in human hepatoma cells

In this study, we examined Korean red ginseng (KRG) extract affects on the lipid metabolism in HepG2 cells. Increase in AMP-activated protein kinase (AMPK) phosphorylation was observed when the cells were treated with KRG. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA caboxylase (ACC), a substrate of AMPK. KRG down-regulated gene expressions of sterol regulatory element binding protein 1c (SREBP1c) and its target proteins, such as fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD1) in time- and dose-dependent fashions. In contrast, gene expressions of peroxisome proliferator-activated receptor α (PPARα) and CD36 were increased. These effects were reversed in the presence of compound C, an AMPK inhibitor. However, there were no differences in gene expressions of SREBP2, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, and low-density-lipoprotein receptor (LDLR). Taken together, KRG induced supression of SREBP1c and activation of PPARα via AMPK and these effects seem to be one of anti-hyperlipidemic mechanism of KRG in HepG2 cells.

Acute signalling responses to intense endurance training commenced with low or normal muscle glycogen

We have previously demonstrated that well-trained subjects who completed a 3 week training programme in which selected high-intensity interval training (HIT) sessions were commenced with low muscle glycogen content increased the maximal activities of several oxidative enzymes that promote endurance adaptations to a greater extent than subjects who began all training sessions with normal glycogen levels. The aim of the present study was to investigate acute skeletal muscle signalling responses to a single bout of HIT commenced with low or normal muscle glycogen stores in an attempt to elucidate potential mechanism(s) that might underlie our previous observations. Six endurance-trained cyclists/triathletes performed a 100 min ride at approximately 70% peak O(2) uptake (AT) on day 1 and HIT (8 x 5 min work bouts at maximal self-selected effort with 1 min rest) 24 h later (HIGH). Another six subjects, matched for fitness and training history, performed AT on day 1 then 1-2 h later, HIT (LOW). Muscle biopsies were taken before and after HIT. Muscle glycogen concentration was higher in HIGH versus LOW before the HIT (390 +/- 28 versus 256 +/- 67 micromol (g dry wt)(1)). After HIT, glycogen levels were reduced in both groups (P < 0.05) but HIGH was elevated compared with LOW (229 +/- 29 versus 124 +/- 41 micromol (g dry wt)(1); P < 0.05). Phosphorylation of 5 AMP-activated protein kinase (AMPK) increased after HIT, but the magnitude of increase was greater in LOW (P < 0.05). Despite the augmented AMPK response in LOW after HIT, selected downstream AMPK substrates were similar between groups. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was unchanged for both groups before and after the HIT training sessions. We conclude that despite a greater activation AMPK phosphorylation when HIT was commenced with low compared with normal muscle glycogen availability, the localization and phosphorylation state of selected downstream targets of AMPK were similar in response to the two interventions.

Suppression of 5′-AMP-activated protein kinase activity does not impair recovery of contractile function during reperfusion of ischemic hearts

Activation of 5'-AMP-activated protein kinase (AMPK) may benefit the heart during ischemia-reperfusion by increasing energy production. While AMPK stimulates glycolysis, mitochondrial oxidative metabolism is the major source of ATP production during reperfusion of ischemic hearts. Stimulating AMPK increases mitochondrial fatty acid oxidation, but this is usually accompanied by a decrease in glucose oxidation, which can impair the functional recovery of ischemic hearts. To examine the relationship between AMPK and cardiac energy substrate metabolism, we subjected isolated working mouse hearts expressing a dominant negative (DN) alpha(2)-subunit of AMPK (AMPK-alpha(2) DN) to 20 min of global no-flow ischemia and 40 min of reperfusion with Krebs-Henseleit solution containing 5 mM [U-(14)C]glucose, 0.4 mM [9, 10-(3)H]palmitate, and 100 microU/ml insulin. AMPK-alpha(2) DN hearts had reduced AMPK activity at the end of reperfusion (82 +/- 9 vs. 141 +/- 7 pmol.mg(-1).min(-1)) with no changes in high-energy phosphates. Despite this, AMPK-alpha(2) DN hearts had improved recovery of function during reperfusion (14.9 +/- 0.8 vs. 9.4 +/- 1.4 beats.min(-1).mmHg.10(-3)). During reperfusion, fatty acid oxidation provided 44.0 +/- 2.8% of total acetyl-CoA in AMPK-alpha(2) DN hearts compared with 55.0 +/- 3.2% in control hearts. Since insulin can inhibit both AMPK activation and fatty acid oxidation, we also examined functional recovery in the absence of insulin. Functional recovery was similar in both groups despite a decrease in AMPK activity and a decreased reliance on fatty acid oxidation during reperfusion (66.4 +/- 9.4% vs. 85.3 +/- 4.3%). These data demonstrate that the suppression of cardiac AMPK activity does not produce an energetically compromised phenotype and does not impair, but may in fact improve, the recovery of function after ischemia.

Determination of AMP-activated protein kinase phosphorylation sites in recombinant protein expressed using the pET28a vector: A cautionary tale

AMP-activated protein kinase (AMPK) is responsible for sensing of the cell’s energetic status and it phosphorylates numerous substrates involved in anabolic and catabolic processes as well as interacting with signaling cascades. Mutations in the gene encoding the γ2 regulatory subunit have been shown to cause hypertrophic cardiomyopathy (HCM) with conduction abnormalities. As part of a study to examine the role of AMPK in the heart, we tested whether specific domains of the thick filament component cardiac myosin binding protein-C (cMyBP-C) were good in vitro AMPK substrates. The commercially available pET28a expression vector was used to generate a recombinant form of the cMyBP-C C8 domain as a fusion protein with a hexahistidine tag. In vitro phosphorylation with activated kinase showed that the purified fusion protein was a good AMPK substrate, phosphorylated at a similar rate to the control SAMS peptide and with phosphate incorporation specifically in serine residues. However, subsequent analysis of alanine replacement mutants and thrombin digestion revealed that the strong AMPK phosphorylation site was contained within the thrombin cleavage sequence encoded by the vector. As this sequence is common to many commercial pET vectors, caution is advised in the mapping of AMPK phosphorylation sites when this sequence is present.

Compound K, Intestinal Metabolite of Ginsenoside, Attenuates Hepatic Lipid Accumulation via AMPK Activation in Human Hepatoma Cells

Compound K (CK) is a major intestinal metabolite of ginsenosides derived from ginseng radix. Although antidiabetic and antihyperlipidemic activities of CK have been investigated in recent years, action mechanism of CK remains poorly understood. Therefore, we examined whether CK affects the lipid metabolism in insulin-resistant HepG2 human hepatoma cells. In this study, a significant increase in AMP-activated protein kinase (AMPK) was observed when the cells were treated with CK. Activation of AMPK was also demonstrated by measuring the phosphorylation of acetyl-CoA carboxylase (ACC), a substrate of AMPK. CK attenuated gene expression of sterol regulatory element-binding protein 1c (SREBP1c) in time- and dose-dependent manners. Genes for fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1), well-known target molecules of SREBP1c, were also suppressed. In contrast, gene expressions of peroxisome proliferator-activated receptor alpha (PPAR-alpha) and CD36 were increased. These effects were reversed by treatment of compound C, an AMPK inhibitor. However, there were no differences in gene expressions of SREBP2, hydroxymethyl glutaryl CoA reductase (HMGR), and low-density-lipoprotein receptor (LDLR). Taken together, AMPK mediates CK induced suppression and activation of SREBP1c and PPAR-alpha, respectively, and these effects seem to be one of antidiabetic and/or antihyperlipidemic mechanisms of CK in insulin-resistant HepG2 human hepatoma cells.

Inhibition of Contraction-Stimulated AMP-Activated Protein Kinase Inhibits Contraction-Stimulated Increases in PAS-TBC1D1 and Glucose Transport Without Altering PAS-AS160 in Rat Skeletal Muscle

OBJECTIVEPhosphorylation of two members of the TBC1 domain family of proteins, Akt substrate of 160 kDa (AS160, also known as TBC1D4) and TBC1D1, has been implicated in the regulation of glucose transport in skeletal muscle. Insulin-stimulated phosphorylation (measured using the phospho-Akt substrate [PAS] antibody) of AS160 and TBC1D1 appears to occur in an Akt-dependent manner, but the kinases responsible for contraction-stimulated PAS-AS160 and PAS-TBC1D1 remain unclear. AMP-activated protein kinase (AMPK) and Akt, both activated by contraction, can each phosphorylate AS160 and TBC1D1 in cell-free assays.RESEARCH DESIGN AND METHODSTo evaluate the roles of AMPK and Akt on insulin- or contraction-stimulated PAS-AS160, PAS-TBC1D1, and glucose transport, rat epitrochlearis was incubated with and without compound C (inhibitor of AMPK) or Wortmannin (inhibitor of phosphatidylinositol [PI] 3-kinase, which is upstream of Akt) before and during insulin stimulation or contraction.RESULTSInsulin-stimulated glucose transport and phosphorylation of both AS160 and TBC1D1 were completely inhibited by Wortmannin. Wortmannin eliminated contraction stimulation of phospho-Ser21/9glycogen synthase kinase 3α/β (pGSK3; Akt substrate) and PAS-AS160 but did not significantly alter pAMPK, phospho-Ser79acetyl CoA carboxylase (pACC; AMPK substrate), PAS-TBC1D1, or glucose transport in contraction-stimulated muscle. Compound C completely inhibited contraction-stimulated pACC and PAS-TBC1D1 and partially blocked glucose transport, but it did not significantly alter pAkt, pGSK3, or PAS-AS160.CONCLUSIONSThese data suggest that 1) insulin stimulates glucose transport and phosphorylation of AS160 and TBC1D1 in a PI 3-kinase/Akt–dependent manner, 2) contraction stimulates PAS-AS160 (but not PAS-TBC1D1 or glucose transport) in a PI 3-kinase/Akt–dependent manner, and 3) contraction stimulates PAS-TBC1D1 and glucose transport (but not PAS-AS160) in an AMPK-dependent manner.

A myosin II ATPase inhibitor reduces force production, glucose transport, and phosphorylation of AMPK and TBC1D1 in electrically stimulated rat skeletal muscle

Contraction-stimulated glucose transport by skeletal muscle appears to be caused by the cumulative effects of multiple inputs [potentially including AMP-activated protein kinase (AMPK), Ca(2+) flux, and force production], making it challenging to isolate the roles of these putative regulatory factors. To distinguish the effects of force production from the direct consequences of Ca(2+) flux, the predominantly type II rat epitrochlearis muscle was incubated without (vehicle) or with N-benzyl-p-toluenesulfonamide (BTS), a highly specific myosin II ATPase inhibitor that prevents force production by electrically stimulated (ES) type II fibers without altering cytosolic Ca(2+). In ES muscles, BTS vs. vehicle had an 84% reduction in force production and a 57% decrement in contraction-stimulated 3-O-methylglucose transport (3MGT). BTS did not alter the ES increase in phosphorylation of CaMKII (indicative of cytosolic Ca(2+)) or the amount of glycogen depletion. ES caused significant reductions in ATP (48%) and phosphocreatine (67%) concentrations for vehicle-treated muscles. For BTS-treated muscles, ES did not reduce ATP and caused only a 42% decrease in phosphocreatine. There was an ES increase in phosphorylation of AMPK, acetyl-CoA carboxylase (an AMPK substrate), and TBC1D1 for vehicle-treated muscles but not for BTS-treated muscles. These results point toward an essential role for tension-related events, including AMPK activation, in the 57% contraction-stimulated increase in 3MGT that was inhibited by BTS and further suggest a possible role for TBC1D1 phosphorylation. Non-tension-related events (e.g., increased cytosolic Ca(2+) rather than increased AMPK and TBC1D1 phosphorylation) are implicated in the contraction-stimulated increase in 3MGT that persisted in the presence of BTS.

Myosin light chains are not a physiological substrate of AMPK in the control of cell structure changes

The kinetics of myosin regulatory light chain (MLC) phosphorylation by recombinant AMP-activated protein kinase (AMPK) were compared with commercial AMPK from rat liver and smooth muscle myosin light chain kinase (smMLCK). With identical amounts of activity units, initial rates of phosphorylation of MLC were at least 100-fold less with recombinant AMPK compared to smMLCK, whereas with rat liver AMPK significant phosphorylation was seen. In Madin-Darby Canine Kidney cells, AMPK activation led to an increase in MLC phosphorylation, which was decreased by a Rho kinase inhibitor without affecting AMPK activation. Therefore, MLC phosphorylation during energy deprivation does not result from direct phosphorylation by AMPK. Structured summaryMINT-6800264: smMLCK (uniprotkb:P11799) phosphorylates (MI:0217) MLC (uniprotkb:P08590) by protein kinase assay (MI:0424)MINT-6800252: AMPK (uniprotkb:Q13131) phosphorylates (MI:0217) ACC2 (uniprotkb:000763) by protein kinase assay (MI:0424)