Palmitic acid acutely stimulates glucose uptake via activation of Akt and ERK1/2 in skeletal muscle cells

Abstract Chronic exposure to saturated fatty acids can cause insulin resistance. However, the acute effects of fatty acids are not clear and need to be elucidated because plasma fatty acid concentrations fluctuate postprandially. Here, we present the acute effects of palmitate (PA) on skeletal muscle cells and their underlying molecular mechanisms. Immuno­fluorescence results showed that PA rapidly induced GLUT4 translocation and stimulated glucose uptake in rat skeletal muscle cell line L6. Phosphorylation of AMP-activated protein kinase (AMPK), Akt, and extracellular signal-related kinase1/2 (ERK1/2) was enhanced by PA in a time-dependent manner. Cell surface-bound PA was sufficient to stimulate Akt phosphorylation. The inhibitors of PI3 kinase (PI3K), AMPK, Akt, and ERK1/2 could decrease PA-induced glucose uptake, and PI3K inhibitor decreased AMPK, Akt, and ERK1/2 phosphorylation. Weakening AMPK activity reduced phosphorylation of Akt but not ERK1/2, and Akt inhibitor could not affect ERK1/2 activation either. Meanwhile, ERK1/2 inhibitors had no effect on Akt phosphorylation. Taken together, our data suggest that PA-mediated glucose uptake in skeletal muscle cells may be stimulated by the binding of PA to cell surface and followed by PI3K/AMPK/Akt and PI3K/ERK1/2 pathways independently. Abstract Chronic exposure to saturated fatty acids can cause insulin resistance. However, the acute effects of fatty acids are not clear and need to be elucidated because plasma fatty acid concentrations fluctuate postprandially. Here, we present the acute effects of palmitate (PA) on skeletal muscle cells and their underlying molecular mechanisms. Immuno­fluorescence results showed that PA rapidly induced GLUT4 translocation and stimulated glucose uptake in rat skeletal muscle cell line L6. Phosphorylation of AMP-activated protein kinase (AMPK), Akt, and extracellular signal-related kinase1/2 (ERK1/2) was enhanced by PA in a time-dependent manner. Cell surface-bound PA was sufficient to stimulate Akt phosphorylation. The inhibitors of PI3 kinase (PI3K), AMPK, Akt, and ERK1/2 could decrease PA-induced glucose uptake, and PI3K inhibitor decreased AMPK, Akt, and ERK1/2 phosphorylation. Weakening AMPK activity reduced phosphorylation of Akt but not ERK1/2, and Akt inhibitor could not affect ERK1/2 activation either. Meanwhile, ERK1/2 inhibitors had no effect on Akt phosphorylation. Taken together, our data suggest that PA-mediated glucose uptake in skeletal muscle cells may be stimulated by the binding of PA to cell surface and followed by PI3K/AMPK/Akt and PI3K/ERK1/2 pathways independently. Plasma fatty acid concentrations are much higher in patients with type 2 diabetes than in healthy subjects. Previous studies have shown that chronically elevated fatty acids can cause the body to be unresponsive to insulin and in turn reduces insulin-stimulated glucose uptake (1Savage D.B. Petersen K.F. Shulman G.I. Disordered lipid metabolism and the pathogenesis of insulin resistance.Physiol. Rev. 2007; 87: 507-526Crossref PubMed Scopus (776) Google Scholar, 2McGarry J.D. What if Minkowski had been ageusic? An alternative angle on diabetes.Science. 1992; 258: 766-835Crossref PubMed Scopus (578) Google Scholar). Of the tissues and organs that respond to insulin stimulation, skeletal muscle consumes more than 70% of the plasma glucose, suggesting that whole body plasma glucose concentration is tightly associated to the sensitivity of muscle tissue to insulin (3DeFronzo R.A. Jacot E. Jequier E. Maeder E. Wahren J. Felber J.P. The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization.Diabetes. 1981; 30: 1000-1006Crossref PubMed Scopus (1408) Google Scholar, 4Shulman G.I. Rothman D.L. Jue T. Stein P. DeFronzo R.A. Shulman R.G. Quantitation of muscle glycogen synthesis in normal subjects and subjects with non-insulin-dependent diabetes by 13C nuclear magnetic resonance spectroscopy.N. Engl. J. Med. 1990; 322: 223-230Crossref PubMed Scopus (1056) Google Scholar). Muscle cells ingest fatty acids from the blood and store them as intramyocellular triglycerides (IMTGs) in a cellular organelle lipid droplet (5Martin S. Parton R.G. Lipid droplets: a unified view of a dynamic organelle.Nat. Rev. Mol. Cell Biol. 2006; 7: 373-380Crossref PubMed Scopus (929) Google Scholar, 6Zhang S. Du Y. Wang Y. Liu P. Lipid droplet—a cellular organelle for lipid metabolism.Acta Biophisica Sinica. 2010; 26: 97-105Google Scholar). Mounting evidence indicates that chronically elevated fatty acids in the plasma induce excess storage of IMTG, which in turn plays a key role in the development of insulin resistance (7Pan D.A. Lillioja S. Kriketos A.D. Milner M.R. Baur L.A. Bogardus C. Jenkins A.B. Storlien L.H. Skeletal muscle triglyceride levels are inversely related to insulin action.Diabetes. 1997; 46: 983-990Crossref PubMed Google Scholar, 8Manco M. Mingrone G. Greco A.V. Capristo E. Gniuli D. De Gaetano A. Gasbarrini G. Insulin resistance directly correlates with increased saturated fatty acids in skeletal muscle triglycerides.Metabolism. 2000; 49: 220-223Abstract Full Text PDF PubMed Scopus (141) Google Scholar, 9Roden M. Muscle triglycerides and mitochondrial function: possible mechanisms for the development of type 2 diabetes.Int J Obes (Lond). 2005; 29: S111-S115Crossref PubMed Scopus (117) Google Scholar). Furthermore, Manco et al. (8Manco M. Mingrone G. Greco A.V. Capristo E. Gniuli D. De Gaetano A. Gasbarrini G. Insulin resistance directly correlates with increased saturated fatty acids in skeletal muscle triglycerides.Metabolism. 2000; 49: 220-223Abstract Full Text PDF PubMed Scopus (141) Google Scholar) have suggested that, in addition to IMTG concentration, the IMTG fatty acid fraction pattern, i.e., whether palmitic acid is present rather than oleic acid, is significantly correlated to glucose uptake in muscle tissue. This is consistent with other reports suggesting that saturated fatty acids suppress body insulin sensitivity. During fasting, adipocytes release stored lipids as free fatty acids into the blood to provide an energy source in the animal body. As a result, the concentrations of fatty acids in the blood are not consistent. In fact, fatty acid concentrations vary widely from hour to hour and display waves according to nutritional state and the presence of regulators including hormones, as well as metabolic and neural signals (10Singer P. Godicke W. Voigt S. Hajdu I. Weiss M. Postprandial hyperinsulinemia in patients with mild essential hypertension.Hypertension. 1985; 7: 182-187Crossref PubMed Scopus (167) Google Scholar, 11Coppack S.W. Fisher R.M. Gibbons G.F. Humphreys S.M. McDonough M.J. Potts J.L. Frayn K.N. Postprandial substrate deposition in human forearm and adipose tissues in vivo.Clin. Sci. (Lond.). 1990; 79: 339-386Crossref PubMed Scopus (158) Google Scholar). Although a few studies have reported the acute effects of fatty acids, conclusions concerning how energy-consuming tissues, such as skeletal muscle, respond to acute rises in fatty acid levels have been inconsistent and the underlying molecular mechanisms controlling these responses are still unclear. For example, α-lipoic acid has been shown to enhance basal glucose uptake both in normal and ob/ob mice (12Eason R.C. Archer H.E. Akhtar S. Bailey C.J. Lipoic acid increases glucose uptake by skeletal muscles of obese-diabetic ob/ob mice.Diabetes Obes. Metab. 2002; 4: 29-35Crossref PubMed Scopus (45) Google Scholar), whereas palmitic acid (PA) treatment was reported to inhibit insulin-stimulated but not basal glucose uptake (13Hardy R.W. Ladenson J.H. Henriksen E.J. Holloszy J.O. McDonald J.M. Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT4).Biochem. Biophys. Res. Commun. 1991; 177: 343-351Crossref PubMed Scopus (45) Google Scholar). To elucidate the mechanism underlying the response of skeletal muscle to acute increases in fatty acids, we examined the acute effects of PA on glucose uptake and on phosphorylation of AMP-activated protein kinase (AMPK), Akt, and extracellular signal-related kinase (ERK) 1/2 in skeletal cell lines. We found that PA stimulated GLUT4 translocation to the plasma membrane and glucose uptake enhancement. Our data also suggest that PI3 kinase (PI3K), AMPK, Akt, and ERK1/2 play critical roles in PA-induced glucose uptake and that cell surface-bound PA is sufficient to trigger the signal transduction. Sodium PA, insulin, myc polyclonal antibody, fatty acid-free BSA, 5-aminoimidazole-4 carboxamide 1-β-D-ribofuranoside (AICAR), Compound C, API-2, Lipid Standard, and 2-deoxy-D-glucose were purchased from Sigma-Aldrich (St. Louis, MO). 2-Deoxy-D-[3H]glucose and TLC plates were obtained from GE Healthcare (UK). Antibodies against phosphorylated Akt (Ser 473), total Akt, phosphorylated AMPK (Thr172), phosphorylated ERK1/2 (p44/42 MAPK) (Thr202/Tyr204), and GAPDH were from Cell Signaling Technology (Beverly, MA). Myc monoclonal antibody was purchased from Upstate (Billerica, MA). 3H-PA was from PerkinElmer Life Sciences (Waltham, MA). LY294002 and API-2 were from Biomol (Plymouth Meeting, PA). PD98058 and U0126 were from Beyotime Institute of Biotechnology (Haimen, China). Short interference RNA (siRNA) duplex targeting Akt (siAkt) and AMPK and negative control siRNA were from GenePharma (Shanghai, China). The L6-GLUT4myc (L6) cell line, a rat skeletal muscle cell line which stably over-expresses GLUT4, was obtained from Dr. Amira Klip, Division of Cell Biology, Hospital for Sick Children, Toronto, Canada. The myc-tagged AMPK dominant negative mutant constructed into pcDNA3.1 was a kind gift from Dr. David Carling, Cellular Stress Group, MRC Clinical Sciences Centre, London, UK. L6-GLUT4myc cells were maintained in α-minimal essential medium (α-MEM) supplemented with 10% (v/v) FBS, 2 µg/ml blasticidin S, 100 units/ml penicillin, and 100 units/ml streptomycin at 37°C with 5% CO2. L6 cells were differentiated into myotubes within 7 days in medium supplemented with 2% FBS. Mouse C2C12 myoblasts (American Type Culture Collection) were maintained in DMEM supplemented with 10% (v/v) FBS, 100 units/ml penicillin, and 100 units/ml streptomycin at 37°C with 5% CO2. Sodium palmitate was prepared according to our previously published method (14Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic.J. Biol. Chem. 2004; 279: 3787-3878Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). Briefly, sodium palmitate was added to ethanol to a final concentration of 100 mM and sonicated on ice at 200 W using 10 s on, 3 s off pulses until the mixture became a milky homogenous solution. The PA stock solution was added to growth medium containing 10% FBS at 60°C and the medium was cooled to 37°C for cell treatment unless otherwise stated. For the control, the same amount of ethanol was added to the growth medium containing 10% FBS as a vehicle. The sequence of the siAkt was that given by Katome et al. (15Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions.J. Biol. Chem. 2003; 278: 28312-28334Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), and AMPK α by Konrad et al. (16Konrad D. Rudich A. Bilan P.J. Patel N. Richardson C. Witters L.A. Klip A. Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells.Diabetologia. 2005; 48: 954-1019Crossref PubMed Scopus (99) Google Scholar). L6 cells were nucleofected with siRNA or plasmids following the manufacturer's procedure. or the the was was to GLUT4 on the cell surface using a previously published method with Kishi K. Ebina Y. Klip A. GLUT4 translocation by insulin in muscle by a and PubMed Scopus Google Scholar). L6-GLUT4myc cells were on a treatment with PA or insulin, cells were on ice and with mM mM mM mM mM and mM with 5% in at for 10 cells were with polyclonal antibody at for with in at for and with mM for 10 with cells were with for at with and examined in with an L6-GLUT4myc cells were with PA, with and in containing and at 37°C for were with and with was using a was at with independently. the cells were directly with and sonicated s at 200 W. tissue were prepared according to a previously published method Liu P. Anderson R.G. targeting of to or Cell Sci. PubMed Google Scholar). Briefly, tissues were sonicated in to cellular and total protein was using were with and in by were by 10% to a using the and using were with body and to with 2 mM PA in containing 10% FBS. The of was by of in the the muscle were in 2 mM PA in containing 10% FBS at 37°C for the in and stored at until animal were by the and Use and followed the Use of the Institute of Chinese of The cells were with treatment with PA and total lipids were with lipid acid by for were with the of acid by and the TLC plates were in at for The of the plates was following the lipid from Lipid and to using a were using and were using was are To the acute effect of PA on glucose uptake in skeletal muscle, a rat skeletal muscle cell line that stably GLUT4 with a myc in L6-GLUT4myc was with PA or 100 As shown in insulin increased plasma GLUT4 as PA treatment also stimulated GLUT4 translocation to the plasma We glucose uptake in L6 cells with of in L6 cells was increased by and and of treatment with PA, The increased glucose uptake in skeletal muscle cells to PA correlated with the of GLUT4 on the plasma the effect of PA on GLUT4 translocation and glucose uptake was to that of insulin, we examined the activation of Akt, a key protein involved in insulin-stimulated GLUT4 Akt phosphorylation (Ser was stimulated by PA in a and and activation of Akt in L6 myoblasts was 10 and at decreased and became 3 suggesting that a signal by PA to the activation of In PA also could stimulate Akt activation in differentiated L6 myotubes but a of Akt activation with L6 myoblasts To the effect of PA on Akt we using skeletal muscle cell line as a result, PA also Akt rapidly to was using rat skeletal were with 2 mM Skeletal muscle were and in with 2 mM PA and to the results obtained from using cell PA stimulated Akt phosphorylation in rat skeletal muscle tissue these data suggest that acute response of PA may be because results were for and rat skeletal muscle cell as well as rat skeletal muscle tissue. We PA-mediated in more Previous studies have shown that treatment with PA can induce AMPK phosphorylation S. of AMP-activated protein kinase and phosphorylation by palmitate in skeletal muscle Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. S. M. M. R. exposure to palmitate fatty acid activation of AMP-activated protein kinase in skeletal muscle PubMed Scopus Google Scholar) and that AMPK is to induce Akt activation N. H. S. M. K. T. T. K. stimulates by AMP-activated protein kinase and Akt in Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). we whether AMPK can also be by PA Our data that treatment with PA is also to stimulate AMPK phosphorylation as as and a at the signal could not be As AMPK activation more rapidly than Akt is possible that AMPK may PA-induced Akt In phosphorylation of AMPK and Akt was stimulated by PA in a PA, we also examined the acute effects of other fatty acids on skeletal muscle acid, oleic acid, and acid could stimulate Akt and AMPK phosphorylation in a in L6 cells C, and Furthermore, the of PA, acid, oleic acid, and acid had effects of which the fatty acid concentration in rat plasma K. T. S. T. in fatty acid in rat blood and organs of acid J. 1991; PubMed Scopus Google Scholar). During PA AMPK and Akt, ERK1/2 was also The results showed that phosphorylation of both and was increased and to basal ERK1/2 that PA-induced glucose responses of rat skeletal muscle cells to PA of AMPK, Akt, ERK1/2, and of glucose that PA may as a that can stimulate skeletal muscle cells by binding to the surface membrane in to To PA binding to skeletal muscle C2C12 cells was by cells with PA for at to PA were with to PA and to 37°C to signal transduction. Akt phosphorylation a at 10 and to the time-dependent of Akt activation at However, fatty acid-free was to the Akt phosphorylation could not be suggesting that binding of PA to the cell surface is for acute Akt We the same with of PA and found that the amount of PA was as as of the amount of PA To the metabolism of PA the PA the total lipids were and by The data showed that fatty acid is the of the total lipids from the cells by at and still the 10 at 37°C The of PA was and whereas and triglyceride not significantly data that PA is the to induce Akt The showed that PA could stimulate glucose uptake and Akt suggesting that Akt may be involved in PA-induced glucose We an Akt inhibitor API-2 M. Liu A.D. M. M. et kinase B a inhibitor of Akt with activity in cells Res. 2004; PubMed Scopus Google Scholar) to Akt As a result, API-2 could Akt phosphorylation as well as decrease PA-induced glucose uptake significantly In Akt was reduced by RNA interference to the results from using siRNA were nucleofected into L6 cells and total Akt levels were at and with negative control siRNA siAkt could Akt As in at siAkt decreased PA-induced glucose uptake, whereas these data suggest that signal glucose uptake in skeletal muscle cells Akt AMPK and Akt activation was in cells we the AMPK and Akt and the role of AMPK in PA-induced glucose AMPK was to stimulate AMPK phosphorylation in L6 and the showed that not AMPK but also Akt was stimulated rapidly by treatment in a time-dependent which that is possible to stimulate Akt AMPK activation in L6 the we AMPK inhibitor Compound to suppress AMPK we found Compound could decrease PA-induced Akt phosphorylation and glucose uptake the myc-tagged AMPK dominant negative was nucleofected into L6 cells and was PA-induced Akt phosphorylation and glucose uptake were also Furthermore, in the L6 cells siRNA duplex mixture targeting AMPK and AMPK α was decreased as and Akt phosphorylation was also decreased consistent with the inhibitor and data showed that AMPK phosphorylation to Akt activity and was involved in PA-induced glucose PI3K is of Akt in the insulin signal we a PI3K to the cells to whether PI3K in glucose AMPK and Akt inhibitors that decreased glucose uptake, PI3K inhibitor could suggesting that PI3K an essential role in the cell response to The results showed that PI3K inhibitor could PA-induced AMPK activation and Akt phosphorylation the data that PA could rapidly trigger signal by binding to cell plasma membrane and stimulate glucose uptake activation of PI3K/AMPK/Akt in skeletal muscle The of the PI3K/AMPK/Akt is consistent with published by et Wang T. H. An Res. PubMed Scopus Google Scholar). AMPK or Akt activity decreased PA-induced glucose uptake, we other possible The role of ERK1/2 in signal was examined by the kinase 1/2 inhibitors and inhibitors could decrease basal and PA-induced ERK1/2 whereas U0126 more ERK1/2 activity was by PA-induced glucose uptake was reduced significantly data showed that ERK1/2 phosphorylation may to PA-induced glucose To the ERK1/2 and PI3K/AMPK/Akt PI3K inhibitor LY294002 was The results showed that ERK1/2 phosphorylation was decreased suggesting PI3K was the of ERK1/2 in PA-induced signal transduction. in AMPK siRNA ERK1/2 phosphorylation was still increased as cells PA treatment Akt inhibitor API-2 was but not affect PA-induced ERK1/2 phosphorylation and inhibitors and U0126 not affect PA-induced Akt results suggest that in PA acute effects ERK1/2 was involved in PA-induced glucose uptake and by PI3K but of AMPK and plasma free fatty acids respond to insulin P.J. J.O. N. of free fatty acid metabolism by insulin in role of and J. 1992; Google Scholar), protein J. K. A.D. Frayn K.N. release of protein and by human adipose tissue in in the Lipid Res. Full Text Full Text PDF PubMed Google Scholar), and other rapidly to basal levels within 2 free fatty acids are from adipocytes into the blood as an energy The plasma fatty acid concentration increases and may within (10Singer P. Godicke W. Voigt S. Hajdu I. Weiss M. Postprandial hyperinsulinemia in patients with mild essential hypertension.Hypertension. 1985; 7: 182-187Crossref PubMed Scopus (167) Google Scholar). the free fatty acid concentration in blood from hour to hour and a in plasma fatty acids also vary but display for example, have higher plasma fatty acid concentrations of with healthy subjects N. M. P.J. insulin resistance in subjects and in type 2 Res. 2002; PubMed Scopus Google Scholar). However, how body tissues respond to the rapidly fatty acid levels unclear. studies have that chronically elevated plasma free fatty acids play a critical role in the development of insulin resistance. Chronic fatty acid may of fatty acid studies on acute fatty acid effects and the effects of acute fatty acid may a view for the of type 2 Results from studies on adipocytes that treatment with fatty acids increases basal glucose uptake (13Hardy R.W. Ladenson J.H. Henriksen E.J. Holloszy J.O. McDonald J.M. Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT4).Biochem. Biophys. Res. Commun. 1991; 177: 343-351Crossref PubMed Scopus (45) Google Scholar, C. J.P. J.M. S. acid stimulates glucose uptake in adipocytes by and GLUT4 levels at the plasma for of and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R.W. J. McDonald J.M. fatty insulin resistance in rat PubMed Scopus Google Scholar). reports provide evidence that fatty acids at of a with insulin (13Hardy R.W. Ladenson J.H. Henriksen E.J. Holloszy J.O. McDonald J.M. Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT4).Biochem. Biophys. Res. Commun. 1991; 177: 343-351Crossref PubMed Scopus (45) Google Scholar), whereas other reports the of but not GLUT4 Liu D. acid stimulates the activity of glucose transporter in adipocytes by a protein kinase PubMed Scopus Google a consistent of the mechanisms involved has not is the in skeletal muscle Here, we the effects of PA in skeletal muscle cell and tissues, and results that glucose uptake activation of the PI3K/AMPK/Akt and PI3K/ERK1/2 pathways to GLUT4 translocation Insulin has been acute PA treatment in adipocytes (13Hardy R.W. Ladenson J.H. Henriksen E.J. Holloszy J.O. McDonald J.M. Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT4).Biochem. Biophys. Res. Commun. 1991; 177: 343-351Crossref PubMed Scopus (45) Google Scholar), and in our we found that plasma PA was sufficient to induce Akt that PA with on the plasma membrane to trigger the However, the of still to be Phosphorylation of Akt was stimulated the concentrations of PA a or 200 in C2C12 we that phosphorylation of Akt may concentrations of fatty acids to our Akt phosphorylation was fatty acid of to 3 and fatty acids were the Akt phosphorylation signal 3 in C2C12 cells not suggesting that fatty phosphorylation and of Akt can be within fatty acid fatty acid to the development of insulin resistance the of The of the results is that PA can play roles in skeletal can inhibit glucose uptake by Akt and on the other cells are to PA for a can enhance glucose uptake by This of PA in skeletal muscle be and to be The and for their critical and The also to and for their We are to Dr. David for with AMP-activated protein kinase AMPK dominant negative extracellular signal-related kinase intramyocellular triglyceride kinase negative control palmitic acid or palmitate phosphorylated Akt AMPK PI3 kinase interference RNA targeting Akt interference RNA

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