p38gamma MAPK regulation of glucose transporter expression and glucose uptake in L6 myotubes and mouse skeletal muscle

Skeletal muscle expresses at least three p38 MAPKs (alpha, beta, gamma). However, no studies have examined the potential regulation of glucose uptake by p38gamma, the isoform predominantly expressed in skeletal muscle and highly regulated by exercise. L6 myotubes were transfected with empty vector (pCAGGS), activating MKK6 (MKK6CA), or p38gamma-specific siRNA. MKK6CA-transfected cells had higher rates of basal 2-deoxy-d-[3H]glucose (2-DG) uptake (P < 0.05) but lower rates of 2,4-dinitrophenol (DNP)-stimulated glucose uptake, an uncoupler of oxidative phosphorylation that operates through an insulin-independent mechanism (P < 0.05). These effects were reversed when MKK6CA cells were cotransfected with p38gamma-specific siRNA. To determine whether the p38gamma isoform is involved in the regulation of contraction-stimulated glucose uptake in adult skeletal muscle, the tibialis anterior muscles of mice were injected with pCAGGS or wild-type p38gamma (p38gammaWT) followed by intramuscular electroporation. Basal and contraction-stimulated 2-DG uptake in vivo was determined 14 days later. Overexpression of p38gammaWT resulted in higher basal rates of glucose uptake compared with pCAGGS (P < 0.05). Muscles overexpressing p38gammaWT showed a trend for lower in situ contraction-mediated glucose uptake (P = 0.08) and significantly lower total GLUT4 levels (P < 0.05). These data suggest that p38gamma increases basal glucose uptake and decreases DNP- and contraction-stimulated glucose uptake, partially by affecting levels of glucose transporter expression in skeletal muscle. These findings are consistent with the hypothesis that activation of stress kinases such as p38 are negative regulators of stimulated glucose uptake in peripheral tissues.     

Curcumin stimulates glucose uptake through AMPK‐p38 MAPK pathways in L6 myotube cells

Curcumin has been shown to exert a variety of beneficial human health effects. However, mechanisms by which curcumin acts are poorly understood. In this study, we report that curcumin activated AMP‐activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. In addition, curcumin activated the mitogen‐activated protein kinase kinase (MEK)3/6‐p38 mitogen‐activated protein kinase (MAPK) signaling pathways in the downstream of the AMPK cascade. Moreover, inhibition of either AMPK or p38 MAPK resulted in blockage of curcumin‐induced glucose uptake. Furthermore, the administration of curcumin to mice increased AMPK phosphorylation in the skeletal muscles. Taken together, these results indicate that the beneficial health effect of curcumin can be explained by its ability to activate AMPK‐p38 MAPK pathways in skeletal muscles. J. Cell. Physiol. 223:771–778, 2010. © 2010 Wiley‐Liss, Inc.     

Arsenite stimulated glucose transport in 3T3-L1 adipocytes involves both Glut4 translocation and p38 MAPK activity.

The protein-modifying agent arsenite stimulates glucose uptake in 3T3-L1 adipocytes. In the current study we have analysed the signalling pathways that contribute to this response. By subcellular fractionation we observed that arsenite, like insulin, induces translocation of the GLUT1 and GLUT4 glucose transporters from the low-density membrane fraction to the plasma membrane. Arsenite did not activate early steps of the insulin receptor (IR)-signalling pathway and the response was insensitive to inhibition of phosphatidylinositol-3'-kinase (PI-3') kinase by wortmannin. These findings indicate that the 'classical' IR-IR substrate-PI-3' kinase pathway, that is essential for insulin-induced GLUT4 translocation, is not activated by arsenite. However, arsenite-treatment did induce tyrosine-phosphorylation of c-Cbl. Furthermore, treatment of the cells with the tyrosine kinase inhibitor, tyrphostin A25, abolished arsenite-induced glucose uptake, suggesting that the induction of a tyrosine kinase by arsenite is essential for glucose uptake. Both arsenite and insulin-induced glucose uptake were inhibited partially by the p38 MAP kinase inhibitor, SB203580. This compound had no effect on the magnitude of translocation of glucose transporters indicating that the level of glucose transport is determined by additional factors. Arsenite- and insulin-induced glucose uptake responded in a remarkably similar dose-dependent fashion to a range of pharmacological- and peptide-inhibitors for atypical PKC-lambda, a downstream target of PI-3' kinase signalling in insulin-induced glucose uptake. These data show that in 3T3-L1 adipocytes both arsenite- and insulin-induced signalling pathways project towards a similar cellular response, namely GLUT1 and GLUT4 translocation and glucose uptake. This response to arsenite is not functionally linked to early steps of the IR-IRS-PI-3' kinase pathway, but does coincide with c-Cbl phosphorylation, basal levels of PKC-lambda activity and p38 MAPK activation.     

Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes

BackgroundLowering blood glucose levels by increasing glucose uptake in insulin target tissues, such as skeletal muscle and adipose tissue, is one strategy to discover and develop antidiabetic drugs from natural products used as traditional medicines.PurposeOur goal was to reveal the mechanism and activity of acacetin (5,7-dihydroxy-4′-methoxyflavone), one of the major compounds in Agastache rugose, in stimulating glucose uptake in muscle cells.MethodsTo determine whether acacetin promotes GLUT4-dependent glucose uptake in cultured L6 skeletal muscle cells, we performed a [¹⁴C] 2-deoxy-_D-glucose (2-DG) uptake assay after treating differentiated L6-GLUT4myc cells with acacetin.ResultsAcacetin dose-dependently increased 2-DG uptake by enhancing GLUT4 translocation to the plasma membrane. Our results revealed that acacetin activated the CaMKII-AMPK pathway by increasing intracellular calcium concentrations. We also found that aPKCλ/ζ phosphorylation and intracellular reactive oxygen species (ROS) production were involved in acacetin-induced GLUT4 translocation. Moreover, acacetin-activated AMPK inhibited intracellular lipid accumulation and increased 2-DG uptake in HepG2 cells.ConclusionTaken together, these results suggest that acacetin might be useful as an antidiabetic functional ingredient. Subsequent experiments using disease model animals are needed to verify our results.     

Interaction of phytochemicals with hypoglycemic drugs on glucose uptake in L6 myotubes

The present study analyses the effect of eugenol, arecoline and vanillic acid alone and in combination with two oral hypoglycemic drugs (OHD), namely, metformin and 2,4-thiazolodinedione (THZ), on 2-deoxyglucose (2DG) uptake in L6 myotubes. 2DG uptake in L6 myotubes was determined using an enzymatic assay developed by Yamamoto et al. (2006). Lipid content inside the cells has been estimated with oil red O assay. The absorption, distribution, metabolism, and excretion (ADME) and drug likeness properties of these phytochemicals are estimated using software QikProp^®. All the three phytochemicals enhance 2DG uptake both in time- and dose-dependent manner. Eugenol and arecoline enhances 2DG uptake synergistically with both the OHD; whereas vanillic acid showing partly synergy with THZ and antagonistic activity with metformin on 2DG uptake. Eugenol and arecoline significantly increase the expressions of the glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3K) genes, but not the peroxisome proliferator-activated receptor (PPAR) gamma. Whereas vanillic acid does not has any significant effect on the expressions of these genes, the ADME results indicate that these phytochemicals are satisfying all the conditions to have a good oral bioavailability. These findings suggest that these phytochemicals can replace the commercial drugs in part, which could lead to a reduction in toxicity and side effects caused by the later as well as reduce the secondary complications.     

MKK6/3 and p38 MAPK pathway activation is not necessary for insulin-induced glucose uptake but regulates glucose transporter expression

p38 mitogen-activated protein kinase (MAPK), which is situated downstream of MAPK kinase (MKK) 6 and MKK3, is activated by mitogenic or stress-inducing stimuli, as well as by insulin. To clarify the role of the MKK6/3-p38 MAPK pathway in the regulation of glucose transport, dominant negative p38 MAPK and MKK6 mutants and constitutively active MKK6 and MKK3 mutants were overexpressed in 3T3-L1 adipocytes and L6 myotubes using an adenovirus-mediated transfection procedure. Constitutively active MKK6/3 mutants up-regulated GLUT1 expression and down-regulated GLUT4 expression, thereby significantly increasing basal glucose transport but diminishing transport induced by insulin. Similar effects were elicited by chronic (24 h) exposure to tumor necrosis factor alpha, interleukin-1beta, or 200 mm sorbitol, all activate the MKK6/3-p38 MAPK pathway. SB203580, a specific p38 MAPK inhibitor, attenuated these effects, further confirming that both MMK6 and MMK3 act via p38 MAPK, whereas they had no effect on the increase in glucose transport induced by a constitutively active MAPK kinase 1 (MEK1) mutant or by myristoylated Akt. In addition, suppression of p38 MAPK activation by overexpression of a dominant negative p38 MAPK or MKK6 mutant did not diminish insulin-induced glucose uptake by 3T3-L1 adipocytes. It is thus apparent that activation of p38 MAPK is not essential for insulin-induced increases in glucose uptake. Rather, p38 MAPK activation leads to a marked down-regulation of insulin-induced glucose uptake via GLUT4, which may underlie cellular stress-induced insulin resistance caused by tumor necrosis factor alpha and other factors.     

Oleylethanolamide impairs glucose tolerance and inhibits insulin-stimulated glucose uptake in rat adipocytes through p38 and JNK MAPK pathways

Oleylethanolamide (OEA) is a lipid mediator that inhibits food intake and body weight gain and also exhibits hypolipemiant actions. OEA exerts its anorectic effects peripherally through the stimulation of C-fibers. OEA is synthesized in the intestine in response to feeding, increasing its levels in portal blood after the meal. Moreover, OEA is produced by adipose tissue, and a lipolytic effect has been found. In this work, we have examined the effect of OEA on glucose metabolism in rats in vivo and in isolated adipocytes. In vivo studies showed that acute administration (30 min and 6 h) of OEA produced glucose intolerance without decreasing insulin levels. Ex vivo, we found that 10 min of preincubation with OEA inhibited 30% insulin-stimulated glucose uptake in isolated adipocytes. Maximal effect was achieved at 1 microM OEA. The related compounds palmitylethanolamide and oleic acid had no effect, suggesting a specific mechanism. Insulin-stimulated GLUT4 translocation was not affected, but OEA promoted Ser/Thr phosphorylation of GLUT4, which may impair transport activity. This phosphorylation may be partly mediated by p38 and JNK kinases, since specific inhibitors (SB-203580 and SP-600125) partly reverted the inhibitory effect of OEA on insulin-stimulated glucose uptake. These results suggest that the lipid mediator OEA inhibits insulin action in the adipocyte, impairing glucose uptake via p38 and JNK kinases, and these effects may at least in part explain the glucose intolerance produced in rats in vivo. These effects of OEA may contribute to the anorectic effects induced by this mediator, and they might be also relevant for insulin resistance in adipose tissue.     

Capsaicin stimulates glucose uptake in C2C12 muscle cells via the reactive oxygen species (ROS)/AMPK/p38 MAPK pathway

Capsaicin has been reported to regulate blood glucose levels and to ameliorate insulin resistance in obese mice. This study demonstrates that capsaicin increases glucose uptake directly by activating AMP-activated protein kinase (AMPK) in C2C12 muscle cells, which manifested as an attenuation of glucose uptake when compound C, an AMPK inhibitor, was co-administered with capsaicin. However, the insulin signaling molecules insulin receptor substrate-1 (IRS-1) and Akt were not affected by capsaicin. Additional results showed that p38 mitogen-activated protein kinase (MAPK) is also involved in capsaicin-induced glucose transport downstream of AMPK because capsaicin increased p38 MAPK phosphorylation significantly and its specific inhibitor SB203580 inhibited capsaicin-mediated glucose uptake. Treatment with an AMPK inhibitor reduced p38 MAPK phosphorylation, but the p38 MAPK inhibitor had no effect on AMPK. Capsaicin stimulated ROS generation in C2C12 muscle cells, and when ROS were captured using the nonspecific antioxidant NAC, the increase in both capsaicin-induced AMPK phosphorylation and capsaicin-induced glucose uptake was attenuated, suggesting that ROS function as an upstream activator of AMPK. Taken together, these results suggest that capsaicin, independent of insulin, increases glucose uptake via ROS generation and consequent AMPK and p38 MAPK activations.     

Inhibition of p38 MAPK and AMPK restores adenosine-induced cardioprotection in hearts stressed by antecedent ischemia by altering glucose utilization

p38 mitogen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) are activated by metabolic stresses and are implicated in the regulation of glucose utilization and ischemia-reperfusion (IR) injury. This study tested the hypothesis that inhibition of p38 MAPK restores the cardioprotective effects of adenosine in stressed hearts by preventing activation of AMPK and the uncoupling of glycolysis from glucose oxidation. Working rat hearts were perfused with Krebs solution (1.2 mM palmitate, 11 mM [(3)H/(14)C]glucose, and 100 mU/l insulin). Hearts were stressed by transient antecedent IR (2 x 10 min I/5 min R) before severe IR (30 min I/30 min R). Hearts were treated with vehicle, p38 MAPK inhibitor (SB-202190, 10 microM), adenosine (500 microM), or their combination before severe IR. After severe IR, the phosphorylation (arbitrary density units) of p38 MAPK and AMPK, rates of glucose metabolism (micromol x g dry wt(-1) x min(-1)), and recovery of left ventricular (LV) work (Joules) were similar in vehicle-, SB-202190- and adenosine-treated hearts. Treatment with SB-202190 + adenosine versus adenosine alone decreased p38 MAPK (0.03 +/- 0.01, n = 3 vs. 0.48 +/- 0.10, n = 3, P < 0.05) and AMPK (0.00 +/- 0.00, n = 3 vs. 0.26 +/- 0.08, n = 3 P < 0.05) phosphorylation. This was accompanied by attenuated rates of glycolysis (1.51 +/- 0.40, n = 7 vs. 3.95 +/- 0.65, n = 7, P < 0.05) and H(+) production (2.12 +/- 0.76, n = 7 vs. 6.96 +/- 1.48, n = 7, P < 0.05), and increased glycogen synthesis (1.91 +/- 0.25, n = 6 vs. 0.27 +/- 0.28, n = 6, P < 0.05) and improved recovery of LV work (0.81 +/- 0.08, n = 7 vs. 0.30 +/- 0.15, n = 8, P < 0.05). These data indicate that inhibition of p38 MAPK abolishes subsequent phosphorylation of AMPK and improves the coupling of glucose metabolism, thereby restoring adenosine-induced cardioprotection.     

A dominant-negative p38 MAPK mutant and novel selective inhibitors of p38 MAPK reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes without affecting GLUT4 translocation

Participation of p38 mitogen-activated protein kinase (p38) in insulin-induced glucose uptake was suggested using pyridinylimidazole p38 inhibitors (e.g. SB203580). However, the role of p38 in insulin action remains controversial. We further test p38 participation in glucose uptake using a dominant-negative p38 mutant and two novel pharmacological p38 inhibitors related to but different from SB203580. We present the structures and activities of the azaazulene pharmacophores A291077 and A304000. p38 kinase activity was inhibited in vitro by A291077 and A304000 (IC(50) = 0.6 and 4.7 microm). At higher concentrations A291077 but not A304000 inhibited JNK2alpha (IC(50) = 3.5 microm). Pretreatment of 3T3-L1 adipocytes and L6 myotubes expressing GLUT4myc (L6-GLUT4myc myotubes) with A291077, A304000, SB202190, or SB203580 reduced insulin-stimulated glucose uptake by 50-60%, whereas chemical analogues inert toward p38 were ineffective. Expression of an inducible, dominant-negative p38 mutant in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. GLUT4 translocation to the cell surface, immunodetected on plasma membrane lawns of 3T3-L1 adipocytes or on intact L6-GLUT4myc myotubes, was not altered by chemical or molecular inhibition of p38. We propose that p38 contributes to enhancing GLUT4 activity, thereby increasing glucose uptake. In addition, the azaazulene class of inhibitors described will be useful to decipher cellular actions of p38 and JNK.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

AICAR stimulates IL-6 production via p38 MAPK in cardiac fibroblasts in adult mice: a possible role for AMPK

Though known as a sensor of energy balance, AMP-activated protein kinase (AMPK) was recently shown to limit damage and apoptotic activity and contribute to the late preconditioning in heart. Interleukin-6 was also reported to involve in anti-apoptosis and cardio-protection in myocardium. Interestingly, both AMPK activity and IL-6 level were increased in response to ischemia, hypertrophy and oxidative stress. To determine whether AMPK activation will promote IL-6 production, cardiac fibroblasts (CFs) from mice were incubated with AMPK activator, 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR). The results demonstrated that AICAR time and dose-dependently stimulated IL-6 production by ELISA and immunofluorescence. Pretreatment with p38 mitogen-activated protein kinase (MAPK) inhibitor blocked AICAR-induced IL-6 production; furthermore, AICAR-activated p38 MAPK phosphorylation by Western blot. To confirm that the increase in IL-6 production is ascribed to AMPK activation, we used another known AMPK activator, metformin. It also dose-dependently potentiated IL-6 production in CFs, and this potentiation could be reversed by p38 MAPK inhibitor. In conclusion, AMPK activation promoted IL-6 production in CFs via p38 MAPK-dependent pathway.     

AMPK/SIRT1 signaling through p38MAPK mediates Interleukin-6 induced neuroendocrine differentiation of LNCaP prostate cancer cells

Neuroendocrine Prostate Cancer (NEPC) is an aggressive form of androgen independent prostate cancer (AIPC), correlated with therapeutic resistance. Interleukin (IL)-6 promotes proliferation and neuroendocrine differentiation (NED) of androgen dependent LNCaP cells. We treated LNCaP cells with IL-6 and observed for in vitro NED of cells and also expression of NE markers βIII tubulin, neuron-specific enolase (NSE) and chromogranin A (ChA). Here we investigated the proteins and/or pathways involved in NED of LNCaP cells induced by IL-6 and characterized their role in NED of PCa cells. We found that the altered proteins modulated AMPK signaling pathway in NE cells. Remarkably, IL-6 induces NED of LNCaP cells through activation of AMPK and SIRT1 and also both of these are co-regulated while playing a predominant role in NED of LNCaP cells. Of the few requirements of AMPK-SIRT1 activation, increased eNOS is essential for NED by elevating Nitric oxide (NO) levels. Pleiotropic effects of NO ultimately regulate p38MAPK in IL-6 induced NED. Hence, IL-6 induced AMPK-SIRT1 activation eventually transfers its activation signals through p38MAPK for advancing NED of LNCaP cells. Moreover, inactivation of p38MAPK with specific inhibitor (SB203580) attenuated IL-6 induced NED of LNCaP cells. Therefore, IL-6 promotes NED of PCa cells via AMPK/SIRT1/p38MAPK signaling. Finally, targeting AMPK-SIRT1 or p38MAPK in androgen independent PC3 cells with neuroendocrine features reversed their neuroendocrine characteristics. Taken together these novel findings reveal that targeting p38MAPK mitigated NED of PCa cells, and thus it can be a favorable target to overcome progression of NEPC.     

AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.     

4-Amino-3-acetylquinoline-induced apoptosis of murine L1210 leukemia cells involves ROS-mitochondrial-mediated death signaling and activation of p38 MAPK

Quinolines are known to be multitarget agents with a broad spectrum of biological activity. In a previous study, we showed that newly prepared 4-amino-3-acetylquinoline (AAQ) possesses strong anticancer activities. In this study, we investigated whether AAQ has cytotoxicity in murine L1210 leukemia cells. Results from cell proliferation assays showed that AAQ caused significant decrease in cell number in a dose-dependent manner. The cell death induced by AAQ appeared to involve apoptosis, based on evidence from apoptotic DNA fragmentation, flow cytometry, fluorescence microscopy, and Western blot analyses. We found that AAQ-treated cells had activated p38 MAPK and that apoptosis was processed through a reactive oxygen species (ROS)-dependent mitochondrial pathway. In summary, our results suggest that AAQ can induce apoptosis, at least in part, through the activation of the p38 MAPK pathway in L1210 leukemia cells.