Overexpression or ablation of JNK in skeletal muscle has no effect on glycogen synthase activity

c-Jun NH₂-terminal kinase (JNK) is highly expressed in skeletal muscle and is robustly activated in response to muscle contraction. Little is known about the biological functions of JNK signaling in terminally differentiated muscle cells, although this protein has been proposed to regulate insulin-stimulated glycogen synthase activity in mouse skeletal muscle. To determine whether JNK signaling regulates contraction-stimulated glycogen synthase activation, we applied an electroporation technique to induce JNK overexpression (O/E) in mouse skeletal muscle. Ten days after electroporation, in situ muscle contraction increased JNK activity 2.6-fold in control muscles and 15-fold in the JNK O/E muscles. Despite the enormous activation of JNK activity in JNK O/E muscles, contraction resulted in similar increases in glycogen synthase activity in control and JNK O/E muscles. Consistent with these findings, basal and contraction-induced glycogen synthase activity was normal in muscles of both JNK1- and JNK2-deficient mice. JNK overexpression in muscle resulted in significant alterations in the basal phosphorylation state of several signaling proteins, such as extracellular signal-regulated kinase 1/2, p90 S6 kinase, glycogen synthase kinase 3, protein kinase B/Akt, and p70 S6 kinase, in the absence of changes in the expression of these proteins. These data suggest that JNK signaling regulates the phosphorylation state of several kinases in skeletal muscle. JNK activation is unlikely to be the major mechanism by which contractile activity increases glycogen synthase activity in skeletal muscle. electroporation; gene delivery; muscle contraction; exercise     

Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle

The serine/threonine kinase Akt/PKB plays diverse roles in cells, and genetic studies have indicated distinct roles for the three Akt isoforms expressed in mammalian cells and tissues. Akt2 is a key signaling intermediate for insulin-stimulated glucose uptake and glycogen synthesis in skeletal muscle. Akt2 has also been shown to be activated by exercise and muscle contraction in both rodents and humans. In this study, we used Akt2 knockout mice to explore the role of Akt2 in exercise-stimulated glucose uptake and glycogen synthesis as well as intracellular signaling pathways that regulate glycogen metabolism in skeletal muscle. We found that Akt2 deficiency does not affect basal or exercise-stimulated glucose uptake or intracellular glycogen content in the soleus muscle. In addition, lack of Akt2 did not result in alterations in basal Akt Thr(308) or basal and contraction-stimulated glycogen synthase kinase-3beta (GSK-3beta) Ser(9) phosphorylation, glycogen synthase phosphorylation, or glycogen synthase activity. In contrast, in situ contraction failed to elicit normal increases in Akt T-loop Thr(308) phosphorylation and GSK-3alpha Ser(21) phosphorylation in tibialis anterior muscles from Akt2-deficient animals. Our data establish a key role for Akt2 in the regulation of GSK-3alpha Ser(21) phosphorylation with contraction and add genetic evidence to support the separation of the intracellular pathways regulated by insulin and exercise that converge on glucose uptake and glycogen synthesis in skeletal muscle.     

Akt signaling in skeletal muscle: regulation by exercise and passive stretch

Akt/protein kinase B is a serine/threonine kinase that has emerged as a critical signaling component for mediating numerous cellular responses. Contractile activity has recently been demonstrated to stimulate Akt signaling in skeletal muscle. Whether physiological exercise in vivo activates Akt is controversial, and the initiating factors that result in the stimulation of Akt during contractile activity are unknown. In the current study, we demonstrate that treadmill running exercise of rats using two different protocols (intermediate high or high-intensity exhaustive exercise) significantly increases Akt activity and phosphorylation in skeletal muscle composed of various fiber types. To determine if Akt activation during contractile activity is triggered by mechanical forces applied to the skeletal muscle, isolated skeletal muscles were incubated and passively stretched. Passive stretch for 10 min significantly increased Akt activity (2-fold) in the fast-twitch extensor digitorum longus (EDL) muscle. However, stretch had no effect on Akt in the slow-twitch soleus muscle, although there was a robust phosphorylation of the stress-activated protein kinase p38. Similar to contraction, stretch-induced Akt activation in the EDL was fully inhibited in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin, whereas glycogen synthase kinase-3 (GSK3) phosphorylation was only partially inhibited. Stretch did not cause dephosphorylation of glycogen synthase on GSK3-targeted sites in the absence or presence of wortmannin. We conclude that physiological exercise in vivo activates Akt in multiple skeletal muscle fiber types and that mechanical tension may be a part of the mechanism by which contraction activates Akt in fast-twitch muscles.     

Insulin and exercise decrease glycogen synthase kinase-3 activity by different mechanisms in rat skeletal muscle.

Glycogen synthase activity is increased in response to insulin and exercise in skeletal muscle. Part of the mechanism by which insulin stimulates glycogen synthesis may involve phosphorylation and activation of Akt, serine phosphorylation and deactivation of glycogen synthase kinase-3 (GSK-3), leading to dephosphorylation and activation of glycogen synthase. To study Akt and GSK-3 regulation in muscle, time course experiments on the effects of insulin injection and treadmill running exercise were performed in hindlimb skeletal muscle from male rats. Both insulin and exercise increased glycogen synthase activity (%I-form) by 2-3-fold over basal. Insulin stimulation significantly increased Akt phosphorylation and activity, whereas exercise had no effect. The time course of the insulin-stimulated increase in Akt was closely matched by GSK-3alpha Ser(21) phosphorylation and a 40-60% decrease in GSK-3alpha and GSK-3beta activity. Exercise also deactivated GSK-3alpha and beta activity by 40-60%. However, in contrast to the effects of insulin, there was no change in Ser(21) phosphorylation in response to exercise. Tyrosine dephosphorylation of GSK-3, another putative mechanism for GSK-3 deactivation, did not occur with insulin or exercise. These data suggest the following: 1) GSK-3 is constitutively active and tyrosine phosphorylated under basal conditions in skeletal muscle, 2) both exercise and insulin are effective regulators of GSK-3 activity in vivo, 3) the insulin-induced deactivation of GSK-3 occurs in response to increased Akt activity and GSK-3 serine phosphorylation, and 4) there is an Akt-independent mechanism for deactivation of GSK-3 in skeletal muscle.     

Effect of acute activation of 5'-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle.

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.     

Possible CaMKK-dependent regulation of AMPK phosphorylation and glucose uptake at the onset of mild tetanic skeletal muscle contraction

The Ca(2+)/calmodulin (CaM) competitive inhibitor KN-93 has previously been used to evaluate 5'-AMP-activated protein kinase (AMPK)-independent Ca(2+)-signaling to contraction-stimulated glucose uptake in muscle during intense electrical stimulation ex vivo. With the use of low-intensity tetanic contraction of mouse soleus and extensor digitorum longus (EDL) muscles ex vivo, this study demonstrates that KN-93 can potently inhibit AMPK phosphorylation and activity after 2 min but not 10 min of contraction while strongly inhibiting contraction-stimulated 2-deoxyglucose uptake at both the 2- and 10-min time points. These data suggest inhibition of Ca(2+)/CaM-dependent signaling events upstream of AMPK, the most likely candidate being the novel AMPK kinase CaM-dependent protein kinase kinase (CaMKK). CaMKK protein expression was detected in mouse skeletal muscle. Similar to KN-93, the CaMKK inhibitor STO-609 strongly reduced AMPK phosphorylation and activity at 2 min and less potently at 10 min. Pretreatment with STO-609 inhibited contraction-stimulated glucose uptake at 2 min in soleus, but not EDL, and in both muscles after 10 min. Neither KN-93 nor STO-609 inhibited 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside-stimulated glucose uptake, AMPK phosphorylation, or recombinant LKB1 activity, suggestive of an LKB1-independent effect. Finally, neither KN-93 nor STO-609 had effects on the reductions in glucose uptake seen in mice overexpressing a kinase-dead AMPK construct, indicating that the effects of KN-93 and STO-609 on glucose uptake require inhibition of AMPK activity. We propose that CaMKKs act in mouse skeletal muscle regulating AMPK phosphorylation and glucose uptake at the onset of mild tetanic contraction and that an intensity- and/or time-dependent switch occurs in the relative importance of AMPKKs during contraction.     

Invited review: effect of acute exercise on insulin signaling and action in humans.

After a single bout of exercise, insulin action is increased in the muscles that were active during exercise. The increased insulin action has been shown to involve glucose transport, glycogen synthesis, and glycogen synthase (GS) activation as well as amino acid transport. A major mechanism involved in increased insulin stimulation of glucose uptake after exercise seems to be the exercise-associated decrease in muscle glycogen content. Muscle glycogen content also plays a pivotal role for the activity of GS and for the ability of insulin to increase GS activity. Insulin signaling in human skeletal muscle is activated by physiological insulin concentrations, but the increase in insulin action after exercise does not seem to be related to increased insulin signaling [insulin receptor tyrosine kinase activity, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (RS1), IRS-1-associated phosphatidylinositol 3-kinase activity, Akt phosphorylation (Ser(473)), glycogen synthase kinase 3 (GSK3) phosphorylation (Ser(21)), and GSK3alpha activity], as measured in muscle lysates. Furthermore, insulin signaling is also largely unaffected by exercise itself. This, however, does not preclude that exercise influences insulin signaling through changes in the spatial arrangement of the signaling compounds or by affecting unidentified signaling intermediates. Finally, 5'-AMP-activated protein kinase has recently entered the stage as a promising player in explaining at least a part of the mechanism by which exercise enhances insulin action.     

Eph B4 receptor signaling mediates endothelial cell migration and proliferation via the phosphatidylinositol 3-kinase pathway

The goals of this study were 2-fold: 1) to determine whether stimulation of Eph B4 receptors promotes microvascular endothelial cell migration and/or proliferation, and 2) to elucidate signaling pathways involved in these responses. The human endothelial cells used possessed abundant Eph B4 receptors with no endogenous ephrin B2 expression. Stimulation of these receptors with ephrin B2/Fc chimera resulted in dose- and time-dependent phosphorylation of Akt. These responses were inhibited by LY294002 and ML-9, blockers of phosphatidylinositol 3-kinase (PI3K) and Akt, respectively. Eph B4 receptor activation increased proliferation by 38%, which was prevented by prior blockade with LY294002, ML-9, and inhibitors of protein kinase G (KT5823) and MEK (PD98059). Nitrite levels increased over 170% after Eph B4 stimulation, indicating increased nitric oxide production. Signaling of endothelial cell proliferation appears to be mediated by a PI3K/Akt/endothelial nitric-oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. Stimulation with ephrin B2 also increased migration by 63% versus controls. This effect was inhibited by blockade with PP2 (Src inhibitor), LY294002 or ML-9 but was unaffected by the PKG and MEK blockers. Eph B4 receptor stimulation increased activation of both matrix metalloproteinase-2 and -9. The results from these studies indicate that Eph B4 stimulates migration and proliferation and may play a role in angiogenesis.     

Glucagon-like peptide-2 receptor activation engages bad and glycogen synthase kinase-3 in a protein kinase A-dependent manner and prevents apoptosis following inhibition of phosphatidylinositol 3-kinase

Activation of glucagon-like peptide-2 receptor (GLP-2R) signaling promotes expansion of the mucosal epithelium indirectly via activation of growth and anti-apoptotic pathways; however, the cellular mechanisms coupling direct GLP-2R activation to cell survival remain poorly understood. We now demonstrate that GLP-2, in a cycloheximide-insensitive manner, enhanced survival in baby hamster kidney cells stably transfected with the rat GLP-2R; reduced mitochondrial cytochrome c efflux; and attenuated the caspase-dependent cleavage of Akt, poly(ADP-ribose) polymerase, and beta-catenin following inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002. The prosurvival effects of GLP-2 on LY294002-induced cell death were independent of Akt, p90(Rsk), or p70 S6 kinase activation; were mimicked by forskolin; and were abrogated by inhibition of protein kinase A (PKA) activity. GLP-2 inhibited activation of glycogen synthase kinase-3 (GSK-3) through phosphorylation at Ser(21) in GSK-3alpha and at Ser(9) in GSK-3beta in a PI3K-independent, PKA-dependent manner. GLP-2 reduced LY294002-induced mitochondrial association of endogenous Bad and Bax and stimulated phosphorylation of a transfected Bad fusion protein at Ser(155) in a PI3K-independent, but H89-sensitive manner, a modification known to suppress Bad pro-apoptotic activity. These results suggest that GLP-2R signaling enhances cell survival independently of PI3K/Akt by inhibiting the activity of a subset of pro-apoptotic downstream targets of Akt in a PKA-dependent manner.     

Influence of muscle glycogen availability on ERK1/2 and Akt signaling after resistance exercise in human skeletal muscle.

Two pathways that have been implicated for cellular growth and development in response to muscle contraction are the extracellular signal-regulated kinase (ERK1/2) and Akt signaling pathways. Although these pathways are readily stimulated after exercise, little is known about how nutritional status may affect stimulation of these pathways in response to resistance exercise in human skeletal muscle. To investigate this, experienced cyclists performed 30 repetitions of knee extension exercise at 70% of one repetition maximum after a low (2%) or high (77%) carbohydrate (LCHO or HCHO) diet, which resulted in low or high (approximately 174 or approximately 591 mmol/kg dry wt) preexercise muscle glycogen content. Muscle biopsies were taken from the vastus lateralis before, approximately 20 s after, and 10 min after exercise. ERK1/2 and p90 ribosomal S6 kinase phosphorylation increased (P < or = 0.05) 10 min after exercise, regardless of muscle glycogen availability. Akt phosphorylation was elevated (P < 0.05) 10 min after exercise in the HCHO trial but was unaffected after exercise in the LCHO trial. Mammalian target of rapamycin phosphorylation was similar to that of Akt during each trial; however, change or lack of change was not significant. In conclusion, the ERK1/2 pathway appears to be unaffected by muscle glycogen content. However, muscle glycogen availability appears to contribute to regulation of the Akt pathway, which may influence cellular growth and adaptation in response to resistance exercise in a low-glycogen state.     

Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.     

Dose-dependent activation of antiapoptotic and proapoptotic pathways by ethanol treatment in human vascular endothelial cells: differential involvement of adenosine

Moderate but not heavy drinking has been found to have a protective effect against cardiovascular morbidity. We investigated the effects of ethanol (EtOH) treatment on the cell survival-promoting phosphatidylinositol 3-kinase (PI3K)/Akt pathway in cultured human umbilical vein endothelial cells (HUVEC). Exposure of cells to 2-20 mm EtOH resulted in rapid (<10 min) induction of Akt phosphorylation that could be prevented by pertussis toxin or the PI3K inhibitors wortmannin and LY294002. Among the downstream effectors of PI3K/Akt, p70S6 kinase, glycogen synthase kinase 3alpha/beta, and IkappaB-alpha were phosphorylated, the latter resulting in 3-fold activation of NF-kappaB. EtOH also activated p44/42 mitogen-activated protein kinase in a PI3K-dependent manner. Low concentrations of EtOH increased endothelial nitric-oxide synthase activity, which could be blocked by transfection of HUVEC with dominant-negative Akt, implicating the PI3K/Akt pathway in this effect. The adenosine A1 receptor antagonist 1,3-dipopylcyclopentylxanthine prevented the phosphorylation of Akt observed in the presence of EtOH, adenosine, or the A1 agonist N(6)-cyclopentyladenosine. Incubation of HUVEC with 50-100 mm EtOH resulted in mitochondrial permeability transition and caspase-3 activation followed by apoptosis, as documented by DNA fragmentation and TUNEL assays. EtOH-induced apoptosis was unaffected by DPCPX and was potentiated by wortmannin or LY294002. We conclude that treatment with low concentrations of EtOH activates the cell survival promoting PI3K/Akt pathway in endothelial cells by an adenosine receptor-dependent mechanism and activation of the proapoptotic caspase pathway by higher concentrations of EtOH via an adenosine-independent mechanism can mask or counteract such effects.     

Burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle

The molecular bases underlying burn- or critical illness-induced insulin resistance still remain unclarified. Muscle protein catabolism is a ubiquitous feature of critical illness. Akt/PKB plays a central role in the metabolic actions of insulin and is a pivotal regulator of hypertrophy and atrophy of skeletal muscle. We therefore examined the effects of burn injury on insulin-stimulated Akt/PKB activation in skeletal muscle. Insulin-stimulated phosphorylation of Akt/PKB was significantly attenuated in burned compared with sham-burned rats. Insulin-stimulated Akt/PKB kinase activity, as judged by immune complex kinase assay and phosphorylation status of the endogenous substrate of Akt/PKB, glycogen synthase kinase-3beta (GSK-3beta), was significantly impaired in burned rats. Furthermore, insulin consistently failed to increase the phosphorylation of p70 S6 kinase, another downstream effector of Akt/PKB, in rats with burn injury, whereas phosphorylation of p70 S6 kinase was increased by insulin in controls. The protein expression of Akt/PKB, GSK-3beta, and p70 S6 kinase was unaltered by burn injury. However, insulin-stimulated activation of ERK, a signaling pathway parallel to Akt/PKB, was not affected by burn injury. These results demonstrate that burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle and suggest that attenuated Akt/PKB activation may be involved in deranged metabolism and muscle wasting observed after burn injury.     

Norepinephrine Stimulates Mobilization of Endothelial Progenitor Cells after Limb Ischemia

Objective

During several pathological processes such as cancer progression, thermal injury, wound healing and hindlimb ischemia, the mobilization of endothelial progenitor cells (EPCs) mobilization was enhanced with an increase of sympathetic nerve activity and norepinephrine (NE) secretion, yet the cellular and molecular mechanisms involved in the effects of NE on EPCs has less been investigated.

Methods and Results

EPCs from BMs, peripheral circulation and spleens, the VEGF concentration in BM, skeletal muscle, peripheral circulation and spleen and angiogenesis in ischemic gastrocnemius were quantified in mice with hindlimbs ischemia. Systemic treatment of NE significantly increased EPCs number in BM, peripheral circulation and spleen, VEGF concentration in BM and skeletal muscle and angiogenesis in ischemic gastrocnemius in mice with hind limb ischemia, but did not affair VEGF concentration in peripheral circulation and spleen. EPCs isolated from healthy adults were cultured with NE in vitro to evaluate proliferation potential, migration capacity and phosphorylations of Akt and eNOS signal moleculars. Treatment of NE induced a significant increase in number of EPCs in the S-phase in a dose-dependent manner, as well as migrative activity of EPCs in vitro (p<0.05). The co-treatment of Phentolamine, I127, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and L-NAME with NE blocked the effects of NE on EPCs proliferation and migration. Treatment with NE significantly increased phosphorylation of Akt and eNOS of EPCs. Addition of phentolamine and I127 attenuated the activation of Akt/eNOS pathway, but metoprolol could not. Pretreatment of mice with either Phentolamine or I127 significantly attenuated the effects of NE on EPCs in vivo, VEGF concentration in BM, skeletal muscle and angiogenesis in ischemic gastrocnemius, but Metoprolol did not.

Conclusion

These results unravel that sympathetic nervous system regulate EPCs mobilization and their pro-angiogenic capacity via α adrenoceptor, β 2 adrenoceptor and meanwhile Akt/eNOS signaling pathway.     

Alterations of nPKC distribution,but normal Akt/PKB activation in denervated rat soleus muscle

Denervation has been shown to impair the ability of insulin to stimulate glycogen synthesis and, to a lesser extent, glucose transport in rat skeletal muscle. Insulin binding to its receptor, activation of the receptor tyrosine kinase and phosphatidylinositol 3'-kinase do not appear to be involved. On the other hand, it has been shown that denervation causes an increase in the total diacylglycerol (DAG) content and membrane-associated protein kinase C (PKC) activity. In this study, we further characterize these changes in PKC and assess other possible signaling abnormalities that might be related to the decrease of glycogen synthesis. The results reveal that PKC-epsilon and -theta;, but not -alpha or -zeta, are increased in the membrane fraction 24 h after denervation and that the timing of these changes parallels the impaired ability of insulin to stimulate glycogen synthesis. At 24 h, these changes were associated with a 65% decrease in glycogen synthase (GS) activity ratio and decreased electrophoretic mobility, indicative of phosphorylation in GS in muscles incubated in the absence of insulin. Incubation of the denervated soleus with insulin for 30 min minimally increased glucose incorporation into glycogen; however, it increased GS activity threefold, to a value still less than that of control muscle, and it eliminated the gel shift. In addition, insulin increased the apparent abundance of GS kinase (GSK)-3 and protein phosphatase (PP)1 alpha in the supernatant fraction of muscle homogenate to control values, and it caused the same increases in GSK-3 and Akt/protein kinase B (PKB) phosphorylation and Akt/PKB activity that it did in nondenervated muscle. No alterations in hexokinase I or II activity were observed after denervation; however, in agreement with a previous report, glucose 6-phosphate levels were diminished in 24-h-denervated soleus, and they did not increase after insulin stimulation. These results indicate that alterations in the distribution of PKC-epsilon and -theta; accompany the impairment of glycogen synthesis in the 24-h-denervated soleus. They also indicate that the basal rate of glycogen synthesis and its stimulation by insulin in these muscles are diminished despite a normal activation of Akt/PKB and phosphorylation of GSK-3. The significance of the observed alterations to GSK-3 and PP1 alpha distribution remain to be determined.     

Insulin-like growth factor-I-induced androgen receptor activation is mediated by the PI3K/Akt pathway in C2C12 skeletal muscle cells

Although insulin-like growth factor-I (IGF-I) and androgen receptor (AR) are well known effectors of skeletal muscle, the molecular mechanism by which signaling pathways integrating AR and IGF-I in skeletal muscle cells has not been previously examined. In this study, the role of PI3K/Akt on IGF-I-induced gene expression and activation of AR in skeletal muscle cells was investigated. C2C12 cells were treated with IGF-I in the absence or presence of inhibitors of PI3K/Akt pathway (LY294002 and Wortmannin). Inhibition of the PI3K/Akt pathway with LY294002 or Wortmannin led to a significant decrease in IGF-I-induced AR phosphorylation and total AR protein expression. Furthermore, IGF-I-induced AR mRNA and skeletal α-actin mRNA were blocked by LY294002 or Wortmannin. Confocal images showed that IGF-I-induced AR translocation from cytosol to nucleus was inhibited significantly in response to treatment with LY294002 or Wortmannin. The present results suggest that modulating effect of IGF-I on AR gene expression and activation in C2C12 mouse skeletal muscle cells is mediated at least in part by the PI3K/Akt pathway.