Palmitate contributes to insulin resistance through downregulation of the Src-mediated phosphorylation of Akt in C2C12 myotubes

The mechanisms of free fatty acid (FFA)-induced peripheral insulin resistance remain elusive. This study aimed to investigate the effect of palmitate, a saturated fatty acid, on glucose metabolism in C2C12 myotubes, and to explore the underlying mechanisms. In it, palmitate decreased insulin-stimulated glucose uptake and consumption in a dose-dependent manner, and it reduced the insulin-stimulated phosphorylation of Akt at Thr308 and Ser473, but had no effect on the protein expression of PI3K-p85 or the activity of PI3K. Additionally, it inhibited the insulin-stimulated phosphorylation of Src at Tyr416, causing a reduction in the Src-mediated phosphorylation of Akt. Inhibition of Src by PP2 resulted in decreases in insulin-stimulated glucose uptake and phosphorylation of Src at Tyr416 and Akt at Thr308 and Ser473. The findings indicate that palmitate contributes to insulin resistance by inhibiting the Src-mediated phosphorylation of Akt in C2C12 myotubes, and this provides insight into the molecular mechanisms of FFA-induced insulin resistance.     

Palmitate Contributes to Insulin Resistance through Downregulation of the Src-Mediated Phosphorylation of Akt in C2C12 Myotubes

The mechanisms of free fatty acid (FFA)-induced peripheral insulin resistance remain elusive. This study aimed to investigate the effect of palmitate, a saturated fatty acid, on glucose metabolism in C2C12 myotubes, and to explore the underlying mechanisms. In it, palmitate decreased insulin-stimulated glucose uptake and consumption in a dose-dependent manner, and it reduced the insulin-stimulated phosphorylation of Akt at Thr308 and Ser473, but had no effect on the protein expression of PI3K-p85 or the activity of PI3K. Additionally, it inhibited the insulin-stimulated phosphorylation of Src at Tyr416, causing a reduction in the Src-mediated phosphorylation of Akt. Inhibition of Src by PP2 resulted in decreases in insulin-stimulated glucose uptake and phosphorylation of Src at Tyr416 and Akt at Thr308 and Ser473. The findings indicate that palmitate contributes to insulin resistance by inhibiting the Src-mediated phosphorylation of Akt in C2C12 myotubes, and this provides insight into the molecular mechanisms of FFA-induced insulin resistance.     

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.     

Tissue-specific responses of IGF-1/insulin and mTOR signaling in calorie restricted rats

Moderate calorie restriction (CR) (～60% of ad libitum, AL, intake) has been associated with numerous favorable physiological outcomes in many species, and the insulin/IGF-1 and mTOR signaling pathways have each been proposed as potential mediators for many of CR's bioeffects. However, few studies have assessed the widely held idea that CR induces the down-regulation of the insulin/IGF-1 and/or mTOR pathways in multiple tissues. Accordingly, we analyzed the phosphorylation status of 11 key signaling proteins from the insulin/IGF-1 (IR(Tyr1162/1163), IGF-1R(Tyr1135/1136), IRS-1(Ser312), PTEN(Ser380), Akt(Ser473), GSK3α(Ser21), GSK3β(Ser9)) and mTOR (TSC2(Ser939), mTOR(Ser2448), P70S6K(Thr412), RPS6(Ser235/236)) pathways in 11 diverse tissues [liver, kidney, lung, aorta, two brain regions (cortex and cerebellum), and two slow-twitch and three fast-twitch skeletal muscles] from 9-month-old male AL and CR Fischer 344 x Brown Norway rats. The rats were studied under two conditions: with endogenous insulin levels (i.e., AL>CR) and with insulin infused during a hyperinsulinemic-euglycemic clamp so that plasma insulin concentrations were matched between the two diet groups. The most striking and consistent effect of CR was greater pAkt in 3 of the 5 skeletal muscles of CR vs. AL rats. There were no significant CR effects on the mTOR signaling pathway and no evidence that CR caused a general attenuation of mTOR signaling across the tissues studied. Rather than supporting the premise of a global downregulation of insulin/IGF-1 and/or mTOR signaling in many tissues, the current results revealed clear tissue-specific CR effects for the insulin signaling pathway without CR effects on the mTOR signaling pathway.     

Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats

Calorie restriction [CR; ~65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[(3)H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr(308) and Ser(473)) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser(588) and Thr(642)) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.     

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.     

A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle

PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40?kDa and Akt substrate of 160?kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206?in a dose-dependent manner. Incubation with high doses of MK-2206 (10?μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.     

Interleukin-6 acts as insulin sensitizer on glycogen synthesis in human skeletal muscle cells by phosphorylation of Ser473 of Akt

Previous studies showed an insulin-"desensitizing" action of IL-6 on glycogen synthesis in hepatocytes. We recently found no inhibition of the proximal steps of the insulin signal cascade in human skeletal muscle cells. Because these data indicate a possible tissue-specific effect of IL-6, we investigated the influence of IL-6 on insulin-stimulated glycogen synthesis in these cells. At first, we found that incubation of the cells with 20 ng/ml IL-6 alone induced phosphorylation of Ser473 of Akt, but not of Thr308 time dependently and we observed that IL-6 augments insulin-induced Ser473 and Thr308 phosphorylation in the low nanomolar range of insulin. Moreover, IL-6 increased insulin-stimulated phosphorylation of glycogen synthase kinase-3. Accordingly, IL-6 enhanced glycogen synthesis in the presence of 3 and 10 nM insulin, whereas IL-6 alone had only a marginal effect. IL-6 treatment of C57Bl/6 mice readily stimulated phosphorylation of Ser473 in skeletal muscle. Our result that IL-6 did not induce Ser473 phosphorylation in the liver of these mice suggests a tissue-specific effect. Together, our data demonstrate a novel insulin-sensitizing function of IL-6 on glycogen synthesis in skeletal muscle cells and indicate that IL-6 exerts cell/tissue-specific effects on insulin action.     

The phosphorylation of Ser318 of insulin receptor substrate 1 is not per se inhibitory in skeletal muscle cells but is necessary to trigger the attenuation of the insulin-stimulated signal

The Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS) is one key mechanism to stimulate and/or attenuate insulin signal transduction. Using a phospho-specific polyclonal antibody directed against phosphorylated Ser(318) of IRS-1, we found a rapid and transient insulin-stimulated phosphorylation of Ser(318) in human and rodent skeletal muscle cell models and in muscle tissue of insulin-treated mice. None of the investigated insulin resistance-associated factors (e.g. high glucose, tumor necrosis factor-alpha, adrenaline) stimulated the phosphorylation of Ser(318). Studying the function of this phosphorylation, we found that replacing Ser(318) by alanine completely prevented both the attenuation of insulin-stimulated Akt/protein kinase B Ser(473) phosphorylation and glucose uptake after 60 min of insulin stimulation. Unexpectedly, after acute insulin stimulation, we observed that phosphorylation of Ser(318) is not inhibitory but rather enhances insulin signal transduction because introduction of Ala(318) led to a reduction of the insulin-stimulated Akt/protein kinase B phosphorylation. Furthermore, replacing Ser(318) by glutamate, i.e. mimicking phosphorylation, improved glucose uptake after acute insulin stimulation. These data suggest that phosphorylation of Ser(318) is not per se inhibitory but is necessary to trigger the attenuation of the insulin-stimulated signal in skeletal muscle cells. Investigating the molecular mechanism of insulin-stimulated Ser(318) phosphorylation, we found that phosphatidylinositol 3-kinase-mediated activation of atypical protein kinase C-zeta and recruitment of protein kinase C-zeta to IRS-1 was responsible for this phosphorylation. We conclude that Ser(318) phosphorylation of IRS-1 is an early physiological event in insulin-stimulated signal transduction, which attenuates the continuing action of insulin.     

Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor

Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.     

Caffeine modulates phosphorylation of insulin receptor substrate-1 and impairs insulin signal transduction in rat skeletal muscle

Caffeine decreases insulin sensitivity and insulin-stimulated glucose transport in skeletal muscle; however, the precise mechanism responsible for this deleterious effect is not understood fully. We investigated the effects of incubation with caffeine on insulin signaling in rat epitrochlearis muscle. Caffeine (≥1 mM, ≥15 min) suppressed insulin-stimulated insulin receptor substrate (IRS)-1 Tyr(612) phosphorylation in a dose- and time-dependent manner. These responses were associated with inhibition of the insulin-stimulated phosphorylation of phosphatidylinositol 3-kinase (PI3K) Tyr(458), Akt Ser(473), and glycogen synthase kinase-3β Ser(9) and with inhibition of insulin-stimulated 3-O-methyl-d-glucose (3MG) transport but not with inhibition of the phosphorylation of insulin receptor-β Tyr(1158/62/63). Furthermore, caffeine enhanced phosphorylation of IRS-1 Ser(307) and an IRS-1 Ser(307) kinase, inhibitor-κB kinase (IKK)-α/β Ser(176/180). Blockade of IKK/IRS-1 Ser(307) by caffeic acid ameliorated the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation and 3MG transport. Caffeine also increased the phosphorylation of IRS-1 Ser(789) and an IRS-1 Ser(789) kinase, 5'-AMP-activated protein kinase (AMPK). However, inhibition of IRS-1 Ser(789) and AMPK phosphorylation by dantrolene did not rescue the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation or 3MG transport. In addition, caffeine suppressed the phosphorylation of insulin-stimulated IRS-1 Ser(636/639) and upstream kinases, including the mammalian target of rapamycin and p70S6 kinase. Intravenous injection of caffeine at a physiological dose (5 mg/kg) in rats inhibited the phosphorylation of insulin-stimulated IRS-1 Tyr(612) and Akt Ser(473) in epitrochlearis muscle. Our results indicate that caffeine inhibits insulin signaling partly through the IKK/IRS-1 Ser(307) pathway, via a Ca(2+)- and AMPK-independent mechanism in skeletal muscle.     

Activation of AMP-activated protein kinase, inhibition of pyruvate dehydrogenase activity, and redistribution of substrate partitioning mediate the acute insulin-sensitizing effects of troglitazone in skeletal muscle cells

The aim of this study was to investigate the acute effects of troglitazone on several pathways of glucose and fatty acid (FA) partitioning and the molecular mechanisms involved in these processes in skeletal muscle. Exposure of L6 myotubes to troglitazone for 1 h significantly increased phosphorylation of AMPK and ACC, which was followed by approximately 30% and approximately 60% increases in palmitate oxidation and carnitine palmitoyl transferase-1 (CPT-1) activity, respectively. Troglitazone inhibited basal ( approximately 25%) and insulin-stimulated ( approximately 35%) palmitate uptake but significantly increased basal and insulin-stimulated glucose uptake by approximately 2.2- and 2.7-fold, respectively. Pharmacological inhibition of AMPK completely prevented the effects of troglitazone on palmitate oxidation and glucose uptake. Interestingly, even though troglitazone exerted an insulin sensitizing effect, it reduced basal and insulin-stimulated rates of glycogen synthesis, incorporation of glucose into lipids, and glucose oxidation to values corresponding to approximately 30%, approximately 60%, and 30% of the controls, respectively. These effects were accompanied by an increase in basal and insulin-stimulated phosphorylation of Akt(Thr308), Akt(Ser473), and GSK3alpha/beta. Troglitazone also powerfully suppressed pyruvate decarboxylation, which was followed by a significant increase in basal ( approximately 3.5-fold) and insulin-stimulated ( approximately 5.5-fold) rates of lactate production by muscle cells. In summary, we provide novel evidence that troglitazone exerts acute insulin sensitizing effects by increasing FA oxidation, reducing FA uptake, suppressing pyruvate dehydrogenase activity, and shifting glucose metabolism toward lactate production in muscle cells. These effects seem to be at least partially dependent on AMPK activation and may account for potential acute PPAR-gamma-independent anti-diabetic effects of thiazolidinediones in skeletal muscle.     

Next-generation Akt inhibitors provide greater specificity: effects on glucose metabolism in adipocytes

Many human tumours exhibit activation of the PI3K (phosphoinositide 3-kinase)/Akt pathway, and inhibition of this pathway slows tumour growth. This led to the development of specific Akt inhibitors for in vivo use. However, activation of Akt is also necessary for processes including glucose metabolism. Therefore a potential complication of such anticancer drugs is insulin resistance and/or diabetes. In the process of characterizing the metabolic effects of early-phase Akt inhibitors, we discovered an off-target inhibitory effect on mammalian facilitative glucose transporters. In view of the crucial role of glucose transport for all mammalian cells, such an off-target effect would have major implications for further development of this family of compounds. In the present study, we have characterized a next-generation Akt inhibitor, MK-2206. MK-2206 is an orally active allosteric Akt inhibitor under development for treating solid tumours. We report that MK-2206 potently inhibits Thr308Akt and Ser473Akt phosphorylation in 3T3-L1 adipocytes (IC50 0.11 and 0.18 μM respectively) as well as downstream effects of insulin on GLUT4 (glucose transporter 4) translocation (IC50 0.47 μM) and glucose transport (IC50 0.14 μM). Notably, the potency of MK-2206 is approximately 1 log higher than previous inhibitors and its specificity is significantly improved with modest inhibitory effects on glucose transport in GLUT4-expressing adipocytes and GLUT1-rich human erythrocytes, independently of Akt. Nevertheless, MK-2206 clearly has potent effects on Akt2, the principal isoform involved in peripheral insulin action, in which case insulin resistance will probably be a major complication following in vivo administration. We conclude that MK-2206 provides an optimal tool for studying the effects of Akt in vitro.     

Calorie restriction prevents the development of insulin resistance and impaired insulin signaling in skeletal muscle of ovariectomized rats

Insulin resistance of skeletal muscle glucose transport due to prolonged loss of ovarian function in ovariectomized (OVX) rats is accompanied by other features of the metabolic syndrome and may be confounded by increased calorie consumption. In this study, we investigated the role of calorie consumption in the development of insulin resistance in OVX rats. In addition, we examined the cellular mechanisms underlying skeletal muscle insulin resistance in OVX rats. Female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated (SHAM). OVX rats either had free access to food, pair feeding (PF) with SHAM or received a 35% reduction in food intake (calorie restriction; CR) for 12weeks. Compared with SHAM, ovariectomy induced skeletal muscle insulin resistance, which was associated with decreases (32-70%) in tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), IRS-1 associated p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), and Akt Ser(473) phosphorylation whereas insulin-stimulated phosphorylation of IRS-1 Ser(307), SAPK/JNK Thr(183)/Tyr(185), and p38 mitogen-activated protein kinase (MAPK) Thr(180)/Tyr(182) was increased (24-62%). PF improved the serum lipid profile but did not restore insulin-stimulated glucose transport, indicating that insulin resistance in OVX rats is a consequence of ovarian hormone deprivation. In contrast, impaired insulin sensitivity and defective insulin signaling were not observed in the skeletal muscle of OVX+CR rats. Therefore, we provide evidence for the first time that CR effectively prevents the development of insulin resistance and impaired insulin signaling in the skeletal muscle of OVX rats.     

Calorie restriction increases the ratio of phosphatidylinositol 3-kinase catalytic to regulatory subunits in rat skeletal muscle

Calorie restriction [CR; 60% of ad libitum (AL) intake] improves insulin-stimulated glucose transport, concomitant with enhanced phosphorylation of Akt. The mechanism(s) for the CR-induced increase in Akt phosphorylation of insulin-stimulated muscle is unknown. The purpose of this study was to determine whether CR increased the ratio of catalytic to regulatory subunits favoring enhanced phosphatidylinositol (PI) 3-kinase signaling, which may contribute to increases in Akt phosphorylation and glucose transport in insulin-stimulated muscles. We measured the PI 3-kinase regulatory (p85alpha/beta, p50alpha, and p55alpha) and catalytic (p110) subunits abundance in skeletal muscle from male F344B/N rats after 8 wk of AL or CR treatment. In CR compared with AL muscles, regulatory isoforms, p50alpha and p55alpha abundance were approximately 40% lower (P < 0.01) with unchanged p85alpha/beta levels. There was no diet-related change in catalytic subunit abundance. Despite lower IRS-1 levels ( approximately 35%) for CR vs. AL, IRS-1-p110 association in insulin-stimulated muscles was significantly (P < 0.05) enhanced by approximately 50%. Downstream of PI 3-kinase, CR compared with AL significantly enhanced Akt serine phosphorylation by 1.5-fold higher (P = 0.01) and 3-O-methylglucose transport by approximately 20% in muscles incubated with insulin. The increased ratio of PI 3-kinase catalytic to regulatory subunits favors enhanced insulin signaling, which likely contributes to greater Akt phosphorylation and improved insulin sensitivity associated with CR in skeletal muscle.     

Acute and chronic leptin treatment mediate contrasting effects on signaling,glucose uptake,and GLUT4 translocation in L6-GLUT4myc myotubes

We have previously shown that in L6-GLUT4myc rat skeletal muscle cells, acute treatment with leptin reduced insulin-stimulated glucose uptake without altering insulin-stimulated GLUT4 translocation. In contrast, we show here that the ability of leptin to increase phosphorylation of its receptor and to reduce insulin-stimulated glucose uptake was lost in cells that were continuously exposed to leptin for 24 h. This desensitization correlated with an increase in expression of suppressor of cytokine signaling-3 (SOCS-3). Time course analysis demonstrated that the transition from acute to chronic effects of leptin occurs after 2 h. The desensitization of leptin action at 24 h was not reversed by 30 min washout period prior to re-exposing cells to leptin. However, despite insulin-stimulated glucose uptake being unaffected upon 24 h preincubation with leptin, a small but significant decrease (37%) in insulin-stimulated GLUT4 translocation and phosphorylation of Akt on T308 was detected. Insulin-stimulated phosphorylation of Akt on S473 or of p38 MAPK were unaffected. These results suggest that the chronic leptin treatment leads to desensitization of leptin signaling yet can simultaneously decrease the ability of insulin to phosphorylate Akt on T308 and translocate GLUT4. However, this does not manifest as a reduction in total glucose uptake into L6 myotubes.     

Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle

Skeletal muscle insulin sensitivity improves with short-term reduction in calorie intake. The goal of this study was to evaluate changes in the abundance and phosphorylation of Akt1 and Akt2 as potential mechanisms for enhanced insulin action after 20 days of moderate calorie restriction [CR; 60% of ad libitum (AL) intake] in rat skeletal muscle. We also assessed changes in the abundance of SH2 domain-containing inositol phosphatase (SHIP2), a negative regulator of insulin signaling. Fisher 344 x Brown Norway rats were assigned to an AL control group or a CR treatment group for 20 days. Epitrochlearis muscles were dissected and incubated with or without insulin (500 microU/ml). Total Akt serine and threonine phosphorylation was significantly increased by 32 (P < 0.01) and 30% (P < 0.005) in insulin-stimulated muscles from CR vs. AL. Despite an increase in total Akt phosphorylation, there was no difference in Akt1 serine or Akt1 threonine phosphorylation between CR and AL insulin-treated muscles. However, there was a 30% decrease (P < 0.05) in Akt1 abundance for CR vs. AL. In contrast, there was no change in Akt2 protein abundance, and there was a 94% increase (P < 0.05) in Akt2 serine phosphorylation and an increase of 75% (P < 0.05) in Akt2 threonine phosphorylation of insulin-stimulated CR muscles compared with AL. There was no diet effect on SHIP2 abundance in skeletal muscle. These results suggest that, with brief CR, enhanced Akt2 phosphorylation may play a role in increasing insulin sensitivity in rat skeletal muscles.     

Rapamycin does not improve insulin sensitivity despite elevated mammalian target of rapamycin complex 1 activity in muscles of ob/ob mice

Studies of cultured cells have indicated that the mammalian target of rapamycin complex 1 (mTORC1) mediates the development of insulin resistance. Because a role for mTORC1 in the development of skeletal muscle insulin resistance has not been established, we studied mTORC1 activity in skeletal muscles of ob/ob (OB) mice and wild-type (WT) mice. In vivo insulin action was assessed in muscles of mice 15 min following an intraperitoneal injection of insulin or an equivalent volume of saline. In the basal state, the phosphorylation of S6K on Thr(389), mTOR on Ser(2448), and PRAS40 on Thr(246) were increased significantly in muscles from OB mice compared with WT mice. The increase in basal mTORC1 signaling was associated with an increase in basal PKB phosphorylation on Thr(308) and Ser(473). In the insulin-stimulated state, no differences existed in the phosphorylation of S6K on Thr(389), but PKB phosphorylation on Thr(308) and Ser(473) was significantly reduced in muscles of OB compared with WT mice. Despite elevated mTORC1 activity in OB mice, rapamycin treatment did not improve either glucose tolerance or insulin tolerance. These results indicate that the insulin resistance of OB mice is mediated, in part, by factors other than mTORC1.     

HM-chromanone,a component of Portulaca oleracea L., stimulates glucose uptake and glycogen synthesis in skeletal muscle cell

BackgroundDiabetes mellitus is a chronic metabolic disease characterized by increased blood glucose levels. In order to lower blood glucose, it is important to stimulate glucose uptake and glycogen synthesis in the muscle. (E)-5-hydroxy-7-methoxy-3-(2′-hydroxybenzyl)-4-chromanone (HM-chromanone), a constituent isolated from Portulaca oleracea L., exhibits anti-diabetic effects; however, its mechanisms are not yet clearly understood on glucose uptake and glycogen synthesis in muscle cells.PurposeIn the present study, we examined the effects of HM-chromanone on glucose uptake into L6 skeletal muscle cells and elucidated the underlying mechanisms.MethodsThe effects of HM-chromanone on glucose uptake into L6 skeletal muscle cells were assessed by 2-Deoxyglucose uptake assay. Western blot analysis was carried out to elucidate the underlying molecular mechanisms.ResultsWe found that HM-chromanone promoted glucose uptake into L6 skeletal muscle cells in a dose-dependent manner. Moreover, HM-chromanone induced the phosphorylation of IRS-1^Tyr612 and AKT^Ser473, and the activation of PI3K. HM-chromanone also stimulated the phosphorylation of AMPK^Thr172, AS160^Thr642, TBC1D1^Ser237, and ACC via the CaMKKβ pathway. Furthermore, HM-chromanone increased glycogen synthesis through the inactivation of glycogen synthase kinase 3 α/β.ConclusionThe results of this study indicate that HM-chromanone stimulates glucose uptake through the activation of the PI3K/AKT and CaMKKβ-AMPK pathways and glycogen synthesis via the GSK3 α/β pathway in L6 skeletal muscle cells.     

mTORC2 protein complex-mediated Akt (Protein Kinase B) Serine 473 Phosphorylation is not required for Akt1 activity in human platelets [corrected

Protein kinase B (PKB, Akt) is a Ser/Thr kinase involved in the regulation of cell survival, proliferation, and metabolism and is activated by dual phosphorylation on Thr(308) in the activation loop and Ser(473) in the hydrophobic motif. It plays a contributory role to platelet function, although little is known about its regulation. In this study, we investigated the role of the mammalian target of rapamycin complex (mTORC)-2 in Akt regulation using the recently identified small molecule ATP competitive mTOR inhibitors PP242 and Torin1. Both PP242 and Torin1 blocked thrombin and insulin-like growth factor 1-mediated Akt Ser(473) phosphorylation with an IC(50) between 1 and 5 nm, whereas the mTORC1 inhibitor rapamycin had no effect. Interestingly, PP242 and Torin1 had no effect on Akt Thr(308) phosphorylation, Akt1 activity, and phosphorylation of the Akt substrate glycogen synthase kinase 3β, indicating that Ser(473) phosphorylation is not necessary for Thr(308) phosphorylation and maximal Akt1 activity. In contrast, Akt2 activity was significantly reduced, concurrent with inhibition of PRAS40 phosphorylation, in the presence of PP242 and Torin1. Other signaling pathways, including phospholipase C/PKC and the MAPK pathway, were unaffected by PP242 and Torin1. Together, these results demonstrate that mTORC2 is the kinase that phosphorylates Akt Ser(473) in human platelets but that this phosphorylation is dispensable for Thr(308) phosphorylation and Akt1 activity.