07-802 Sigma-AldrichAnti-phospho-AS160 (Thr642) Antibody

A single bout of exercise can enhance insulin-stimulated glucose uptake in both fast-twitch (type II) and slow-twitch (type I) skeletal muscle for several hours postexercise. Akt substrate of 160 kDa (AS160) is most distal insulin signaling proteins that have been proposed to contribute to the postexercise enhancement of insulin action in fast-twitch muscle. In this study, we examined whether the postexercise increase in insulin action of glucose uptake in slow-twitch muscle is accompanied by increased phosphorylation of AS160 and its paralog TBC1D1. Male Wistar rats (~1-month-old) were exercised on a treadmill for 180 min (9 m/min). Insulin (50 μU/mL)-stimulated glucose uptake was increased at 2 h after cessation of exercise in soleus muscle composed of predominantly slow-twitch fibers. This postexercise increase in insulin action of glucose uptake was accompanied by increased phosphorylation of AS160 (detected by phospho-Thr642 and phospho-Ser588 antibody). On the other hand, prior exercise did not increase phosphorylation of TBC1D1 (detected by phospho-Thr590) at 2 h postexercise. These results suggest the possibility that an enhancement in AS160 phosphorylation but not TBC1D1 phosphorylation is involved with increased postexercise insulin action of glucose uptake in slow-twitch muscle.

Exercise has been demonstrated to enhance subsequent insulin-stimulated glucose uptake (GU) by predominantly type II (fast-twitch) muscle of old rats, but previous research has not evaluated exercise effects on GU by type I (slow-twitch) muscle from old rats. Accordingly, we studied male Fischer 344/Brown Norway rats (24 months old) and determined GU (0, 100, 200, and 5,000 μU/ml insulin) of isolated soleus (predominantly type I) and epitrochlearis (predominantly type II) muscles after one exercise session. Epitrochlearis (100, 200, and 5,000 μU/ml insulin) and soleus (100 and 200 μU/ml insulin) GU were greater at 3-h postexercise vs. age-matched sedentary controls. Insulin receptor tyrosine phosphorylation (Tyr1162/1163) was unaltered by exercise in either muscle. Akt phosphorylation (pAkt) was greater for exercised vs. sedentary rats in the epitrochlearis (Ser473 and Thr308 with 100 and 200 μU/ml, respectively) and soleus (Ser473 with 200 μU/ml). AS160 phosphorylation (pAS160) was greater for exercised vs. sedentary rats in the epitrochlearis (Thr642 with 100 μU/ml), but not the soleus. Exercised vs. sedentary rats did not differ for total protein abundance of insulin receptor, Akt, AS160, or GLUT4 in either muscle. These results demonstrate that both predominantly type I and type II muscles from old rats are susceptible to exercise-induced improvement in insulin-mediated GU by mechanisms that are independent of enhanced insulin receptor tyrosine phosphorylation or altered abundance of important signaling proteins or GLUT4. Exercise-induced elevation in pAkt, and possibly pAS160, may contribute to this effect in the epitrochlearis of old rats, but other mechanisms are likely important for the soleus.

Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems level mechanistic understanding of insulin resistance, using systems wide and internally consistent data from human adipocytes. Based on quantitative steady-state and dynamic time course data on signaling intermediaries, normally and in diabetes, we developed a dynamic mathematical model of insulin signaling. The model structure and parameters are identical in the normal and diabetic states of the model, except for three parameters that change in diabetes: (i) reduced concentration of insulin receptor, (ii) reduced concentration of insulin-regulated glucose transporter GLUT4, and (iii) changed feedback from mammalian target of rapamycin in complex with raptor (mTORC1). Modeling reveals that at the core of insulin resistance in human adipocytes is attenuation of a positive feedback from mTORC1 to the insulin receptor substrate-1, which explains reduced sensitivity and signal strength throughout the signaling network. Model simulations with inhibition of mTORC1 are comparable with experimental data on inhibition of mTORC1 using rapamycin in human adipocytes. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling, both at the cellular level and, using a multilevel model, at the whole body level. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.

Prior exercise by rats can induce a sustained increase in muscle Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) (pAS160(Thr642)). Because phosphorylation of AS160 on both AS160(Thr642) and AS160(Ser588) is important for insulin-stimulated glucose transport (GT), we determined if exercise would also induce a sustained increase in pAS160(Ser588) concomitant with persistently elevated pAS160(Thr642) and GT. Given that the mechanisms for sustained postexercise (PEX) effects on pAS160 were uncertain, we also studied the four kinases known to phosphorylate AS160 (Akt, AMPK, RSK, and SGK1). In addition, because the serine/threonine phosphatase(s) that dephosphorylate muscle AS160 were previously unidentified, we assessed the ability of four serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) to dephosphorylate AS160. We also evaluated exercise effects on posttranslational modifications (Tyr(307) and Leu(309)) that regulate PP2A. In isolated epitrochlearis muscles from rats, GT at 3hPEX with insulin significantly (P less than 0.05) exceeded SED controls. Muscles from 0hPEX vs. 0hSED and 3hPEX vs. 3hSED rats had greater pAS160(Thr642) and pAS160(Ser588). AMPK was the only kinase with greater phosphorylation at 0hPEX vs. 0hSED, and none had greater phosphorylation at 3hPEX vs. 3hSED. Each phosphatase was able to dephosphorylate pAS160(Thr642) and pAS160(Ser588) in cell-free assays. Exercise did not alter posttranslational modifications of PP2A. Our results revealed: 1) pAMPK as a potential trigger for increased pAS160(Thr642) and pAS160(Ser588) at 0hPEX; 2) PP1, PP2A, PP2B, and PP2C were each able to dephosphorylate AS160; and 3) sustained PEX-induced elevations of pAS160(Thr642) and pAS160(Ser588) were attributable to mechanisms other than persistent phosphorylation of known AS160 kinases or altered posttranslational modifications of PP2A.

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.

The cellular mechanisms whereby prior exercise enhances insulin-stimulated glucose transport (GT) are not well understood. Previous studies suggested that a prolonged increase in phosphorylation of Akt substrate of 160 kDa (AS160) may be important for the postexercise increase in insulin sensitivity. In the current study, the effects of in vivo exercise and in vitro contraction on subsequent insulin-stimulated GT were studied separately and together. Consistent with results from previous studies, prior exercise resulted in an increase in AS160 (642)Thr phosphorylation immediately after exercise in rat epitrochlearis muscles, and this increase remained 3 h postexercise concomitant with enhanced insulin-stimulated GT. For experiments with in vitro contraction, isolated rat epitrochlearis muscles were electrically stimulated to contract in the presence or absence of rat serum. As expected, insulin-stimulated GT measured 3 h after electrical stimulation in serum, but not after electrical stimulation without serum, exceeded resting controls. Immediately after electrical stimulation with or without serum, phosphorylation of both AS160 (detected by phospho-Akt substrate, PAS, antibody, or phospho-(642)Thr antibody) and its paralog TBC1D1 (detected by phospho-(237)Ser antibody) was increased. However, both AS160 and TBC1D1 phosphorylation had reversed to resting values at 3 h poststimulation with or without serum. Increasing the amount of exercise (from 1 to 2 h) or electrical stimulation (from 5 to 10 tetani) did not further elevate insulin-stimulated GT. In contrast, the combination of prior exercise and electrical stimulation had an additive effect on the subsequent increase in insulin-stimulated GT, suggesting that these exercise and electrical stimulation protocols may amplify insulin-stimulated GT through distinct mechanisms, with a persistent increase in AS160 phosphorylation potentially important for increased insulin sensitivity after exercise, but not after in vitro contraction.

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism and insulin resistance, possibly reflecting defects in skeletal muscle and adipocyte insulin signaling. Low-frequency (2 Hz) electroacupuncture (EA) increases insulin sensitivity in female rats with dihydrotestosterone (DHT)-induced PCOS, but the mechanism is unclear. We hypothesized that low-frequency EA regulates mediators involved in skeletal muscle glucose uptake and metabolism and alters the lipid profile in rats with DHT-induced PCOS. To test this hypothesis, we implanted in prepubescent female rats 90-day continuous-release pellets containing DHT (PCOS). At 70 days of age, the rats were randomly subdivided into two groups: one received low-frequency EA (evoking muscle twitches) for 20-25 min five times/wk for 4-5 wk; the other did not. Controls were implanted with pellets containing vehicle only. All three groups were otherwise handled similarly. Lipid profile was measured in fasting blood samples. Insulin sensitivity was determined by euglycemic hyperinsulinemic clamp, soleus muscle protein expression of glucose transporter 4 (GLUT4), and phosphorylated and nonphosphorylated Akt, and Akt substrate of 160 kDa was determined by Western blot analysis and GLUT4 location by immunofluorescence staining. PCOS EA rats had normalized insulin sensitivity, lower levels of total high-density lipoprotein and low-density lipoprotein cholesterol, and increased expression of GLUT4 in different compartments of skeletal muscle compared with PCOS rats. Total weight and body composition did not differ in the groups. Thus, in rats with DHT-induced PCOS, low-frequency EA has systemic and local effects involving intracellular signaling pathways in muscle that may, at least in part, account for the marked improved insulin sensitivity.

A single exercise bout can increase insulin-independent glucose transport immediately postexercise and insulin-dependent glucose transport (GT) for several hours postexercise. Akt substrate of 160 kDa (AS160) and TBC1D1 are paralog Rab GTPase-activating proteins that have been proposed to contribute to these exercise effects. Previous research demonstrated greater AS160 and Akt threonine phosphorylation in rat skeletal muscle at 3-4 h postexercise concomitant with enhanced insulin-stimulated GT. To further probe whether these signaling events or TBC1D1 phosphorylation were important for the enhanced postexercise insulin-stimulated GT, male Wistar rats were studied using four experimental protocols (2-h swim exercise, differing with regard to timing of muscle sampling and whether food was provided postexercise) that were known to vary in their influence of insulin-independent and insulin-dependent GT postexercise. The results indicated that, in isolated rat epitrochlearis muscle, 1) elevated phosphorylation of AS160 (measured using anti-phospho-Akt substrate, PAS-AS160, and phosphospecific anti-Thr(642)-AS160, pThr(642)-AS160) consistently tracked with elevated insulin-stimulated GT; 2) PAS-TBC1D1 was not different from sedentary values at 3 or 27 h postexercise, when insulin sensitivity was increased; 3) insulin-stimulated Akt activity was not increased postexercise in muscles with increased insulin sensitivity; 4) PAS-TBC1D1 was increased immediately postexercise, when insulin-independent GT was elevated, and reversed at 3 and 27 h postexercise, when insulin-independent GT was also reversed; and 5) there was no significant effect of exercise or insulin on total abundance of AS160, TBC1D1, Akt, or GLUT4 protein with any of the protocols. The results are consistent with increased AS160 phosphorylation (PAS-AS160 or pThr(642)-AS160) but not increased PAS-TBC1D1 or Akt activity, which is important for increased postexercise insulin-stimulated GT in rat skeletal muscle. They also support the idea that increased TBC1D1 phosphorylation may play a role in the insulin-independent increase in GT postexercise.