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1
Kim, Hyo-Young, Hyo-Jung Choi, Jung-Suk Lim, Eui-Jung Park, Hyun Jun Jung, Yu-Jung Lee, Sang-Yeob Kim, and Tae-Hwan Kwon. "Emerging role of Akt substrate protein AS160 in the regulation of AQP2 translocation." American Journal of Physiology-Renal Physiology 301, no. 1 (July 2011): F151—F161. http://dx.doi.org/10.1152/ajprenal.00519.2010.
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AS160, a novel Akt substrate of 160 kDa, contains a Rab GTPase-activating protein (GAP) domain. The present study examined the role of Akt and AS160 in aquaporin-2 (AQP2) trafficking. The main strategy was to examine the changes in AQP2 translocation in response to small interfering RNA (siRNA)-mediated AS160 knockdown in mouse cortical collecting duct cells (M-1 cells and mpkCCDc14 cells). Short-term dDAVP treatment in M-1 cells stimulated phosphorylation of Akt (S473) and AS160, which was also seen in mpkCCDc14 cells. Conversely, the phosphoinositide 3-kinase (PI3K) inhibitor LY 294002 diminished phosphorylation of Akt (S473) and AS160. Moreover, siRNA-mediated Akt1 knockdown was associated with unchanged total AS160 but decreased phospho-AS160 expression, indicating that phosphorylation of AS160 is dependent on PI3K/Akt pathways. siRNA-mediated AS160 knockdown significantly decreased total AS160 and phospho-AS160 expression. Immunocytochemistry revealed that AS160 knockdown in mpkCCDc14 cells was associated with increased AQP2 density in the plasma membrane [135 ± 3% of control mpkCCDc14 cells ( n = 65), P < 0.05, n = 64] despite the absence of dDAVP stimulation. Moreover, cell surface biotinylation assays of mpkCCDc14 cells with AS160 knockdown exhibited significantly higher AQP2 expression [150 ± 15% of control mpkCCDc14 cells ( n = 3), P < 0.05, n = 3]. Taken together, PI3K/Akt pathways mediate the dDAVP-induced AS160 phosphorylation, and AS160 knockdown is associated with higher AQP2 expression in the plasma membrane. Since AS160 contains a GAP domain leading to a decrease in the active GTP-bound form of AS160 target Rab proteins for vesicle trafficking, decreased expression of AS160 is likely to play a role in the translocation of AQP2 to the plasma membrane.
2
Hargett, Stefan R., Natalie N. Walker, and Susanna R. Keller. "Rab GAPs AS160 and Tbc1d1 play nonredundant roles in the regulation of glucose and energy homeostasis in mice." American Journal of Physiology-Endocrinology and Metabolism 310, no. 4 (February 15, 2016): E276—E288. http://dx.doi.org/10.1152/ajpendo.00342.2015.
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The related Rab GTPase-activating proteins (Rab GAPs) AS160 and Tbc1d1 regulate the trafficking of the glucose transporter GLUT4 that controls glucose uptake in muscle and fat cells and glucose homeostasis. AS160- and Tbc1d1-deficient mice exhibit different adipocyte- and skeletal muscle-specific defects in glucose uptake, GLUT4 expression and trafficking, and glucose homeostasis. A recent study analyzed male mice with simultaneous deletion of AS160 and Tbc1d1 (AS160−/−/Tbc1d1−/− mice). Herein, we describe abnormalities in male and female AS160−/−/Tbc1d1−/− mice on another strain background. We confirm the earlier observation that GLUT4 expression and glucose uptake defects of single-knockout mice join in AS160−/−/Tbc1d1−/− mice to affect all skeletal muscle and adipose tissues. In large mixed fiber-type skeletal muscles, changes in relative basal GLUT4 plasma membrane association in AS160−/− and Tbc1d1−/− mice also combine in AS160−/−/Tbc1d1−/− mice. However, we found different glucose uptake abnormalities in isolated skeletal muscles and adipocytes than reported previously, resulting in different interpretations of how AS160 and Tbc1d1 regulate GLUT4 translocation to the cell surface. In support of a larger role for AS160 in glucose homeostasis, in contrast with the previous study, we find similarly impaired glucose and insulin tolerance in AS160−/−/Tbc1d1−/− and AS160−/− mice. However, in vivo glucose uptake abnormalities in AS160−/−/Tbc1d1−/− skeletal muscles differ from those observed previously in AS160−/− mice, indicating additional defects due to Tbc1d1 deletion. Similar to AS160- and Tbc1d1-deficient mice, AS160−/−/Tbc1d1−/− mice show sex-specific abnormalities in glucose and energy homeostasis. In conclusion, our study supports nonredundant functions for AS160 and Tbc1d1.
3
Lansey, Melissa N., Natalie N. Walker, Stefan R. Hargett, Joseph R. Stevens, and Susanna R. Keller. "Deletion of Rab GAP AS160 modifies glucose uptake and GLUT4 translocation in primary skeletal muscles and adipocytes and impairs glucose homeostasis." American Journal of Physiology-Endocrinology and Metabolism 303, no. 10 (November 15, 2012): E1273—E1286. http://dx.doi.org/10.1152/ajpendo.00316.2012.
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Tight control of glucose uptake in skeletal muscles and adipocytes is crucial to glucose homeostasis and is mediated by regulating glucose transporter GLUT4 subcellular distribution. In cultured cells, Rab GAP AS160 controls GLUT4 intracellular retention and release to the cell surface and consequently regulates glucose uptake into cells. To determine AS160 function in GLUT4 trafficking in primary skeletal muscles and adipocytes and investigate its role in glucose homeostasis, we characterized AS160 knockout (AS160−/−) mice. We observed increased and normal basal glucose uptake in isolated AS160−/− adipocytes and soleus, respectively, while insulin-stimulated glucose uptake was impaired and GLUT4 expression decreased in both. No such abnormalities were found in isolated AS160−/− extensor digitorum longus muscles. In plasma membranes isolated from AS160−/− adipose tissue and gastrocnemius/quadriceps, relative GLUT4 levels were increased under basal conditions and remained the same after insulin treatment. Concomitantly, relative levels of cell surface-exposed GLUT4, determined with a glucose transporter photoaffinity label, were increased in AS160−/− adipocytes and normal in AS160−/− soleus under basal conditions. Insulin augmented cell surface-exposed GLUT4 in both. These observations suggest that AS160 is essential for GLUT4 intracellular retention and regulation of glucose uptake in adipocytes and skeletal muscles in which it is normally expressed. In vivo studies revealed impaired insulin tolerance in the presence of normal (male) and impaired (female) glucose tolerance. Concurrently, insulin-elicited increases in glucose disposal were abolished in all AS160−/− skeletal muscles and liver but not in AS160−/− adipose tissues. This suggests AS160 as a target for differential manipulation of glucose homeostasis.
4
Alkhateeb, Hakam, Adrian Chabowski, Jan F. C. Glatz, Brendon Gurd, Joost J. F. P. Luiken, and Arend Bonen. "Restoring AS160 phosphorylation rescues skeletal muscle insulin resistance and fatty acid oxidation while not reducing intramuscular lipids." American Journal of Physiology-Endocrinology and Metabolism 297, no. 5 (November 2009): E1056—E1066. http://dx.doi.org/10.1152/ajpendo.90908.2008.
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We examined whether AICAR or leptin rapidly rescued skeletal muscle insulin resistance via increased palmitate oxidation, reductions in intramuscular lipids, and/or restoration of insulin-stimulated AS60 phosphorylation. Incubation with palmitate (2 mM, 0–18 h) induced insulin resistance in soleus muscle. From 12–18 h, palmitate was removed or AICAR or leptin was provided while 2 mM palmitate was maintained. Palmitate oxidation, intramuscular triacylglycerol, diacylglycerol, ceramide, AMPK phosphorylation, basal and insulin-stimulated glucose transport, plasmalemmal GLUT4, and Akt and AS160 phosphorylation were examined at 0, 6, 12, and 18 h. Palmitate treatment (12 h) increased intramuscular lipids (triacylglycerol +54%, diacylglycerol +11%, total ceramide +18%, C16:0 ceramide +60%) and AMPK phosphorylation (+118%), whereas it reduced fatty acid oxidation (−60%) and insulin-stimulated glucose transport (−70%), GLUT4 translocation (−50%), and AS160 phosphorylation (−40%). Palmitate removal did not rescue insulin resistance or associated parameters. The AICAR and leptin treatments did not consistently reduce intramuscular lipids, but they did rescue palmitate oxidation and insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation. Increased AMPK phosphorylation was associated with these improvements only when AICAR and leptin were present. Hence, across all experiments, AMPK phosphorylation did not correlate with any parameters. In contrast, palmitate oxidation and insulin-stimulated AS160 phosphorylation were highly correlated ( r = 0.83). We speculate that AICAR and leptin activate both of these processes concomitantly, involving activation of unknown kinases in addition to AMPK. In conclusion, despite the maintenance of high concentrations of palmitate (2 mM), as well as increased concentrations of intramuscular lipids (triacylglycerol, diacylglycerol, and ceramide), the rapid AICAR- and leptin-mediated rescue of palmitate-induced insulin resistance is attributable to the restoration of insulin-stimulated AS160 phosphorylation and GLUT4 translocation.
5
Cartee, Gregory D., and Jørgen F. P. Wojtaszewski. "Role of Akt substrate of 160 kDa in insulin-stimulated and contraction-stimulated glucose transport." Applied Physiology, Nutrition, and Metabolism 32, no. 3 (March 2007): 557–66. http://dx.doi.org/10.1139/h07-026.
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Insulin and exercise, the most important physiological stimuli to increase glucose transport in skeletal muscle, trigger a redistribution of GLUT4 glucose transporter proteins from the cell interior to the cell surface, thereby increasing glucose transport capacity. The most distal insulin signaling protein that has been linked to GLUT4 translocation, Akt substrate of 160 kDa (AS160), becomes phosphorylated in insulin-stimulated 3T3-L1 adipocytes; this is important for insulin-stimulated GLUT4 translocation and glucose transport. Insulin also induces a rapid and dose-dependent increase in AS160 phosphorylation in skeletal muscle. Available data from skeletal muscle support the concepts developed in adipocytes with regard to the role AS160 plays in the regulation of insulin-stimulated glucose transport. In vivo exercise, in vitro contractions, or in situ contractions can also stimulate AS160 phosphorylation. AMP-activated protein kinase (AMPK) is likely important for phosphorylating AS160 in response to exercise/contractile activity, whereas Akt2 appears to be important for insulin-stimulated AS160 phosphorylation in muscle. Evidence of a role for AS160 in exercise/contraction-stimulated glucose uptake is currently inconclusive. The distinct signaling pathways that are stimulated by insulin and exercise/contraction converge at AS160. Although AS160 phosphorylation is apparently important for insulin-stimulated GLUT4 translocation and glucose transport, it is uncertain whether elevated AS160 phosphorylation plays a similar role with exercise/contraction.
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Liang, Xiubin, Michael B. Butterworth, Kathryn W. Peters, and Raymond A. Frizzell. "AS160 Modulates Aldosterone-stimulated Epithelial Sodium Channel Forward Trafficking." Molecular Biology of the Cell 21, no. 12 (June 15, 2010): 2024–33. http://dx.doi.org/10.1091/mbc.e10-01-0042.
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Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (Isc). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and Isc 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and Isc under basal conditions to ∼80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, β and ε. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3β and ε and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.
7
Ducommun, Serge, Hong Yu Wang, Kei Sakamoto, Carol MacKintosh, and Shuai Chen. "Thr649Ala-AS160 knock-in mutation does not impair contraction/AICAR-induced glucose transport in mouse muscle." American Journal of Physiology-Endocrinology and Metabolism 302, no. 9 (May 1, 2012): E1036—E1043. http://dx.doi.org/10.1152/ajpendo.00379.2011.
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AS160 and its closely related protein TBC1D1 have emerged as key mediators for both insulin- and contraction-stimulated muscle glucose uptake through regulating GLUT4 trafficking. Insulin increases AS160 phosphorylation at multiple Akt/PKB consensus sites, including Thr649, and promotes its binding to 14-3-3 proteins through phospho-Thr649. We recently provided genetic evidence that AS160-Thr649 phosphorylation/14-3-3 binding plays a key role in mediating insulin-stimulated glucose uptake in muscle. Contraction has also been proposed to increase phosphorylation of AS160 and TBC1D1 via AMPK, which could be detected by a generic phospho-Akt substrate (PAS) antibody. Here, analysis of AS160 immunoprecipitates from muscle extracts with site-specific phospho-antibodies revealed that contraction and AICAR caused no increase but rather a slight decrease in phosphorylation of the major PAS recognition site AS160-Thr649. In line with this, contraction failed to enhance 14-3-3 binding to AS160. Consistent with previous reports, we also observed that in situ contraction stimulated the signal intensity of PAS antibody immunoreactive protein of ∼150–160 kDa in muscle extracts. Using a TBC1D1 deletion mutant mouse, we showed that TBC1D1 protein accounted for the majority of the PAS antibody immunoreactive signals of ∼150–160 kDa in extracts of contracted muscles. Consistent with the proposed role of AS160-Thr649 phosphorylation/14-3-3 binding in mediating glucose uptake, AS160-Thr649Ala knock-in mice displayed normal glucose uptake upon contraction and AICAR in isolated muscles. We conclude that the previously reported PAS antibody immunoreactive band ∼150–160 kDa, which were increased upon contraction, does not represent AS160 but TBC1D1, and that AS160-Thr649Ala substitution impairs insulin- but neither contraction- nor AICAR-stimulated glucose uptake in mouse skeletal muscle.
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Dreyer, Hans C., Micah J. Drummond, Erin L. Glynn, Satoshi Fujita, David L. Chinkes, Elena Volpi, and Blake B. Rasmussen. "Resistance exercise increases human skeletal muscle AS160/TBC1D4 phosphorylation in association with enhanced leg glucose uptake during postexercise recovery." Journal of Applied Physiology 105, no. 6 (December 2008): 1967–74. http://dx.doi.org/10.1152/japplphysiol.90562.2008.
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Akt substrate of 160 kDa (AS160/TBC1D4) is associated with insulin and contraction-mediated glucose uptake. Human skeletal muscle AS160 phosphorylation is increased during aerobic exercise but not immediately following resistance exercise. It is not known whether AS160 phosphorylation is altered during recovery from resistance exercise. Therefore, we hypothesized that muscle AS160/TBC1D4 phosphorylation and glucose uptake across the leg would be increased during recovery following resistance exercise. We studied 9 male subjects before, during, and for 2 h of postexercise recovery. We utilized femoral catheterizations and muscle biopsies in combination with indirect calorimetry and immunoblotting to determine whole body glucose and fat oxidation, leg glucose uptake, muscle AMPKα2 activity, and the phosphorylation of muscle Akt and AS160/TBC1D4. Glucose oxidation was reduced while fat oxidation increased (∼35%) during postexercise recovery ( P ≤ 0.05). Glucose uptake increased during exercise and postexercise recovery ( P ≤ 0.05). Akt phosphorylation was increased at 1 h and AMPKα2 activity increased at 2 h postexercise ( P ≤ 0.05). Phospho(Ser/Thr)-Akt substrate (PAS) phosphorylation (often used as a marker for AS160) was unchanged immediately postexercise and increased at 1 h ( P ≤ 0.05) and 2 h postexercise ( P = 0.07). The PAS antibody is not always specific for AS160/TBC1D4 and can detect proteins at a similar molecular weight. Therefore, we immunoprecipitated AS160/TBC1D4 and then blotted with the PAS antibody, which confirmed that PAS phosphorylation is occurring on AS160/TBC1D4. There was also a positive correlation between PAS phosphorylation and leg glucose uptake during recovery ( P < 0.05). We conclude that resistance exercise increases AS160/TBC1D4 phosphorylation in association with an increase in leg glucose uptake during postexercise recovery.
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Wang, Hong Yu, Serge Ducommun, Chao Quan, Bingxian Xie, Min Li, David H. Wasserman, Kei Sakamoto, Carol Mackintosh, and Shuai Chen. "AS160 deficiency causes whole-body insulin resistance via composite effects in multiple tissues." Biochemical Journal 449, no. 2 (December 14, 2012): 479–89. http://dx.doi.org/10.1042/bj20120702.
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AS160 (Akt substrate of 160 kDa) is a Rab GTPase-activating protein implicated in insulin control of GLUT4 (glucose transporter 4) trafficking. In humans, a truncation mutation (R363X) in one allele of AS160 decreased the expression of the protein and caused severe postprandial hyperinsulinaemia during puberty. To complement the limited studies possible in humans, we generated an AS160-knockout mouse. In wild-type mice, AS160 expression is relatively high in adipose tissue and soleus muscle, low in EDL (extensor digitorum longus) muscle and detectable in liver only after enrichment. Despite having lower blood glucose levels under both fasted and random-fed conditions, the AS160-knockout mice exhibited insulin resistance in both muscle and liver in a euglycaemic clamp study. Consistent with this paradoxical phenotype, basal glucose uptake was higher in AS160-knockout primary adipocytes and normal in isolated soleus muscle, but their insulin-stimulated glucose uptake and overall GLUT4 levels were markedly decreased. In contrast, insulin-stimulated glucose uptake and GLUT4 levels were normal in EDL muscle. The liver also contributes to the AS160-knockout phenotype via hepatic insulin resistance, elevated hepatic expression of phosphoenolpyruvate carboxykinase isoforms and pyruvate intolerance, which are indicative of increased gluconeogenesis. Overall, as well as its catalytic function, AS160 influences expression of other proteins, and its loss deregulates basal and insulin-regulated glucose homoeostasis, not only in tissues that normally express AS160, but also by influencing liver function.
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Howlett, Kirsten F., Alicia Mathews, Andrew Garnham, and Kei Sakamoto. "The effect of exercise and insulin on AS160 phosphorylation and 14-3-3 binding capacity in human skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 294, no. 2 (February 2008): E401—E407. http://dx.doi.org/10.1152/ajpendo.00542.2007.
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AS160 is an Akt substrate of 160 kDa implicated in the regulation of both insulin- and contraction-mediated GLUT4 translocation and glucose uptake. The effects of aerobic exercise and subsequent insulin stimulation on AS160 phosphorylation and the binding capacity of 14-3-3, a novel protein involved in the dissociation of AS160 from GLUT4 vesicles, in human skeletal muscle are unknown. Hyperinsulinemic-euglycemic clamps were performed on seven men at rest and immediately and 3 h after a single bout of cycling exercise. Skeletal muscle biopsies were taken before and after the clamps. The insulin sensitivity index calculated during the final 30 min of the clamp was 8.0 ± 0.8, 9.1 ± 0.5, and 9.2 ± 0.8 for the rest, postexercise, and 3-h postexercise trials, respectively. AS160 phosphorylation increased immediately after exercise and remained elevated 3 h after exercise. In contrast, the 14-3-3 binding capacity of AS160 and phosphorylation of Akt and AMP-activated protein kinase were only increased immediately after exercise. Insulin increased AS160 phosphorylation and 14-3-3 binding capacity and insulin receptor substrate-1 and Akt phosphorylation, but the response to insulin was not enhanced by prior exercise. In conclusion, the 14-3-3 binding capacity of AS160 is increased immediately after acute exercise in human skeletal muscle, but this is not maintained 3 h after exercise completion despite sustained AS160 phosphorylation. Insulin increases AS160 phosphorylation and 14-3-3 binding capacity, but prior exercise does not appear to enhance the response to insulin.
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Funai, Katsuhiko, and Gregory D. Cartee. "Contraction-stimulated glucose transport in rat skeletal muscle is sustained despite reversal of increased PAS-phosphorylation of AS160 and TBC1D1." Journal of Applied Physiology 105, no. 6 (December 2008): 1788–95. http://dx.doi.org/10.1152/japplphysiol.90838.2008.
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Akt substrate of 160 kDa (AS160), the most distal insulin signaling protein known to be important for insulin-stimulated glucose transport, becomes phosphorylated with skeletal muscle contraction. Akt, AMP-activated protein kinase (AMPK), and Ca2+/calmodulin-dependent kinase II (CaMKII) have been implicated in regulating AS160 and/or glucose transport. Our primary aim was to assess time courses for contraction's effects on glucose transport and phosphorylation of Akt, AMPK, CaMKII, and AS160. Isolated rat epitrochlearis muscles were studied without or with contraction (5, 10, 20, 40, 60 min). Phospho-Akt substrate (PAS) antibody was used to measure AS160 PAS phosphorylation by quantifying the ∼160-kDa band on PAS immunoblots (PAS-160); a separate band at 150 kDa (PAS-150) that responded similarly to contraction was also identified. Using specific antibodies for AS160 or TBC1D1 on immunoblots, the molecular mass of PAS-160 was found to correspond with that of AS160 and not TBC1D1, whereas PAS-150 corresponded with TBC1D1 and not AS160. Furthermore, supernatant of sample immunodepleted with anti-AS160 had greatly reduced PAS-160, whereas supernatant of sample immunodepleted with anti-TBC1D1 had greatly reduced PAS-150, providing further evidence that PAS-160 and PAS-150 correspond with PAS-AS160 and PAS-TBC1D1, respectively. Contraction induced transient increases in PAS-160, PAS-150, phospho-glycogen synthase kinase 3 (an Akt substrate) and phospho-CaMKII; glucose transport and phospho-AMPK increases were maintained for 60 min of contraction. These data suggest the following: 1) PAS-160 (AS160) and PAS-150 (TBC1D1) respond to contraction transiently, despite sustained stimulation; 2) continual AMPK activation was insufficient for sustained increase in PAS-160 or PAS-150; and 3) sustained elevation of PAS-160 or PAS-150 was unnecessary to maintain contraction-stimulated glucose transport for up to 60 min.
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Vendelbo, M. H., A. B. Møller, J. T. Treebak, L. C. Gormsen, L. J. Goodyear, J. F. P. Wojtaszewski, J. O. L. Jørgensen, N. Møller, and N. Jessen. "Sustained AS160 and TBC1D1 phosphorylations in human skeletal muscle 30 min after a single bout of exercise." Journal of Applied Physiology 117, no. 3 (August 1, 2014): 289–96. http://dx.doi.org/10.1152/japplphysiol.00044.2014.
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Background: phosphorylation of AS160 and TBC1D1 plays an important role for GLUT4 mobilization to the cell surface. The phosphorylation of AS160 and TBC1D1 in humans in response to acute exercise is not fully characterized. Objective: to study AS160 and TBC1D1 phosphorylation in human skeletal muscle after aerobic exercise followed by a hyperinsulinemic euglycemic clamp. Design: eight healthy men were studied on two occasions: 1) in the resting state and 2) in the hours after a 1-h bout of ergometer cycling. A hyperinsulinemic euglycemic clamp was initiated 240 min after exercise and in a time-matched nonexercised control condition. We obtained muscle biopsies 30 min after exercise and in a time-matched nonexercised control condition ( t = 30) and after 30 min of insulin stimulation ( t = 270) and investigated site-specific phosphorylation of AS160 and TBC1D1. Results: phosphorylation on AS160 and TBC1D1 was increased 30 min after the exercise bout, whereas phosphorylation of the putative upstream kinases, Akt and AMPK, was unchanged compared with resting control condition. Exercise augmented insulin-stimulated phosphorylation on AS160 at Ser341and Ser704270 min after exercise. No additional exercise effects were observed on insulin-stimulated phosphorylation of Thr642and Ser588on AS160 or Ser237and Thr596on TBC1D1. Conclusions: AS160 and TBC1D1 phosphorylations were evident 30 min after exercise without simultaneously increased Akt and AMPK phosphorylation. Unlike TBC1D1, insulin-stimulated site-specific AS160 phosphorylation is modified by prior exercise, but these sites do not include Thr642and Ser588. Together, these data provide new insights into phosphorylation of key regulators of glucose transport in human skeletal muscle.
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Ishikura, Shuhei, and Amira Klip. "Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation." American Journal of Physiology-Cell Physiology 295, no. 4 (October 2008): C1016—C1025. http://dx.doi.org/10.1152/ajpcell.00277.2008.
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Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiring Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade.
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Arias, Edward B., Junghoon Kim, Katsuhiko Funai, and Gregory D. Cartee. "Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 292, no. 4 (April 2007): E1191—E1200. http://dx.doi.org/10.1152/ajpendo.00602.2006.
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The main purpose of this study was to determine whether the increased glucose transport (GT) found immediately postexercise (IPEX) or 4 h postexercise (4hPEX) is accompanied by increased phosphorylation of Akt substrate of 160 kDa (AS160, a protein regulator of GLUT4 translocation). Paired epitrochlearis muscles were dissected from rats (sedentary or IPEX, 2-h swim) and used to measure protein phosphorylation and insulin-independent GT. IPEX values exceeded sedentary values for GT and phosphorylations of AS160, AMP-activated protein kinase (pAMPK) and acetyl-CoA carboxylase (pACC) but not for AS160 abundance or phosphorylation of Akt serine (pSerAkt), Akt threonine (pThrAkt), or glycogen synthase kinase-3 (pGSK3). AS160 phosphorylation was significantly correlated with GT ( R = 0.801, P < 0.01) and pAMPK ( R = 0.655, P < 0.05). Muscles from other rats were studied 4hPEX along with sedentary controls. One muscle per rat was incubated without insulin, and the contralateral muscle was incubated with insulin. 4hPEX values exceeded sedentary values for insulin-stimulated GT. The elevated pAMPK and pACC found IPEX had reversed by 4hPEX. Insulin caused a significant increase in pSerAkt, pThrAkt, pGSK3, and AS160 phosphorylation with or without exercise. Exercise significantly increased AS160 phosphorylation, regardless of insulin, with unchanged AS160 abundance. Among the signaling proteins studied, insulin-stimulated GT was significantly correlated only with insulin-stimulated pThrAkt ( R = 0.720, P < 0.0005). The results are consistent with a role for increased AS160 phosphorylation in the increased insulin-independent GT IPEX, and the exercise effects on AS160 phosphorylation and/or pThrAkt at 4hPEX are potentially relevant to the increased insulin-stimulated glucose transport at this time.
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Funai, Katsuhiko, George G. Schweitzer, Naveen Sharma, Makoto Kanzaki, and Gregory D. Cartee. "Increased AS160 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 297, no. 1 (July 2009): E242—E251. http://dx.doi.org/10.1152/ajpendo.00194.2009.
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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-Thr642-AS160, pThr642-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 pThr642-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.
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Jessen, Niels, Ding An, Aina S. Lihn, Jonas Nygren, Michael F. Hirshman, Anders Thorell, and Laurie J. Goodyear. "Exercise increases TBC1D1 phosphorylation in human skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 301, no. 1 (July 2011): E164—E171. http://dx.doi.org/10.1152/ajpendo.00042.2011.
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Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% V̇o2 max). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser711 (AMPK), TBC1D1 Ser231 (AMPK), TBC1D1 Ser660 (AMPK), TBC1D1 Ser700 (AMPK), and TBC1D1 Thr590 (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss ( P < 0.001) and a significant increase in insulin sensitivity ( M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser711, TBC1D1 Ser231, and TBC1D1 Ser660 but had no effect on TBC1D1 Ser700. Exercise did not increase TBC1D1 Thr590 phosphorylation or TBC1D1/AS160 PAS phosphorylation, consistent with the lack of Akt activation. These data demonstrate that a single bout of exercise regulates TBC1D1 and AS160 phosphorylation on multiple sites in human skeletal muscle.
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Alves, Daiane S., Glen A. Farr, Patricia Seo-Mayer, and Michael J. Caplan. "AS160 Associates with the Na+,K+-ATPase and Mediates the Adenosine Monophosphate-stimulated Protein Kinase-dependent Regulation of Sodium Pump Surface Expression." Molecular Biology of the Cell 21, no. 24 (December 15, 2010): 4400–4408. http://dx.doi.org/10.1091/mbc.e10-06-0507.
Full textAbstract:
The Na+,K+-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na+,K+-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na+,K+-ATPase binding partners revealed a direct association between the Na+,K+-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na+,K+-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na+,K+-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na+,K+-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na+,K+-ATPase.
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Randhawa, V. K., I. Talior-Volodarsky, and A. Klip. "Stepping up regulatory mechanisms of GLUT4 traffic in L6 skeletal muscle cellss." Clinical & Investigative Medicine 30, no. 4 (August 1, 2007): 92. http://dx.doi.org/10.25011/cim.v30i4.2871.
Full textAbstract:
Insulin increases glucose uptake into muscle and fat by enhancing GLUT4 glucose transporter externalization; a process requiring input from Akt and actin. Downstream of phosphatidylinositol-3-kinase, insulin signaling bifurcates into Akt and actin activating arms. Akt-mediated phosphorylation of the Rab-GAP AS160 is required for gain in surface GLUT4 by insulin. However, little is known of the mechanism(s) by which AS160 and/or actin dynamics modulate GLUT4 traffic in muscle. We recently showed that GLUT4 arrival and/or fusion can be regulated by insulin signaling molecules and phospholipids. Using ‘rounded up’ L6 myoblasts stably expressing GLUT4myc, we find that transient expression of a non-phosphorylatable mutant of AS160 (AS160-4P) abrogates the surface fusion of GLUT4myc and partially reduces its sub-membranous accumulation. In contrast, tetanus toxin-mediated cleavage of VAMP2 inhibits GLUT4myc fusion but not arrival to the plasma membrane. Conversely, disrupting actin dynamics with Latrunculin B or silencing expression of a cytoskeletal protein a-actinin4 precludes the insulin-induced cortical build-up of GLUT4myc. These data suggest that AS160 and actin dynamics impinge on distinct stages of insulin-regulated GLUT4 traffic: AS160 may contribute to peripheral retention and is essential for GLUT4myc vesicle docking/fusion. It will be interesting to note which Rabs facilitate these AS160-dependent events. Actin dynamics instead may allow GLUT4 vesicle movement to the cell surface and/or its retention, presumably via cortical anchoring mechanisms involving a-actinin4, whilst VAMP2 has a major role in GLUT4 vesicle fusion. Indeed, defects in AS160 phosphorylation and actin dynamics are associated with insulin resistant states. Thus, discerning which steps of GLUT4 traffic are modulated by these inputs may help elucidate strategies to bypass insulin resistance.
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Schweitzer, George G., Edward B. Arias, and Gregory D. Cartee. "Sustained postexercise increases in AS160 Thr642 and Ser588 phosphorylation in skeletal muscle without sustained increases in kinase phosphorylation." Journal of Applied Physiology 113, no. 12 (December 15, 2012): 1852–61. http://dx.doi.org/10.1152/japplphysiol.00619.2012.
Full textAbstract:
Prior exercise by rats can induce a sustained increase in muscle Akt substrate of 160 kDa (AS160) phosphorylation on Thr642 (pAS160Thr642). Because phosphorylation of AS160 on both AS160Thr642 and AS160Ser588 is important for insulin-stimulated glucose transport (GT), we determined if exercise would also induce a sustained increase in pAS160Ser588 concomitant with persistently elevated pAS160Thr642 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 (Tyr307 and Leu309) that regulate PP2A. In isolated epitrochlearis muscles from rats, GT at 3hPEX with insulin significantly ( P < 0.05) exceeded SED controls. Muscles from 0hPEX vs. 0hSED and 3hPEX vs. 3hSED rats had greater pAS160Thr642 and pAS160Ser588. 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 pAS160Thr642 and pAS160Ser588 in cell-free assays. Exercise did not alter posttranslational modifications of PP2A. Our results revealed: 1) pAMPK as a potential trigger for increased pAS160Thr642 and pAS160Ser588 at 0hPEX; 2) PP1, PP2A, PP2B, and PP2C were each able to dephosphorylate AS160; and 3) sustained PEX-induced elevations of pAS160Thr642 and pAS160Ser588 were attributable to mechanisms other than persistent phosphorylation of known AS160 kinases or altered posttranslational modifications of PP2A.
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Geraghty, Kathryn M., Shuai Chen, Jean E. Harthill, Adel F. Ibrahim, Rachel Toth, Nick A. Morrice, Franck Vandermoere, Greg B. Moorhead, D. Grahame Hardie, and Carol MacKintosh. "Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR." Biochemical Journal 407, no. 2 (September 25, 2007): 231–41. http://dx.doi.org/10.1042/bj20070649.
Full textAbstract:
AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA–AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.
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Treebak, Jonas T., Jesper B. Birk, Adam J. Rose, Bente Kiens, Erik A. Richter, and Jørgen F. P. Wojtaszewski. "AS160 phosphorylation is associated with activation of α2β2γ1- but not α2β2γ3-AMPK trimeric complex in skeletal muscle during exercise in humans." American Journal of Physiology-Endocrinology and Metabolism 292, no. 3 (March 2007): E715—E722. http://dx.doi.org/10.1152/ajpendo.00380.2006.
Full textAbstract:
We investigated time- and intensity-dependent effects of exercise on phosphorylation of Akt substrate of 160 kDa (AS160) in human skeletal muscle. Subjects performed cycle exercise for 90 min (67% V̇o2 peak, n = 8), 20 min (80% V̇o2 peak, n = 11), 2 min (110% of peak work rate, n = 9), or 30 s (maximal sprint, n = 10). Muscle biopsies were obtained before, during, and after exercise. In trial 1, AS160 phosphorylation increased at 60 min (60%, P = 0.06) and further at 90 min of exercise (120%, P < 0.05). α2β2γ3-AMP-activated protein kinase (AMPK) activity increased significantly to a steady-state level after 30 min, whereas α2β2γ1-AMPK activity increased after 60 min of exercise with a further significant increase after 90 min. α2β2γ1-AMPK activity and AS160 phosphorylation correlated positively ( r2 = 0.55). In exercise trials 2, 3, and 4, α2β2γ3-AMPK activity but neither AS160 phosphorylation nor α2β2γ1-AMPK activity increased. Akt Ser473 phosphorylation was unchanged in all trials, whereas Akt Thr308 phosphorylation increased significantly in trial 3 and 4 only. These results show that AS160 is phosphorylated in a time-dependent manner during moderate-intensity exercise and suggest that α2β2γ1- but not α2β2γ3-AMPK may act in a pathway responsible for exercise-induced AS160 phosphorylation. Furthermore, we show that AMPK complexes in skeletal muscle are activated differently depending on exercise intensity and duration.
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Capilla, Encarnación, Naoko Suzuki, Jeffrey E. Pessin, and June Chunqiu Hou. "The Glucose Transporter 4 FQQI Motif Is Necessary for Akt Substrate of 160-Kilodalton-Dependent Plasma Membrane Translocation But Not Golgi-Localized γ-Ear-Containing Arf-Binding Protein-Dependent Entry into the Insulin-Responsive Storage Compartment." Molecular Endocrinology 21, no. 12 (December 1, 2007): 3087–99. http://dx.doi.org/10.1210/me.2006-0476.
Full textAbstract:
Abstract Newly synthesized glucose transporter 4 (GLUT4) enters into the insulin-responsive storage compartment in a process that is Golgi-localized γ-ear-containing Arf-binding protein (GGA) dependent, whereas insulin-stimulated translocation is regulated by Akt substrate of 160 kDa (AS160). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminus and the large intracellular loop of GLUT4 into GLUT1 (chimera 1-441) fully recapitulated the basal state retention, insulin-stimulated translocation, and GGA and AS160 sensitivity of wild-type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when coexpressed with both wild-type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by coexpression of dominant-interfering GGA. In addition, coexpression with a dominant-interfering AS160 (AS160-4P) abolished insulin-stimulated GLUT4-WT but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminus and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane; however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.
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Protzek, André O. P., José M. Costa-Júnior, Luiz F. Rezende, Gustavo J. Santos, Tiago Gomes Araújo, Jean F. Vettorazzi, Fernanda Ortis, Everardo M. Carneiro, Alex Rafacho, and Antonio C. Boschero. "Augmentedβ-Cell Function and Mass in Glucocorticoid-Treated Rodents Are Associated with Increased Islet Ir-β/AKT/mTOR and Decreased AMPK/ACC and AS160 Signaling." International Journal of Endocrinology 2014 (2014): 1–14. http://dx.doi.org/10.1155/2014/983453.
Full textAbstract:
Glucocorticoid (GC) therapies may adversely cause insulin resistance (IR) that lead to a compensatory hyperinsulinemia due to insulin hypersecretion. The increasedβ-cell function is associated with increased insulin signaling that has the protein kinase B (AKT) substrate with 160 kDa (AS160) as an important downstream AKT effector. In muscle, both insulin and AMP-activated protein kinase (AMPK) signaling phosphorylate and inactivate AS160, which favors the glucose transporter (GLUT)-4 translocation to plasma membrane. Whether AS160 phosphorylation is modulated in islets from GC-treated subjects is unknown. For this, two animal models, Swiss mice and Wistar rats, were treated with dexamethasone (DEX) (1 mg/kg body weight) for 5 consecutive days. DEX treatment induced IR, hyperinsulinemia, and dyslipidemia in both species, but glucose intolerance and hyperglycemia only in rats. DEX treatment caused increased insulin secretion in response to glucose and augmentedβ-cell mass in both species that were associated with increased islet content and increased phosphorylation of the AS160 protein. Protein AKT phosphorylation, but not AMPK phosphorylation, was found significantly enhanced in islets from DEX-treated animals. We conclude that the augmentedβ-cell function developed in response to the GC-induced IR involves inhibition of the islet AS160 protein activity.
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Mikłosz, Agnieszka, Bartłomiej Łukaszuk, Małgorzata Żendzian-Piotrowska, Krzysztof Kurek, and Adrian Chabowski. "The Effects of AS160 Modulation on Fatty Acid Transporters Expression and Lipid Profile in L6 Myotubes." Cellular Physiology and Biochemistry 38, no. 1 (2016): 267–82. http://dx.doi.org/10.1159/000438628.
Full textAbstract:
Background/Aims: AS160 is a key intracellular regulator of energy utilization in cells. It was shown to regulate GLUT4 translocation from intracellular depots to the plasma membrane, with subsequent changes in facilitated glucose uptake into the skeletal muscles. Similarly, also free fatty acids (FFAs) transmembrane transport seems to be largely protein-mediated. Therefore, the objective of this study was to examine the effects of moderate AS160 depletion (-82% mRNA, -25% of protein content) on the expression of fatty acid transporters and subsequent changes in lipid profile in L6 myotubes. Results: Surprisingly, moderate down regulation of AS160 expression was followed by increased AS160 phosphorylation (∼40%). These resulted in a greater expression of fatty acid transporters, namely FABPpm and FAT/CD36, with subsequently increased FAs cellular influx. No changes in the expression of FATP1 and 4 were noticed. Accordingly, we have observed a reduction in total TAG content. This was mainly caused by a significant changes in TAG fatty acids composition favouring a decrease in the amount of palmitic and stearic fatty acid moieties. In contrast, our experimental intervention led to distinctively increased total content of DAG and PL, but concomitantly decreased the content of all measured sphingolipids, e.g. SFA, SA1P, CER, SFO and S1P, in the AS160 knockdown group. Conclusions: Modulation of AS160 level and activity led to significant increase in the concentration of DAG and PL, which was associated with changes in FAs composition and expression of fatty acid transporters. Interestingly, the intervention also simultaneously decreased the content of sphingolipids.
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Mîinea, Cristinel P., Hiroyuki Sano, Susan Kane, Eiko Sano, Mitsunori Fukuda, Johan Peränen, William S. Lane, and Gustav E. Lienhard. "AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain." Biochemical Journal 391, no. 1 (September 26, 2005): 87–93. http://dx.doi.org/10.1042/bj20050887.
Full textAbstract:
Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.
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Sadacca, L. Amanda, Joanne Bruno, Jennifer Wen, Wenyong Xiong, and Timothy E. McGraw. "Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs." Molecular Biology of the Cell 24, no. 16 (August 15, 2013): 2544–57. http://dx.doi.org/10.1091/mbc.e13-02-0103.
Full textAbstract:
Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.
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Zeigerer, Anja, Mary Kate McBrayer, and Timothy E. McGraw. "Insulin Stimulation of GLUT4 Exocytosis, but Not Its Inhibition of Endocytosis, Is Dependent on RabGAP AS160." Molecular Biology of the Cell 15, no. 10 (October 2004): 4406–15. http://dx.doi.org/10.1091/mbc.e04-04-0333.
Full textAbstract:
Insulin maintains whole body blood glucose homeostasis, in part, by regulating the amount of the GLUT4 glucose transporter on the cell surface of fat and muscle cells. Insulin induces the redistribution of GLUT4 from intracellular compartments to the plasma membrane, by stimulating a large increase in exocytosis and a smaller inhibition of endocytosis. A considerable amount is known about the molecular events of insulin signaling and the complex itinerary of GLUT4 trafficking, but less is known about how insulin signaling is transmitted to GLUT4 trafficking. Here, we show that the AS160 RabGAP, a substrate of Akt, is required for insulin stimulation of GLUT4 exocytosis. A dominant-inhibitory mutant of AS160 blocks insulin stimulation of exocytosis at a step before the fusion of GLUT4-containing vesicles with the plasma membrane. This mutant, however, does not block insulin-induced inhibition of GLUT4 endocytosis. These data support a model in which insulin signaling to the exocytosis machinery (AS160 dependent) is distinct from its signaling to the internalization machinery (AS160 independent).
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Castorena, Carlos M., James G. MacKrell, Jonathan S. Bogan, Makoto Kanzaki, and Gregory D. Cartee. "Clustering of GLUT4, TUG, and RUVBL2 protein levels correlate with myosin heavy chain isoform pattern in skeletal muscles, but AS160 and TBC1D1 levels do not." Journal of Applied Physiology 111, no. 4 (October 2011): 1106–17. http://dx.doi.org/10.1152/japplphysiol.00631.2011.
Full textAbstract:
Skeletal muscle is a heterogeneous tissue. To further elucidate this heterogeneity, we probed relationships between myosin heavy chain (MHC) isoform composition and abundance of GLUT4 and four other proteins that are established or putative GLUT4 regulators [Akt substrate of 160 kDa (AS160), Tre-2/Bub2/Cdc 16-domain member 1 (TBC1D1), Tethering protein containing an UBX-domain for GLUT4 (TUG), and RuvB-like protein two (RUVBL2)] in 12 skeletal muscles or muscle regions from Wistar rats [adductor longus, extensor digitorum longus, epitrochlearis, gastrocnemius (mixed, red, and white), plantaris, soleus, tibialis anterior (red and white), tensor fasciae latae, and white vastus lateralis]. Key results were 1) significant differences found among the muscles (range of muscle expression values) for GLUT4 (2.5-fold), TUG (1.7-fold), RUVBL2 (2.0-fold), and TBC1D1 (2.7-fold), but not AS160; 2) significant positive correlations for pairs of proteins: GLUT4 vs. TUG ( R = 0.699), GLUT4 vs. RUVBL2 ( R = 0.613), TUG vs. RUVBL2 ( R = 0.564), AS160 vs. TBC1D1 ( R = 0.293), and AS160 vs. TUG ( R = 0.246); 3) significant positive correlations for %MHC-I: GLUT4 ( R = 0.460), TUG ( R = 0.538), and RUVBL2 ( R = 0.511); 4) significant positive correlations for %MHC-IIa: GLUT4 ( R = 0.293) and RUVBL2 ( R = 0.204); 5) significant negative correlations for %MHC-IIb vs. GLUT4 ( R = −0.642), TUG ( R = −0.626), and RUVBL2 ( R = −0.692); and 6) neither AS160 nor TBC1D1 significantly correlated with MHC isoforms. In 12 rat muscles, GLUT4 abundance tracked with TUG and RUVBL2 and correlated with MHC isoform expression, but was unrelated to AS160 or TBC1D1. Our working hypothesis is that some of the mechanisms that regulate GLUT4 abundance in rat skeletal muscle also influence TUG and RUVBL2 abundance.
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Wang, Haiyan, Edward B. Arias, Mark W. Pataky, Laurie J. Goodyear, and Gregory D. Cartee. "Postexercise improvement in glucose uptake occurs concomitant with greater γ3-AMPK activation and AS160 phosphorylation in rat skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 315, no. 5 (November 1, 2018): E859—E871. http://dx.doi.org/10.1152/ajpendo.00020.2018.
Full textAbstract:
A single exercise session can increase insulin-stimulated glucose uptake (GU) by skeletal muscle, concomitant with greater Akt substrate of 160 kDa (AS160) phosphorylation on Akt-phosphosites (Thr642 and Ser588) that regulate insulin-stimulated GU. Recent research using mouse skeletal muscle suggested that ex vivo 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or electrically stimulated contractile activity-inducing increased γ3-AMPK activity and AS160 phosphorylation on a consensus AMPK-motif (Ser704) resulted in greater AS160 Thr642 phosphorylation and GU by insulin-stimulated muscle. Our primary goal was to determine whether in vivo exercise that increases insulin-stimulated GU in rat skeletal muscle would also increase γ3-AMPK activity and AS160 site-selective phosphorylation (Ser588, Thr642, and Ser704) immediately postexercise (IPEX) and/or 3 h postexercise (3hPEX). Epitrochlearis muscles isolated from sedentary and exercised (2-h swim exercise; studied IPEX and 3hPEX) rats were incubated with 2-deoxyglucose to determine GU (without insulin at IPEX; without or with insulin at 3hPEX). Muscles were also assessed for γ1-AMPK activity, γ3-AMPK activity, phosphorylated AMPK (pAMPK), and phosphorylated AS160 (pAS160). IPEX versus sedentary had greater γ3-AMPK activity, pAS160 (Ser588, Thr642, Ser704), and GU with unaltered γ1-AMPK activity. 3hPEX versus sedentary had greater γ3-AMPK activity, pAS160 Ser704, and GU with or without insulin; greater pAS160 Thr642 only with insulin; and unaltered γ1-AMPK activity. These results using an in vivo exercise protocol that increased insulin-stimulated GU in rat skeletal muscle are consistent with the hypothesis that in vivo exercise-induced enhancement of γ3-AMPK activation and AS160 Ser704 IPEX and 3hPEX are important for greater pAS160 Thr642 and enhanced insulin-stimulated GU by skeletal muscle.
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Jordens, Ingrid, Dorothee Molle, Wenyong Xiong, Susanna R. Keller, and Timothy E. McGraw. "Insulin-regulated Aminopeptidase Is a Key Regulator of GLUT4 Trafficking by Controlling the Sorting of GLUT4 from Endosomes to Specialized Insulin-regulated Vesicles." Molecular Biology of the Cell 21, no. 12 (June 15, 2010): 2034–44. http://dx.doi.org/10.1091/mbc.e10-02-0158.
Full textAbstract:
Insulin stimulates glucose uptake by regulating translocation of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane. In the absence of insulin GLUT4 is actively sequestered away from the general endosomes into GLUT4-specialized compartments, thereby controlling the amount of GLUT4 at the plasma membrane. Here, we investigated the role of the aminopeptidase IRAP in GLUT4 trafficking. In unstimulated IRAP knockdown adipocytes, plasma membrane GLUT4 levels are elevated because of increased exocytosis, demonstrating an essential role of IRAP in GLUT4 retention. Current evidence supports the model that AS160 RabGAP, which is required for basal GLUT4 retention, is recruited to GLUT4 compartments via an interaction with IRAP. However, here we show that AS160 recruitment to GLUT4 compartments and AS160 regulation of GLUT4 trafficking were unaffected by IRAP knockdown. These results demonstrate that AS160 is recruited to membranes by an IRAP-independent mechanism. Consistent with a role independent of AS160, we showed that IRAP functions in GLUT4 sorting from endosomes to GLUT4-specialized compartments. This is revealed by the relocalization of GLUT4 to endosomes in IRAP knockdown cells. Although IRAP knockdown has profound effects on GLUT4 traffic, GLUT4 knockdown does not affect IRAP trafficking, demonstrating that IRAP traffics independent of GLUT4. In sum, we show that IRAP is both cargo and a key regulator of the insulin-regulated pathway.
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Peck, Grantley R., Siying Ye, Vi Pham, Ruani N. Fernando, S. Lance Macaulay, Siew Yeen Chai, and Anthony L. Albiston. "Interaction of the Akt Substrate, AS160, with the Glucose Transporter 4 Vesicle Marker Protein, Insulin-Regulated Aminopeptidase." Molecular Endocrinology 20, no. 10 (October 1, 2006): 2576–83. http://dx.doi.org/10.1210/me.2005-0476.
Full textAbstract:
Abstract Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.
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Vendelbo, M. H., B. F. F. Clasen, J. T. Treebak, L. Møller, T. Krusenstjerna-Hafstrøm, M. Madsen, T. S. Nielsen, et al. "Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 302, no. 2 (January 15, 2012): E190—E200. http://dx.doi.org/10.1152/ajpendo.00207.2011.
Full textAbstract:
During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast.
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Kim, Nami, Jung Ok Lee, Hye Jeong Lee, Yong Woo Lee, Hyung Ip Kim, Su Jin Kim, Sun Hwa Park, et al. "AMPK, a metabolic sensor, is involved in isoeugenol-induced glucose uptake in muscle cells." Journal of Endocrinology 228, no. 2 (November 19, 2015): 105–14. http://dx.doi.org/10.1530/joe-15-0302.
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Isoeugenol exerts various beneficial effects on human health. However, the mechanisms underlying these effects are poorly understood. In this study, we observed that isoeugenol activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. Isoeugenol-induced increase in intracellular calcium concentration and glucose uptake was inhibited by STO-609, an inhibitor of calcium/calmodulin-dependent protein kinase kinase (CaMKK). Isoeugenol also increased the phosphorylation of protein kinase C-α (PKCα). Chelation of calcium with BAPTA-AM blocked isoeugenol-induced AMPK phosphorylation and glucose uptake. Isoeugenol stimulated p38MAPK phosphorylation that was inhibited after pretreatment with compound C, an AMPK inhibitor. Isoeugenol also increased glucose transporter type 4 (GLUT4) expression and its translocation to the plasma membrane. GLUT4 translocation was not observed after the inhibition of AMPK and CaMKK. In addition, isoeugenol activated the Akt substrate 160 (AS160) pathway, which is downstream of the p38MAPK pathway. Knockdown of the gene encoding AS160 inhibited isoeugenol-induced glucose uptake. Together, these results indicate that isoeugenol exerts beneficial health effects by activating the AMPK/p38MAPK/AS160 pathways in skeletal muscle.
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Sharma, Naveen, Haiyan Wang, Edward B. Arias, Carlos M. Castorena, and Gregory D. Cartee. "Mechanisms for independent and combined effects of calorie restriction and acute exercise on insulin-stimulated glucose uptake by skeletal muscle of old rats." American Journal of Physiology-Endocrinology and Metabolism 308, no. 7 (April 1, 2015): E603—E612. http://dx.doi.org/10.1152/ajpendo.00618.2014.
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Either calorie restriction [CR; consuming 60–65% of ad libitum (AL) intake] or acute exercise can independently improve insulin sensitivity in old age, but their combined effects on muscle insulin signaling and glucose uptake have previously been unknown. Accordingly, we assessed the independent and combined effects of CR (beginning at 14 wk old) and acute exercise (3–4 h postexercise) on insulin signaling and glucose uptake in insulin-stimulated epitrochlearis muscles from 30-mo-old rats. Either CR alone or exercise alone vs. AL sedentary controls induced greater insulin-stimulated glucose uptake. Combined CR and exercise vs. either treatment alone caused an additional increase in insulin-stimulated glucose uptake. Either CR or exercise alone vs. AL sedentary controls increased Akt Ser473and Akt Thr308phosphorylation. Combined CR and exercise further elevated Akt phosphorylation on both sites. CR alone, but not exercise alone, vs. AL sedentary controls significantly increased Akt substrate of 160 kDa (AS160) Ser588and Thr642phosphorylation. Combined CR and exercise did not further enhance AS160 phosphorylation. Exercise alone, but not CR alone, modestly increased GLUT4 abundance. Combined CR and exercise did not further elevate GLUT4 content. These results suggest that CR or acute exercise independently increases insulin-stimulated glucose uptake via overlapping (greater Akt phosphorylation) and distinct (greater AS160 phosphorylation for CR, greater GLUT4 for exercise) mechanisms. Our working hypothesis is that greater insulin-stimulated glucose uptake in the combined CR and exercise group vs. CR or exercise alone relies on greater Akt activation, leading to greater phosphorylation of one or more Akt substrates other than AS160.
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Jiang, Xiao-Hua, Jian-Wen Sun, Ming Xu, Xiao-Fei Jiang, Chun-Fang Liu, and Yuan Lu. "Frequent hyperphosphorylation of AS160 in breast cancer." Cancer Biology & Therapy 10, no. 4 (August 15, 2010): 362–67. http://dx.doi.org/10.4161/cbt.10.4.12426.
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Ren, Wenying, Sarwat Cheema, and Keyong Du. "The Association of ClipR-59 Protein with AS160 Modulates AS160 Protein Phosphorylation and Adipocyte Glut4 Protein Membrane Translocation." Journal of Biological Chemistry 287, no. 32 (June 11, 2012): 26890–900. http://dx.doi.org/10.1074/jbc.m112.357699.
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Alkhateeb, Hakam, and Arend Bonen. "Thujone, a component of medicinal herbs, rescues palmitate-induced insulin resistance in skeletal muscle." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 299, no. 3 (September 2010): R804—R812. http://dx.doi.org/10.1152/ajpregu.00216.2010.
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Thujone is thought to be the main constituent of medicinal herbs that have antidiabetic properties. Therefore, we examined whether thujone ameliorated palmitate-induced insulin resistance in skeletal muscle. Soleus muscles were incubated for ≤12 h without or with palmitate (2 mM). Thujone (0.01 mg/ml), in the presence of palmitate, was provided in the last 6 h of incubation. Palmitate oxidation, AMPK/acetyl-CoA carboxylase (ACC) phosphorylation and insulin-stimulated glucose transport, plasmalemmal GLUT4, and AS160 phosphorylation were examined at 0, 6, and 12 h. Palmitate treatment for 12 h reduced fatty acid oxidation (−47%), and insulin-stimulated glucose transport (−71%), GLUT4 translocation (−40%), and AS160 phosphorylation (−26%), but it increased AMPK (+51%) and ACC phosphorylations (+44%). Thujone (6–12 h) fully rescued palmitate oxidation and insulin-stimulated glucose transport, but only partially restored GLUT4 translocation and AS160 phosphorylation, raising the possibility that an increased GLUT4 intrinsic activity may also have contributed to the restoration of glucose transport. Thujone also further increased AMPK phosphorylation but had no further effect on ACC phosphorylation. Inhibition of AMPK phosphorylation with adenine 9-β-d-arabinofuranoside (Ara) (2.5 mM) or compound C (50 μM) inhibited the thujone-induced improvement in insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation. In contrast, the thujone-induced improvement in palmitate oxidation was only slightly inhibited (≤20%) by Ara or compound C. Thus, while thujone, a medicinal herb component, rescues palmitate-induced insulin resistance in muscle, the improvement in fatty acid oxidation cannot account for this thujone-mediated effect. Instead, the rescue of palmitate-induced insulin resistance appears to occur via an AMPK-dependent mechanism involving partial restoration of insulin-stimulated GLUT4 translocation.
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Ginion, Audrey, Julien Auquier, Carley R. Benton, Céline Mouton, Jean-Louis Vanoverschelde, Louis Hue, Sandrine Horman, Christophe Beauloye, and Luc Bertrand. "Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake." American Journal of Physiology-Heart and Circulatory Physiology 301, no. 2 (August 2011): H469—H477. http://dx.doi.org/10.1152/ajpheart.00986.2010.
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The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.
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Montessuit, Christophe, Irène Papageorgiou, and René Lerch. "Nuclear Receptor Agonists Improve Insulin Responsiveness in Cultured Cardiomyocytes through Enhanced Signaling and Preserved Cytoskeletal Architecture." Endocrinology 149, no. 3 (December 6, 2007): 1064–74. http://dx.doi.org/10.1210/en.2007-0656.
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Insulin resistance is the failure of insulin to stimulate the transport of glucose into its target cells. A highly regulatable supply of glucose is important for cardiomyocytes to cope with situations of metabolic stress. We recently observed that isolated adult rat cardiomyocytes become insulin resistant in vitro. Insulin resistance is combated at the whole body level with agonists of the nuclear receptor complex peroxisome proliferator-activated receptor γ (PPARγ)/retinoid X receptor (RXR). We investigated the effects of PPARγ/RXR agonists on the insulin-stimulated glucose transport and on insulin signaling in insulin-resistant adult rat cardiomyocytes. Treatment of cardiomyocytes with ciglitazone, a PPARγ agonist, or 9-cis retinoic acid (RA), a RXR agonist, increased insulin- and metabolic stress-stimulated glucose transport, whereas agonists of PPARα or PPARβ/δ had no effect. Stimulation of glucose transport in response to insulin requires the phosphorylation of the signaling intermediate Akt on the residues Thr308 and Ser473 and, downstream of Akt, AS160 on several Thr and Ser residues. Phosphorylation of Akt and AS160 in response to insulin was lower in insulin-resistant cardiomyocytes. However, treatment with 9-cis RA markedly increased phosphorylation of both proteins. Treatment with 9-cis RA also led to better preservation of microtubules in cultured cardiomyocytes. Disruption of microtubules in insulin-responsive cardiomyocytes abolished insulin-stimulated glucose transport and reduced phosphorylation of AS160 but not Akt. Metabolic stress-stimulated glucose transport also involved AS160 phosphorylation in a microtubule-dependent manner. Thus, the stimulation of glucose uptake in response to insulin or metabolic stress is dependent in cardiomyocytes on the presence of intact microtubules.
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Chen, Shuai, Jane Murphy, Rachel Toth, David G. Campbell, Nick A. Morrice, and Carol Mackintosh. "Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators." Biochemical Journal 409, no. 2 (December 21, 2007): 449–59. http://dx.doi.org/10.1042/bj20071114.
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AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking of GLUT4 (glucose transporter 4) storage vesicles to the cell surface. All animal species examined contain TBC1D1, whereas AS160 evolved with the vertebrates. TBC1D1 has two clusters of phosphorylated residues, either side of the second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated in HEK (human embryonic kidney)-293 cells, 14-3-3s bind primarily to pSer237 (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr596 (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer237 and pThr596 contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr596 of TBC1D1, and promotes phosphorylation of AMPK and Ser237 of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser235 prevents subsequent phosphorylation of Ser237. In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser237 and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr596 but not 14-3-3 binding. In contrast, AS160 is phosphorylated on its 14-3-3-binding sites (Ser341 and Thr642) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle.
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Blesson, Chellakkan S., Kunju Sathishkumar, Vijayakumar Chinnathambi, and Chandrasekhar Yallampalli. "Gestational Protein Restriction Impairs Insulin-Regulated Glucose Transport Mechanisms in Gastrocnemius Muscles of Adult Male Offspring." Endocrinology 155, no. 8 (August 1, 2014): 3036–46. http://dx.doi.org/10.1210/en.2014-1094.
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Type II diabetes originates from various genetic and environmental factors. Recent studies showed that an adverse uterine environment such as that caused by a gestational low-protein (LP) diet can cause insulin resistance in adult offspring. The mechanism of insulin resistance induced by gestational protein restriction is not clearly understood. Our aim was to investigate the role of insulin signaling molecules in gastrocnemius muscles of gestational LP diet–exposed male offspring to understand their role in LP-induced insulin resistance. Pregnant Wistar rats were fed a control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery and a normal diet after weaning. Only male offspring were used in this study. Glucose and insulin responses were assessed after a glucose tolerance test. mRNA and protein levels of molecules involved in insulin signaling were assessed at 4 months in gastrocnemius muscles. Muscles were incubated ex vivo with insulin to evaluate insulin-induced phosphorylation of insulin receptor (IR), Insulin receptor substrate-1, Akt, and AS160. LP diet-fed rats gained less weight than controls during pregnancy. Male pups from LP diet–fed mothers were smaller but exhibited catch-up growth. Plasma glucose and insulin levels were elevated in LP offspring when subjected to a glucose tolerance test; however, fasting levels were comparable. LP offspring showed increased expression of IR and AS160 in gastrocnemius muscles. Ex vivo treatment of muscles with insulin showed increased phosphorylation of IR (Tyr972) in controls, but LP rats showed higher basal phosphorylation. Phosphorylation of Insulin receptor substrate-1 (Tyr608, Tyr895, Ser307, and Ser318) and AS160 (Thr642) were defective in LP offspring. Further, glucose transporter type 4 translocation in LP offspring was also impaired. A gestational LP diet leads to insulin resistance in adult offspring by a mechanism involving inefficient insulin-induced IR, Insulin receptor substrate-1, and AS160 phosphorylation and impaired glucose transporter type 4 translocation.
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Jing, Ming, Vinay K. Cheruvu, and Faramarz Ismail-Beigi. "Stimulation of glucose transport in response to activation of distinct AMPK signaling pathways." American Journal of Physiology-Cell Physiology 295, no. 5 (November 2008): C1071—C1082. http://dx.doi.org/10.1152/ajpcell.00040.2008.
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AMP-activated protein kinase (AMPK) plays a critical role in the stimulation of glucose transport in response to hypoxia and inhibition of oxidative phosphorylation. In the present study, we examined the signaling pathway(s) mediating the glucose transport response following activation of AMPK. Using mouse fibroblasts of AMPK wild type and AMPK knockout, we documented that the expression of AMPK is essential for the glucose transport response to both azide and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). In Clone 9 cells, the stimulation of glucose transport by a combination of azide and AICAR was not additive, whereas there was an additive increase in the abundance of phosphorylated AMPK (p-AMPK). In Clone 9 cells, AMPK wild-type fibroblasts, and H9c2 heart cells, azide or hypoxia selectively increased p-ERK1/2, whereas, in contrast, AICAR selectively stimulated p-p38; phosphorylation of JNK was unaffected. Azide's effect on p-ERK1/2 abundance and glucose transport in Clone 9 cells was partially abolished by the MEK1/2 inhibitor U0126. SB 203580, an inhibitor of p38, prevented the phosphorylation of p38 and the glucose transport response to AICAR and, unexpectedly, to azide. Hypoxia, azide, and AICAR all led to increased phosphorylation of Akt substrate of 160 kDa (AS160) in Clone 9 cells. Employing small interference RNA directed against AS160 did not inhibit the glucose transport response to azide or AICAR, whereas the content of P-AS160 was reduced by ∼80%. Finally, we found no evidence for coimmunoprecipitation of Glut1 and p-AS160. We conclude that although azide, hypoxia, and AICAR all activate AMPK, the downstream signaling pathways are distinct, with azide and hypoxia stimulating ERK1/2 and AICAR stimulating the p38 pathway.
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Lehnen, Alexandre M., Graziela H. Pinto, Júlia Borges, Melissa M. Markoski, and Beatriz D. Schaan. "Adaptations in GLUT4 Expression in Response to Exercise Detraining Linked to Downregulation of Insulin-Dependent Pathways in Cardiac but not in Skeletal Muscle Tissue." International Journal of Sport Nutrition and Exercise Metabolism 30, no. 4 (July 1, 2020): 272–79. http://dx.doi.org/10.1123/ijsnem.2019-0337.
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Insulin resistance is associated with cardiometabolic risk factors, and exercise training can improve insulin-mediated glucose uptake. However, few studies have demonstrated the reversibility of exercise-induced benefits. Thus, the authors examine the time–response effects of exercise training and detraining on glucose transporter 4 (GLUT4) content, insulin-dependent and insulin-independent pathways in cardiac and gastrocnemius muscle tissues of spontaneously hypertensive rats. Thirty-two male spontaneously hypertensive rats, 4 months old, were assigned to (n = 8/group): T (exercise training: 10-week treadmill exercise, 50–70% maximum effort capacity, 1 hr/day, 5 days/week); D2 (exercise training + 2-day detraining), D4 (exercise training + 4-day detraining); and S (no exercise). The authors evaluated insulin resistance, maximum effort capacity, GLUT4 content, p-IRS-1Tyr1179, p-AS160Ser588, p-AMPKα1Thr172, and p-CaMKIIThr286 in cardiac and gastrocnemius muscle tissues (Western blot). In response to exercise training, there were improvements in insulin resistance (15.4%; p = .010), increased GLUT4 content (microsomal, 29.4%; p = .012; plasma membrane, 27.1%; p < .001), p-IRS-1 (42.2%; p < .001), p-AS160 (60.0%; p < .001) in cardiac tissue, and increased GLUT4 content (microsomal, 29.4%; p = .009; plasma membrane, 55.5%; p < .001), p-IRS-1 (28.1%; p = .018), p-AS160 (76.0%; p < .001), p-AMPK-α1 (37.5%; p = .026), and p-CaMKII (30.0%; p = .040) in the gastrocnemius tissue. In D4 group, the exercise-induced increase in GLUT4 was reversed (plasma membrane, −21.3%; p = .027), p-IRS1 (−37.1%; p = .008), and p-AS160 (−82.6%; p < .001) in the cardiac tissue; p-AS160 expression (−35.7%; p = .034) was reduced in the gastrocnemius. In conclusion, the cardiac tissue is more susceptible to exercise adaptations in the GLUT4 content and signaling pathways than the gastrocnemius muscle. This finding may be explained by particular characteristics of insulin-dependent and insulin-independent pathways in the muscle tissues studied.
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Funai, Katsuhiko, George G. Schweitzer, Carlos M. Castorena, Makoto Kanzaki, and Gregory D. Cartee. "In vivo exercise followed by in vitro contraction additively elevates subsequent insulin-stimulated glucose transport by rat skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 298, no. 5 (May 2010): E999—E1010. http://dx.doi.org/10.1152/ajpendo.00758.2009.
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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 AS160642Thr 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-642Thr antibody) and its paralog TBC1D1 (detected by phospho-237Ser 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.
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Roach, William G., Jose A. Chavez, Cristinel P. Mîinea, and Gustav E. Lienhard. "Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1." Biochemical Journal 403, no. 2 (March 26, 2007): 353–58. http://dx.doi.org/10.1042/bj20061798.
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Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this protein by Akt (protein kinase B) leads to suppression of its GAP activity and results in the elevation of the GTP form of a critical Rab. The present study examines a similar Rab GAP, Tbc1d1, about which very little is known. We found that the Rab specificity of the Tbc1d1 GAP domain is identical with that of AS160. Ectopic expression of Tbc1d1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect. Insulin treatment led to the phosphorylation of Tbc1d1 on an Akt site that is conserved between Tbc1d1 and AS160. These results show that Tbc1d1 regulates GLUT4 translocation through its GAP activity, and is a likely Akt substrate. An allele of Tbc1d1 in which Arg125 is replaced by tryptophan has very recently been implicated in susceptibility to obesity by genetic analysis. We found that this form of Tbc1d1 also inhibited GLUT4 translocation and that this effect also required a functional GAP domain.
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Wang, Haiyan, Edward B. Arias, Kentaro Oki, Mark W. Pataky, Jalal A. Almallouhi, and Gregory D. Cartee. "Fiber type-selective exercise effects on AS160 phosphorylation." American Journal of Physiology-Endocrinology and Metabolism 316, no. 5 (May 1, 2019): E837—E851. http://dx.doi.org/10.1152/ajpendo.00528.2018.
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Earlier research using muscle tissue demonstrated that postexercise elevation in insulin-stimulated glucose uptake (ISGU) occurs concomitant with greater insulin-stimulated Akt substrate of 160 kDa (AS160) phosphorylation (pAS160) on sites that regulate ISGU. Because skeletal muscle is a heterogeneous tissue, we previously isolated myofibers from rat epitrochlearis to assess fiber type-selective ISGU. Exercise induced greater ISGU in type I, IIA, IIB, and IIBX but not IIX fibers. This study tested if exercise effects on pAS160 correspond with previously published fiber type-selective exercise effects on ISGU. Rats were studied immediately postexercise (IPEX) or 3.5 h postexercise (3.5hPEX) with time-matched sedentary controls. Myofibers dissected from the IPEX experiment were analyzed for fiber type (myosin heavy chain isoform expression) and key phosphoproteins. Isolated muscles from the 3.5hPEX experiment were incubated with or without insulin. Myofibers (3.5hPEX) were analyzed for fiber type, key phosphoproteins, and GLUT4 protein abundance. We hypothesized that insulin-stimulated pAS160 at 3.5hPEX would exceed sedentary controls only in fiber types characterized by greater ISGU postexercise. Values for phosphorylation of AMP-activated kinase substrates (acetyl CoA carboxylaseSer79 and AS160Ser704) from IPEX muscles exceeded sedentary values in each fiber type, suggesting exercise recruitment of all fiber types. Values for pAS160Thr642 and pAS160Ser704 from insulin-stimulated muscles 3.5hPEX exceeded sedentary values for type I, IIA, IIB, and IIBX but not IIX fibers. GLUT4 abundance was unaltered 3.5hPEX in any fiber type. These results advanced understanding of exercise-induced insulin sensitization by providing compelling support for the hypothesis that enhanced insulin-stimulated phosphorylation of AS160 is linked to elevated ISGU postexercise at a fiber type-specific level independent of altered GLUT4 expression.
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Marko, Daniel M., Gregory Foran, Filip Vlavcheski, David C. Baron, Grant C. Hayward, Bradley J. Baranowski, Aleksander Necakov, Evangelia Tsiani, and Rebecca E. K. MacPherson. "Interleukin-6 Treatment Results in GLUT4 Translocation and AMPK Phosphorylation in Neuronal SH-SY5Y Cells." Cells 9, no. 5 (April 30, 2020): 1114. http://dx.doi.org/10.3390/cells9051114.
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Interleukin-6 (IL-6) is a pleiotropic cytokine that can be released from the brain during prolonged exercise. In peripheral tissues, exercise induced IL-6 can result in GLUT4 translocation and increased glucose uptake through AMPK activation. GLUT4 is expressed in the brain and can be recruited to axonal plasma membranes with neuronal activity through AMPK activation. The aim of this study is to examine if IL-6 treatment: (1) results in AMPK activation in neuronal cells, (2) increases the activation of proteins involved in GLUT4 translocation, and (3) increases neuronal glucose uptake. Retinoic acid was used to differentiate SH-SY5Y neuronal cells. Treatment with 100 nM of insulin increased the phosphorylation of Akt and AS160 (p < 0.05). Treatment with 20 ng/mL of IL-6 resulted in the phosphorylation of STAT3 at Tyr705 (p ≤ 0.05) as well as AS160 (p < 0.05). Fluorescent Glut4GFP imaging revealed treatment with 20ng/mL of IL-6 resulted in a significant mobilization towards the plasma membrane after 5 min until 30 min. There was no difference in GLUT4 mobilization between the insulin and IL-6 treated groups. Importantly, IL-6 treatment increased glucose uptake. Our findings demonstrate that IL-6 and insulin can phosphorylate AS160 via different signaling pathways (AMPK and PI3K/Akt, respectively) and promote GLUT4 translocation towards the neuronal plasma membrane, resulting in increased neuronal glucose uptake in SH-SY5Y cells.
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Ching, James Kain, Pooja Rajguru, Nandhini Marupudi, Sankha Banerjee, and Jonathan S. Fisher. "A role for AMPK in increased insulin action after serum starvation." American Journal of Physiology-Cell Physiology 299, no. 5 (November 2010): C1171—C1179. http://dx.doi.org/10.1152/ajpcell.00514.2009.
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Serum starvation is a common cell culture procedure for increasing cellular response to insulin, though the mechanism for the serum starvation effect is not understood. We hypothesized that factors known to potentiate insulin action [e.g., AMP-activated protein kinase (AMPK) and p38] or to be involved in insulin signaling leading to glucose transport [e.g., Akt, PKCζ, AS160, and ataxia telangiectasia mutated (ATM)] would be phosphorylated during serum starvation and would be responsible for increased insulin action after serum starvation. L6 myotubes were incubated in serum-containing or serum-free medium for 3 h. Levels of phosphorylated AMPK, Akt, and ATM were greater in serum-starved cells than in control cells. Serum starvation did not affect p38, PKCζ, or AS160 phosphorylation or insulin-stimulated Akt or AS160 phosphorylation. Insulin had no effect on glucose transport in control cells but caused an increase in glucose uptake for serum-starved cells that was preventable by compound C (an AMPK inhibitor), by expression of dominant negative AMPK (AMPK-DN), and by KU55933 (an ATM inhibitor). ATM protein levels increased during serum starvation, and this increase in ATM was prevented by compound C and AMPK-DN. Thus, it appears that AMPK is required for the serum starvation-related increase in insulin-stimulated glucose transport, with ATM as a possible downstream effector.
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Gallo, Maria Pia, Saveria Femminò, Susanna Antoniotti, Giulia Querio, Giuseppe Alloatti, and Renzo Levi. "Catestatin Induces Glucose Uptake and GLUT4 Trafficking in Adult Rat Cardiomyocytes." BioMed Research International 2018 (October 2, 2018): 1–7. http://dx.doi.org/10.1155/2018/2086109.
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Catestatin is a cationic and hydrophobic peptide derived from the enzymatic cleavage of the prohormone Chromogranin A. Initially identified as a potent endogenous nicotinic–cholinergic antagonist, Catestatin has recently been shown to act as a novel regulator of cardiac function and blood pressure and as a cardioprotective agent in both pre- and postconditioning through AKT-dependent mechanisms. The aim of this study is to investigate the potential role of Catestatin also on cardiac metabolism modulation, particularly on cardiomyocytes glucose uptake. Experiments were performed on isolated adult rat cardiomyocytes. Glucose uptake was assessed by fluorescent glucose incubation and confocal microscope analysis. Glut4 plasma membrane translocation was studied by immunofluorescence experiments and evaluation of the ratio peripheral vs internal Glut4 staining. Furthermore, we performed immunoblot experiments to investigate the involvement of the intracellular pathway AKT/AS160 in the Catestatin dependent Glut4 trafficking. Our results show that 10 nM Catestatin induces a significant increase in the fluorescent glucose uptake, comparable to that exerted by 100 nM Insulin. Moreover, Catestatin stimulates Glut4 translocation to plasma membrane and both AKT and AS160 phosphorylation. All these effects were inhibited by Wortmannin. On the whole, we show for the first time that Catestatin is able to modulate cardiac glucose metabolism, by inducing an increase in glucose uptake through Glut4 translocation to the plasma membrane and that this mechanism is mediated by the AKT/AS160 intracellular pathway.
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Gusba, Jenny E. "The roles of IL-6 in the regulation of glucose homeostasis." Applied Physiology, Nutrition, and Metabolism 34, no. 1 (February 2009): 83–84. http://dx.doi.org/10.1139/h08-105.
Full textAbstract:
This thesis examined the roles of interleukin (IL)-6 in the regulation of glucose homeostasis, with a specific focus on skeletal muscle. Study 1 sought to determine whether muscle glycogen content is a stimulus for the production of IL-6, examining the periods during and after exercise. The relationship between IL-6 and muscle glycogen content was measured during similar bouts of exhaustive exercise on 2 occasions that resulted in large increases in muscle messenger (m)RNA for IL-6 and circulating levels of IL-6. On 1 occasion, subjects received carbohydrate during recovery to facilitate rates of glycogen resynthesis. During exercise, subjects performed similar bouts of exercise, such that differences in an individual’s glycogen levels between trials could be compared with differences in IL-6. No correlation was detected between the net change in glycogen content and the net change in plasma IL-6 or IL-6 mRNA from rest to exhaustion. Moreover, when the difference within subjects at exhaustion in IL-6 and glycogen was compared, there was no correlation between the 2 variables. During recovery, although carbohydrate intake significantly increased glycogen resynthesis, there was no change in postexercise IL-6 mRNA level or plasma IL-6 concentration. Therefore, glycogen was not the sole regulator of IL-6 production in skeletal muscle. Study 2 examined the direct effect of IL-6 and tumor necrosis factor (TNF)-α on glucose transport and the phosphorylation of key signalling proteins with or without insulin and during rodent muscle contraction. Under basal conditions, IL-6 increased glucose transport in association with an increase in 5′AMP-activated protein kinase (AMPK) and AS160 phosphorylation, but IL-6 decreased insulin-stimulated glucose transport via a reduction in phosphorylation of calcium–calmodulin-dependent protein kinase (CaMK)II and AS160. A novel finding generated from these experiments was the direct involvement of IL-6 in contraction-mediated glucose transport. In the case of muscle contraction, IL-6 was found to increase the phosphorylation of CaMKII and AS160. This research suggests that the activation of CaMKII is involved in the actions of IL-6 under insulin-stimulated and contraction-mediated conditions. Furthermore, AS160 was identified as a common signalling intermediate, influenced by IL-6. It also suggests that AS160 may be a point of convergence for multiple signalling pathways. Finally, the actions of TNF-α mimicked those of IL-6, except during contraction, where TNF-α had no significant effect on glucose transport and attenuated the effects of IL-6.