Regulation of Glycogen Synthesis by Amino Acids in Cultured Human Muscle Cells (original) (raw)
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Running Title: Amino acids, Insulin, GSK-3, and Glycogen Synthase Address correspondence to
2013
Insulin stimulates muscle glucose disposal via both glycolysis and glycogen synthesis. Insulin activates glycogen synthase (GS) in skeletal muscle by phosphorylating protein kinase B (PKB or Akt) which in turn phosphorylates and inactivates glycogen synthase kinase 3 (GSK-3), with subsequent activation of GS. A rapamycin-sensitive pathway, most likely acting via ribosomal protein S6 kinase (p70 S6K), has also been implicated in the regulation of GSK-3 and GS by insulin. Amino acids potently stimulate p70 S6K and recent studies on cultured muscle cells suggest that amino acids also inactivate GSK-3 and/or activate GS via activating p70 S6K. To assess the physiological relevance of these findings to normal human physiology, we compared the effects of amino acids and insulin on whole body glucose disposal, p70 S6K and GSK-3 phosphorylation, and the activity of GS in vivo in skeletal muscle of 24 healthy human volunteers. After an overnight fast, subjects received intravenously either a...
Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle
American Journal of Physiology-endocrinology and Metabolism, 2007
Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3 through phosphorylation. Insulin also promotes glucose uptake and glucose-6-phosphate (G6P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphoryltion of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knock-in mice in which wild-type GSK3 and genes are replaced with mutant forms (GSK3 / S21A/S21A/S9A/S9A ), which are non-responsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3 / S21A/S21A/S9A/S9A mice, glycogen content in different muscles from these mice was similar compared to wild-type mice. Basal and adrenaline-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knock-in mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G6P, the rate of [ 14 C]glucose incorporation into glycogen, as well as an increase in total glycogen content were similar between wild-type and GSK3 knock-in mice. Injection of glucose containing [ 3 H]2-deoxy-glucose and [ 14 C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knock-in mice. These results suggest that insulin-mediated inhibition of GSK3
Journal of Biological Chemistry, 1999
The role of increased glucose transport in the hormonal regulation of glycogen synthase by insulin was investigated in 3T3-L1 adipocytes. Insulin treatment stimulated glycogen synthase activity 4 -5-fold in these cells. Cytosolic glycogen synthase levels decreased by 75% in response to insulin, whereas, conversely, the glycogenolytic agent isoproterenol increased cytosolic enzyme levels by 200%. Removal of extracellular glucose reduced glycogen synthase activation by 40% and completely blocked enzymatic translocation. Addition of 5 mM 2-deoxyglucose did not restore glycogen synthase translocation but did augment dephosphorylation of the protein by insulin. The translocation event could be reconstituted in vitro only by the addition of UDP-glucose to basal cell lysates. Amylase pretreatment of the extracts suppressed glycogen synthase translocation, indicating that the enzyme was binding to glycogen. Incubation of 3T3-L1 adipocytes with 10 mM glucosamine induced a state of insulin resistance, blocked the translocation of glycogen synthase, and inhibited insulinstimulated glycogen synthesis by 50%. Surprisingly, glycogen synthase activation by insulin was enhanced 4-fold, in part due to allosteric activation by a glucosamine metabolite. In vitro, glucosamine 6-phosphate and glucose 6-phosphate stimulated glycogen synthase activity with similar concentration curves. These results indicate that glucose metabolites have an impact on the regulation of glycogen synthase activation and localization by insulin.
AJP: Endocrinology and Metabolism, 2004
Insulin stimulates muscle glucose disposal via both glycolysis and glycogen synthesis. Insulin activates glycogen synthase (GS) in skeletal muscle by phosphorylating PKB (or Akt), which in turn phosphorylates and inactivates glycogen synthase kinase 3 (GSK-3), with subsequent activation of GS. A rapamycin-sensitive pathway, most likely acting via ribosomal 70-kDa protein S6 kinase (p70S6K), has also been implicated in the regulation of GSK-3 and GS by insulin. Amino acids potently stimulate p70S6K, and recent studies on cultured muscle cells suggest that amino acids also inactivate GSK-3 and/or activate GS via activating p70S6K. To assess the physiological relevance of these findings to normal human physiology, we compared the effects of amino acids and insulin on whole body glucose disposal, p70S6K, and GSK-3 phosphorylation, and on the activity of GS in vivo in skeletal muscle of 24 healthy human volunteers. After an overnight fast, subjects received intravenously either a mixed a...
Diabetes, 2002
Glycogen synthase kinase (GSK)-3 has been implicated in the regulation of multiple cellular physiological processes in skeletal muscle. Selective cell-permeable reversible inhibitors (INHs) of GSK-3 (CT98014 and CHIR98023 [Chiron, Emeryville, CA] and LiCl) were used to evaluate the role of GSK-3 in controlling glucose metabolism. Acute treatment (30 min) of cultured human skeletal muscle cells with either INH resulted in a dose-dependent activation of glycogen synthase (GS) with a maximally effective concentration of ∼2 μmol/l. The maximal acute effect of either INH on GS (103 ± 25% stimulation over basal) was greater than the maximal insulin response (48 ± 9%, P < 0.05 vs. INH); LiCl was as effective as insulin. The GSK-3 inhibitor effect, like that of insulin, was on the activation state (fractional velocity [FV]) of GS. Cotreatment of muscle cells with submaximal doses of INH and insulin resulted in an additive effect on GS FV (103 ± 10% stimulation, P < 0.05 vs. either age...
Journal of Biological Chemistry, 2005
The effects of inhibition or constitutive activation of glycogen synthase kinase-3 (GSK3) on glycogen synthase (GS) activity, abundance, and glycogen deposition in L6 rat skeletal muscle cells were investigated. GS protein expression increased ϳ5-fold during differentiation of L6 cells (comparing cells at the end of day 5 with those at the beginning of day 3). However, exposure of undifferentiated myoblasts (day 3) to 50 M SB-415286, a GSK3 inhibitor, led to a significant elevation in GS protein that was not accompanied by changes in the abundance of GLUT4, another late differentiation marker. In contrast, stable expression of a constitutively active form of GSK3 (GSK3 S9A ) led to a significant reduction (ϳ80%) in GS protein that was antagonized by SB-415286. Inhibition of GSK3 or expression of the constitutively active GSK3 S9A did not result in any detectable changes in GS mRNA abundance. However, the increase in GS protein in undifferentiated myoblasts or that seen following incubation of cells expressing GSK3 S9A with GSK3 inhibitors was blocked by cycloheximide suggesting that GSK3 influences GS abundance possibly via control of mRNA translation. Consistent with the reduction in GS protein, cells expressing GSK3 S9A were severely glycogen depleted as judged using a specific glycogen-staining antibody. Inhibiting GSK3 in wild-type or GSK3 S9A -expressing cells using SB-415286 resulted in an attendant activation of GS, but not that of glucose transport. However, GS activation alone was insufficient for stimulating glycogen deposition. Only when muscle cells were incubated simultaneously with insulin and SB-415286 or with lithium (which stimulates GS and glucose transport) was an increase in glycogen accretion observed. Our findings suggest that GSK3 activity is an important determinant of GS protein expression and that while glycogen deposition in muscle cells is inherently dependent upon the activity/expression of GS, glucose transport is a key rate-determining step in this process.
Bioscience, Biotechnology, and Biochemistry, 2005
Insulin receptor substrate-1 (IRS-1) plays a pivotal role in insulin signal transduction. It has been shown that the amino acids modulate insulin signaling at the level of IRS-1. Here we show that an amino acid unbalanced diet causes a reduction in serine phosphorylation as well as an elevation in insulin-induced tyrosine phosphorylation of IRS-1 in rat muscle. In fibroblasts and myotube cells, the effect of amino acid deprivation on IRS-1 phosphorylation was evident only when cells were pretreated with reagents causing hyperphosphorylation of serines of IRS-1. But, the target kinases of these reagents were not inactivated by amino acid deprivation, suggesting that amino acid deprivation activates serine/threonine phosphatase(s) of IRS-1. The phosphatases regulated by mammalian target of rapamycin do not appear to participate in the dephosphorylation either. These results suggest that amino acid deprivation dephosphorylates IRS-1 through unidentified serine/threonine phosphatases and thereby potentiates insulin signaling.