Role of glycogen synthase kinase-3 in skeletal muscle insulin resistance in Type 2 diabetes

Journal of Diabetes and Its Complications 16 (2002) 69 – 71

Role of glycogen synthase kinase-3 in skeletal muscle insulin resistance in Type 2 diabetes T.P. Ciaraldi, S.E. Nikoulina, R.R. Henry* UCSD/VA Medical Center, 111G, 3350 La Jolla Village Drive, San Diego, CA 92161, USA Received 25 June 2001; accepted 2 July 2001

Characteristic features of Type 2 diabetes include resistance in peripheral tissues to the ability of insulin to stimulate glucose uptake (GU) and disposal (GDR). In skeletal muscle, this is expressed as impairments in both glucose oxidation and nonoxidative glucose disposal (GDR) (Thornburn, Gumbiner, Bulacan, Wallace, & Henry, 1990). The major component of nonoxidative glucose metabolism is glucose storage in glycogen; this process has been shown to be impaired in diabetic skeletal muscle (Shulman et al., 1990). The rate-determining enzyme for glucose incorporation into glycogen is glycogen synthase (GS). GS activity can be acutely modulated by allosteric regulators and phosphorylation/dephosphorylation (Roach & Larner, 1997), as well as by control of protein expression. Dephosphorylation of specific residues on GS in response to insulin increases activity at physiologic substrate levels (Thornburn, Gumbinger, Bulacan, Brechtel, & Henry, 1991). Several investigators, ourselves included, have found insulin activation of GS to be impaired in Type 2 diabetic muscle (Thornburn et al., 1991, 1990). GS activity in diabetic skeletal muscle is also reduced under baseline fasting conditions (Thornburn et al., 1990). Multiple defects in GS activity are present in diabetes that contribute to reduced glycogen formation. For the most part, these do not involve mutations in GS itself (Bjorbaek et al., 1994). Total activity of the enzyme has been found to be reduced in diabetic muscle and correlated with glycogen content (Thornburn et al., 1991). Differences in GS expression, either mRNA or protein, are more variable but essentially normal (Vestergaard, Lund, Larsen, Bjerrum, & Pedersen, 1993). The most consistent defects in GS in diabetes have been seen for activity measured at physiologic levels of substrate (G6P), also expressed as the

* Corresponding author. Tel.: +1-858-552-8585 ext 3648; fax: +1-858642-6242. E-mail address: [email protected] (R.R. Henry).

fractional velocity (FV = ratio of activity measured at 0.1 mM G6P/10 mM G6P). GS FV in diabetic muscle is reduced by  50% under baseline conditions and insulin stimulation is greatly impaired (Thornburn et al., 1991). There is a strong correlation between whole-body GDR and GS FV determined in muscle biopsies after insulin infusion (Bogardus, Lillioja, Stone, & Mott, 1984), indicative that muscle GS is a strong determinant of glucose utilization. Impairments in GS activation in diabetic muscle could be the result of genetic factors or acquired from the hyperglycemia, hyperinsulinemia, and hyperlipidemia, constituting the diabetic metabolic environment. However, it has been difficult to rigorously evaluate the relative roles of these factors. We have recently established a system for the growth of human skeletal muscle cells in culture (Henry, Abrams, Nikoulina, & Ciaraldi, 1995). Under specific conditions, these cells can differentiate into myotubes that display the morphological (multinucleation, myosin striations), biochemical (differentiation-associated increases in expression of myosin, sarcomeric-specific a-actin and the M isoform of creatine phosphokinase), and metabolic (insulin stimulation of GU and GS) properties of mature skeletal muscle. Most importantly, defects in glucose metabolism that are present in vivo are retained in cultured muscle cells. Insulin-stimulated GU in muscle cells from diabetic subjects is reduced by 40– 50% compared to cells from nondiabetic subjects and is correlated with whole-body GDR determined by the euglycemic clamp technique, performed at the same time as muscle tissue was obtained for cell culture (Table 1). We also compared GS activity in skeletal muscle tissue and muscle cells obtained from the same individuals. The activity from the skeletal muscle biopsy will be reflective of the in vivo metabolic milieu, while the cultured muscle cells were grown under controlled conditions more closely resembling the nondiabetic environment. GS FV were quite similar in tissue and cultured cells from the same subjects (Table 1). Defects in GS in diabetic muscle seen in biopsies were retained in cultured cells. Both basal and insulin-

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T.P. Ciaraldi et al. / Journal of Diabetes and Its Complications 16 (2002) 69–71

Table 1 In vivo and in vitro comparisons of insulin-stimulated GDR – GU and GS in skeletal muscle biopsies and cultured muscle cells GS FV

Nondiabetic Type 2

Skeletal muscle biopsies

Cultured human skeletal muscle cells

GDR (mg/kg/min)

GU (pmol/mg/min)

Basal

With insulin

Basal

With insulin

9.5 ± 0.5 5.0 ± 0.5

22 ± 1 13 ± 3

0.09 ± 0.01 0.05 ± 0.01

0.17 ± 0.03 0.09 ± 0.02

0.09 ± 0.01 0.05 ± 0.01

0.16 ± 0.02 0.09 ± 0.02

stimulated GS FV were reduced by  50% (Henry, Ciaraldi, Abrams-Carter, et al., 1996). Thus, impaired regulation of skeletal muscle GS in Type 2 diabetes is not completely reversible in culture and represents an intrinsic, possibly genetic, property of diabetes. The major differences between nondiabetic and diabetic GS were in the kinetic characteristics of the enzyme. The affinities of the enzyme for UDP-glucose and G6P were lower in diabetic cells. As these are the properties of the enzyme that are influenced by phosphorylation, it is possible that in diabetic muscle GS is hyperphosphorylated compared to nondiabetics. In addition to revealing intrinsic defects in GS in diabetes, the muscle culture system also permits independent evaluation of the effect of various components of the diabetic environment on GS. Incubation of muscle cells from nondiabetic subjects under hyperglycemic conditions (20 mM) had no effect on GS activity in either the absence or presence of insulin (Henry, Ciaraldi, Mudaliar, Abrams-Carter, & Nikoulina, 1996). Prolonged exposure to hyperinsulinemia did not influence basal GS activity but eliminated acute insulin responsiveness. The effects were seen on GS activation state, altering the kinetic affinity of GS for UDP-glucose in the same direction as with diabetes. GS mRNA and protein abundance were unaltered so the defects in GS generated by hyperinsulinemia were posttranslational in origin. Diabetic muscle cells responded differently to manipulation of media conditions. Hyperglycemia was able to both impair insulin stimulation of GS and reduce GS protein expression (Nikoulina et al., 1997). In vitro hyperinsulinemia normalized basal GS activity and GS expression, yet also abolished insulin responsiveness of GS and glycogen synthesis. Thus, in diabetic muscle, hyperinsulinemia may serve to partially compensate for the impaired basal GS activity and for the adverse effects of hyperglycemia on GS protein content and activity. However, in addition to these beneficial effects, hyperinsulinemia also induces severe impairment of insulin-stimulated GS activity and glycogen formation, which may contribute to the acquired component of insulin resistance in Type 2 diabetes.

Beyond defects in GS itself, insulin signaling represents other potential sites for insulin resistance in diabetes. As best characterized, insulin signaling to activation of GS is initiated by insulin binding to its cell surface receptor, followed by receptor autophosphorylation and activation of the receptor tyrosine kinase. Subsequent is tyrosine phosphorylation of a series of insulin receptor substrates, including IRS-1 and IRS-2. Phosphorylated IRS-1/2 can associate with the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-K), activating the enzyme. PI 3-K is essential for insulin stimulation of GS. Downstream of PI 3-K, and activated by it, is the serine/threonine kinase Akt (PKB). Akt has been shown to directly phosphorylate GS kinase-3 (GSK3), reducing its activity (Roach & Larner, 1997). As GSK3 is a negative regulator of GS, Akt could represent an important step in insulin signaling to GS. In collaboration with Dr. Barbara Kahn et al., we investigated key steps in insulin signaling in skeletal muscle from Type 2 diabetic and nondiabetic subjects. Diabetic muscle displayed defects in insulin-stimulated PI 3-K activity associated with both IRS-1 (50% reduction, P < .05) and IRS-2 (38 – 39% reduction, P < .05) (Kim, Nikoulina, Ciaraldi, Henry, & Kahn, 1999); there was no defect in Akt activity in the muscle. From these results, we conclude that Akt may not be the major downstream mediator of these metabolic actions of insulin. We investigated one enzyme potentially responsible for hyperphosphorylation of GS. GSK3 is a serine kinase that phosphorylates specific residues on GS, reducing GS activity (Eldar-Finkelman, Argast, Foord, Fischer, & Krebs, 1996). Insulin has been shown to cause serine phosphorylation of GSK3, reducing GSK3 activity, which would remove an inhibitory influence on GS. GSK3 exists as two highly homologous isoforms (GSK3a-51 kDa and GSK3b-46 kDa). We found both isoforms to be present in human skeletal muscle (Table 2), along with an additional form (53 kDa) that was immunologically identified as GSK3a, possibly a product of differences in posttranslational processing (Nikoulina et al., 2000). The expression

Table 2 GSK3 protein and activity in skeletal muscle GSK3a

GSK3b Total activity (pmol

Nondiabetic Type 2 diabetic

32

Total activity (32P-I2-AU/min/mg protein)

P/min/mg protein)

Protein (AU/20 mg protein)

Basal

With insulin

Protein

Basal

With insulin

681 ± 82 1006 ± 80

36 ± 4 65 ± 7

26 ± 4 43 ± 5

488 ± 86 898 ± 82

50 ± 12 183 ± 32

32 ± 8 119 ± 32

T.P. Ciaraldi et al. / Journal of Diabetes and Its Complications 16 (2002) 69–71

of all isoforms of GSK3 was significantly elevated in diabetic muscle (Table 2). The specific activities of each isoform in the basal condition were similar between nondiabetic and diabetic muscle. Insulin infusion during the clamp procedure resulted in a significant reduction in the specific activity of GSK3a that was similar in nondiabetic (35% drop) and diabetic (36%) subjects. Thus, total activity was elevated by approximately twofold in diabetic subjects in both the basal and insulin-stimulated conditions. Insulin infusion increased the serine phosphorylation of this isoform and to a similar extent in nondiabetic and diabetic subjects. These data indicate that GSK3 is qualitatively normal in diabetic skeletal muscle. Rather, the defect in GSK3 in diabetes is a quantitative one, an overexpression of the enzyme. Strong inverse correlations are seen between GSK3a and GSK3b protein and activity expression and both GS FV in muscle biopsies and wholebody GDR (Nikoulina et al., 2000): the more GSK3 the subject has, the worse their glucose tolerance. In order to learn if GSK3 could be a causative factor for insulin resistance, we returned to the cultured human muscle system. Cells were treated with a series of reversible, highly selective, cell permeable inhibitors of GSK3 developed at Chiron Corp. Acute (15 – 60 min) exposure of cells stimulated GS FV. Maximal concentrations of inhibitors were more effective ( 100% increase) than insulin (  50%), yet at submaximal doses, inhibitor + insulin resulted in additive or synergistic effects on GS. LiCl, a commonly employed, yet less specific, inhibitor of GSK3 also stimulated GS. Insulin and LiCl also acutely stimulated GU, while the Chiron inhibitors did not. The effects of insulin and LiCl on both GS and GU were transient; activities returned to basal by 6 h. Prolonged (24 h) insulin treatment led to a desensitization of both responses. Meanwhile, with exposure of cells to the Chiron inhibitors, GS activity continued to increase by nearly threefold at 24 h of treatment; a response that was sustained for up to 96 h. GS protein expression was unaltered. However, both GSK3 protein and activity were reduced by  65%, so these inhibitors worked on two levels to reduce GSK3 activity. Unlike the situation with acute treatment, GU was also elevated during chronic inhibitor exposure. Both basal ( 150% increase) and insulin-stimulated  (220%) uptake were increased and insulin action was augmented. The general conclusion from the studies using GSK3 inhibitors in muscle cells is that a reduction in GSK3 activity can have multiple effects to improve GDR and insulin action. These results provide a complement to the studies in muscle biopsies where elevations in GSK3 are associated with glucose intolerance and insulin resistance. In summary, a considerable body of evidence points to GS as an important determinant of glucose tolerance and insulin responsiveness, especially in Type 2 diabetes. Given its effects on GS, GSK3 can also be expected to play a critical role in the pathogenesis of insulin resistance. Inter-

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ventions that improve GS activity, possibly through GSK3, represent an area of potential therapeutic benefit.

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