Insulin activation of glycogen synthase in cultured human fibroblasts is not mediated solely via the insulin receptor

Insulin binds to its specific cell surface receptor in cultured human fibroblasts and also stimulates the conversion of glycogen synthase from the glucose-6-phosphate (G-6-P) dependent to the G-6-P independent form. Although these two processes are tightly coupled in most target tissues for insulin action, in the fibroblast a variety of findings question the relationship of these two events to one another. In human fibroblasts the amount of insulin required to displace half of the 125I-insulin bound to the insulin receptor is 4 ng/ml (6.6 X 10(-10)M), but the activation of glycogen synthase is not maximal until 1-10 micrograms/ml with an ED50 of 30 ng/ml insulin. Antibodies directed against the insulin receptor, which activate glycogen synthase in both fat and muscle, do not stimulate the activation of glycogen synthase in the fibroblast. Fab fragments from anti-insulin receptor antibody compete for insulin binding, but do not inhibit the insulin-stimulated rise in independent activity. The insulin-like growth factor, MSA, which is 1% as potent as insulin in stimulating glucose oxidation in rat fat cells and in inhibiting 125I-insulin binding to human fibroblasts, is 25% as potent as insulin in stimulating glycogen synthase. Proinsulin is 2-10% as potent as insulin, but behaves as a "partial agonist" of insulin action in the fibroblast, i.e. proinsulin is able to elicit only 60% of the maximal response of insulin in the glycogen synthase assay, even at high concentrations. Finally, cell lines from patients with clearly defective insulin receptors exhibit normal insulin dose response curves for the activation of glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interconversion between multiple glucose 6-phosphate-dependent forms of glycogen synthase in intact adipose tissue.

We have tested the hypothesis that interconversion between multiple glucose-6-P-dependent forms of glycogen synthase helps regulate glycogen synthesis in adipose tissue. Our results indicate that interconversion of glycogen synthase in adipose tissue involves primarily dependent forms and that these interconversions were measured better by monitoring the activation constant (A0.5) for glucose-6-P than measuring the -: + glucose-6-P activity ratio. Insulin decreased and epinephrine increased the A0.5 for glucose-6-P without significant change in the activity ratio. Insulin consistently decreased the A0.5 in either the presence or absence of glucose, indicating that the insulin-promoted interconversion did not require increased hexose transport. Isoproterenol increased the A0.5 for glucose-6-P, while methoxamine was without effect, indicating beta receptors mediate adrenergic control of interconversion between glucose-6-P-dependent forms. The changes in the A0.5 produced by incubations with insulin or epinephrine were mutually reversible. We conclude that 1) glycogen synthesis in adipose tissue is catalyzed by multiple glucose-6-P-dependent forms of glycogen synthase, 2) hormones regulate glycogen metabolism by promoting reversible interconversions between these forms, and 3) there is no evidence that a glucose-6-P-independent form of glycogen synthase exists in intact adipose tissue.     

Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling

Insulin-stimulated glucose uptake and incorporation of glucose into skeletal muscle glycogen contribute to physiological regulation of blood glucose concentration. In the present study, glucose handling and insulin signaling in isolated rat muscles with low glycogen (LG, 24-h fasting) and high glycogen (HG, refed for 24 h) content were compared with muscles with normal glycogen (NG, rats kept on their normal diet). In LG, basal and insulin-stimulated glycogen synthesis and glycogen synthase activation were higher and glycogen synthase phosphorylation (Ser(645), Ser(649), Ser(653), Ser(657)) lower than in NG. GLUT4 expression, insulin-stimulated glucose uptake, and PKB phosphorylation were higher in LG than in NG, whereas insulin receptor tyrosyl phosphorylation, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, and GSK-3 phosphorylation were unchanged. Muscles with HG showed lower insulin-stimulated glycogen synthesis and glycogen synthase activation than NG despite similar dephosphorylation. Insulin signaling, glucose uptake, and GLUT4 expression were similar in HG and NG. This discordant regulation of glucose uptake and glycogen synthesis in HG resulted in higher insulin-stimulated glucose 6-phosphate concentration, higher glycolytic flux, and intracellular accumulation of nonphosphorylated 2-deoxyglucose. In conclusion, elevated glycogen synthase activation, glucose uptake, and GLUT4 expression enhance glycogen resynthesis in muscles with low glycogen. High glycogen concentration per se does not impair proximal insulin signaling or glucose uptake. "Insulin resistance" is observed at the level of glycogen synthase, and the reduced glycogen synthesis leads to increased levels of glucose 6-phosphate, glycolytic flux, and accumulation of nonphosphorylated 2-deoxyglucose.     

Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating glycogen synthase kinase (GSK) 3 through phosphorylation. Insulin also promotes glucose uptake and glucose 6-phosphate (G-6-P) 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 dephosphorylation of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knockin mice in which wild-type GSK3 alpha and -beta genes are replaced with mutant forms (GSK3 alpha/beta S21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3 alpha/beta S21A/S21A/S9A/S9A mice, glycogen content in different muscles from these mice was similar compared with wild-type mice. Basal and epinephrine-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knockin 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 G-6-P, the rate of [14C]glucose incorporation into glycogen, and an increase in total glycogen content were similar between wild-type and GSK3 knockin mice. Injection of glucose containing 2-deoxy-[3H]glucose and [14C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knockin mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G-6-P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.     

Kinetic properties of glycogen synthase from skeletal muscle after phosphorylation by glycogen synthase kinase 4

Glycogen synthase I (EC 2.4.1.11) from rat and from rabbit skeletal muscle was phosphorylated in vitro by glycogen synthase kinase 4 (EC 2.7.1.37) to the extent of 0.8 phosphates/subunit. For both phosphorylated enzymes, the activity ratio (activity without glucose 6-P divided by activity with 8 mM glucose 6-P) was 0.8 when determined with low concentrations of glycogen synthase and/or short incubation times. However, the activity ratio was 0.5 with high enzyme concentrations and longer incubation times. It was found that the lower activity ratios result largely from UDP inhibition of activity measured in the absence of glucose 6-P. Inhibition by UDP was much less pronounced for glycogen synthase I, indicating that a major consequence of phosphorylation by glycogen synthase kinase 4 is an increased sensitivity to UDP inhibition.     

Control of glycogen synthase by insulin and isoproterenol in rat adipocytes. Changes in the distribution of phosphate in the synthase subunit in response to insulin and beta-adrenergic receptor activation

Rat adipocytes were incubated with [32P]phosphate to label glycogen synthase, which was rapidly immunoprecipitated from cellular extracts and cleaved using either CNBr or trypsin. All of the [32P]phosphate in synthase was recovered in two CNBr fragments, denoted CB-1 and CB-2. Isoproterenol (1 microM) rapidly decreased the synthase activity ratio (-glucose-6-P/+glucose-6-P) and stimulated the phosphorylation of both CB-1 and CB-2 by approximately 30%. Insulin opposed the decrease in activity ratio and blocked the stimulation of phosphorylation by isoproterenol. Incubating cells with insulin alone changed the 32P content of neither CB-1 nor CB-2. Trypsin fragments were separated by reverse phase liquid chromatography and divided into peak fractions, denoted F-I-F-VII in order of increasing hydrophobicity. F-V contained almost half of the [32P]phosphate and was phosphorylated when synthase was immunoprecipitated from unlabeled fat cells and incubated with [gamma-32P]ATP and the cAMP-independent protein kinase, FA/GSK-3. That F-V also had the same retention time as the skeletal muscle synthase fragment containing sites 3(a + b + c) suggests that it contains sites 3. Muscle sites 1a, 5, 1b, and 2 eluted with F-I, F-II, F-VI, and F-VII, respectively. F-V was increased approximately 25% by isoproterenol, but the largest relative increases were observed in F-I (4-fold), F-III (4-fold), and F-VI (2-fold). These results indicate that beta-adrenergic receptor activation results in increased phosphorylation of multiple sites on glycogen synthase. Insulin plus glucose decreased the overall 32P content of synthase by approximately 30%, with the largest decrease (40%) occurring in F-V. Without glucose, insulin decreased the [32P]phosphate in F-V by 17%, an effect which was balanced by increases in F-I, F-II, and F-III so that no net change in the total 32P contents of the fractions was observed. Thus, activation of glycogen synthase by the glucose transport-independent pathway seems to involve a redistribution of phosphate in the synthase subunit.     

Specific desensitization of glycogen synthase activation by insulin in 3T3-L1 adipocytes. Connection between enzymatic activation and subcellular localization

A protocol was developed in 3T3-L1 adipocytes that resulted in the specific desensitization of glycogen synthase activation by insulin. Cells were pretreated for 15 min with 100 nm insulin, and then recovered for 1.5 h in the absence of hormone. Subsequent basal and insulin-induced phosphorylation of the insulin receptor, IRS-1, MAPK, Akt kinase, and GSK-3 were similar in control and pretreated cells. Additionally, enhanced glucose transport and incorporation into lipid in response to insulin were unaffected. However, pretreatment reduced insulin-stimulated glycogen synthesis by over 50%, due to a nearly complete inhibition of glycogen synthase activation. Removal of extracellular glucose during the recovery period blocked the increase in glycogen levels, and restored insulin-induced glycogen synthase activation. Furthermore, incubation of pretreated 3T3-L1 adipocytes with glycogenolytic agents reversed the desensitization event. Separation of cellular lysates on sucrose gradients revealed that glycogen synthase was primarily located in the dense pellet fraction, with lesser amounts in the lighter fractions. Insulin induced glycogen synthase translocation from the lighter to the denser glycogen-containing fractions. Interestingly, insulin preferentially activated translocated enzyme while having little effect on the majority of glycogen synthase activity in the pellet fraction. In insulin-pretreated cells, glycogen synthase did not return to the lighter fractions during recovery, and thus did not move in response to the second insulin exposure. These results suggest that, in 3T3-L1 adipocytes, the translocation of glycogen synthase may be an important step in the regulation of glycogen synthesis by insulin. Furthermore, intracellular glycogen levels can regulate glycogen synthase activation, potentially through modulation of enzymatic localization.     

The role of glucose metabolites in the activation and translocation of glycogen synthase by insulin in 3T3-L1 adipocytes.

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 insulin-stimulated 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.     

Insulin regulation of glycogen synthase phosphatase in primary cultures of hepatocytes

Activation of glycogen synthase in the perfused rat liver is defective in severely diabetic rats. In the present study, activation of glycogen synthase by glucose and increased incorporation of [14C]glucose into glycogen by insulin are defective in hepatocytes isolated from alloxan diabetic rats. Acute activation of glycogen synthase in hepatocytes isolated from diabetic rats was restored by treatment of the rats with insulin in vivo. Restoration of synthase activation was not achieved by incubation of hepatocytes in the presence of insulin in vitro for up to 12 h. When isolated hepatocytes from diabetic rats were placed in primary culture in a serum-free defined medium over a 3-day period, glycogen synthesis was partially restored by cortisol and triiodothyronine and dramatically increased by insulin. Concomitant with restoration of [14C]glycogen synthesis was an insulin-mediated increase in glycogen synthase I and synthase phosphatase activity. Restoration of regulation of glycogen synthesis in primary cultures of hepatocytes from diabetic rats by insulin required the presence of cortisol and triiodothyronine. Primary cultures of hepatocytes from normal rats did not require triiodothyronine for insulin to effect glycogenesis over a 3-day period. These data demonstrate that insulin acts in a chronic manner in concert with other hormones to control synthase phosphatase activity, an effect which may be influencing acute control of hepatic glycogen synthesis.     

Activation of rat adipocyte glycogen synthase by insulins.

Incubation of fat cells with insulin increased glycogen synthase I activity without changing total synthase activity. This effect of insulin was dependent upon the particular lot of albumin present in the medium and was abolished by incubating cells with trypsin. Half-maximal activation of glycogen synthase was obtained with 8 microunits/ml of insulin, a concentration very similar to that which half-maximally stimulated 3-O-methylglucose uptake. The basal percentage of phosphorylase a activity was not detectably altered by insulin, although it was decreased by incubating cells with 5 mM glucose. Insulin (50 microunits/ml) markedly opposed actions of epinephrine (0.05 to 10 muM) to increase phosphorylase a activity and decrease glycogen synthase I activity, effects which were observed without glucose. Partial activation of glycogen synthase by insulin was seen after 1 min and complete activation after 4 min. Glucose alone produced a transient increase in synthase I activity. When cells were incubated with insulin plus glucose for 4 min, the increase in the percent synthase I activity was much greater than the additive effects of insulin and glucose alone. This potentiation of the effect of insulin on glucogen synthase I activity depended on the time of incubation with glucose and on the concentration of the hexose. If cells were incubated with cytochalasin B before insulin plus glucose, the effect of glucose was abolished. These results suggest that there are at least two mechanisms by which insulin can increase fat cell glycogen synthase I activity. One requires glucose and activation occurs secondary to an increase in glucose transport; where another mechanism(s) is operative even in the absence of glucose.     

Activation of glycogen synthase by insulin in rat adipocytes. Evidence of hormonal stimulation of multisite dephosphorylation by glucose transport-dependent and -independent pathways

Adipocytes were incubated with [32P]phosphate to achieve steady state labeling of glycogen synthase. The enzyme was then rapidly immunoprecipitated and subjected to electrophoresis on polyacrylamide slab gels in the presence of sodium dodecyl sulfate. The 32P-labeled glycogen synthase had an apparent molecular weight ( Mapp ) equal to 90,000. All of the [32P]phosphate could be recovered in two cyanogen bromide fragments. The larger fragment, CB-2 ( Mapp = 28,000), contained about five times more [32P]phosphate than the smaller fragment, CB-1 ( Mapp = 15,500). Insulin increased the activity ratio (-glucose-6-P/+glucose-6-P) of glycogen synthase from 0.12 to 0.26, but did not decrease the amount of [32P]phosphate in the enzyme. However, insulin promoted the formation of species of CB-2 of lower Mapp , suggesting dephosphorylation of sites that affected the electrophoretic mobility of the fragment. Glucose did not affect the mobility of CB-2, but slightly increased the activity ratio and decreased the [32P] phosphate by approximately 20%. With insulin plus glucose, the increase in activity ratio was much greater than the additive effects of either agent alone. The combination decreased the [32P]phosphate in each cyanogen bromide fragment by approximately 60%, indicating that the synergistic activation was due to enhanced dephosphorylation of multiple sites. 2-Deoxyglucose also promoted dephosphorylation of glycogen synthase, decreasing the 32P content of CB-1 and CB-2 by approximately 40% each. 3-O-Methylglucose was without effect. The results presented suggest that the activation of glycogen synthase by insulin via a glucose transport-dependent pathway may involve increased intracellular glucose-6-P which promotes dephosphorylation of sites in both CB-1 and CB-2. Activation by a glucose transport-independent pathway appears to be confined to sites located in CB-2.     

Effects of lithium and insulin on glycogen synthesis in L6 myocytes: additive effects on inactivation of glycogen synthase kinase-3

In insulin-sensitive L6 myocytes, insulin stimulated glycogen synthesis in a dose-dependent manner and lithium further stimulated glycogen synthesis at all insulin concentrations. Lithium alone at 20 mM stimulated glycogen synthesis to the degree similar to the maximal insulin response. Effects of lithium and insulin were fully additive for both glycogen synthesis and glycogen synthase activity. In L6 myocytes, insulin increased phosphorylation of Akt1 and glycogen synthase kinase-3 alpha and beta (GSK-3 alpha and beta), resulting in its activation and inactivation, respectively. Unlike insulin, lithium directly inhibited GSK-3 (both alpha and beta) without affecting phosphorylation of GSK-3. Moreover, lithium in vitro could further inhibit enzyme activity of GSK-3 (both alpha and beta) that was isolated from insulin-stimulated cells (thus already phosphorylated and inactivated by insulin). In summary, insulin increases glycogen synthesis by the Akt1/GSK-3/glycogen synthase pathway, but lithium increases glycogen synthesis by direct inhibition of GSK-3 in L6 myocytes. Inhibitory effects of lithium and insulin on GSK-3 (both alpha and beta) were additive, which may account, at least in part, for their additive effects on glycogen synthase activity and glycogen synthesis in L6 myocytes.     

Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R² = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70^s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.     

Insulin resistance of glycogen synthase mediated by o-linked N-acetylglucosamine

We have investigated the mechanism by which high concentrations of glucose inhibit insulin stimulation of glycogen synthase. In NIH-3T3-L1 adipocytes cultured in low glucose (LG; 2.5 mm), the half-maximal activation concentration (A(0.5)) of glucose 6-phosphate was 162 +/- 15 microm. Exposure to either high glucose (HG; 20 mm) or glucosamine (GlcN; 10 mm) increased the A(0.5) to 558 +/- 61 or 612 +/- 34 microm. Insulin treatment with LG reduced the A(0.5) to 96 +/- 10 microm, but cells cultured with HG or GlcN were insulin-resistant (A(0.5) = 287 +/- 27 or 561 +/- 77 microm). Insulin resistance was not explained by increased phosphorylation of synthase. In fact, culture with GlcN decreased phosphorylation to 61% of the levels seen in cells cultured in LG. Hexosamine flux and subsequent enzymatic protein O-glycosylation have been postulated to mediate nutrient sensing and insulin resistance. Glycogen synthase is modified by O-linked N-acetylglucosamine, and the level of glycosylation increased in cells treated with HG or GlcN. Treatment of synthase in vitro with protein phosphatase 1 increased basal synthase activity from cells cultured in LG to 54% of total activity but was less effective with synthase from cells cultured in HG or GlcN, increasing basal activity to only 13 or 16%. After enzymatic removal of O-GlcNAc, however, subsequent digestion with phosphatase increased basal activity to over 73% for LG, HG, and GlcN. We conclude that O-GlcNAc modification of glycogen synthase results in the retention of the enzyme in a glucose 6-phosphate-dependent state and contributes to the reduced activation of the enzyme in insulin resistance.     

Control of glycogen content in retina: allosteric regulation of glycogen synthase

Retinal tissue is exceptional because it shows a high level of energy metabolism. Glycogen content represents the only energy reserve in retina, but its levels are limited. Therefore, elucidation of the mechanisms controlling glycogen content in retina will allow us to understand retina response under local energy demands that can occur under normal and pathological conditions. Thus, we studied retina glycogen levels under different experimental conditions and correlated them with glucose-6-phosphate (G-6-P) content and glycogen synthase (GS) activity. Glycogen and G-6-P content were studied in ex vivo retinas from normal, fasted, streptozotocin-treated, and insulin-induced hypoglycemic rats. Expression levels of GS and its phosphorylated form were also analyzed. Ex vivo retina from normal rats showed low G-6-P (14±2 pmol/mg protein) and glycogen levels (43±3 nmol glycosyl residues/mg protein), which were increased 6 and 3 times, respectively, in streptozotocin diabetic rats. While no changes in phosphorylated GS levels were observed in any condition tested, a positive correlation was found between G-6-P levels with GS activity and glycogen content. The results indicated that in vivo, retina glycogen may act as an immediately accessible energy reserve and that its content was controlled primarily by G-6-P allosteric activation of GS. Therefore, under hypoglycemic situations retina energy supply is strongly compromised and could lead to the alterations observed in type 1 diabetes.     

Activation of hepatocyte glycogen synthase by metabolic inhibitors

Incubation of isolated rat hepatocytes with metabolic inhibitors causes an increase in the -glucose 6-P/+glucose 6-P activity ratio of glycogen synthase after decreasing ATP and increasing AMP levels. Concomitantly, the activity of phosphorylase is increased six-fold by the same treatment. This activation of both enzymes remains after gel filtration of the hepatocyte extracts. Addition of metabolic inhibitors to cells pretreated with an inhibitor of AMP-deaminase results in an accumulation of AMP and, simultaneously, in a further increase in the activation state of glycogen synthase. The correlation coefficient between the intracellular concentration of AMP and glycogen synthase activity is r = 0.93. It is proposed that the covalent activation of glycogen synthase by metabolic inhibitors can be triggered by changes in the level of the intracellular concentrations of adenine nucleotides.     

Inhibitor-1 is not required for the activation of glycogen synthase by insulin in skeletal muscle.

Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle.     

Hormonal regulation of glycogen synthase: Insulin decreases protein kinase sensitivity to cyclic AMP

Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1–2 μM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 μM. Insulin (0–100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.     

Role of glycogen content in insulin resistance in human muscle cells

We have used primary human muscle cell cultures to investigate the role of glycogen loading in cellular insulin resistance. Insulin pre-treatment for 2 h markedly impaired insulin signaling, as assessed by protein kinase B (PKB) phosphorylation. In contrast, insulin-dependent glycogen synthesis, glycogen synthase (GS) activation, and GS sites 3 de-phosphorylation were impaired only after 5 h of insulin pre-treatment, whereas 2-deoxyglucose transport was only decreased after 18 h pre-treatment. Insulin-resistant glycogen synthesis was associated closely with maximal glycogen loading. Both glucose limitation and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) treatment during insulin pre-treatment curtailed glycogen accumulation, and concomitantly restored insulin-sensitive glycogen synthesis and GS activation, although GS de-phosphorylation and PKB phosphorylation remained impaired. Conversely, glycogen super-compensation diminished insulin-sensitive glycogen synthesis and GS activity. Insulin acutely promoted GS translocation to particulate subcellular fractions; this was abolished by insulin pre-treatment, as was GS dephosphorylation therein. Limiting glycogen accumulation during insulin pre-treatment re-instated GS dephosphorylation in particulate fractions, whereas glycogen super-compensation prevented insulin-stimulated GS translocation and dephosphorylation. Our data suggest that diminished insulin signaling alone is insufficient to impair glucose disposal, and indicate a role for glycogen accumulation in inducing insulin resistance in human muscle cells.     

Insulin stimulation of heart glycogen synthase D phosphatase (protein phosphatase).

Insulin rapidly produced an increase in per cent of total heart glycogen synthase in the I form in fed rats. In fasted rats the response was diminished and delayed. In diabetic animals there was no response over the 15-min time period studied. Since synthase phosphatase activity is necessary for synthase D to I conversion, the phosphatase activity was determined in extracts from these groups of animals. In the fasted and diabetic rats phosphatase activity was less than one-half of that in fed animals. Administration of insulin to fasting animals increased synthase phosphatase activity to a level approaching that of fed animals by 15 min. In diabetic animals insulin also stimulated an increase in synthase phosphatase activity but 30 min were required for full activation. Insulin had no effect in normal fed animals. Insulin activation of synthase phosphatase activity in heart extracts from fasted animals was still present after Sephadex G-25 chromatography and ammonium sulfate precipitation. Thus insulin had induced a stable modification of the phosphatase itself or of its substrate synthase D rendering the latter a more favorable substrate for the reaction. A difference in sensitivity of the reaction to glycogen inhibition was present between fed and fasted animals. Increasing concentrations of glycogen had only a slight inhibitory effect in extracts from fed animals but considerably reduced activity in extracts from fasted animals. Insulin administration reduced the sensitivity of the phosphatase reaction to glycogen inhibition. This could explain, at least in part, the increased phosphatase activity noted in the insulin-treated, fasted rats since glycogen was routinely added to the homogenizing buffer.