Regulation of glycogen synthesis from glucose and gluconeogenic precursors by insulin in periportal and perivenous rat hepatocytes.

Glycogen synthesis in hepatocyte cultures is dependent on: (1) the nutritional state of the donor rat, (2) the acinar origin of the hepatocytes, (3) the concentrations of glucose and gluconeogenic precursors, and (4) insulin. High concentrations of glucose (15-25 mM) and gluconeogenic precursors (10 mM-lactate and 1 mM-pyruvate) had a synergistic effect on glycogen deposition in both periportal and perivenous hepatocytes. When hepatocytes were challenged with glucose, lactate and pyruvate in the absence of insulin, glycogen was deposited at a linear rate for 2 h and then reached a plateau. However, in the presence of insulin, the initial rate of glycogen deposition was increased (20-40%) and glycogen deposition continued for more than 4 h. Consequently, insulin had a more marked effect on the glycogen accumulated in the cell after 4 h (100-200% increase) than on the initial rate of glycogen deposition. Glycogen accumulation in hepatocyte cultures prepared from rats that were fasted for 24 h and then re-fed for 3 h before liver perfusion was 2-fold higher than in hepatocytes from rats fed ad libitum and 4-fold higher than in hepatocytes from fasted rats. The incorporation of [14C]lactate into glycogen was 2-4-fold higher in periportal than in perivenous hepatocytes in both the absence and the presence of insulin, whereas the incorporation of [14C]glucose into glycogen was similar in periportal and perivenous hepatocytes in the absence of insulin, but higher in perivenous hepatocytes in the presence of insulin. Rates of glycogen deposition in the combined presence of glucose and gluconeogenic precursors were similar in periportal and perivenous hepatocytes, whereas in the presence of glucose alone, rates of glycogen deposition paralleled the incorporation of [14C]glucose into glycogen and were higher in perivenous hepatocytes in the presence of insulin. It is concluded that periportal and perivenous hepatocytes utilize different substrates for glycogen synthesis, but differences between the two cell populations in the relative utilization of glucose and gluconeogenic precursors are dependent on the presence of insulin and on the nutritional state of the rat.     

Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes

Regulation of insulin-binding and basal (insulin-independent) as well as insulin-stimulated glycogen synthesis from [14C]glucose, net glycogen deposition and glycogen synthase activation by insulin and dexamethasone were studied in primary cultures of adult rat hepatocytes maintained under chemically defined conditions. Insulin receptor number was increased in a dose-dependent fashion by dexamethasone added to the medium between 24 and 48 h of culture and reduced by insulin, whereas ligand affinity remained unaltered. Insulin-induced down-regulation of insulin receptors was not affected by the glucocorticoid. Although the changes in the sensitivity to insulin of glycogen synthesis from glucose and net glycogen deposition paralleled the modulation of the number of insulin receptors, postbinding events appear to be implicated also in the regulation of insulin-sensitivity. Alterations of the responsiveness of glycogen synthesis to insulin caused by the glucocorticoid and/or insulin and by variation between individual rats were inversely related to cellular glycogen contents, suggesting that hepatocellular glycogen content participates in the regulation of insulin-responsiveness of this metabolic pathway. Regulation of insulin-dependent glycogen synthesis were different. Since the effects of this 'physiological' increase in exogenous glucose were small compared to the acute action of insulin, insulin rather than portal venous glucose is considered to represent the prime stimulator of hepatic glycogen synthesis.     

Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes

Regulation of insulin-binding and basal (insulin-independent) as well as insulin-stimulated glycogen synthesis from [¹⁴C]glucose, net glycogen deposition and glycogen synthase activation by insulin and dexamethasone were studied in primary cultures of adult rat hepatocytes maintained under chemically defined conditions. (1) Insulin receptor number was increased in a dose-dependent fashion by dexamethasone added to the medium between 24 and 48 h of culture and reduced by insulin, whereas ligand affinity remained unaltered. Insulin-induced down-regulation of insulin receptors was not affected by the glucocorticoid. (2) Although the changes in the sensitivity to insulin of glycogen synthesis from glucose and net glycogen deposition paralleled the modulation of the number of insulin receptors, postbinding events appear to be implicated also in the regulation of insulin-sensitivity. (3) Alterations of the responsiveness of glycogen synthesis to insulin caused by the glucocorticoid and/or insulin and by variation between individual rats were inversely related to cellular glycogen contents, suggesting that hepatocellular glycogen content participates in the regulation of insulin-responsiveness of this metabolic pathway. (4) Regulation of insulin-independent glycogenesis in response to an increase from 5 to 10 mM glucose, and of insulin-dependent glycogen synthesis were different. Since the effects of this ‘physiological’ increase in exogenous glucose were small compared to the acute action of insulin, insulin rather than portal venous glucose is considered to represent the prime stimulator of hepatic glycogen synthesis.     

Regulation of glycogen synthesis in rat-hepatocyte cultures by glucose, insulin and glucocorticoids.

The changes in glycogen content and in its rate of synthesis in two-day-old primary cultures of rat hepatocytes were assessed under various conditions. Hepatocytes cultivated in serum-free and hormone-free medium switch from glycogen degradation to glycogen deposition at 10.3 mM glucose. After pretreatment of the cells with glucocorticoids this threshold was reduced, in the absence or presence of insulin, to 5.4 or 1.2 mM glucose, respectively. The rate of glycogen synthesis in the presence of 10 mM glucose was amplified from 5 nmol x h-1 x mg protein-1 to 20 nmol glucose x h-1 x mg protein-1 after pretreatment with triamcinolone. Glucagon pretreatment also significantly increased the subsequent glycogen synthesis rate. Insulin addition accelerated glycogen synthesis about twofold regardless of the pretreatment. The dose-response relationship between insulin concentration and glycogen synthesis rate showed half-maximal effect at 0.62 +/- 0.22 nM (mean +/- S.D.) insulin. Pretreatment of hepatocytes with glucocorticoids, glucagon, insulin or combinations of these hormones did not significantly change the concentration which gives the half-maximal effect.     

Interactions between insulin and alpha 1-adrenergic agents in the regulation of glycogen metabolism in isolated hepatocytes

Addition of insulin to liver cells from fed rats incubated in the absence of other hormones resulted in a 2-fold increase in glycogen synthase activity. This direct effect of insulin has been characterized and compared with the antagonism by insulin of alpha 1-adrenergic effects on glycogen metabolism. The activation of glycogen synthase by insulin developed slowly (20-25 min) and was most effective when the enzyme was partially preactivated by glucose. With glucose concentrations above 15 mM the effects of insulin and glucose were additive. In contrast to glucose, which caused inverse changes in phosphorylase and glycogen synthase activity, insulin activated glycogen synthase without affecting phosphorylase a. Treatment of hepatocytes with phenylephrine led to an activation of phosphorylase and inactivation of glycogen synthase, which could be partially blocked by insulin. This antagonistic effect of insulin was rapid (complete within 5 min of insulin addition) and showed an identical time course for both enzymes. The activation of glycogen synthase by insulin and inactivation by phenylephrine both resulted principally from alterations in the Vmax. Insulin added alone did not alter the basal cytosolic free Ca2+ concentration, which was 160 nM as measured with Quin 2 as an intracellular Ca2+ indicator. Both the magnitude and the initial rate of cytosolic free Ca2+ increase induced by phenylephrine were reduced by about 50% in cells pretreated with insulin. It is concluded that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase, whereas insulin antagonizes the effects of alpha 1-agonists by interfering with their ability to elevate cytosolic free Ca2+.     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

Glucose transport rate and glycogen synthase activity both limit skeletal muscle glycogen accumulation

We varied rates of glucose transport and glycogen synthase I (GS-I) activity (%GS-I) in isolated rat epitrochlearis muscle to examine the role of each process in determining the rate of glycogen accumulation. %GS-I was maintained at or above the fasting basal range during 3 h of incubation with 36 mM glucose and 60 microU/ml insulin. Lithium (2 mM LiCl) added to insulin increased glucose transport rate and muscle glycogen content compared with insulin alone. The glycogen synthase kinase-3beta inhibitor GF-109203 x (GF; 10 microM) maintained %GS-I about twofold higher than insulin with or without lithium but did not increase glycogen accumulation. When %GS-I was lowered below the fasting range by prolonged incubation with 36 mM glucose and 2 mU/ml insulin, raising rates of glucose transport with bpV(phen) or of %GS-I with GF produced additive increases in glycogen concentration. Phosphorylase activity was unaffected by GF or bpV(phen). In muscles of fed animals, %GS-I was approximately 30% lower than in those of fasted rats, and insulin-stimulated glycogen accumulation did not occur unless %GS-I was raised with GF. We conclude that the rate of glucose transport is rate limiting for glycogen accumulation unless %GS-I is below the fasting range, in which case both glucose transport rate and GS activity can limit glycogen accumulation.     

Insulin regulation of hepatic glycogen synthase and phosphorylase.

The relative roles of insulin and glucose in the regulation of hepatic glycogen synthase and phosphorylase were studied in hepatocytes from fed rats. Elevation of extra-cellular glucose led to a rapid decrease in phosphorylase a activity followed by a slower increase in glycogen synthase I activity. A reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose was observed; following initial glucose-induced inactivation of phosphorylase, there was a highly significant linear inverse relationship between residual phosphorylase activity and glycogen synthase activation. Insulin led to a further decrease in phosphorylase activity and a 30-50% additional increase in glycogen synthase activity over that caused by glucose. The effects of insulin required the presence of glucose and served to augment acute glucose stimulation of glycogen synthase and inhibition of phosphorylase. Insulin did not perturb the reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose. The results suggest that the ability of insulin to activate hepatic glycogen synthase can be entirely accounted for by its ability to inactivate phosphorylase.     

Contribution of glucose and gluconeogenic substrates to insulin-stimulated glycogen synthesis in cultured fetal hepatocytes

The influence of medium composition on basal and insulin-stimulated glycogenesis was studied in cultured 17-day-old rat fetal hepatocytes, which contain no glycogen at the time of transplantation. Continuous-labeling ¹⁴C-glucose experiments were used to determine both glycogen content and glycogen labeling. The specific activity of glucose units in the newly formed glycogen (a) was compared to that of the medium glucose (b): the ratio a/b expresses the contribution of medium glucose to glycogen formation. In standard medium (5.5 mM glucose), this ratio averaged 0.60. Variations of glucose concentration in the medium from 1 to 40 mM were accompanied by a progressive increase in both glycogen content and the ratio a/b (up to 0.80). Supplementation of standard medium with fructose, galactose, glycerol, or lactate-pyruvate decreased the hepatocyte glucose uptake from the medium. Galactose (1 to 5 mM) or lactate-pyruvate (5 mM) enhanced the glycogen content whereas glycerol or fructose (1 to 5 mM) had no effect. The ratio a/b, not modified by glycerol or lactate-pyruvate, was decreased to 0.45 by fructose (5 mM). Galactose at concentrations as low as 1 to 2 mM brought the ratio down to 0.30, indicating that it is a superior precursor of glycogen as compared to glucose. When the hepatocytes were grown in the presence of 10 nM insulin, the glycogen content was constantly higher than in the absence of the hormone (2-fold stimulation). Also the amplitude of the glycogenic effect of insulin was similar whatever the modifications of the medium, whereas ratio a/b and glucose uptake were hardly increased by insulin. Thus several substrates can contribute to glycogen formation (especially galactose) in cultured fetal hepatocytes and the essential effect of insulin is a stimulation of the final step of the glycogenosynthetic pathway.     

Short-term stimulation of net glycogen production by insulin in rat hepatocytes

Isolated liver cells from 24 h starved rats were incubated in Krebs-Ringer buffer containing 4% albumin. In the presence of 10, 20 and 30 mM glucose, addition of insulin stimulated net glycogen production by 52, 39 and 20%, respectively. 2 . 10(-9) M insulin was required for half-maximal stimulation. Increases of glycogen production and of glycogen synthase a activity were observed after 15-30 min of incubation with insulin. The stimulatory effect of insulin was additive to that of lithium. In agreement with the literature, insulin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen deposition whereby a decrease of glucagon-elevated cyclic AMP levels was observed. In addition, we found that insulin also decreased the basal cyclic AMP levels in the absence of added glucagon by 22%. It is concluded that physiological concentrations of insulin stimulate net glycogen deposition in hepatocytes from fasted rats; the decrease of basal cyclic AMP levels upon insulin addition may play a role in the mechanism of the hormone action.     

Effect of down-regulation and return of insulin receptors on glycogen synthesis in cultured rat hepatocytes

The dependence of the regulation of insulin receptors by insulin on the time hepatocytes were maintained in culture and the relationship between the return of down-regulated receptors and glycogen synthesis from labelled glucose were investigated in primary cultures of adult rat hepatocytes. Insulin receptor numbers, but not ligand affinity, decreased significantly within the first 24 h of culture, even in the absence of insulin, and then returned to the immediate 'post-attachment' level during 24-48 h. Therefore, down-regulation of insulin receptors by 10 nmol/l insulin was only minor during the 1st day in culture, but amounted to 50% of control levels after the 2nd day, whereas the rate of insulin degradation remained unaltered throughout the entire period of culture. When down-regulated monolayers were switched to insulin-free medium, receptors returned to control levels within 5-10 h. The reduced basal rate of glycogenesis as well as insulin-sensitivity and insulin responsiveness of this metabolic pathway also gradually increased to control levels. However, the time-dependent receptor return was dissociated from the increase in insulin-sensitivity, emphasising the importance of postbinding events. Since the changes both in basal rates and in insulin responsiveness of glycogenesis during the period of receptor return were inversely related to differences in the actual glycogen content between control and down-regulated cells, cellular glycogen content might participate in the regulation of glycogenesis as a 'feedback inhibitor'.     

Hormonal and nutritional factors influencing glycogen deposition in primary cultures of rat liver parenchymal cells

The effect of the glucocorticoids, insulin, and glucose concentration on glycogen deposition in adult rat liver parenchymal cells maintained in a chemically defined, serum-free medium has been studied. Increasing the medium concentration of glucose from 5.6 mM to 30.6mM in the absence of hormones increased cellular glycogen content from 6.5 to 51 μg of glycogen per mg of cell protein. Treatment of the cells with insulin increased the glycogen content by 15 to 30% at medium glucose concentrations above 10.6 mM. The addition of the synthetic glucocorticoid, dexamethasone, to the culture medium resulted in 40 to 105% increases in glycogen content at glucose concentrations greater than 5.6 mM. The addition of dexamethasone and insulin together in the culture medium resulted in an increase in glycogen content that was greater than the additive effect of each hormone alone. This established that glucose concentrations above 10.6 mM stimulate glycogen deposition in the absence of any hormonal stimulus. In addition, glucocorticoids directly stimulate glycogen deposition at glucose concentrations which are greater than physiological (5.6 mM).     

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.     

Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes

Protein phosphatase-1 (PP1) plays an important role in the regulation of glycogen synthesis by insulin. Protein targeting to glycogen (PTG) enhances glycogen accumulation by increasing PP1 activity against glycogen-metabolizing enzymes. However, the specificity of PTG's effects on cellular dephosphorylation and glucose metabolism is unclear. Overexpression of PTG in 3T3-L1 adipocytes using a doxycycline-controllable adenoviral construct resulted in a 10-20-fold increase in PTG levels and an 8-fold increase in glycogen levels. Inclusion of 1 microg/ml doxycycline in the media suppressed PTG expression, and fully reversed all PTG-dependent effects. Infection of 3T3-L1 adipocytes with the PTG adenovirus caused a marked dephosphorylation and activation of glycogen synthase. The effects of PTG seemed specific, because basal and insulin-stimulated phosphorylation of a variety of signaling proteins was unaffected. Indeed, glycogen synthase was the predominant protein whose phosphorylation state was decreased in 32P-labeled cells. PTG overexpression did not alter PP1 protein levels but increased PP1 activity 6-fold against phosphorylase in vitro. In contrast, there was no change in PP1 activity measured using myelin basic protein, suggesting that PTG overexpression specifically directed PP1 activity against glycogen-metabolizing enzymes. To investigate the metabolic consequences of altering PTG levels, glucose uptake and storage in 3T3-L1 adipocytes was measured. PTG overexpression did not affect 2-deoxy-glucose transport rates in basal and insulin-stimulated cells but dramatically enhanced glycogen synthesis rates under both conditions. Despite the large increases in cellular glucose flux upon PTG overexpression, basal and insulin-stimulated glucose incorporation into lipid were unchanged. Cumulatively, these data indicate that PTG overexpression in 3T3-L1 adipocytes discretely stimulates PP1 activity against glycogen synthase and phosphorylase, resulting in a marked and specific increase in glucose uptake and storage as glycogen.     

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.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism.

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.     

Adaptations of glycogen metabolism in rat epididymal adipose tissue during fasting and refeeding.

It is well documented that adipose tissue glycogen content decreases during fasting and increases above control during refeeding. We now present evidence that these fluctuations result from adaptations intrinsic to adipose tissue glycogen metabolism that persist in vitro: in response to insulin (1 milliunit/ml), [3H]glucose incorporation into rat fat pad glycogen was reduced to 10% of control after a 3-day fast; incorporation increased 6-fold over fed control on the 4th day of refeeding following a 3-day fast. We have characterized this adaptation with regard to alterations in glycogen synthase and phosphorylase activity. In addition, we found that incubation of fat pads from fasted rats with insulin (1 milliunit/ml) increased glucose-6-P content, indicating that glucose transport was not the rate-limiting step for glucose incorporation into glycogen in the presence of insulin. In contrast, feeding a fat-free diet resulted in dramatic increases in glycogen content of fat pads without a concomitant increase in glucose incorporation into glycogen in response to insulin (1 milliunit/ml). Thus, fasting and refeeding appeared to alter insulin action on adipose tissue glycogen metabolism more than this dietary manipulation.     

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.     

Enhancement of glycogen concentrations in primary cultures of rat hepatocytes exposed to glucose and fructose.

Glycogen synthesis in isolated hepatocytes can occur from glucose both by a direct mechanism and by an indirect process in which glucose is first metabolized to C3 intermediates before use for glycogenesis via gluconeogenesis. We studied the incorporation into glycogen of glucose and the gluconeogenic substrate, fructose, in primary cultures of hepatocytes from fasted rats. In the presence of insulin, both glucose and fructose promoted net deposition of glycogen; however, fructose carbon was incorporated into glycogen to a greater extent than that from glucose. When glucose and fructose were administered simultaneously, the glycogenic utilization of glucose was stimulated 2-3-fold, and that of fructose was increased by about 50%. At constant hexose concentrations, the total incorporation of carbon, and the total accumulation of glycogen mass, from glucose and fructose when present together exceeded that from either substrate alone. Fructose did not change the relative proportion of glucose carbon incorporated into glycogen via the indirect (gluconeogenic) mechanism. The synergism of glucose and fructose in glycogen synthesis in isolated rat hepatocytes in primary culture appears to result from a decrease in the rate of degradation of newly deposited glycogen, owing to (i) decreased amount of phosphorylase a mediated by glucose and (ii) noncovalent inhibition of residual phosphorylase activity by some intermediate arising from the metabolism of fructose, presumably fructose 1-phosphate.