Altered liver glycogen metabolism in fed genetically obese mice.

The cyclic AMP and glycogen concentrations and the activities of phosphorylase kinase, phosphorylase a and glycogen synthase a were not different in livers from lean or ob/ob mice despite increased plasma glucose and insulin in the obese group. The liver water content was decreased by 10% in the obese mice. In hepatocytes isolated from lean mice and incubated with increasing glucose concentrations (14-112 mM), a sequential inactivation of phosphorylase and activation of glycogen synthase was observed. In hepatocytes from obese mice the inactivation of phosphorylase was not followed by an activation of synthase. The inactivation of phosphorylase occurred more rapidly and was followed by an activation of synthase in hepatocytes isolated from both groups of mice when in the incubation medium Na+ was replaced by K+ or when Ca2+ was omitted and 2.5 mM-EGTA included. The inactivation of phosphorylase and activation of synthase were not different in broken-liver-cell preparations from lean and obese animals. The re-activation of phosphorylase in liver filtrates in the presence of 0.1 microM-cyclic AMP and MgATP was inhibited by about 70% by EGTA and stimulated by Ca2+ and was always greater in preparations from ob/ob mice. The apparent paradox between the impairment of glycogen metabolism in isolated liver preparations and the situation in vivo in obese mice is discussed.     

Hepatic CCAAT/enhancer binding protein alpha mediates induction of lipogenesis and regulation of glucose homeostasis in leptin-deficient mice

CCAAT/enhancer binding protein alpha (C/EBP alpha) is a critical factor in glucose metabolism in the neonate as revealed by conventional C/EBP alpha-null mice that do not survive beyond the first day after birth because of severe hypoglycemia and a deficiency in hepatic glycogen accumulation. To elucidate the function of C/EBP alpha in leptin-deficient mouse (ob/ob) liver, a C/EBP alpha-liver null mouse on an ob/ob background (ob/ob-C/EBP alpha/Cre(+)) was produced using a floxed C/EBP alpha allele and Cre recombinase under control of the albumin promoter (AlbCre). The C/EBP alpha-deficient liver in ob/ob mice had significantly decreased triglyceride content compared with equivalent mice lacking the AlbCre transgene (ob/ob-C/EBP alpha/Cre(-)). Expression of genes involved in lipogenesis including fatty acid synthase, acetyl-coenzyme A carboxylase, stearoyl-coenzyme A desaturase 1 and ATP-citrate lyase dramatically decreased in ob/ob-C/EBP alpha/Cre(+) mouse liver. Induction of these lipogenic genes by a high-carbohydrate diet caused an exacerbation in the development of fatty liver and an increase in liver size, hepatic triglyceride, and cholesterol contents in ob/ob-C/EBP alpha/Cre(-) mice but not in ob/ob-C/EBP alpha/Cre(+) mice. Deficiency in hepatic C/EBP alpha expression caused an exacerbation of hyperglycemia because of decreased insulin secretion. Taken together, these results indicate that hepatic C/EBP alpha plays a critical role in the acceleration of lipogenesis in ob/ob mice and in glucose homeostasis by the indirect regulation of insulin secretion.     

The effects of fasting and refeeding on liver glycogen synthase and phosphorylase in obese and lean mice.

The responses of hepatic glycogen synthase and phosphorylase to fasting and refeeding were assessed as part of an investigation into possible sites of insulin resistance in gold thioglucose (GTG) obese mice. The active forms glycogen synthase and phosphorylase (synthase I and phosphorylase a) and the total activity of these enzymes were estimated in lean and GTG mice over 48 h of food deprivation, and for 120 min after glucose gavage (1 g/kg wt). In lean mice there was a maximal reduction in hepatic glycogen content after 12 h of starvation and the activity of phosphorylase a decreased from 23.8 +/- 1.9 to 6.8 +/- 0.7 mumol/g protein/min. These changes were accompanied by an increase in the activity of synthase I (from 0.14 +/- 0.01 to 0.46 +/- 0.04 mumol/g protein/min). In obese mice, similar changes in enzyme activity occurred after 48 h of starvation. These changes were accompanied by a significant reduction in the hyperinsulinemia and hyperglycemia of the GTG mice. After glucose gavage in both lean and obese mice, the activity of synthase I further increased over the first 30 min and declined thereafter. The activity of phosphorylase a increased progressively after refeeding. Results from this study suggest that despite increased hepatic glycogen deposition, the responses of glycogen synthase and phosphorylase, in livers of obese mice, to fasting and refeeding are similar to those of control mice even in the presence of insulin resistance.     

Liver glycogen metabolism during intestinal perfusion in the rat.

The effects of metabolic constituents on glycogen metabolism in the liver can be studied by perfusing the small intestine with those constituents capable of being absorbed across the gut mucosa. Perfusion of the gut with glucose to give concentrations of approx. 20 mM in the portal vein produced a 5-fold increase in liver glycogen after 60 min. The rate of synthesis over the 15–30 min period of perfusion of 1.24 μmol/min/g liver fell to 0.34 in the 30–60 min period. This stimulation was accompanied by a transient activation of glycogen synthase which occurred in the first 30 min period of perfusion but which had disappeared by 60 min. Glucose perfusion had no effect on total and active phosphorylase at the time intervals studied. While the pattern of insulin secretion during perfusion could not be ascertained, the activation of glycogen synthase and increase in glycogen synthesis correlated with the production of gastric inhibitory polypeptide (GIP), a potent stimulator of insulin secretion. Perfusion of the gut with fructose did not stimulate glycogen synthesis or activate glycogen synthase or stimulate GIP secretion, but instead it produced a marked activation of phosphorylase after 30 min of perfusion.     

Synergism of glucose and fructose in net glycogen synthesis in perfused rat livers

Synergism of glucose and fructose in net glycogen synthesis was studied in perfused livers from 24-h fasted rats. With either glucose or fructose alone, net glycogen deposition did not occur (p greater than 0.10 for each), whereas the addition of both together resulted in significant glycogen accumulation (net glycogen accumulation was 0.21 +/- 0.03 mumol of glucose/g of liver/min at 2 mM fructose and 30 mM glucose, p less than 0.001). To better understand this synergism, intermediary substrate levels were compared at steady state with various glucose levels in the absence and in the presence of 2 mM fructose. Independent of fructose, hepatic glucose and glucose 6-phosphate increased proportionally when glucose level in the medium was raised (r = 0.86, p less than 0.001). Unlike glucose 6-phosphate, UDP-glucose did not consistently increase with glucose (p greater than 0.10); in fact, there was a small decrease at a very high glucose level (30 mM), a result consistent with the well-established activation of glycogen synthase by glucose. With elevated glucose, the level of glucose 6-phosphate was strongly correlated with glycogen content (r = 0.71, p less than 0.01, slope = 32). Adding fructose increased the "efficiency" of glucose 6-phosphate to glycogen conversion: the effect of a given increment in glucose 6-phosphate upon glycogen accumulation was increased 2.6-fold (r = 0.73, p less than 0.01, slope = 86). A kinetic modeling approach was used to investigate the mechanisms by which fructose synergized glycogen accumulation when glucose was elevated. Based on steady-state hepatic substrate levels, net hepatic glucose output, and net glycogen synthesis rate, the model estimated the rate constants of major enzymes and individual fluxes in the glycogen metabolic pathway. Modeling analysis is consistent with the following scenario: glycogen synthase is activated by glucose, whereas glucose-6-phosphatase was inhibited. In addition, the model supports the hypothesis that fructose synergizes net glycogen accumulation due to suppression of phosphorylase. Overall, our analysis suggests that glucose enhances the metabolic flux to glycogen by inducing a build up of glucose 6-phosphate via combined effects of mass action and glucose-6-phosphatase inhibition and activating glycogen synthase and that fructose enhances glycogen accumulation by retaining glycogen via phosphorylase inhibition.     

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.     

Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.     

Deficiency of a Glycogen Synthase-associated Protein,Epm2aip1, Causes Decreased Glycogen Synthesis and Hepatic Insulin Resistance

Glycogen synthesis is a major component of the insulin response, and defective glycogen synthesis is a major portion of insulin resistance. Insulin regulates glycogen synthase (GS) through incompletely defined pathways that activate the enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs allosteric activation of GS by glucose 6-phosphate, decreases hepatic glycogen synthesis, increases liver fat, causes hepatic insulin resistance, and protects against age-related obesity. Our work identifies a novel GS-associated GS activity-modulating component of insulin resistance.     

Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.     

Hepatic glycogen metabolism in the db/db mouse

Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in V_max, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.     

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.     

Transforming growth factor alpha inhibits glycogen synthesis and counteracts the stimulation by insulin in hepatocytes.

Rat transforming growth factor alpha (TGF alpha) inhibits glycogen synthesis in rat and guinea pig hepatocyte cultures and counteracts the stimulation of glycogen deposition and activation of glycogen synthase caused by insulin. The EC50 for inhibition of glycogen deposition was 0.2nM. The inhibition of glycogen synthesis was also observed in the absence of extracellular Ca2+ and was not blocked by indomethacin, suggesting that it is not mediated by production of prostaglandins. Since TGF alpha is produced by hepatocytes during liver regeneration and by macrophages during endotoxin stimulation, it may have an autocrine/paracrine effect on hepatic carbohydrate metabolism in these states, and may account for the low hepatic glycogen levels during liver regeneration and the impaired glucose tolerance associated with sepsis.     

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.     

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.     

Cyclic AMP-mediated activation of hepatic glycogenolysis by fructose

Isolated livers from fed and fasted rats were perfused for 30 min with recirculating blood-buffer medium containing no added substrate and then switched to a flow-through perfusion using the same medium for an additional 5, 10 and 30 min. Continous infusion of fructose for the final 5, 10 or 30 min resulted in activation of glycogen phosphorylase, an increase in the activity of protein kinase, elevated levels of tissue adenosine 3′,5′-monphosphate (cylic AMP), and no consistent effect on glycogen synthase. Infusion of glucose under the same conditions resulted in activation of glycogen synthase, inactivation of glycogen phosphorylase, no change in protein kinase, and no consistent change in tissue cyclic AMP. These results demonstrate that while glucose promotes hepatic glycogen synthesis, fructose promotes activation of the enzymatic cascade responsible for glycogen breakdown.     

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.     

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.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

Control of glycogen deposition

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.     

Mechanism of hepatic insulin resistance in non-alcoholic fatty liver disease

Short term high fat feeding in rats results specifically in hepatic fat accumulation and provides a model of non-alcoholic fatty liver disease in which to study the mechanism of hepatic insulin resistance. Short term fat feeding (FF) caused a approximately 3-fold increase in liver triglyceride and total fatty acyl-CoA content without any significant increase in visceral or skeletal muscle fat content. Suppression of endogenous glucose production (EGP) by insulin was diminished in the FF group, despite normal basal EGP and insulin-stimulated peripheral glucose disposal. Hepatic insulin resistance could be attributed to impaired insulin-stimulated IRS-1 and IRS-2 tyrosine phosphorylation. These changes were associated with activation of PKC-epsilon and JNK1. Ultimately, hepatic fat accumulation decreased insulin activation of glycogen synthase and increased gluconeogenesis. Treatment of the FF group with low dose 2,4-dinitrophenol to increase energy expenditure abrogated the development of fatty liver, hepatic insulin resistance, activation of PKC-epsilon and JNK1, and defects in insulin signaling. In conclusion, these data support the hypothesis hepatic steatosis leads to hepatic insulin resistance by stimulating gluconeogenesis and activating PKC-epsilon and JNK1, which may interfere with tyrosine phosphorylation of IRS-1 and IRS-2 and impair the ability of insulin to activate glycogen synthase.