Differences between glucose and insulin stimulation of glycogenesis in cultured fetal hepatocytes

The glycogenic effects of a glucose load (15 mM) and/or insulin (10 nM) were studied in 18-day-old fetal rat hepatocytes after 2 days of culture when medium contained 4 mM glucose. A glucose load led to a stimulation of [¹⁴C]glucose glycogen labelling (20 min) earlier than with insulin (30–40 min); maximal stimulations were 3-fold after 1 h for the glucose load and 5-fold after 2–3 h for insulin. Simultaneous addition of the two agents produced synergic effects. When insulin was added 4 h after a glucose load (or vice versa), a second glycogenic response was elicited: a further addition of the same glycogenic agent was ineffective. The early glycogenic effects (up to 2 h) also occurred in the presence of 10 μM cycloheximide, with, however, some decrease of insulin stimulation. The contribution of medium glucose to the glycogen formed for 2 days (67% in the absence of glycogenic agent) was clearly enhanced by a glucose load and to a lesser degree by insulin after a 4-h exposure (83 and 71%, respectively). This was accompanied by a related modification of the participation of glucogenic precursors such as fructose and galactose. Thus, acute glycogenic response to glucose and insulin appeared both synergic and independent, and quite different in several aspects in cultured fetal hepatocytes.     

Metabolic response to anisoosmolarity of rat skeletal muscle in vitro.

Isolated rat hepatocytes exhibit an insulin-like anabolic response to hypoosmotic incubation and a glucagon-like catabolic response to hyperosmotic incubation. Recently, a distinct glycogenic response to hypoosmotic treatment was likewise reported for cultured rat myotubes. The present study examines the effects of anisoosmolar exposure on glucose metabolism in freshly isolated rat soleus muscle strips. Under the same experimental conditions as used for cultured myotubes, hypoosmolarity reduced net glycogen synthesis to 52%, while hyperosmolarity stimulated glycogen storage to 231% of isoosmolar control (nmol glucose incorporated into glycogen g(-1) x h(-1): hypoosmolar, 34+/-3; isoosmolar, 65+/-8; hyperosmolar, 150+/-11; p<0.01 each vs. isoosmolar). The responses of native skeletal muscle to anisoosmolarity are therefore in opposition to what has been described for hepatocytes and cultured myotubes. Further experiments on rat skeletal muscle revealed that the observed lack of a glycogenic response to hypoosmolarity persisted independent of medium composition, specifically with regard to prevailing glucose and K+ concentrations. In conclusion, hypoosmotic exposure inhibits glycogen synthesis in isolated rat soleus muscle, which clearly argues against the hypothesis that osmotic changes and cell swelling may be physiologically relevant stimulators of muscle glycogen synthesis.     

Improved hepatocyte culture system for studying the regulation of glycogen synthase and the effects of diabetes

When 3–4-week-old rats (young rats) are used as a source of hepatocytes, primary culture cells express the adult, differentiated, liver-specific isoform of glycogen synthase. Synthase enzyme protein levels are relatively stable over a 3 day culture period in young but not in adult (>150 g rat) hepatocyte cultures. Corresponding synthase enzyme activity and mRNA levels decrease over time in culture in adult but not in young hepatocyte cultures. Young rat hepatocytes also have the ability to proliferate in chemically defined medium in the absence of added mitogens. A diabetes-induced increase in total synthase activity has been demonstrated by our lab and others, using cultured hepatocytes, liver homogenates, and perfused livers. In the present study, utilizing synthase-specific antibody and primary cultures of cells from young normal and alloxan diabetic rats, we found that greater total synthase activity in the diabetic cells was associated with higher levels of enzyme protein. Immuneprecipitaion of ³⁵S methionine-labeled freshly plated cells demonstrates an increase in the rate of protein synthesis in diabetic as compared with normal cells. Synthase mRNA levels are correspondingly increased in the diabetic relative to normal cells. Chronic exposure of young, normal hepatocytes to increasing levels of glucose induces a dose-dependent increase in total synthase activity, total synthase protein, and synthase message levels. By comparison, cells from diabetic animals do not respond by any of these measures to increased glucose concentrations. We conclude that this defined primary culture system represents a useful model for investigating the regulation of hepatic glycogen synthase and the defects which occur as a result of diabetes. © 1996 Wiley-Liss, Inc.     

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.     

Role of cortisol on the glycogenolytic effect of glucagon and on the glycogenic response to insulin in fetal hepatocyte culture.

The effects of insulin and glucagon on glycogen metabolism were studied in cultured fetal hepatocytes transplanted from 15-day-old fetuses. The effects of these hormones were examined just after transplantation, when the cells contained only minute amounts of glycogen, and during the 3 to 4 day culture period, when the hepatocytes were exposed to 10 muM cortisol and actively accumulated glycogen. At all stages of the culture, glucagon addition (10 nM) was followed by a rapid depletion of labeled glycogen, previously synthesized during a pulse labeling with [14C]glucose: this effect was mimicked by N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) (0.3 to 1 nM). Such a glycogenolytic effect of glucagon was observed even 6 hours after transplantation, i.e. at a time when cortisol was not present. In addition, glucagon clearly induced cyclic adenosine 3':5'-monosphosphate (cyclic AMP) accumulation in cells grown for 18 hours in the absence of cortisol. With cells grown for 3 days in the presence of cortisol, glucagon-dependent glycogenolysis was also obtained when cortisol was removed from the medium 20 hours before hormone addition. Thus the presence of cortisol is not necessary either to maintain a response to glucagon or for the onset of the glycogenolytic effect of glucagon. Insulin addition (10 nM) stimulated [14C]glucose incorporation into glycogen at all stages of the culture when grown in the presence of cortisol; no glycogenic response to insulin was observed 6 hours after transplantation where cortisol was not previously introduced. In addition, if the hepatocytes were grown in the presence of insulin alone (i.e. in the absence of cortisol) no significant storage of glycogen occurred. Maximal storage (or labeling) of glycogen was observed when hepatocytes were grown in the presence of both cortisol and insulin. The presence of cortisol was therefore necessary for the expression of the glycogenic effect of insulin. These data show that marked difference exist between the onset of developmental responses towards glucagon and insulin. The glucagon-dependent regulatory pathway should be present very early in fetal development and should not depend on cortisol. On the contrary, the onset of the insulin-dependent regulatory pathway seems to be induced during culture, and it is likely that this is caused by cortisol.     

Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).     

The glycogenic action of protein targeting to glycogen in hepatocytes involves multiple mechanisms including phosphorylase inactivation and glycogen synthase translocation

Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counter-acted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.     

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.     

Effects of glucocorticoid and thyroid hormones on regulatory enzymes of fatty acid synthesis and glycogen metabolism in developing fetal rat lung

Although glucocorticoid and thyroid hormones are known to act synergistically to stimulate surfactant production, they have opposite effects on other parameters of fetal lung maturation. We recently reported that the developmental increases in de novo fatty acid synthesis and glycogen accumulation in fetal rat lung were accelerated by dexamethasone but prevented by triiodothyronine and that the dexamethasone-induced increases were diminished when the two hormones were administered together. We have now examined the effects of maternal administration of these hormones on activities of enzymes of lung fatty acid synthesis and glycogen metabolism in the rat. There was a developmental increase in fatty-acid synthase activity between 19 and 21 days gestation. This activity was increased by dexamethasone but decreased by triiodothyronine. When the two hormones were administered together the stimulatory effect of dexamethasone was decreased from 56% to 29%. The stimulatory effect on fatty-acid synthase was also observed in fetal lung explants cultured in the presence of dexamethasone. This shows that the effect of the hormone was directly on the fetal lung. Dexamethasone had no effect on liver fatty-acid synthase. There was a developmental decrease in acetyl-CoA carboxylase activity but it was not affected by the hormones. These data show that the developmental and hormone-induced changes in fetal lung de novo fatty acid synthesis are mediated by fatty-acid synthase. Although there were developmental changes in fetal lung 6-phosphofructokinase, glycogen synthase and glycogen phosphorylase activities, these enzymes were not affected by the hormones.     

Glycogen metabolism in rat liver during transition form the fed to fasted states

Hepatic glycogen metabolism was studied in rats during the period of transition from the fed to fasted states. Glycogenic activity was measured in vivo based on the incorporation of [¹⁴C]glucose into liver glycogen. Its changes were almost parallel to the changes in glucogen synthase activity. Progressive accumulation of liver glycogen that occurred in the fed state was associated with a proportional increase in glycogenic activity. Within 4 h after the cessation of food intake, glycogenic activity showd a precipitous fall from the peak to its nadir without significant changes in glycogen content. Meanwhile, the glucose concentration in the portal vein decreased. Upon further development of fasting, glycogenic activity displayed a progressive regain, reciprocally as glycogen contents gradually decreased. The precipitous fall of glycogenic activity during the transition from the fed to fasted states was associated with a transient increase in plasma glucagon, and was partly overcome by the injection of anti-glucagon serum. It is concluded that the fall of portal venous concentration of glucose and secretion of glucagon act as a signal to initiate liver glycogen metabolism characteristics of the fasted or postabsorptive state.     

Oxytocin inactivates and phosphorylates rat hepatocyte glycogen synthase.

Incubation of isolated rat hepatocytes with oxytocin produces a time- and dose-dependent inactivation of glycogen synthase. Such inactivation is associated with an increase in the phosphorylation state of the 88 kDa subunit of the enzyme, as observed after electrophoretic analysis of the 32P-labelled enzyme isolated by immunoprecipitation from cells incubated with [32P]phosphate. CNBr cleavage of the immunoprecipitated glycogen synthase showed that multiple sites were phosphorylated after exposure of the cells to the hormone. The effect of oxytocin on hepatocyte glycogen synthase activity was not observed in the absence of extracellular Ca2+. Inactivation of glycogen synthase by oxytocin was partially abolished in the presence of insulin. These results indicate that the effects of oxytocin on glycogen synthase from rat hepatocytes are similar to those observed for other Ca2+-mediated glycogenolytic hormones, such as vasopressin.     

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.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Effects of mild heat shock on glycogenesis and its regulation by insulin in cultured fetal hepatocytes

The effects of a mild heat shock were investigated using cultured 15-day-old fetal rat hepatocytes in which an acute glucocorticoid-dependent glycogenic response to insulin was present. After exposure from 15 min to 2 h at 42.5°C, cell surface [¹²⁵I]insulin binding progressively decreased down to 60% of the value shown in cells kept at 37°C, due to a decrease in the apparent number of insulin binding sites with little change in insulin receptor affinity. In parallel cultures, protein labeling with [³⁵S]methionine exhibited stimulated synthesis of specific proteins, in particular, 73-kDa Hsc (heat shock cognate) and 72-kDa Hsp (heat shock protein). When cells were returned to 37°C after 2 h at 42.5°C, cell surface insulin binding showed a two-third restoration within 3 h (insulin receptor half-life = 13 h), with similar concomitant return of Hsps72,73 synthesis to preinduction levels. The rate of [¹⁴C]glucose incorporation into glycogen measured at 37°C after 1- to 2-h heat treatment revealed a striking yet transient increase in basal glycogenesis (up to 5-fold). At the same time, the glycogenesis stimulation by insulin was reduced (from 3.2 to 1.4—fold), whereas that induced by a glucose load was maintained. Induction of thermotolerance after a first heating was obtained for the heat shock-dependent events except for the enhanced basal glycogenesis. In insulin-unresponsive cells grown in the absence of glucocorticoids, heat shock decreased the glycogenic capacity without modifying the glucose load stimulation, supporting the hypothesis that insulin and thermal stimulation of glycogenesis share at least part of the same pathway. Inverse variations were observed between Hsps72,73 synthesis and both cell surface insulin receptor level and insulin glycogenic response in fetal hepatocytes experiencing heat stress. © 1995 Wiley-Liss, Inc.     

Distinctive regulatory and metabolic properties of glycogen-targeting subunits of protein phosphatase-1 (PTG, GL, GM/RGl) expressed in hepatocytes

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.     

Increased sensitivity of glycogen synthesis to phosphorylase-a and impaired expression of the glycogen-targeting protein R6 in hepatocytes from insulin-resistant Zucker fa/fa rats

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.     

Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures.

Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action.     

Effects of lithium ions on glycogen synthase and phosphorylase in rat hepatocytes

Incubation of hepatocytes from fasted rats with LiCl provoked a concentration- and time-dependent activation of glycogen synthase. This effect was observed in the absence of glucose in the incubation medium. No changes in the intracellular concentrations of ATP or glucose-6-phosphate were detected. Lithium was also able to activate glycogen synthase in the absence of extracellular calcium. If hepatocytes were incubated with lithium and insulin, an additive effect of both agents on glycogen synthase activity was observed. LiCl was also effective in activating the enzyme in hepatocytes obtained from fed rats. When hepatocytes were incubated with [33P]phosphate and then treated with LiCl, a decrease in the amount of [32P]phosphate incorporated in the enzyme was observed. This dephosphorylation affected two CNBr fragments of the enzyme (CB-2 and CB-1), suggesting that several phosphorylation sites were involved. Lithium was also able to activate glycogen phosphorylase from both fasted and fed rats. Phosphorylase activation was concentration- and time-dependent, either in the presence or absence of calcium in the incubation medium. These findings demonstrate that although lithium appears to mimic the effects of insulin on glycogen synthase activity, its mechanism of action must be different from that of the hormone.     

Hepatic glycogen synthase phosphatase and phosphorylase phosphatase activities are increased in obese (fa/fa) hyperinsulinemic Zucker rats: effects of glyburide administration

The chronically hyperinsulinemic Zucker fatty rat, with peripheral insulin resistance and glucose intolerance, represents a model of noninsulin dependent diabetes mellitus (NIDDM). These animals have elevated hepatic glycogen levels. Hepatic levels of synthase phosphatase and phosphorylase phosphatase, which are diminished in the IDDM rat, were markedly increased in the obese rats. Glyburide, a sulfonylurea used in treatment of NIDDM, resulted in reduced levels of glycemia and increased insulin levels in Zucker rats. Hepatic glycogen levels were increased, as was the activation of glycogen synthase, although there were no effects of drug administration on synthase phosphatase or phosphorylase phosphatase activities. G6P levels were increased by glyburide in lean rats but not in obese animals. These effects of glyburide on liver glycogen metabolism are accounted for via potentiation of the glycogenic effects of insulin.     

Effect of bemythyl on carbohydrate metabolism in cirrhotic rat liver

Effect of actoprotector bemitil (2-ethylthiobenzimidazole hydrobromide) on glycogen content and activities of glycogen synthase, glycogen phosphorylase, and glucose-6-phosphatase was studied in cirrhotically altered rat liver. The contents of glycogen and its fraction were determined a cytofluorimetrically (Kudryavtseva et al., 1974). In cirrhosis, the total glycogen content in hepatocytes increases by nearly 3 times, while the amount of a stable fraction of glycogen rises by 7.5 times. Glucose-6-phosphatase activity fell to the level of 25% compare to the norm. Activities of glycogen synthase and glycogen phosphorylase in the cirrhotic liver did not differ from the norm. In cirrhotically altered liver, bemitil produced a decrease in the total glycogen content due to a decrease in glycogen synthase activity in an increase in glucose-6-phosphatase and glycogen phosphorylase activities. The above results suggest a favorable effect of bemitil on cirrhotic liver.