Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.     

Reversal of diet-induced glucose intolerance by hepatic expression of a variant glycogen-targeting subunit of protein phosphatase-1.

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. Expression of one family member, PTG, in the liver of normal rats improves glucose tolerance without affecting other plasma variables but leaves animals unable to reduce hepatic glycogen stores in response to fasting. In the current study, we have tested whether expression of other targeting subunit isoforms, such as the liver isoform G(L), the muscle isoform G(M)/R(Gl), or a truncated version of G(M)/R(Gl) termed G(M)DeltaC in liver ameliorates glucose intolerance in rats fed on a high fat diet (HF). HF animals overexpressing G(M)DeltaC, but not G(L) or G(M)/R(Gl), exhibited a decline in blood glucose of 35-44 mg/dl relative to control HF animals during an oral glucose tolerance test (OGTT) such that levels were indistinguishable from those of normal rats fed on standard chow at all but one time point. Hepatic glycogen levels were 2.1-2.4-fold greater in G(L)- and G(M)DeltaC-overexpressing HF rats compared with control HF animals following OGTT. In a second set of studies on fed and 20-h fasted HF animals, G(M)DeltaC-overexpressing rats lowered their liver glycogen levels by 57% (from 402 +/- 54 to 173 +/- 27 microg of glycogen/mg of protein) in the fasted versus fed states compared with only 44% in G(L)-overexpressing animals (from 740 +/- 35 to 413 +/- 141 microg of glycogen/mg of protein). Since the OGTT studies were performed on 20-h fasted rats, this meant that G(M)DeltaC-overexpressing rats synthesized much more glycogen than G(L)-overexpressing HF rats during the OGTT (419 versus 117 microg of glycogen/mg of protein, respectively), helping to explain why G(M)DeltaC preferentially enhanced glucose clearance. We conclude that G(M)DeltaC has a unique combination of glycogenic potency and responsiveness to glycogenolytic signals that allows it to be used to lower blood glucose levels in diabetes.     

Expression and glycogenic effect of glycogen-targeting protein phosphatase 1 regulatory subunit GL in cultured human muscle

Glycogen-targeting PP1 (protein phosphatase 1) subunit G(L) (coded for by the PPP1R3B gene) is expressed in human, but not rodent, skeletal muscle. Its effects on muscle glycogen metabolism are unknown. We show that G(L) mRNA levels in primary cultured human myotubes are similar to those in freshly excised muscle, unlike subunits G(M) (gene PPP1R3A) or PTG (protein targeting to glycogen; gene PPP1R3C), which decrease strikingly. In cultured myotubes, expression of the genes coding for G(L), G(M) and PTG is not regulated by glucose or insulin. Overexpression of G(L) activates myotube GS (glycogen synthase), glycogenesis in glucose-replete and -depleted cells and glycogen accumulation. Compared with overexpressed G(M), G(L) has a more potent activating effect on glycogenesis, while marked enhancement of their combined action is only observed in glucose-replete cells. G(L) does not affect GP (glycogen phosphorylase) activity, while co-overexpression with muscle GP impairs G(L) activation of GS in glucose-replete cells. G(L) enhances long-term glycogenesis additively to glucose depletion and insulin, although G(L) does not change the phosphorylation of GSK3 (GS kinase 3) on Ser9 or its upstream regulator kinase Akt/protein kinase B on Ser473, nor its response to insulin. In conclusion, in cultured human myotubes, the G(L) gene is expressed as in muscle tissue and is unresponsive to glucose or insulin, as are G(M) and PTG genes. G(L) activates GS regardless of glucose, does not regulate GP and stimulates glycogenesis in combination with insulin and glucose depletion.     

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.     

Identification of binding sites on protein targeting to glycogen for enzymes of glycogen metabolism

The activation of protein phosphastase-1 (PP1) by insulin plays a critical role in the regulation of glycogen metabolism. PTG is a PP1 glycogen-targeting protein, which also binds the PP1 substrates glycogen synthase, glycogen phosphorylase, and phosphorylase kinase (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Through a combination of deletion analysis and site-directed mutagenesis, the regions on PTG responsible for binding PP1 and its substrates have been delineated. Mutagenesis of Val-62 and Phe-64 in the highly conserved (K/R)VXF PP1-binding motif to alanine was sufficient to ablate PP1 binding to PTG. Phosphorylase kinase, glycogen synthase, and phosphorylase binding all mapped to the same C-terminal region of PTG. Mutagenesis of Asp-225 and Glu-228 to alanine completely blocked the interaction between PTG and these three enzymes, without affecting PP1 binding. Disruption of either PP1 or substrate binding to PTG blocked the stimulation of PP1 activity in vitro against phosphorylase, indicating that both binding sites may be important in PTG action. Transient overexpression of wild-type PTG in Chinese hamster ovary cells overexpressing the insulin receptor caused a 50-fold increase in glycogen levels. Expression of PTG mutants that do not bind PP1 had no effect on glycogen accumulation, indicating that PP1 targeting is essential for PTG function. Likewise, expression of the PTG mutants that do not bind PP1 substrates did not increase glycogen levels, indicating that PP1 targeting glycogen is not sufficient for the metabolic effects of PTG. These results cumulatively demonstrate that PTG serves as a molecular scaffold, allowing PP1 to recognize its substrates at the glycogen particle.     

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.     

A novel glycogen-targeting subunit of protein phosphatase 1 that is regulated by insulin and shows differential tissue distribution in humans and rodents

Stimulation of glycogen-targeted protein phosphatase 1 (PP1) activity by insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis. The glycogen-targeting subunits of PP1, GL and R5/PTG, are downregulated in the livers of diabetic rodents and restored by insulin treatment. We show here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver. The phosphatase activity associated with R3E is slightly higher than that associated with R5/PTG and it is downregulated in streptozotocin-induced diabetes by 60-70% and restored by insulin treatment. Surprisingly, although mRNA for R3E is most highly expressed in rat liver and heart muscle, with only low levels in skeletal muscle, R3E mRNA is most abundant in human skeletal muscle and heart tissues with barely detectable levels in human liver. This species-specific difference in R3E mRNA expression has similarities to the high level of expression of GL mRNA in human but not rodent skeletal muscle. The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents and humans.     

Overexpression of protein targeting to glycogen in cultured human muscle cells stimulates glycogen synthesis independent of glycogen and glucose 6-phosphate levels

There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.     

Metabolic impact of overexpression of liver glycogen synthase with serine-to-alanine substitutions in rat primary hepatocytes

To investigate the effect of elevation of liver glycogen synthase (GYS2) activity on glucose and glycogen metabolism, we performed adenoviral overexpression of the mutant GYS2 with six serine-to-alanine substitutions in rat primary hepatocytes. Cell-free assays demonstrated that the serine-to-alanine substitutions caused constitutive activity and electrophoretic mobility shift. In rat primary hepatocytes, overexpression of the mutant GYS2 significantly reduced glucose production by 40% and dramatically induced glycogen synthesis via the indirect pathway rather than the direct pathway. Thus, we conclude that elevation of glycogen synthase activity has an inhibitory effect on glucose production in hepatocytes by shunting gluconeogenic precursors into glycogen. In addition, although intracellular compartmentation of glucose-6-phosphate (G6P) remains unclear in hepatocytes, our results imply that there are at least two G6P pools via gluconeogenesis and due to glucose phosphorylation, and that G6P via gluconeogenesis is preferentially used for glycogen synthesis in hepatocytes.     

Transgenic overexpression of protein targeting to glycogen markedly increases adipocytic glycogen storage in mice

Adipocytes express the rate-limiting enzymes required for glycogen metabolism and increase glycogen synthesis in response to insulin. However, the physiological function of adipocytic glycogen in vivo is unclear, due in part to the low absolute levels and the apparent biophysical constraints of adipocyte morphology on glycogen accumulation. To further study the regulation of glycogen metabolism in adipose tissue, transgenic mice were generated that overexpressed the protein phosphatase-1 (PP1) glycogen-targeting subunit (PTG) driven by the adipocyte fatty acid binding protein (aP2) promoter. Exogenous PTG was detected in gonadal, perirenal, and brown fat depots, but it was not detected in any other tissue examined. PTG overexpression resulted in a modest redistribution of PP1 to glycogen particles, corresponding to a threefold increase in the glycogen synthase activity ratio. Glycogen synthase protein levels were also increased twofold, resulting in a combined greater than sixfold enhancement of basal glycogen synthase specific activity. Adipocytic glycogen levels were increased 200- to 400-fold in transgenic animals, and this increase was maintained to 1 yr of age. In contrast, lipid metabolism in transgenic adipose tissue was not significantly altered, as assessed by lipogenic rates, weight gain on normal or high-fat diets, or circulating free fatty acid levels after a fast. However, circulating and adipocytic leptin levels were doubled in transgenic animals, whereas adiponectin expression was unchanged. Cumulatively, these data indicate that murine adipocytes are capable of storing far higher levels of glycogen than previously reported. Furthermore, these results were obtained by overexpression of an endogenous adipocytic protein, suggesting that mechanisms may exist in vivo to maintain adipocytic glycogen storage at a physiological set point.     

Insulin-stimulated phosphorylation of the protein phosphatase-1 striated muscle glycogen-targeting subunit and activation of glycogen synthase

Protein phosphatase-1 (PP-1) in heart and skeletal muscle binds to a glycogen-targeting subunit (G(M)) in the sarcoplasmic reticulum. Phosphorylation of G(M) has been postulated to govern activity of PP1 in response to adrenaline and insulin. In this study, we used biochemical assays and G(M) expression in living cells to examine the effects of insulin on the phosphorylation of G(M), and the binding of PP-1 to G(M). We also assayed glycogen synthase activation in cells expressing wild type G(M) and G(M) mutated at the phosphorylation sites. In biochemical assays kinase(s) prepared from insulin-stimulated Chinese hamster ovary (CHO-IR) cells and C2C12 myotubes phosphorylated a glutathione S-transferase (GST) fusion protein, GST-G(M)(1-240), at both site 1 (Ser(48)) and site 2 (Ser(67)). Phosphorylation of both sites was dependent on activation of the mitogen-activated protein kinase pathway, involving in particular ribosomal protein S6 kinase. Full-length G(M) was expressed in CHO-IR cells and metabolic (32)P labeling at sites 1 and 2 was increased by insulin treatment. The G(M) expressed in CHO-IR cells or in C2C12 myotubes co-immunoprecipitated endogenous PP-1, and association was transiently lost following treatment of the cells with insulin. In contrast PP-1 binding to G(M)(S67T), a version of G(M) not phosphorylated at site 2, was unaffected by insulin treatment. Expression of G(M) increased basal activity of endogenous glycogen synthase in CHO-IR cells. Insulin stimulated glycogen synthase activity the same extent in cells expressing wild type G(M) or G(M) mutated to eliminate phosphorylation site 1 and/or site 2. Phosphorylation of G(M) is stimulated by insulin, but this phosphorylation is not involved in insulin control of glycogen metabolism. We speculate that other functions of G(M) at the sarcoplasmic reticulum membrane might be affected by insulin.     

Hepatic glycogen synthesis in the absence of glucokinase: the case of embryonic liver

Glucokinase (GK, hexokinase type IV) is required for the accumulation of glycogen in adult liver and hepatoma cells. Paradoxically, mammalian embryonic livers store glycogen successfully in the absence of GK. Here we address how mammalian embryonic livers, but not adult livers or hepatoma cells, manage to accumulate glycogen in the absence of this enzyme. Hexokinase type I or II (HKI, HKII) substitutes for GK in hepatomas and in embryonic livers. We engineered FTO2B cells, a hepatoma cell line in which GK is not expressed, to unveil the modifications required to allow them to accumulate glycogen. In the light of these results, we then examined glycogen metabolism in embryonic liver. Glycogen accumulation in FTO2B cells can be triggered through elevated expression of HKI or either of the protein phosphatase 1 regulatory subunits, namely PTG or G L. Between these two strategies to activate glycogen deposition in the absence of GK, embryonic livers choose to express massive levels of HKI and HKII. We conclude that although the GK/liver glycogen synthase tandem is ideally suited to store glycogen in liver when blood glucose is high, the substitution of HKI for GK in embryonic livers allows the HKI/liver glycogen synthase tandem to make glycogen independently of the glucose concentration in blood, although it requires huge levels of HK. Moreover, the physiological consequence of the HK isoform switch is that the embryonic liver safeguards its glycogen deposits, required as the main source of energy at birth, from maternal starvation.     

Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes

The liver is a major insulin‐responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin‐induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin‐induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin‐induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML‐12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin‐induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin‐induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin‐induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin‐induced glucose uptake and glycogenesis in hepatocytes. J. Cell. Biochem. 113: 2064–2076, 2012. © 2012 Wiley Periodicals, Inc.     

The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction, in vivo treadmill running and in situ electrical stimulation. Both procedures resulted in a 2-fold increase in the GS -/+ glucose-6-P activity ratio in WT mice, but this response was completely absent in the KO mice. The KO mice, which also have a reduced GS activity associated with significantly reduced basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R(GL) OE mice are characterized by enhanced GS activity ratio and an approximately 3-4-fold increase in glycogen content in skeletal muscle. These animals were able to tolerate exercise normally. Stimulation of GS and glucose uptake following muscle contraction was not significantly different as compared with WT littermates. These results indicate that although PP1G/R(GL) is not necessary for activation of GS by insulin, it is essential for regulation of glycogen metabolism under basal conditions and in response to contractile activity, and may explain the reduced muscle glycogen content in the R(GL) KO mice, despite the normal insulin activation of GS.     

Insulin action on protein phosphatase-1 activation is enhanced by the antidiabetic agent pioglitazone in cultured diabetic hepatocytes

Effect of the antidiabetic agent pioglitazone on the insulin-mediated activation of protein phosphatase-1 was examined in diabetic hepatocytes. Streptozotocin-induced diabetes in Sprague Dawley rats caused a significant decrease in the activation of glycogen synthase in hepatocytes isolated from these animals. There was an inverse correlation between the in vivo hyperglycemic condition and the in vitro activation of glycogen synthase in liver cells (r = 0.93, p > 0.001). Long term incubation of diabetic hepatocytes with insulin and dexamethasone caused significant (p > 0.001) improvement in the activation of glycogen synthase activation. When incubated along with hormones, pioglitazone enhanced their action (p > 0.05-0.01). Diabetic hepatocytes were also characterized by 50% decrease in the activity of protein phosphatase-1, the enzyme which dephosphorylates and activates glycogen synthase. Pioglitazone potentiated the acute stimulatory effect of insulin on protein phosphatase-1 in normal hepatocytes but not in diabetic hepatocytes. Long term incubation of diabetic hepatocytes with insulin ameliorated the decrease in the protein phosphatase -1 activity in these cells. This stimulatory long-term effect of insulin was significantly (p > 0.05) enhanced by the antidiabetic agent pioglitazone.     

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.     

Glucokinase overexpression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats.

Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats. Glucokinase overexpression using adenovirus restored glucose metabolism in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose 6-phosphate levels increased. Moreover, glycogen deposition was greatly enhanced due to the activation of glycogen synthase. Pyruvate kinase was also activated, and fructose-2,6-bisphosphate concentration and lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but glucokinase overexpression improved their glucose utilization and storage.     

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.     

Muscle glycogen synthase isoform is responsible for testicular glycogen synthesis: Glycogen overproduction induces apoptosis in male germ cells

Glycogen is the main source of glucose for many biological events. However, this molecule may have other functions, including those that have deleterious effects on cells. The rate‐limiting enzyme in glycogen synthesis is glycogen synthase (GS). It is encoded by two genes, GYS1, expressed in muscle (muscle glycogen synthase, MGS) and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase, LGS). Expression of GS and its activity have been widely studied in many tissues. To date, it is not clear which GS isoform is responsible for glycogen synthesis and the role of glycogen in testis. Using RT‐PCR, Western blot and immunofluorescence, we have detected expression of MGS but not LGS in mice testis during development. We have also evaluated GS activity and glycogen storage at different days after birth and we show that both GS activity and levels of glycogen are higher during the first days of development. Using RT‐PCR, we have also shown that malin and laforin are expressed in testis, key enzymes for regulation of GS activity. These proteins form an active complex that regulates MGS by poly‐ubiquitination in both Sertoli cell and male germ cell lines. In addition, PTG overexpression in male germ cell line triggered apoptosis by caspase3 activation, proposing a proapoptotic role of glycogen in testis. These findings suggest that GS activity and glycogen synthesis in testis could be regulated and a disruption of this process may be responsible for the apoptosis and degeneration of seminiferous tubules and possible cause of infertility. J. Cell. Biochem. 114: 1653–1664, 2013. © 2013 Wiley Periodicals, Inc.     

Ethanol-induced hepatic steatosis is modulated by glycogen level in the liver

Alcoholic liver disease (ALD) is a major health problem worldwide and hepatic steatosis is an early response to alcohol consumption. Fat and glycogen are two major forms of energy storage in the liver; however, whether glycogen metabolism in the liver impacts alcohol-induced steatosis has been elusive. In this study, we used a mouse model with overexpression of PPP1R3G in the liver to dissect the potential role of glycogen on alcohol-induced fatty liver formation. PPP1R3G is a regulatory subunit of protein phosphatase 1 and stimulates glycogenesis in the liver. Chronic and binge ethanol (EtOH) feeding reduced glycogen level in the mouse liver and such inhibitory effect of EtOH was reversed by PPP1R3G overexpression. In addition, PPP1R3G overexpression abrogated EtOH-induced elevation of serum levels of alanine aminotransferase and aspartate aminotransferase, increase in liver triglyceride concentration, and lipid deposition in the liver. EtOH-stimulated sterol regulatory element-binding protein (SREBP)-1c, a master regulator of lipogenesis, was also reduced by PPP1R3G overexpression in vivo. In AML-12 mouse hepatocytes, PPP1R3G overexpression could relieve EtOH-induced lipid accumulation and SREBP-1c stimulation. In conclusion, our data indicate that glycogen metabolism is closely linked to EtOH-induced liver injury and fatty liver formation.