Phosphorylase synthesis in diabetic hepatocytes and cardiomyocytes

Rulfs, Jill; Jaspers, Stephen R.; Garnache, Alice K.; Miller, Thomas B.

doi:10.1152/ajpendo.1989.257.1.e74

Cited by 6 publications

(7 citation statements)

References 4 publications

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“…The decrease in total hepatic phosphorylase activity in diabetic rats has also been observed by other investigators (9,15,18,34). Rulfs et al (18) have further reported that the decrease in total phosphorylase activity is related to the reduction in phosphorylase protein as a result of the decreased synthesis of this protein. In contrast to these results, Bahnak and Gold (9) have observed that the decrease in total phosphorylase protein is due to a more pronounced increase in the degradation rate of phosphorylase.…”

Section: Discussionsupporting

confidence: 70%

“…Previous studies in our laboratory (8,10,16) and others (9,18) have demonstrated that the activities of phosphorylase and glycogen synthase are altered in the livers of animal models of type 1 insulin-dependent diabetes mellitus. In streptozotocin-or alloxan-induced diabetic animals, the activities of both the active form and the total (i.e.…”

Section: Discussionmentioning

confidence: 90%

“…A similar decrease in total phosphorylase activity has been observed in cultured hepatocytes prepared from alloxan-induced diabetic rats (17). The decrease in the total glycogen phosphorylase activity in diabetic liver is due to a reduction in the enzymic protein level (10,18), and it is probably due to a decrease in its synthesis (18). The reason for the decreased synthesis of phosphorylase and whether insulin plays any role in this process or in regulation of the phosphorylase gene are not known.…”

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confidence: 99%

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The Effects of Streptozotocin-induced Diabetes and Insulin Supplementation on Expression of the Glycogen Phosphorylase Gene in Rat Liver

Rao

Pugazhenthi

Khandelwal

1995

Journal of Biological Chemistry

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We have previously observed that the chronic effects of streptozotocin-induced diabetes cause a decrease in the total hepatic glycogen phosphorylase activity with a corresponding reduction in the phosphorylase protein levels. These effects were normalized by insulin administration to diabetic rats. There was no change in the total glycogen synthase activity as a result of diabetes or insulin supplementation. These results are extended to examine the effects of diabetes and insulin administration to diabetic animals on the expression of phosphorylase and glycogen synthase enzymes. The expression (i.e. mRNA levels) of phosphorylase was down-regulated (45% of normal levels) in diabetic livers, and this was normalized by insulin supplementation to diabetic animals. Diabetes or insulin supplementation to diabetic rats showed no effect on the transcription rate of phosphorylase. As expected, diabetes (or insulin administration to diabetic animals) did not cause any alteration in the mRNA levels or in the transcription rate of hepatic glycogen synthase. The stability of phosphorylase mRNA was then examined using hepatocytes prepared from normal and diabetic rats. Diabetes caused a decrease in the half-life of phosphorylase mRNA from 14 h in normal hepatocytes to 6.5 h in diabetic hepatocytes. Insulin supplementation to the medium of diabetic hepatocytes increased the half-life of phosphorylase mRNA to a level comparable with normal values. This study indicates that the chronic effect of insulin on the activation of the total hepatic phosphorylase activity (and protein) is mediated through the stabilization of its mRNA levels.It is now well established that reversible phosphorylation is one of the major mechanisms by which hormones exert their effects on the regulation of metabolic pathways. Glycogen metabolism is one of the well studied metabolic processes regulated by such a mechanism involving phosphorylation-dephosphorylation of many proteins (or enzymes) (1-5). Glycogen phosphorylase and glycogen synthase are the two key regulatory enzymes that catalyze the rate-limiting steps of glycogen degradation and synthesis, respectively. Both of these enzymes exist in two interconvertible phosphorylated and dephosphorylated forms. The level of the active phosphorylated form of phosphorylase (a-form) is balanced by the opposing actions of phosphorylase phosphatase and phosphorylase kinase. Unlike phosphorylase, the dephosphorylated form of glycogen synthase (a-form) is active, and its levels are regulated by several protein kinases (including protein kinase A, protein kinase C, glycogen synthase kinase-3, casein kinase II, and phosphorylase kinase) and synthase phosphatase (protein phosphatase type 1).The liver plays a central role in the maintenance of blood glucose homeostasis. Glycogen metabolism in hepatic tissue is one of the major metabolic processes involved in such a homeostasis. It has been well established that the balance between levels of insulin and glucagon is vital for this regulation. Insulin is involved in b...

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Section: Discussionsupporting

confidence: 70%

Section: Discussionmentioning

confidence: 90%

mentioning

confidence: 99%

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The Effects of Streptozotocin-induced Diabetes and Insulin Supplementation on Expression of the Glycogen Phosphorylase Gene in Rat Liver

Rao

Pugazhenthi

Khandelwal

1995

Journal of Biological Chemistry

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“…In our results experimental diabetes caused a decrease of GPa. It was most obvious that lower enzyme activity was caused by a reduction in the enzyme protein level and was presumably due to a decrease in synthesis [48].…”

Section: Discussionmentioning

confidence: 99%

Prior heat stress induces moderation of diabetic alterations in glycogen metabolism of rats

Miova

Dinevska-Ќovkarovska

Djimrevska³

et al. 2013

Open Life Sciences

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Adaptation to one environmental stressor sometimes provides protection against additional, more intensive type of stress, a phenomenon called cross-tolerance. We aimed to estimate theprotection provided by acute heat stress (AHS) over carbohydrate disturbances in streptozotocin-diabetic rats. We investigated changes in activity of some hepatic glycolitic and gluconeogenic enzymes, and concentration of some substrates in control and diabetic animals exposed to AHS (41±0.5°C / 1 h), with 1 h and 24 h recovery at room temperature before sacrifice or induction of streptozotocin (STZ)-diabetes, respectively. AHS with 1 h-recovery before sacrifice resulted in intensive glycogenolysis, directed to endogenous glucose production and further utilization of glucose by peripheral tissues, while 24 h recovery resulted in a slight tendency towards normalization of metabolic disturbances caused by AHS. Experimental diabetes caused a significant decrease of substrates and glycolytic enzymes, but an increase of gluconeogenic enzymes. In diabetic animals previously exposed to AHS we measured a less intensive decrease of liver glycogen and glucose-6-phosphate concentration and hexokinase activity, as well as less intensive increase of liver glucose concentration, glucose-6-phosphatase and fructose-1,6-bisphosphatase activity compared to control diabetic animals that had been maintained at room temperature. Prior AHS provided some protection over diabetes-induced alterations in carbohydrate-related parameters (see graphical apstract), indicating a possible development of cross-tolerance phenomenon between the two stressors, AHS and STZ-diabetes.

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“…Total homogenate protein (100 pg) or 50 pg samples of the 30,OOOg supernatants were run on 8% SDS-polyacrylamide gels and transferred to Immobilon transfer membranes (Millipore, Bedford, MA) overnight at 30 V in 25 mM Tris, 192 mM glycine, pH 8.3, and 20% methanol or for 90 min at 300 mamps in a semidry blotting apparatus (Owl Scientific, Cambridge, MA) in 48 mM Tris, 39 mM glycine, and 10% methanol. Immunodetection was carried out as previously reported (Rulfs et al, 1989) using sheep anti-rat liver synthase as the primary antibody and donkey anti-sheep IgG conjugated to alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN) as secondary antibody. Blots were developed using alkaline phosphate chromogenic substrate (BCIPNBT; Pierce, Rockford, IL) for visualization.…”

Section: Immunoblottingmentioning

confidence: 99%

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

et al. 1996

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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. Immuneprecipitation 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.

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Phosphorylase synthesis in diabetic hepatocytes and cardiomyocytes

Cited by 6 publications

References 4 publications

The Effects of Streptozotocin-induced Diabetes and Insulin Supplementation on Expression of the Glycogen Phosphorylase Gene in Rat Liver

The Effects of Streptozotocin-induced Diabetes and Insulin Supplementation on Expression of the Glycogen Phosphorylase Gene in Rat Liver

Prior heat stress induces moderation of diabetic alterations in glycogen metabolism of rats

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

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