Isolation and characterization of liver glycogen synthase from diabetic rats

Bahnak, Bruce R.; Gold, Alvin H.

doi:10.1016/0003-9861(82)90575-6

Cited by 22 publications

(8 citation statements)

References 32 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.…”

Section: Discussionsupporting

confidence: 55%

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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: 55%

“…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: 99%

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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“…This was associated with an increased synthesis and increased turnover rate. They could not confirm the presence of an altered form of the enzyme [9].The purpose of the present study was to determine whether the increase in total synthase activity observed previously in diabetic rats was associated with an increase in enzyme protein mass and whether the reported increase in enzyme turnover was associated with an increase in mRNA abundance. Therefore, Diabetologia (1997) Summary Hepatic glycogen synthase activity is increased in diabetic animals.…”

mentioning

confidence: 72%

“…This was associated with an increased synthesis and increased turnover rate. They could not confirm the presence of an altered form of the enzyme [9].…”

mentioning

confidence: 86%

Effect of feeding, fasting, and diabetes on liver glycogen synthase activity, protein, and mRNA in rats

Gannon

Nuttall

1997

Diabetologia

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Our laboratory [1], as well as many others [2][3][4][5][6][7][8] , have reported an increase in total synthase activity in liver from diabetic rats. The increase in hepatic synthase activity could be due to an increased mass of the enzyme or to the presence of a more catalytically efficient form(s) of the enzyme. Akatsuka et al. [5] reported changes in the M r of synthase in liver from streptozotocin diabetic rats. They attributed this to an increase in the phosphorylation state of synthase, which in turn resulted in greater total synthase activity. In contrast, in alloxan diabetic rats, Bahnak and Gold [3] reported that the increase in hepatic synthase activity was due to an increased mass of the enzyme. This was associated with an increased synthesis and increased turnover rate. They could not confirm the presence of an altered form of the enzyme [9].The purpose of the present study was to determine whether the increase in total synthase activity observed previously in diabetic rats was associated with an increase in enzyme protein mass and whether the reported increase in enzyme turnover was associated with an increase in mRNA abundance. Therefore, Diabetologia (1997) Summary Hepatic glycogen synthase activity is increased in diabetic animals. However, the relationship between enzymic activity, enzyme protein mass, and mRNA abundance has not been well characterized. In the present study, these relationships were determined in 3-and 8-day diabetic, fed and fasted rats. The results were compared to data obtained in normal fed and fasted animals. In normal rats, total synthase specific activity and protein mass were similar in the fed and fasted state. However, in fed animals, the synthase mRNA abundance was increased 1.7-fold. In 3-day diabetic rats, total synthase specific activity was increased approximately 29 % compared to normal controls. It was unaffected by feeding and fasting and was associated with an approximate 15 % increase in enzyme mass. Synthase mRNA was increased 1.8 and 2.6-fold in fasted and fed animals, respectively. In 8-day diabetic rats, total synthase specific activity was increased more than 2-fold compared to controls. However, the enzyme protein mass was decreased by approximately 20 %. The mRNA abundance in 8-day diabetic fasted rats was only 30 % of controls, while in fed rats it was increased by 40 %. These data indicate that feeding and fasting have a major effect on synthase mRNA abundance which is independent of synthase activity, or protein mass, or both, in normal and diabetic animals. Total synthase specific activity increased with duration of diabetes. This was associated with only a modest change in protein mass. Thus, diabetes induces an increase in synthase catalytic efficacy. The specific activity of phosphorylase is decreased in diabetic rats. [Diabetologia (1997) 40: 758-763]

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“…Purified glycogen phosphorylase was isolated from rat liver as described by Roesler & Khandelval (1986) and Bahnak & Gold (1982). Briefly, rat liver samples were homogenized, and the crude homogenate was centrifugated at 8000 g for 10 min.…”

Section: Antigensmentioning

confidence: 99%

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase

Klein

Berg

1990

Clinical and Experimental Immunology

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SUMMARYAnti-M9 antibodies in sera from patients with primary biliary cirrhosis (PBC) were previously found to recognize two antigenic determinants at 98 and 59 kD, using a purified antigen fraction derived from rat liver mitochondria in the Western blot. Here we show that these antibodies are directed against an epitope of the enzyme glycogen phosphorylase. By Western blotting, a determinant at 98 kD was obtained testing anti-M9 positive sera against phosphorylase from skeletal muscle, and after plasmin treatment a degradation product appeared at 59 kD. Both determinants were identical to the M9-specific determinants 98 and 59 kD as shown by absorption studies. When these antibodies were eluted from the 98 and 59 kD determinants of the M9 antigen after immunoblotting, they again recognized the same epitopes on plasmin-treated phosphorylase. Furthermore, phosphorylase enzyme activity could be also demonstrated in the purified M9 fraction, and anti-M9-positive/anti-M2-negative but not anti-M9-negative/anti-M2-positive sera could be shown to stimulate phosphorylase activity. Testing sera from 1189 patients with different hepatic and non-hepatic disorders against M9 and phosphorylase from skeletal muscle by ELISA, 20% were positive with phosphorylase and only 2% with the M9 fraction. These data indicate that the commercially available phosphorylase from skeletal muscle cannot be recommended as M9 source. It may still contain non-PBC-specific epitopes which are probably recognized by naturally occurring antibodies directed against this highly conserved protein.

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Isolation and characterization of liver glycogen synthase from diabetic rats

Cited by 22 publications

References 32 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

Effect of feeding, fasting, and diabetes on liver glycogen synthase activity, protein, and mRNA in rats

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase

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