An improved procedure for the assay of glycogen synthase and phosphorylase in rat liver homogenates

Golden, Sybil; Wals, P.A.; Katz, Joseph

doi:10.1016/0003-2697(77)90257-3

Cited by 81 publications

(49 citation statements)

References 10 publications

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“…GK and G-6-Pase activities were measured in the supernatant and sedimentary fractions, respectively, after centrifugation. Glycogen phosphorylase activities in the liver were measured using the method described by Golden et al (31).…”

Section: Methodsmentioning

confidence: 99%

Defect in glucokinase translocation in Zucker diabetic fatty rats

Fujimoto¹,

Donahue²,

Shiota³

2004

American Journal of Physiology-Endocrinology and Metabolism

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Hepatic glucose fluxes and intracellular movement of glucokinase (GK) in response to increased plasma glucose and insulin were examined in 10-wk-old, 6-h-fasted, conscious Zucker diabetic fatty (ZDF) rats and lean littermates. Under basal conditions, plasma glucose (mmol/l) and glucose turnover rate (GTR; mol ⅐ kg Ϫ1 ⅐ min Ϫ1 ) were slightly higher in ZDF (8.4 Ϯ 0.3 and 53 Ϯ 7, respectively) than in lean rats (6.2 Ϯ 0.2 and 45 Ϯ 4, respectively), whereas plasma insulin (pmol/l) was higher in ZDF (1,800 Ϯ 350) than in lean rats (150 Ϯ 14). ) were higher in ZDF (35 Ϯ 5, 87 Ϯ 16, and 33 Ϯ 10, respectively) compared with lean rats (18 Ϯ 3, 56 Ϯ 6, and 11 Ϯ 2, respectively). [3 H]glucose incorporation into glycogen (mol glucose/g liver) was similar in lean (1.0 Ϯ 0.7) and ZDF (1.6 Ϯ 0.8) rats. GK was predominantly located in the nucleus in both rats. With elevated plasma glucose and insulin, GTR (mol ⅐ kg3 H]G (%), and [ 3 H]glucose incorporation into glycogen (mol glucose/g liver) were markedly higher in lean (191 Ϯ 22, 62 Ϯ 3, and 5.0 Ϯ 1.4, respectively) but similar in ZDF rats (100 Ϯ 6, 37 Ϯ 3, and 1.4 Ϯ 0.4, respectively) compared with basal conditions. GK translocation from the nucleus to the cytoplasm occurred in lean but not in ZDF rats. The unresponsiveness of hepatic glucose flux to the rise in plasma glucose and insulin seen in prediabetic ZDF rats was associated with impaired GK translocation.liver; glucose flux; insulin; hyperglycemia PATIENTS WITH TYPE 2 DIABETES exhibit preprandial hyperglycemia and excessive postprandial hyperglycemia. The preprandial hyperglycemia is due to impaired suppression of endogenous glucose production in response to increased plasma glucose and/or insulin as well as impaired glucose uptake in peripheral tissues (19,41,42). In addition to these impairments, a defect of splanchnic glucose uptake has been reported to contribute to the occurrence of excessive postprandial hyperglycemia (9, 10, 24). Changes in plasma glucose and insulin are major factors regulating net hepatic glucose flux (8, 52). The flux is the balance between the rate of glucose phosphorylation catalyzed by glucokinase (GK) and the rate of dephosphorylation of glucose 6-phosphate (G-6-P) catalyzed by glucose-6-phosphatase (G-6-Pase). In studies using normal rats, glucose-induced suppression of net hepatic glucose production (HGP) was associated with increased glucose phosphorylation (53), and intact GK activity was required for the normal suppression of HGP by hyperglycemia (7). Basu et al. (9,10) showed that lower net splanchnic glucose uptake during a hyperglycemic hyperinsulinemic clamp in type 2 diabetic subjects was associated with a proportionate decrease in both the flux through the uridine 5Ј-diphosphate (UDP)-glucose pool and the percentage of contribution of extracellular glucose to glycogen synthesis by the direct pathway (glucose 3 G-6-P 3 G-1-P 3 UDP-glucose 3 glycogen) compared with nondiabetic subjects. Mevorach et al. (42) reported a lack of increasing glucose cycling (GC) in the presence of u...

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Section: Methodsmentioning

confidence: 99%

Defect in glucokinase translocation in Zucker diabetic fatty rats

Fujimoto¹,

Donahue²,

Shiota³

2004

American Journal of Physiology-Endocrinology and Metabolism

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“…GK activity was assessed as described by Barzilai and Rossetti (2), and glycogen synthase (GS) and phosphorylase activities were determined by the method of Golden et al (21).…”

Section: Analysesmentioning

confidence: 99%

Chronic overeating impairs hepatic glucose uptake and disposition

Coate

Kraft

Shiota

et al. 2015

American Journal of Physiology-Endocrinology and Metabolism

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Dogs consuming a hypercaloric high-fat and -fructose diet (52 and 17% of total energy, respectively) or a diet high in either fructose or fat for 4 wk exhibited blunted net hepatic glucose uptake (NHGU) and glycogen deposition in response to hyperinsulinemia, hyperglycemia, and portal glucose delivery. The effect of a hypercaloric diet containing neither fructose nor excessive fat has not been examined. Dogs with an initial weight of Ϸ25 kg consumed a chow and meat diet (31% protein, 44% carbohydrate, and 26% fat) in weight-maintaining (CTR; n ϭ 6) or excessive (Hkcal; n ϭ 7) amounts for 4 wk (cumulative weight gain 0.0 Ϯ 0.3 and 1.5 Ϯ 0.5 kg, respectively, P Ͻ 0.05). They then underwent clamp studies with infusions of somatostatin and intraportal insulin (4ϫ basal) and glucagon (basal). The hepatic glucose load was doubled with peripheral (Pe) glucose infusion for 90 min (P1) and intraportal glucose at 4 mg·kg Ϫ1 ·min Ϫ1 plus Pe glucose for the final 90 min (P2). NHGU was blunted (P Ͻ 0.05) in Hkcal during both periods (mg·kg Ϫ1 ·min Ϫ1 ; P1: 1.7 Ϯ 0.2 vs. 0.3 Ϯ 0.4; P2: 3.6 Ϯ 0.3 vs. 2.3 Ϯ 0.4, CTR vs. Hkcal, respectively). Terminal hepatic glucokinase catalytic activity was reduced nearly 50% in Hkcal vs. CTR (P Ͻ 0.05), although glucokinase protein did not differ between groups. In Hkcal vs. CTR, liver glycogen was reduced 27% (P Ͻ 0.05), with a 91% increase in glycogen phosphorylase activity (P Ͻ 0.05) but no significant difference in glycogen synthase activity. Thus, Hkcal impaired NHGU and glycogen synthesis compared with CTR, indicating that excessive energy intake, even if the diet is balanced and nutritious, negatively impacts hepatic glucose metabolism. glycogen; glycogen phosphorylase; hypercaloric diet; liver THE LIVER IS AN ESPECIALLY IMPORTANT ORGAN in regard to glucose homeostasis because it is able to both export glucose for use by other tissues and extract glucose to reduce glycemia. Failure of the liver to make the transition from net output to uptake of glucose in the postprandial state is a major contributor to the development of impaired glucose tolerance and hyperglycemia (3,25). It is difficult to quantify hepatic glucose uptake (HGU) in the human because of the invasiveness of the hepatic portal vein catheterization required. Portal and hepatic vein catheterization is feasible in the dog, and the canine model provides a useful method for examining the liver's role in glucose disposal under postprandial conditions (31). Both high-fat and high-fructose diets and a diet with a combination of high fat and fructose are associated with marked impairment of HGU under hyperinsulinemic hyperglycemic (HIHG) clamp conditions in the dog (11,12). Under all of these circumstances, however, the modified diets were hypercaloric compared with that of the control group.The impact of excessive energy intake per se, i.e., excessive intake of foods normally viewed as nutritious, on the liver's role in glucose disposal remains unknown. The present studies were thus carried out to determine whether chronic con...

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“…The cell suspension was homogenized for 1 min on ice using a Tekmar Tissumizer (Tekmar Co., Cincinnati, OH). This homogenate was used within 1 h after preparation for both synthase and phosphorylase assays performed using the method of Golden et al (10). Synthase a was assayed in the presence of 15 mM Na2S04, and total synthase in the presence of 3 mM glucose-6-phosphate.…”

Section: Isolation Of Hepatocytesmentioning

confidence: 99%