Direct enzymatic procedure for the determination of liver glycogen

Roehrig, Karia L.; Allred, John B.

doi:10.1016/0003-2697(74)90210-3

Cited by 258 publications

(97 citation statements)

References 14 publications

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“…Glucose and Glycogen Determinations-Plasma glucose was analyzed using colorimetric glucose oxidase method (Sigma), and glycogen content was measured according to the methods of Roehrig and Allred (49).…”

Section: Methodsmentioning

confidence: 99%

Stearoyl-CoA Desaturase 1 Gene Expression Is Necessary for Fructose-mediated Induction of Lipogenic Gene Expression by Sterol Regulatory Element-binding Protein-1c-dependent and -independent Mechanisms

Miyazaki

Dobrzyń²,

Man³

et al. 2004

Journal of Biological Chemistry

261

215

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Stearoyl-CoA desaturase (SCD) synthesizes oleate necessary for the biosynthesis of triglycerides and other lipids. Mice with a targeted disruption of the SCD1 gene are deficient in tissue oleate and have reduced expression of the sterol regulatory element-binding protein (SREBP) and its target genes. The SREBP-1c isoform is a known mediator of insulin action on hepatic gene expression, but its transcriptional effects due to glucose or fructose are still unclear. We found that fructose compared with glucose is a stronger inducer of SREBP-1c and lipogenic gene expression, causing a dramatic increase in hepatic triglyceride levels. However, when fed to the SCD1؊/؊ mice, fructose failed to induce SREBP-1 or lipogenic genes and the triglyceride levels were not increased. Instead fructose feeding caused a decrease in hepatic glycogen and plasma glucose levels. The induction of SREBP-1 and lipogenic gene expression as well as the levels of liver triglycerides, glycogen, and plasma glucose was partially restored when the fructose diet was supplemented with very high levels of oleate (20% by weight) but not with palmitate, stearate, or linoleate. Fructose in a long term feeding induced the expression of SCD1 and that of other lipogenic genes in the liver of SREBP-1c؊/؊ mice, and a further increase in expression of these genes occurred when the fructose diet was supplemented with oleate. Our observations demonstrated that oleate produced by SCD is necessary for fructosemediated induction of lipogenic gene expression through SREBP-1c-dependent and -independent mechanisms and suggested that SCD1 gene expression is important in lipid and carbohydrate homeostasis.

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Section: Methodsmentioning

confidence: 99%

Stearoyl-CoA Desaturase 1 Gene Expression Is Necessary for Fructose-mediated Induction of Lipogenic Gene Expression by Sterol Regulatory Element-binding Protein-1c-dependent and -independent Mechanisms

Miyazaki

Dobrzyń²,

Man³

et al. 2004

Journal of Biological Chemistry

261

215

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“…Amino acids were estimated by the ninhydrin method following the procedure of Rosen (1957). Glycogen in liver homogenates was assayed as glucose following treatment with amyloglucosidase (Roehrig and Allred, 1974). Hepatic alanine aminotrasferase was assayed in liver cytosol by colorimetric measurement of phenylhydrazones derived proportionately from the reaction of 2,4-dinitrophenylhydrazine and the pyruvic acid formed in transaminase activity (Sigma Diagnostics,Kit No.…”

Section: Assay Proceduresmentioning

confidence: 99%

Divergence of endocrine and metabolic responses to stress in two rainbow trout lines selected for differing cortisol responsiveness to stress

Trenzado

Carrick

Pottinger

2003

General and Comparative Endocrinology

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Abstract. Rainbow trout (Oncorhynchus mykiss) of two lines selected for low (LR) and high (HR) cortisol stress-responsiveness were subjected to confinement for a period of 336 hours.Endocrine (plasma cortisol, hepatic cortisol binding) and metabolic (plasma glucose, lactate, amino acids; hepatic glycogen and alanine aminotransferase levels) indices of stress were measured at intervals in confined and unconfined fish of both lines. During confinement plasma cortisol concentration reached maximum values earlier in HR fish (2 hours) than in LR fish (6 hours) returning to control values within 336 hours in both lines. Paradoxically, although both HR and LR lines displayed a characteristic metabolic stress response, these changes were more pronounced in LR fish. Plasma glucose and lactate levels increased during confinement in both lines but to a significantly greater extent in LR fish. Confinement significantly elevated plasma amino acids to a greater extent in LR fish than in HR fish. Liver glycogen concentration was depleted most rapidly in LR fish but was significantly higher in confined fish of both lines than controls at the end of the experiment. No significant changes were observed in hepatic alanine aminotransferase activity during confinement. Confined fish of both lines displayed a decrease in hepatic cortisol receptor abundance within 24 h and this was more sustained in HR fish. The more pronounced disturbance of a broad range of indicators of stress in confined LR fish, compared to HR fish, throws doubt on the magnitude of the cortisol response being the primary driver of these differences.

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“…For glycogen determination cells were washed twice with glucose free PBS [9], scraped out of the culture flasks by a rubber policeman with i ml 0.6 mol/1 HC104, and homogenized on ice. Cellular glycogen was then assayed by a direct enzymatic procedure [11] and protein content was estimated according to Lowry et al [12].…”

Section: Analytical Proceduresmentioning

confidence: 99%

Effect of insulin on glycogen and protein synthesis in monolayer cultures of hepatocytes from normal and alloxan diabetic rats

et al. 1977

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Summary. The effects of insulin on net glycogen synthesis and amino acid incorporation into protein were studied in cultured hepatocytes from adult normal and alloxan diabetic rats. Insulin stimulated glycogen synthesis in monolayer cells throughout a four day culture period and enhanced leucine incorporation into protein more effectively in normal cells with high glycogen levels than in cultured diabetic cells. These differences correlate well with the observed cellular ultrastructures which were maintained much better in the presence of insulin. Restoration of the morphological changes of alloxan diabetic hepatocytes to normal liver cell structures can be observed at any time during the culture period by giving insulin continuously.Key words: Insulin, glycogen, amino acid, alloxan diabetes, cultured hepatocytes.It has been generally accepted that insulin plays an essential role in the regulation of carbohydrate and protein metabolism in liver tissue. The specific nature of its action at cellular level has been previously investigated in perfused liver [1,2] and in hepatocyte suspensions [3,4], but various attempts to obtain hepatocytes capable of efficient glycogen synthesis during a several day culture period have not been successful. Cultured hepatocytes have, however, become increasingly popular as a valuable system for investigating many liver biochemical runePresent address. Department of Biochemistry, University of Surrey, Gufldford, Surrey GU2 5XH, England 2 Pathologisches Instltut der Universitat Tfibmgen, D-7400 Ttibingen 9 tions in vitro [5,6]. We set out to develop a hepatocyte preparation capable of glycogen synthesis from normal gluconeogenic substrates. It was, therefore, of considerable interest to see if these cultured hepatocytes, especially from diabetic animals could synthesize cellular glycogen in the presence of insulin, and if amino acid incorporation into protein could be stimulated by the hormone even after several days in culture. In the present study we report the stimulatory effect of insulin on hepatic glycogen and protein synthesis and interrelate it with ultrastructural alterations in monolayer cultures of normal and diabetic rat hepatocytes. Materials and Methods Isolation and Cultivation of HepatocytesMale Sprague-Dawley rats weighing 100-150g were used throughout. All rats were kept on a standard laboratory diet (Altromin | and tap water ad libitum. The animals had easy access to food from a dish placed on the floor. This procedure was recently reported to enhance significantly liver glycogen content [7]. Hepatocytes were isolated by a modified method of collagenase/hyaluronidase (50 and 100 rag, respectively, per 100 ml Hank's solution) digestion of liver slices, which is especially suitable for harvesting a large number of viable cells from small liver pieces, e.g. biopsy samples [8]. The total cell yield and the viability index (percentage of viable cells) were calculated in duplicate with a haemocytometer; viability was based on the ability of cells to exclude the dye, tryp...

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Direct enzymatic procedure for the determination of liver glycogen

Cited by 258 publications

References 14 publications

Stearoyl-CoA Desaturase 1 Gene Expression Is Necessary for Fructose-mediated Induction of Lipogenic Gene Expression by Sterol Regulatory Element-binding Protein-1c-dependent and -independent Mechanisms

Stearoyl-CoA Desaturase 1 Gene Expression Is Necessary for Fructose-mediated Induction of Lipogenic Gene Expression by Sterol Regulatory Element-binding Protein-1c-dependent and -independent Mechanisms

Divergence of endocrine and metabolic responses to stress in two rainbow trout lines selected for differing cortisol responsiveness to stress

Effect of insulin on glycogen and protein synthesis in monolayer cultures of hepatocytes from normal and alloxan diabetic rats

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