Determination of phosphoenolpyruvate carboxykinase activity with deoxyguanosine 5′-diphosphate as nucleotide substrate

Petrescu, Ioan; Bojan, O.; Saied, M.; Bârzu, Octavian; Schmidt, Felix; Kühnle, H.F.

doi:10.1016/0003-2697(79)90582-7

Cited by 106 publications

(48 citation statements)

References 9 publications

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“…For the Western blotting, proteins were extracted using ∼50 mg liver. Liver proteins were compartmentalized into three fractions, namely mitochondria plus nucleus, cytoplasm, and microsomes as previously reported (49,50). Detailed methods for experiments are provided in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease

Kumashiro

Erion

Zhang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

503

409

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Insulin resistance is associated with nonalcoholic fatty liver disease (NAFLD) and is a major factor in the pathogenesis of type 2 diabetes. The development of hepatic insulin resistance has been ascribed to multiple causes, including inflammation, endoplasmic reticulum (ER) stress, and accumulation of hepatocellular lipids in animal models of NAFLD. However, it is unknown whether these same cellular mechanisms link insulin resistance to hepatic steatosis in humans. To examine the cellular mechanisms that link hepatic steatosis to insulin resistance, we comprehensively assessed each of these pathways by using flash-frozen liver biopsies obtained from 37 obese, nondiabetic individuals and correlating key hepatic and plasma markers of inflammation, ER stress, and lipids with the homeostatic model assessment of insulin resistance index. We found that hepatic diacylglycerol (DAG) content in cytoplasmic lipid droplets was the best predictor of insulin resistance (R = 0.80, P < 0.001), and it was responsible for 64% of the variability in insulin sensitivity. Hepatic DAG content was also strongly correlated with activation of hepatic PKCε (R = 0.67, P < 0.001), which impairs insulin signaling. In contrast, there was no significant association between insulin resistance and other putative lipid metabolites or plasma or hepatic markers of inflammation. ER stress markers were only partly correlated with insulin resistance. In conclusion, these data show that hepatic DAG content in lipid droplets is the best predictor of insulin resistance in humans, and they support the hypothesis that NAFLD-associated hepatic insulin resistance is caused by an increase in hepatic DAG content, which results in activation of PKCε.

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Section: Methodsmentioning

confidence: 99%

Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease

Kumashiro

Erion

Zhang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

503

409

View full text Add to dashboard Cite

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“…Protein content of the preparations was determined by the method of Peterson (1977). PEPCK activity was assayed at 30 C by coupling the formation of oxalacetate with NADH oxidation in the presence of excess malate dehydrogenase (Boehringer Mannheim UK, Lewes, East Sussex, UK), as described by Petrescu et al (1979). The reaction mixture (1 ml final volume) contained 50 mM Hepes (pH 6·5), 50 mM sodium bicarbonate, 1 mM MnCl 2 , 0·25 mM NADH, 1 mM phosphoenolpyruvate, 1·5 U malate dehydrogenase and 10 µl of liver cytosolic preparation.…”

Section: Pepck Assaymentioning

confidence: 99%

Programming hyperglycaemia in the rat through prenatal exposure to glucocorticoids-fetal effect or maternal influence?

Nyirenda¹,

Welberg²,

Seckl³

2001

Journal of Endocrinology

141

127

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In a previous study, we showed that exposure of rats to dexamethasone (Dex) selectively in late pregnancy produces permanent induction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) expression and hyperglycaemia in the adult offspring. The mechanisms by which glucocorticoids cause this programming are unclear but may involve direct actions on the fetus/neonate, or glucocorticoids may act indirectly by affecting maternal postnatal nursing behaviour. Using a cross-fostering paradigm, the present data demonstrate that switching the offspring at birth from Dex-treated dams to control dams does not prevent induction of PEPCK or hyperglycaemia. Similarly, offspring born to control dams but reared by Dex-treated dams from birth maintain normal glycaemic control. During the neonatal period, injection of saline per se was sufficient to cause exaggeration in adult offspring responses to an oral glucose load, with no additional effect from Dex. However, postnatal treatment with either saline or Dex did not alter hepatic PEPCK activity. Prenatal Dex permanently raised basal plasma corticosterone levels, but under stress conditions there were no differences in circulating corticosterone levels. Likewise, Dex-exposed rats had similar plasma catecholamine concentrations to control animals. These findings show that glucocorticoids programme hyperglycaemia through mechanisms that operate on the fetus or directly on the neonate, rather than via effects that alter maternal postnatal behaviour during the suckling period. The hyperglycaemic response does not appear to result from abnormal sympathoadrenal activity or hypothalamic-pituitary-adrenal response during stress.

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“…The resulting supernatant (cytosol) was collected and stored at 70 C until PEPCK assay. Following protein determination, PEPCK activity was assayed at 30 C by coupling the formation of oxaloacetate with NADH oxidation in the presence of excess malate dehydrogenase (BoehringerMannheim UK, Lewes, East Sussex, UK), as described by Petrescu et al (1979). The reaction mixture (1 ml final volume) contained 50 mmol/l Hepes (pH 6·5), 50 mmol/l NaHCO 3 , 1 mmol/l MnCl 2 , 0·25 mmol/l NADH, 1 mmol/l phosphoenolpyruvate, 1·5 U malate dehydrogenase and 10 µl cytosolic liver preparation.…”

Section: Quantification Of Pepck Activitymentioning

confidence: 99%

Interactions between oestradiol and glucocorticoid regulatory effects on liver-specific glucocorticoid-inducible genes: possible evidence for a role of hepatic 11beta-hydroxysteroid dehydrogenase type 1

Jamieson¹,

Nyirenda²,

Walker³

et al. 1999

Journal of Endocrinology

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In vitro, 11 -hydroxysteroid dehydrogenase type 1 (11 -HSD-1) catalyses the interconversion of active corticosterone and inert 11-dehydrocorticosterone. 11 -HSD-1 is highly expressed in liver, where the reaction direction is 11 -reduction, thus potentially increasing intrahepatic active glucocorticoid levels. Inhibition of 11 -HSD-1 increases insulin sensitivity in humans in vivo suggesting that hepatic 11 -HSD-1 plays a role in the maintenance or control of key glucocorticoid-regulated metabolic functions.We have selectively repressed hepatic 11 -HSD-1 in rats by oestradiol administration for 42 days. This nearly completely repressed hepatic 11 -HSD-1 mRNA expression and enzyme activity and reduced expression of hepatic glucocorticoid-inducible genes including phosphoenolpyruvate carboxykinase (PEPCK), the ratelimiting step in gluconeogenesis. Similar effects were seen after 3 weeks of oestradiol treatment. To examine whether this was due to any direct effect of oestradiol upon PEPCK, the experiment was repeated in adrenalectomised rats glucocorticoid replacement. In adrenalectomised rats, oestradiol did not attenuate hepatic PEPCK, whilst glucocorticoid replacement restored this action. Oestradiol did not alter hepatic metabolism of corticosterone by pathways other than 11 -HSD-1.These data suggest 11 -HSD-1 plays an important role in maintaining expression of key glucocorticoid-regulated hepatic transcripts. Enzyme inhibition may provide a useful therapeutic target for manipulating glucose homeostasis.

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Determination of phosphoenolpyruvate carboxykinase activity with deoxyguanosine 5′-diphosphate as nucleotide substrate

Cited by 106 publications

References 9 publications

Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease

Cellular mechanism of insulin resistance in nonalcoholic fatty liver disease

Programming hyperglycaemia in the rat through prenatal exposure to glucocorticoids-fetal effect or maternal influence?

Interactions between oestradiol and glucocorticoid regulatory effects on liver-specific glucocorticoid-inducible genes: possible evidence for a role of hepatic 11beta-hydroxysteroid dehydrogenase type 1

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