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Regulation of glucose-6-phosphate dehydrogenase synthesis and mRNA abundance in cultured rat hepatocytes.

ABSTRACT: Conditions were identified which, for the first time, demonstrate that primary hepatocytes can express the same range of glucose-6-phosphate dehydrogenase (G6PD) synthesis and mRNA as in live rats. Primary hepatocytes were cultured without prior exposure to serum, hormones or carbohydrates. Five modulators implicated in G6PD induction in vivo were examined: insulin, dexamethasone, tri-iodothyronine (T3), glucose and fructose, T3 did not affect G6PD activity, and did not interact with carbohydrate to affect the activity of G6PD. Neither glucose nor fructose alone affected G6PD activity, and they did not interact with insulin to increase G6PD activity. Hepatocytes isolated from fasted rats and cultured in serum-free media with amino acids ad the only energy source how a 12-fold increase in G6PD synthesis and mRNA (measured by a solution-hybridization assay). This induction does not require added hormones or carbohydrate. The addition of insulin alone caused another increase in G6PD synthesis and mRNA. There are at least three distinct phases to G6PD induction under these conditions. The largest increase in G6PD synthesis (12-fold) occurs in the absence of any hormones and with amino acids as the only energy source. This phase is due to increased G6PD mRNA. Insulin causes an additional 2-3-fold increase in G6PD synthesis and mRNA. However, dexamethasone and insulin are both required before G6PD synthesis is equal to that in rats which are fasted and refed on a high-carbohydrate diet.

SUBMITTER: Manos P

PROVIDER: S-EPMC1151171 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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Project description:The effect of inhibition of NADP-specific isocitrate dehydrogenase (EC 1.1.1.42) by DL-threo-alpha-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylase) on urea synthesis was studied in isolated rat hepatocytes. alpha-Methylisocitrate substantially inhibited the rate of urea synthesis (35--84%) with substrates requiring net reductive amination of 2-oxoglutarate to glutamate for aspartate synthesis (i.e., L-serine, D-alanine, or NH4Cl + L-lactate). alpha-Methylisocitrate did not inhibit synthesis of urea from substrates not requiring reductive formation of glutamate (i.e. L-alanine, L-glutamine, L-asparagine, or NH4Cl + L-ornithine). The rate-limiting role of NADPH in urea synthesis was correlated with the decrease in NADPH content that occurred upon addition of NH4Cl or of alpha-methylisocitrate to hepatocytes incubated with lactate and pyruvate, indicating utilization of NADPH for reductive amination of 2-oxoglutarate and inhibition of NADPH generation via NADP-isocitrate dehydrogenase, respectively. Similar results were obtained with D-alanine and L-serine; however, alpha-methylisocitrate or NH4Cl did not substantially decrease NADPH content when L-alanine was the substrate. Inhibitors or ornithine--2-oxo acid transaminase (L-canaline or gabaculine) decreased the uptake of ornithine by hepatocytes and inhibited the alpha-methylisocitrate insensitive urea synthesis from ornithine and NH4Cl. Canaline did not inhibit urea synthesis from lactate, ornithine, and NH4Cl but the inhibition by alpha-methylisocitrate of urea formation from this combination was appreciably larger with canaline (approx. 82%) than without canaline (approx. 48%). Inhibition of urea synthesis from NH4Cl + lactate by alpha-methylisocitrate was partially prevented by oleate, octanoate, or 3-hydroxybutyrate. When the NADH content of hepatocytes was increased by 3-hydroxybutyrate, the addition of NH4Cl and/or alpha-methylisocitrate caused a decline in NADH (and NADPH) content, suggesting that reducing equivalents from NADH as well as from NADPH can support net reductive amination of 2-oxoglutarate when required for urea synthesis.

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Regulation of glucose-6-phosphate dehydrogenase synthesis and mRNA abundance in cultured rat hepatocytes.

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