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.

Insulin and cyclic AMP act at different levels on transcription of the L‐type pyruvate kinase gene

“…As a first hypothesis, insulin might remain necessary to induce expression of, or to activate, other enzymes involved in glucose metabolism. Insulin is known to be involved in the short‐term and long‐term regulation of several other enzymes of the glycolytic pathway [22], and, in the pentose phosphate pathway, is involved in the transcriptional activation of the key enzyme glucose 6‐phosphate dehydrogenase [23, 24]. A puzzling element, however, is the low concentration of insulin (0.2 nM) sufficient to achieve expression of the L‐PK gene when active glucokinase is already present.…”

Glucose‐6‐phosphate dehydrogenase: a “housekeeping” enzyme subject to tissue‐specific regulation by hormones, nutrients, and oxidant stress

“…Further refinements in this type of analysis have demonstrated that hepatocytes from fasted animals contain as little as one molecule of G6PDH mRNA per cell and the level increases in excess of 30 Fritz et al (12), using molecular probes, showed that insulin increased the levels of mRNA encoding G6PDH, confirming earlier studies using in vitro translation assays (28). Subsequent studies (7,9) agreed with these observations and led to the conclusion that insulin is a primary hormone in upregulating expression of G6PDH, presumably at the level of transcription since studies employing the fasting/refeeding paradigm have shown that transcriptional activation of the gene occurs (5,6). A major challenge to molecular endocrinologists is to define, at a molecular level, how insulin increases transcription of this and other insulinregulated genes.…”

“…The glucocorticoids have a well-established role in hepatic lipogenesis (reviewed in ref 26) and the hepatocyte cell culture system has been useful in defining the interaction of these hormones with insulin. The glucocorticoids by themselves appear only to modestly increase the activity of the enzyme in hepatocytes (7,9,12,29). However, they clearly in-crease G6PDH mRNA in the presence (7,9,12) or absence (9, 12) of insulin while apparently not affecting the relative rate of enzyme synthesis (28).…”

“…For example, hepatic enzyme activity has long been known to respond to several hormonal and dietary manipulations (3,4). Recent studies (5)(6)(7)(8)(9)(10)(11)(12) have demonstrated that changes in hepatic enzyme activity are the result of increases in gene transcriptional activity, mRNA stability, and in the rate of enzyme protein synthesis, a pattern typical of inducible gene expression. A major focus of this review is to present the available evidence for adaptive, tissue-specific regulation of G6PDH expression.…”

A circadian study of liver antioxidant enzyme systems of female Fischer-344 rats subjected to dietary restriction for six weeks.

“…Since maximal velocity would rarely, if ever, be obtained, much of the G6PDH potential in AL animals is likely never realized. This may be the result of altered post-translational modifications of G6PDH which are known to occur (35). Accordingly, an increase or decrease in NADPH requirement over a sufficient period of time would be expected to yield a fluctuation in the amount of total enzyme.…”