An insulin response element in the glyceraldehyde-3-phosphate dehydrogenase gene binds a nuclear protein induced by insulin in cultured cells and by nutritional manipulations in vivo.

Abstract

Two independent cis-acting insulin response elements (IREs) in the gene encoding glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12], designated IRE-A and IRE-B, are sufficient to direct insulininducible gene expression. Using the electrophoretic mobility shift assay, a 4-fold increase in the amount of IRE-A DNA bound to nuclear proteins was detected when extracts isolated from insulin-stimulated differentiated 3T3-L1 cells or from the liver of rats refed a high-carbohydrate/low-fat diet after a 72-hr fast were compared to control nuclear extracts. The points of contact between protein and IRE-A DNA may represent a sequence recognized by at least one class of insulinsensitive transcription factor(s).The interaction of insulin with its cell surface receptor initiates changes in the activity and cellular content of metabolic enzymes that promote energy storage and cell growth. Although many genes have been described recently to be regulated by insulin at the level of transcription, the molecular mechanism by which insulin mediates its effects is unknown. In an ongoing attempt to elucidate the signal transduction pathway of insulin action on gene expression, we have attempted to define cis-acting sequences that mediate the effect of insulin on gene transcription and work backward to define the mechanism by which the trans-acting factors that interact with these sequences are regulated.We previously isolated an insulin-responsive gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1) and showed that the expression of a hybrid reporter gene containing bases -488 to + 21 of the human gene for GAPDH and the coding region of the gene encoding chloramphenicol acetyltransferase (CAT), designated plasmid HGAPDH-CAT, was increased 3-fold in insulin-treated differentiated 3T3-L1 cells and 5-fold in H35 hepatoma cells that were stably transfected with this hybrid gene (2). An increase in HGAPDH-CAT mRNA was detected within 4 hr after insulin exposure and was maintained overnight, a pattern that parallels the time course of GAPDH mRNA induction (3).These results indicated that the human gene for GAPDH contained a cis-acting element capable of conferring insulin inducibility to a marker gene. As it appears that binding of cellular transcription factors to inducible cis-acting elements is required for function, we used the gel shift assay to detect nuclear factors that bind this element in a specific manner. This paper describes the regulation of an insulin-inducible factor detected in vitro with the differentiated 3T3-L1 cultured cell line and in vivo in the liver of fasted rats refed a high-carbohydrate/low-fat diet, a model in which circulating insulin levels are high and many metabolic enzymes required to promote energy storage are induced.

The human hexokinase II gene promoter: functional characterization and detection of variants among patients with NIDDM

“…Various cis-acting sequences that mediate insulindependent regulation of transcription have been reported in promoters of genes encoding phosphoenolpyruvate carboxykinase (PEPCK) [45], c-fos [51,52], amylase [53], glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [54,55], glugacon [56,57], malic enzyme [58], delta 1-crystallin [59], prolactin [60], b-actin [61], insulin-like growth factor I-binding protein [62], tyrosine aminotransferase [46], thyrotropin receptor [63] and fatty acid synthase [64]. There seems to be no unique sequence found so far to mediate the effects of insulin.…”

Effects of ketoacidosis on rat apolipoprotein A1 gene expression: a link with acidosis but not with ketones

“…Glucose and insulin regulate expression of the apoA1 gene in the liver (Murao et al 1998) through an insulin response core element (IRCE) located in the 5 -flanking region of the rat apoA1 gene. The IRCE present in the apoAI promoter is related to the IRCEs identified in the G3PDH gene and glucagon gene (Nasrin et al 1990, Philippe 1991. This process may have a role in reducing apoA1 mRNA expression in diabetic animals (Fig.…”

Transcriptional control of genes that regulate glycolysis and gluconeogenesis in adult liver

“…Secondly, it is differentiation-dependent since it occurs in mature adipocytes but not in pre-adipocytes [69]. Thirdly, both stable and transient transfection experiments in 3T3 adipocytes and hepatoma cells demonstrated that insulin stimulates transcription from the GAPDH promoter [71,72]. Run-on experiments showed that the insulin effect occurs within 30 min and results from an increase in the transcription rate [71].…”

“…Study of the proteins that bind to IRE-A revealed that a protein, IRP-A, was induced in insulin-treated adipocytes as well as in liver upon refeeding after fasting. IRP-A binds to the 5' half of IRE-A and recognizes the CCCGCCTC core sequence [72]. By screening an expression library prepared from rat adipocytes with an IRE-A oligonucleotide, Nasrin et al [73] …”