Abstract. A heterotrimeric Gai subunit, «i-3, is localized on Golgi membranes in LLC-PKI and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence . The M-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The a;_3 subunit is found on isolated rat liver Golgi membranes by Western blotting and Gai-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PKI cells were stably transfected with GO ;-3 on an MT1, inducible promoter in order to overexpress ai-3 on Golgi membranes. The intracellular processing and

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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.