Polyunsaturated fatty acids inhibit the expression of hepatic glucose-6-phosphate dehydrogenase (G6PD) by changes in the amount of G6PD pre-mRNA in the nucleus in the absence of changes in the transcription rate of the gene. We have compared the nuclear accumulation of partially and fully spliced mRNA for G6PD in the livers of mice fed diets high versus low in polyunsaturated fat. Consumption of a diet high in polyunsaturated fat decreased the accumulation of partially spliced forms of the G6PD pre-mRNA. Examining the fate of multiple introns within the G6PD primary transcript indicated that in mice fed a high fat diet, G6PD pre-mRNA containing intron 11 accumulated within the nucleus, whereas G6PD mature mRNA abundance was inhibited 50% or more within the same livers. Transient transfection of RNA reporters into primary hepatocyte cultures was used to localize the cis-acting RNA element involved in this regulated splicing. Reporter RNA produced from constructs containing exon 12 were decreased in amount by arachidonic acid. The extent of this decrease paralleled that seen in the expression of the endogenous G6PD mRNA. The presence of both exon 12 and a neighboring intron within the G6PD reporter RNA was essential for regulation by polyunsaturated fatty acid. Inhibition was not dependent on the presence of the G6PD polyadenylation signal and the 3-untranslated region, but substitution with the SV40 poly(A) signal attenuated the inhibition by arachidonic acid. Thus, exon 12 contains a putative splicing regulatory element involved in the inhibition of G6PD expression by polyunsaturated fat.Polyunsaturated fatty acids are potent regulators of cellular metabolism. As components of phospholipids, they are involved in signal transduction from specific plasma membrane receptors and are precursors for the synthesis of eicosanoids. When present in the diet of animals or the medium of cells in culture, polyunsaturated fats can both increase and decrease the activity or amount of specific cellular proteins. In this regard, polyunsaturated fatty acids have long been known to decrease the capacity of liver cells to synthesize fatty acids de novo.The pathway of de novo fatty acid biosynthesis is essential for the conversion of energy substrates, such as glucose, which are in excess of immediate needs, to fatty acids that can be stored as triacylglycerols. This pathway is most active in liver and adipose tissue and involves a family of enzymes referred to as the lipogenic enzymes (for review, see Ref. 1). These enzymes include ATP-citrate lyase, acetyl-CoA carboxylase, fatty acids synthase, malic enzyme, and glucose-6-phosphate dehydrogenase (G6PD).1 Consistent with their role in energy metabolism, the activities of these enzymes are induced when animals are fed a high carbohydrate diet and decreased during starvation. Likewise, lipogenic enzyme activity is decreased by a high fat diet, particularly a diet rich in polyunsaturated fatty acids. Regulation of the activities of ATP-citrate lyase, acetylCoA carboxylase, fatty acid ...

Background: Regulation of G6PD expression by nutrients occurs by changes in accumulation of spliced mRNA without changes in transcriptional activity of the gene. Results: Refeeding enhances SRSF3 binding to G6PD mRNA. Loss of SRSF3 inhibits G6PD expression. Conclusion: SRSF3 is a target for nutritional regulation of splicing. Significance: Regulation of RNA splicing is a novel target for nutrient action.

Insulin is the central hormone required for the activation of lipogenic genes in the liver. Feeding animals a high carbohydrate diet enhances the expression of the lipogenic genes. This effect involves the stimulatory actions of both dietary glucose and insulin (1, 2). In contrast, dietary polyunsaturated fats attenuate the stimulatory effect of feeding a high carbohydrate diet (3, 4). We have used glucose-6-phosphate dehydrogenase (G6PD), 2 a member of the lipogenic gene family, as a model system for studying the mechanism of action of fatty acids. The advantage of this model is that insulin is the primary inducer of G6PD expression, and fatty acids such as arachidonic acid are the primary inhibitors of G6PD expression; this regulation is independent of other hormonal requirements (5, 6). The intracellular mechanisms by which polyunsaturated fats inhibit G6PD or other lipogenic genes are not completely understood. Inhibition by polyunsaturated fatty acid may represent a direct action of fatty acids on factors involved in gene expression. Alternatively, fatty acids may act indirectly via the inhibition of stimulatory signal transduction pathways of glucose or insulin. We hypothesized that fatty acids inhibit G6PD expression by inhibition of the insulin induction.Insulin transduces its signal upon binding to the insulin receptor. Transduction of this signal in liver involves phosphorylation of two intracellular substrates, insulin receptor substrate (IRS)-1 and IRS-2 (7). These proteins play complementary roles in insulin signaling (8). Activation of phosphoinositide (PI) 3-kinase is associated with the stimulatory effects of insulin on metabolic pathways, including lipogenesis (9 -11).The IRS proteins can be phosphorylated on both tyrosines and serines. A known mechanism for the inhibition of IRS-1 activation is by phosphorylation at serines 307, 612, and 632 (12). These serine residues, when phosphorylated might interfere with the interaction between IRS-1 and the insulin receptor or PI 3-kinase (13,14). Among the factors known to cause serine phosphorylation of IRS-1 are the mitogen-activated protein (MAP) kinases. Activation of the MAP kinases extracellular regulated kinase (ERK) (15, 16), c-Jun NH 2 -terminal kinase (JNK) (17-19), or p38 MAP kinase (MAPK) (17,20) is associated with the development of insulin resistance in muscle and adipose tissue.Known activators of MAP kinases include tumor necrosis factor ␣ (TNF␣) and very high fat diets. TNF␣, a potent mediator of insulin resistance, activates all three of the MAP kinases. Phosphorylation and activation of p38 MAPK by TNF␣ correlates with IRS-1 serine phosphorylation and a decrease in PI 3-kinase activity (17,20). In muscle and adipose tissue, this results in the decrease in glucose uptake associated with insulin resistance. Likewise, diets containing 40% or more of the energy content as fat also decrease PI 3-kinase activation and result in an insulin-resistant phenotype in intact animals (21-23). This may involve activation of MAP kinases (18,19). In c...

The inhibition of glucose-6-phosphate dehydrogenase (G6PD) expression by arachidonic acid occurs by changes in the rate of pre-mRNA splicing. Here, we have identified a cis-acting RNA element required for regulated splicing of G6PD mRNA. Using transfection of G6PD RNA reporter constructs into rat hepatocytes, the cis-acting RNA element involved in this regulation was localized to nucleotides 43-72 of exon 12 in the G6PD mRNA. In in vitro splicing assays, RNA substrates containing exon 12 were not spliced. In contrast, RNA substrates containing other regions (exons 8 and 9 or exons 10 and 11) of the G6PD mRNA were efficiently spliced. Furthermore, exon 12 can inhibit splicing when substituted for other exons in RNA substrates that are readily spliced. This activity of the exon 12 regulatory element suggests that it is an exonic splicing silencer. Consistent with its activity as a splicing silencer, spliceosome assembly was inhibited on RNA substrates containing exon 12 compared with RNAs representing other regions of the G6PD transcript. Elimination of nucleotides 43-72 of exon 12 did not restore splicing of exon 12-containing RNA; thus, the 30-nucleotide element may not be exclusively a silencer. The binding of heterogeneous nuclear ribonucleoproteins K, L, and A2/B1 from both HeLa and hepatocyte nuclear extracts to the element further supports its activity as a silencer. In addition, SR proteins bind to the element, consistent with the presence of enhancer activity within this sequence. Thus, an exonic splicing silencer is involved in the inhibition of splicing of a constitutively spliced exon in the G6PD mRNA.Glucose-6-phosphate dehydrogenase (G6PD) 2 is a member of a family of enzymes that catalyze the de novo synthesis of fatty acids. In liver, the lipogenic pathway plays an essential role in converting excess dietary energy into a storage form. Consistent with this role in energy homeostasis, the capacity of this pathway is regulated by dietary changes, such as fasting, feeding, and the amount and type of carbohydrate and polyunsaturated fat in the diet (1). For many of the lipogenic enzymes, regulation of enzyme amount occurs primarily by changes in the transcription rate of the gene, but posttranscriptional regulation via mRNA stability has also been implicated (1). G6PD differs from the other family members in that dietary regulation occurs exclusively at a posttranscriptional step (2-4).G6PD expression is inhibited by polyunsaturated fatty acids, such as arachidonic acid; this occurs at a unique posttranscriptional step involving a decrease in the rate of splicing of the nascent G6PD transcript. Several lines of evidence indicate that changes in mature mRNA accumulation are caused by changes in the efficiency of splicing of the G6PD transcript. First, changes in the cytoplasmic accumulation of G6PD mRNA are preceded by changes in the accumulation of mRNA in the nucleus in the absence of changes in transcriptional activity of the gene (3-5). Second, stimulatory treatments, such as refeeding, enhance the ...

Understanding how a cell adapts to dietary energy in the form of carbohydrate versus energy in the form of triacylglycerol requires knowledge of how the activity of the enzymes involved in lipogenesis is regulated. Changes in the activity of these enzymes are largely caused by changes in the rate at which their proteins are synthesized. Nutrients within the diet can signal these changes either via altering hormone concentrations or via their own unique signal transduction pathways. Most of the lipogenic genes are regulated by changes in the rate of their transcription. Glucose-6-phosphate dehydrogenase (G6PD) is unique in this group of enzymes in that nutritional regulation of its synthesis involves steps exclusively at a posttranscriptional level. G6PD activity is enhanced by the consumption of diets high in carbohydrate and is inhibited by the consumption of polyunsaturated fat. In this review, evidence is presented that changes in the rate of synthesis of the mature G6PD mRNA involves regulation of the efficiency of splicing of the nascent G6PD transcript. Furthermore, this regulation involves the activity of a cis-acting sequence in the G6PD primary transcript. This sequence in exon 12 is essential for the inhibition of G6PD mRNA splicing by PUFA. Understanding the mechanisms by which nutrients alter nuclear posttranscriptional events will provide new information on the breadth of mechanisms involved in gene regulation.