Abstract
Mechanisms underlying dietary nutrient regulation of glucose-6-phosphatase (Glc-6-Pase) gene expression are not well understood. Here we investigated the effects of short-chain fatty acids on the expression of this gene in primary cultures of rat hepatocytes and H4IIE hepatoma cells. Propionate, butyrate, valerate, and caproate induced severalfold increases in the expression of Glc-6-Pase mRNA. In reporter gene assays, propionate, valerate, caproate, and also octanoate increased Glc-6-Pase promoter activity by 6-16-fold. Butyrate, by itself, had little or no effect on promoter activity, but it induced a robust increase (45-fold) in promoter activity in cells co-transfected with a plasmid expressing the transcription factor HNF-4alpha (alpha isoforms of hepatic nuclear factor 4). HNF-4alpha also enhanced promoter activity induced by other short-chain fatty acids. A dominant negative form of HNF-4alpha abrogated the fatty acid-induced promoter activity, a finding that accentuates a role for HNF-4alpha in the transcription process studied here. In cells transfected with HNF-4alpha, short-chain fatty acids and trichostatin A, an inhibitor of histone deacetylase, synergistically enhanced promoter activity, suggesting that hyperacetylation of histones is an important component of the transactivation of the Glc-6-Pase gene promoter by HNF-4alpha. Region-751/-466 of this promoter contains seven putative HNF-4alpha-binding motifs. Binding of HNF-4alpha to this region was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, indicating that HNF-4alpha is recruited to the Glc-6-Pase gene promoter during short-chain fatty acid-induced transcription from this promoter.
Highlights
Mechanisms underlying dietary nutrient regulation of glucose-6-phosphatase (Glc-6-Pase) gene expression are not well understood
In cells transfected with HNF-4␣, short-chain fatty acids and trichostatin A, an inhibitor of histone deacetylase, synergistically enhanced promoter activity, suggesting that hyperacetylation of histones is an important component of the transactivation of the Glc-6-Pase gene promoter by HNF-4␣
Using primary cultures of rat hepatocytes and the hepatoma cell line H4IIE, we show that short-chain fatty acids induce Glc-6-Pase gene transcription and that this induction occurs via recruitment of the transcription factor HNF-4␣ to the Glc-6-Pase promoter
Summary
Glc-6-Pase, glucose-6-phosphatase; DR, direct repeat; EMSA, electrophoretic mobility shift assay; HDAC, histone deacetylase; HNF-4␣, ␣ isoform of hepatic nuclear factor 4; LUC, luciferase; PBS, phosphate-buffered saline; PEPCK, phosphoenolpyruvate carboxykinase; PMSF, phenylmethylsulfonyl fluoride; TSA, trichostatin A; MOPS, 4-morpholinepropanesulfonic acid. Hepatic Glc-6-Pase is a multicomponent complex located in the endoplasmic reticulum and consists of at least five different proteins (2, 3) that include three transporters termed T1, T2, and T3, the catalytic unit, and a stabilizer protein (2, 4 – 6). Hormones and nutrients such as glucose and fatty acids, which are elevated in poorly controlled diabetes, profoundly modulate the expression of the gene for the catalytic unit (Glc-6-Pase) (7–9). Stimulation of Glc-6-Pase gene transcription by long-chain saturated fatty acids has been attributed to stabilization of the mRNA (1). Using primary cultures of rat hepatocytes and the hepatoma cell line H4IIE, we show that short-chain fatty acids induce Glc-6-Pase gene transcription and that this induction occurs via recruitment of the transcription factor HNF-4␣ to the Glc-6-Pase promoter
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