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Identification of Insulin-Responsive Transcription Factors That Regulate Glucose Production by Hepatocytes.

ABSTRACT: Hepatocyte glucose production is a complex process that integrates cell-autonomous mechanisms with cellular signaling, enzyme activity modulation, and gene transcription. Transcriptional mechanisms controlling glucose production are redundant and involve nuclear hormone receptors and unliganded transcription factors (TFs). Our knowledge of this circuitry is incomplete. Here we used DNA affinity purification followed by mass spectrometry to probe the network of hormone-regulated TFs by using phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase (G6pc) in liver and primary hepatocytes as model systems. The repertoire of insulin-regulated TFs is unexpectedly broad and diverse. Whereas in liver the two test promoters are regulated by largely overlapping sets of TFs, in primary hepatocytes Pck1 and G6pc regulation diverges. Insulin treatment preferentially results in increased occupancy by the two promoters, consistent with a model in which the hormone's primary role is to recruit corepressors rather than to clear activators. Nine insulin-responsive TFs are present in both models, but only FoxK1, FoxA2, ZFP91, and ZHX3 require an intact Pck1p insulin response sequence for binding. Knockdown of FoxK1 in primary hepatocytes decreased both glucose production and insulin's ability to suppress it. The findings expand the repertoire of insulin-dependent TFs and identify FoxK1 as a contributor to insulin signaling.

SUBMITTER: Wang L

PROVIDER: S-EPMC6610019 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The entomopathogenic fungus Beauveria bassiana can produce the secondary metabolite oosporein under alkaline conditions or in fungus-killed cadavers. However, the regulatory mechanism of oosporein synthesis is not fully understood. In thisstudy, we found that the pH signaling transcription factor BbPacC is involved in the regulation of oosporein production. Overexpression of BbPacC promotes oosporein production in B. bassiana at pH 6.0 or under alkaline conditions (pH 8.0), but deletion of this gene abolished oosporein production. Under acidic conditions (pH 4.0), no oosporein production was observed in the wild-type and BbPacC overexpression strains. Yeast one-hybrid assays and electrophoretic mobility shift assay (EMSA) confirmed the binding ability of BbPacC with 4 putative PacC-binding sites in the promoter region of BbOpS3, a transcription factor located in the oosporein synthetic gene cluster regulating the expression of oosporein synthetic genes. Overexpression of Bbmsn2, a previously reported negative regulator of oosporein synthesis, in OEPacC or wild-type strains abolished oosporein production in all tested conditions. However, deletion of Bbmsn2 in the BbPacC overexpression strain significantly improved oosporein production even at pH 4.0. These results indicated that BbPacC is a positive regulator of oosporein production and functions jointly with Bbmsn2 to regulate oosporein production in different environments and particularly under alkaline conditions. IMPORTANCE B. bassiana produces the red dibenzoquinone pigment oosporein under certain specific conditions, such as alkaline conditions and fungus-killed cadavers. Ooporein possesses antibiotic and insect immune inhibition activities and plays multiple roles during the infection process of B. bassiana against insect hosts. Several negative regulators involved in oosporein synthesis have been reported; however, we know little about the positive regulators outside the biosynthetic gene cluster. Here, we found that the pH signaling transcription factor BbPacC positively regulates oosporein production by binding to several PacC-binding sites. In addition, our results also indicate that BbPacC jointly acts with the negative regulator Bbmsn2 to regulate oosporein synthesis. Our results provide insight into understanding the regulatory mechanism of oosporein production as well as targets to engineer B. bassiana strains producing high levels of oosporein.

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Identification of Insulin-Responsive Transcription Factors That Regulate Glucose Production by Hepatocytes.

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