Regulation of hepatic gluconeogenesis by nuclear factor Y transcription factor in mice.

Hepatic gluconeogenesis is essential to maintain blood glucose levels, and its abnormal activation leads to hyperglycemia and type 2 diabetes. However, the molecular mechanisms in the regulation of hepatic gluconeogenesis remain to be fully defined. In this study, using murine hepatocytes and a liver-specific knockout mouse model, we explored the physiological role of nuclear factor Y (NF-Y) in regulating hepatic glucose metabolism and the underlying mechanism. We found that NF-Y targets the gluconeogenesis pathway in the liver. Hepatic NF-Y expression was effectively induced by cAMP, glucagon, and fasting in vivo Lentivirus-mediated NF-Y overexpression in Hepa1-6 hepatocytes markedly raised the gluconeogenic gene expression and cellular glucose production compared with empty vector control cells. Conversely, CRISPR/Cas9-mediated knockdown of NF-Y subunit A (NF-YA) attenuated gluconeogenic gene expression and glucose production. We also provide evidence indicating that CRE-loxP-mediated, liver-specific NF-YA knockout compromises hepatic glucose production. Mechanistically, luciferase reporter gene assays and ChIP analysis indicated that NF-Y activates transcription of the gluconeogenic genes Pck1 and G6pc, by encoding phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase), respectively, via directly binding to the CCAAT regulatory sequence motif in their promoters. Of note, NF-Y enhanced gluconeogenesis by interacting with cAMP-responsive element-binding protein (CREB). Overall, our results reveal a previously unrecognized physiological function of NF-Y in controlling glucose metabolism by up-regulating the gluconeogenic genes Pck1 and G6pc Modulation of hepatic NF-Y expression may therefore offer an attractive therapeutic approach to manage type 2 diabetes.

Increased ethanol production by deletion of HAP4 in recombinant xylose-assimilating Saccharomyces cerevisiae.

The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.

CCAAT/enhancer binding protein ß regulates prostaglandin E synthase expression and prostaglandin E2 production in activated microglial cells.

The eicosanoid prostaglandin E2 (PGE2 ) plays important roles in neuroinflammation and it is produced by the sequential action of the enzymes cyclooxygenase-2 (COX-2) and prostaglandin E synthase (PTGES). The expression of both enzymes and the production of PGE2 are increased in neuroinflammation. The objective of this study was to elucidate whether the transcription factor CCAAT/enhancer binding protein ß (C/EBPß) regulates the expression of prostaglandin synthesis enzymes in neuroinflammation. To this aim, the expression of these enzymes in wild-type and C/EBPß-null mice was analyzed in vitro and in vivo. In mixed glial cultures, lipopolysaccharide (LPS) ± interferon γ (IFN-γ) induced C/EBPß binding to COX-2 and PTGES promoters. LPS ± IFN-γ-induced increases in PTGES expression and in PGE2 production in mixed glial and microglial cultures were abrogated in the absence of C/EBPß. Also, increased brain PTGES expression induced by systemic LPS administration was markedly reduced in C/EBPß-null mice. In contrast to PTGES, the induction of COX-2 expression in vitro or in vivo was not markedly affected by the absence of C/EBPß. These results demonstrate that C/EBPß regulates PTGES expression and PGE2 production by activated microglial cells in vitro and point to C/EBPß as a regulator of PTGES expression in vivo in the inflamed central nervous system. Altogether, these findings strengthen the proposed role of C/EBPß as a key player in the orchestration of neuroinflammatory gene response.

Inactivation of CBF/NF-Y in postnatal liver causes hepatocellular degeneration, lipid deposition, and endoplasmic reticulum stress.

We previously demonstrated that CBF activity is needed for cell proliferation and early embryonic development. To examine the in vivo function of CBF in differentiated hepatocytes, we conditionally deleted CBF-B in hepatocytes after birth. Deletion of CBF-B resulted in progressive liver injury and severe hepatocellular degeneration 4 weeks after birth. Electron microscopic examination demonstrated pleiotropic changes of hepatocytes including enlarged cell and nuclear size, intracellular lipid deposition, disorganized endoplasmic reticulum, and mitochondrial abnormalities. Gene expression analyses showed that deletion of CBF-B activated expression of specific endoplasmic reticulum (ER) stress-regulated genes. Inactivation of CBF-B also inhibited expression of C/EBP alpha, an important transcription factor controlling various metabolic processes in adult hepatocytes. Altogether, our study reveals for the first time that CBF is a key transcription factor controlling ER function and metabolic processes in mature hepatocytes.

A novel Hansenula polymorpha transcriptional factor HpHAP4-B, able to functionally replace the S. cerevisiae HAP4 gene, contains an additional bZip motif.

The transcriptional regulator HAP4, whose expression is induced on respiratory substrates, has been shown to be involved in the balance between fermentation and respiration in Saccharomyces cerevisiae. We have previously identified a HAP4 orthologue in the yeast Hansenula polymorpha, called HpHAP4-A, which, despite its very limited sequence conservation (a 16 amino acid N-terminal motif), is fully functional in S. cerevisiae. Based on the same N-terminal motif, a second gene has now been identified in the same organism. It was shown to contain an additional cis-binding motif of the bZip type. We report on the cloning, heterologous expression and analysis in S. cerevisiae of this novel ScHAP4 orthologue. From these experiments we could conclude that, as with HpHAP4-A, the novel orthologue, designated HpHAP4-B, could functionally replace the S. cerevisiae gene but to a lesser extent. The relationship between the presence of the additional cis-binding motif and the weaker potential as a HAP4 functional homologue is discussed.