Phosphoenolpyruvate Carboxykinase, a Key Enzyme That Controls Blood Glucose, Is a Target of Retinoic Acid Receptor-Related Orphan Receptor α

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes a committed and rate-limiting step in hepatic gluconeogenesis, and its activity is tightly regulated to maintain blood glucose levels within normal limits. PEPCK activity is primarily regulated through hormonal control of gene transcription. Transcription is additionally regulated via a cAMP response unit, which includes a cAMP response element and four binding sites for CCAAT/enhancer-binding protein (C/EBP). Notably, the cAMP response unit also contains a putative response element for retinoic acid receptor-related orphan receptor α (RORα). In this paper, we characterize the effect of the RORα response element on cAMP-induced transcription. Electrophoresis mobility shift assay indicates that RORα binds this response element in a sequence-specific manner. Furthermore, luciferase reporter assays indicate that RORα interacts with C/EBP at the PEPCK promoter to synergistically enhance transcription. We also found that cAMP-induced transcription depends in part on RORα and its response element. In addition, we show that suppression of RORα by siRNA significantly decreased PEPCK transcription. Finally, we found that a RORα antagonist inhibits hepatic gluconeogenesis in an in vitro glucose production assay. Taken together, the data strongly suggest that PEPCK is a direct RORα target. These results define possible new roles for RORα in hepatic gluconeogenesis.

Retinoic acid receptor-related orphan receptor α reduces lipid droplets by upregulating neutral cholesterol ester hydrolase 1 in macrophages

“…In summary, the data from this study suggested that human NCEH1 is a direct target of RORα, containing two functional response elements in the NCEH1 promoter region. EMSA EMSAs were performed as described previously [23]. The IκB probe was prepared by radiolabeling synthetic double-stranded DNA using [g-32 P] ATP (Perkin-Elmer, Waltham, MA, USA).…”

“…After 24 h at 37 °C and 5% CO 2 , THP1 cells were differentiated into macrophages via treatment with 100 nM PMA for 12 h. In RORα agonist-treated experiments, THP1 cells were differentiated into macrophages by treating them with 100 nM PMA for 72 h and were then treated with 5 μM SR1078, an RORα agonist, for 24 h. HepG2 cells were seeded at 1 × 10 5 cells/well in D-MEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. After 24 h at 37 °C and 5% CO 2 , HepG2 cells were induced by treatment with 5 μM SR1078 as an RORα agonist for 48 or 72 h. qRT-PCR using a SYBR green reaction was performed as described previously [23]. RORα and NCEH1 expression levels were measured, with CD11b and MMP9…”

“…Data were collected from at least three independent experiments. The lysate was assayed for qRT-PCR using a SYBR green reaction performed as described previously [23]. siRNA experiments siRNAs targeting different sequences in RORα (siRORa-258 and siRORa-1388) were generated using an in vitro transcription T7 kit (Takara Bio).…”

Lactate-Fortified Puerariae Radix Fermented by Bifidobacterium breve Improved Diet-Induced Metabolic Dysregulation via Alteration of Gut Microbial Communities

“…To explore the probable molecular mechanism(s) underlying such beneficial effects of XEN, PR, and fPR, we evaluated the effects of these agents in HFD+F-fed mice on the gene expressions associated with three different proteins PEPCK, PPARγ, and PGC1α, which have key roles in metabolic processes ( Figure 4A,B). Among them, PEPCK, which catalyzes a committed, rate-limiting step in hepatic gluconeogenesis by converting oxaloacetate to phosphoenolpyruvate, has a vital role in maintaining a normal blood glucose level [58]. The nuclear receptor PPARγ is a regulator of lipid and glucose metabolism [59], whereas PGC1α, a multifunctional regulatory factor originally identified as a coactivator of PPARγ, has a central role in the regulatory network of glucose metabolism [60] and has been shown to be a metabolic regulator of intestinal epithelial cell fate [61].…”

Retinoic acid receptor-related orphan receptor α reduces lipid droplets by upregulating neutral cholesterol ester hydrolase 1 in macrophages

“…The lysate was assayed for qRT-PCR using a SYBR green reaction performed as described previously [23]. siRNA experiments siRNAs targeting different sequences in RORα (siRORa-258 and siRORa-1388) were generated using an in vitro transcription T7 kit (Takara Bio).…”