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

<div><p>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 <i>in vitro</i> 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.</p></div

RORα binds to a putative response element in PEPCK.

<p>(A) Sequence of the putative RORα response element (in boldface) in human PEPCK. The probe mt is equivalent to this motif, except that underlined nucleotides have been mutated. The I-kb probe contains the known RORα response element in NF-kappaB inhibitor α. (B) EMSA was used to test the ability of unlabeled wild type and mutated probes, at 5- and 20-fold excess, to inhibit binding of RORα to the putative response element (open arrowhead). The positions of free probe (free) and nonspecific probe of RORα for crude protein (nonspecific) were indicated. All reactions except lane 1 contain crude products from <i>in vitro</i> translation. In lane 2, the probe was incubated with the crude product obtained from <i>in vitro</i> translation in the absence of the RORα expression vector.</p

cAMP-dependent stimulation of the PEPCK promoter as mimicked by forskolin.

<p>(A) HepG2 cells were treated with (closed bars) or without 10 μM forskolin (open bars). Expression of RORα and PEPCK was analyzed by qRT-PCR, and normalized to 18S rRNA. Data are mean ± S.E. (n = 3). (B) HepG2 cells were transfected with luciferase under the control of wild type or mutated PEPCK promoters, and treated with (closed bars) or without 10 μM forskolin (open bars). Data are shown as mean ± S.E. of three experiments. *, <i>p</i> < 0.05.</p

<i>In vitro</i> gluconeogenesis is suppressed by a RORα antagonist.

<p>(A) Glucose production in HepG2 cells incubated for 4 h in 10 μM SR1001, a RORα antagonist, or in DMSO. (B) Effect of 10 μM SR1001 on PEPCK mRNA expression in HepG2 cultures 7 h after treatment. Data are mean ± S.E. of triplicate experiments. *, <i>p</i> < 0.05.</p

The PEPCK promoter is directly activated by C/EBPβ and coactivated by RORα.

<p>(A) Transactivation of the PEPCK promoter by the nuclear receptors CREB, C/EBPβ, and glucocorticoid receptor with or without RORα. HepG2 cells were transfected with luciferase under the control of the PEPCK promoter (-1305 to +51). Data are fold transactivation over basal activity, and are reported as mean ± S.E. (n = 3). (B) HepG2 cells was transfected with RORα (dotted bars), C/EBPβ (closed bars), or both (hatched bars), along with luciferase driven by wild type or mutated PEPCK promoters. Data are mean ± S.E. (n = 3). Mutations in the promoter include deletions of the C1, C4, and C5 binding sites, as well as deletion of both C2 and C3 and mutation of C3 and of the RORα response element. *, <i>p</i> < 0.05.</p

Effect of RORα deficiency on PEPCK expression.

<p>Suppression of endogenous RORα by siRNA significantly decreased PEPCK mRNA in HepG2 cells. HepG2 cells were transfected with 50 nM siRNA, and analyzed by qRT-PCR to measure expression of RORα and PEPCK. siRNAs that target sequences around 258 bp (siRORa-258, dotted bars) and 1388 bp (siRORa-1388, hatched bars) downstream of the RORα start codon decreased RORα and PEPCK mRNA levels, while a scrambled siRNA (siRORa-mt, closed bars) did not. siRNA against green fluorescent protein was used as negative control (siGFP, open bars). Data are mean ± S.E. of three experiments, and are normalized to 18S rRNA. *, <i>p</i> < 0.05.</p

Additional file 1 of Safety and feasibility of minimally invasive gastrectomy following preoperative chemotherapy for highly advanced gastric cancer

Additional file 1: Supplementary Figure 1. Consort diagram. Supplementary Figure 2. Kaplan–Meier survival curves for overall survival (subgroups). Supplementary Table 1. Adverse events during preoperative chemotherapy. Supplementary Table 2. Short-term outcomes and risk factors for postoperative complications following gastrectomy. Supplementary Table 3. Prognostic risk factors (All cases)

Relationship between c-D-index and oral complications.

<p>Relationship between c-D-index and oral complications.</p

Relationship between parameters and oral complications.

<p>Relationship between parameters and oral complications.</p

Box plot of the D-index in each grade of chemotherapy.

<p>Box plot of the D-index in each grade of chemotherapy.</p