Fasting-induced hepatic production of DHEA is regulated by PGC-1alpha, ERRalpha, and HNF4alpha

Abstract

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha is involved in the coordinate induction of changes in gene expression in the liver that enable a homeostatic response to alterations in metabolic state, environmental cues, and nutrient availability. In exploring the specific pathways under PGC-1alpha regulation in the liver, we have made the surprising observation that this coactivator can induce the expression of CYP11A1 and CYP17A1, key rate-limiting enzymes involved in the initial steps of steroidogenesis. Both of these enzymes function to produce C(19)-steroids, converting cholesterol into pregnenolone, and then to dehydroepiandrosterone (DHEA). Estrogen-related receptor (ERR)-alpha mediates PGC-1alpha's induction of CYP11A1 and binds within the first intron of the CYP11A1 gene. Both ERR-alpha and hepatocyte nuclear factor-4alpha are required for PGC-1alpha-mediated induction of CYP17A1, and specific binding sites for these receptors have been identified in the regulatory regions of this gene. The potential physiological significance of these observations was highlighted in rats where fasting induced hepatic expression of PGC-1alpha and CYP17A1 and was associated with an increase in hepatic levels of DHEA. These data suggest that DHEA could be playing a role as an intracellular signaling molecule involved in modulating hepatic activity in response to fasting conditions.

Figure 1

PGC-1α and PGC-1α 2x9 induce gene expression of CYP11A1 and CYP17A1 in hepatic cells. Gene expression of CYP11A1 and CYP17A1 was measured by quantitative PCR in HepG2 cells (A), Hep3B cells (B), or primary human hepatocytes (C) infected with adenoviruses expressing β-gal, PGC-1α, PGC-1α 2x9, or PGC-1α L2L3M. Gene expression was normalized to 36B4 expression. Error bars represent sem of three replicates, and each graph is representative of at least three independent experiments.

Figure 2

PGC-1α and PGC-1α 2x9 increase the functional activity of CYP11A1 and CYP17A1. A, HepG2 cells were infected with adenoviruses expressing β-gal, PGC-1α, PGC-1α 2x9, or PGC-1α L2L3M and then were incubated with 200 nm [³H]pregnenolone (Preg) for 6 h. Steroids were extracted and separated by HPLC. Bar graph represents area under the curve. B and C, HepG2 cells were infected as above and incubated with 200 nm pregnenolone (B) or 10 μm 22(R)-OH cholesterol (C), and DHEA was measured using a RIA. Error bars represent sem of three replicates, and each RIA is representative of three independent experiments.

Figure 3

ERRα mediates PGC-1α induction of CYP11A1. A and B, HepG2 cells were transduced with lentiviruses expressing siRNA to either a nonspecific control si (Csi), or to ERRα (A) or HNF4α (B), followed by adenoviral delivery of β-gal, PGC-1α, PGC-1α 2x9, or PGC-1α L2L3M. Gene expression of CYP11A1 was measured by qPCR and normalized to 36B4 expression, and relevant protein expression is shown by Western blot. C, Schematic of the putative ERRE sites around the CYP11A1 gene tested by ChIP. Regions not bound by ERRα are indicated by boxes with black hash marks. The white box indicates the site identified by ChIP scanning in D. Numbering is relative to the start site. D, ChIP of ERRα and amplification of putative ERREs by qPCR, where the ERRα promoter was used as a positive control.

Figure 4

CYP17A1 is regulated by PGC-1α through ERRα. A, HepG2 cells were transduced with lentiviruses expressing either a nonspecific control si (Csi), or siERRα, followed by adenoviral transduction of β-gal, PGC-1α, PGC-1α 2x9, or PGC-1α L2L3M. Gene expression of CYP17A1 was measured by qPCR and normalized to 36B4 expression. B, Schematic of the putative ERRE sites found in and around the CYP17A1 gene region. Regions not bound by ERRα are indicated by boxes with black hash marks. White boxes indicate the position of sites identified by ChIP scanning in C. Numbering is relative to the start site. C, ChIP of ERRα and amplification of putative ERREs by qPCR, where the ERRα promoter serves as a positive control.

Figure 5

CYP17A1 is regulated by PGC-1α through HNF4α. A, HepG2 cells were transduced with lentiviruses expressing either a nonspecific control si (Csi), or siHNF4α, followed by adenoviral transduction of β-gal, PGC-1α, PGC-1α 2x9, or PGC-1α L2L3M. Gene expression of CYP17A1 was measured by qPCR and normalized to 36B4 expression. B, Schematic of the DR-1 sites found in and around the CYP17A1 gene region. Regions not bound by HNF4α are indicated by boxes with black hash marks. White box indicates the position of site identified by ChIP scanning shown in C. C, ChIP of HNF4α and amplification of regions by qPCR, where HNF4α binding to the CYP7A1 promoter served as a positive control. D, Activity of a reporter gene fused to 3.2-kb promoter region upstream of CYP17A1 tested in combination with HNF4α fused to a VP16 activation domain. VP16-SF-1 is a positive control. Each DR-1 site was mutated to examine binding activity. mut, Mutant; wt, wild type.

Figure 6

Fasting induces Cyp17A1 expression and function. A and B, Rats were fasted for 14–16 h. RNA was collected for gene expression analysis by qPCR, gene expression was normalized to cyclophilin (A), and liver samples were collected for steroid extraction and analysis of DHEA concentration by RIA (B). Boxes represent the interquartile range (25–75th percentile), with median value in the center. Whiskers mark the 10th and 90th percentiles, and dots represent measurements less than the 10th percentile or more than the 90th percentile. Statistical significance was calculated by a Student’s t test.

Figure 7

DHEA reduces the concentration of free amino acids in HepG2 cells. HepG2 cells were treated with 1 nm DHEA. Cell lysates were collected for analysis of free amino acid concentrations and normalized to total protein concentration. Error bars represent the se of three replicates, and the graph is representative of three independent experiments. Asterisks indicate significant difference (P < 0.05) by independent Student’s t test.

Figure 8

Model for fasting-induced expression of steroidogenic enzymes and synthesis of hepatic steroids. chol, Cholesterol; preg, pregnenelone.