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. 2003 Apr 1;100(7):4012-7.
doi: 10.1073/pnas.0730870100. Epub 2003 Mar 21.

Regulation of hepatic fasting response by PPARgamma coactivator-1alpha (PGC-1): requirement for hepatocyte nuclear factor 4alpha in gluconeogenesis

James Rhee  1 , Yusuke Inoue, J Cliff Yoon, Pere Puigserver, Melina Fan, Frank J Gonzalez, Bruce M Spiegelman
Affiliations

Regulation of hepatic fasting response by PPARgamma coactivator-1alpha (PGC-1): requirement for hepatocyte nuclear factor 4alpha in gluconeogenesis

James Rhee et al. Proc Natl Acad Sci U S A. .

Abstract

The liver plays several critical roles in the metabolic adaptation to fasting. We have shown previously that the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) is induced in fasted or diabetic liver and activates the entire program of gluconeogenesis. PGC-1alpha interacts with several nuclear receptors known to bind gluconeogenic promoters including the glucocorticoid receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), and the peroxisome proliferator-activated receptors. However, the genetic requirement for any of these interactions has not been determined. Using hepatocytes from mice lacking HNF4alpha in the liver, we show here that PGC-1alpha completely loses its ability to activate key genes of gluconeogenesis such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase when HNF4alpha is absent. It is also shown that PGC-1alpha can induce genes of beta-oxidation and ketogenesis in hepatocytes, but these effects do not require HNF4alpha. Analysis of the glucose-6-phosphatase promoter indicates a key role for HNF4alpha-binding sites that function robustly only when HNF4alpha is coactivated by PGC-1alpha. These data illustrate the involvement of PGC-1alpha in several aspects of the hepatic fasting response and show that HNF4alpha is a critical component of PGC-1alpha-mediated gluconeogenesis.

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Figures

Figure 1
Figure 1
HNF4α is required for gluconeogenesis. Control (HNF4α flox) mice or mice lacking liver HNF4α were fed ad libitum or fasted for 14, 24, or 48 h. RNA taken from the livers of these mice was analyzed for HNF4α, PEPCK, G6Pase, and PGC-1α. GAPDH was blotted to verify equal loading.
Figure 2
Figure 2
PGC-1α requires HNF4α to induce the genes of gluconeogenesis. Primary hepatocytes taken from control and liver HNF4α knockout mice were infected with adenovirus encoding either GFP or PGC-1α as described in Materials and Methods. RNA was subsequently harvested and analyzed for the expression of gluconeogenic, β-oxidative, and ketogenic markers. 36B4 was blotted as an equal loading control. CPT1, carnitine palmitoyl transferase 1; MCAD, medium-chain acyl-CoA dehydrogenase; HMG-CoA lyase, 3-hydroxy-3-methylglutaryl-CoA lyase.
Figure 3
Figure 3
PGC-1α strongly coactivates HNF4α on the G6Pase promoter. (A) Coactivation is independent of the IRU. SV40-transformed hepatocytes were transfected with HNF4α ± PGC-1α. Transcriptional activity was measured by using a reporter construct containing the G6Pase promoter upstream of luciferase. A wild-type version of the reporter (−1227/+57 G6Pase Luc wt) was compared with one lacking an intact IRU (−1227/+57 G6Pase Luc mIRU). (B) PGC-1α coactivates HNF4α on a region of the promoter between nucleotides −298 and −180. Reporter constructs representing various truncations of the promoter were cotransfected with HNF4α ± PGC-1α. These graphs are representative of at least three independent trials.
Figure 4
Figure 4
Identification of HNF4α-binding sites in the G6Pase promoter. (A) Computer algorithm search for HNF4α-binding sites. The four highest-scoring potential HNF4α-binding sites (DR1s) between −298 and −180 of the G6Pase promoter are depicted with their position and sequence. The nucleotides in bold correspond to the consensus HNF4α-binding site. Boxed nucleotides were mutated within the context of the parent promoter construct. (B) HNF4α protein binds in vitro to sites A and B. Electrophoretic mobility-shift assays were conducted in which in vitro-translated HNF4α was incubated with radiolabeled probes corresponding to the sites depicted in A. The single arrow marks the level of the HNF4α-specific complex. A slower migrating complex is visible after the addition of an antibody against HNF4α. Free probe was in excess and is not shown. (C) Mutation of site B, and to a lesser extent of site A, impairs PGC-1α coactivation of HNF4α on the G6Pase promoter. Sites A–D on the G6Pase promoter were mutated as depicted in A. SV40-transformed hepatocytes were cotransfected with HNF4α and PGC-1α in the presence of these mutant reporters. The graph is representative of three independent trials.
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