Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Oct 2;284(40):27042-53.
doi: 10.1074/jbc.M109.047340. Epub 2009 Aug 3.

Activation of SIRT1 by resveratrol represses transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) by deacetylating hepatic nuclear factor 4alpha

Jianqi Yang  1 , Xiaoying Kong, Maria Emilia S Martins-Santos, Gabriela Aleman, Ernestine Chaco, George E Liu, Shwu-Yuan Wu, David Samols, Parvin Hakimi, Cheng-Ming Chiang, Richard W Hanson
Affiliations

Activation of SIRT1 by resveratrol represses transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) by deacetylating hepatic nuclear factor 4alpha

Jianqi Yang et al. J Biol Chem. .

Abstract

The SIRT1 activators isonicotinamide (IsoNAM), resveratrol, fisetin, and butein repressed transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C). An evolutionarily conserved binding site for hepatic nuclear factor (HNF) 4alpha (-272/-252) was identified, which was required for transcriptional repression of the PEPCK-C gene promoter caused by these compounds. This site contains an overlapping AP-1 binding site and is adjacent to the C/EBP binding element (-248/-234); the latter is necessary for hepatic transcription of PEPCK-C. AP-1 competed with HNF4alpha for binding to this site and also decreased HNF4alpha stimulation of transcription from the PEPCK-C gene promoter. Chromatin immunoprecipitation experiments demonstrated that HNF4alpha and AP-1, but not C/EBPbeta, reciprocally bound to this site prior to and after treating HepG2 cells with IsoNAM. IsoNAM treatment resulted in deacetylation of HNF4alpha, which decreased its binding affinity to the PEPCK-C gene promoter. In HNF4alpha-null Chinese hamster ovary cells, IsoNAM and resveratrol failed to repress transcription from the PEPCK-C gene promoter; overexpression of HNF4alpha in Chinese hamster ovary cells re-established transcriptional inhibition. Exogenous SIRT1 expression repressed transcription, whereas knockdown of SIRT1 by RNA interference reversed this effect. IsoNAM decreased the level of mRNA for PEPCK-C but had no effect on mRNA for glucose-6-phosphatase in AML12 mouse hepatocytes. We conclude that SIRT1 activation inhibited transcription of the gene for PEPCK-C in part by deacetylation of HNF4alpha. However, SIRT1 deacetylation of other key regulatory proteins that control PEPCK-C gene transcription also likely contributed to the inhibitory effect.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Transcriptional repression of the PEPCK-C gene promoter by SIRT1 activators. HepG2 cells were co-transfected with 200 ng of p2000-Luc plasmid DNA and 50 ng of PKAc plasmid DNA or 50 ng of empty vector DNA, using the protocol described under “Experimental Procedures.” Cells were then treated with the indicated concentrations of SIRT1 activators, namely IsoNAM (panel A), resveratrol (panel B), and fisetin and butein (panel C) for 24 h. The inset in panel A was redrawn from data in Table 5 of Reshef and Hanson (58), in which fed rats were given 5 mmol of nicotinamide (NAM)/kg of body weight for 5 h and the activity of hepatic PEPCK-C determined. A unit of enzyme activity represents the conversion of 1 μmol of substrate to product per min. The IC50 was the concentration of IsoNAM required to achieve 50% inhibition of transcription from the PEPCK-C gene promoter. Values were expressed as mean ± S.E. of triplicate determinations. *, p < 0.001 and **, p < 0.023 as compared with untreated controls. Northern blotting (panel D) and RT-PCR (panel E) were used to determine endogenous mRNA levels of PEPCK-C in FTO-2B and HepG2 cells treated with IsoNAM for various times, or in AML12 mouse hepatocytes treated with IsoNAM at the indicated concentrations. The 18S rRNA stained with ethidium bromide or glyceraldehyde-3-phosphate dehydrogenase mRNA was used as loading controls. Panel F, IsoNAM-induced inhibition of PEPCK-C was reversible. FTO-2B cells were cultured in Krebs-Ringer bicarbonate buffer, supplemented with 2% of bovine serum albumin and 5 mm glucose in the absence (untreated) or presence of 5 mm IsoNAM (treated). After 6 h of treatment, IsoNAM was withdrawn from culture medium. Cells were collected at the time points indicated. PEPCK-C mRNA was measured using RT-PCR; the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as a control.
FIGURE 2.
FIGURE 2.
A novel HNF4α binding site in the PEPCK-C gene promoter is required for IsoNAM-induced transcriptional repression. Panel A, HepG2 cells were treated with 5 mm IsoNAM or water (untreated control) after co-transfection of PKAc DNA and luciferase reporter constructs, which harbored various segments of the PEPCK-C gene promoter from rat. IsoNAM-dependent repression of transcription from the PEPCK-C gene promoter was expressed as a percent inhibition of treated as compared with untreated cells. Values were expressed as mean ± S.E. of four separate tests, with each test performed in triplicate. Panel B, the sequence from −272 to −208 of the rat PEPCK-C gene promoter was aligned with corresponding sequences from the mouse (Mou), human (Hum), chimpanzee (Chp), monkey (Mon), dog (Dog), cow (Cow), and chicken (Chn). Evolutionary conserved sites are indicated by boxes. Panel C, HNF4α binding site-2 interacted with HNF4α. Purified FLAG-HNF4α was used to perform an EMSA with the wild type or mutated DNA oligonucleotides corresponding to the HNF4α binding site-2 sequence, or with the wild type DNA corresponding to AF1, as described under “Experimental Procedures.” For the supershift assays, either anti-HNF4α serum or anti-β-actin serum was added to the reactions. Panel D, HNF4α binding sites were mutated individually or jointly with other conserved binding sites for AP1 and C/EBP in the p2000-Luc plasmid. The effect of IsoNAM on transcription from the PEPCK-C gene promoter, in the presence of co-transfected PKAc was measured using the procedure described under panel A. Values were expressed as mean ± S.E. of three separated tests, with each test done in triplicate. *, p < 0.001, and **, p < 0.016 as compared with the wild type gene promoter control. Panel E, an artificial promoter, composed of three copies of HNF4α-AP1-C/EBP binding sequences (−277/−218) and the TK minimal gene promoter, was cloned into pGL3-Basic vector to generate a reporter plasmid (inset). When this reporter plasmid was co-transfected with PKAc into HepG2 cells, IsoNAM caused a dose-dependent inhibition of transcription from the PEPCK-C gene promoter. Values are expressed as mean ± S.E. of three separate analyses.
FIGURE 3.
FIGURE 3.
HNF4α is required for SIRT1 activator-induced transcriptional repression of PEPCK-C. Chinese hamster ovary cells were co-transfected with p2000-Luc, PKAc, and various amounts of plasmid overexpressing HNF4α. Cells were then treated with 5 mm IsoNAM or 20 μm resveratrol. The total amount of DNA for each transfection was adjusted to the same value by adding empty vector DNA. The results were expressed as the mean ± S.E. of triplicate measurements. *, p < 0.001 and **, p < 0.004. Insets show levels of HNF4α in cells as determined by Western blotting.
FIGURE 4.
FIGURE 4.
IsoNAM-induced deacetylation of HNF4α attenuates its binding to PEPCK-C gene promoter. Panel A, IsoNAM treatment resulted in a time-dependent deacetylation of nuclear HNF4α. HepG2 cells were treated with 5 mm IsoNAM or water for the times indicated in the panel. Total nuclear HNF4α was measured using Western blotting; lamin B was used as a loading control. The acetylated species of endogenous HNF4α was assayed using immunoprecipitation of HNF4α, followed by Western blotting of acetyl-lysine. Panel B, SIRT1 deacetylated HNF4α in vitro. Purified FLAG-HNF4α was mixed with reaction buffer or reaction buffer plus SIRT1. The acetylation status of HNF4α was then assayed using Western blotting for acetyl-lysine. The band density was quantified by densitometry and the value of the control was set as 1. Panel C, deacetylation of HNF4α attenuated its binding to DNA. Purified FLAG-HNF4α was deacetylated in vitro by SIRT1 in the presence or absence of NAD+. The resultant proteins were used in EMSA reactions. Panel D, IsoNAM treatment attenuated the binding of HNF4α to the HNF4α site-2 in the PEPCK-C gene promoter as determined by a ChIP assay. HepG2 cells were treated with water or 5 mm IsoNAM for the times indicated. Binding of HNF4α to the HNF4α site-2 was assessed using a ChIP assay, as described under “Experimental Procedures.”
FIGURE 5.
FIGURE 5.
IsoNAM enhances binding of AP-1 to the PEPCK-C gene promoter. Panel A, an EMSA of the purified AP-1 components bound to the overlapping HNF4α site-2/AP-1 region in the PEPCK-C gene promoter. Panel B, purified AP-1 and HNF4α compete for the overlapping HNF4α site-2/AP-1 region in the PEPCK-C gene promoter. Panel C, IsoNAM enhanced the interaction of AP-1, but decreased the binding of HNF4α, to the PEPCK-C gene promoter. HepG2 cells were treated 5 mm IsoNAM for 10 h. A ChIP assay was used to monitor the binding of AP-1, HNF4α, and C/EBPβ to the overlapping HNF4α site-2/AP-1 region (target region). Panel D, AP-1 and HNF4α competitively regulated the CRE-mutated PEPCK-C gene promoter. HepG2 cells were co-transfected with a CRE-mutated p2000-Luc plasmid and with 0.05 and 0.1 μg of plasmids expressing HNF4α or AP-1 and luciferase activity was measured. The values are expressed as the mean ± S.E. of triplicate measurements. *, p < 0.05 and **, p < 0.01. BSA, bovine serum albumin.
FIGURE 6.
FIGURE 6.
SIRT1 represses transcription of the PEPCK-C gene in HepG2 cells. Panel A, mouse SIRT1 was overexpressed in HepG2 cells in the absence (open bar) or presence of PKAc (black bar). Transcription of the PEPCK-C gene promoter was determined using a luciferase reporter assay. Panel B, RNAi knock-down of SIRT1 attenuated IsoNAM-induced transcriptional repression of PEPCK-C. HepG2 cells were co-transfected with p2000-Luc and no RNAi, control RNAi, or Sirt1 RNAi, and then treated with water (open bar) or 5 mm IsoNAM (black bar). Luciferase activity was measured 24 h later. The insert is a Western blot of SIRT1 in the lysates of the transfected cells. The luciferase activities were expressed as the mean ± S.E. from triplicate measurements.
FIGURE 7.
FIGURE 7.
A model for the SIRT1-induced transcriptional repression of PEPCK-C gene promoter. PEPCK-C gene promoter and several key transcription factor binding sites are drawn to scale.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hanson R. W., Patel Y. M. (eds) (1994) P-enolpyruvate Carboxykinase: The Gene and the Enzyme, John Wiley and Sons, New York
    1. Olswang Y., Cohen H., Papo O., Cassuto H., Croniger C. M., Hakimi P., Tilghman S. M., Hanson R. W., Reshef L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 625–630 - PMC - PubMed
    1. Franckhauser S., Muñoz S., Pujol A., Casellas A., Riu E., Otaegui P., Su B., Bosch F. (2002) Diabetes 51, 624–630 - PubMed
    1. Franckhauser S., Muñoz S., Elias I., Ferre T., Bosch F. (2006) Diabetes 55, 273–280 - PubMed
    1. Reshef L., Olswang Y., Cassuto H., Blum B., Croniger C. M., Kalhan S. C., Tilghman S. M., Hanson R. W. (2003) J. Biol. Chem. 278, 30413–30416 - PubMed

Publication types

MeSH terms

LinkOut - more resources