SIR-2.1 is essential for proper fasting-dependent down-regulation of lipid synthesis and fat storage in C. elegans (A) Sudan Black staining reveals that fat storage is decreased in wild-type animals upon fasting, but is maintained in fasted sir-2.1(lof) animals. (B) Thin-liquid chromatography/gas chromatography analysis shows that triacylglycerols (TAGs) normally reduced during fasting are still present in sir-2.1(lof) animals. (C) Intestinal expression of fat-7p∷GFP is strongly decreased during fasting. (D) Chemical interference with sirtuin activity (nicotinamide [NAM], 12.5 mM; sirtinol, 0.1 mM) causes a retention of fat-7p∷GFP expression in fasted animals. (E) Fasting-dependent decreases in expression of endogenous fat-7 do not occur in sir-2.1(lof) animals. fat-7 expression is normalized to act-1. (F) The SBP-1 target gene lbp-6 is abnormally regulated in the fasting response of sir-2.1(lof) animals. Gene expression was measured by qRT–PCR normalized to act-1. Error bars represent standard deviations between parallel reactions. (G) Increased SIR-2.1 activity in nematodes results in lower levels of stored fat, as measured by Sudan Black staining. (H) C. elegans overexpressing sir-2.1 exhibit lower levels of SBP-1 target gene expression. Relative mRNA amounts of the SBP-1 target genes fat-7 or lbp-6 from fed animals were measured by qRT–PCR. Error bars represent standard deviations from parallel reactions. (*) P < 0.05; (**) P < 0.01.

SIR-2.1 is essential for proper fasting-dependent down-regulation of SBP-1-dependent gene expression and localization in C. elegans. (A) fat-7p∷GFP expressed during fasting in response to sirtuin inactivation is also dependent on sbp-1. (B) fat-7 expression is decreased in sbp-1(ep79) animals at the nonpermissive temperature in both fed and fasted conditions. (C) In contrast with fat-7 expression, mRNA expression of sbp-1 or the NHR-49 target cpt-4 was very modestly altered in sir-2.1(lof) animals. C. elegans were placed in fed or fasted conditions for 7 h. Gene expression was measured by qRT–PCR normalized to act-1. Error bars represent standard deviations between parallel reactions. (*) P < 0.05; (**) P < 0.01. (D) Nuclear levels of SBP-1∷GFP are robust in fasted SBP-1∷GFP;sir-2.1(lof) intestines. White arrows represent positions of nuclei as visualized by light microscopy.

SIRT1 regulation of SREBP target gene expression during fasting in the mouse liver. qRT–PCR was used to analyze the mRNA levels of SREBP target genes FASN](A), ELOVL6 (B), and SREBP-1c (C) in the mouse liver. The fasting-dependent changes of gene expression are compared between livers of mice subjected to tail-vein injection of adenoviruses programmed to express control shRNA (C; n = 7) and SIRT1 shRNA (shSIRT1; n = 7) (fasted for 20 h). All data are normalized to 18s rRNA expression. Error bars represent SEM. (*) P < 0.05; (**) P < 0.01. (D) Hierarchical clustering of mouse liver transcriptomes with or without SIRT1 knockout. The heat map demonstrates that SREBP target genes are among the most significantly up-regulated genes in SIRT1 knockout livers.

Mammalian SIRT1 represses SREBP-1 and SREBP-2 target gene expression. (A) SREBP target genes are expressed at higher levels in SIRT1 knockout MEFs when compared with the wild-type cells, as judged by qRT–PCR; β-actin served as the control of total RNA. (MK1) Mevalonate kinase-1; (SCD2) stearoyl-CoA desaturase-2; (SCD3) stearoyl-CoA desaturase-3. (B) Treatment of human IMR-90 fibroblasts with the sirtuin inhibitors nicotinamide (NAM; 20 mM) or sirtinol (0.1 mM) for 5 h resulted in increased expression of SREBP target genes by qRT–PCR. Data were first normalized by β-actin, then normalized by the control (0.1% DMSO), and expressed as mean ± SD. (SCD1) Stearoyl-CoA desaturase-1. (C) Expression of a dominant-negative form of SIRT1 (H363Y) in HEK293T cells increased the transcription of the LDLR, HMG-CR, HMG-CS, and SCD1 genes as assessed by qRT–PCR. SREBP-1a or SREBP-2 transcriptional levels were unchanged. GAPDH served as a loading control of total RNA. (D) Overexpression of SIRT1 decreases expression of SREBP target genes in HEK293T cells. SIRT1 and SIRT1(H363Y) (Fig. 4C) were expressed at similar levels. Error bars represent standard deviations between cDNA made from independently treated cells. (*) P < 0.05; (**) P < 0.01.

SIRT1 directly deacetylates SREBP and controls SREBP protein stability. (A) HeLa cells were transfected with Flag-tagged SREBP-1a, and then were treated with (+) or without (−) 20 mM NAM for 20 h. Flag-SREBP-1a was purified by immunoprecipitation using anti-Flag antibodies, and was eluted with Flag peptide. The presence of acetylated SREBP-1a was detected by immunoblotting using an acetyl lysine-specific antibody. Flag-SREBP-1a served as the loading control. (B) HeLa cells were treated with proteasome inhibitors alone or with proteasome inhibitors and 25 mM NAM for 6 h. Endogenous SREBP-1a was immunoprecipitated by antibodies specific to SREBP-1a. The presence of acetylated SREBP-1a was detected by immunoblotting using an acetyl lysine-specific antibody. (C) HeLa cells were treated with proteasome inhibitors alone or with proteasome inhibitors and 25 mM NAM for 6 h. Endogenous SREBP-2 was immunoprecipitated. The presence of acetylated SREBP-2 was detected by immunoblotting using an acetyl lysine-specific antibody. (D) Overexpressed Flag-SREBP-1a proteins were purified from HeLa cells treated with NAM, and were incubated with GST fusion proteins as indicated in the in vitro deacetylation assay. The degree of SREBP-1a acetylation was detected by immunoblotting using an acetyl-lysine-specific antibody. Flag-SREBP-1a served as the loading control. (E) HeLa cells were transfected with Flag-tagged SREBP-1a, and then were treated with (+) or without (−) 20 mM NAM for 20 h. After the indicated time of cycloheximide (0.1 mM) treatment, whole-cell extracts were prepared, and Flag-SREBP-1a was detected by immunoblotting using anti-Flag antibodies. Equal amounts of proteins were loaded in each lane. (F) Overexpressed Flag-SREBP-1a or Flag-SREBP-1a K324R, K333R were treated with nicotinamide and cycloheximide as in E. (G) IMR-90 cells were placed in 1% lipid-depleted serum with or without NAM for 16 h. Nuclear extracts were prepared and detected by immunoblotting with an antibody to SREBP-1. (FL) Full-length SREBP-1 precursor; (M) mature, nuclear SREBP-1. (H) Endogenous SREBP-2 in IMR-90 cells was detected as in G.

Sirtuin inhibitors regulate nuclear abundance of SREBP-1a, SREBP-2, and SREBP-1c in primary cells. IMR-90 cells were placed in 1% lipid-depleted serum and proteasome inhibitors and then treated with NAM or sirtinol for 6 h. Cells were stained with antibodies to SREBP-1a (A) or SREBP-2 (B) along with DAPI, and were visualized by immunofluorescence. (C) Hepatocytes from wild-type or SIRT1 LKO mice were stained with antibodies specific for SREBP-1c, and were stained with DAPI to visualize nuclei.

SIRT1 activators promote increased SREBP ubiquitination, decreased SREBP nuclear levels, and inhibition of SREBP target genes, and ameliorate aberrant lipogenesis in vivo. (A) HeLa cells were transfected with Flag-SREBP-1a and HA-Ubiquitin, and were treated with DMSO (vehicle) or the SIRT1 activator SRT2183. Extracts were immunoprecipitated with Flag antibodies, and then were probed by immunoblotting with antibodies to Flag or HA. (B) HeLa cells were treated with the SIRT1 activator SRT2183, and extracts were immunoblotted with an antibody to SREBP-1a. (FL) Full-length SREBP-1a precursor; (M) mature, nuclear SREBP-1a. (C) HeLa cells were placed in medium containing 1% lipid-depleted serum, low glucose, and proteasome inhibitors. Cells were treated with DMSO or SRT2183 for 6 h, fixed, and stained with an antibody to SREBP-1a. (D) SIRT1 activation decreases SREBP-dependent gene activity in human cells. HeLa cells were placed in low-glucose DMEM, supplemented with 1% fetal bovine serum with or without SRT2183. Target gene expression was analyzed by qRT–PCR normalized to β-actin levels, and error bars represent standard deviations from three independently treated wells. (*) P < 0.05; (**) P < 0.01. (E) Livers from ob/ob mice fed a high-fat diet and treated daily with vehicle or the SIRT1 activator SRT1720 for 2 or 4 wk were analyzed by qRT–PCR. Expression of SREBP-1c target genes was analyzed. Expression was normalized to 18s rRNA. Error bars show standard error for five mice. (F) Livers from ob/ob mice fed a high-fat diet and treated with vehicle or SRT1720 for 4 wk were analyzed for lipid content. Total lipid content was normalized to body weight, and error bars represent standard error. Significance was determined by Student's t-test. (**) P < 0.01.