SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation

Abstract

The family of mammalian Sirtuin proteins comprises seven members homologous to yeast Sir2. Here we show that SIRT2, a cytoplasmic sirtuin, is the most abundant sirtuin in adipocytes. Sirt2 expression is downregulated during preadipocyte differentiation in 3T3-L1 cells. Overexpression of SIRT2 inhibits differentiation, whereas reducing SIRT2 expression promotes adipogenesis. Both effects are accompanied by corresponding changes in the expression of PPARgamma, C/EBPalpha, and genes marking terminal adipocyte differentiation, including Glut4, aP2, and fatty acid synthase. The mechanism underlying the effects of reduced SIRT2 in 3T3-L1 adipocytes includes increased acetylation of FOXO1, with direct interaction between SIRT2 and FOXO1. This interaction enhances insulin-stimulated phosphorylation of FOXO1, which in turn regulates FOXO1 nuclear and cytosolic localization. Thus, Sirt2 acts as an important regulator of adipocyte differentiation through modulation of FOXO1 acetylation/phosphorylation and activity and may play a role in controlling adipose tissue mass and function.

Figure 1

Expression of Sirt2 and stable Sirt2 knockdown in 3T3-L1 preadipocytes. (A) Affymetrix microarray analysis was performed using mRNA isolated from epididymal adipocytes as described previously (Gesta et al., 2006). To confirm these finding, quantitative realtime PCR was performed as described in Material and Methods. (B) Using Realtime PCR, changes in expression of the different Sirt mRNA was assessed during 3T3-L1 white adipocyte differentiation. Sirt2 mRNA was the most abundant in adipocytes and both Sirt1 and Sirt2 had similar pattern of diminishing expression during adipocyte differentiation. (C) shRNA overexpression constructs were generated with pSuper-Retro vector. Two shRNA constructs (S-1 and S-2) were tested targeting different exons of Sirt2 genomic sequence. An shGFP RNAi sequence was used as control. After retroviral infection and selection, 3T3-L1 preadipocytes carrying either shGFP or shSirt2 overexpression constructs were grown to confluence then RNA was extracted to synthesize cDNA and realtime PCR was performed for Sirt 1-3. (D) Transient transfection experiments were done using two different shRNA and control shRNA constructs along with either control pBabe or Sirt2-FLAG overexpression. Both shSirt2 (S1 and S2) effectively knockdown the overexpression of Sirt2-FLAG protein. Endogenous Sirt2 knockdown was also detected by western blot.

Figure 2

Sirt2 knockdown promotes 3T3L1 adipocyte differentiation. Stable shRNA transfected 3T3-L1 preadipocytes were subjected to differentiation using the standard protocol. Oil Red O staining of shGFP and shSirt2 cells on day 4 of differentiation indicated that shSirt2 had accelerated differentiation with enhanced lipid staining. During 3T3-L1 adipocyte differentiation, shSirt2 cells (empty circles) had consistently lower endogenous Sirt2 mRNA expression compared with shGFP cells (solid circles). The mRNA expression for various differentiation markers was also determined by realtime PCR. The protein expression of different adipocyte differentiation markers was determined by Western blotting.

Figure 3

Sirt2 overexpression inhibits 3T3-L1 adipocyte differentiation without affecting insulin signaling in preadipocytes. (A). Following the differentiation protocol described in Material and Methods, Oil Red O staining of stably transfected 3T3-L1 cells with either control pBabe vector or Sirt2-FLAG-pBabe overexpression construct showed that exogenous Sirt2 overexpression inhibited adipocyte differentiation as compared with control cells. (B). Insulin signaling was assessed by western blotting of phospho-Akt, phospho-p38 and phospho-MAP kinase in confluent 3T3L1 preadipocytes. Stimulation was performed using 10 nM and 100 nM insulin for 5 min.

Figure 4

Sirt2 knockdown promotes FoxO1 acetylation. Non-denaturing total protein extracts from either shGFP or shSirt2 cells were immunoprecipitated with anti-acetylated-lysine antibody and precipitated lysates were blotted with anti-FoxO1 antibody. Total lysate input was detected by western blotting.

Figure 5

Sirt2 interacts with FoxO1 in vitro and Sirt2 knockdown promotes FoxO1 phosphorylation and cytosolic localization. (A) The non-denaturing lysates from either pBabe control or Sirt2-FLAG overexpression cell lines were immunoprecipitated with anti-FLAG-agarose. The precipitated lysates were blotted with anti-FoxO1 antibody. Markedly more FoxO1 protein was precipitated with anti-FLAG-agarose from Sirt2-FLAG overexpressing cells. (B) The non-denaturing lysates from HEK293 cells transiently transfected with Sirt2-HA and/or FoxO1-FLAG overexpressing constructs were subjected to immunoprecipitation with anti-HA agarose. Western blot of protein eluted from HA-Agarose shows that there is interaction between Sirt2 and FoxO1 in vitro. (C) The shGFP or shSirt2 cells were acutely (5 or 15 minutes) stimulated with different concentrations of insulin (10 nM and 100 nM) after serum deprivation. Insulin stimulated Akt and GSK3β phosphorylation (5 min stimulation) and FoxO1 phosphorylation (15 min stimulation) were assessed by western blotting. (D) Following a modified protocol for cytosolic and nuclear extract described previously (Emanuelli et al., 2000), lysates from both shGFP and shSirt2 cells were subjected to western blot analysis with anti-FoxO1 antibody. There was more FoxO1 protein translocated to the cytosol in shSirt2 3T3L1 cells. SOD4 and LaminA (LmnA) bands showed effective separation of nuclear and cytosolic proteins. Immunocytochemistry was done with cells carrying FoxO1-FLAG overexpressing construct with either stably transfected shGFP or shSirt2. Cells were fixed 48 hours after being plated in 10% FBS DMEM media. The anti-FLAG-FITC was used to detect subcellular localization of the recombinant FoxO1 in the cells.

Figure 6

FoxO1 acetylation/deacetylation mimics regulate 3T3-L1 adipocyte differentiation and FoxO1 phosphorylation. (A) Different FoxO1 overexpression constructs were made with either wild type FoxO1 amino acid sequence or replacing all three lysine residues surrounding Ser-253 with Glutamine (KQ) or Arginine (KR). The FoxO1 WT, KQ, and KR overexpression constructs were all FLAG tagged. Overexpression was determined by western blotting using anti-FLAG antibody. Quantitative PCR with primers targeting the FoxO1 coding region showed that the level of overexpression of different constructs was similar and was about 5 times the level of endogenous FoxO1 observed in control cells (Supplemental figure2). Cell lines carrying various FoxO1 overexpression constructs or control cells were subjected to the differentiation protocol described in Material and Methods. Oil Red O staining of cells eight days after differentiation induction showed differences among different cell lines. Realtime PCR quantification of different adipocyte markers was consistent with the degree of adipocyte differentiation as accessed by bright light microscopic image and Oil Red O staining. Cells overexpressing wildtype FoxO1 had significantly decreased mRNA expression of different adipocyte differentiation markers comparing with control cells, as indicated with “a”; while cells expressing the KR mutant had significantly decreased mRNA expression compared with that of wild type FoxO1 overexpression, as indicated with “b”. (B) FoxO1 mutations mimicking different lysine acetylation states affect Ser-253 phosphorylation of FoxO1 and 3T3L1 adipocyte differentiation. After serum deprivation for 12 hours, 3T3L1 cells carrying wild type FoxO1, KQ, and KR mutant overexpression constructs were acutely stimulated with different concentrations (10 nM or 100 nM) of insulin for 10 minutes. Total cell extracts were subjected to western blot analysis to assess insulin stimulated phosphorylation status of FoxO1, Akt, and GSK3β. KQ mutant overexpression promoted both basal and insulin stimulated FoxO1 phosphorylation, whereas cells overexpressing the KR mutant had decreasedvFoxO1 phosphorylation in response to insulin, both as compared to cells overexpressing wildtype FoxO1. (C) Immunocytochemistry of different FoxO1 wildtype and mutants overexpressing cells was performed using anti-FLAG-FITC. Different subcellular localization patterns are observed for FoxO1 mutants.