Serum- and glucocorticoid-inducible kinase 1 (SGK1) regulates adipocyte differentiation via forkhead box O1

Abstract

The serum and glucocorticoid-inducible kinase 1 (SGK1) is an inducible kinase the physiological function of which has been characterized primarily in the kidney. Here we show that SGK1 is expressed in white adipose tissue and that its levels are induced in the conversion of preadipocytes into fat cells. Adipocyte differentiation is significantly diminished via small interfering RNA inhibition of endogenous SGK1 expression, whereas ectopic expression of SGK1 in mesenchymal precursor cells promotes adipogenesis. The SGK1-mediated phenotypic effects on differentiation parallel changes in the mRNA levels for critical regulators and markers of adipogenesis, such as peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, and fatty acid binding protein aP2. We demonstrate that SGK1 affects differentiation by direct phosphorylation of Foxo1, thereby changing its cellular localization from the nucleus to the cytosol. In addition we show that SGK1-/- cells are unable to relocalize Foxo1 to the cytosol in response to dexamethasone. Together these results show that SGK1 influences adipocyte differentiation by regulating Foxo1 phosphorylation and reveal a potentially important function for this kinase in the control of fat mass and function.

Figure 1

SGK1 is expressed in fat tissue and induced during adipogenesis. A, SGK1 mRNA levels in tissues obtained from C57BL/6J 8-wk-old male mice. Relative mRNA levels were determined by real-time PCR and normalized using 18S. B, mRNA levels of SGK1 were evaluated during differentiation (0–6 d) after MDI induction or after treatment with rosiglitazone and insulin (3T3-L1 cells) or after MDI or troglitazone and insulin treatment (10T1/2 cells) (*,#, P < 0.05; **,##, P < 0.01; ***,###, P < 0.001; ns, nonsignificant). C, SGK1 mRNA levels in 3T3-L1 after stimulation with a specific inducer (*, P < 0.05; **, P < 0.01, ***, P < 0.001; ns, nonsignificant). BAT, Brown adipose tissue; IBMX, isobutylmethylxanthine; WAT, white adipose tissue.

Figure 2

SGK1 modulates adipogenesis. A, SGK1 mRNA levels were measured by RT-PCR in constitutively active SGK1 (^S422DSGK1)-expressing cells and compared with control vector cells (***, P < 0.001). B, Oil Red O staining of 10T1/2 ectopically expressing ^S422DSGK1 after 6 d of differentiation showed increased lipid accumulation. C, Ectopic expression of ^S422DSGK1 in 10T1/2 enhanced PPARγ, CEBPα, and aP2 mRNA levels in comparison to vector during MDI-induced differentiation. (***, P < 0.001). D, Analysis of SGK1 mRNA levels in 10T1/2 cells expressing either control siRNA or si-SGK1 (***, P < 0.001). E, Oil Red O staining of 10T1/2 cells expressing si-SGK1 showed decreased lipid accumulation compared with si-ctl expressing cells at d 6 of differentiation. F, PPARγ, CEBPα, and aP2 mRNA levels were reduced in SGK1 knockdown 10T1/2 cells compared with control-expressing cells induced to differentiate with MDI (***, P < 0.001). si-ctl, Small interfering control.

Figure 3

SGK1 phosphorylates Foxo1. A, In vitro kinase assays performed in the presence of [γ-³²P]ATP and purified GST-Foxo1 or GST proteins. B, Western blot analysis of U2OS cells cotransfected with GFP-Foxo1 WT, GFP-Foxo1AAA, or control vector, in the presence or absence of either constitutively active SGK1 (^S422DSGK1) or SGK1 kinase-dead (^K127NSGK1). Phospho-specific antibodies recognizing phosphorylated Foxo1 protein at residues T-24, S-256, and S-319 were used to visualize phosphorylation. GFP and β-actin antibodies were used as controls. C, Phosphorylation levels of endogenous Foxo1 in 10T1/2 cells expressing constitutively active ^S422DSGK1 or kinase-dead ^K127NSGK1 or vector, using phospho-specific antibodies. D, Total and phosphoryated levels of endogenous SGK1 (P-SGK1) and Foxo1 (P-Foxo1) proteins during a time course of 3T3-L1 differentiation.

Figure 4

Foxo1 subcellular localization is SGK1 dependent. Panel A, U2OS and 10T1/2 cells were transfected with GFP-Foxo1 in combination with constitutively active ^S422DSGK1 or vector and nuclear /cytoplasmic localization monitored by fluorescence. Panel B, GFP-Foxo1 WT or GFP-Foxo1AAA (T24A, S256A, S319A) were cotransfected in U2OS cells with constitutively active ^S422DSGK1 or vector. Quantification of the number of cells expressing GFP-Foxo1 WT or GFP-Foxo1AAA in the nucleus or in the cytoplasm were expressed as percentage relative to the total number of cells counted per subcellular compartment (***, P < 0.001; ns, not significant; N, nuclear localization; C, cytoplasmic localization). Panel C, Quantification of the number of cells showing endogenous Foxo1 localized either in the nucleus or in the cytoplasm, in WT or in SGK1−/− MEF cells in the presence of dexamethasone or vehicle alone. The values are expressed as percentage of number of cells with nuclear or cytoplasmic Foxo1 vs. total number of cells counted per field (***, P < 0.001; ns, not significant; N, nuclear localization; C, cytoplasmic localization; Dex, dexamethasone). Panel D, Subcellular localization of endogenous Foxo1 in WT or SGK1−/− MEF cells. Endogenous Foxo1 is visualized in red and nuclei with 4′,6-diamidino-2-phenylindole staining.

Figure 5

SGK1 rescues Foxo1-inhibitory effect on adipogenesis. Oil Red O staining of 10T1/2 cells expressing (A) vector, Foxo1 WT, or Foxo1AAA or (B) si-control vs. si-Foxo1. C, PPARγ, C/EBPα, and aP2 levels in 10T1/2 cells expressing Foxo1 WT, Foxo1AAA, or control vector, at 3 d of differentiation. (***, P < 0.001). D, aP2 mRNA levels in 10T1/2 cells expressing si-Foxo1 or si-control. E, PPARγ, C/EBPα, and aP2 mRNA levels in 10T1/2 expressing Foxo1 WT or Foxo1AAA and constitutively active ^S422DSGK1 or inactive ^K127NSGK1 (***, P < 0.001; ns, nonsignificant). F, PPARγ, C/EBPα, and aP2 mRNA levels determined after 3 d of differentiation in 10T1/2 cells expressing si-control or si-SGK1 in the presence of vector, GFP-Foxo1 WT, or GFP-Foxo1AAA. (***, P < 0.001; ns, not significant). si-ctl, si-control.