Akt3 inhibits adipogenesis and protects from diet-induced obesity via WNK1/SGK1 signaling

Abstract

Three Akt isoforms, encoded by 3 separate genes, are expressed in mammals. While the roles of Akt1 and Akt2 in metabolism are well established, it is not yet known whether Akt3 plays a role in metabolic diseases. We now report that Akt3 protects mice from high-fat diet-induced obesity by suppressing an alternative pathway of adipogenesis via with no lysine protein kinase-1 (WNK1) and serum/glucocorticoid-inducible kinase 1 (SGK1). We demonstrate that Akt3 specifically phosphorylates WNK1 at T58 and promotes its degradation via the ubiquitin-proteasome pathway. A lack of Akt3 in adipocytes increases the WNK1 protein level, leading to activation of SGK1. SGK1, in turn, promotes adipogenesis by phosphorylating and inhibiting transcription factor FOXO1 and, subsequently, activating the transcription of PPARγ in adipocytes. Akt3-deficient mice have an increased number of adipocytes and, when fed a high-fat diet, display increased weight gain, white adipose tissue expansion, and impaired glucose homeostasis. Pharmacological blockade of SGK1 in high-fat diet-fed Akt3-deficient mice suppressed adipogenesis, prevented excessive weight gain and adiposity, and ameliorated metabolic parameters. Thus, Akt3/WNK1/SGK1 represents a potentially novel signaling pathway controlling the development of obesity.

Keywords: Adipose tissue; Metabolism; Molecular biology; Obesity.

Figure 1. Lack of Akt3 activity promotes preadipocyte differentiation into adipocytes.

(A) Microscopic images of WT, Akt1^–/–, Akt2^–/–, and Akt3^–/– MEF at day 8 after induction of adipogenesis. Scale bars: 500 μm. n = 5. (B) Oil red O staining of Akt3^–/– MEF–differentiated adipocytes after treatment with plasmid encoding Akt3 gene. Scale bar: 200 μm. n = 3. (C) Oil red O staining of 3T3 differentiated adipocytes after treatment with Akt1-specific siRNA, Akt3-specific siRNA, WT Akt3 plasmid, or mutant Akt3 (Akt3^nmf350) plasmid. 3T3-L1 cells were transfected with indicated siRNA or plasmids for 24 hours before addition of differentiation medium. Scale bar: 50 μm. n = 5. (D) BODIPY 493/503 staining of 3T3 differentiated adipocytes after treatment with Akt1-specific siRNA, Akt3-specific siRNA, WT Akt3 plasmid, or mutant Akt3 (Akt3^nmf350) plasmid. Scale bar: 100 μm. n = 5. (E) Adipogenesis in human s.c. preadipocytes treated with Akt3 siRNA. Scale bar: 200 μm. n = 3. (F) Western blot analysis of FABP4, C/EBPα, PPARγ, FAS, HSL, ATGL, MGL, and phosphorylation of HSL (Ser563) in WT and Akt3^–/– MEF–differentiated adipocytes. Right graphs show densitometric quantification. n = 4. (G) The content of triglyceride in WT and Akt3^–/– MEF before (control) or after (Diff) 1-week incubation in adipogenic media containing dexamethasone, insulin, and IBMX. n = 5. Data represent means ± SEM. *P < 0.05 by 2-tailed Student’s t test.

Figure 2. Akt3 regulates adipogenesis via a FOXO1 pathway.

(A) Plasma adiponectin levels in 20-week-old WT and Akt3^–/– mice on a chow diet (n = 10, 5 male, 5 female) were assessed by a mouse adiponectin ELISA kit. (B) Western blot analyses of FOXO1, phospho-FOXO1, and PPARγ expression in mouse embryonic fibroblasts (MEF) from WT, Akt1^–/–, and Akt3^–/– MEF. n = 4. Lower graphs show densitometric quantification. (C) Western blot analysis of FOXO1, phospho-FOXO1, and adiponectin expression in MEF differentiated adipocytes. n = 4. Lower graphs show densitometric quantification. (D) Immunofluorescent staining of WT and Akt3^–/– MEF using anti-FOXO1 or anti–phospho-FOXO1 (Ser256) antibody. Scale bar: 25 μm. n = 5. (E) Expression of FOXO1, PPARγ, Akt1, and Akt3 in 3T3 differentiated adipocytes after treatment with indicated siRNA. n = 3. Right graph shows densitometric quantification. (F) Oil red O staining of ferentiated adipocytes after treatment with plasmid encoding FOXO1 gene. Scale bar: 200 μm. n = 3. (G) The cytoplasmic and nuclear expression of Akt1, Akt3, p-Akt (thr308), and total Akt in WT and Akt3^–/– MEF. Lamin B1 was used as loading controls. Right panel shows subcellular localization of Akt1 and Akt3 in WT MEF. n = 3. C, cytoplasm; N, nucleus. (H) Subcellular localization of Akt1 and Akt3 in 3T3 differentiated adipocytes. Lipid was stained with BODIPY 493/503 (green) or HCS LipidTOX Red Neutral Lipid Stain (red). DAPI (blue) was used as the nuclear marker. Scale bar: 10 μm. n = 4. (I) Expression of Akt1, Akt2, Akt3, pan-Akt, and phospho-Akt (Ser473) in WT, Akt1^–/–, and Akt3^–/– MEF. Graphs on the right show densitometric quantification. n = 4. Right graph shows densitometric quantification. Data represent means ± SEM. *P < 0.05 by 2-tailed Student’s t test.

Figure 3. Akt3 regulates adipogenesis via a SGK1/FOXO1 pathway.

(A) Western blot analysis of SGK1 and phospho-SGK1 in WT and Akt3^–/– MEF (n = 3). (B) SGK1 mRNA expression in WT and Akt3^–/– MEF. (C) The expression of NDRG1 (a SGK1 substrate) and phospho-NDRG1 in WT and Akt3^–/– MEF (n = 3). (D) The cytoplasmic and nuclear expression of SGK1 and phospho-NDRG1 in WT and Akt3^–/– MEF. GAPDH and Lamin B1 as loading controls. n = 4. (E) Western blot analysis of SGK1, phospho-SGK1, NDRG1, and phospho-NDRG1 in WAT of female/male WT and Akt3^–/– mice fed a chow diet (n = 8, 4 females, 4 males). Graphs show SGK1 activity (phosphorylation of NDRG1) in adipocytes isolated from WAT of female mice on a chow diet (n = 6). (F) The expression of phospho-FOXO1 and FOXO1 in WT and Akt3^–/– MEF in the presence of SGK1 inhibitor (GSK650394) at different time points. n = 3. Right graph shows densitometric quantification of phospho-FOXO1 levels relative to total FOXO1 levels. (G) Phosphorylation of NDRG1 in WT and Akt3^–/– MEF in the presence of GSK650394 at different time points. n = 3. (H) The differentiation of WT and Akt3^–/– MEF into adipocytes was substantially reduced in the presence of GSK650394. Scale bar=100 μm. n = 5. Data represent means ± SEM. *P < 0.05 by 2-tailed Student’s t test.

Figure 4. Akt3 regulates SGK1 activity via WNK1.

(A) Western blot analysis of WNK1 expression in Akt3^–/– MEF and Akt3^–/– adipocytes of female mice; WNK1 and Akt3 expression in 3T3-L1 cells treated with Akt3 siRNA or WNK1 siRNA; WNK1 and phospho-NDRG1/NDRG1 ratio (SGK1 activity) in human preadipocytes treated with Akt3-specific siRNA. n = 3. (B) Western blot analysis of WNK1, NDRG1, Phospho-NDRG1 expression in Akt3^–/– MEF transfected with WNK1 siRNA for 24 hours. n = 4. (C) Adipogenesis assay using 3T3-L1 cells transfected with Akt3 siRNA or WNK1 siRNA, or cotransfected with Akt3/WNK1 siRNA for 24 hours. Scale bar: 100 μm. n = 5. (D) Comparable WNK1 mRNA expression in WT and Akt3^–/– MEF using quantitative PCR; β-actin mRNA used as a loading control. n = 7. (E) Immunoprecipitation of WNK1 from WT and Akt3^–/– MEF lysates after a 2-hour pulse-label with [³⁵S] methionine. Experiment was repeated 3 times. n = 4. (F) Decreased phosphorylation of WNK1 (Thr58) in Akt3-deficient MEF. n = 4. (G) WNK1 expression in WT and Akt3^–/– MEF treated with cycloheximide (10 μg/ml) for 6, 12, and 24 hours; β-actin used as a loading control. n = 3. (H) Immunoprecipitation of WNK1 from WT and Akt3^–/– MEF lysates with treatment of MG132 (MG, 5 μM) for 20 hours, followed by SDS-PAGE and immunoblotting using anti-ubiquitin antibody and anti-WNK1 antibody. n = 4. (I) WNK1 expression in WT and Akt3^–/– MEF treated with proteasome inhibitor MG132 (MG, 5 μM) or Bortezomib (Bor, 10 nM) for 24 hours; β-actin used as a loading control. n = 3. (J) Expression of WNK1, SGK1, NDRG1, and phosphorylation of NDRG1 in WT and Akt1^–/– MEF cells. Scale bar: 100 μm. Data represent means ± SEM. *P < 0.05 by 2-tailed Student’s t test.

Figure 5. Deficiency of Akt3 promotes white adipose tissue deposition and weight gain in mice.

(A) Body weight of sex-matched WT and Akt3^–/– mice fed a chow diet at 4, 8, 10, 12, 14, 16, 18, 20, and 22 weeks of age. n = 20 (10 male, 10 female). (B) Body weight changes in sex-matched WT and Akt3^–/– mice fed an HFD. n = 16 (8 male, 8 female). (C) Body weight of male and female WT and Akt3^–/– mice fed an HFD for 0 and 16 weeks. n = 8 (4 male, 4 female). (D) Body fat percentage of WT and Akt3^–/– mice fed a chow diet or HFD. n = 10 (5 male, 5 female). (E) Weight of white adipose tissue (WAT) of WT and Akt3^–/– mice on a chow diet or an HFD. n = 10 (5 male, 5 female). (F) Weight of gonadal (gon), retroperitoneal (retro), and s.c. (sub) white adipose tissue from WT and Akt3^–/– mice fed a chow diet or an HFD. n = 10 (5 male, 5 female). (G) Abdominal fat image of representative 10-month-old female WT and Akt3^–/– mice on a chow diet (upper panel). Lower panel shows gonadal (gon), retroperitoneal (retro), and s.c. (sub) fat pad isolated from WT and Akt3^–/– mice on a chow diet. (H) Lean mass was calculated by subtracting the fat mass from body weight of mice fed a chow diet or HFD. n = 10. (I) H&E-stained paraffin-embedded sections of gonadal WAT from 8-week-old female WT and Akt3^–/– mice fed a chow diet. Graph to the right shows size distribution of adipocytes. n = 5. Data represent means ± SEM. *P < 0.05 by ANOVA with Bonferroni post-hoc test (A and B) and 2-tailed Student’s t test (C–H).

Figure 6. SGK1 inhibitor EMD638683 prevents diet-induced obesity in Akt3^–/– mice.

Female Akt3^–/– mice fed HFD orally received EMD638683 (EMD mice, n = 9) or DMSO (control, n = 9). (A) Relative body weight (% of day 0) in EMD and control group. Treatment was terminated at day 21. (B) Lean mass of EMD and control mice. (C) Plasma liver transaminases (AST and ALT) in EMD and control mice. (D) Weight of gonadal (gon), retroperitoneal (retro), and s.c. (sub) white adipose tissue from EMD and control mice. (E) Body fat percentage in EMD mice and control mice. (F) Western blotting analyses of phospho-NDRG1 and NDRG1 expression in WAT from EMD mice and control mice. (G) H&E staining of gonadal WAT from EMD and control mice. Graphs to the right show size distribution of adipocytes. Scale bar: 50 μm. (H) Plasma free fatty acids level and glucose level in EMD and control mice. (I) Absolute adiponectin and relative adiponectin levels (relative to WAT weight) in EMD mice and control mice. Data represent means ± SEM. *P < 0.05 by ANOVA with Bonferroni post-hoc test (A) and 2-tailed Student’s t test (B–F, H, I).