Withaferin A inhibits adipogenesis in 3T3-F442A cell line, improves insulin sensitivity and promotes weight loss in high fat diet-induced obese mice

Abstract

The increased prevalence of obesity and associated insulin resistance calls for effective therapeutic treatment of metabolic diseases. The current PPARγ-targeting antidiabetic drugs have undesirable side effects. The present study investigated the anti-diabetic and anti-obesity effects of withaferin A (WFA) in diet-induced obese (DIO) C57BL/6J mice and also the anti-adipogenic effect of WFA in differentiating 3T3- F442A cells. DIO mice were treated with WFA (6 mg/kg) or rosiglitazone (10 mg/kg) for 8 weeks. At the end of the treatment period, metabolic profile, liver function and inflammatory parameters were obtained. Expression of selective genes controlling insulin signaling, inflammation, adipogenesis, energy expenditure and PPARγ phosphorylation-regulated genes in epididymal fats were analyzed. Furthermore, the anti-adipogenic effect of WFA was evaluated in 3T3- F442A cell line. WFA treatment prevented weight gain without affecting food or caloric intake in DIO mice. WFA-treated group also exhibited lower epididymal and mesenteric fat pad mass, an improvement in lipid profile and hepatic steatosis and a reduction in serum inflammatory cytokines. Insulin resistance was reduced as shown by an improvement in glucose and insulin tolerance and serum adiponectin. WFA treatment upregulated selective insulin signaling (insr, irs1, slc2a4 and pi3k) and PPARγ phosphorylation-regulated (car3, selenbp1, aplp2, txnip, and adipoq) genes, downregulated inflammatory (tnf-α and il-6) genes and altered energy expenditure controlling (tph2 and adrb3) genes. In 3T3- F442A cell line, withaferin A inhibited adipogenesis as indicated by a decrease in lipid accumulation in differentiating adipocytes and protein expression of PPARγ and C/EBPα. The effect of rosiglitazone on physiological and lipid profiles, insulin resistance, some genes expression and differentiating adipocytes were markedly different. Our data suggest that WFA is a promising therapeutic agent for both diabetes and obesity.

Fig 1. Effect of WFA treatment on diet-induced obese C57BL/6J mice.

(A-D) Mice fed with ND received vehicle and mice fed with HFD received WFA (6 mg/kg/three times a week), RSG (10 mg/kg/day) or vehicle for 8 weeks. (A) body weight (B) percentage change in body weight. (C) daily food intake and (D) daily caloric intake. Values are represented as the mean ± SD, n = 8–10. **p < .01 and ***p < .001 vs HFD (vehicle) and ^###p < .001 vs ND (vehicle).

Fig 2. Effect of WFA treatment on liver and adipose tissue in diet-induced obese C57BL/6J mice.

(A-G) ND group received vehicle and HFD group received WFA (6 mg/kg/three times a week), RSG (10 mg/kg/day) or vehicle for 8 weeks. The mass of (A) epididymal fat, (B) perirenal fat, (C) mesenteric fat, and (D) liver were normalized to body weight. Histopathological images of H&E stained sections of (E) liver and (F) adipose tissues. Values are represented as the mean ± SD, n = 8–10. *p < .05, **p < .01 and ***p < .001 vs HFD (vehicle) and ^###p< .001 vs ND (vehicle). Black arrows indicate fat deposition in the mice livers. (Magnification: 400×, Scale bar: 50 μm) (E) and (F). Adipocyte size was measured using the ImageJ software with Adiposoft plugin (G).

Fig 3. Effect of WFA treatment on glucose homeostasis in diet-induced obese C57BL/6J mice.

(A-E) Mice fed with ND received vehicle and mice fed with HFD received WFA (6 mg/kg/three times a week), RSG (10 mg/kg/day) or vehicle for 8 weeks. (A) OGTT curve and its (B) area under the curve and (C) ITT curve and its (D) area under the curve and (E) insulin resistance index. Values are represented as the mean ± SD, n = 8–10. *p < .05, **p < .01 and ***p < .001 vs HFD (vehicle) and ^###p < .001 vs ND (vehicle).

Fig 4. Effect of WFA treatment on serum adipokines and insulin in diet-induced obese C57BL/6J mice.

(A-G) Mice fed with ND received vehicle and mice fed with HFD received WFA (6 mg/kg/three times a week), RSG (10 mg/kg/day) or vehicle for 8 weeks. Serum levels of (A) TNF-α, (B) IL-6, (C) MCP-1, (D) resistin, (E) leptin (F) adiponectin and (G) insulin. Values are represented as the mean ± SD, n = 8–10. *p < .05, **p < .01 and ***p < .001 vs HFD (vehicle) and ^###p < .001 vs ND (vehicle).

Fig 5. Inhibitory effect of WFA on adipogenesis.

The effect of WFA on viability of (A) preadipocytes and (B) differentiating adipocytes. (C) Representative images of Oil Red O stained differentiating 3T3‐F442A preadipocytes treated with WFA (0.25–1 μM) and RSG (2 μM). Intracellular fat droplets were stained with Oil Red O and examined using light microscopy (magnification: 50x, scale bar: 200 μm). (D) The percentage of differentiation was calculated using the intensity of Oil Red O relative to that of control cells, assigned as 100% differentiation. (E) Protein expressions of PPARγ, and C/EBPα in differentiating 3T3-F442A cells on day 8 of differentiation. (F) The relative protein expression of PPARγ and C/EBPα. The values are represented as mean ± SD of three independent experiments. *p < .05, **p < .01 and ***p<0.001 vs vehicle control. ND = non differentiated cells, VC = vehicle control, WFA = withaferin A, RSG = rosiglitazone.