pNaKtide Inhibits Na/K-ATPase Signaling and Attenuates Obesity

Abstract

Obesity is a growing public health crisis across the world and has been recognized as an underlying risk factor for metabolic syndrome. Growing evidence demonstrates the critical role of oxidative stress in the pathophysiological mechanisms of obesity and related metabolic dysfunction. As we have established previously that Na/K-ATPase can amplify oxidative stress signaling, we aimed to explore the effect of inhibition of this pathway on obesity phenotype using the peptide antagonist, pNaKtide. The experiments performed in murine preadipocytes showed the dose-dependent effect of pNaKtide in attenuating oxidant stress and lipid accumulation. Furthermore, these in vitro findings were confirmed in C57Bl6 mice fed a high-fat diet. Interestingly, pNaKtide could significantly reduce body weight, ameliorate systemic oxidative and inflammatory milieu and improve insulin sensitivity in obese mice. Hence the study demonstrates the therapeutic utility of pNaKtide as an inhibitor of Na/K-ATPase oxidant amplification signaling to alleviate obesity and associated comorbidities.

Keywords: Adipose tissue; Na/K-ATPase; Obesity; Oxidative stress.

Figure 1:

pNaKtide improves adipogenesis in murine adipocytes. To evaluate the effect of pNaKtide on adipogenesis and determine optimal concentration, murine preadipocytes were cultured with varying concentrations of pNaKtide. (A) Quantitative assessment of relative absorbance by Oil Red O staining to measure adipogenesis in murine preadipocytes cultured in adipogenic media with varying concentrations of pNaKtide, (n=7/group); (B) Dihydroethidium (DHE) staining to measure superoxide levels, (n=6/group); Western blot analysis in murine adipocyte treated with 0.7 μM of pNaKtide for protein expression of adipogenic markers, (C) FAS (n=8/group), (D) MEST (n=8/group) and (E) PPARγ (n=7/group), with mean band density normalized to actin. Note: Each box and whisker plot represents values as maximum and minimum range, upper quartile, median and lower quartile. *p<0.05 vs. CTR, **p<0.01 vs. CTR.

Figure 2:

pNaKtide improves body weight and adipose tissue mass in dose-dependent manner in high fat diet-fed mice. Mice were fed either normal chow or high fat diet with or without varying concentrations of pNaKtide, including 1 mg/kg, 5 mg/kg and 25 mg/kg. (A) Body weight of mice measured over 12 weeks of study period (n=7–21/group); Line plot representing mean values, with their standard errors represented by vertical bars. *p<0.05 and **p<0.01 CTR vs. HF, ##p<0.01 HF vs. HF+pNaKtide 25 mg (B) Visceral adipose tissue mass (n = 7–20/group) and (C) Subcutaneous adipose tissue mass (n=7–20/group), in mice fed a high fat diet with or without varying concentration of pNaKtide. Note: Each box and whisker plot represents values as maximum and minimum range, upper quartile, median and lower quartile. *p<0.05 vs. CTR, **p<0.01 vs. CTR, #p<0.05 vs. HF, ##p<0.01 vs. HF.

Figure 3:

pNaKtide improves adipogenesis and adiponectin expression in dose-dependent manner in high fat diet-fed mice. Western blot analysis in mice fed a high diet with or without varying concentration of pNaKtide for protein expression of (A) FAS (n=6/group), (B) MEST (n=6/group) and (C) adiponectin (n=6/group), with mean band density normalized to actin. Note: Each box and whisker plot represents values as maximum and minimum range, upper quartile, median and lower quartile. *p<0.05 vs. CTR, **p<0.01 vs. CTR, #p<0.05 vs. HF, ##p<0.01 vs. HF.

Figure 4:

pNaKtide improves metabolic profile, oxidative stress and inhibits Na/K-ATPase signaling cascade in mice fed a high fat diet. Dose- dependent effect of pNaKtide was determined by (A) Estimation of insulin resistance by HOMA-IR score, (n=6–7/group); (B) Blood glucose levels measured in glucose tolerance test, (n=6–8/group). Line plot representing mean values, with their standard errors represented by vertical bars. **p<0.01 CTR vs. HF, #p<0.05 and ##p<0.01 HF vs. HF+pNaKtide 25 mg; (C) Quantitative assessment of TBARS in visceral adipose tissue, (n=7/group). (D) Plasma adiponectin levels measured by ELISA, (n=7–19/group); (E) Quantitative assessment of protein carbonylation levels shown as DNP expression, normalized with Coomassie loading control, (n=10/group); Immunoblot analysis of (F) pSrc, with mean band density normalized to total Src, (n=8/group) and (G) pERK1/2 with mean band density normalized to total ERK, (n=6–12/group) to demonstrate antagonism of Na/K-ATPase signaling and inhibition of downstream mediators by pNaKtide. Note: Each box and whisker plot represents values as maximum and minimum range, upper quartile, median and lower quartile. *p<0.05 vs. CTR, **p<0.01 vs. CTR, #p<0.05 vs. HF, ##p<0.01 vs. HF.

Figure 5:

pNaKtide improves adipogenesis, oxidative stress and inhibits Na/K-ATPase signaling cascade in murine adipocytes exposed to glucose oxidase. Murine preadipocytes were cultured in adipogenic medium and exposed to glucose oxidase with or without 0.7 μM of pNaKtide. (A) Quantitative assessment of relative absorbance by Oil Red O staining to measure adipogenesis, (n=8–16/group); Western blot analysis for protein expression of adipogenic markers, (B) FAS (n=6/group) and (C) MEST (n=7/group), with mean band density normalized to actin. Note: Each box and whisker plot represents values as maximum and minimum range, upper quartile, median and lower quartile. *p<0.05 vs. CTR, **p<0.01 vs. CTR, #p<0.05 vs. GO, ##p<0.01 vs. GO.

Figure 6:

pNaKtide improves adipogenesis in murine adipocytes exposed to fructose. Murine preadipocytes were cultured in adipogenic medium and exposed to fructose (500 μM concentration) with or without 0.7 μM of pNaKtide. (A) Quantitative assessment of relative absorbance by Oil Red O staining to measure adipogenesis, (n=4/group); Western blot analysis for protein expression of adipogenic markers, (B) FAS (n=4/group) and (C) MEST (n=6/group), with mean band density normalized to actin. Note: Each box and whisker plot represent values as maximum and minimum range, upper quartile, median and lower quartile. *p<0.05 vs. CTR, **p<0.01 vs. CTR, #p<0.05 vs. Fr, ##p<0.01 vs. Fr.