Mitochondriogenesis and apoptosis: possible cause of vitamin A-mediated adipose loss in WNIN/Ob-obese rats

Abstract

Background: Previously, we reported that vitamin A-enriched diet (129 mg/kg diet) intake reduces the adiposity development in obese rats of WNIN/Ob strain. Here, we hypothesize that dose lesser than 129 mg of vitamin A/kg diet would also be effective in ameliorating the development of obesity in these rats.

Methods: Five-month-old male lean and obese rats designated as A & B were divided into four subgroups (I, II, III and IV) consisting of 8 rats from each phenotype and received diets containing 2.6 mg (control group), 26 mg, 52 mg and 129 mg vitamin A/kg diet as retinyl palmitate for 20 weeks. Body composition and morphological analysis of brown adipose tissue (BAT) was analyzed. Expression of uncoupling protein 1 (UCP1), retinoic acid receptor α (RARα) and retinoid X receptor α (RXRα) in BAT and levels of Bcl2 and Bax in epididymal white adipose tissue (eWAT) were determined by immunoblotting.

Results: Vitamin A supplementation to obese rats at doses of 52 and 129 mg/kg diet showed reduced body weight gain and adiposity compared to control diet-fed obese rats receiving 2.6 mg of vitamin A/kg diet. In BAT of obese rats, vitamin A supplementation at doses of 26 and 52 mg of vitamin A/kg diet resulted in increased UCP1 expression with concomitant decrease in RARα and RXRα levels compared to control diet-fed obese rats. Further, transmission electron microscopy study revealed an increase in number of BAT mitochondria of obese rats supplemented with 26 and 52 mg of vitamin A/kg diet. Also, obese rats fed on 52 mg/kg diet resulted in increased apoptosis by altering the ratio of Bcl2 to Bax protein levels in eWAT. Notably, most of these changes were not observed in lean rats fed vitamin A-enriched diets.

Conclusion: In conclusion, chronic consumption of 52 mg of vitamin A/kg diet seems to be an effective dose in ameliorating obesity possibly through mitochondriogenesis, UCP1-mediated thermogenesis in BAT and apoptosis in eWAT of obese rats. Therefore, the role of dietary vitamin A in correcting human obesity would be of unquestionable relevance and can only be addressed by future studies.

Keywords: Adipose tissue; Apoptosis; Dietary supplementation; Nuclear receptors; Thermogenesis; Uncoupling protein; Vitamin A.

Figure 1

Effect of vitamin A supplementation on BAT-UCP1 protein levels in lean and obese rats. Five-month-old male lean (A) and obese (B) rats were fed vitamin A at a dose of 2.6 (I), 26 (II), 52 (III) and 129 (IV) mg/kg diet for a period of 20 weeks. Representative western blot showing the levels of UCP1 in BAT of lean and obese rats supplemented with various doses of vitamin A. Equal loading of the protein was ensured by staining the membranes with Ponceau S (image not shown). Bar graph shows the densitometric analysis of 3 rats, representing each of the dietary group. Bars are mean ± SE and are expressed relative to the mean value of lean control group (AI), which was set as 1. ^a-d Bars not sharing a common superscript are significantly different by one-way ANOVA and LSD post hoc comparison (P < 0.05).

Figure 2

Effect of vitamin A on BAT RARα and RXRα protein expressions in lean and obese rats of WNIN/Ob strain. A. i) Representative western blot showing the levels of RARα and RXRα in BAT of lean rats & ii) Histogram represents the densitometric (arbitrary units) values of blot relative to control diet-fed lean rats (AI). B. i) Representative western blot showing the levels of RARα and RXRα in BAT of obese rats & ii) Histogram represents the densitometric (arbitrary units) values of blot relative to control diet-fed obese rats (BI). Equal loading of the protein was ensured by staining the membranes with Ponceau S (image not shown). Bars are given as means ± SE of 4 rats. Vitamin A-enriched diet was compared to stock diet of respective phenotypes. P < 0.05 was considered significant (one-way ANOVA). AI, BI- control diet, AII, BII- 2.6 mg/kg diet, AIII, BIII- 52 mg/kg diet & AIV, BIV- 129 mg/kg diet fed groups. ^a-c Bars not sharing a common superscript are significantly different by one-way ANOVA and LSD post hoc comparison (P < 0.05).

Figure 3

Effect of vitamin A supplementation on BAT morphology. Five-month-old male lean (A) and obese (B) rats were fed vitamin A at a dose of 2.6 (I), 26 (II), 52 (III) and 129 (IV) mg/kg diet for a period of 20 weeks. AI served as the control group for lean phenotype, while BI was the control group for obese phenotype. The BAT samples were fixed, and sections were stained with uranyl citrate and lead citrate for morphologic analysis. Photomicrographs were taken at 15000x magnification. Scale bar, 667 nm. The ruptured membranes observed in obese group (BI) are depicted by arrow heads. The intact mitochondria in BII and BIII are depicted by arrow. M- mitochondria, LD- lipid droplet.

Figure 4

Effect of vitamin A on epididymal WAT (eWAT) Bcl2 and Bax protein expressions in lean and obese rats of WNIN/Ob strain. A. i) Representative western blot showing the levels of Bcl2 and Bax in eWAT of lean rats & ii) Histogram represents eWAT Bcl2–Bax ratio quantified densitometric values expressed relative to a value of 1 for control diet-fed lean rats (AI). B. i) Representative western blot showing the levels of Bcl2 and Bax in eWAT of obese rats & ii) Histogram represents eWAT Bcl2–Bax ratio quantified densitometric values expressed relative to a value of 1 for control diet-fed obese rats (BI). Equal loading of the protein was ensured by staining the membranes with Ponceau S (image not shown). Bars are given as means ± SE of 4 rats. Vitamin A-enriched diet was compared to stock diet of respective phenotypes. P < 0.05 was considered significant (one-way ANOVA). AI, BI- control diet, AII, BII- 2.6 mg/kg diet, AIII, BIII- 52 mg/kg diet & AIV, BIV- 129 mg/kg diet fed groups. ^a-bBars not sharing a common superscript are significantly different by one-way ANOVA and LSD post hoc comparison (P < 0.05).

Figure 5

Nucleosomal DNA fragmentation. Representative picture of genomic DNA (A) and fragmented DNA (B) run on an agarose gel stained with ethidium bromide. M – DNA marker containing 100 bp ladder, AI and BI - lean and obese rats respectively fed on 2.6 mg of vitamin A/kg diet, AII and BII - lean and obese rats respectively fed on 26 mg of vitamin A/kg diet, AIII and BIII - lean and obese rats respectively fed on 52 mg of vitamin A/kg diet, AIV and BIV - lean and obese rats respectively fed on 129 mg of vitamin A/kg diet.