Taurine supplementation prevents ethanol-induced decrease in serum adiponectin and reduces hepatic steatosis in rats

Abstract

Chronic ethanol feeding decreases expression of adiponectin by adipocytes and circulating adiponectin. Adiponectin treatment during chronic ethanol feeding prevents liver injury in mice. Chronic ethanol feeding also increases oxidative and endoplasmic reticulum (ER) stress in adipose tissue. Here we tested the hypothesis that supplemental taurine, an amino acid that functions as a chemical chaperone/osmolyte and enhances cellular antioxidant activity, would prevent ethanol-induced decreases in adiponectin expression and attenuate liver injury. Serum adiponectin concentrations decreased as early as 4 to 7 days after feeding rats a 36% ethanol diet. This rapid decrease was associated with increased oxidative, but not ER, stress in subcutaneous adipose tissue. Taurine prevented ethanol-induced oxidative stress and increased inflammatory cytokine expression in adipose tissue. Ethanol feeding also rapidly decreased expression of transcription factors regulating adiponectin expression (CCAAT/enhancer binding protein alpha; peroxisome proliferator-activated receptor alpha/gamma) in subcutaneous adipose tissue. Taurine prevented the ethanol-induced decrease in CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor alpha, normalizing adiponectin messenger (m)RNA and serum adiponectin concentrations. In the liver, taurine prevented ethanol-induced oxidative stress and attenuated tumor necrosis factor alpha expression and steatosis, at least in part, by increasing expression of genes involved in fatty acid oxidation.

Conclusion: In subcutaneous adipose tissue, taurine decreased ethanol-induced oxidative stress and cytokine expression, as well as normalized expression of adiponectin mRNA. Taurine prevented ethanol-induced decreases in serum adiponectin; normalized adiponectin was associated with a reduction in hepatic oxidative stress, tumor necrosis factor alpha expression, and steatosis. Taken together, these data demonstrate that taurine has important protective effects against ethanol-induced tissue injury in both adipose and liver tissue.

Figure 1. Effects of ethanol feeding on serum adiponectin concentration and adiponectin oligomer distribution

(A) Rats were allowed free access to an ethanol-containing diet or pair-fed a control diet for 4 weeks. Adiponectin oligomers in serum from pair-fed and ethanol-fed rats were separated by FPLC and quantified by adiponectin ELISA. Values represent means ± SEM, n=3. (B) Rats were allowed free access to an ethanol-containing diet or pair-fed control diet for 4–7 days. Serum adiponectin concentrations were measured by ELISA. Values represent means ± SEM, n=9–14. Values with different superscripts are different from each other, p<0.05..

Figure 2. Early effects of ethanol feeding include oxidative stress, but not ER stress, in subcutaneous adipose tissue

Rats were allowed free access to an ethanol-containing diet or pair-fed a control diet for 4 and 7 days. (A) Formalin-fixed subcutaneous adipose sections were stained with an antibody against 4-HNE protein adducts. (B) Immunoreactive CYP2E1 was measured in subcutaneous adipose tissue homogenates by Western blotting. ERK1/2 was used as loading control. The values (means ± SEM, arbitrary units of density for CYP2E1/ERK1/2, n=4) were 0.16 ± 0.04 for pair-fed, 0.40 ± 0.08 at day 4 and 0.57 ± 0.14 at day 7. CYP2E1 expression was increased at day 7 compared to pair-fed, p<0.05. (C) XBP-1 splicing was detected by RT-PCR. (D) Immunoreactive phospho-eIF2α, CHOP and grp78 in subcutaneous adipose tissue homogenates were measured by Western blotting. ERK1/2 was used as loading control. Images are representative of 3 (A) and 4–6 (B, C and D) rats in each experimental group. P: pair-fed, E: ethanol-fed, Tg: thapsigargin.

Figure 3. Taurine prevented ethanol-induced oxidative stress in subcutaneous adipose

Rats were allowed free access to an ethanol-containing diet or pair-fed a control diet supplemented or not with 30g/L taurine. (A) Taurine concentration in plasma, liver and subcutaneous adipose tissue was measured by HPLC. Values represent means ± SEM, n=4–7. Values with different superscripts are different from each other, p<0.05. (B) Formalin-fixed subcutaneous adipose sections were stained with an antibody against 4-HNE protein adducts. The relative quantity of 4-HNE adducts was measured using the Image J program. Value represent means ± SEM, n=8–10. Values with different superscripts are different from each other, p<0.05. (C) Immunoreactive of CYP2E1 in subcutaneous adipose tissue homogenates was measured by Western blotting. ERK1/2 was used as loading control. Images are representative of 4 rats in each experimental group. The values (means ± SEM, arbitrary units of density for CYP2E1/ERK1/2, n=5–6) were 0.13 ± 0.02 for pair-fed, 0.08 ± 0.02 for pair-fed/taurine, 0.47 ± 0.09 for ethanol-fed and 0.39 ± 0.0.12 for ethanol-fed/taurine. CYP2E1 expression was increased by ethanol feeding with or without taurine supplementation compared to pair-fed, p<0.05. (D) Catalase mRNA level in subcutaneous adipose tissue was measured by real-time PCR and then normalized to β-actin mRNA level. Values represent means ± SEM, n=6. Values with different letters are significantly different from each other, p<0.05.

Figure 4. Taurine prevented ethanol-induced increases in Egr-1 and IL-6 mRNA in subcutaneous adipose

Rats were allowed free access to an ethanol-containing diet or pair-fed a control diet supplemented or not with 30g/L taurine. Egr-1 (A), TNF-α (B), IL-6 (C) and MCP1 (D) mRNA levels in subcutaneous adipose tissue were measured by real-time PCR and then normalized to β-actin mRNA level. Values represent means ± SEM, n=6–8. Values with different letters are significantly different from each other, p<0.05.

Figure 5. Taurine prevented ethanol-induced decreases in adiponectin expression

Rats were allowed free access to an ethanol-containing diet or pair-fed a control diet supplemented or not with 30g/L taurine. (A) Serum adiponectin concentrations were measured by ELISA. Values represent means ± SEM, n=11–12. Values with different letters are significantly different from each other, p<0.05. (B) Adiponectin mRNA levels in subcutaneous adipose tissues were measure by real-time PCR and then normalized to β-actin mRNA level. Values represent means ± SEM, n=6–7. Values with different letters are significantly different from each other, p<0.05.

Figure 6. Taurine prevented the decrease of C/EBPα and PPARα mRNA in subcutaneous adipose tissue after ethanol feeding

Rats were allowed free access to an ethanol-containing diet or pair-fed a control diet supplemented or not with 30g/L taurine. C/EBPα (A), C/EBPβ (B), SREBP1c (C), PPARγ2 (D) and PPARα (E) mRNA levels in subcutaneous adipose tissue were measured by real-time PCR and then normalized to β-actin mRNA level. Values represent means ± SEM, n=5–8. Values with different letters are significantly different from each other, p<0.05.

Figure 7. Taurine prevented/attenuated ethanol-induced oxidative stress and hepatic steatosis

Rats were allowed free access to an ethanol-containing diet or pair-fed a control diet supplemented or not with 30g/L taurine. (A) Formalin-fixed liver sections were stained with an antibody against 4-HNE protein adducts. The relative quantity of 4-HNE adducts was measured using the Image J program. Values represent means ± SEM, n=4. (B) Concentration of hepatic malondialdehyde (MDA) was measured by ELISA. Values represent means ± SEM, n=7–8. (C) Frozen liver OCT sections were prepared and stained in Oil Red O. Images were taken at 100x magnification. Total triglyceride content in the liver was quantified using the Trinder reagent. Values represent means ± SEM, n=11. (D) Plasma ALT activity was measured by enzymatic assay. Values represent means ± SEM, n=7–8. (E/F) Formalin-fixed liver sections were stained with antibody against TNF-α (E) or ED2 (F), a marker for macrophages. The relative quantity of TNF-α was measured using the Image J program. Values represent means ± SEM, n=4. For all panels, values with different letters are significantly different from each other, p<0.05.

Figure 8. Taurine partially reversed ethanol-induced decrease in fatty acid oxidation

SREBP1 (A), FAS (B), ACC (C), PPARα (D), CPT1α (E) and CD36 (F) mRNA levels in liver were measured by real-time PCR and then normalized to β-actin mRNA level. Values represent means ± SEM, n=5–6. Values with different letters are significantly different from each other, p<0.05.