Oxidative stress regulates adipocyte apolipoprotein e and suppresses its expression in obesity

Abstract

Objective: Endogenous expression of apolipoprotein E (apoE) has a significant impact on adipocyte lipid metabolism and is markedly suppressed in obesity. Adipose tissue oxidant stress is emerging as an important mediator of adipocyte dysfunction. These studies were undertaken to evaluate the role of oxidant stress for regulation of adipocyte apoE.

Research design and methods: ApoE gene and protein expression in 3T3-L1 adipocytes or mature adipocytes and adipose tissue from C57/BL6 mice was evaluated after induction of oxidant stress. The response of adipose tissue and adipocytes from obese compared with lean mice to antioxidants was also assessed.

Results: Oxidant stress in 3T3-L1 cells or adipocytes and adipose tissue from lean mice significantly reduced apoE mRNA and protein level. Inclusion of an antioxidant eliminated this reduction. Oxidant stress was accompanied by activation of the nuclear factor-kappaB (NF-kappaB) transcription complex, and its effect on apoE was eliminated by an NF-kappaB activation inhibitor. Treatment of freshly isolated adipose tissue or mature adipocytes from obese mice with antioxidant increased apoE expression but had no effect on cells or tissue from lean mice. Incubation of freshly isolated adipocytes from lean mice with stromovascular cells from obese mice significantly suppressed adipocyte apoE compared with incubation with stromovascular cells from lean mice, but this suppression was reversed by inclusion of antioxidant or a neutralizing antibody to tumor necrosis factor-alpha.

Conclusions: Oxidant stress significantly modulates adipose tissue and adipocyte apoE expression. Furthermore, oxidant stress contributes to suppression of adipocyte apoE in obesity. This suppression depends on interaction between adipose tissue stromovascular cells and adipocytes.

FIG. 1.

Oxidative stress reduces apoE mRNA level in adipocytes. A: 3T3-L1 adipocytes were preincubated for 6 h in serum-free medium and then treated with 0, 0.5, or 1 mmol/l H₂O₂ for 10 min_. Cells were then incubated in serum-free medium for an additional 4 or 18 h before being harvested for measurement of apoE mRNA. □, 0.0 mmol/l; , 0.5 mmol/l; ▪, 1.0 mmol/l. Adipocytes were treated with or without 25 mU/ml glucose oxidase (B) or 10 mU/ml xanthine oxidase (C) with 0.6 mmol/l hypoxanthine for 18 h in serum-free medium. Total RNA was extracted and apoE mRNA level was measured using RT-PCR. Each experiment was performed using triplicate samples and was repeated three times with similar results. Results shown are from a representative experiment as means ± SD. *P < 0.001 for the difference compared with untreated control.

FIG. 2.

NAC abrogates the effect oxidative stress on apoE expression in adipocytes. 3T3-L1 adipocytes in serum-free medium were incubated with 20 mmol/l NAC for 1 h before incubation with the indicated oxidative stress agents. A: Cells were treated with 1 mmol/l H₂O₂ for 10 min in the presence or absence of NAC and then harvested 4 h later for measurement of apoE mRNA. B: Cells were treated with 25 mU/ml glucose oxidase (GO) for 18 h in the presence or absence of NAC and then harvested for measurement of apoE mRNA. C: Western blot showing apoE protein expression level in adipocytes treated with 1 mmol/l H₂O₂ for 10 min in the presence or absence of NAC. Cells were then harvested 18 h after treatment with H₂O₂. Signal quantitation is shown in parentheses. Experiments were performed using triplicate samples and repeated three times with similar results. Results are shown as means ± SD, *P < 0.002, ⁺P < 0.02 for the difference compared with untreated control.

FIG. 3.

NF-κB pathway mediates the effect of oxidative stress on apoE expression in adipocytes. A: 3T3-L1 adipocytes in serum-free medium for 6 h were treated with 1 mmol/l H₂O₂ for 0, 1, 5, and 10 min. Cells were then immediately lysed in the presence of phosphatase inhibitor cocktail. Fifty micrograms of protein were resolved on SDS-PAGE and probed with phospho-IκBα (Ser 32/36) antibody. The nitrocellulose membrane was stripped and reprobed with anti-IκBα antibody. The blots shown are representative of three separate experiments. B: 3T3-L1 adipocytes were placed in serum-free medium for 6 h and then preincubated with 100 nmol/l NF-κB activation inhibitor QNZ for 1 h before treatment with 1 mmol/l H₂O₂ for 10 min. Cells were washed and incubated for an additional 4 h without additions. The results shown are from a representative experiment performed using triplicate samples and repeated two times with similar results. Results are shown as means ± SD. *P < 0.001 for the difference compared with untreated control.

FIG. 4.

Effect of oxidative stress on apoE mRNA level in freshly isolated adipose tissue, mature adipocytes, and stromovascular cells from C57Bl/6J mice. A: Intra-abdominal fat pads were isolated and adipose tissue, mature adipocytes, and the stromovascular fraction (SVC) were separated and treated with 1 mmol/l H₂O₂ for 10 min. Cells and tissue were washed and incubated in serum-free medium for an additional 4 h before harvest. All results are expressed as fold change compared with apoE mRNA level in untreated adipose tissue. Results shown are means ± SD of cells and adipose tissue from six mice, *P < 0.005 for the difference compared with untreated control. B: Western blot for the effect of 1 mmol/l H₂O₂ (10 min) on apoE protein expression in isolated adipose tissue harvested 18 h after treatment with H₂O₂. Signal quantitation is shown in parentheses. ⁺P < 0.02 for the difference compared with control. □, no addition; ▪, H₂O₂.

FIG. 5.

NAC increases apoE mRNA level in freshly isolated adipose tissue and mature adipocytes from ob/ob mice. Freshly isolated adipose tissue (A) or freshly isolated mature adipocytes (B) from fat pads of ob/ob mice or lean littermate controls were isolated as described in research design and methods and incubated alone or with 20 mmol/l NAC for 4 h. Total RNA was extracted, and apoE mRNA was measured. Results are expressed as fold change compared with apoE level in untreated lean mice and are means ± SD of cells and adipose tissue from six mice. □, no addition; , NAC. *P < 0.0005 for comparison of untreated vs. NAC treated.

FIG. 6.

Importance of stromovascular-adipocyte cross-talk for regulating adipocyte apoE expression in obesity. A: Mature adipocytes from lean mice were incubated alone or with 1.0 × 10⁶ cells from the stromovascular fraction (SVF) of lean mice or ob/ob mice for 4 h as described in research design and methods. Adipocytes were harvested for measurement of apoE mRNA. B: Mature adipocytes from lean mice were incubated alone or with 1 × 10⁶ cells isolated from the stromovascular fraction of visceral or subcutaneous fat from ob/ob mice. After 4 h, adipocytes were harvested for measurement of apoE mRNA. Results are means ± SD from five lean mice and are representative of two experiments with similar results. *P < 0.01, **P < 0.001.

FIG. 7.

Role of ROS and TNF-α in suppression of adipocyte apoE by the obese stromovascular fraction. Adipocytes from lean mice were incubated alone or with 1.0 × 10⁶ stromovascular fraction (SVF) cells from ob/ob mice alone or with the indicated additions. After 4 h, adipocytes were harvested for measurement of apoE mRNA. Results are means ± SD of adipocytes from five lean mice and are representative of two experiments with similar results. *P < 0.01.