Increased oxidative stress in obesity and its impact on metabolic syndrome

Abstract

Obesity is a principal causative factor in the development of metabolic syndrome. Here we report that increased oxidative stress in accumulated fat is an important pathogenic mechanism of obesity-associated metabolic syndrome. Fat accumulation correlated with systemic oxidative stress in humans and mice. Production of ROS increased selectively in adipose tissue of obese mice, accompanied by augmented expression of NADPH oxidase and decreased expression of antioxidative enzymes. In cultured adipocytes, elevated levels of fatty acids increased oxidative stress via NADPH oxidase activation, and oxidative stress caused dysregulated production of adipocytokines (fat-derived hormones), including adiponectin, plasminogen activator inhibitor-1, IL-6, and monocyte chemotactic protein-1. Finally, in obese mice, treatment with NADPH oxidase inhibitor reduced ROS production in adipose tissue, attenuated the dysregulation of adipocytokines, and improved diabetes, hyperlipidemia, and hepatic steatosis. Collectively, our results suggest that increased oxidative stress in accumulated fat is an early instigator of metabolic syndrome and that the redox state in adipose tissue is a potentially useful therapeutic target for obesity-associated metabolic syndrome.

Figure 1

Levels of lipid peroxidation and plasma adiponectin in nondiabetic subjects. (A) Correlation of plasma TBARS, urinary 8-epi-PGF2α, and plasma adiponectin with BMI and waist circumference. (B) Correlation of plasma adiponectin with plasma TBARS and urinary 8-epi-PGF2α. Pearson’s correlation coefficient (r) is shown for each relationship. MDA, malondialdehyde.

Figure 2

Increased oxidative stress in plasma and WAT of obese KKAy mice. (A) Body weight, parametrial WAT weight, plasma levels of glucose, lipid peroxidation (TBARS), and H₂O₂ in C57BL/6 and KKAy mice at 7 and 13 weeks of age. Values are expressed as mean ± SEM (n = 6–8). (B) Tissue levels of lipid peroxidation in WAT, liver, and skeletal muscle of C57BL/6 and KKAy mice. Values are expressed as mean ± SEM (n = 6–8). (C) The release of H₂O₂ from WAT, skeletal muscle, and aorta of C57BL/6 and KKAy mice at 7 weeks of age. Values are expressed as mean ± SEM (n = 5). ***P < 0.001 compared with C57BL/6 mice.

Figure 3

Dysregulated expressions of adipose genes and increased expressions of NADPH oxidase subunits in WAT of obese KKAy mice. (A) The mRNA expressions of adiponectin, TNF-α, PAI-1, and PPARγ in WAT of C57BL/6 (white bars) and KKAy (black bars) mice. The mRNA amounts were quantified by real-time PCR. Values are normalized to the level of cyclophilin mRNA, and expressed as mean ± SEM (n = 6–8). (B) The mRNA expressions of NADPH oxidase subunits in various mouse tissues. The mRNA amounts were quantified by real-time PCR, using total RNA extracted from 8 tissues of C57BL/6 mice at 12 weeks of age. Values are normalized to the level of 18S ribosomal RNA. (C and D) The mRNA expressions of NADPH oxidase subunits and PU.1 in WAT (C), liver (D, left) and skeletal muscle (D, right) of C57BL/6 (white bars) and KKAy (black bars) mice at 7 and 13 weeks of age. Values are normalized to the level of cyclophilin mRNA, and expressed as mean ± SEM (n = 6–8). **P < 0.01 and ***P < 0.001 compared with C57BL/6 mice. BAT, brown adipose tissue.

Figure 4

Decreased mRNA expressions and activities of antioxidant enzymes in WAT of obese KKAy mice. (A) The mRNA expressions of Cu,Zn-SOD, Gpx, and catalase in WAT, liver, and skeletal muscle of C57BL/6 (white bars) and KKAy (black and gray bars) mice at 7 and 13 weeks of age. Values are normalized to the level of cyclophilin mRNA, and expressed as mean ± SEM (n = 6–8). (B) Immunoblot analysis of the amount of Cu,Zn-SOD protein in WAT, liver, and skeletal muscle of C57BL/6 and KKAy mice at 7 and 13 weeks of age. (C) Total SOD activities (left) and GPx activities (right) in WAT, liver, and skeletal muscle of C57BL/6 (white bars) and KKAy (black and gray bars) mice at 7 and 13 weeks of age. Values are expressed as mean ± SEM (n = 6–8). (D) Model illustrating increased production of oxidative stress in accumulated fat. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with C57BL/6 mice.

Figure 5

Production of ROS in 3T3-L1 adipocytes. (A) ROS production during differentiation of 3T3-L1 cells into adipocytes. ROS production was measured by NBT reduction. Oil red O staining (top) and NBT treatment (middle) of the cells. Dark-blue formazan was dissolved and the absorbance was determined at 560 nm (bottom). (B) Effect of linoleate and inhibitors of ROS production in fully differentiated 3T3-L1 adipocytes. 3T3-L1 adipocytes were incubated with (FFA⁺) or without (FFA^–) 200 μM linoleate for 24 hours. In the final hour of incubation, 10 mM NAC, 10 μM DPI, 200 μM apocynin, 100 μM oxypurinol, 100 μM rotenone, or 100 μM thenoyltrifluoroacetone (TTFA) was added, and ROS production was measured by NBT reduction. Values are expressed as mean ± SEM (n = 3). ***P < 0.001 compared with cells without linoleate or inhibitors. ^###P < 0.001 compared with cells treated with 200 μM linoleate.

Figure 6

Effects of ROS on gene expressions in 3T3-L1 adipocytes. (A and B) The mRNA expression levels of adiponectin, PAI-1, PPARγ, IL-6, and MCP-1 in 3T3-L1 adipocytes exposed to ROS, with or without antioxidant NAC. Fully differentiated 3T3-L1 adipocytes were exposed to ROS by incubation with H₂O₂. Values are normalized to the level of cyclophilin mRNA and expressed as mean ± SEM (n = 3). (C) The mRNA expression levels of NADPH oxidase subunits and PU.1 in 3T3-L1 adipocytes exposed to ROS. Values are normalized to the level of cyclophilin mRNA. (D) Effects of H₂O₂ and NAC on adiponectin secretion from 3T3-L1 adipocytes. Adiponectin levels in the media were determined by Western blotting (inset) and the values were quantified using a densitometer. (E) Effects of ROS and NAC on the transcriptional activity of adiponectin promoter in 3T3-L1 adipocytes. RLU, relative luciferase units. Values are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control.

Figure 7

In vivo effects of an NADPH oxidase inhibitor, apocynin, on adipocytokine expression, and glucose and lipid metabolism in KKAy mice. (A) Body weight of C57BL/6 and KKAy mice, treated with or without apocynin for 6 weeks. (B) Lipid peroxidation in WAT, and (C) release of H₂O₂ from WAT of C57BL/6 and KKAy mice, treated with or without apocynin for 6 weeks. (D) The mRNA expression levels of adiponectin and TNF-α in WAT of KKAy mice. Values are normalized to the level of cyclophilin mRNA. (E) Plasma adiponectin concentrations in C57BL/6 and KKAy mice. (F) Plasma glucose, insulin, and TG concentrations in C57BL/6 and KKAy mice. (G) Liver TG content in C57BL/6 and KKAy mice. Values are expressed as mean ± SEM (n = 8). *P < 0.05 and **P < 0.01 compared with vehicle-treated group.

Figure 8

A working model illustrating how increased ROS production in accumulated fat contributes to metabolic syndrome.