Obesity is closely associated with metabolic syndrome (1
). Recent studies have shown that the dysregulated production of “offensive” adipocytokines, such as PAI-1 (10
), TNF-α (11
), IL-6 (45
), MCP-1 (46
), and angiotensinogen (47
), and of “defensive” adipocytokines, such as adiponectin (18
) and leptin (15
), is critically involved in the pathogenesis of metabolic syndrome. However, the mechanisms by which fat accumulation leads to abnormal expression of adipocytokines and development of metabolic syndrome have not been fully elucidated.
In the present study, we have demonstrated that, in nondiabetic human subjects, fat accumulation closely correlated with the markers of systemic oxidative stress. These data are in good agreement with recent studies suggesting that systemic oxidative stress correlates with BMI (48
). In addition, we demonstrated that plasma adiponectin levels correlated inversely with systemic oxidative stress. We then reproduced the results of our human studies in several mouse models of obesity, including KKAy, db/db
, and DIO mice. The main finding of the present study is that oxidative stress in accumulated fat mediates the obesity-associated development of metabolic syndrome by the following potential mechanisms: (a) increased oxidative stress in accumulated fat leads to dysregulated production of adipocytokines, and (b) the selective increase in ROS production in accumulated fat leads to elevation of systemic oxidative stress.
Plasma adiponectin levels correlated inversely with the markers of systemic oxidative stress in nondiabetic human subjects. In cultured adipocytes, addition of oxidative stress suppressed mRNA expression and secretion of adiponectin, and it also increased PAI-1, IL-6, and MCP-1 mRNA expression. Furthermore, treatment with the NADPH oxidase inhibitor apocynin reduced oxidative stress in WAT and increased plasma adiponectin levels in KKAy mice. These results indicate that a local increase in oxidative stress in accumulated fat causes dysregulated production of adipocytokines. Recently, we reported that PPARγ positively regulates the transcription of the adiponectin gene via PPARγ-responsive element in the promoter (40
). We showed that oxidative stress suppressed PPARγ mRNA expression in 3T3-L1 adipocytes. It was also shown that nuclear translocation of PPARγ was inhibited by nitration associated with oxidative stress (50
). Therefore, downregulation of adiponectin expression may be partially attributed to the decreased gene expression and smaller amount of nuclear PPARγ under conditions of oxidative stress.
In the present study, H2
production was increased only in adipose tissue of obese mice, but not in other tissues examined, including the liver, skeletal muscle, and aorta. These results suggest that adipose tissue is the major source of the elevated plasma ROS. Oxidative stress is known to impair both insulin secretion by pancreatic β cells (32
) and glucose transport in muscle (30
) and adipose tissue (31
). Increased oxidative stress in vascular walls is involved in the pathogenesis of hypertension (33
) and atherosclerosis (34
). Oxidative stress also underlies the pathophysiology of hepatic steatosis (51
). Thus, oxidative stress locally produced in each of the above tissues seems to be involved in the pathogenesis of these diseases. Our results suggest that increased ROS secretion into peripheral blood from accumulated fat in obesity is also involved in induction of insulin resistance in skeletal muscle and adipose tissue, impaired insulin secretion by β cells, and pathogenesis of various vascular diseases such as atherosclerosis and hypertension.
Why is oxidative stress increased only in accumulated fat? We found that in WAT but not other tissues of obese mice, the mRNA expression levels of NADPH oxidase subunits increased, and mRNA expression levels and activities of antioxidant enzymes decreased. We also found a high level of mRNA expression of the transcription factor PU.1, which upregulates the transcription of the NADPH oxidase gene (37
) in adipose tissue of obese mice. Recently, Weisberg et al. (52
) and Xu et al. (53
) reported that macrophages infiltrated the obese adipose tissues and were an important source of inflammatory cytokines. Since macrophages are also known to produce ROS, it is possible that infiltrated macrophages are involved in augmented NADPH oxidase and elevated ROS production in the obese adipose tissue. In this regard, a family of gp91phox
homologs, termed NOX (NADPH oxidase) proteins, has been reported to be expressed in nonphagocytic cells, not in macrophages (54
). Recent studies of adipocytes found that NOX4, a member of the NOX family, plays a role in the generation of H2
). The expression of NOX4 was not detected in macrophages (57
). In contrast, we found high expression levels of NOX4 in WAT, as well as gp91phox
, and mRNA expression of NOX4 was significantly increased in WAT of obese mice (Supplemental Figure 3, A and B). These results suggest that adipose NADPH oxidase is elevated and contributes to ROS production in accumulated fat.
We demonstrated that ROS production was increased in 3T3-L1 adipocytes, in parallel with fat accumulation and by incubation with linoleic acid, in a NADPH oxidase–dependent manner. These results suggest that in accumulated fat, elevated levels of fatty acids activate NADPH oxidase and induce ROS production. We also demonstrated that ROS itself augmented mRNA expressions of NADPH oxidase subunits, including NOX4 (Supplemental Figure 3C) and PU.1 in adipocytes. Therefore, in accumulated fat of obesity, elevated ROS appear to upregulate mRNA expression of NADPH oxidase, establishing a vicious cycle that augments oxidative stress in WAT and blood. We found that ROS increased the expression of MCP-1, a chemoattractant for monocytes and macrophages, in adipocytes. Byproducts of lipid peroxidation by ROS, such as trans
-4-hydroxy-2-nonenal and malondialdehyde, are themselves potent chemoattractants (59
). Hence, it is possible that increased ROS production and MCP-1 secretion from accumulated fat should cause infiltration of macrophages and inflammation in adipose tissue of obesity.
Importantly, our in vivo study revealed that treatment with the NADPH oxidase inhibitor apocynin reduced ROS production in adipose tissue of KKAy mice. It also improved hyperinsulinemia, hyperglycemia, hypertriglyceridemia, and hepatic steatosis. Increased expression of adiponectin and decreased expression of TNF-α were observed in WAT of apocynin-treated KKAy mice, demonstrating that reduction of oxidative stress in accumulated fat could improve the dysregulation of adipocytokines in vivo. Our results demonstrate that treatment with NADPH oxidase inhibitor is effective in ameliorating the development of obesity-associated metabolic syndrome.
It has been reported that prolonged exposure of 3T3-L1 adipocytes to ROS results in impairments of insulin-induced activation of PI3-kinase and Akt, insulin-stimulated lipogenesis, glucose uptake, and GLUT4 translocation to the plasma membrane (31
). Thus, NADPH oxidase inhibitors might improve insulin sensitivity via suppression of these effects induced by chronic exposure to ROS. Our current results are consistent with several previous studies demonstrating that antioxidant treatment improves insulin function in diabetic subjects (61
Recent studies, on the other hand, have proposed that ROS such as H2
are produced transiently in response to insulin stimulation and also act as a second messenger for insulin signaling in adipocytes (63
). NADPH oxidase is thought to be involved in insulin-induced ROS generation (63
). We assume that a transient increase of intracellular ROS is important for the insulin signaling pathway, while excessive and long-term exposure to ROS reduces insulin sensitivity and impairs glucose and lipid metabolism.
In the present study, we observed stimulation of ROS production by fatty acids via NADPH oxidase activation. In addition, we also found that ROS suppressed the mRNA expressions of lipogenic genes, such as fatty acid synthase and sterol regulatory element binding protein-1c, in 3T3-L1 adipocytes (data not shown). These results suggest that increased ROS production caused by fat accumulation may prevent further lipid storage, but may simultaneously cause dysregulated expression of adipocytokines and insulin resistance. Conceivably, inhibition of NADPH oxidase should improve the dysregulation of adipocytokines and insulin sensitivity via restoration of normal ROS production in obese adipocytes.
Figure illustrates our working hypothesis regarding the role of ROS in metabolic syndrome. Increased oxidative stress in accumulated fat, via increased NADPH oxidase and decreased antioxidant enzymes, causes dysregulated production of adipocytokines, locally. Increased ROS production from accumulated fat also leads to increased oxidative stress in blood, hazardously affecting other organs including the liver, skeletal muscle, and aorta. We propose that increased oxidative stress in accumulated fat is an early instigator and one of the important underlying causes of obesity-associated metabolic syndrome, hence, the redox state in adipose tissue is a potentially useful target in new therapies against obesity-associated metabolic syndrome, as we demonstrated in the mouse in vivo study.
A working model illustrating how increased ROS production in accumulated fat contributes to metabolic syndrome.