Adipose tissue-specific dysregulation of angiotensinogen by oxidative stress in obesity

Abstract

Adipose tissue expresses all components of the renin-angiotensin system including angiotensinogen (AGT). Recent studies have highlighted a potential role of AGT in adipose tissue function and homeostasis. However, some controversies surround the regulatory mechanisms of AGT in obese adipose tissue. In this context, we here demonstrated that the AGT messenger RNA (mRNA) level in human subcutaneous adipose tissue was significantly reduced in obese subjects as compared with nonobese subjects. Adipose tissue AGT mRNA level in obese mice was also lower as compared with their lean littermates; however, the hepatic AGT mRNA level remained unchanged. When 3T3-L1 adipocytes were cultured for a long period, the adipocytes became hypertrophic with a marked increase in the production of reactive oxygen species. Expression and secretion of AGT continued to decrease during the course of adipocyte hypertrophy. Treatment of the 3T3-L1 and primary adipocytes with reactive oxygen species (hydrogen peroxide) or tumor necrosis factor alpha caused a significant decrease in the expression and secretion of AGT. On the other hand, treatment with the antioxidant N-acetyl cysteine suppressed the decrease in the expression and secretion of AGT in the hypertrophied 3T3-L1 adipocytes. Finally, treatment of obese db/db mice with N-acetyl cysteine augmented the expression of AGT in the adipose tissue, but not in the liver. The present study demonstrates for the first time that oxidative stress dysregulates AGT in obese adipose tissue, providing a novel insight into the adipose tissue-specific interaction between the regulation of AGT and oxidative stress in the pathophysiology of obesity.

Fig. 1

The AGT mRNA levels in obese adipose tissue from humans and mice. A, The relation between the AGT mRNA level in subcutaneous abdominal adipose tissue and the degree of obesity in humans: nonobese (BMI <25), n = 20; overweight (25 ≤ BMI < 30), n = 13; obese (BMI ≥30), n = 13. B, Comparison of the adipose tissue AGT mRNA levels in 12-week-old male ob/ob mice (n = 4; mean body weight, 60 ± 0.7 g) and their lean littermates (n = 4; mean body weight, 29 ± 0.3 g). Left: epididymal adipose tissue depots. Right: subcutaneous abdominal adipose tissue depots. C, Comparison of the hepatic AGT mRNA level between the ob/ob mice (n = 4) and their lean littermates (n = 4). The mRNA level was examined by real-time PCR and normalized to that of 18S ribosomal RNA (rRNA). The data are expressed as the mean ± SE. *P <.05 as compared with the nonobese subjects or the lean littermates.

Fig. 2

The AGT expression during the course of hypertrophy in the 3T3-L1 adipocytes. A, Oil red O staining of the 3T3-L1 adipocytes on days 8, 18, and 28 after induction of differentiation. Bar = 30 µm. B, Size of the 3T3-L1 adipocytes on days 8 and 30. Adipocyte size was measured using a Coulter Multisizer III. C, The ROS production during adipocyte hypertrophy. The ROS production was assessed by the NBT assay. Dark blue formazan was dissolved, and the absorbance was determined at 560 nm (n = 3). D, The MCP-1, IL-6, and AGT mRNA levels in the 3T3-L1 adipocytes on days 8, 18, and 28 (n = 4). The mRNA level was examined by real-time PCR and normalized to that of 18S rRNA. E, The AGT protein concentration in the culture media. The MCP-1, IL-6, and AGT concentrations in the 3T3-L1 adipocytes on days 8, 18, and 28 were analyzed by ELISA (n = 4). Results are representatives of at least 3 independent experiments. The data are expressed as the mean ± SE. *P < .05 and **P < .01 as compared with the value of day 8. U.D. indicates undetectable.

Fig. 3

Impact of TNFα on the expression and secretion of AGT in the 3T3-L1 adipocytes. A, The AGT, MCP-1, and IL-6 mRNA level in the 3T3-L1 adipocytes (day 8) treated with TNFα for 24 hours (n = 4). The mRNA level was examined by real-time PCR and normalized to that of 18S rRNA. B, The AGT protein concentration in the culture media in the 3T3-L1 adipocytes (day 8) treated with TNFα for 24 hours (n = 4). The protein level was assessed by ELISA. C, The AGT and MCP-1 mRNA level in the primary adipocytes treated with TNFα (10 ng/mL) for 24 hours (n = 4). The mRNA level was examined by real-time PCR and normalized to that of 18S rRNA. Results are representatives of at least 3 independent experiments. The data are expressed as the mean ± SE. *P < .05 and **P < .01 as compared with the control value.

Fig. 4

Impact of oxidative stress on the expression and secretion of AGT in the 3T3-L1 adipocytes. A, The AGT and MCP-1 mRNA level in the 3T3-L1 adipocytes (day 8) treated with H₂O₂ for 24 hours (n = 4). The mRNA level was examined by real-time PCR and normalized to that of 18S rRNA. B, The AGT protein level in the culture media of the 3T3-L1 adipocytes (day 8) treated with H₂O₂ for 24 hours (n = 4). The protein concentration was assessed by ELISA. C, The AGT and MCP-1 mRNA level in the primary adipocytes treated with H₂O₂ (1 mmol/L) for 24 hours (n = 4). The mRNA level was examined by real-time PCR and normalized to that of 18S rRNA. Results are representatives of at least 3 independent experiments. The data are expressed as the mean ± SE. *P < .05 and **P < .01 as compared with the control value.

Fig. 5

Effect of antioxidant treatment on the expression and secretion of AGT in the adipocytes. A, Suppression of ROS generation in the 3T3-L1 adipocytes treated with NAC (10 mmol/L) for 10 days (n = 3). The ROS was estimated by the NBT assay. B, The MCP-1 and AGT mRNA levels in the 3T3-L1 adipocytes incubated with NAC (10 mmol/L) (n = 8). The mRNA level was examined by real-time PCR and normalized to that of 18S rRNA. Results are representatives of at least 3 independent experiments. C, Comparison of the AGT and TNFα mRNA levels between 10-week-old male db/db mice (n = 4; mean body weight, 48 ± 1.5 g) and their lean littermates (n = 4; mean body weight, 28 ± 1.0 g) in epididymal adipose tissue. D, The level of IL-6, TNFα, and AGT mRNA in the epididymal adipose tissue depots of obese db/db mice treated with NAC (150 mg/kg body weight) or vehicle (phosphate-buffered saline) once daily for 1 week (n = 3). E, The AGT mRNA level in the liver of obese db/db mice treated with NAC or vehicle for 1 week (n = 3). The mRNA level was examined by real-time PCR and normalized to that of cyclophilin mRNA. The data are expressed as the mean ± SE. *P < .05 and **P < .01 as compared with the control value.