We applied a quantitative proteomic approach to detect serum proteins that might be differentially expressed during obesity, and identified A1AT as a major serum protein that is significantly reduced in both leptin-deficient ob/ob and HFD-induced obese mice. We showed that leptin stimulated A1AT expression through the Jak2-Stat3 pathway in cultured hepatocytes. This finding is consistent with previous reports that a Stat3 consensus sequence is present in the 3′ enhancer region of the serpina1
gene (Kalsheker et al., 2002
). Moreover, leptin infusion into ob/ob mice increased serum A1AT levels, validating the significance of the in vitro
findings. Importantly, studies with clinical samples from lean and obese South Asian and U.S. Caucasian subjects revealed that serum A1AT levels were significantly reduced in obese leptin-resistant subjects. Thus, it is possible that leptin is a physiological regulator of A1AT expression and function. Our data suggests for the first time that leptin deficiency and leptin resistance are both related to the observed obesity-related reduction in A1AT expression and secretion.
NE plays an important role in host defense and inflammation (Pham, 2006
). NE also breaks down a range of matrix substrates including elastin and other circulating proteins, such as adiponectin (Waki et al., 2005
). The catalytic function of NE is blocked by A1AT through covalent binding to each other; thus, A1AT protects tissues from serine protease-induced damage. Here, we report that serum NE activity was significantly increased in leptin-deficient ob/ob mice, HFD-induced obese mice, and obese human subjects, consistent with the reciprocal reductions in the level of the NE inhibitor A1AT. Of noted, we also observed that NE activity increase in the sera of mice fed with a HFD for only three days (Figure S7D
) before the development of leptin resistance. Therefore, the imbalance between NE and A1AT occurs in HFD-induced obesity both at the early stage due to the increase of NE activity and at the later stage due to the decrease of A1AT expression. It is possible that increased NE activity is related to neutrophil activation in the early stage (Elgazar-Carmon et al., 2008
) and the decrease A1AT expression is related to the development of leptin resistance and fatty liver at the later stage of HFD feeding. We hypothesized that this imbalance might be related to the pathological changes observed in obesity. This hypothesis was tested using NE null mice, hA1AT transgenic mice and NE chemical inhibitor. Both NE null mice and hA1AT transgenic mice were resistant to multiple HFD-induced metabolic disturbances, including weight gain, glucose intolerance, insulin resistance, fatty liver and macrophage infiltration in adipose tissues. Our data support the concept that the imbalance between NE and A1AT is a key factor contributing to the development of HFD-induced obesity, insulin resistance, and liver steatosis. This is particularly important because increased NE activity is observed at an early stage of over-nutrition in WT mice. Our data suggest that NE and A1AT are potential drug targets for treatment of obesity and insulin resistance. Thus, reversing the ratio of NE to A1AT could be beneficial for obesity and insulin resistance due to over-nutrition. This notion is further supported by the benefits of overexpression of hA1AT and treatment with a NE inhibitor on metabolic profiles in HFD-fed mice.
It is well established that obesity is accompanied by low-grade inflammation of adipose tissue, which manifests as infiltration of inflammatory cells such as macrophages, lymphocytes, mast cells, and neutrophils, as well as production of inflammatory cytokines and chemokines that contribute to leukocyte infiltration and insulin resistance (Lumeng and Saltiel, 2011
; Osborn and Olefsky, 2012
). Notably, we observed that visceral fat of Ela2−/−
mice fed an HFD contained less than 10% of the number of macrophages present in fat of WT mice. The expression of proinflammatory M1 macrophage markers, but not M2 macrophage markers, was significantly lower in adipose tissue of Ela2−/−
mice. This is consistent with the observation of classically activated M1 macrophages in inflamed adipose tissue in obesity (Lumeng et al., 2007
). In addition, we observed no increase in early inflammation markers such as chemoattractant MCP-1, and adhesion molecule ICAM-1, after 3 days of HFD feeding of Ela2−/−
mice. Furthermore, our data demonstrated that NE accumulated in eWAT in a similar pattern as macrophage infiltration and fibrosis. These data therefore suggest that NE is a key regulator of HFD-induced inflammation and tissue damage in eWAT throughout the development of obesity. It is possible that activation of NE by HFD feeding may induce vascular damage, which allows entry of neutrophils and M1 macrophages. Thus, this study supports the notion that the imbalance of NE and A1AT is a key pathological mechanism linking over-nutrition to leukocyte infiltration, adipose fibrosis and subsequent insulin resistance in obesity.
Adiponectin is a cytokine secreted by adipose tissue that exists in high, medium, and low molecular weight forms in the circulation. Compelling evidence suggests that adiponectin plays an important role in the regulation of insulin sensitivity, lipid and glucose metabolism, and inflammation (Kadowaki et al., 2006
; Ohashi et al., 2010
; Shetty et al., 2009
). Multiple studies have reported that circulating adiponectin levels, particularly the HMW adipokine, are significantly reduced in obese and type II diabetic patients compared with lean subjects (Pajvani et al., 2003
; Semple et al., 2007
; Waki et al., 2003
). HMW adiponectin has been shown to regulate expression of IRS2 and phosphorylation of AMPK and ACC2 in the liver, which are thought to be important for HMW adiponectin-mediated insulin sensitization and fatty acid oxidation (Awazawa et al., 2011
; Kadowaki et al., 2006
; Shetty et al., 2012
; Yamauchi et al., 2002
). In this study, Ela2−/−
mice were resistant to the HFD-induced decrease in serum HMW adiponectin, suggesting that NE is a regulator of HMW adiponectin homeostasis. Interestingly, Ela2−/−
mice showed increased phosphorylation of AMPK and FAO in the liver. These results are consistent not only with the observation that circulating HMW adiponectin levels are increased in Ela2−/−
mice, but also with the functional analyses showing that those mice have improved insulin sensitivity and are resistant to HFD-induced liver steatosis. The reduced expression of PEPCK and glucose-6-phosphatase in the liver of Ela2−/−
mice further supports these observations. Thus, our data suggest an additional mechanism by which NE regulates insulin sensitivity and obesity by increasing HMW adiponectin expression and AMPK activation in the liver. It is possible that NE directly cleaves adiponectin resulting in lower levels of HMW adiponectin. Alternatively, the inflammatory damage of adipose tissue may lead to less HMW adiponectin secretion and therefore, less serum HMW adiponectin in WT mice than in Ela2−/−
Our data also provides a potential molecular mechanism whereby the imbalance of NE and A1AT affects HFD-induced bodyweight gain. We observed that both AMPK protein expression and phosphorylation were significantly increased in the BAT of Ela2−/− mice. The increase of the inhibitory phosphorylation of ACC2 by the activated AMPK may contribute to the increase in FAO in the BAT of NE null mice. Therefore, our data for the first time provides the link of the NE-A1AT system to AMPK signaling – FAO – energy expenditure axis. In addition, UCP1 protein levels were significantly increased in the BAT of NE null mice after HFD feeding. These data are consistent with the observation that Ela2−/− mice had higher body temperature, more energy expenditure and less bodyweight gain than WT mice fed with a HFD. It is possible that increased UCP1 expression in the BAT from NE null mice may be due to the decrease of inflammation and chemokine production in the BAT (data not shown). However, it remains to be evaluated how depletion of NE leads to the activation of the AMPK pathway and increase of UCP1 protein levels in the BAT.
Taken together, our data suggest that elevation of serum and tissue NE and reduction of A1AT are key events in obesity. Furthermore, the imbalance between NE and A1AT is associated with leptin resistance and it also precedes the decrease of HMW adiponectin. This study also suggests that the imbalance of NE and A1AT affects energy expenditure through regulating the AMPK pathway and contributes to the development obese-related inflammation, adipose tissue remodeling, insulin resistance and liver steatosis. Hence, both NE and A1AT are important molecular markers for obesity and may be potential drug targets for treatment of obesity and related diseases. Our proteomic profiling-based study is validated with clinical samples and is consistent with an independent recent report suggesting that elastase is involved in the development of inflammation and insulin resistance (Talukdar et al., 2012