Accumulating evidence suggests an important role for low-grade inflammation and adipose tissue remodeling in the development of obesity. In the present study we investigated the adipose tissue cytokine and angiogenesis-related protein profiles from obese and lean mice by using sensitive high-throughput protein arrays. Furthermore, we examined the influence of calorie restriction (CR) on adipose tissue protein profiles. The important finding from the present study was that obesity is associated with simultaneous induction of several cytokines and angiogenesis-related proteins in adipose tissue. CR decreased body weight and body fat percentage to a similar extent in obese and lean mice. However, CR showed opposite effects on protein profiles between obese and lean mice. CR largely ameliorated cytokine and angiogenesis-related protein expression in obese mice, while in lean mice marked upregulation of several proteins was seen.
Accumulating evidence suggests a close relationship between the amount of visceral fat, metabolic disturbances and cardiovascular diseases [4, 6]. Adipose tissue dysfunction leads abnormal cytokine secretion thus inducing the development of low-grade inflammatory state that contributes to obesity-linked metabolic disorders such as type 2 diabetes [4, 6]. To study further the molecular mechanisms mediating adipose tissue inflammation in obesity, we characterized the cytokine expression profiles from visceral fat. We were able to demonstrate that obesity is associated with up-regulation of several pro-inflammatory cytokines, including IL-1ra, IL-2, IL-16, MCP-1, MIG, RANTES, C5a and sICAM-1. It is of great interest that CR in obese mice markedly attenuated cytokine overexpression, whereas in lean mice CR actually increased the levels of most of the above mentioned pro-inflammatory cytokines in the adipose tissue. Distinct effects of CR on cytokine expression profiles in obese and lean mice can not been explained by differences in the study design as both the body weight and body fat percentage were decreased by CR to a similar extent in obese and lean mice. Our findings are in good agreement with the study by Fenton et al.  demonstrating that CR increases serum cytokine levels in lean mice. Our findings are also consistent with the recent report by Wang et al.  showing that CR ameliorates adipose tissue inflammation in diet-induced obese mice, in particular when CR is carried out by restricting intake of HFD. Further studies are thus warranted to investigate the cellular mechanisms mediating the opposite effects of CR on adipose tissue inflammatory response between obese and lean mice.
Adipose tissue is the highly vascularised tissue, and fat mass expansion is closely linked to angiogenesis [7, 8]. Although the cellular mechanisms regulating adipose tissue-related angiogenesis are still poorly understood, several pro- and anti-angiogenic components have been identified . As adipose tissue angiogenesis is known to be critical for adipogenesis, a more deep understanding of the regulation of adipose tissue angiogenesis may provide novel drug targets for obesity and obesity-related disorders. We therefore examined the expression of 53 different pro- and anti-angiogenic factors in adipose tissue. We were able to demonstrate that obesity is associated with marked alterations in the protein expression of cell growth regulators, angiogenic growth factors and proteases as well as their inhibitors. The present study also revealed that CR has a pronounced modulating effect on adipose tissue protein expression profiles. However, inclusive nature of our angiogenic findings should be underlined; we did not perform histological analyses to characterize the vasculature, endothelial cells or ECM proteins in adipose tissue. Further studies are thus warranted to investigate how the altered adipose tissue protein expression profiles influence the vasculature. Furthermore, as obesity has been shown to alter collagen and elastin expression in adipose tissue , it would be important to examine the influence of CR on collagen metabolism in future.
Our study showed that leptin was one of the angiogenic growth factor that is highly sensitive to body weight changes. Leptin is an adipocyte-derived hormone that regulates food intake and energy homeostasis. Leptin is also a potent angiogenic factor. Leptin induces angiogenesis through activation of its own receptor in endothelial cells leading to activation of Stat3 pathway and enhancement of its DNA-binding activity . Leptin also indirectly activates angiogenesis by up-regulating VEGF mRNA expression via activation of the Jak/Stat3 signaling pathway . In addition, leptin has a synergistic effect with FGF basic (also called FGF-2) and VEGF on stimulation of new blood vessel formation . In the present study, leptin was high expressed in obese mice compared to lean mice. Interestingly, higher protein expression of leptin in obese mice associated with lower expression of FGF basic, but there was trend toward increased in PlGF-2 (VEGF homologue) and VEGF-B (see in supplemental data) protein expression between obese and lean mice. In obese mice CR down-regulated leptin expression and up-regulated VEGF expression. In lean mice the effect of CR on leptin expression was opposite; CR up-regulated leptin expression, down-regulated FGF basic and up-regulated VEGF expression. These findings indicate distinct effects of CR on adipose tissue leptin expression between obese and lean mice and suggest also interaction between leptin, FGF basic and VEGF family members.
In the present study angiogenic growth factors endostatin and endoglin were up-regulated by CR both in obese and lean mice. Endostatin is an endogenous angiogenesis inhibitor , and treatment with endostatin reduces body weight of obese mice . Silha et al. showed recently that plasma levels of vascular growth factors as well as the angiogenesis inhibitor endostatin are increased in obese individuals . Endoglin in turn is a membrane glycoprotein that serves as a receptor for members of the TGF-β superfamily proteins. It is highly expressed on proliferating vascular endothelial cells and it has crucial role in vascular development and disease . However, the effects of endoglin on adipose tissue remodeling in obesity are still elusive. In the present study we demonstrated that endothelin-1 level in the adipose tissue was increased in obese mice. Previous studies have revealed that endothelin-1 induces insulin resistance by suppressing glucose uptake [29, 30] and lipolysis in adipocytes [31–34] through ETA receptors. Increased plasma endothelin-1 levels have also been reported in obese subjects with metabolic syndrome . However, the present study revealed that CR does not reduce adipose tissue endothelin-1 levels.
Pericellular proteases have been shown to play an important role in regulating angiogenesis. Proteases participate in extracellular matrix (ECM) remodeling and in angiogenic processes by generating pro- and anti-angiogenic factors from ECM proteins and by processing growth factors and receptors . Plasminogen activator-plasmin system (fibrinolytic system) and matrix metalloproteinases (MMPs) are two major component of proteolytic system . Plasminogen activator inhibitor-1 (PAI-1, also known as serpine E1) is an inhibitor of fibrinolytic system exerting several physiological and pathophysiologial effects related to tumorigenesis, inflammation, thrombosis and metabolic disturbances such as obesity and insulin resistance . Data from studies investigating the effects of PAI-1 on adipogenesis are controversial; some studies using a diet-induced obese mouse models suggest that PAI-1 deficiency has little if any effect on the development of obesity [37, 38], while other studies report prevention of obesity and insulin resistance in mice lacking PAI-1 . Furthermore, PAI-1 inhibitor tiplaxtinin has been shown to prevent adipogenesis and diet-induced obesity [40, 41]. In the present study PAI-1 expression correlated with body weight, and significantly higher PAI-1 expression were found in obese mice. We also noticed that CR down-regulated PAI-1 expression only in obese mice. Our findings thus suggest an important role for PAI-1 in the development of adipose tissue.
The expression of matrix metallopeptidases (MMPs) in the adipose tissue were also altered in diet-induced obese mice. We report here increased MMP-3 expression in obese mice and down-regulation of MMP-3 in the adipose tissue by CR. It is of great interest that CR down-regulated MMP-9 expression both in obese and lean mice, although no difference was detected when the mice were fed ad libitum. Up-regulation of MMP-3 and down-regulation of MMP-9 mRNA expression have been reported recently in expanding adipose tissue . Enhanced adipose tissue development and increased adipose tissue blood vessel density have been demonstrated in MMP-3 deficient mice kept on high-fat diet . Moreover, MMPs inhibitors have been shown to inhibit angiogenesis and to reduce body weight in diet-induced obese mice [14, 44–46].
MMPs are inhibited by endogenous tissue inhibitors (TIMPs), and we here demonstrated upregulation of tissue inhibitors of metalloproteinases TIMP-1 and TIMP-4 with obesity. CR increased TIMP-1 expression both in obese and lean mice, whereas TIMP-4 expression was down-regulated by CR in obese mice and up-regulated in lean mice. TIMP-1 deficient mice has been shown to gain less weight and develop less adipose tissue when fed with high-fat diet and this was related to lower leptin levels detected in TIMP-1 deficient mice . These findings suggest an important role for proteolytic system in adipose tissue development during diet-induced obesity and during weight reduction induced by CR.
Recent studies suggest an important role for osteopontin in the development of HFD-induced insulin resistance and, regulation of vascular and adipose tissue inflammation [48, 49]. Weight loss has been shown to decrease plasma osteopontin levels . We also demonstrated that CR decreased adipose tissue osteopontin expression both in obese and lean mice. Surprisingly, in contrast to some previous studies [50, 51], we were unable to demonstrate obesity-induced osteopontin overexpression in the adipose tissue. Finally, we here reported increased expression of CXCL16 in obese mice. Furthermore, we were able to demonstrate that CR decreased adipose tissue CXCL16 expression both in lean and obese mice. Previous studies have linked CXCL16 and its receptor CXCR6 to inflammation-associated cancers , renal fibrosis , and vascular inflammatory diseases, such as atherosclerosis . Further studies are warranted to investigate the role of CXCL16/CXCR6 axis in adipose tissue remodeling.