OPN is a chemokine-like, extracellular matrix–associated protein involved in monocyte motility and the inflammatory immune response (10
). Since its initial cloning in 1986 (25
), OPN has been associated with a remarkable range of pathologic responses (10
); however, its role in obesity and metabolic disorders has not, to our knowledge, previously been investigated. In the present study, we demonstrate expression of OPN in adipose tissue and outline what we believe to be a previously unrecognized role for OPN to mediate obesity-associated ATM recruitment, adipose tissue inflammation, and resulting insulin resistance.
A host of adipose tissue–generated adipokines and cytokines have been identified that have emerged as an important source of systemic inflammation in obesity (2
). We report here that OPN expression in adipose tissue increases with obesity and that plasma levels increase during the development of DIO OPN, suggesting a potential novel function of OPN for obesity-associated inflammatory and metabolic changes in adipose tissue. OPN plasma levels are elevated in various inflammatory diseases, including atherosclerosis (26
), inflammatory bowel disease (27
), granulomatous inflammatory diseases (28
), rheumatoid arthritis (29
), and multiple sclerosis (30
). OPN is rapidly expressed after cellular activation, and it is abundantly secreted by activated macrophages but not resting macrophages or monocytes (31
). Consistent with this notion, we observed that OPN expression in obese adipose tissue colocalized with macrophages and that OPN mRNA was highly expressed in macrophages isolated from the SVF. In contrast, we observed only negligible OPN mRNA in adipocytes and a decline in OPN transcript levels with differentiation of 3T3-L1 adipocytes. Although OPN is expressed in proliferating fibroblasts (33
), these observations confirm earlier reports documenting decreased OPN mRNA levels in differentiated adipocytes as compared with preadipocytes (34
). Interestingly, OPN has previously been characterized as a PPARγ target gene in macrophages, and overexpression of PPARγ or ligand treatment with a thiazolidinedione suppresses OPN transcription (17
). PPARγ expression increases with differentiation of adipocytes, and when we overexpressed PPARγ in 3T3-L1 fibroblasts, thiazolidinedione treatment decreased OPN promoter activity. Similarly, overexpression of a constitutively active PPARγ mutant suppressed basal OPN promoter activity. Thus PPARγ-mediated downregulation of OPN could provide a mechanism for the observed decline in OPN mRNA expression during the differentiation process of preadipocytes into mature adipocytes.
Using a model of DIO, our studies revealed that OPN has no effect on the development of obesity and does not affect food intake or energy metabolism. Consistent with several recent studies (9
), total body weights were similar in wild-type and OPN-deficient mice. However, in previous studies body composition was not analyzed, and an unexpected but interesting and consistent observation in both lean and obese animals was a slightly decreased lean body mass in OPN–/–
mice. Our experiments employed NMR technology to provide measures of body fat mass, fat-free mass, and water content (37
). Considering the limitation of this technique to distinguish different fat-free tissue masses including bone and muscle, the reason for the slightly decreased lean body mass in OPN–/–
mice remains elusive. OPN deficiency renders mice less sensitive to bone resorption (36
). Thus this knowledge and the observation that bone density analyzed by micro-CT is not affected by OPN deficiency in unstressed mice (38
) argues against a contribution of OPN deficiency in bone tissue to the observed decrease in lean body mass. To date no studies have determined whether OPN affects total body muscle mass. Therefore, it will be important for the determination of the mechanisms by which OPN deletion modulates lean body mass to characterize fat-free tissue masses in OPN-deficient mice and perform further studies using newly available techniques including NMR to analyze total body composition.
In bone marrow transplantation studies, Weisberg et al. have recently provided evidence that the majority of ATMs are derived from the circulation (3
). The MCP-1/C-C motif chemokine receptor 2 (MCP-1/CCR2) axis is well established to regulate macrophage recruitment to sites of inflammation (39
), and MCP-1 is secreted from adipocytes and plasma levels are increased in obesity (41
). In addition, MCP-1 levels secreted from adipocytes correlate with adipocyte size (42
), indicating that the MCP-1/CCR2 cascade is likely among the earliest mechanisms involved in the recruitment of monocytes to adipose tissue. This is further supported by recent studies demonstrating that adipocyte-specific MCP-1 overexpression results in enhanced macrophage infiltration of adipose tissue (43
), while MCP-1 or CCR2 deficiency decreases macrophage content in obese adipose tissue (19
). However, in both models obesity-induced macrophage accumulation in adipose tissue is not normalized and macrophages remain accumulating in the adipose tissue, suggesting that additional mechanisms are involved in this process. In this study, we demonstrate decreased macrophage content in obese adipose tissue from OPN–/–
mice in the absence of any differences in total fat mass, thus providing an additional mechanism by which macrophages infiltrate adipose tissue. This requirement of OPN for macrophage recruitment to adipose tissue is consistent with our data examining the role of OPN for macrophage chemotaxis. In wild-type macrophages OPN amplifies macrophage migration and exerts additive effects on chemotaxis in the presence of MCP-1. In contrast, OPN-deficient macrophages are hypomotile and less responsive to MCP-1, a phenotype that exogenous OPN is unable to completely correct. These observations suggest that OPN augments the MCP-1 response and functions primarily in an autocrine mechanism to promote macrophage chemotaxis. This concept is in agreement with recent studies demonstrating that endogenous OPN expression in macrophages is important to maintain macrophage function, including chemotaxis, differentiation, and inflammation (13
). Moreover, intracellular OPN forms a complex with the CD44 receptor and ezrin/radixin/moesin proteins at the cell membrane of cell processes (45
) that is required for cell fusion and chemotaxis of macrophages (22
). Finally, it is well recognized that OPN–/–
mice have defective granulomatous responses (8
) that likely involve abnormal macrophage function.
The observation that OPN is primarily expressed by macrophages in obese adipose tissue combined with the important autocrine role of OPN in macrophage function suggest a model in which endogenous OPN amplifies macrophage recruitment through the MCP-1/CCR2 cascade in the early stages of obesity. Our ex vivo experiments demonstrate increased macrophage chemotaxis toward the SVF isolated from obese mice, indicating that the continued recruitment of macrophages within the SVF may further exacerbate macrophage infiltration. In contrast, migration toward the SVF isolated from obese OPN–/– mice was substantially decreased. Our findings that macrophage content in the SVF from OPN–/– mice is considerably less compared with that from OPN+/+ mice may indicate that fewer macrophages accumulating during the course of obesity ultimately secrete fewer migratory signals, which would likely contribute to decreased macrophage recruitment during later stages of obesity. In concert, these studies support an important role for OPN to promote macrophage infiltration into obese adipose tissue. However, confirming the contribution macrophage-dervied OPN to adipose tissue inflammation in vivo requires studies that will depend on the specific deletion of OPN in macrophages using either bone marrow transplantation approaches or conditional OPN deletion strategies.
Concomitant with the attenuated macrophage content in obese adipose tissue from OPN–/–
mice, we documented decreased inflammatory gene expression in adipose tissue and decreased systemic levels of the proinflammatory cytokines IL-6, MCP-1, and PAI-1. MCP-1– and CCR2-deficient mice develop less obesity-induced insulin resistance (19
), and the current understanding of the role of ATMs and adipose tissue inflammation in obesity-induced insulin resistance would suggest that prevention of macrophage accumulation in adipose tissue preserves insulin sensitivity. In further support of this concept, we demonstrate that decreased ATM accumulation in OPN-deficient mice is associated with increased insulin sensitivity: OPN–/–
mice developed less obesity-associated hyperinsulinemia, cleared glucose more rapidly following an intraperitoneal glucose challenge, and exhibited an enhanced insulin response after an intraperitoneal injection of insulin. Importantly, these effects of OPN deficiency were observed despite the same level of obesity in OPN–/–
and wild-type mice. Increased insulin sensitivity associated with OPN deficiency was unlikely a result of altered adipokine secretion, since plasma levels of 3 adipokines implicated in insulin resistance (adiponectin, resistin, and leptin) were not significantly different in OPN–/–
mice. Based on the strong recent evidence that ATMs are both necessary and sufficient for the development of obesity-associated insulin resistance (4
), the observed decrease in ATM content in obese OPN–/–
mice provides a likely mechanism for the increased insulin sensitivity in these mice. Macrophages present in adipose tissue directly interfere with insulin signaling and insulin-stimulated glucose uptake in adipocytes by decreasing GLUT4 and insulin receptor substrate 1 (IRS-1) expression, leading to a decrease in Akt phosphorylation and impaired insulin-stimulated GLUT4 translocation to the plasma membrane (47
). Interestingly, blocking TNF-α, which is well established to contribute to insulin resistance (48
), prevents macrophage-induced alterations in adipocyte insulin signaling (47
). These observations confirm important cross-talk between ATMs and adipocytes in mediating insulin resistance and indicate that ATMs affect insulin signaling by perpetuating inflammatory pathways in adipocytes. While the concept of decreased ATM content in OPN–/–
mice as a mechanism for improved insulin sensitivity is intriguing and supported by these studies, we cannot exclude that OPN may also affect hepatic insulin sensitivity. OPN deficiency has previously been associated with decreased hepatic fibrosis in experimental nonalcoholic steatohepatitis (49
), and since hepatic OPN expression increases during nonalcoholic steatohepatitis in obese mice (50
), OPN deficiency may also modulate obesity-induced hepatic insulin resistance.
In summary, in the present study we characterize OPN, a versatile mediator of macrophage motility involved in cell-mediated inflammation (9
), as a novel cytokine expressed by ATMs and secreted during DIO. OPN expression is required for macrophage recruitment into adipose tissue and for obesity-associated adipose tissue and systemic inflammation. Finally, we demonstrate that decreased adipose tissue inflammation in OPN–/–
mice is associated with improved obesity-associated insulin resistance without significantly altering body mass. These data therefore identify OPN as a previously unappreciated link between obesity, adipose tissue inflammation, and insulin resistance.