Under normal circumstances, adipose tissue is considered a connective tissue of low density and high plasticity. However, here we have demonstrated that during progression to an obese state, the connective fiber content of adipose tissue increases dramatically, due to an upregulation of several collagens. As the collagen content increases, the overall rigidity of adipose tissue also increases, likely contributing to an increase in its mechanical strength. The term fibrosis has been defined as the formation of fibrous tissue as a reparative or reactive process; it has been widely utilized in the context of the liver, lung, and kidney, in addition to several other tissues. In adipose tissue, fibrosis appears to be initiated in response to adipocyte hypertrophy, which occurs as the initial step toward fat pad expansion through enlargement of the lipid droplet size in existing adipocytes. This cellular expansion seems to constitute the initial “insult,” in response to which the upregulation of extracellular matrix components is triggered. We are currently investigating which cellular factors couple the cellular expansion of the adipocyte with increased transcriptional activity at the level of collagens; our preliminary data suggest that hypoxia-inducible factor 1α plays a critical role in this process (N. Halberg et al., submitted for publication).
The level of extracellular matrix constituents in all tissues is a reflection of the balance between the rate of synthesis of matrix proteins and their degradation. Degradation is achieved through a family of proteins termed MMPs, which in turn are regulated by their inhibitors, called tissue inhibitors of MMPs. During tissue remodeling, the balance between these molecules can shift to accommodate the tissue's immediate needs for expansion. The altered expression of the proteoglycans lumican and decorin in col6KOob/ob mice has fundamental implications in the antifibrotic nature of the fat.
Lumican has been implicated in various processes, such as cell migration, proliferation, wound healing, and inflammation. One of its more unique roles is its ability to bind and interact with macrophages, an interaction for which its keratan sulfate side chains are critical (19
). Decorin is well-known for its antifibrotic role in lung, kidney, muscle, and liver through its ability to inhibit TGF-β (25
). TGF-β, in turn, plays a central role in modulating the balance between the rate of synthesis and degradation of matrix proteins. TGF-β contributes to fibrosis of several tissues: by increasing expression of matrix components, such as collagens, fibronectin, and matrix proteolgycans, and by inducing inhibitors of matrix-degrading MMPs. Therefore, despite the fact that lumican and decorin are both collagen cross-linking proteins, these two proteins have opposing roles in fibrosis. This could be due to their specific roles during collagen fibrillogenesis or due to the diverse interactions that their unique proteoglycan side chains enable (19
). The downregulation of lumican and TGF-β and the upregulation of decorin suggest a mechanism of reduced fat fibrosis in collagen VI-deficient ob/ob
Recent reports have described a role for PPARγ as a potent antifibrotic factor. PPARγ agonists can reduce fibrosis in several organs, including kidneys, liver, heart, and lungs (1
). It has been suggested that the PPARγ agonist tesaglitazar, a member of the thiazolidinedione (TZD) family, reduces glomerulofibrosis and collagen deposition by lowering TGF-β1 levels in the kidney (8
). Xu and colleagues have demonstrated that PPARγ can act as a repressor of type I collagen synthesis by increasing gamma interferon-induced repression of collagen synthesis (57
). We have demonstrated here that COOH, a selective non-TZD-based PPARγ agonist, reduces the expression levels of a vast majority of collagens in adipose tissue. This provides an alternative mechanism of action, by which TZDs can improve the diabetic phenotype through the lowering of the overall extracellular matrix content of adipose tissue.
Since TGF-β1 plays a critical role in collagen deposition, it is likely a target of PPARγ agonist-induced reductions in collagen levels. In fact, we found that wild-type mice treated with a PPARγ agonist have reduced levels of TGF-β1 in adipose tissue (Fig. ). The reduction of TGF-β1 levels in col6KOob/ob
mice is consistent with a possible role of TGF-β in inducing fibrosis of adipose tissue as well. TGF-β can also be upregulated by mechanical stress in many cell types (6
). Mechanical stress on the adipocyte membrane, triggered by the expanding lipid droplet, may also be the source of shear stress at the level of the plasma membrane. Similar to what has been reported for many other tissues in response to mechanical stress, TGF-β is likely to upregulate collagens and other extracellular matrix proteins to counteract this expansion. During times of positive energy balance, there is, however, continued pressure on the adipocyte to expand further. The increased rigidity of the adipose tissue matrix counteracts this trend for further expansion. Such a tug-of-war between cellular expansion of adipocytes and the rigid extracellular environment exerts massive pressure on the plasma membrane that can result in cell death by necrosis. Demeulemeester and colleagues demonstrated that mice treated with an MMP inhibitor while receiving a high-fat diet challenge had less adipose tissue, increased adipocyte cell number, and decreased average adipocyte diameter. Their increased adipose collagen content coupled with reduced adipocyte size is consistent with the notion that increased collagen restricts expansion of adipocytes (17
). A reduced level of elastin in col6KOob/ob
adipose tissue is also consistent with a more flexible microenvironment of the adipocyte.
One major characteristic of obese adipose tissue is an increased frequency of adipocyte cell death. Work by Cinti et al. demonstrated that macrophages aggregate around these dead adipocytes and become increasingly activated in their attempt to clear the potentially cytotoxic remnant lipid droplet (13
). These adipose tissue macrophages fuse to form multinucleated cells and surround each dead adipocyte in a crown-like structure. In addition to this phenomenon, the number of necrotic adipocytes positively correlates with average adipocyte size in obese mice and other mouse models of adipocyte hypertrophy (13
). Hormone-sensitive lipase (HSL) knockout mice exhibit adipocyte hypertrophy and macrophage recruitment without any other characteristics of obesity. However, since HSL-deficient mice possess the typical rigid environment, in contrast to our col6KOob/ob
mice, it is likely that adipocytes in HSL-deficient mice encounter increased shear stress, cell death, and inflammation.
Shear stress and membrane stretching have been shown to activate members of the MAPK family in several cell types (23
). In response to shear stress, large adipocytes exhibit an increased level of activation of the β1-integrin/ERK signaling pathway (18
). In some circumstances, mechanical stretching-induced activation of JNK can also result in apoptosis of cells (41
). Therefore, shear stress in large adipocytes appears to be the trigger for adipocyte death and the impending inflammation. The association of adipocyte hypertrophy with increased macrophage recruitment has led to the current dogma that an increase in adipocyte size is indicative of inflamed adipose tissue and is generally associated with an unfavorable metabolic profile. In contrast, in the absence of collagen VI, the adipocyte matrix has more flexibility and allows adipocytes to continue to expand without the associated necrosis and inflammation. This may be one of the reasons why the col6KOob/ob
mice defy the currently established rule that larger adipocytes are associated with increased inflammation.
Since an array of signaling molecules, including many upstream regulators of ERK and JNK, reside in caveolae, it has been suggested that shear stress-induced MAPK activation involves caveolae (5
). Park and colleagues demonstrated that exposure of endothelial cells to shear stress dramatically increases the number of invaginated caveolae. ob/ob
mice deficient in collagen VI have an accumulation of uninvaginated, vesicular caveolae in their adipocytes. The differences in caveolar structures between the col6KOob/ob
mice are therefore a direct reflection of the reduced levels of shear stress in the col6KOob/ob
The data presented here are consistent with the concept that reduced local inflammation in the adipose tissue of col6KOob/ob mice is the major contributing factor to their overall improvement in the metabolic phenotype. The weakened extracellular matrix environment in col6KOob/ob adipose tissue enables their adipocytes to expand unhindered, thus facilitating storage of excess lipids and reducing ectopic lipid deposition. col6KOob/ob mice also become more insulin sensitive, as depicted in their reduced islet hyperplasia and improvements in overall islet morphology.
The extracellular matrix is extremely important for structure and function of almost any cell type; furthermore, it is involved in numerous processes such as cell adhesion, proliferation, differentiation, migration, apoptosis, and gene induction (20
). In adipose tissue, it is particularly crucial for maintaining structural integrity of adipocytes and plays a critical role in adipogenesis. There are specific sequential alterations in the extracellular milieu during adipocyte differentiation, with a cascade of activation and deactivation of various MMPs, complementing the creation and destruction of the collagen network (31
). Treatment of 3T3-L1 preadipocytes with a broad-specificity MMP inhibitor hinders the formation of fully differentiated lipid-laden adipocytes (11
). The extracellular matrix also undergoes numerous changes during obesity, with the MMP/tissue inhibitors of MMP balance being shifted toward increased matrix degradation, presumably as a compensatory response to the increased collagen content during obesity (11
). Despite the clear relevance of the matrix on adipocytes, very few studies have focused on a thorough characterization of the metabolic impact of the extracellular matrix during obesity or during other metabolic conditions.
One of the first available examples of a metabolic study devoted to an extracellular matrix protein in adipose tissue was a study on SPARC, a matricellular protein involved in cell-extracellular matrix interactions. SPARC-null mice have a larger epididymal fat pad and an increased number of adipocytes in skin. Even though we lack information on any metabolic consequences that the absence of SPARC causes, this was the first time that an extracellular matrix protein was implicated in an effect on adipose tissue. More recently, Chun and colleagues demonstrated that the absence of matrix metalloproteinase MT1-MMP causes lipodystrophy in mice by impairing white adipose tissue development, due to a direct impact of MT1-MMP on adipocyte differentiation (12
). Interestingly, these effects could only be observed in vitro, when cells were grown in a three-dimensional matrix.
We selected collagen VI as a candidate matrix component and studied its impact on adipose tissue physiology. Collagen VI is an ideal candidate due to its relatively abundant and highly enriched expression in adipose tissue. It is the most highly expressed collagen in differentiated adipocytes and undergoes dramatic structural changes during adipogenesis (38
). Collagen VI fibrils alter their appearance during differentiation of 3T3-L1 preadipocytes, switching from a thin fibril texture at day 4 of differentiation to a very thick appearance in fully differentiated adipocytes. Several studies have further demonstrated that collagen VI levels correlate with conditions of hyperglycemia and insulin resistance (2
). We have previously demonstrated a role for adipocyte-derived collagen VI in progression of mammary tumor growth (26
). During tumor growth, a proteolytic cleavage event occurs and the resulting C-terminal collagen VI fragments accumulate in tumor cells, activating the NG2 receptor and promoting a mitogenic response in the cancer cells. Our data describing increased mammary epithelial apoptosis in the absence of collagen VI protein are compatible with previous data that have demonstrated the requirement of collagen VI in the prevention of apoptosis and its stimulatory role in proliferation through AKT signaling events (22
). The absence of collagen VI results in a proapoptotic signal event, thus reducing the likelihood of tumor proliferation.
Our observations have highlighted a number of changes at the level of food intake and energy expenditure: the lack of collagen VI triggers reduced food intake and reduced energy expenditure, with a higher rate of fatty acid consumption. Since these changes are not only observed during high fat diet exposure but also in the ob/ob background, these phenomena cannot exclusively be driven by leptin. It is not clear how the reported changes in adipose tissue trigger the systemic alterations in food intake and energy expenditure. These are questions of obvious interest that we are currently pursuing. In addition, we cannot formally rule out that collagen VI exerts its impact on metabolic control through action in tissues other than fat.
In summary, our study highlights the fact that collagen VI, and possibly additional extracellular matrix constituents, are extremely important in modulating adipocyte physiology. Collagen VI has an essential role in the fibrotic component of obesity and directly affects the ability of adipocytes to expand. The involvement of collagen VI in obesity opens a new chapter in which the adipocyte itself affects overall metabolic function; however, it is now apparent that the extracellular matrix environment surrounding the adipocyte can also play an essential role. Further studies will be required to explore the physiological consequences of adipose tissue fibrosis and whether collagen VI plays a critical role in other fibrotic tissues.