Adipose tissue from obese individuals displays several prominent features not typically observed in the lean state; to accommodate an excessive triglyceride load, adipose tissue expands through both adipocyte hypertrophy and hyperplasia. This expansion is furthermore associated with hypoxia, fibrosis, local inflammation, and concomitant insulin resistance. While local inflammation and insulin resistance in adipose tissue are causally related to systemic metabolic dysfunction and type II diabetes, the temporal and mechanistic connections among the processes prior to inflammation have not, as yet, been characterized. Nevertheless, late-stage processes related to the interaction between adipose tissue inflammation and insulin sensitivity, however, are better understood. Several components of the inflammatory pathways have been implicated in reducing insulin sensitivity, such as TNF-α (21
), c-jun N-terminal kinase (19
), and NF-κB (54
). The associated infiltration of monocytes and macrophages into adipose tissue has been extensively studied (50
). Whether such an infiltration is strictly secondary to concomitant necrosis of adipocytes during fat expansion or is the result of enhanced chemokine secretion by enlarged adipocytes, or is a combination of the two, remains unclear. From cell culture studies, we know that the three-dimensional network of the extracellular matrix surrounding the adipocyte is functionally very important (5
). We have recently demonstrated that fibrosis of the adipose tissue plays an important role in adipose tissue dysfunction (28
). Here, we show that obese adipose tissue contains large streaks that stain positively for fibrillar collagens, interspersed in between adipocytes. In Khan et al. (28
), we have furthermore demonstrated that the genetic removal of a key constituent of the adipose tissue extracellular matrix, collagen VI, leads to a significant improvement in the metabolic phenotype of mice challenged with a dietary intervention or in the ob/ob
background. Reduced macrophage infiltration in these collagen VI null mice indicates a connection among alterations in the adipose tissue extracellular matrix, adipocyte survival, and inflammation. Here, we sought to identify the upstream mechanisms that trigger the accumulation of extracellular matrix components that ultimately lead to fibrosis. We found that local adipose tissue hypoxia may be the most important driving force for the downstream events associated with adipose tissue dysfunction.
As oxygen diffuses away from the capillary bed, its partial pressure falls from approximately 100 mmHg inside the vessel to almost zero within as little as 100 μm into the tissue (14
). Considering adipocytes are rather large cells, with diameters of up to 200 μm (42
), the hypoxic phenomenon is prone to be relevant based on the sheer size of the adipocyte. Additionally, obese adipose tissue displays an attenuated increase in postprandial blood flow (7
), which is in part due to reduced insulin sensitivity of the cells in the vessels (26
). Consequently, obesity-associated adipose tissue hypoxia has been demonstrated by several groups in human adipose tissue (3
) and in rodent adipose tissue (20
). These initial findings led to the hypothesis that local adipose tissue hypoxia may underlie the inflammatory response (47
); however, direct evidence for such a mechanism has been lacking to date.
Hypoxia in adipose tissue results in the stabilization of the transcription factor HIF1 (3
). This master regulator of the hypoxia response has been thoroughly investigated in the context of tumor biology. Its major effect is the induction of an angiogenic response through binding to the hypoxia response element of target genes, such as VEGFa and angiopoietin 2. This, in turn, allows the tumor to establish a better oxygenated and nutritionally-enriched microenvironment (1
). Tumors can divert their pyruvate metabolism away from the mitochondrial electron transport chain toward an anaerobic conversion into lactate through a process that is largely mediated by HIF1α (40
). Here, our objectives were to characterize the role of HIF1α in large adipocytes and to investigate any possible connections between HIF1α and fibrosis. In order to achieve that, we took advantage of the overexpression of a dominant active deletion mutant of HIF1α (HIF1α-ΔODD). With this transgene, we failed to detect any transcriptional increase in some of the classical HIF1α targets such as VEGF-A as well as failed to observe an accompanying increase in angiogenesis and anaerobic glycolysis. However, HIF1α-ΔODD overexpression did result in a transgene-dependent global glucose intolerance that could be enhanced by age, diet, and genetically induced obesity. Further profiling of the transgenic fat highlighted the critical transcriptional targets. Such targets included a general HIF1α-induced increase in fibrotic proteins such as LOX, elastin, collagen I and III, TIMP1, and CTGF. Induction of such a fibrotic program resulted in increased fibrillar collagen (I and III) accumulation in the extracellular matrix of the adipose tissue in the transgenic animals, thereby turning wild-type fat pads into tissue resembling fat from ob/ob
mice. This is consistent with several cell culture studies that have previously demonstrated that hypoxia increases the expression of extracellular proteins, such as collagen I, fibronectin, and TIMP1, in various mesenchymal cell lines as well as in human renal fibroblasts (8
). More importantly, however, to the production of collagens and other extracellular matrix constituents, the strength of this matrix is highly dependent on further processing of the components. One such enzyme that plays a critical role in the stabilization of the extracellular matrix components is LOX, which can cross-link elastins and collagens in the extracellular matrix and thus increase extracellular tensile strength. Since LOX is a known HIF1 target gene, we decided to investigate the functional role of LOX in rapidly expanding adipose tissue. The LOX gene is highly responsive to metabolic cues and is generally upregulated in situations characterized by dysfunctional adipose tissue such as obesity or exposure to endotoxin. On the other hand, PPARγ agonist treatment and adipogenesis are characterized by a significant reduction in LOX gene expression. Within as little as 5 weeks of HFD exposure, prior to any weight differences between our transgenic HIF1α-ΔODD and wild-type mice, the transgenic mice displayed increased expression of LOX as well as a decrease in glucose tolerance and increased adipose tissue fibrosis. The increased fibrosis was even more pronounced as the HIF1α-ΔODD mice were genetically challenged by crossing them into the ob/ob
background. Pharmacological inhibition of LOX activity resulted in an increase in the insulin sensitivity of HIF1α-overexpressing animals. Microarray analysis furthermore showed that approximately half the genes upregulated by HIF1α are dependent on HIF1α-induced LOX expression. As such, our studies pinpoint LOX as a key player in HIF1α-mediated fibrosis and associated insulin resistance. However, this does not rule out important contributions of other HIF1α targets. As such, another interesting candidate could be CTGF, which has been shown to be regulated in a fashion similar to that of LOX in the adipocyte (46
). Like the findings presented here, Higgins et al. have demonstrated that renal hypoxia leads to a HIF1α-mediated fibrosis through induction of LOX (18
). Interestingly, aside from its effects on collagens and elastins in the extracellular space, LOX has been detected inside the nuclear compartment of the cell as well (31
), where it may function as a transcription factor for elastin and collagen III (15
). Nuclear LOX may further compound the effects of HIF1α on fibrosis in adipose tissue.
Throughout all experiments performed, we noticed that the effects we observed were strictly dependent on the local levels of expression of the transgene. We observe this phenomenon of differential transgene expression driven by the aP2 promoter in different fat pads quite frequently in several unrelated instances. This phenomenon is a function of the integration site of the transgene. While uniform transgene expression in different fat pads can be achieved with this promoter (resulting in more-severe systemic phenotypes), we decided to further characterize a transgenic line with differential expression levels in different fat pads, such that we can use fat pads with low transgene expression levels as internal controls for comparisons with fat pads with high transgene expression levels.
The induction of collagen I- and III-laden “trichrome-positive streaks” during the early stages of adipose tissue dysfunction is intriguing. Such structures are highly obvious when comparing adipose tissue from ob/ob
animals to that of wild-type animals. They can, however, further be detected under conditions with much milder metabolic alterations associated with HIF1α overexpression in younger mice. Furthermore, these structures are distinct from the “crown-like structures” previously defined by Strissel and colleagues (44
) and are apparent before these crown-like structures start to appear. It is technically very difficult to establish a direct relationship between trichrome-positive streaks and crown-like structures, but it is tempting to speculate that such “fibrotic streaks” reflect local hypoxic pockets that will ultimately be associated with increased necrosis of the surrounding adipocytes that will eventually attract macrophages to form crown-like structures. Our detailed time course analysis over the first 20 days of an HFD showed that the fibrotic program (e.g., HIF1α, LOX, collagen I and III) is induced shortly after initiation of the HFD challenge. Macrophages and the associated inflammation appear much later in the process.
It is unlikely that hypoxia affects an expanding fat pad uniformly but rather develops from pockets that are initially less vascularized. Additionally, it is by now well established that the epididymal fat pad in rodents expands asymmetrically, with the tip of this fat pad expanding most dramatically (4
As evident by Fig. S3 in the supplemental material, the HIF1α transgene is expressed in brown adipose tissue as well as in white adipose tissue. However, we have not been able to detect any local phenotypic changes with respect to histology (H&E and trichrome staining), gene expression, or Western blotting in brown adipose tissue (data not shown). We therefore cannot draw any conclusions with respect to the function of HIF1α in brown adipose tissue and have consequently chosen to focus solely on white adipose tissue in the present study. However, this does not mean than HIF1α does not exert potentially important functions in brown adipose tissue. The lack of a phenotypic change in brown adipocytes is most likely a reflection of a low level of transgene expression in this particular fat pad.
In light of our data presented here, we would like to propose the following model for the early stages of adipose tissue dysfunction (Fig. ): excess caloric intake results in an expansion of adipose tissue. Such expansion causes local adipose tissue hypoxia, which triggers increased expression and stabilization of HIF1α. Despite its enhanced expression HIF1α fails to alleviate hypoxia, due to its inability to induce proangiogenic factors. However, HIF1α does stimulate a host of extracellular factors, such as collagens, in addition to components involved in establishing and remodeling the extracellular matrix. Most notably, LOX exerts a prominent role in this remodeling that leads to an increased deposition of fibrillar collagen in the adipose tissue. This global upregulation of extracellular matrix constituents subsequently causes fibrosis, and it is this fibrosis per se that results in increased stress of expanding adipocytes as well as necrosis. As a consequence, this triggers an increased infiltration of macrophages that ultimately mediates higher levels of local inflammation and a concomitant reduction in insulin sensitivity.
FIG. 9. Schematic representation of the suggested hypothesis. During periods of a positive-energy balance, white adipose tissue expands to meet the need for extra triglyceride storage. As a consequence, adipose tissue becomes hypoxic, thereby activating the transcription (more ...)