Hypoxia has been linked to the pathological changes associated with obesity (4
). HIF1α is an essential mediator of the antihypoxic response under hypoxic stress in many cell types. It is also induced over the course of progression toward an obese fat pad. As one of the earliest events during adipose tissue expansion, HIF1α induction presents a critical step in the sequential process of obesity-associated adipose tissue dysfunction (10
). Intriguingly, unlike its action in most other tissues, including tumor tissues, HIF1α stabilization in adipose tissue does not lead to the induction of proangiogenic factors, such as VEGF (7
). Therefore, hypoxic fat pads are very ineffective at mounting a proangiogenic response. Even though quantitatively less prominent, this is also true in obese human adipose tissue (7
). On the other hand, an alternative HIF1α-mediated transcriptional program is significantly induced, mainly leading to an enhanced fibrotic response. The transcription of many extracellular matrix constituents (ECM) ultimately leads to an inflexible “shell” around individual adipocytes, which results in an increased rate of adipocyte necrosis, ultimately triggering an increased rate of macrophage infiltration and inflammation (10
). In this study, we used an in vivo
oxygen sensor probe in an HFD-fed obese model and not only demonstrated low oxygen pressure in rapidly expanding WAT but also confirmed that HIF1α is induced under these conditions. A number of recent studies implicate HIF1α in the pathophysiology of obesity (49
). This prevailing obesity-associated hypoxia (and concomitant HIF1α induction) is mainly observed in white fat pads, while the induction in other tissues, such as brown adipose tissue (BAT) and liver, is only marginal. This highlights that adipose tissue is uniquely affected under these conditions and suggests the possibility that systemic HIF1α inhibition may primarily exert its therapeutic effects through action in adipose tissue. The data presented establish this phenomenon for the first time.
Furthermore, given the central role of HIF1α in the activation of numerous pathways responsible for metabolic dysfunction demonstrated in gain-of-function studies (10
), we wanted to test whether the inactivation of HIF1α action specifically targeted to adipocytes is a reasonable therapeutic strategy for obesity-associated metabolic dysfunction. Surprisingly, there are no reports in the literature that focus on targeted therapies in this area despite widespread efforts in the context of cancer (21
). We therefore investigated the effects of one of the more promising HIF1α inhibitors, PX-478, on obesity-related metabolic dysfunction. PX-478 has been well characterized both in cancer cell lines and in cancer mouse models (26
). The main impact of PX-478 in different tumor models is to downregulate Glut1 levels (29
), suggesting that the thrust of its antimitogenic activity is mediated through its regulation of glucose metabolism. Consistent with this model, PX-478 has very limited effects on tumor angiogenesis (21
PX-478 is orally available (29
). We therefore treated the mice with PX-478 by gastric gavage. During the 5 weeks of treatment, we observed a lower rate of weight gain compared to placebo treatment, mainly due to differences in fat mass in PX-478-treated mice. PX-478 treatment also exhibited improved glucose tolerance. In addition, circulating glucose and triglyceride levels were decreased in the PX-478-treated mice. These metabolic improvements are likely due to the reduced overall adiposity in these mice, which is reflected by the smaller adipocyte size, the reduced fat pads, and the altered circulating adipokine levels. Therefore, we expect these PX-478-treated mice to display a full metabolic improvement across the board, and all parameters tested suggest that this is indeed the case. Our metabolic studies also show that PX-478-treated mice display an increased basal metabolic rate and energy expenditure, without any changes observed with respect to food intake. Furthermore, the “browning” markers UCP-1 and PGC-1α were significantly upregulated in SWATs of PX-478-treated mice, suggesting a browning program in these mice. We do not know what the underlying mechanistic basis is for the increased energy expenditure upon HIF1α inhibition. Since this is a phenomenon seen both with systemic pharmacological inhibition and with adipocyte-specific manipulation of the pathway, it suggests that an adipocyte-derived signal is responsible for this phenomenon. Leptin would be an excellent candidate as a mediator of these effects. Leptin is a critical adipokine that regulates energy homeostasis through the regulation of food intake and energy expenditure (53
). However, circulating leptin levels are decreased upon HIF1α inhibition, making it an unlikely effector. As a direct target of HIF1α, leptin levels were dramatically decreased in both mouse models, yet food intake was normal. This suggests that these models are more leptin sensitive under this HFD insult. We were surprised to see that the manipulation of HIF1α levels had such a profound impact on leptin expression and release. Previously, Wang et al. reported that leptin is expressed only in well-differentiated adipocytes under normoxic conditions (46
). However, under hypoxic conditions, preadipocytes express and secrete leptin as well (46
). Our data suggest that the unique secretion pattern in preadipocytes is regulated by HIF1α and that the effect can be inhibited by PX-478 treatment or overexpression of a dn-HIF1α. Indeed, Ambrosini and colleagues suggest that the leptin gene is a direct target for HIF1α. These authors have identified a hypoxia response element (HRE) in the leptin promoter (64
). Our studies highlight the importance of these findings in the in vivo
setting. Leptin levels in circulation are generally directly proportional to fat mass. An unresolved issue is how an individual fat cell gauges its own size and consequently adjusts its leptin expression levels. A HIF1α-mediated mechanism would be an excellent mediator of these effects. Large cells are chronically hypoxic and hence will enjoy higher HIF1α levels, leading to increased leptin transcription and release. Therefore, we can manipulate leptin levels very effectively in vivo
by manipulating HIF1α activity, independent of fat mass. To our surprise, we did not observe significant changes in circulating adiponectin levels by either HIF1α inhibition paradigm employed. However, the dramatic decrease in leptin levels, together with downregulation of other adipokines, such as IL-6, suggests decreased adiposity in these models.
Fibrosis in adipose tissue plays critical roles in downstream events, which lead to further metabolic dysfunction in fat pads (20
). Reduced adipose tissue fibrosis can be achieved through a genetic disruption of collagen VI. This results in an overall improved metabolic phenotype (20
). Since we previously found that hypoxia-induced HIF1α in adipose tissue mainly upregulates fibrillar collagens and the enzyme LOX (which plays an important role in collagen fiber formation [10
]), we focused on the regulation of these HIF1α target genes by HIF1α inhibition. As expected, collagens I and III and the collagen cross-linking LOX enzyme induced by HFD feeding were dramatically downregulated by PX-478 and dn-HIF1α. Trichrome staining further confirms that a local state of fibrosis induced by HFD was suppressed by HIF1α inhibition.
The development of inflammation in adipose tissue has been linked to many other metabolic syndromes (55
). During fat pad expansion, monocytes and macrophages infiltrate into adipose tissue (57
). The local state of fibrosis caused by HIF1α within adipose tissue can be the initiating factor for monocyte and macrophage infiltration and inflammation, even though it is not firmly established what the key signal is for this process (10
). We observed significantly decreased expression of inflammatory factors such as IL-6 by qPCR in EWAT. Along with these transcriptional changes, a reduced frequency of crown-like structures surrounding adipocytes in the PX-478-treated group could be seen.
We repeated the studies performed with the pharmacological inhibitor with a local overexpression of a dn-HIF1α protein. The dn-HIF1α protein is a fragment of wild-type HIF1α lacking the DNA-binding domain, the transactivation domain, and the oxygen-dependent domain (ODD) (34
). This mutant has been demonstrated to effectively reduce HIF1α activity in neurons and pancreatic cancer cells. In our system, we took advantage of the inducible and tissue-specific nature of our construct. We initiated the adipose tissue-specific overexpression only upon endogenous HIF1α induction with the HFD challenge. In that way, we selectively blocked the function of endogenous HIF1α only upon HFD induction. By using this model, we were able to observe metabolically beneficial outcomes similar to those with PX-478 treatment. We did not achieve in all instances quantitatively the same effects as with the pharmacological inhibitor, even though qualitatively, we obtained the same results across the board. This is likely due to the lack of a complete inhibition of endogenous HIF1α activity using a dominant negative version of HIF1α. Complete inhibition can be achieved more effectively using pharmacological agents, such as PX-478.
Although we confirmed all the metabolic effects obtained pharmacologically with PX-478 with an adipose tissue-specific dn-HIF1α overexpression model, we noticed that the magnitude of the effects in the transgenic mouse model were milder than in the PX-478-treated mice. This suggests that in addition to the local effects in adipose tissue, PX-478 may also suppress HIF1α functions in other tissues. Of note, as an active metabolic site, skeletal muscle experiences dramatic oxygen level fluctuations during endurance exercise. As a result, hypoxic conditions may develop in that setting, and HIF1α-mediated pathways play an important metabolic role (54
). Moreover, HIF1α has also been shown to play a role in liver metabolism by regulating hepatic glucose homeostasis (61
). Even though neither of these organs displays hypoxia under conditions of HFD exposure, we cannot exclude the possibility that PX-478-mediated effects in muscle and liver contribute to the metabolic improvements observed.
In conclusion, our findings in combination with reports from others (10
) suggest that HIF1α, a factor induced early in adipose tissue during the development of obesity and critically involved in the pathogenesis of insulin resistance in adipose tissue, is an attractive drug target. However, even though several recent reports confirmed that adipose tissue in humans is also poorly oxygenated in the obese (62
), we do not know whether these hypoxic conditions are sufficient to induce high levels of HIF1α. Human adipose tissue expansion in most instances occurs over much more prolonged periods, allowing even modest proangiogenic activity to at least partially vascularize expanding pads. Nevertheless, many common features between rodent and human adipose tissue expansion are shared, and the basic lessons learned from the studies here warrant further investigation in a clinical setting as well.