The data presented here demonstrate that HIF-1 is a mediator of apelin transcription in cultured adipocytes, and appears to participate in insulin induction of the expression of this gene. Hypoxia strongly induced apelin expression in differentiated 3T3-L1 adipocytes, an effect that was duplicated by CoCl2, a prototypic chemical inducer of normoxic HIF-1α stabilization, and DMOG, a well-described inhibitor of prolyl hydroxylase activity. Furthermore, insulin-activated apelin expression was abolished by rotenone, a well-known inhibitor of HIF activation. Extension of these experiments in mouse embryonic fibroblasts containing a targeted deletion of the HIF-1α gene showed clear involvement of HIF in the hypoxic and insulin-mediated induction of apelin.
Our results are complementary to those of a recently published paper reporting hypoxia-induced apelin expression in cultured cardiomyocytes (33
). In this study, Ronkainen et al demonstrated that expression of a constitutively active form of HIF-1α increased apelin expression in the absence of other stimuli, whereas expression of IPAS, an inhibitor of HIF-mediated transcription, decreased apelin induction in the setting of hypoxia. Our findings extend these observations by showing that (1
) HIF is involved in apelin upregulation in response to insulin, and (2
) ablation of HIF-1α diminishes both hypoxia- and insulin-induced apelin expression.
Of note, apelin elaboration in the HIF- KO MEFs was significantly reduced by CoCl2
, and to a lesser extent, by DMOG and hypoxia. This effect was not apparent at the transcriptional level. While we are unable to provide a definitive explanation for this phenomenon, it is possible that these treatments may exert an effect on post-translational processing and/or secretion of the apelin peptide. In any event, it remains apparent that none of these stimuli induce apelin expression in cells lacking the HIF-1α gene. Apelin possesses functionality consistent with a response to hypoxia. Recruitment of vasculature to a hypoxic or ischemic region is critical to establishing an environment in which the metabolic demands of that tissue can be met. A number of HIF-inducible factors, most notably VEGF, have been demonstrated to play integral roles in the proliferation, migration, and tube-formation of endothelial cells that is characteristic of angiogenesis (39
). Apelin demonstrates similar properties. For example, Masri et al demonstrated that apelin stimulates the proliferation of human umbilical vein endothelial cells in a p70 S6 protein kinase-sensitive fashion (27
). Additionally, Kasai et al determined that apelin not only is a mitogenic stimulus to retinal endothelial cells, but also promotes migration and tube formation in these cells (21
In the in vivo
setting, it has been reported that apelin is crucial for the normal development of the Xenopus embryonic vasculature. Anti-sense silencing of either the apelin mRNA transcript, or that of its receptor, APJ, results in absent or disrupted formation of intersegmental vessels (9
). Furthermore, implantation of apelin soaked beads produces ectopic vascularization of the implanted area. Finally, apelin-APJ signaling has also been implicated in normal cardiac development of the Xenopus embryo (17
). Whether apelin modulates cardiomyocyte development directly, or whether this effect occurs via malformation of the cardiac vasculature remains to be elucidated. Taken together, these studies, coupled with the data presented here, are consistent with a central role for apelin as an effector of HIF mediated angiogenesis. In addition to its angiogenic actions, HIF is known to modulate vascular tone via transactivation of several factors, including endothelial nitric oxide synthase (eNOS), endothelin, and adrenomedullin (8
) . Interestingly, apelin is also involved in the regulation of vascular tone, though its precise role is controversial. A decrease in mean arterial blood pressure following apelin administration in anesthetized rats was first demonstrated by Tatemoto et al (43
). This effect was abolished by of NOS inhibition, and was accompanied by an increase in plasma nitrate/nitrite, suggesting that apelin modulates eNOS activity. However, other authors have reported that apelin has vasoconstrictive properties in certain circumstances (12
). Nevertheless, the predominant systemic effect in vivo
appears to be arterial and venous vasodilation (7
). Dilatation of collateral vessels is an important means of increasing perfusion to hypoxic regions of the myocardium; thus the vasoactive effects of apelin are also consistent with a response to tissue hypoxia.
Given its identity as an adipokine, the expression of apelin in adipose tissue is induced by insulin in culture (4
), in animals (4
), and in human subjects (4
). Our data demonstrate that insulin induction of apelin is significantly reduced in cells lacking a functioning HIF-1α subunit. Furthermore, we show that insulin stimulates HIF-1α stabilization and nuclear translocation in differentiated 3T3-L1 cells, and that insulin stimulation of apelin in these cells is inhibited by rotenone. These data indicate that insulin stimulates apelin in adipocytes, at least in part, via its ability stabilize HIF-1α and promote HIF-1 transcriptional activity. Further analysis of the apelin promoter is required to fully elucidate the relative contributions of individual HREs to the transcriptional response of apelin to insulin signaling.
Our results suggest a potential novel role for HIF in the insulin-mediated induction of apelin expression in the adipocytes of obese subjects. Obesity is associated with hyperinsulinemia, and HIF-1α expression in adipose tissue has also been shown to be increased in preoperative gastric bypass patients (5
). A number of HIF-responsive genes are upregulated in the adipose tissue of this population, including VEGF, PAI-1, leptin, and visfatin (2
), and data suggests that apelin exhibits a similar pattern of expression (4
). It is therefore possible that insulin mediates the expression of these genes in adipose tissue via a HIF-dependent mechanism.
How, then, do we reconcile the response-to-hypoxia role of HIF-1 with its expression in adipose tissue? HIF-1 is essential for normal vascular development in embryogenesis; targeted deletion of HIF-1α severely restricts vascularization of the developing embryo, resulting in lethality by day E11 (19
). Given the absolute requirement for HIF-mediated angiogenesis in embryonic organ development, it is not unreasonable to hypothesize that the same mechanism is involved in adipogenesis throughout the life of the organism. In ontogeny, putative fetal fat depots exist as vascular beds prior to the appearance of fat cells (13
). Angiogenesis is also implicated in the maintenance and expansion of adipose tissue in adult animals. Rupnick, et al, demonstrated that in genetically obese db/db
mice, weight gain, weight maintenance, and adipose tissue mass are all inhibited by the antiangiogenic agents TNP-490, angiostatin, and endostatin (34
). In this regard, the function of HIF as a central mediator of angiogenesis is consistent with a role in adipogenesis. We speculate that, as a pro-angiogenic target of HIF-1, apelin may promote the development of new vasculature to a developing or expanding fat depot, augmenting VEGF and PAI-1 function in this process.
Obesity and its attendant complications represent a burgeoning public health crisis in the Western world. Despite this urgency, very little is known regarding the normal and pathophysiologic molecular processes governing adipose tissue development, expansion, and progression to insulin resistance. Similarly, it is only recently that the endocrine functionalities of this organ have come to be appreciated. It is certainly possible that the expression of apelin, as well as that of other adipokines that demonstrate similar patterns of expression in the adipose tissue of obese individuals, are integral regulators of the molecular events that govern development and expansion of fat depots. It is also conceivable that some of these adipokines may be implicated in the progression from obesity to insulin resistance, either as contributors, mitigating factors, or merely as passive markers. Further examination of the roles of apelin, HIF, and other HIF-mediated adipokines in adipose tissue angiogenesis, as well as preadipocyte proliferation, migration, and differentiation, may provide valuable insight into the regulation and dysregulation of this tissue as it applies to the pathogenesis of obesity and insulin resistance.