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Non-hypoxic induction of HIF-3alpha by 2-deoxy-D-glucose and insulin
Abstract
Hypoxia-inducible factors (HIFs) are key mediators of cellular adaptation to hypoxia, but also respond to non-hypoxic stimuli. To clarify involvement in metabolic disturbances, HIFs were characterised in rats subjected to insulin-induced hypoglycaemia or cellular glucoprivation provoked by 2-deoxy-D-glucose (2-DG). Using real-time qPCR, organ-specific expression of HIF-1alpha, -2alpha, -3alpha, -1beta, and of the target gene GLUT-1 was determined. Distribution of HIF-3alpha proteins was examined by immunohistochemistry. Both, insulin and 2-DG resulted in a widespread increase in HIF-3alpha mRNA. HIF-2alpha mRNA increased in lung and heart after 2-DG only, whereas other HIFs remained unaffected. A pronounced increase of protein levels in cerebral cortex was observed for HIF-3alpha. Functional significance of HIF induction was reflected in enhancement of GLUT-1 mRNA. Transcriptional up-regulation of HIF-3alpha represents a typical response to in vivo hypoglycaemia and glucoprivation. These data suggest an involvement of the HIF system in metabolic derangements as for instance caused by diabetes.
... While the landscape of post-translational modification of HIF-1α and HIF-2α is steadily expanding, current knowledge with respect to HIF-3α is limited. To date, it is known that 2-deoxy-D-glucose and insulin can increase HIF-3α expression in rat kidney, cerebral cortex (Heidbreder et al., 2007) and primary hepatocytes (Kietzmann et al., 2003). Since insulin is a major activator of the phosphoinositide 3-kinase-protein kinase B/AKT (PI3K-PKB/ AKT) pathway, this may imply that insulin exerts its effect on HIF-3α via the PI3K-PKB/AKT axis, similar to FOXO transcription factors. ...
... To investigate the potential role of the PI3K/PKB signaling pathway in insulin-mediated modulation of HIF-3α1 protein expression, we conducted experiments using MCF-7 cells because they express insulin receptors and respond to insulin stimulation (Kietzmann et al., 1999). First, we treated MCF-7 cells with insulin and demonstrated by qRT-PCR analysis that insulin upregulated HIF3A mRNA levels under both normoxia and mild hypoxia, which is in line with previous studies (Kietzmann et al., 2003;Heidbreder et al., 2007) (Supplementary Figure S1). Next, we transfected MCF-7 with wild-type V5-tagged HIF-3α1 and subsequently treated for a duration of 4 h with insulin, wortmannin (a potent PI3K inhibitor), and BML-257 (a specific PKB inhibitor) alone or in combination under normoxia or mild hypoxia. ...
... Although HIF-1α and HIF-2α have been extensively studied, the function of HIF-3α isoforms remains poorly characterized. Initial findings suggested that hypoxia can induce the expression of some HIF-3α variants via HIF-1α but not HIF-2α in a cell and tissue-specific manner (Li et al., 2006;Heidbreder et al., 2007;Tanaka et al., 2009;Yang et al., 2015) and that these HIF-3α isoforms act as negative regulators of the hypoxia response by competing with HIF-1α for binding to HIF-1β/ARNT (Hara et al., 2001;Heikkilä et al., 2011). However, recent studies have shown that full-length HIF-3α1 and the long splicing variant HIF-3α2 can act as positive regulators and induce the expression of some genes. ...
Hypoxia-inducible factors (HIFs) are best known for their roles in the adaptation to low oxygen environments. Besides hypoxia, HIF-1/2 α-subunits are also regulated by various non-hypoxic stimuli including insulin which can act via the PI3K/protein kinase B (PKB) signaling pathway. However, with respect to insulin little is known about HIF-3α. We aimed to investigate this relationship and found that insulin stimulates HIF-3α expression under both normal and low oxygen conditions. Blocking PKB activity reversed the effects of insulin, indicating that HIF-3α is a direct target of PKB. We identified serine 524, located in the oxygen-dependent degradation domain of HIF-3α, as a phosphorylation site of PKB. Mutating serine 524 impaired binding of PKB to HIF-3α and its ubiquitination, suggesting that PKB regulates HIF-3α stability through phosphorylation, thereby affecting important cellular processes such as cell viability and cell adhesion. Importantly, we discovered that this phosphorylation site also influenced insulin-dependent cell migration. These findings shed light on a novel mechanism by which insulin affects PKB-dependent HIF-3α expression and activity, with potential implications in metabolic diseases and cancer.
... Further analysis will be needed to decipher the specific role of NADPH oxidases (Nox 2 and Nox4) in the mechanisms induced by low glucose in the 661W photoreceptor cell line and in hypoglycemic animals. We also tested protein expression of hypoxia-inducible factor (HIF) 3 a, because it has been described as key regulator in glucose metabolism [41]. Heidbreder et al. recently described an in vivo induction of this gene by insulin-induced hypoglycemia and glucoprivation. ...
... This may explain the difference with our results and justify further analysis. However, in low glucose (2 mM) cultured 661W cells we observed an increase of HIF3 a (Figure S3B ), which is similar to Heidbreder's observation in HT- 22 cells cultured at 5 mM [41]. We clearly need further studies to implicate this protein in GSH depletion, ROS production and caspase 3-induced apoptosis. ...
Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Glycemic excursions could lead to cardiovascular disease, nephropathy, neuropathy and retinopathy. A vast body of literature exists on hyperglycemia namely in the field of diabetic retinopathy, but very little is known about the deleterious effect of hypoglycemia. Therefore, we decided to study the role of acute hypoglycemia in mouse retina.
To test effects of hypoglycemia, we performed a 5-hour hyperinsulinemic/hypoglycemic clamp; to exclude an effect of insulin, we made a hyperinsulinemic/euglycemic clamp as control. We then isolated retinas from each group at different time-points after the clamp to analyze cells apoptosis and genes regulation. In parallel, we used 661W photoreceptor cells to confirm in vivo results. We showed herein that hypoglycemia induced retinal cell death in mouse via caspase 3 activation. We then tested the mRNA expression of glutathione transferase omega 1 (Gsto1) and glutathione peroxidase 3 (Gpx3), two genes involved in glutathione (GSH) homeostasis. The expression of both genes was up-regulated by low glucose, leading to a decrease of reduced glutathione (GSH). In vitro experiments confirmed the low-glucose induction of 661W cell death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. Moreover, decrease of GSH content by inhibition with buthionine sulphoximine (BSO) at high glucose induced apoptosis, while complementation with extracellular glutathione ethyl ester (GSHee) at low glucose restored GSH level and reduced apoptosis.
We showed, for the first time, that acute insulin-induced hypoglycemia leads to caspase 3-dependant retinal cell death with a predominant role of GSH content.
... Hypertrophy of adipocytes, as well as insufficient vascularization in relation to the needs, promotes the occurrence of local hypoxia in adipose tissue, which in turn leads to an increase in the level of HIF-1α, which mediates the development of insulin resistance [45]. Insulin is one of the factors that upregulates HIF3A expression [46], so it was expected that patients with higher BMI would show greater HIF3A gene expression. However, the results of our study proved the opposite; the level of HIF3A gene expression was lower. ...
Hypoxia-inducible factor 1 (HIF-1) may play a role in mammary gland development, milk production and secretion in mammals. Due to the limited number of scientific reports on the expression of HIF genes in colostrum cells, it was decided to examine the expression of HIF1A, HIF3A and EPAS1 in the these cells, collected from 35 patients who voluntarily agreed to provide their biological material for research, were informed about the purpose of the study and signed a consent to participate in it. The expression of HIF genes was assessed using qPCR. Additionally, the influence of clinical parameters (method of delivery, occurrence of stillbirths in previous pregnancies, BMI level before pregnancy and at the moment of delivery, presence of hypertension during pregnancy, presence of Escherichia coli in vaginal culture, iron supplement and heparin intake during pregnancy) on the gene expression was assessed, revealing statistically significant correlations. The expression of HIF1A was 3.5-fold higher in the case of patients with the presence of E. coli in vaginal culture (p = 0.041) and 2.5 times higher (p = 0.031) in samples from women who used heparin during pregnancy. Approximately 1.7-fold higher expression of the EPAS1 was observed in women who did not supplement iron during pregnancy (p = 0.046). To our knowledge, these are the first studies showing the relationship between HIF expression in cells from breast milk and the method of delivery and health condition of women giving birth. The assessment of HIF expression requires deeper examination in a larger study group, and the results of further studies will allow to determine whether HIF can become biomarkers in pregnancy pathology states.
... The results reported in this review showed that CpG islands in the promoter region of HIF3a were more methylated in women with GDM compared to women without GDM in European and Chinese populations (23,46), and that higher methylation was correlated with decreased HIF3a gene expression (23). Hypoxia-inducible factors (HIFs) are transcription factors that mediate hypoxia in many tissues (98) and HIF3a has been shown to play a role in glucose metabolism, adipocyte differentiation and inflammation, which are important pathways associated with GDM (68,99). Previous studies have reported that increased HIF3a promoter methylation is associated with obesity and higher plasma glucose levels and waist-hip ratio (71,100) and adipose tissue dysfunction, T2DM and lower insulin sensitivity (70, 71, 101). ...
Diabetes in pregnancy is associated with adverse pregnancy outcomes and poses a serious threat to the health of mother and child. Although the pathophysiological mechanisms that underlie the association between maternal diabetes and pregnancy complications have not yet been elucidated, it has been suggested that the frequency and severity of pregnancy complications are linked to the degree of hyperglycemia. Epigenetic mechanisms reflect gene-environment interactions and have emerged as key players in metabolic adaptation to pregnancy and the development of complications. DNA methylation, the best characterized epigenetic mechanism, has been reported to be dysregulated during various pregnancy complications, including pre-eclampsia, hypertension, diabetes, early pregnancy loss and preterm birth. The identification of altered DNA methylation patterns may serve to elucidate the pathophysiological mechanisms that underlie the different types of maternal diabetes during pregnancy. This review aims to provide a summary of existing knowledge on DNA methylation patterns in pregnancies complicated by pregestational type 1 (T1DM) and type 2 diabetes mellitus (T2DM), and gestational diabetes mellitus (GDM). Four databases, CINAHL, Scopus, PubMed and Google Scholar, were searched for studies on DNA methylation profiling in pregnancies complicated with diabetes. A total of 1985 articles were identified, of which 32 met the inclusion criteria and are included in this review. All studies profiled DNA methylation during GDM or impaired glucose tolerance (IGT), while no studies investigated T1DM or T2DM. We highlight the increased methylation of two genes, Hypoxia‐inducible Factor‐3α (HIF3α) and Peroxisome Proliferator-activated Receptor Gamma-coactivator-Alpha (PGC1-α), and the decreased methylation of one gene, Peroxisome Proliferator Activated Receptor Alpha (PPARα), in women with GDM compared to pregnant women with normoglycemia that were consistently methylated across diverse populations with varying pregnancy durations, and using different diagnostic criteria, methodologies and biological sources. These findings support the candidacy of these three differentially methylated genes as biomarkers for GDM. Furthermore, these genes may provide insight into the pathways that are epigenetically influenced during maternal diabetes and which should be prioritized and replicated in longitudinal studies and in larger populations to ensure their clinical applicability. Finally, we discuss the challenges and limitations of DNA methylation analysis, and the need for DNA methylation profiling to be conducted in different types of maternal diabetes in pregnancy.
... Consistent with our results, Epas1 and Hif3a were rapidly activated in rat brains after exposure to systemic hypoxia 70 and glucose deprivation. 73 In contrast, lasting mild hypoxia training led to normalization of gene expression of HIF complex subunits and to a decrease of Vegfa, whereas expression of genes involved in cellular growth and differention processes elicited by PI3K-Akt-mTOR signaling were increased in bulk brain tissue and in cerebral microvessel isolates, suggesting resolution and adaptation of the vascular network remodeling in frontal cortex in response to moderate oxygen deprivation. Extrapolation of results from mice to humans must be approached with caution as recent comparative aging studies found little overlap between genes differentially expressed between human and the mouse brains, suggesting that that physiologically aged mice do not necessary recapitulate the effect of aging on human brain responses. ...
Introduction:
Molecular responses in the brains of persons with mild cognitive impairment (MCI), the earliest transitional state between normal aging and early Alzheimer's disease (AD), are poorly understood.
Methods:
We examined AD-related neuropathology and transcriptome changes in the neocortex of individuals with MCI relative to controls and temporal responses to the mild hypoxia in mouse brains.
Results:
Subsets of vascular early response to hypoxia genes were upregulated in MCI prior to the buildup of AD neuropathology. Early activation of pro-angiogenic hypoxia-inducible factor signaling in response to mild hypoxia was detected in mouse brains similar to those that were altered in MCI. Protracted responses to hypoxia were characterized by activation of phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt)-the mammalian target of rapamycin (mTOR) pathways in brain microvessel isolates.
Discussion:
These findings suggest that cerebrovascular remodeling is an important antecedent to the development of dementia and a component of the homeostatic response to reduced oxygen tension in aging prior to the onset of AD.
... Activation of SIRT1 alleviates oxidative stress, promotes autophagic flux, and prevents cardiomyocyte dysfunction and demise in diabetic hearts [13,[52][53][54]. Similarly, a high-fat diet acts to suppress (whereas glucose deprivation activates) HIF-2α [55][56][57], whereas upregulation of HIF-2α reduces oxidative stress and promotes autophagy in the heart [38,39]. Thus, changes in nutrient and oxygen deprivation signaling can influence organellar stability, oxidative stress and inflammasome activation and modulate cellular dysfunction in diabetic hearts by mechanisms that are autophagy-dependent and -independent. ...
Autophagy is a lysosome-dependent intracellular degradative pathway, which mediates the cellular adaptation to nutrient and oxygen depletion as well as to oxidative and endoplasmic reticulum stress. The molecular mechanisms that stimulate autophagy include the activation of energy deprivation sensors, sirtuin-1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK). These enzymes not only promote organellar integrity directly, but they also enhance autophagic flux, which leads to the removal of dysfunctional mitochondria and peroxisomes. Type 2 diabetes is characterized by suppression of SIRT1 and AMPK signaling as well as an impairment of autophagy; these derangements contribute to an increase in oxidative stress and the development of cardiomyopathy. Antihyperglycemic drugs that signal through insulin may further suppress autophagy and worsen heart failure. In contrast, metformin and SGLT2 inhibitors activate SIRT1 and/or AMPK and promote autophagic flux to varying degrees in cardiomyocytes, which may explain their benefits in experimental cardiomyopathy. However, metformin and SGLT2 inhibitors differ meaningfully in the molecular mechanisms that underlie their effects on the heart. Whereas metformin primarily acts as an agonist of AMPK, SGLT2 inhibitors induce a fasting-like state that is accompanied by ketogenesis, a biomarker of enhanced SIRT1 signaling. Preferential SIRT1 activation may also explain the ability of SGLT2 inhibitors to stimulate erythropoiesis and reduce uric acid (a biomarker of oxidative stress)-effects that are not seen with metformin. Changes in both hematocrit and serum urate are the most important predictors of the ability of SGLT2 inhibitors to reduce the risk of cardiovascular death and hospitalization for heart failure in large-scale trials. Metformin and SGLT2 inhibitors may also differ in their ability to mitigate diabetes-related increases in intracellular sodium concentration and its adverse effects on mitochondrial functional integrity. Differences in the actions of SGLT2 inhibitors and metformin may reflect the distinctive molecular pathways that explain differences in the cardioprotective effects of these drugs.
... Furthermore, the influence of HIF3A on lipolysis could go beyond the genetic effects. In addition to hypoxia, HIF3A expression has also been reported to be regulated by the anti-lipolytic hormone insulin [46]. Furthermore, SAT CpG methylation of HIF3A has been associated with gene expression and BMI [47]. ...
Objectives
Lipolysis, hydrolysis of triglycerides to fatty acids in adipocytes, is tightly regulated, poorly understood, and, if perturbed, can lead to metabolic diseases including obesity and type 2 diabetes. The goal of this study was to identify the genetic regulators of lipolysis and elucidate their molecular mechanisms.
Methods
Adipocytes from abdominal subcutaneous adipose tissue biopsies were isolated and were incubated without (spontaneous lipolysis) or with a catecholamine (stimulated lipolysis) to analyze lipolysis. DNA was extracted and genome-wide genotyping and imputation conducted. After quality control, 939 samples with genetic and lipolysis data were available. Genome-wide association studies of spontaneous and stimulated lipolysis were conducted. Subsequent in vitro gene expression analyses were used to identify candidate genes and explore their regulation of adipose tissue biology.
Results
One locus on chromosome 19 demonstrated genome-wide significance with spontaneous lipolysis. 60 loci showed suggestive associations with spontaneous or stimulated lipolysis, of which many influenced both traits. In the chromosome 19 locus, only HIF3A was expressed in the adipocytes and displayed genotype-dependent gene expression. HIF3A knockdown in vitro increased lipolysis and the expression of key lipolysis-regulating genes.
Conclusions
In conclusion, we identified a genetic regulator of spontaneous lipolysis and provided evidence of HIF3A as a novel key regulator of lipolysis in subcutaneous adipocytes as the mechanism through which the locus influences adipose tissue biology.
... demonstrated that the methylation of insulin receptor promoter in adipose tissues contributes critically to the GDM pathophysiology, especially in visceral adipose tissues (VAT) [16]. HIF3A is usually high-expressed in the adipocytes, and the upregulation of HIF3A is a typical response to hypoglycemia and glucoprivation in vivo [51]. Meanwhile, the aberrant expression of HIF3A could noticeably induce the expressions of multiple adipocytokines and accelerate adipose differentiation [22]. ...
Methylation of hypoxia-inducible factor-3α (HIF3A) was previously demonstrated to be highly associated with insulin resistance (IR) in patients with gestational diabetes mellitus (GDM). We aimed to study the therapeutic effects of Berberine (BBR) on GDM and the possible mechanisms. The expressions and methylated states of HIF3A in pregnant women with GDM were compared with that in healthy controls. The IR cell models of 3T3-L1 adipocytes was constructed by 1 μmol/L dexamethasone (Dex) and 1 μmol/L insulin (Ins). To evaluate the effects of BBR on IR adipocyte models, cells were subjected to BBR treatment at different concentrations. Transfection of HIF3A siRNA further confirmed the role of HIF3A in the BBR-induced improving effects. Low expression and high methylation of HIF3A gene were frequent in the GDM pregnancies. BBR treatment noticeably increased the glucose usage rates, adiponectin secretion and cell differentiation of IR 3T3-L1 adipocytes. Increased HIF3A expression and decreased methylated state of HIF3A were also found in IR adipocytes. Furthermore, HIF3A silencing not only reversed the effects of BBR on improving insulin sensibility, but also partially abolished the expression alterations of insulin-related genes in IR adipocytes induced by BBR treatment. Our results suggest that BBR improves insulin sensibility in IR adipocyte models, and the improving effects of BBR are possibly realized through the inhibition of HIF3A methylation.
... As such, we were powered for identifying larger effects in HIF3A.1 (80% power for a 3.37% increase in methylation) than the small magnitude of association previously reported (a 1.3% increase in univariate analysis, for which we had 19.2% power). HIF3A plays a role in glucose metabolism [27] and induces adipocyte-related gene expression [28] so may be involved in mediating the effects of maternal GDM on the infant's development. ...
Background:
Methylation of the hypoxia-inducible factor 3α gene (HIF3A) has been linked to pregnancy exposures, infant adiposity and later BMI. Genetic variation influences HIF3A methylation levels and may modify these relationships. However, data in very early life are limited, particularly in association with adverse pregnancy outcomes. We investigated the relationship between maternal and gestational factors, infant anthropometry, genetic variation and HIF3A DNA methylation in the Barwon Infant Study, a population-based birth cohort. Methylation of two previously studied regions of HIF3A were tested in the cord blood mononuclear cells of 938 infants.
Results:
No compelling evidence was found of an association between birth weight, adiposity or maternal gestational diabetes with methylation at the most widely studied HIF3A region. Male sex (- 4.3%, p < 0.001) and pre-eclampsia (- 5.4%, p = 0.02) negatively associated with methylation at a second region of HIF3A; while positive associations were identified for gestational diabetes (4.8%, p = 0.01) and gestational age (1.2% increase per week, p < 0.001). HIF3A genetic variation also associated strongly with methylation at this region (p < 0.001).
Conclusions:
Pre- and perinatal factors impact HIF3A methylation, including pre-eclampsia. This provides evidence that specific pregnancy complications, previously linked to adverse outcomes for both mother and child, impact the infant epigenome in a molecular pathway critical to several vascular and metabolic conditions. Further work is required to understand the mechanisms and clinical relevance, particularly the differing effects of in utero exposure to gestational diabetes or pre-eclampsia.
... vessel growth and are involved in adipose tissue inflammation and obesity-related insulin resistance 43 . Hif3a was reported as the most significantly associated gene in a genome-wide study of DNA methylation and BMI 44 , and our up-regulation of this gene after hypoxia exposure parallels the role of Hif3a in the cellular response to glucose and insulin 45 . Combined, these examples underscore the departure from homeostatic equilibrium of the placenta under late gestational hypoxia. ...
Intrauterine growth restriction (IUGR) enhances risk for adult onset cardiovascular disease (CVD). The mechanisms underlying IUGR are poorly understood, though inadequate blood flow and oxygen/nutrient provision are considered common endpoints. Based on evidence in humans linking IUGR to adult CVD, we hypothesized that in murine pregnancy, maternal late gestational hypoxia (LG-H) exposure resulting in IUGR would result in (1) placental transcriptome changes linked to risk for later CVD, and 2) adult phenotypes of CVD in the IUGR offspring. After subjecting pregnant mice to hypoxia (10.5% oxygen) from gestational day (GD) 14.5 to 18.5, we undertook RNA sequencing from GD19 placentas. Functional analysis suggested multiple changes in structural and functional genes important for placental health and function, with maximal dysregulation involving vascular and nutrient transport pathways. Concordantly, a ~10% decrease in birthweights and ~30% decrease in litter size was observed, supportive of placental insufficiency. We also found that the LG-H IUGR offspring exhibit increased risk for CVD at 4 months of age, manifesting as hypertension, increased abdominal fat, elevated leptin and total cholesterol concentrations. In summary, this animal model of IUGR links the placental transcriptional response to the stressor of gestational hypoxia to increased risk of developing cardiometabolic disease.
... In 1998, Gu et al 52 identified another HIF-α isoform in mice sharing a ~55% amino acid sequence identity with HIF-1α and HIF-2α in its bHLH PAS domain, which was categorized as the third member of the HIF transcription factor family: HIF-3α 53 . However, to date, the regulation mechanism of HIF-3α levels within cells by hypoxia is still poorly understood 54 . Human HIF-3α was identified in 2001 by Hara et al 33 its open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa. ...
Osteoarthritis (OA) is the most common joint disease, and in recent years has become a major public health problem. The hallmark of OA is cartilage destruction with local commitment of subchondral bone and the synovial
membrane. Hypoxia-inducible factors (HIFs) are transcriptional factors and key regulators of the cellular response to hypoxia. To date, three members of the human HIF-α protein family have been described: HIF-1α, HIF-2α, and HIF-
3α. HIF-1α plays an essential role in the articular cartilage (a hypoxic tissue), as it has a protective effect in the maintenance of the articular cartilage matrix, HIF-2α has a harmful effect on the articular cartilage matrix, and HIF-3α acts as a negative regulator of HIF-1α and HIF-2α. Due to the recent growing interest in the role of HIFs in rheumatic diseases, we focused this review on the potential role of these key regulators in articular cartilage maintenance as the central axis in OA development.
... Data showed that the methylation level at three HIF3A sites was strongly correlated to BMI in adipose tissue, i.e., an increase in BMI was associated with an increase in methylation level at HIF3A sites. The HIF3A gene could not only respond to oxygen content locally, but it also played a role in cellular response to insulin and glucose and accelerated adipocyte differentiation in acquired obesity (Heidbreder et al., 2007;Hatanaka et al., 2009;Robciuc et al., 2011). Obesity has been reported to increase a global mean DNA methylation in adipocytes (Arner et al., 2015;Benton et al., 2015). ...
Liver cancer is the third most common cancer type and the second leading cause of deaths in men. Large population studies have demonstrated remarkable gender disparities in the incidence and the cumulative risk of liver cancer. A number of emerging risk factors regarding metabolic alterations associated with obesity, diabetes and dyslipidemia have been ascribed to the progression of non-alcoholic fatty liver diseases (NAFLD) and ultimately liver cancer. The deregulation of fat metabolism derived from excessive insulin, glucose, and lipid promotes cancer-causing inflammatory signaling and oxidative stress, which eventually triggers the uncontrolled hepatocellular proliferation. This review presents the current standing on the gender differences in body fat compositions and their mechanistic linkage with the development of NAFLD-related liver cancer, with an emphasis on genetic, epigenetic and microRNA control. The potential roles of sex hormones in instructing adipocyte metabolic programs may help unravel the mechanisms underlying gender dimorphism in liver cancer and identify the metabolic targets for disease management.
... It has been shown that adipose tissue-specific Hif3a knockout mice are resistant to weight gain and have a better glucose tolerance and insulin sensitivity [3]. HIF-3α is highly expressed in adipocytes and acts as an accelerator of adipogenesis [4] and may also be involved in the regulation of glucose metabolism since it is upregulated by both insulin and 2-deoxy-D-glucose-induced glucoprivation [5]. ...
Background
Associations between BMI and DNA methylation of hypoxia-inducible factor 3-alpha (HIF3A) in both blood cells and subcutaneous adipose tissue (SAT) have been reported. In this study, we investigated associations between BMI and HIF3A DNA methylation in the blood and SAT from the same individuals, and whether HIF3A gene expression in SAT and skeletal muscle biopsies showed associations with BMI and insulin resistance. Furthermore, we aimed to investigate gender specificity and heritability of these traits. Methods
We studied 137 first-degree relatives of type 2 diabetes (T2D) patients from 48 families, from whom we had SAT and muscle biopsies. DNA methylation of four CpG sites in the HIF3A promoter was analyzed in the blood and SAT by pyrosequencing, and HIF3A gene expression was analyzed in SAT and muscle by qPCR. An index of whole-body insulin sensitivity was estimated from oral glucose tolerance tests. ResultsBMI was associated with HIF3A methylation at one CpG site in the blood, and there was a positive association between the blood and SAT methylation levels at a different CpG site within the individuals. The SAT methylation level did not correlate with HIF3A gene expression. Interestingly, HIF3A expression in SAT, but not in muscle, associated negatively with BMI and whole-body insulin resistance. We found a significant effect of familiality on HIF3A methylation levels in the blood and HIF3A expression levels in skeletal muscle. Conclusions
Our findings are in line with the previously reported link between BMI and DNA methylation of HIF3A in the blood. The tissue-specific results of HIF3A gene expression indicate that SAT is the more functional tissue in which a low expression may adversely affect whole-body insulin sensitivity.
... 1,4 Different HIF-3α splice variants exist, which are able to activate or repress HIF signalling depending on the cellular context. 4,12,13 The beta subunits Aryl hydrocarbon receptor nuclear translocator (ARNT) and ARNT2, also designated as HIF-1β and HIF-2β, respectively, serve as obligatory binding partners for HIF-α subunits in order to form functional complexes. 4 ARNT is present in all tissues and regarded to be constitutively expressed, meaning to be unaffected by oxygen tension (despite the name HIF-1β). ...
Oxygen-deprived (hypoxic) areas are commonly found within neoplasms caused by excessive cell proliferation. The transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT) is part of the hypoxia-inducible factor (HIF) pathway, which mediates adaptive responses to ensure cellular survival under hypoxic conditions. HIF signalling leads to metabolic alterations, invasion/metastasis and the induction of angiogenesis in addition to radio-chemoresistance of tumour cells. Activation of the HIF pathway is based on the abundance of HIF-α subunits, which are regulated in an oxygen-dependent manner and form transcriptional active complexes with ARNT or ARNT2 (also referred as HIF-1β and HIF-2β, respectively). ARNT is considered to be unaffected by hypoxia but certain cell lines, including Hep3B cells, are capable to elevate this transcription factor in response to oxygen deprivation, which implies an advantage. Therefore, the aim of this study was to elucidate the mechanism of hypoxia-dependent ARNT upregulation and to determine implications on HIF signalling. Gene silencing and overexpression techniques were used to alter the expression pattern of HIF transcription factors under normoxic and hypoxic conditions. qRT-PCR and western blotting were performed to measure gene and protein expression, respectively. HIF activity was determined by reporter gene assays. The results revealed a HIF-1α-dependent mechanism leading to ARNT upregulation in hypoxia. Forced expression of ARNT increased reporter activity under normoxic and hypoxic conditions. In conclusion, these findings indicate a novel feed-forward loop and suggest that ARNT might be a limiting factor. Augmented HIF signalling in terms of elevated target gene expression might be advantageous for tumour cells.
... For example, PPARγ , CCAAT/enhancer binding protein alpha (CEBPA) and R-spondin 3 homologue (RSPO3) are candidate genes for adipogenesis, but also play an important role in white adipose tissue differentiation [401] and may influence expansion or loss of adipose tissue [255,266]. Induction of HIF-3α, can accelerate adipogenesis through the activation of adipocytes-related genes [402,403]. Studies on the FTO and adrenoceptor beta 3 (ADRβ3) genes have highlighted biological pathways that relate to the browning of white adipocytes, lipid storage gene expression, repression of basal mito-chondrial respiration, decrease in thermogenesis in response to stimulus, and an increase in adipocyte size [252,399]. In lipodystrophy, the fat accumulation in skeletal muscles is associated with IR and T2D [201,202]. ...
In high-, middle- and low-income countries, the rising prevalence of obesity is the underlying cause of numerous health complications and increased mortality. Being a complex and heritable disorder, obesity results from the interplay between genetic susceptibility, epigenetics, metagenomics and the environment. Attempts at understanding the genetic basis of obesity have identified numerous genes associated with syndromic monogenic, non-syndromic monogenic, oligogenic and polygenic obesity. The genetics of leanness are also considered relevant as it mirrors some of obesity's aetiologies. In this report, we summarize ten genetically elucidated obesity syndromes, some of which are involved in ciliary functioning. We comprehensively review 11 monogenic obesity genes identified to date and their role in energy maintenance as part of the leptin-melanocortin pathway. With the emergence of genome-wide association studies over the last decade, 227 genetic variants involved in different biological pathways (central nervous system, food sensing and digestion, adipocyte differentiation, insulin signalling, lipid metabolism, muscle and liver biology, gut microbiota) have been associated with polygenic obesity. Advances in obligatory and facilitated epigenetic variation, and gene-environment interaction studies have partly accounted for the missing heritability of obesity and provided additional insight into its aetiology. The role of gut microbiota in obesity pathophysiology, as well as the 12 genes associated with lipodystrophies is discussed. Furthermore, in an attempt to improve future studies and merge the gap between research and clinical practice, we provide suggestions on how high-throughput '-omic' data can be integrated in order to get closer to the new age of personalized medicine.
... In accordance with these observations, expression of inflammatory genes (IL-1b, TNF, CCL2) tended to decrease in response to ozone in most organs except the kidney, effects that were predominantly blocked or reduced by metyrapone (Supplementary Figure 2). Levels of hypoxia inducible factor (HIF)-3a, involved in the response to hypoxia and glucoprivation (Heidbreder et al., 2007) were uniformly increased across organs by ozone and blocked by metyrapone treatment ( Figure 4B). In contrast, mRNA levels of two other genes involved in metabolic processes, BCL2/ adenovirus E1B 19 kDa-interacting protein (BNIP)-3, a stress-responsive protein involved in mitochondrial function and lipid metabolism (Glick et al., 2012), and sterol regulatory elementbinding protein (SREBP)-1, a transcription factor involved in cholesterol and fatty acid biosynthesis and uptake and insulin signaling (Horton et al., 2002), were altered by ozone and metyrapone in a tissue-dependent manner (Supplementary Figure 2). ...
Growing evidence implicates air pollutants in adverse health effects beyond respiratory and cardiovascular disease, including metabolic impacts (diabetes, metabolic syndrome, obesity) and neurological/neurobehavioural outcomes (neurodegenerative disease, cognitive decline, perceived stress, depression, suicide). We have shown that inhalation of particulate matter or ozone activates the hypothalamic-pituitary-adrenal axis in rats and increases plasma levels of the glucocorticoid corticosterone. To investigate the role of corticosterone in mediating inflammatory and metabolic effects of pollutant exposure, in the present study male Fischer 344 rats were administered the 11β-hydroxylase inhibitor metyrapone (0, 50, 150 mg/kg body weight) and exposed by nose-only inhalation for 4 h to air or 0.8 ppm ozone. Ozone inhalation provoked a 2-fold increase in plasma corticosterone, an effect blocked by metyrapone, but did not alter epinephrine levels. Inhibition of corticosterone production was associated with increased inflammatory signalling in the lungs and plasma in response to ozone, consistent with a role for glucocorticoids in limiting local and systemic inflammatory responses. Effects of ozone on insulin and glucagon, but not ghrelin or plasminogen activator inhibitor-1, were modified by metyrapone, revealing glucocorticoid-dependent and -independent effects of ozone on circulating metabolic and hemostatic factors. Several immunosuppressive and metabolic impacts of ozone in the lungs, heart, liver, kidney, and spleen were blocked by metyrapone and reproduced through exogenous administration of corticosterone (10 mg/kg body weight), demonstrating glucocorticoid-dependent effects in target tissues. Our results support involvement of endogenous glucocorticoids in ozone-induced inflammatory and metabolic effects, providing insight into potential biological mechanisms underlying health impacts and susceptibility.
... This implicates that HIF3A may act as a transcription regulator of some adipocytes-related genes. Heidbreder et al. [25] conducted a study in rats subjected to insulin-dependent hypoglycemia or insulinindependent cytoglucopenia provoked by 2-deoxy-D-glucose (2-DG), and suggested that the hypoglycemia or cytoglucopenia were fully effective to cause a rapid increase of HIF-3αmRNA levels, whereas other HIFs remained unaffected. ...
Gene polymorphisms associated so far with body mass index (BMI) can explain only 1.18-1.45% of observed variation in BMI. Recent studies suggest that epigenetic modifications, especially DNA methylation, could contribute to explain part of the missing heritability, and two epigenetic genome-wide analysis studies (EWAS) have reported that Hypoxia Inducible Factor 3 Alpha Subunit (HIF3A) methylation was associated with BMI or BMI change. We therefore assessed whether the HIF3A methylation is associated with obesity and other obesity-related phenotypes in Chinese children. The subjects included 110 severe obese cases aged 7-17y and 110 normal-weight controls matched by age and gender for measurement of blood DNA methylation levels at the HIF3A gene locus using the Sequenom's MassARRAY system. We observed significantly higher methylation levels in obese children than in controls at positions 46801642 and 46801699 in HIF3A gene (P<0.05), and found positive associations between methylation and alanine aminotransferase (ALT) levels adjusted by gender, age and BMI at the position 46801699 (r = 0.226, P = 0.007). These results suggest that HIF3A DNA methylation is associated with childhood obesity, and has a BMI-independent association with ALT. The results provide evidence for identifying epigenetic factors of elivated ALT and may be useful for risk assessment and personalized medicine of liver diseases such as non-alcoholic fatty liver disease (NAFLD).
... Furthermore, it is a one of strongest GR targets in both male and female "development" networks that include Akt as a central node, although identity and connectivity of components of this network are sexually dimorphic. HIF3␣, is one of 3 hypoxia inducible factors, whose expression is elevated under hypoxic conditions in multiple organs including brain (cortex and hippocampus) (33) but also functions outside of its known hypoxia response role (34). It is necessary for proper lung epithelium development (35) but does not respond to dex in human fetal lung epithelial cells (36). ...
Exposure to excess glucocorticoids during fetal development has long-lasting physiological and behavioral consequences, although the mechanisms are poorly understood. The impact of prenatal glucocorticoids exposure on stress responses in juvenile and adult offspring implicates the developing hypothalamus as a target of adverse prenatal glucocorticoid action. Therefore, primary cultures of hypothalamic-neural progenitor/stem cells (NPSCs) derived from mouse embryos (E14.5) were used to identify the glucocorticoid transcriptome in both males and females. NPSCs were treated with vehicle or the synthetic glucocorticoid dexamethasone (dex, 100 nM) for 4 hours and total RNA analyzed using RNA-Seq. Bioinformatic analysis demonstrated that primary hypothalamic NPSC cultures expressed relatively high levels of a number of genes regulating stem cell proliferation and hypothalamic progenitor function. Interesting, while these cells express glucocorticoid receptors (GR), only low levels of sex-steroid receptors are expressed, which suggested that sex-specific differentially regulated genes identified are mediated by genetic and not hormonal influences. We also identified known or novel GR target coding and noncoding genes that are either regulated equivalently in male and female NPSCs or differential responsiveness in one sex. Using gene ontology analysis, the top functional network identified was cell proliferation and using BrdU incorporation observed a reduction in proliferation of hypothalamic NPSCs after dex treatment. Our studies provide the first characterization and description of glucocorticoid-regulated pathways in male and female embryonically-derived hypothalamic NPSCs and identified GR-target genes during hypothalamic development. These findings may provide insight into potential mechanisms responsible for the long-term consequences of fetal glucocorticoid exposure in adulthood.
... As described previously by Dick et al. (27), these probes are within likely regulatory elements (open chromatin regions) and are potentially functional; moreover methylation level was inversely associated with HIF3A gene expression at one of the five expression probes examined. HIF3A is a component of the hypoxia inducible transcription factor (HIF) involved in the physiological response to hypoxia but is also implicated in adipocyte differentiation (50) and is expressed in response to glucose and insulin changes (51). However, we found that these HIF3A methylation probes were not associated with BMI in either the Framingham Heart Study or the GOLDN study. ...
Obesity is an important component of the pathophysiology of chronic diseases. Identifying epigenetic modifications associated with elevated adiposity, including DNA methylation variation, may point to genomic pathways that are dysregulated in numerous conditions.
The Illumina 450K Bead Chip array was used to assay DNA methylation in leukocyte DNA obtained from 2,097 African American adults in the Atherosclerosis Risk in Communities (ARIC) study. Mixed effects regression models were used to test the association of methylation beta value with concurrent BMI and waist circumference (WC), and BMI change, adjusting for batch effects and potential confounders. Replication using whole blood DNA from 2,377 White adults in the Framingham Heart Study and CD4+ T cell DNA from 991 Whites in the GOLDN Study was followed by testing using adipose tissue DNA from 648 women in the MuTHER cohort.
Seventy-six (76) BMI-related probes, 164 WC-related probes, and 8 BMI change-related probes passed the threshold for significance in ARIC (p<1 x 10(-7); Bonferroni), including probes in the recently reported HIF3A, CPT1A, and ABCG1 regions. Replication using blood DNA was achieved for 37 BMI probes and 1 additional WC probe. Sixteen (16) of these also replicated in adipose tissue, including 15 novel methylation findings near genes involved in lipid metabolism, immune response/cytokine signaling, and other diverse pathways, including LGALS3BP, KDM2B, PBX1, and BBS2, among others.
Adiposity traits are associated with DNA methylation at numerous CpG sites that replicate across studies despite variation in tissue type, ethnicity, and analytic approaches.
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... It is thought that factors containing the alpha-3 subunit are negative regulators of hypoxia-inducible gene expression (Makino et al., 2001). Transcriptional up-regulation of HIF-3alpha as found in CHF mice in hippocampus may therefore represent a typical response to in vivo hypoxia, hypoglycaemia, and glucoprivation in the brain after MI and CHF development (Heidbreder et al., 2007). ...
Background: Depression and anxiety are common and independent outcome predictors in patients with chronic heart failure (CHF). However, it is unclear whether CHF causes depression. Thus, we investigated whether mice develop anxiety- and depression-like behavior after induction of ischemic CHF by myocardial infarction (MI).
Methods and Results: In order to assess depression-like behavior, anhedonia was investigated by repeatedly testing sucrose preference for 8 weeks after coronary artery ligation or sham operation. Mice with large MI and increased left ventricular dimensions on echocardiography (termed CHF mice) showed reduced preference for sucrose, indicating depression-like behavior. 6 weeks after MI, mice were tested for exploratory activity, anxiety-like behavior and cognitive function using the elevated plus maze (EPM), light-dark box (LDB), open field (OF), and object recognition (OR) tests. In the EPM and OF, CHF mice exhibited diminished exploratory behavior and motivation despite similar movement capability. In the OR, CHF mice had reduced preference for novelty and impaired short-term memory. On histology, CHF mice had unaltered overall cerebral morphology. However, analysis of gene expression by RNA-sequencing in prefrontal cortical, hippocampal, and left ventricular tissue revealed changes in genes related to inflammation and cofactors of neuronal signal transduction in CHF mice, with Nr4a1 being dysregulated both in prefrontal cortex and myocardium after MI.
Conclusions: After induction of ischemic CHF, mice exhibited anhedonic behavior, decreased exploratory activity and interest in novelty, and cognitive impairment. Thus, ischemic CHF leads to distinct behavioral changes in mice analogous to symptoms observed in humans with CHF and comorbid depression.
... 35 In the hypothalamus, HIF signalling (primarily via EPAS1) has a role in glucose sensing and regulation of energy balance and weight by aff ecting expression of proopiomelanocortin. 36 Although HIF3A has not been investigated as thoroughly as the other α subunits in this context, it has been shown to have a role in the cellular response to glucose and insulin, and functions as an accelerator of adipocyte diff erentiation. 38,39 Furthermore, siRNA inhibition of HIF3A in Hep3B cells signifi cantly downregulates mRNA expression of ANGPTL4, 31 which could have a role in acquired obesity. 40 The cross-sectional nature of our analysis means that we cannot assign a cause-eff ect association directly from the association we observed between HIF3A methylation and BMI. ...
Obesity is a major health problem that is determined by interactions between lifestyle and environmental and genetic factors. Although associations between several genetic variants and body-mass index (BMI) have been identified, little is known about epigenetic changes related to BMI. We undertook a genome-wide analysis of methylation at CpG sites in relation to BMI.
479 individuals of European origin recruited by the Cardiogenics Consortium formed our discovery cohort. We typed their whole-blood DNA with the Infinium HumanMethylation450 array. After quality control, methylation levels were tested for association with BMI. Methylation sites showing an association with BMI at a false discovery rate q value of 0·05 or less were taken forward for replication in a cohort of 339 unrelated white patients of northern European origin from the MARTHA cohort. Sites that remained significant in this primary replication cohort were tested in a second replication cohort of 1789 white patients of European origin from the KORA cohort. We examined whether methylation levels at identified sites also showed an association with BMI in DNA from adipose tissue (n=635) and skin (n=395) obtained from white female individuals participating in the MuTHER study. Finally, we examined the association of methylation at BMI-associated sites with genetic variants and with gene expression.
20 individuals from the discovery cohort were excluded from analyses after quality-control checks, leaving 459 participants. After adjustment for covariates, we identified an association (q value ≤0·05) between methylation at five probes across three different genes and BMI. The associations with three of these probes-cg22891070, cg27146050, and cg16672562, all of which are in intron 1 of HIF3A-were confirmed in both the primary and second replication cohorts. For every 0·1 increase in methylation β value at cg22891070, BMI was 3·6% (95% CI 2·4-4·9) higher in the discovery cohort, 2·7% (1·2-4·2) higher in the primary replication cohort, and 0·8% (0·2-1·4) higher in the second replication cohort. For the MuTHER cohort, methylation at cg22891070 was associated with BMI in adipose tissue (p=1·72 × 10(-5)) but not in skin (p=0·882). We observed a significant inverse correlation (p=0·005) between methylation at cg22891070 and expression of one HIF3A gene-expression probe in adipose tissue. Two single nucleotide polymorphisms-rs8102595 and rs3826795-had independent associations with methylation at cg22891070 in all cohorts. However, these single nucleotide polymorphisms were not significantly associated with BMI.
Increased BMI in adults of European origin is associated with increased methylation at the HIF3A locus in blood cells and in adipose tissue. Our findings suggest that perturbation of hypoxia inducible transcription factor pathways could have an important role in the response to increased weight in people.
The European Commission, National Institute for Health Research, British Heart Foundation, and Wellcome Trust.
... SGK, another classical glucocorticoid-inducible gene increased in this study primarily in the kidney, heart, and brain, is implicated in a host of (patho)physiological processes, including metabolism, kidney function, gastrointestinal function, and control of blood pressure (Lang et al., 2006). Metabolic disturbance may explain the systemic increase in HIF-3α following pollutant inhalation, as in addition to its responsiveness to hypoxia HIF-3α is sensitive to glucose and insulin (Heidbreder et al., 2007). ...
Recent epidemiological studies have demonstrated associations between air pollution and adverse effects that extend beyond respiratory and cardiovascular disease, including low birth weight, appendicitis, stroke, and neurological/neurobehavioural outcomes (e.g. neurodegenerative disease, cognitive decline, depression, suicide). To gain insight into mechanisms underlying such effects, we mapped gene profiles in the lungs, heart, liver, kidney, spleen, cerebral hemisphere, and pituitary of male Fischer-344 rats immediately and 24 h after a 4 h exposure by inhalation to particulate matter (0, 5, 50 mg/m(3) EHC-93 urban particles) and ozone (0, 0.4, 0.8 ppm). Pollutant exposure provoked differential expression of genes involved in a number of pathways, including antioxidant response, xenobiotic metabolism, inflammatory signalling, and endothelial dysfunction. The mRNA profiles, while exhibiting some inter-organ and pollutant-specific differences, were remarkably similar across organs for a set of genes, including increased expression of redox/glucocorticoid-sensitive genes and decreased expression of inflammatory genes, suggesting a possible hormonal effect. Pollutant exposure increased plasma levels of adrenocorticotropic hormone and the glucocorticoid corticosterone, confirming activation of the hypothalamic-pituitary-adrenal axis, and there was a corresponding increase in markers of glucocorticoid activity. Although effects were transient and presumably represent an adaptive response to acute exposure in these healthy animals, chronic activation and inappropriate regulation of the hypothalamic-pituitary-adrenal axis is associated with adverse neurobehavioural, metabolic, immune, developmental, and cardiovascular effects. The experimental data are consistent with epidemiological associations of air pollutants with extrapulmonary health outcomes, and suggest a mechanism through which such health effects may be induced.
... Istniej¹ dane dowiadczalne sugeruj¹ce, ¿e czynnikiem odpowiedzialnym za ekspresjê tego genu nie jest bezporednie obni¿enie pO 2 , a zaburzenie potencja³u energetycznego komórki. [20,48]. ...
Although tissues may exist regardless of reduced oxygen pressure, this requires glycolytic ATP generation, which is very expensive from the energetic viewpoint. Hypoxia is defined as the condition in which oxygen pressure is reduced at the level of bodily tissues. There are many clinical situations during which decreased tissue oxygenation may occur. It may be transient or chronic, as well as systemic or local. An emergent need exists for monitoring and diagnosis with respect to numerous possible clinical circumstances leading to hypoxia and its life-threatening consequences. The assessment of global oxygen homeo-stasis relies on blood gas analysis and lactate concentration, but such an approach does not fully reflect the local oxygenation of tissues. Oxygen needle microelectrode measurements reveal great differences in tissue pO2 levels. Local pO2 levels depend on many factors, among which the most important are: the distance to the nearest capillary, the extracellular and intracellular fluid diffusion rates and intracellular measurements of the number and activity levels of mitochondria. Thus, nowadays, it is impossible to establish an accurate normal value ranges for local tissue pO2. Oxygen deficiency is an important gene regulator. A sequence-specific DNA-binding factor, the hypoxia induced factor (HIF), is the fundamental hypoxia response protein. 70 genes identified so far have been found to be HIF-dependent. They are responsible for increased oxygen delivery, i.e. by boosting angiogensis due to vascular endothelial growth factor (VEGF) release and the enhancement of red blood cell production by erythropoietin (EPO). VEGF-induced angiogenesis is one of several key hypoxia adaptations. An enhanced vascular bed in response to hypoxia affects almost every bodily tissue and organ. This was observed particularly in skeletal muscles as well as in the brain. The expression of a few hypoxia markers does not require HIF activation. An especially interesting member of this group is osteopontin (OPN), whose synthesis increases during hypoxia. OPN was originally linked to bone remodeling, but currently it seems to posses an important role in immunity, inflammation and tumor pathogenesis. Quantification of hypoxia is clinically essential both for therapy and prognosis. Taking account of the fact that the concept of oxygen pressure at the tissue level is not quantitative (norms do not exist, results are incomparable), biochemical markers are preferable. Particularly significant in this context are hypoxia-induced proteins such as HIF, EPO, VEGF or potentially OPN.
... HIFs have previously been shown to be sensitive to nonhypoxic stimuli, such as hypoglycemia. 20 Therefore, it is possible that impaired nutritional support due to choriocapillaris fenestration loss and subsequent endothelial wall thickening may account for the increase in HIF-1␣ observed. ...
Despite a lack of active angiogenesis, VEGF is expressed in nearly every adult tissue, and recent evidence suggests that VEGF may serve as a survival factor for both vascular and nonvascular tissues. VEGF blockade is a widely used treatment for neovascular diseases such as wet age-related macular degeneration (AMD). Therefore, it was sought in this study to evaluate the expression and role of endogenous VEGF in RPE.
VEGF and VEGFR2 expression in the murine retina were assessed during development. Bevacizumab was used to neutralize VEGF in ARPE-19 cells, and the effects on cell survival and apical microvill were assessed by TUNEL and SEM, respectively. VEGF was systemically neutralized in vivo by adenoviral-mediated overexpression of soluble VEGFR1 (sFlt). RPE and choriocapillaris were analyzed by transmission electron microscopy (TEM). Changes in gene expression were evaluated by quantitative real-time PCR.
VEGF expression was detected in the developing RPE as early as embryonic day (E) 9.5, whereas VEGFR2 expression by RPE began nonuniformly between postnatal (P) day 6.5 and P8.5. VEGF neutralization in vitro led to increased apoptosis and reduced microvilli density and length. Systemic VEGF neutralization led to transient degenerative changes; RPE were vacuolated and separated from photoreceptor outer segments, and choriocapillaris fenestrations were decreased. VEGF levels were elevated in RPE of Ad-sFlt1 mice at day 4 postinfection, and there was increased expression of the neurotrophic factor CD59a at day 14.
These results indicate that VEGF plays a critical role in survival and maintenance of RPE integrity. Potential undesired off-target effects should be considered with chronic use of anti-VEGF agents.
... HIFs are proteins that mediate a protective response to hypoxia. Certain HIF subunits are also activated during hypoglycaemia (6). When activated, HIF promotes the formation of EPO and VEGF, but also the glucose transporter-1 (GLUT1; reviewed in (7)). ...
Circulating erythropoietin (EPO) and vascular endothelial growth factor (VEGF) increase during hypoglycaemia and may represent protective hormonal counter-regulatory responses. We tested the hypothesis that low levels of EPO and VEGF are associated with a higher frequency of severe hypoglycaemia in a cohort of patients with type 1 diabetes.
Prospective observational follow-up study.
Totally 219 patients with type 1 diabetes (41% females, age 46+/-13 years (mean+/-s.d.), duration of diabetes 21+/-12 years, and HbAlc 8.5+/-1.1%) were followed in a 1-year observational study. Plasma EPO and serum VEGF levels were measured at baseline with ELISA. Events of severe hypoglycaemia defined by third party assistance were recorded and validated in telephone interviews within 24 h.
Totally 235 episodes of severe hypoglycaemia (1.1 episodes per patient-year) were reported by 82 patients (37%). At baseline, plasma EPO was 8.6 (3.1-34.3) U/l (median (range)), and serum VEGF was 52.2 (6.6-337) pg/ml. The levels of EPO and VEGF were not associated with frequency of severe and mild hypoglycaemia. The levels of EPO were not associated with age, sex, duration of diabetes, body mass index, HbAlc, C-peptide level or hypoglycaemia awareness status. The levels of VEGF were positively associated with age and female sex.
Although several studies suggest that VEGF and EPO may affect brain function during hypoglycaemia, this study does not support random VEGF or EPO levels to determine future risk of severe hypoglycaemia in people with type 1 diabetes.
... Although insulin-like growth factor (IGF)-II is known to be a target gene of HIF-1 (Feldser et al., 1999), insulin, IGF-I and IGF-II are all able to induce HIF-1a protein expression in various cell lines (Zelzer et al., 1998; Feldser et al., 1999; Jiang et al., 2001; Chavez and LaManna, 2002; Treins et al., 2002; Thomas and Kim, 2008; Alam et al., 2009). Similarly, both insulin and 2-deoxy-D-glucose treatment resulted in a widespread increase in HIF-3a mRNA and protein (Heidbreder et al., 2007). IGF-I can also induce VEGF expression in human osteoblast-like cells through transcriptional activation involving the HIF-2a/ARNT complex (Akeno et al., 2002 ). ...
In the first trimester the extravillous cytotrophoblast cells occlude the uterine spiral arterioles creating a low oxygen environment early in pregnancy, which is essential for pregnancy success. Paradoxically, shallow trophoblast invasion and defective vascular remodelling of the uterine spiral arteries in the first trimester may result in impaired placental perfusion and chronic placental ischemia and hypoxia later in gestation leading to adverse pregnancy outcomes. The hypoxia inducible factors (HIFs) are key mediators of the response to low oxygen. We aimed to elucidate mechanisms of regulation of HIFs and the role these may play in the control of placental differentiation, growth and function in both normal and pathological pregnancies. The Pubmed database was consulted for identification of the most relevant published articles. Search terms used were oxygen, placenta, trophoblast, pregnancy, HIF and hypoxia. The HIFs are able to function throughout all aspects of normal and abnormal placental differentiation, growth and function; during the first trimester (physiologically low oxygen), during mid-late gestation (where there is adequate supply of blood and oxygen to the placenta) and in pathological pregnancies complicated by placental hypoxia/ischemia. During normal pregnancy HIFs may respond to complex alterations in oxygen, hormones, cytokines and growth factors to regulate placental invasion, differentiation, transport and vascularization. In the ever-changing environment created during pregnancy, the HIFs appear to act as key mediators of placental development and function and thereby are likely to be important contributors to both normal and adverse pregnancy outcomes.
... There are, however, a broad variety of inflammatory mediators (ANGII, thrombin, TGFbII, bacterial lipopolysaccharides, IL-1b, TNFa) that regulate HIF-1a at transcriptional or at translational level (Frede et al., 2007) so that HIF1 is found in normoxic conditions. The isoform HIF-3a, which has been much less studied than HIF-1a, is regulated at the transcriptional level in hypoxia also in man (Heidbreder et al., 2003Heidbreder et al., , 2007). In addition, ROS affect the activity of HIF1. ...
Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the elderly throughout the world. AMD is attributed to a complex interaction of genetic and environmental factors. It is characterized by degeneration involving the retinal photoreceptors, retinal pigment epithelium (RPE), and Bruch's membrane, as well as alterations in choroidal capillaries. Aging and age-associated degenerative diseases, such as AMD, are intimately associated with decreased levels of tissue oxygenation and hypoxia that may induce accumulation of detrimental RPE-associated deposits, inflammation and neovascularization processes in retina. Hypoxia-inducible factor (HIF) is the master regulator for hypoxia-induced cellular adaptation that is involved in NF-kappaB signaling and the autophagic protein clearance system. In this review, we discuss role of HIF in AMD pathology and as a possible therapeutic target.
... Overall, it is most likely (as noted from gene transcript data) that insulin deprivation leads the cascade of events that cause HIF activation in normoxic condition and promote mitochondrial dysfunction. Recent studies suggest that nonhypoxic induction of HIF-1␣ occurs during altered glucose metabolism (37). Another explanation might be that in the normoxic condition or normal oxygen concentration, cells can promote abnormal TCA cycle because of lack of insulin action, leading to stabilization of HIF-␣ and promoting muscle mitochondria dysfunction. ...
Muscle mitochondrial dysfunction occurs in many insulin-resistant states, such as type 2 diabetes, prompting a hypothesis that mitochondrial dysfunction may cause insulin resistance. We determined the impact of insulin deficiency on muscle mitochondrial ATP production by temporarily depriving type 1 diabetic patients of insulin treatment.
We withdrew insulin for 8.6 +/- 0.6 h in nine C-peptide-negative type 1 diabetic subjects and measured muscle mitochondrial ATP production and gene transcript levels (gene array and real-time quantitative PCR) and compared with insulin-treated state. We also measured oxygen consumption (indirect calorimetry); plasma levels of glucagon, bicarbonate, and other substrates; and urinary nitrogen.
Withdrawal of insulin resulted in increased plasma glucose, branched chain amino acids, nonesterified fatty acids, beta-hydroxybutyrate, and urinary nitrogen but no change in bicarbonate. Insulin deprivation decreased muscle mitochondrial ATP production rate (MAPR) despite an increase in whole-body oxygen consumption and altered expression of many muscle mitochondrial gene transcripts. Transcript levels of genes involved in oxidative phosphorylation were decreased, whereas those involved in vascular endothelial growth factor (VEGF) signaling, inflammation, cytoskeleton signaling, and integrin signaling pathways were increased.
Insulin deficiency and associated metabolic changes reduce muscle MAPR and expression of oxidative phosphorylation genes in type 1 diabetes despite an increase in whole-body oxygen consumption. Increase in transcript levels of genes involved in VEGF, inflammation, cytoskeleton, and integrin signaling pathways suggest that vascular factors and cell proliferation that may interact with mitochondrial changes occurred.
Complex interplay of genetics and environmental factors in developing obesity has attracted a lot of attention in recent years. Different approaches in genetic analysis has illustrated numerous genetic loci that contribute in adiposity traits as monogenic obesity, syndromic obesity, and polygenic obesity. However, current studies are mostly on coding genes. Studies propose that epigenetic modifications such as DNA methylation plays a substantial role in the regulation of genes, involved in obesity-related processes. Also, miRNAs determine the adipocyte fate. Mechanistically they have impact on adipogenesis, adipocyte differentiation, lipid metabolism, glucose homeostasis, and insulin resistance. On the other hand, long non-coding RNAs play a protective role in metabolic dysfunction during obesity. Even though there are huge amount of evidence regarding genetic and epigenetic factors involved in development of obesity, there are lots of questions yet to be answered.
The heart forms early in development and delivers oxygenated blood to the rest of the embryo. After birth, the heart requires kilograms of ATP each day to support contractility for the circulation. Cardiac metabolism is omnivorous, utilizing multiple substrates and metabolic pathways to produce this energy. Cardiac development, metabolic tuning, and the response to ischemia are all regulated in part by the hypoxia-inducible factors (HIFs), central components of essential signaling pathways that respond to hypoxia. Here we review the actions of HIF1, HIF2, and HIF3 in the heart, from their roles in development and metabolism to their activity in regeneration and preconditioning strategies. We also discuss recent work on the role of HIFs in atherosclerosis, the precipitating cause of myocardial ischemia and the leading cause of death in the developed world.
Objective
In previous epigenome-wide association studies, Hypoxia inducible Factor 3 Alpha Subunit (HIF3A) DNA methylation has been reported to be associated with body mass index (BMI) and weight change. However, none of these studies have included Mexican Americans.
Methods
In the current study, we assessed levels of HIF3A methylation in 927 Mexican American women identified from Mano-A-Mano, the Mexican American Cohort study.
Results
Significantly higher methylation levels at three CpG sites (position 46801557, 46801642, and 46801699) were observed in obese women compared to non-obese women (P < 0.05). Furthermore, we found that elevated methylation levels at those three CpG sites were associated with significant weight gain (P < 0.05), defined as an increase in BMI by at least one category between the baseline and the follow-up, with a median follow-up time of 39 months. Then, using pre-diagnostic blood DNA samples, we found increased DNA methylation at CpG 46801642 to be associated with a 1.35-fold increased risk of breast cancer (Hazard Ratio (HR) = 1.35, 95% Confidence Interval (CI): 1.02, 3.01), with a median follow-up time of 127 months. Using the Cancer Genome Atlas (TCGA) data, we further found that levels of HIF3A were significantly higher-methylated and down-regulated in breast tumor than in normal tissues (P < 1 × 10¹² for both).
Conclusion
Thus, our results provide evidence to support the role of HIF3A in obesity, weight gain, and the development of breast cancer.
Human mesenchymal stromal/stem cells (hMSCs) emerged as a promising therapeutic tool for ischemic disorders, due to their ability to regenerate damaged tissues, promote angiogenesis and reduce inflammation, leading to encouraging, but still limited results. The outcomes in clinical trials exploring hMSC therapy are influenced by low cell retention and survival in affected tissues, partially influenced by lesion’s microenvironment, where low oxygen conditions (i.e. hypoxia) and inflammation coexist. Hypoxia and inflammation are pathophysiological stresses, sharing common activators, such as hypoxia-inducible factors (HIFs) and NF-κB. HIF1α and HIF2α respond essentially to hypoxia, activating pathways involved in tissue repair. Little is known about the regulation of HIF3α. Here we investigated the role of HIF3α in vitro and in vivo. Human MSCs expressed HIF3α, differentially regulated by pro-inflammatory cytokines in an oxygen-independent manner, a novel and still uncharacterized mechanism, where NF-κB is critical for its expression. We investigated if epigenetic modifications are involved in HIF3α expression by methylation-specific PCR and histone modifications. Robust hypermethylation of histone H3 was observed across HIF3A locus driven by pro-inflammatory cytokines. Experiments in a murine model of arteriotomy highlighted the activation of Hif3α expression in infiltrated inflammatory cells, suggesting a new role for Hif3α in inflammation in vivo.
Eine intrauterine Gestationsdiabetes (GDM) Exposition induziert in den betroffenen Nachkommen eine lebenslang erhöhte Prädisposition für metabolische und komplexe Erkrankungen. Die Krankheitssuszeptibilität wird dabei durch epigenetische Veränderungen vermittelt, die sich über die Regulation der Genaktivität auch auf das Expressionsniveau und den Phänotypen auswirken. Um neue Gene zu finden, die eine Rolle in der fetalen Programmierung spielen, wurden in dieser Arbeit genomweite Methylierungsmuster von Nabelschnurbluten (FCBs) aus GDM-Schwangerschaften und Kontrollen miteinander verglichen. Mit Illumina Infinium HumanMethylation 450K Arrays konnten signifikante Gruppenunterschiede für insgesamt 65 CpG-Stellen (52 davon genassoziiert) festgestellt werden, die multiplem Testen standhielten. Mittels Pyrosequenzierung wurden vier dieser Kandidaten-Loci (ATP5A1, MFAP4, PRKCH, SLC17A4), sowie ein Gen aus der Literatur (HIF3A) genauer untersucht und die Effekte erfolgreich validiert. Für das zugrundeliegende multivariate Regressionsmodell wurden die potenziellen Störfaktoren Gestationsalter, kindliches Geschlecht und mütterlicher BMI berücksichtigt. Der GDM-Effekt zeigte sich stärker in der insulinbehandelten Subgruppe (I-GDM) als in der diätisch behandelten (D GDM) und scheint insgesamt multifaktoriell bedingt zu sein, da viele Gene betroffen waren, jedoch alle mit einer vergleichsweise niedrigen Effekt-Größe. Zusätzlich konnten für den MEG3 Promotor, MEST und PEG3, drei von vier geprägten Genen, die mittels Deep Bisulfite Sequencings (DBS) analysiert wurden, ebenfalls signifikante Methylierungs-unterschiede zwischen der GDM- und Kontroll-Gruppe detektiert werden. Die identifizierten Gene stellen labile Zielregionen für die GDM-induzierte intrauterine Programmierung dar und können zukünftig nützliche Biomarker für Krankheitsdiagnosen und Prognosen sein. Mittels DBS können darüber hinaus Einzelmolekül-Analysen durchgeführt werden, für die in differentiell methylierten Regionen (DMRs) anhand eines informativen SNPs die parentale Allel-Herkunft bestimmt und bei der Berechnung von Epimutationsraten einbezogen werden kann. Epimutationen wurde als solche gewertet, wenn sie ein > 50 % abnormal (de)methyliertes Methylierungsprofil aufwiesen. Die DBS-Daten wurden mit zwei verschiedenen Sequenzierplattformen generiert (Roche GS Junior und Illumina MiSeq). Für Zweitere wurde ein eigenes, unabhängiges Library-Präparations-Protokoll entwickelt. In Nabelschnurblut, adultem Blut und Viszeralfett wurden für die paternal exprimierte MEST Promotor DMR und die maternal exprimierte MEG3 intergenic (IG) DMR hohe Epimutationsraten für das jeweils unmethylierte Allel detektiert. Die geprägten (methylierten) Allele wiesen dagegen nur niedrige Epimutationsraten auf. Da MEST und MEG3 invers geprägte Gene sind, war die Hypermethylierung des nicht geprägten Allels (HNA) demnach unabhängig von der parentalen Allel-Herkunft. Die HNA scheint außerdem erst nach der Fertilisation aufzutreten, da in Spermien nur sehr wenige Epimutationen gefunden wurden. Für die sekundäre MEG3 Promotor DMR (deren Prägung von der primären MEG3 IG-DMR reguliert wird) wurde ein deutlich schwächerer, wenngleich signifikanter HNA-Effekt im FCB gemessen, für die paternal exprimierte PEG3 Promotor DMR konnte dagegen kein signifikanter Unterschied zwischen den beiden parentalen Epimutationsraten festgestellt werden. Der HNA-Effekt für die MEST DMR, MEG3 IG-DMR und MEG3 Promotor DMR war weder mit GDM noch mit Adipositas assoziiert und zeigte allgemein eine große interindividuelle Varianz. Die Aufrechterhaltung differenzieller Methylierungsmuster in Imprinting Kontrollregionen (ICRs) scheint in manchen Entwicklungs-Zeitspannen von großer Bedeutung und damit streng kontrolliert zu sein, später jedoch redundant zu werden, was sich in der Anreicherung von stochastischen sowie umweltinduzierten Fehlern auf dem nicht geprägten Allel äußern kann. HNA-suszeptible geprägte Gene ähneln in mancherlei Hinsicht metastabilen Epiallelen. Diese Studie zeigt, dass sowohl stochastische Faktoren als auch Umweltstimuli während der frühen embryonalen Entwicklung u.a. über HNA-Effekte geprägte Gen-Netzwerke programmieren, die in Wachstumsprozesse involviert sind. Um tiefere Einblicke in allelspezifische Prägungsprofile zu erhalten, wären umfangreiche DBS HNA-Längsschnittstudien aller 50-100 human geprägten Gene in unterschiedlichen Gewebetypen und Differenzierungsstadien wünschenswert.
Cancer biomarkers (CB) are biomolecules produced either by the tumor cells or by other cells of the body in response to the tumor. Every cell type has its unique molecular signature and identifiable characteristics such as levels or activities of myriad of genes, proteins, or other molecular features; therefore, biomarkers can facilitate the molecular definition of cancer. Our aim was providing updated knowledge and performing detailed review about CB regarding their molecular and biochemical characterization and their clinical utility in screening, diagnosis, follow-up, or therapeutic stratification for cancer patients. Focusing on conventional, the FDA approved as well as promising future biomarkers in most common cancers. In addition, emphasizing on their prospective role may be of great value in improving the management of cancer patients. The challenge and future prospective of biomarkers, by facilitating the combination of therapeutics with diagnostics, promise to play an important role in the development of personalized medicine.
Recently, a genome-wide analysis identified DNA methylation of the HIF3A (hypoxia-inducible factor 3A) as strongest correlate of BMI. Here we tested the hypothesis that HIF3A mRNA expression and CpG-sites methylation in adipose tissue (AT) and genetic variants in HIF3A are related to parameters of AT distribution and function. In paired samples of subcutaneous AT (SAT) and visceral AT (VAT) from 603 individuals, we measured HIF3A mRNA expression and analyzed its correlation with obesity and related traits. In subgroups of individuals, we investigated the effects on HIF3A genetic variants on its AT expression (N = 603) and methylation of CpG-sites (N = 87). HIF3A expression was significantly higher in SAT compared to VAT and correlated with obesity and parameters of AT dysfunction (including CRP and leucocytes count). HIF3A methylation at cg22891070 was significantly higher in VAT compared to SAT and correlated with BMI, abdominal SAT and VAT area. Rs8102595 showed a nominal significant association with AT HIF3A methylation levels as well as with obesity and fat distribution. HIF3A expression and methylation in AT are fat depot specific, related to obesity and AT dysfunction. Our data support the hypothesis that HIF pathways may play an important role in the development of AT dysfunction in obesity.
Background: Under hypoxic environment carbohydrate is consumed preferentially to supply energy. Long-term hypoxic stress has profound influence on the metabolic process such as the intake, utilization and oxidization of glucose. Therefore, it is necessary to explore the effects and mechanism of hypoxic stress on glucose metabolism. Objective: To explore the possible mechanism of the effect of hypoxic acclimatization on glycometabolism, so as to provide theoretical fundament for related studies. Retrieve Strategy: The relevant English and Chinese articles dated between January 1990 and July 2007 were retrieved from PubMed and China Journal Full-text Database (CJFD) by using the keywords of "hypoxia, metabolic enzyme, insulin, GLUT, HIF-1 and AMPK" in English and Chinese. 338 literatures were retrieved, including 215 Chinese, and 123 English. Articles with close association and published in recently or in the authoritative journals were included. Repetitive articles and Meta analysis articles were excluded. Forty literatures were selected. Literature Evaluation: Of 40 included articles, 8 were review articles, and the others were clinical and experimental researches. Data Synthesis: With hypoxic acclimatization, the amount of saccharophosphorylase and glycogen synthetase is increased, the activity enhanced and the ability to transfer glucose promoted. The possible mechanism of the effect of hypoxic acclimatization on glycometabolism is that hypoxia inducible factor-1 up-regulates the expression of hypoxia responsive genes about glycometabolism and the activation of the AMP-activated protein kinase regulates the process of glycometabolism through an unknown pathway. Conclusion: The enhancement of glycometabolism is one of the important signs of hypoxic acclimatization. The increase in the capability of glycometabolism under hypoxia has extensive application prospects in the field of the altitude training and the prevention and improvement of insulin-resistant conditions.
Hypoxia-inducible factor (HIF) is an important transcription factor of the cellular response to hypoxia, which transactivates a myriad of genes to active adaptive responses to hypoxia. HIF is a heterodimer comprising an unstable oxygen-labile α (HIF-α) subunit and a stable constitutively expressed β (HIF-β) subunit. In human beings, there are three isoforms of α subunit including HIF-1α, HIF-2α and HIF-3α. In contrast to the indepth studies of HIF-1α and HIF-2α, we have still poorly known about HIF-3α. HIF-3α has been conventionally regarded as a negative regulator of HIF-1α and HIF-2α. HIF-3α gene is subject to extensive alternative spliced variants which have a broad physiological functions. This review systematically discusses the HIF-3α structure, spliced variants, expression and regulation process in response to hypoxia, and it also discusses the future direction of research of HIF-3α.
The hypoxia-inducible factor (HIF) family has three distinct members in most vertebrates. All three HIFs consist of a unique and oxygen-labile α subunit and a common and stable β subunit. While HIF-1 and HIF-2 function as master regulators of the transcriptional response to hypoxia, much less is known about HIF-3. The HIF-3α gene gives rise to multiple HIF-3α variants due to the utilization of different promoters, different transcription initiation sites, and alternative splicing. These HIF-3α variants are expressed in different tissues, at different developmental stages, and that they are differentially regulated by hypoxia and other factors. Recent studies suggest that different HIF-3α variants have different and even opposite functions. There is strong evidence that full-length HIF-3α protein functions as an oxygen-regulated transcription activator and it activates a unique transcriptional program in response to hypoxia. Many HIF-3α target genes have been identified. While some short HIF-3α variants act as dominant negative regulators of HIF-1/2α actions, other HIF-3α variants can inhibit HIF-1/2α actions by competing for the common HIF-β. There are also a number of HIF-3α variants yet to be explored. Future studies of these naturally occurring HIF-3α variants will provide new and important insights into HIF biology and may lead to the development of new therapeutic strategies.
To conduct an epigenome-wide analysis of DNA methylation and obesity traits.
DNA methylation was quantified in CD4+ T-cells using the Illumina Infinium HumanMethylation450 array in 991 participants of the Genetics of Lipid Lowering Drugs and Diet Network. Methylation at individual cytosine-phosphate-guanine (CpG) sites as a function of body mass index (BMI) and waist circumference (WC), adjusting for age, gender, study site, T-cell purity, smoking, and family structure, was modeled.
Epigenome-wide significant associations between eight CpG sites and BMI and five CpG sites and WC, successfully replicating the top hits in whole blood samples from the Framingham Heart Study (n = 2,377) and the Atherosclerosis Risk in Communities study (n = 2,097), were found. Top findings were in CPT1A (meta-analysis P = 2.7 × 10(-43) for BMI and 9.9 × 10(-23) for WC), PHGDH (meta-analysis P = 2.0 × 10(-15) for BMI and 4.0 × 10(-9) for WC), CD38 (meta-analysis P = 6.3 × 10(-11) for BMI and 1.6 × 10(-12) for WC), and long intergenic non-coding RNA 00263 (meta-analysis P = 2.2 × 10(-16) for BMI and 8.9 × 10(-14) for WC), regions with biologically plausible relationships to adiposity.
This large-scale epigenome-wide study discovered and replicated robust associations between DNA methylation at CpG loci and obesity indices, laying the groundwork for future diagnostic and/or therapeutic applications.
© 2015 The Obesity Society.
Hypoxia inducible factors (HIFs) are transcription factors, which are commonly expressed in mammals, including humans. The HIFs consist of hypoxia-regulated α and oxygen‑insensitive β subunits, and are key regulators of gene expression during hypoxia in normal and solid tumor tissues. Three members of the HIF family, HIF‑1α, HIF‑2α, and HIF‑3α, are currently known. HIF‑3α differs from HIF‑1α and HIF‑2α in protein structure and regulation of gene expression. For a long time, HIF‑3α was considered as a negative mediator of HIF‑regulated genes. HIF‑3 has a transcriptional regulatory function, which negatively affects gene expression by competing with HIF‑1α and HIF‑2α in binding to transcriptional elements in target genes during hypoxia. Previously, certain target genes of HIF‑3α have been identified, confirming the role of HIF‑3α as a transcription factor. In this review, the protein structure, gene regulation and biological function of HIF-3 are discussed based on the literature.
Increased age, BMI and HbA1c levels are risk factors for several non-communicable diseases. However, the impact of these factors on the genome-wide DNA methylation pattern in human adipose tissue remains unknown. We analyzed DNA methylation of ∼480,000 sites in human adipose tissue from 96 males and 94 females, and related methylation to age, BMI and HbA1c. We also compared epigenetic signatures in adipose tissue and blood. Age was significantly associated with both altered DNA methylation and expression of 1,050 genes (e.g. FHL2, NOX4 and PLG). Interestingly, many reported epigenetic biomarkers of ageing in blood, including ELOVL2, FHL2, KLF14 and GLRA1, also showed significant correlations between adipose tissue DNA methylation and age in our study. The most significant association between age and adipose tissue DNA methylation was found upstream of ELOVL2. We identified 2,825 genes (e.g. FTO, ITIH5, CCL18, MTCH2, IRS1 and SPP1) where both DNA methylation and expression correlated with BMI. Methylation at previously reported HIF3A sites correlated significantly with BMI in females only. HbA1c (range 28-46 mmol/mol) correlated significantly with methylation of 711 sites, annotated to e.g. RAB37, TICAM1 and HLA-DPB1. Pathway analyses demonstrated that methylation levels associated with age and BMI are overrepresented among genes involved in cancer, type 2 diabetes and cardiovascular disease. Our results highlight the impact of age, BMI and HbA1c on epigenetic variation of candidate genes for metabolic diseases and cancer in human adipose tissue. Importantly, we demonstrate that epigenetic biomarkers in blood can mirror age-related epigenetic signatures in target tissues for metabolic diseases such as adipose tissue.
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Hypoxia-inducible factor (HIF) is a heterodimer composed of HIF-α and -β, and to date, three HIF-α subunits, HIF-1 α, -2α, and -3α, have been identified. Among these HIF-α subunits, HIF-1α represses peroxisome proliferator activator γ2 gene expression and then inhibits adipogenesis, and HIF-2α is induced during adipose differentiation and functions as a positive regulator of adipogenesis. We here found that like HIF-2α, HIF-3α was induced during 3T3-L1 adipose differentiation. Reporter gene analysis revealed that HIF-2α enhanced the promoter activity of the 5′-flanking region of the mouse HIF-3α gene (nucleotides -2710 to +56), while HIF-1α had no substantial effects on the promoter activity. These results suggested that HIF-2α, which was induced during adipogenesis, might regulate the HIF-3α gene expression. Furthermore, the 5′-deletion analysis revealed that the sequence between -251 and -228 in mouse HIF-3α promoter was essential in response to HIF-2α. We further examined the effect of ectopic expression of HIF-3α in 3T3-L1 cells on adipose differentiation and found that ectopic expression of HIF-3α at the early stage of differentiation induced the expression of several kinds of adipocytes-related genes and enhanced adipogenic potential. HIF-3α, which is induced by HIF-2α, might function as an accelerator of adipogenesis.
Hypoxia is one of the fundamental biological phenomena that are intricately associated with the development and aggressiveness of a variety of solid tumors. Hypoxia-inducible factors (HIF) function as a master transcription factor, which regulates hypoxia responsive genes and has been recognized to play critical roles in tumor invasion, metastasis, and chemo-radiation resistance, and contributes to increased cell proliferation, survival, angiogenesis and metastasis. Therefore, tumor hypoxia with deregulated expression of HIF and its biological consequence lead to poor prognosis of patients diagnosed with solid tumors, resulting in higher mortality, suggesting that understanding of the molecular relationship of hypoxia with other cellular features of tumor aggressiveness would be invaluable for developing newer targeted therapy for solid tumors. It has been well recognized that cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) phenotypic cells are associated with therapeutic resistance and contribute to aggressive tumor growth, invasion, metastasis and believed to be the cause of tumor recurrence. Interestingly, hypoxia and HIF signaling pathway are known to play an important role in the regulation and sustenance of CSCs and EMT phenotype. However, the molecular relationship between HIF signaling pathway with the biology of CSCs and EMT remains unclear although NF-κB, PI3K/Akt/mTOR, Notch, Wnt/β-catenin, and Hedgehog signaling pathways have been recognized as important regulators of CSCs and EMT. In this article, we will discuss the state of our knowledge on the role of HIF-hypoxia signaling pathway and its kinship with CSCs and EMT within the tumor microenvironment. We will also discuss the potential role of hypoxia-induced microRNAs (miRNAs) in tumor development and aggressiveness, and finally discuss the potential effects of nutraceuticals on the biology of CSCs and EMT in the context of tumor hypoxia.
The Per-Arnt-Sim (PAS) domain is conserved across the kingdoms of life and found in an ever-growing list of proteins. This domain can bind to and sense endogenous or xenobiotic small molecules such as molecular oxygen, cellular metabolites, or polyaromatic hydrocarbons. Members of this family are often found in pathways that regulate responses to environmental change; in mammals these include the hypoxia, circadian, and dioxin response pathways. These pathways function in development and throughout life to regulate cellular, organ, and whole-organism adaptive responses. Remarkably, in the case of the clock, this adaptation includes anticipation of environmental change. In this review, we summarize the roles of PAS domain-containing proteins in mammals. We provide structural evidence that functionally classifies both known and unknown biological roles. Finally, we discuss the role of PAS proteins in anticipation of and adaptation to environmental change.
Hypoxia-inducible factor (HIF) is a heterodimer composed of HIF-alpha and -beta, and to date, three HIF-alpha subunits, HIF-1alpha, -2alpha, and -3alpha, have been identified. Among these HIF-alpha subunits, HIF-1alpha represses peroxisome proliferator activator gamma2 gene expression and then inhibits adipogenesis, and HIF-2alpha is induced during adipose differentiation and functions as a positive regulator of adipogenesis. We here found that like HIF-2alpha, HIF-3alpha was induced during 3T3-L1 adipose differentiation. Reporter gene analysis revealed that HIF-2alpha enhanced the promoter activity of the 5'-flanking region of the mouse HIF-3alpha gene (nucleotides -2710 to +56), while HIF-1alpha had no substantial effects on the promoter activity. These results suggested that HIF-2alpha, which was induced during adipogenesis, might regulate the HIF-3alpha gene expression. Furthermore, the 5'-deletion analysis revealed that the sequence between -251 and -228 in mouse HIF-3alpha promoter was essential in response to HIF-2alpha. We further examined the effect of ectopic expression of HIF-3alpha in 3T3-L1 cells on adipose differentiation and found that ectopic expression of HIF-3alpha at the early stage of differentiation induced the expression of several kinds of adipocytes-related genes and enhanced adipogenic potential. HIF-3alpha, which is induced by HIF-2alpha, might function as an accelerator of adipogenesis.
2-Deoxy-D-Glucose (2-DG), a synthetic analogue of glucose, is used as an anticancer agent either alone or in combination with other tumor treatment protocols. The present study was conducted to identify the systemic effects of 2-DG on parameters of vital importance. The blood pressure, ECG and respiratory excursions were recorded in anesthetized adult rats. At the end (after 120 min) of experiments, the plasma glucose and serum insulin levels were estimated. Injection of 2-DG (0.5 g/kg) produced an immediate increase in mean arterial pressure (MAP) and respiratory rate. The increase in MAP continued throughout the period of observation (120 min) and the maximal increase was seen at 90 min (27%). Whereas, the respiratory rate decreased by 17% at 15 min which decreased further to 37% by 120 min. Heart rate also decreased after 2-DG in a time-dependent manner and 40% decrease was observed at 120 min. Administration of 2-DG increased the plasma glucose level significantly (30%) as compared to saline control group but did not increase the serum insulin level. The results indicate that 2-DG alters the cardio-respiratory parameters by mechanisms unrelated to plasma insulin activity.
Studies of gene regulation by oxygen have recently defined the existence of a widely operative system that responds to hypoxia
but not mitochondrial inhibitors and involves the induction of a DNA-binding complex termed hypoxia-inducible factor 1. This
system has been implicated in the regulation of erythropoietin, certain angiogenic growth factors, and particular glycolytic
isoenzymes. The glucose transporter Glut-1 is induced by both hypoxia and mitochondrial inhibitors, implying the operation
of a different mechanism of oxygen sensing. To explore that possibility, we analyzed the cis-acting sequences that convey
these responses. An enhancer lying 5′ to the mouse Glut-1 gene was found to convey responses both to hypoxia and to the mitochondrial
inhibitors, azide and rotenone. However, detailed analysis of this enhancer demonstrated that distinct elements responded
to hypoxia and the mitochondrial inhibitors. The response to hypoxia was mediated by sequences that contained a functionally
critical, although atypical, hypoxia-inducible factor 1 binding site, whereas sequences lying approximately 100 nucleotides
5′ to this site, which contained a critical serum response element, conveyed responses to the mitochondrial inhibitors. Thus,
rather than reflecting an entirely different mechanism of oxygen sensing, regulation of Glut-1 gene expression by hypoxia
and mitochondrial inhibitors arises from the function of two different sensing systems. One of these responds to hypoxia alone
and resembles that involved in erythropoietin regulation, while the other responds to mitochondrial inhibitors and involves
activation of a serum response element.
Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix transcription factor which is expressed when mammalian cells are subjected to hypoxia and which activates transcription of genes encoding erythropoietin, vascular endothelial growth factor, and other proteins that are important for maintaining oxygen homeostasis. Previous studies have provided indirect evidence that HIF-1 also regulates transcription of genes encoding glycolytic enzymes. In this paper we characterize hypoxia response elements in the promoters of the ALDA, ENO1, and Ldha genes. We demonstrate that HIF-1 plays an essential role in activating transcription via these elements and show that although absolutely necessary, the presence of a HIF-1 binding site alone is not sufficient to mediate transcriptional responses to hypoxia. Analysis of hypoxia response elements in the ENO1 and Ldha gene promoters revealed that each contains two functionally-essential HIF-1 sites arranged as direct and inverted repeats, respectively. Our data establish that functional hypoxia-response elements consist of a pair of contiguous transcription factor binding sites at least one of which contains the core sequence 5'-RCGTG-3' and is recognized by HIF-1. These results provide further evidence that the coordinate transcriptional activation of genes encoding glycolytic enzymes which occurs in hypoxic cells is mediated by HIF-1.
We have isolated and characterized a cDNA for a novel Per-Arnt/AhR-Sim basic helix-loop-helix (bHLH-PAS) factor that interacts with the Ah receptor nuclear translocator (Arnt), and its predicted amino acid sequence exhibits significant similarity to the hypoxia-inducible factor 1alpha (HIF1alpha) and Drosophila trachealess (dTrh) gene product. The HIF1alpha-like factor (HLF) encoded by the isolated cDNA bound the hypoxia-response element (HRE) found in enhancers of genes for erythropoietin, vascular endothelial growth factor (VEGF), and various glycolytic enzymes, and activated transcription of a reporter gene harboring the HRE. Although transcription-activating properties of HLF were very similar to those reported for HIF1alpha, their expression patterns were quite different between the two factors; HLF mRNA was most abundantly expressed in lung, followed by heart, liver, and other various organs under normoxic conditions, whereas HIF1alpha mRNA was ubiquitously expressed at much lower levels. In lung development around parturition, HLF mRNA expression was markedly enhanced, whereas that of HIF1alpha mRNA remained apparently unchanged at a much lower level. Moreover, HLF mRNA expression was closely correlated with that of VEGF mRNA. Whole mount in situ hybridization experiments demonstrated that HLF mRNA was expressed in vascular endothelial cells at the middle stages (9.5 and 10.5 days postcoitus) of mouse embryo development, where HIF1alpha mRNA was almost undetectable. The high expression level of HLF mRNA in the O2 delivery system of developing embryos and adult organs suggests that in a normoxic state, HLF regulates gene expression of VEGF, various glycolytic enzymes, and others driven by the HRE sequence, and may be involved in development of blood vessels and the tubular system of lung.
Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS (bHLH-PAS) superfamily. In our efforts to identify new bHLH-PAS proteins, we cloned a cDNA encoding a novel alpha-class hypoxia inducible factor, HIF3alpha. The HIF3alpha open reading frame encodes a 662-amino acid protein with a predicted molecular weight of 73 kDa and is expressed in adult thymus, lung, brain, heart, and kidney. The N-terminal bHLH-PAS domain of this protein shares amino acid sequence identity with that of HIF1alpha and HIF2alpha (57% and 53% identity, respectively). The C-terminus of HIF3alpha contains a 36-amino acid sequence that shares 61% identity with the hypoxia responsive domain-1 (HRD1) of HIF1alpha. In transient transfections, this domain confers hypoxia responsiveness when linked to a heterologous transactivation domain. In vitro studies reveal that HIF3alpha dimerizes with a prototype beta-class subunit, ARNT, and that the resultant heterodimer recognizes the hypoxia responsive element (HRE) core sequence, TACGTG. Transient transfection experiments demonstrate that the HIF3alpha-ARNT interaction can occur in vivo, and that the activity of HIF3alpha is upregulated in response to cobalt chloride or low oxygen tension.
Orexins are produced specifically by neurons located in the lateral hypothalamus. Recent results suggested peripheral actions of orexins. Therefore, we analyzed the mRNA expression of prepro-orexin and the orexin receptor subtypes OX(1) and OX(2) in peripheral rat tissues. Using real-time quantitative RT-PCR we detected significant amounts of prepro-orexin mRNA in testis, but not in ovaries. OX(1) receptor mRNA was highly expressed in the brain and at lower levels in the pituitary gland. Only small amounts of OX(1) receptor mRNA were found in other tissues such as kidney, adrenal, thyroid, testis, ovaries, and jejunum. Very high levels of OX(2) receptor mRNA, 4-fold higher than in brain, were found in adrenal glands of male rats. Low amounts of OX(2) receptor mRNA were present in lung and pituitary. In adrenal glands, OX(2) receptor mRNA was localized in the zona glomerulosa and reticularis by in situ hybridization, indicating a role in adrenal steroid synthesis and/or release. OX(1) receptor mRNA in the pituitary and OX(2) receptor mRNA in the adrenal gland were much higher in male than in female rats. In the hypothalamus, OX(1) receptor mRNA was slightly elevated in female rats. The differential mRNA expression of orexin receptor subtypes in peripheral organs indicates discrete peripheral effects of orexins and the existence of a peripheral orexin system. This is supported by the detection of orexin A in rat plasma. Moreover, the sexually dimorphic expression of OX(1) and OX(2) receptors in the hypothalamus, pituitary, and adrenal glands suggests gender-specific roles of orexins in the control of endocrine functions.
One of the most common signatures of highly malignant tumors is their capacity to metabolize more glucose to lactic acid than their tissues of origin. Hepatomas exhibiting this phenotype are dependent on the high expression of type II hexokinase, which supplies such tumors with abundant amounts of glucose 6-phosphate, a significant carbon and energy source especially under hypoxic conditions. Here we report that the distal region of the hepatoma type II hexokinase promoter displays consensus motifs for hypoxia-inducible factor (HIF-1) that overlap E-box sequences known to be related in other gene promoters to glucose response. Moreover, we show that subjecting transfected hepatoma cells to hypoxic conditions activates the type II hexokinase promoter almost 3-fold, a value that approaches 7-fold in the presence of glucose. Consistent with these findings is the induction under hypoxic conditions of the HIF-1 protein. Reporter gene analyses with a series of nested deletion mutants of the hepatoma type II hexokinase promoter show that a significant fraction of the total activation observed under hypoxic conditions localizes to the distal region where the overlapping HIF-1/E-box sequences are located. Finally, DNase I footprint analysis with a segment of the promoter containing these elements reveals the binding of several nuclear proteins. In summary, these novel studies identify and characterize a marked glucose-modulated activation response of the type II hexokinase gene to hypoxic conditions within highly glycolytic hepatoma cells, a property that may help assure that such cells exhibit a growth and survival advantage over their parental cells of origin.
Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.
Hypoxia-inducible factor 1 (HIF-1) is a transcription factor involved in normal mammalian development and in the pathogenesis of several disease states. It consists of two subunits, HIF-1alpha, which is degraded during normoxia, and HIF-1beta, which is constitutively expressed. Activated HIF-1 induces the expression of genes involved in angiogenesis, erythropoiesis, and glucose metabolism. We have previously reported that insulin stimulates vascular endothelial growth factor (VEGF) expression (). In this study, we show that insulin activates HIF-1, leading to VEGF expression in retinal epithelial cells. Insulin activates HIF-1alpha protein expression in a dose-dependent manner with a maximum reached within 6 h. The expression of HIF-1alpha is correlated with the activation of HIF-1 DNA binding activity and the transactivation of a HIF-1-dependent reporter gene. Insulin does not appear to affect HIF-1alpha mRNA transcription but regulates HIF-1alpha protein expression through a translation-dependent pathway. The expression of an active form of protein kinase B and treatment of cells with specific inhibitors of phosphatidylinositol 3-kinase (PI3K), MAPK, and target of rapamycin (TOR) show that mainly PI3K and to a lesser extent TOR are required for insulin-induced HIF-1alpha expression. HIF-1 activity and VEGF expression are also dependent on PI3K- and TOR-dependent signaling. In conclusion, we show here that insulin regulates HIF-1 action through a PI3K/TOR-dependent pathway, resulting in increased VEGF expression.
Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.
The inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, functions as a dominant negative regulator of hypoxia-inducible transcription factors (HIFs) by
forming complexes with those proteins that fail to bind to hypoxia response elements of target genes. We have previously observed
that IPAS is predominantly expressed in mice in Purkinje cells of the cerebellum and in corneal epithelium of the eye where
it appears to play a role in negative regulation of angiogenesis and maintenance of an avascular phenotype. Sequencing of
the mouse IPAS genomic structure revealed that IPAS is a splicing variant of the HIF-3α locus. Thus, in addition to three
unique exons (1a, 4a, and 16) IPAS shares three exons (2, 4, and 5) with HIF-3α as well as alternatively spliced variants
of exons 3 and 6. In experiments using normal mice and mice exposed to hypoxia (6% O2) for 6 h we observed alternative splicing of the HIF-3α transcript in the heart and lung. The alternatively spliced transcript
was only observed under hypoxic conditions, thus defining a novel mechanism of hypoxia-dependent regulation of gene expression.
Importantly, this mechanism may establish negative feedback loop regulation of adaptive responses to hypoxia/ischemia in these
tissues.
Nutrient deprivation has been shown to cause cancer cell death. To exploit nutrient deprivation as anti-cancer therapy, we investigated the effects of the anti-metabolite 2-deoxy-D-glucose on breast cancer cells in vitro. This compound has been shown to inhibit glucose metabolism. Treatment of human breast cancer cell lines with 2-deoxy-D-glucose results in cessation of cell growth in a dose dependent manner. Cell viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide conversion assay and clonogenic survival are decreased with 2-deoxy-D-glucose treatment indicating that 2-deoxy-D-glucose causes breast cancer cell death. The cell death induced by 2-deoxy-D-glucose was found to be due to apoptosis as demonstrated by induction of caspase 3 activity and cleavage of poly (ADP-ribose) polymerase. Breast cancer cells treated with 2-deoxy-D-glucose express higher levels of Glut1 transporter protein as measured by Western blot analysis and have increased glucose uptake compared to non-treated breast cancer cells. From these results we conclude that 2-deoxy-D-glucose treatment causes death in human breast cancer cell lines by the activation of the apoptotic pathway. Our data suggest that breast cancer cells treated with 2-deoxy-D-glucose accelerate their own demise by initially expressing high levels of glucose transporter protein, which allows increased uptake of 2-deoxy-D-glucose, and subsequent induction of cell death. These data support the targeting of glucose metabolism as a site for chemotherapeutic intervention by agents such as 2-deoxy-D-glucose.
British Journal of Cancer (2002) 87, 805–812. doi:10.1038/sj.bjc.6600547 www.bjcancer.com
© 2002 Cancer Research UK
Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.
Hypoxia-induced mitogenic factor (HIMF), also called FIZZ1 or RELMalpha, was a newly found cytokine. Hypoxia caused robust HIMF induction in the lung, and HIMF has potent pulmonary vasoconstrictive, proliferative, and angiogenic properties. To investigate the role of HIMF in lung development, we determined its spatial and temporal expression. From embryonic day (E)16 to postnatal day (P)28, HIMF was strongly expressed in the cytoplasm of bronchial epithelial cells, type II cells, endothelial cells, and primitive mesenchymal cells. Treatment with HIMF resulted in a significant reduction of apoptosis in cultured embryonic lung, thus revealing a previously unknown function of HIMF. Because HIMF gene is upregulated by hypoxia and contains a hypoxia-inducible transcription factor (HIF) binding site, we subsequently investigated whether HIMF was coexpressed with HIF-2alpha or HIF-1alpha. HIF-1alpha expression was temporally distinct from HIMF expression. In contrast, HIF-2alpha was present in endothelial cells, bronchial epithelial cells, and type II cells from E18 to P28. Thus, HIMF and HIF-2alpha were temporally and spatially coexpressed in the developing lung. These results indicate a role for HIMF in lung development, possibly under the control of HIF-2, and suggest that HIMF regulates apoptosis and may participate in lung alveolarization and maturation.
Hyperglycemia and hypoxia are suggested to play essential pathophysiological roles in the complications of diabetes, which may result from a defective response of the tissues to low oxygen tension. In this study, we show that in primary dermal fibroblasts and endothelial cells, hyperglycemia interferes with the function of hypoxia-inducible factor-1 (HIF-1), a transcription factor that is essential for adaptive responses of the cell to hypoxia. Experiments using proteasomal and prolyl hydroxylases inhibitors indicate that hyperglycemia inhibits hypoxia-induced stabilization of HIF-1alpha protein levels against degradation and suggest that mechanisms in addition to proline hydroxylation may be involved. This effect of hyperglycemia was dose dependent and correlates with a lower transcription activation potency of HIF-1alpha, as assessed by transient hypoxia-inducible reporter gene assay. Regulation of HIF-1alpha function by hyperglycemia could be mimicked by mannitol, suggesting hyperosmolarity as one critical parameter. The interference of hyperglycemia with hypoxia-dependent stabilization of HIF-1alpha protein levels was confirmed in vivo, where only very low levels of HIF-1alpha protein could be detected in diabetic wounds, as compared with chronic venous ulcers. In conclusion, our data demonstrate that hyperglycemia impairs hypoxia-dependent protection of HIF-1alpha against proteasomal degradation and suggest a mechanism by which diabetes interferes with cellular responses to hypoxia.
Dietary energy restriction (DER) is a potent inhibitor of carcinogenesis, but chronic DER in human populations is difficult to sustain. Consequently, interest exists in identifying energy restriction mimetic agents (ERMAs), agents that provide the health benefits of DER without reducing caloric intake. The selection of a candidate ERMAs for this study was based on evidence that DER inhibits carcinogenesis by limiting glucose availability. The study objective was to determine if 2-deoxyglucose (2-DG), a glucose analogue that blocks its metabolism, would inhibit mammary carcinogenesis. Pilot studies were done to establish a dietary concentration of 2-DG that would not affect growth. For the carcinogenesis study, ninety 21-day-old female Sprague-Dawley rats were injected i.p. with 50 mg of 1-methyl-1-nitrosourea per kilogram of body weight. Following injection, animals were ad libitum fed AIN-93G diet containing 0.00%, 0.02%, or 0.03% (w/w) 2-DG for 5 weeks. 2-DG decreased the incidence and multiplicity of mammary carcinomas and prolonged cancer latency (P < 0.05). The 0.02% dose of 2-DG had no effect on circulating levels of glucose, insulin, insulin-like growth factor-I, IGF binding protein-3, leptin, or body weight gain. Using MCF-7 human breast cancer cells to investigate the signaling pathways perturbed by disruption of glucose metabolism, 2-DG reduced cell growth and intracellular ATP in a dose- and time-dependent manner (P < 0.01). Treatment with 2-DG increased levels of phosphorylated AMP-activated protein kinase and Sirt-1 and reduced phosphorylated Akt (P < 0.05). These studies support the hypothesis that DER inhibits carcinogenesis, in part, by limiting glucose availability and that energy metabolism is a target for the development of ERMA for chemoprevention.
A universal response to changes in cellular oxygen tension is governed by a family of heterodimeric transcription factors called hypoxia-inducible factor (HIF). Tumor hypoxia, as well as various cancer-causing mutations, has been shown to elevate the level of HIF-1alpha, signifying a critical role of the HIF pathway in cancer development. The recently identified third member of the human HIF-alpha family, HIF-3alpha, produces multiple splice variants that contain extra DNA binding elements and protein-protein interaction motifs not found in HIF-1alpha or HIF-2alpha. Here we report the molecular cloning of the alternatively spliced human HIF-3alpha variant HIF-3alpha4 and show that it attenuates the ability of HIF-1 to bind hypoxia-responsive elements located within the enhancer/promoter of HIF target genes. The overexpression of HIF-3alpha4 suppresses the transcriptional activity of HIF-1 and siRNA-mediated knockdown of the endogenous HIF-3alpha4 increases transcription by hypoxia-inducible genes. HIF-3alpha4 itself is oxygen-regulated, suggesting a novel feedback mechanism of controlling HIF-1 activity. Furthermore, the expression of HIF-3alpha4 is dramatically down-regulated in the majority of primary renal carcinomas. These results demonstrate an important dominant-negative regulation of HIF-1-mediated gene transcription by HIF-3alpha4 in vivo and underscore its potential significance in renal epithelial oncogenesis.
We previously demonstrated that insulin stimulates vascular endothelial growth factor (VEGF) synthesis and secretion via phosphatidylinositol-3 kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways in vascular smooth muscle cells (VSMC) from humans and from insulin-sensitive lean Zucker fa/+ rats. We also showed that this effect is attenuated in VSMC from insulin-resistant obese Zucker fa/fa rats. As it is not known whether the effects of insulin on VEGF involve activation of hypoxia-inducible factor-1 (HIF-1), we aimed to evaluate: (1) whether insulin modulates HIF-1alpha protein synthesis and activity; (2) the insulin signalling pathways involved; and (3) the role of insulin resistance.
Using aortic VSMC taken from humans and Zucker rats and cultured in normoxia, the following were evaluated: (1) dose-dependent (0.5, 1, 2 nmol/l) and time-dependent (2, 4, 6 h) effects exerted by insulin on HIF-1alpha content in both nucleus and cytosol, measured by Western blots; (2) insulin effects on HIF-1 DNA-binding activity on the VEGF gene, measured by electrophoretic mobility shift assay; and (3) involvement of the insulin signalling molecules in these insulin actions, by using the following inhibitors: LY294002 (PI3-K), PD98059 (extracellular signal regulated kinase [ERK]), SP600125 (Jun N terminal kinase [JNK]), SB203580 (p38 mitogen-activated protein kinase) and rapamycin (mammalian target of rapamycin), and by detecting the insulin signalling molecules by Western blots.
In aortic VSMC from humans and Zucker fa/+ rats cultured in normoxia insulin increases the HIF-1alpha content in cytosol and nucleus via dose- and time-dependent mechanisms, and HIF-1 DNA-binding activity on the VEGF gene. The insulin-induced increase of HIF-1alpha is blunted by the translation inhibitor cycloheximide, LY294002, PD98059, SP600125 and rapamycin, but not by SB203580. It is also reduced in Zucker fa/fa rats, which present an impaired ability of insulin to induce Akt, ERK-1/2 and JNK-1/2 phosphorylation.
These results provide a biological mechanism for the impaired collateral vessel formation in obesity.
Tumour cells emerge as a result of genetic alteration of signal circuitries promoting cell growth and survival, whereas their expansion relies on nutrient supply. Oxygen limitation is central in controlling neovascularization, glucose metabolism, survival and tumour spread. This pleiotropic action is orchestrated by hypoxia-inducible factor (HIF), which is a master transcriptional factor in nutrient stress signalling. Understanding the role of HIF in intracellular pH (pH(i)) regulation, metabolism, cell invasion, autophagy and cell death is crucial for developing novel anticancer therapies. There are new approaches to enforce necrotic cell death and tumour regression by targeting tumour metabolism and pH(i)-control systems.
The role of the hypoxia-inducible factor (HIF) subunits 1alpha and 1beta in cellular response to hypoxia is well established, whereas little is known about HIF-2alpha and HIF-3alpha with respect to organ distribution and transcriptional regulation by hypoxia. We investigated mRNA levels of all HIF subunits and of their target genes erythropoietin (EPO) and glucose-transporter 1 (GLUT1) in rats undergoing systemic hypoxia for 30 or 120 min by quantitative real-time RT-PCR. In normoxia, persistently high mRNA levels of all HIF subunits were detected in cerebral cortex, hippocampus, and lung; the heart contained the lowest amounts. Hypoxia did not affect mRNA levels of HIF-1alpha, -1beta, and -2alpha. HIF-3alpha mRNA levels increased in all organs examined after 2 h of hypoxia. A significant rise of EPO and GLUT1 mRNA levels occurred in cortex, heart, liver, and kidney after 2 h of hypoxia, indicating activation of the HIF system. Protein levels of all HIF subunits, determined in brain and lung by immunoblotting, showed a marked increase corresponding to the duration of hypoxia. Our results suggest that induction at the transcriptional level is a unique feature of HIF-3alpha, which therefore may represent a rapidly reacting component of the HIF system in protection against hypoxic damage.
—The time course of effects of 2-deoxy-d-glucose on cerebral glucose metabolism has been studied in vivo and the inhibitory actions of 2-deoxy-d-glucose and 2-deoxy-d-glucose-6-phosphate on cerebral glycolytic enzymes in vitro. Mice were given 2-deoxy-d-glucose 3 g/kg intraperitoneally. Blood 2-deoxy-d-glucose/glucose ratio was 2–3 from 5 to 30 min after injection, the hyperglycaemic response to 2-deoxy-d-glucose having been suppressed with propranolol. Maximal cerebral 2-deoxy-d-glucose uptake observed was 1μ11 μmol/g/min between 5 and 10 min after injection. At 10 min brain concentrations of 2-deoxy-d-glucose and 2-deoxy-d-glucose-6-phosphate were 5·82 and 3·12 μmol/g. Analysis of the fate of d-[U-¹⁴C] glucose given subcutaneously 5 min before death showed that glucose uptake was reduced to 40–60 per cent of control from 5 to 30 min after 2-deoxy-d-glucose. However brain glucose concentration rose three to five-fold 20–30 min after 2-deoxy-d-glucose. The majority of glucose entering the brain after 10 min of 2-deoxy-d-glucose treatment was recovered as glucose. Conversion of brain glucose to other acid soluble components was reduced to 1/3 at 10 min and 1/5 at 20–30 min. Glucose-6-phosphate concentration rose from 5 min onwards and was maintained at twice control concentration from 10–30 min. However, because of the rapid entry of 2-deoxy-d-glucose and its conversion to 2-deoxy-d-glucose-6-phosphate, the 2-deoxy-d-glucose 6-P/glucose 6-P ratio was between 19 and 32. Brain adenosine triphosphate concentration did not change, creatine phosphate concentration fell after 25 min.
Studies of gene regulation by oxygen have recently defined the existence of a widely operative system that responds to hypoxia but not mitochondrial inhibitors and involves the induction of a DNA-binding complex termed hypoxia-inducible factor 1. This system has been implicated in the regulation of erythropoietin, certain angiogenic growth factors, and particular glycolytic isoenzymes. The glucose transporter Glut-1 is induced by both hypoxia and mitochondrial inhibitors, implying the operation of a different mechanism of oxygen sensing. To explore that possibility, we analyzed the cisacting sequences that convey these responses. An enhancer lying 5' to the mouse Glut-1 gene was found to convey responses both to hypoxia and to the mitochondrial inhibitors, azide and rotenone. However, detailed analysis of this enhancer demonstrated that distinct elements responded to hypoxia and the mitochondrial inhibitors. The response to hypoxia was mediated by sequences that contained a functionally critical, although atypical, hypoxia-inducible factor 1 binding site, whereas sequences lying approximately 100 nucleotides 5' to this site, which contained a critical serum response element, conveyed responses to the mitochondrial inhibitors. Thus, rather than reflecting an entirely different mechanism of oxygen sensing, regulation of Glut-1 gene expression by hypoxia and mitochondrial inhibitors arises from the function of two different sensing systems. One of these responds to hypoxia alone and resembles that involved in erythropoietin regulation, while the other responds to mitochondrial inhibitors and involves activation of a serum response element.
Hypoxia inducible factors (HIFs) are heterodimeric transcription factors that regulate a number of adaptive responses to low oxygen tension. They are composed of alpha- and beta-subunits that belong to the basic helix-loop-helix-PAS superfamily. Here we examined the expression of HIF-alpha subunit proteins in the human kidney and found that, in addition to HIF-1alpha and -2alpha, HIF-3alpha was also expressed. The sequence analysis revealed that, like mouse HIF-3alpha, human HIF-3alpha has high similarity with HIF-1alpha and -2alpha in the bHLH and PAS domains, but lacks structures for transactivation found in the C-terminus of HIF-1alpha and -2alpha. Furthermore, we performed reporter gene analysis and showed that HIF-3alpha suppresses hypoxia-inducible HIF-mediated gene expression. HIF-3alpha might be a negative regulator of hypoxia-inducible gene expression in the human kidney.
Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.
Endothelial PAS domain protein 1 (EPAS1) has been identified as a member of the basic helix-loop-helix (bHLH)-PAS protein family, and plays a critical role in the regulation of hypoxia inducible genes. It remains unknown whether physiological stimuli other than hypoxia modulate EPAS1 expression. This study examined the inducible expression of EPAS1 by various cytokines and growth factors, and determined the target gene for EPAS1 in cardiac myocytes. In cultured cardiac myocytes, interleukin-1beta (IL-1beta) but not tumor necrosis factor alpha markedly increased the EPAS1 mRNA and protein levels in a time- and dose-dependent manner, whereas hypoxia increases the expression of EPAS1 protein but not its mRNA. Such an induction of EPAS1 by IL-1beta was efficiently inhibited by the pretreatment of the cells with Src kinase inhibitors, such as herbimycin A and PP1. The expression of adrenomedullin (AM) mRNA, which is also upregulated by IL-1beta, was dramatically increased in cardiac myocytes transduced with adenovirus expressing EPAS1. Transient transfection assays using the site-specific mutation of the AM promoter showed that EPAS1 overexpression increases the transcriptional activity through a sequence similar to the consensus HRE (hypoxia responsive element). These results suggest that IL-1beta induces the EPAS1 at the transcriptional level, which in turn activates the AM gene. Since IL-1beta has been implicated in the pathogenesis of heart failure and AM can ameliorate the cardiac function, our results suggest that EPAS1 plays a role in the adaptation of the cardiac myocytes during heart failure as well as in the regulation of gene expression by hypoxia.
K. Maemura and R. Nagai. Novel Insight into the Role of Endothelial PAS Domain Protein 1 in Congestive Heart Failure. Journal of Molecular and Cellular Cardiology (2002) 34, 703–707.
Iron chelation by deferoxamine (DFO) blocks the Fenton reaction, but also inhibits prolyl hydroxylases and thereby activates certain hypoxia-inducible transcription factors (HIFs) that trigger cellular adaptation to hypoxia. Because both mechanisms may alleviate tissue damage in ischemia and reperfusion, we tried to differentiate their contribution to DFO-induced cardioprotection. Myocardial ischemia and reperfusion were induced in anesthetized Wistar rats. Infarct size was related to the ischemic area. Myocardial mRNA expression was determined by real-time PCR. Radical reactivity was probed in myocardial tissue slices with the redox-sensitive dye CM-H(2)DCFDA. Single ip applications of DFO (200 mg/kg) administered 2 h to 3 days before infarction reduced infarct size from 55 +/- 7% to 22-26%. Protection was abolished by the radical scavenger N-(2-mercaptopropionyl)glycine and the protein kinase C inhibitor chelerythrine when either was given 30 min before DFO, whereas subsequent application was ineffective. DFO did not alter the expression of various HIF target genes, whereas mRNAs of HIF-independent genes, aldose reductase and glucose transporter-4, were increased in infarcted myocardium 2 days after DFO treatment. Enhancement of superoxide activity by DFO could be demonstrated in vitro. Acute and prolonged myocardial preconditioning is triggered by DFO in response to accumulation of oxygen radicals and activation of protein kinase C.
Early kidney development is associated with the coordinated branching of the renal tubular and vascular system and hypoxia has been proposed to be a major regulatory factor in this process. Under low oxygen levels, the hypoxia-inducible transcription factor (HIF) regulates the expression of genes involved in angiogenesis, erythropoiesis and glycolysis. To investigate the role of HIF in kidney development, we analyzed the temporal and spatial expression of the oxygen regulated HIF-1alpha and -2alpha subunits at different stages of rat and human kidney development. Using double-staining procedures, localization of the HIF target geneproducts vascular endothelial growth factor (VEGF) and endoglin was studied in relation to HIFalpha. In both species, we found marked nuclear expression of HIF-1alpha in medullary and cortical collecting ducts and in glomerular cells. In contrast, HIF-2alpha was expressed in interstitial and peritubular cells podocytes of the more mature glomeruli. After completion of glomerulogenesis and nephrogenesis, HIF-1alpha and -2alpha were no longer detectable. The HIF-target gene VEGF colocalized with HIF-1alpha protein in glomeruli and medullary collecting ducts. HIF-2alpha colocalized with the endothelium-associated angiogenic factor, endoglin. Both HIFalpha isoforms are activated in the developing kidney in a cell-specific and temporally controlled manner, indicating a regulatory role of oxygen tension in nephrogenesis. HIF-1alpha seems to be primarily involved in tubulogenesis and HIF-2alpha in renal vasculogenesis. Both isoforms are found in glomerulogenesis, potentially having synergistic effects.
- S B Catrina
- K Okamoto
- T Pereira
- K Brismar
- L Poellinger
S.B. Catrina, K. Okamoto, T. Pereira, K. Brismar, L. Poellinger,
Hyperglycemia regulates hypoxia-inducible factor-1a protein stability
and function, Diabetes 53 (2004) 3226-3232.
Hypoxia signalling in cancer and approaches to enforce tumour regression
- Pouyssegur