Hypoxia-inducible factor prolyl 4-hydroxylases and metabolism

Abstract Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs, also known as PHDs or EglNs) are enzymes that act as cellular oxygen sensors. Inhibition of HIF-P4Hs leads to stabilization of hypoxia-inducible transcription factors (HIFs), which initiates a gene expression program that allows organisms to cope with low oxygen levels and restore tissue oxygenation. This involves, for example, upregulation of erythropoiesis and angiogenesis, modulation of inflammatory responses, and reprogramming of metabolism. Currently, several pharmacological HIF-P4H inhibitors are in clinical trials mainly for renal anemia. However, recent data suggest that HIF-P4H inhibitors could also be considered to treat metabolic disorders. Here, we discuss the potential of targeting HIF-P4Hs and the HIF pathway for the treatment of obesity, metabolic syndrome, atherosclerosis, and fatty liver diseases (FLDs)

HER2 regulates cancer stem-like cell phenotype in ALK translocated NSCLC

Abstract We have previously shown that cancer stem-like cells (CSLCs) can mediate therapy resistance in ALK translocated lung cancers. HER2 has been linked to CSLCs in breast cancers and, therefore, we wanted to assess whether HER2 has a role in CSLCs in ALK translocated cancers. ALK translocated cell lines, H3122 and H2228, with variable sensitivity to ALK inhibition were used in the study. HER2 overexpression or knockdown was induced by retro- or lentiviral infections and cells were treated with pharmacological agents targeting HER2 and ALK signaling. Furthermore, tumorigenic properties of the cells were assessed in vitro using colony and sphere formation assays. In the ALK inhibitor sensitive H3122 cells, HER2 overexpression unaltered the primary response to ALK inhibition, but increased CSLC marker expression and enhanced colony and sphere formation and late AKT and ERK1/2 signaling recovery. In the ALK inhibitor semi-sensitive H2228 cells, HER2 knockdown reduced basal expression of CSLC markers, modestly increased sensitivity to ALK inhibition in colony and sphere formation assays, and reduced late AKT and ERK1/2 signaling recovery. In addition, HER2 induced cross activation of other ErbB-members of which HER3 followed most closely the CSLC marker expression and neuregulin-1, a HER3 ligand, or pan-ErbB inhibitor afatinib, were able to alter CSLC marker expression and colony formation. the present study suggests that HER2 has an important role in the regulation of the CSLC phenotype in ALK translocated lung cancers that is mainly orchestrated by HER2/HER3 heterodimers

2-Oxoglutarate-dependent dioxygenases in cancer

Abstract 2-Oxoglutarate-dependent dioxygenases (2OGDDs) are a superfamily of enzymes that play diverse roles in many biological processes, including regulation of hypoxia-inducible factor-mediated adaptation to hypoxia, extracellular matrix formation, epigenetic regulation of gene transcription and the reprogramming of cellular metabolism. 2OGDDs all require oxygen, reduced iron and 2-oxoglutarate (also known as α-ketoglutarate) to function, although their affinities for each of these co-substrates, and hence their sensitivity to depletion of specific co-substrates, varies widely. Numerous 2OGDDs are recurrently dysregulated in cancer. Moreover, cancer-specific metabolic changes, such as those that occur subsequent to mutations in the genes encoding succinate dehydrogenase, fumarate hydratase or isocitrate dehydrogenase, can dysregulate specific 2OGDDs. This latter observation suggests that the role of 2OGDDs in cancer extends beyond cancers that harbour mutations in the genes encoding members of the 2OGDD superfamily. Herein, we review the regulation of 2OGDDs in normal cells and how that regulation is corrupted in cancer

Cancer-associated 2-oxoglutarate analogues modify histone methylation by inhibiting histone lysine demethylases

Abstract Histone lysine demethylases (KDMs) are 2-oxoglutarate-dependent dioxygenases (2-OGDDs) that regulate gene expression by altering chromatin structure. Their dysregulation has been associated with many cancers. We set out to study the catalytic and inhibitory properties of human KDM4A, KDM4B, KDM5B, KDM6A and KDM6B, aiming in particular to reveal which of these enzymes are targeted by cancer-associated 2-oxoglutarate (2-OG) analogues. We used affinity-purified insect cell-produced enzymes and synthetic peptides with trimethylated lysines as substrates for the in vitro enzyme activity assays. In addition, we treated breast cancer cell lines with cell-permeable forms of 2-OG analogues and studied their effects on the global histone methylation state. Our data show that KDMs have substrate specificity. Among the enzymes studied, KDM5B had the highest affinity for the peptide substrate but the lowest affinity for the 2-OG and the Fe2+ cosubstrate/cofactors. R-2-hydroxyglutarate (R-2HG) was the most efficient inhibitor of KDM6A, KDM4A and KDM4B, followed by S-2HG. This finding was supported by accumulations of the histone H3K9me3 and H3K27me3 marks in cells treated with the cell-permeable forms of these compounds. KDM5B was especially resistant to inhibition by R-2HG, while citrate was the most efficient inhibitor of KDM6B. We conclude that KDM catalytic activity is susceptible to inhibition by tumorigenic 2-OG analogues and suggest that the inhibition of KDMs is involved in the disease mechanism of cancers in which these compounds accumulate, such as the isocitrate dehydrogenase mutations

Higher hemoglobin levels are an independent risk factor for adverse metabolism and higher mortality in a 20-year follow-up

Abstract The aim of this study was to cross-sectionally and longitudinally examine whether higher hemoglobin (Hb) levels within the normal variation associate with key components of metabolic syndrome and total and cardiovascular mortality. The study included 967 Finnish subjects (age 40–59 years) followed for ≥ 20 years. The focus was on Hb levels, cardiovascular diseases (CVDs) and mortality rates. Higher Hb levels associated positively with key anthropometric and metabolic parameters at baseline. At the follow-up similar associations were seen in men. The highest Hb quartile showed higher leptin levels and lower adiponectin levels at baseline and follow-up (p < 0.05) and lower plasma ghrelin levels at baseline (p < 0.05). Higher baseline Hb levels associated independently with prevalence of type 2 diabetes at follow-up (p < 0.01). The highest Hb quartile associated with higher serum alanine aminotransferase levels (p < 0.001) and independently with increased risk for liver fat accumulation (OR 1.63 [1.03; 2.57]) at baseline. The highest Hb quartile showed increased risk for total (HR = 1.48 [1.01; 2.16]) and CVD-related mortality (HR = 2.08 [1.01; 4.29]). Higher Hb levels associated with an adverse metabolic profile, increased prevalence of key components of metabolic syndrome and higher risk for CVD-related and total mortality

Human phytanoyl-CoA dioxygenase domain-containing 1 (PHYHD1) is a putative oxygen sensor associated with RNA and carbohydrate metabolism

Abstract Human phytanoyl-CoA dioxygenase domain-containing 1 (PHYHD1) is a 2-oxoglutarate (2OG)-dependent dioxygenase implicated in Alzheimer’s disease, some cancers, and immune cell functions. The substrate, kinetic and inhibitory properties, function and subcellular localization of PHYHD1 are unknown. We used recombinant expression and enzymatic, biochemical, biophysical, cellular and microscopic assays for their determination. The apparent Km values of PHYHD1 for 2OG, Fe²⁺ and O₂ were 27, 6 and > 200 μm, respectively. PHYHD1 activity was tested in the presence of 2OG analogues, and it was found to be inhibited by succinate and fumarate but not R-2-hydroxyglutarate, whereas citrate acted as an allosteric activator. PHYHD1 bound mRNA, but its catalytic activity was inhibited upon interaction. PHYHD1 was found both in the nucleus and cytoplasm. Interactome analyses linked PHYHD1 to cell division and RNA metabolism, while phenotype analyses linked it to carbohydrate metabolism. Thus, PHYHD1 is a potential novel oxygen sensor regulated by mRNA and citrate

Hypoxia ameliorates maternal diet-induced insulin resistance during pregnancy while having a detrimental effect on the placenta

Abstract Maternal overweight/obesity contributes significantly to the development of gestational diabetes, which causes risks to both mother and fetus and is increasing sharply in prevalence worldwide. Since hypoxia reprograms energy metabolism and can alleviate weight gain, adiposity, insulin resistance (IR), and dyslipidemia, we set out to study the potential of sustained reduced ambient oxygen tension (15% O2) during pregnancy for alleviating the detrimental effects of diet-induced IR in C57Bl/6N mice, taking normal chow-fed and normoxia (21% O2) groups as controls. Our data show that hypoxic intervention reduced maternal weight gain, adiposity, and adipose tissue inflammation, and ameliorated maternal glucose metabolism and IR during gestation in diet-induced IR relative to normoxia. Where diet-induced IR reduced maternal hemoglobin and increased serum erythropoietin levels, hypoxic intervention compensated for these changes. Diet-induced IR reduced fetal growth in normoxia, and even more in hypoxia. Hypoxic intervention reduced liver weight gain during pregnancy in the dams with diet-induced IR, maternal liver weight being positively associated with embryo number. In case of diet-induced IR, the hypoxic intervention compromised placental energy metabolism and vascularization and increased end-pregnancy placental necrosis. Altogether, these data show that although hypoxic intervention mediates several beneficial effects on maternal metabolism, the combination of it with diet-induced IR is even more detrimental to the placental and fetal outcome than diet-induced IR alone

Hypoxia causes reductions in birth weight by altering maternal glucose and lipid metabolism

Abstract Hypoxia of residence at high altitude (>2500 m) decreases birth weight. Lower birth weight associates with infant mortality and morbidity and increased susceptibility to later-in-life cardiovascular and metabolic diseases. We sought to determine the effects of hypoxia on maternal glucose and lipid metabolism and their contributions to fetal weight. C57BL6/NCrl mice, housed throughout gestation in normobaric hypoxia (15% oxygen) or normoxia, were studied at mid (E9.5) or late gestation (E17.5). Fetal weight at E17.5 was 7% lower under hypoxia than normoxia. The hypoxic compared with normoxic dams had ~20% less gonadal white adipose tissue at mid and late gestation. The hypoxic dams had better glucose tolerance and insulin sensitivity compared with normoxic dams and failed to develop insulin resistance in late gestation. They also had increased glucagon levels. Glucose uptake to most maternal tissues was ~2-fold greater in the hypoxic than normoxic dams. The alterations in maternal metabolism in hypoxia were associated with upregulation of hypoxia-inducible factor (HIF) target genes that serve, in turn, to increase glycolytic metabolism. We conclude that environmental hypoxia alters maternal metabolism by upregulating the HIF-pathway, and suggest that interventions that antagonize such changes in metabolism in high-altitude pregnancy may be helpful for preserving fetal growth

Expression and roles of individual HIF prolyl 4-hydroxylase isoenzymes in the regulation of the hypoxia response pathway along the murine gastrointestinal epithelium

Abstract The HIF prolyl 4-hydroxylases (HIF-P4H) control hypoxia-inducible factor (HIF), a powerful mechanism regulating cellular adaptation to decreased oxygenation. The gastrointestinal epithelium subsists in “physiological hypoxia” and should therefore have an especially well-designed control over this adaptation. Thus, we assessed the absolute mRNA expression levels of the HIF pathway components, Hif1a, HIF2a, Hif-p4h-1, 2 and 3 and factor inhibiting HIF (Fih1) in murine jejunum, caecum and colon epithelium using droplet digital PCR. We found a higher expression of all these genes towards the distal end of the gastrointestinal tract. We detected mRNA for Hif-p4h-1, 2 and 3 in all parts of the gastrointestinal tract. Hif-p4h-2 had significantly higher expression levels compared to Hif-p4h-1 and 3 in colon and caecum epithelium. To test the roles each HIF-P4H isoform plays in the gut epithelium, we measured the gene expression of classical HIF target genes in Hif-p4h-1−/−, Hif-p4h-2 hypomorph and Hif-p4h-3−/− mice. Only Hif-p4h-2 hypomorphism led to an upregulation of HIF target genes, confirming a predominant role of HIF-P4H-2. However, the abundance of Hif-p4h-1 and 3 expression in the gastrointestinal epithelium implies that these isoforms may have specific functions as well. Thus, the development of selective inhibitors might be useful for diverging therapeutic needs

Genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) reduces atherosclerotic plaques in mice

Abstract Objective: Atherosclerosis is a key component of cardiovascular diseases. We set out to study here whether genetic ablation of P4H-TM (transmembrane prolyl 4-hydroxylase) could protect against atherosclerosis as does inhibition of the other 3 classical HIF-P4Hs (hypoxia-inducible factor prolyl 4-hydroxylases). Approach and Results: We generated a double knockout mouse line deficient in P4H-TM and LDL (low-density lipoprotein) receptor (P4h-tm−/−/Ldlr−/−) and subjected these mice to a high-fat diet for 13 weeks. The double knockout mice had less atherosclerotic plaques in their full-length aorta than their P4h-tm+/+/Ldlr−/− counterparts and also had lower serum triglyceride levels on standard laboratory diet and high-fat diet, higher levels of IgM autoantibodies against Ox-LDL (oxidized LDL), and significantly higher lipoprotein lipase protein levels in white adipose tissue and sera. RNA-sequencing analysis revealed changes in expression of mRNAs in multiple pathways including lipid metabolism and immunologic response in the P4h-tm−/−/Ldlr−/− livers as compared with P4h-tm+/+/Ldlr−/−. Conclusions: Our data identify P4H-TM inhibition as a potential novel immuno-metabolic mechanism for intervening in the pathology of atherosclerosis, as hypertriglyceridemia is an individual risk factor for atherosclerosis, and IgM antibodies to Ox-LDL and increased lipoprotein lipase have been associated with protection against it