Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells.

Hypoxia-inducible factor 1α (HIF1α) is a master regulator of numerous biological processes under low oxygen tensions. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in primary cells remain elusive. Here, we show that HIF1α signaling is activated in several human primary vascular cells under ambient oxygen tensions, and in vascular smooth muscle cells (VSMCs) of normal human lung tissue, which contributed to a relative resistance to further enhancement of glycolytic activity in hypoxia. Mechanistically, aerobic HIFα activation is mediated by paracrine secretion of three branched chain α-ketoacids (BCKAs), which suppress prolyl hydroxylase domain-containing protein 2 (PHD2) activity via direct inhibition and via lactate dehydrogenase A (LDHA)-mediated generation of L-2-hydroxyglutarate (L2HG). Metabolic dysfunction induced by BCKAs was observed in the lungs of rats with pulmonary arterial hypertension (PAH) and in pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH patients. BCKA supplementation stimulated glycolytic activity and promoted a phenotypic switch to the synthetic phenotype in PASMCs of normal and PAH subjects. In summary, we identify BCKAs as novel signaling metabolites that activate HIF1α signaling in normoxia and that the BCKA-HIF1α pathway modulates VSMC function and may be relevant to pulmonary vascular pathobiology.

Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins.

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD-HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD-HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [14C]O2 release from labeled [14C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.

PHD3 Loss Promotes Exercise Capacity and Fat Oxidation in Skeletal Muscle.

Rapid alterations in cellular metabolism allow tissues to maintain homeostasis during changes in energy availability. The central metabolic regulator acetyl-CoA carboxylase 2 (ACC2) is robustly phosphorylated during cellular energy stress by AMP-activated protein kinase (AMPK) to relieve its suppression of fat oxidation. While ACC2 can also be hydroxylated by prolyl hydroxylase 3 (PHD3), the physiological consequence thereof is poorly understood. We find that ACC2 phosphorylation and hydroxylation occur in an inverse fashion. ACC2 hydroxylation occurs in conditions of high energy and represses fatty acid oxidation. PHD3-null mice demonstrate loss of ACC2 hydroxylation in heart and skeletal muscle and display elevated fatty acid oxidation. Whole body or skeletal muscle-specific PHD3 loss enhances exercise capacity during an endurance exercise challenge. In sum, these data identify an unexpected link between AMPK and PHD3, and a role for PHD3 in acute exercise endurance capacity and skeletal muscle metabolism.

Live offspring from mice lacking the Y chromosome long arm gene complement.

The mouse Y chromosome long arm (Yq) comprises approximately 70 Mb of repetitive, male-specific DNA together with a short (0.7-Mb) pseudoautosomal region (PAR). The repetitive non-PAR region (NPYq) encodes genes whose deficiency leads to subfertility and infertility, resulting from impaired spermiogenesis. In XSxr(a)Y*(X) mice, the only Y-specific material is provided by the Y chromosome short arm-derived sex reversal factor Sxr(a), which is attached to the X chromosome PAR; these males (NPYq- males) produce sperm with severely malformed heads and are infertile. In the present study, we investigated sperm function in these mice in the context of intracytoplasmic sperm injection (ICSI). Of 261 oocytes injected, 103 reached the 2-cell stage, and 46 developed to liveborn offspring. Using Xist RT-PCR genotyping as well as gamete and somatic cell karyotyping, all six predicted genotypes were identified among ICSI-derived progeny. The sex chromosome constitution of NPYq- males does not allow production of offspring with the same genotype, but one of the expected offspring genotypes is XY*(X)Sxr(a) (NPYq-(2)), which has the same Y gene complement as NPYq-. Analysis of NPYq-(2) males revealed they had normal-sized testes with ongoing spermatogenesis. Like NPYq- males, these males were infertile, and their sperm had malformed heads that nevertheless fertilized eggs via ICSI. In vitro fertilization (IVF), however, was unsuccessful. Overall, we demonstrated that a lack of NPYq-encoded genes does not interfere with the ability of sperm to fertilize oocytes via ICSI but does prevent fertilization via IVF. Thus, NPYq-encoded gene functions are not required after the sperm have entered the oocyte. The present work also led to development of a new mouse model lacking NPYq gene complement that will facilitate future studies of Y-encoded gene function.