RESUMEN
AIMS/HYPOTHESIS: Hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) enzymes regulate adaptive cellular responses to low oxygen concentrations. Inhibition of HIF-P4Hs leads to stabilisation of hypoxia-inducible factors (HIFs) and activation of the HIF pathway affecting multiple biological processes to rescue cells from hypoxia. As evidence from animal models suggests that HIF-P4H inhibitors could be used to treat metabolic disorders associated with insulin resistance, we examined whether roxadustat, an HIF-P4H inhibitor approved for the treatment of renal anaemia, would have an effect on glucose metabolism in primary human myotubes. METHODS: Primary skeletal muscle cell cultures, established from biopsies of vastus lateralis muscle from men with normal glucose tolerance (NGT) (n=5) or type 2 diabetes (n=8), were treated with roxadustat. Induction of HIF target gene expression was detected with quantitative real-time PCR. Glucose uptake and glycogen synthesis were investigated with radioactive tracers. Glycolysis and mitochondrial respiration rates were measured with a Seahorse analyser. RESULTS: Exposure to roxadustat stabilised nuclear HIF1α protein expression in human myotubes. Treatment with roxadustat led to induction of HIF target gene mRNAs for GLUT1 (also known as SLC2A1), HK2, MCT4 (also known as SLC16A4) and HIF-P4H-2 (also known as PHD2 or EGLN1) in myotubes from donors with NGT, with a blunted response in myotubes from donors with type 2 diabetes. mRNAs for LDHA, PDK1 and GBE1 were induced to a similar degree in myotubes from donors with NGT or type 2 diabetes. Exposure of myotubes to roxadustat led to a 1.4-fold increase in glycolytic rate in myotubes from men with NGT (p=0.0370) and a 1.7-fold increase in myotubes from donors with type 2 diabetes (p=0.0044), with no difference between the groups (p=0.1391). Exposure to roxadustat led to a reduction in basal mitochondrial respiration in both groups (p<0.01). Basal glucose uptake rates were similar in myotubes from donors with NGT (20.2 ± 2.7 pmol mg-1 min-1) and type 2 diabetes (25.3 ± 4.4 pmol mg-1 min-1, p=0.4205). Treatment with roxadustat enhanced insulin-stimulated glucose uptake in myotubes from donors with NGT (1.4-fold vs insulin-only condition, p=0.0023). The basal rate of glucose incorporation into glycogen was lower in myotubes from donors with NGT (233 ± 12.4 nmol g-1 h-1) than in myotubes from donors with type 2 diabetes (360 ± 40.3 nmol g-1 h-1, p=0.0344). Insulin increased glycogen synthesis by 1.9-fold (p=0.0025) in myotubes from donors with NGT, whereas roxadustat did not affect their basal or insulin-stimulated glycogen synthesis. Insulin increased glycogen synthesis by 1.7-fold (p=0.0031) in myotubes from donors with type 2 diabetes. While basal glycogen synthesis was unaffected by roxadustat, pretreatment with roxadustat enhanced insulin-stimulated glycogen synthesis in myotubes from donors with type 2 diabetes (p=0.0345). CONCLUSIONS/INTERPRETATION: Roxadustat increases glycolysis and inhibits mitochondrial respiration in primary human myotubes regardless of diabetes status. Roxadustat may also improve insulin action on glycogen synthesis in myotubes from donors with type 2 diabetes.
RESUMEN
Transmembrane prolyl 4-hydroxylase (P4H-TM) is an enigmatic enzyme whose cellular function and primary substrate remain to be identified. Its loss-of-function mutations cause a severe neurological HIDEA syndrome with hypotonia, intellectual disability, dysautonomia and hypoventilation. Previously, P4H-TM deficiency in mice was associated with reduced atherogenesis and lower serum triglyceride levels. Here, we characterized the glucose and lipid metabolism of P4h-tm-/- mice in physiological and tissue analyses. P4h-tm-/- mice showed variations in 24-h oscillations of energy expenditure, VO2 and VCO2 and locomotor activity compared to wild-type (WT) mice. Their rearing activity was reduced, and they showed significant muscle weakness and compromised coordination. Sedated P4h-tm-/- mice had better glucose tolerance, lower fasting insulin levels, higher fasting lactate levels and lower fasting free fatty acid levels compared to WT. These alterations were not present in conscious P4h-tm-/- mice. Fasted P4h-tm-/- mice presented with faster hepatic glycogenolysis. The respiratory rate of conscious P4h-tm-/- mice was significantly lower compared to the WT, the decrease being further exacerbated by sedation and associated with acidosis and a reduced ventilatory response to both hypoxia and hypercapnia. P4H-TM deficiency in mice is associated with alterations in whole-body energy metabolism, day-night rhythm of activity, glucose homeostasis and neuromuscular and respiratory functions. Although the underlying mechanism(s) are not yet fully understood, the phenotype appears to have neurological origins, controlled by brain and central nervous system circuits. The phenotype of P4h-tm-/- mice recapitulates some of the symptoms of HIDEA patients, making this mouse model a valuable tool to study and develop tailored therapies.
RESUMEN
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.