Activation of the hypoxia-inducible factor pathway by roxadustat improves glucose metabolism in human primary myotubes from men

Abstract

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.

Keywords: Glucose metabolism; Hypoxia-inducible factor; Insulin resistance; Insulin signalling; Primary human muscle cells; Roxadustat.

Fig. 1

Nuclear HIF1α protein stabilisation by roxadustat in human myotubes. Primary human myotubes from men with NGT (n=4) were exposed to 10 μmol/l roxadustat or 0.1% DMSO as control for 24 h. Protein expression was analysed by western blotting. The quantified intensity of nuclear HIF1α protein was normalised to β-actin. Data are expressed as mean ± SEM. *p<0.05, analysed by paired one-tailed Student’s t test

Fig. 2

HIF target gene mRNA expression in roxadustat-treated myotubes. Primary human myotubes from men with NGT (n=5) or type 2 diabetes (n=5, consisting of three men with a low insulin response and two men with a robust insulin response in glycogen synthesis assays) were exposed to 10 μmol/l roxadustat or 0.1% DMSO as control for 24 h. qPCR was used to detect the induction of HIF-responsive genes. Values were normalised to the control sample for each individual. T2D, type 2 diabetes. Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 for roxadustat vs control; ^†p<0.05, ^†††p<0.001 for type 2 diabetes vs NGT; analysed by two-way ANOVA with repeated measurements, with Holm–Šídák’s post hoc test

Fig. 3

The effect of roxadustat on glucose metabolism in human myotubes. Primary human myotubes were exposed to 10 μmol/l roxadustat or 0.1% DMSO as control for 24 h, followed by metabolic tests with or without stimulation with 100 nmol/l insulin. (a) Basal and insulin-stimulated glucose uptake rate (in pmol mg⁻¹ min⁻¹) was determined in myotubes from men with NGT (n=5) or type 2 diabetes (n=8). (b) Basal glycolytic rate (in pmol min⁻¹ μg⁻¹) was determined in myotubes from men with NGT (n=5) or type 2 diabetes (n=5, consisting of three men with a low insulin response and two men with a robust insulin response in glycogen synthesis assays). (c) Basal and insulin-stimulated rates of glucose incorporation into glycogen (in nmol g⁻¹ h⁻¹) were determined in myotubes from men with NGT (n=5) or type 2 diabetes (n=8). (d) Basal and insulin-stimulated rates of glucose incorporation into glycogen (in nmol g⁻¹ h⁻¹) were determined in myotubes from men with NGT (n=5) and in a subgroup of myotubes that were more insulin-resistant in vitro, obtained from men with type 2 diabetes (n=5). Values were normalised to the basal (a, c, d) or control (b) sample of each participant. Ins, insulin; Roxa, roxadustat; T2D, type 2 diabetes. Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 for roxadustat, a combination of roxadustat and insulin or insulin only vs respective basal, control or insulin only; ^†p<0.05, ^††p<0.01 for type 2 diabetes vs NGT; analysed by two-way ANOVA with repeated measurements, with Holm–Šídák’s post hoc test

Fig. 4

Mitochondrial respiration in roxadustat-treated myotubes. Primary human myotubes from men with NGT (n=5) or type 2 diabetes (n=5, consisting of three men with a low insulin response and two with a robust insulin response in glycogen synthesis assays) were exposed to 10 μmol/l roxadustat or 0.1% DMSO as control for 24 h. (a–c) Basal respiration (a), maximal respiration (b) and ATP-linked respiration (c) (in pmol min⁻¹ μg⁻¹) were analysed. Values were normalised to the control sample for each participant. T2D, type 2 diabetes. Data are expressed as mean ± SEM. **p<0.01, ***p<0.001 for roxadustat vs control; ^†p<0.05, ^††p<0.01 for type 2 diabetes vs NGT; analysed by two-way ANOVA with repeated measurements, with Holm–Šídák’s post hoc test

Fig. 5

Analysis of activation of the insulin signalling pathway in roxadustat-treated myotubes. Primary human myotubes from men with NGT (n=5) or with type 2 diabetes (n=4, consisting of two men with a low insulin response and two men with a robust insulin response in glycogen synthesis assays) were pretreated with 10 μmol/l roxadustat or 0.1% DMSO as control for 24 h, followed by 10 min stimulation with 100 nmol/l insulin. Insulin signalling was analysed by western blotting. (a–c) Quantification of p-Akt-Ser⁴⁷³ (a), p-AS160-Thr⁶⁴² (b) and p-GSK3β-Ser⁹ (c) was normalised to the intensity of their corresponding total proteins. (d) Representative images of the western blots. A.U., arbitrary units; Ins, insulin; Roxa, roxadustat; T2D, type 2 diabetes. Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 vs stimulation with insulin only, analysed by two-way ANOVA with repeated measurements, with Holm–Šídák’s post hoc test