RESUMO
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.
RESUMO
As life expectancy increases, the effectiveness of cervical cancer screening programs needs to be reassessed for the older population. We addressed the effect of test history in and outside organized screening at age 50-64 years on later cervical cancer risk. A case-control study was conducted by deriving 229 cases of 65-79 years old women with invasive cervical cancer in 2010-2019 from the Finnish Cancer Registry. Ten controls were matched for each case by birth year and hospital district. The effect of test uptake and abnormal results in 50-64 year olds on cancer risk was investigated using conditional logistic regression and adjusted for self-selection. Test uptake within the 50-64 years age group showed 75% lower odds of cervical cancer [adjusted OR (aOR) = 0.25; 95% confidence interval (95% CI), 0.18-0.35]. Untested women had 4.9 times higher odds than those tested with normal results (aOR = 4.86; 95% CI, 3.42-6.92). Having at least one abnormal test result increased the odds by 2.5 when compared with only normal results but showed lower odds when compared with untested women. The importance of testing is exhibited by the result showing a reduction of odds of cancer to one-fourth for those tested compared with untested. Similarly, receiving abnormal results was protective of cancer compared with having no tests highlighting the importance of proper follow-up. Therefore, screening history should be considered when further developing cervical cancer screening programs with special interest in non-attenders and those receiving abnormal results at older ages. Significance: To our knowledge, this is the first study from Finnish data describing the effect of test history on later cervical cancer at older ages. Focusing on the cervical tests taken within the Finnish national screening program and outside it highlights the overall importance of having cervical tests and adds this study into the slowly increasing number of studies considering all cervical testing in Finland.