DOI: 10.25345/c59g5gs3k
title: MassIVE MSV000098274 - IRP1 deficiency triggers metabolic reprograming, increases insulin sensitivity and protects mice against metabolic syndrome pathologies
title: Iron regulatory protein 1 (IRP1) is a post-transcriptional regulator of cellular iron metabolism. Irp1-/- mice are viable but develop polycythemia. We report that these animals also exhibit fasting hypoglycemia and are protected against high fat diet-induced hyperglycemia and liver steatosis. Mechanistically, proteomics analysis revealed mitochondrial dysfunction in Irp1-/-livers. Seahorse assays validated impaired respiratory capacity in primary hepatocytes but also differentiated myotubes from Irp1-/- mice, which caused a switch from oxidative phosphorylation to glycolytic metabolism for energy production. This was accompanied by increased insulin sensitivity and glucose uptake by skeletal muscles. However, under conditions of metabolic stress or iron restriction, IRP1 deficiency prevented appropriate mitochondrial iron supply. Mitochondrial dysfunction and inefficient energy production caused metabolic rewiring and accumulation of metabolites in skeletal muscles of Irp1-/- mice, making them unavailable for gluconeogenesis in the liver. Thus, IRP1 emerges as an important metabolic regulator. LC-MS based lipidomics The protein-precipitation liquid extraction protocol described previously (Jenkins et al., 2020). Briefly, to each sample (25 uL of serum or ~25 mg of liver tissue) we added 400 uL of chloroform to each sample, along with a single 5 mm stainless steel ball bearing. The samples were then homogenised using a Bioprep 24-1004 homogenizer (Allsheng, Hangzhou, China) run at speed; 4.5 m/s, time; 30 s for 2 cycles. Following this, 250 uL of chloroform, 250 uL of methanol and 100 uL of the LIPID+CARNITINE internal standard (1-10 uM in methanol), and 400 uL of acetone was added to each sample. The samples were vortexed thoroughly. The samples were then centrifuged for 10 minutes at ~20,000 g to pellet any insoluble material. The supernatant was pipetted into separate 2 mL screw cap amber-glass auto-sampler vials (Agilent Technologies, Cheadle, United Kingdom). The extracts were dried down to dryness using a Concentrator Plus system (Eppendorf, Stevenage, United Kingdom) run for 60 minutes at 60 degree Celsius. For lipidomics LC-MS analysis, the samples were reconstituted in 100 uL of 2: 1: 1 (propan-2-ol, acetonitrile and water, respectively) then thoroughly vortexed. The reconstituted sample was transferred into a 250 uL low-volume vial insert inside a 2 mL amber glass auto-sample vial ready for liquid chromatography with mass spectrometry detection (LC-MS) lipidomics analysis. Full chromatographic separation of intact lipids was achieved using Waters Acquity H-Class HPLC System (Waters, Hertfordshire, United Kingdom) with the injection of 10 uL onto a Waters Acquity Premier UPLC CSH C18 column; 1.7 um, I.D. 2.1 mm X 50 mm, maintained at 55 degrees Celsius. Mobile phase A was 6:4, acetonitrile and water with 10 mM ammonium formate. Mobile phase B was 9:1, propan-2-ol and acetonitrile with 10 mM ammonium formate. The flow was maintained at 500 uL per minute through the following gradient: 0.00 minutes_40% mobile phase B; 1.5 minutes_40% mobile phase B; 8.00 minutes_99% mobile phase B; 10.00 minutes_99% mobile phase B; 10.10 minutes_40% mobile phase B; 12.00 minutes_40% mobile phase B. The sample injection needle was washed using 9:1, propan-2-ol and acetonitrile [strong wash] and 2: 1: 1 (propan-2-ol, acetonitrile and water) [weak wash]. The mass spectrometer used was the Thermo Scientific Q-Exactive Orbitrap with a heated electrospray ionisation source (Thermo Fisher Scientific, Hemel Hempstead, United Kingdom). The mass spectrometer was calibrated immediately before sample analysis using positive and negative ionisation calibration solution (recommended by Thermo Scientific). Additionally, the mass spectrometer scan rate was set at 4 Hz, giving a resolution of 35,000 (at 200 m/z) with a full-scan range of m/z 120 to 1,800 with continuous switching between positive and negative mode. The data processing involved the integration of the extracted ion chromatogram peaks for each target lipid species (circa 1,600 species) and the stable isotope labelled internal standards at their expected retention time. The area ratio response of the target lipid to the corresponding internal standard were converted into uM for the fluid samples or nmole results normalised to the amount of tissue extracted. All results were subjected to blank correction and comprehensive quality checking before further statistical analysis.