Fig. 4.

Protection against IR by PEMT deficiency depends on increased mitochondrial CoQ content. (A) Mitochondrial CoQ₉ content in 3T3-L1 adipocytes treated with control or anti-Pemt ASO, and TNFα (chronic low dose inflammation inducer) or 4-nitrobenzoic acid (4NB, an inhibitor of CoQ biosynthesis) (n = 3). *Significant difference between control and PEMT-ASO in the same treatment group. ^aSignificant difference between TNFα and 4NB treatments compared with vehicle in control-ASO treated cells. ^bSignificant difference between TNFα and 4NB treatments compared with vehicle in PEMT-ASO treated cells. ^#No significant difference between cells treated with PEMT-ASO and TNFα or 4NB compared with vehicle-treated control-ASO cells. (B) Glucose uptake in 3T3-L1 adipocytes treated with control or anti-Pemt ASO, and TNFα (chronic low dose inflammation inducer) or 4-nitrobenzoic acid (4NB, an inhibitor of CoQ biosynthesis) as measured by uptake of ³H-2-deoxyglucose (n = 5). *ns denotes significant difference or lack thereof, respectively, between control and PEMT-ASO in the same treatment group. ^aSignificant difference between TNFα and TNFα+4NB treatments compared with vehicle in control-ASO treated cells. ^#No significant difference between cells treated with PEMT-ASO and TNFα or 4NB compared with vehicle-treated control-ASO cells. (C) PEMT activity in C57BL/6J mice fed HFD treated with either control or anti-PEMT ASO for 10 weeks (n = 5–9). (D) Glucose tolerance test (GTT) in C57BL/6J mice fed HFD treated with either control or anti-PEMT ASO for 10 weeks (n = 5–10). For area under the curve (AUC), each data point represents the area under the curve extrapolated per animal from blood glucose concentrations determined over 3 h post glucose challenge. AUC was determined as outlined in the Methods. (E) Insulin tolerance test (ITT) in C57BL/6J mice fed HFD treated with either control or anti-PEMT ASO for 10 weeks (n = 5–10). For incremental area under the curve (iAUC), blood glucose concentrations per animal were baseline (0 min) corrected, and iAUC calculated from Δ blood glucose concentrations determined over 3 h post insulin challenge. (F) Mitochondrial CoQ₉ concentrations in C57BL/6J mice fed HFD treated with either control or anti-PEMT ASO for 10 weeks (n = 5–12). (G) Mitochondrial SAM concentrations in C57BL/6J mice fed HFD treated with either control or anti-PEMT ASO for 10 weeks (n = 5–12). (H), Mitochondrial SAM/SAH ratio in C57BL/6J mice fed HFD treated with either control or anti-PEMT ASO for 10 weeks (n = 5–12). (I) Schematic summary of results and proposed mechanism. PEMT deficiency increases cytosolic SAM resulting in increased mitochondrial SAM and SAM/SAH. Increased mitochondrial SAM increases CoQ biosynthesis by donating methyl groups to the benzoquinone head group of CoQ formed from 4-hydroxybenzoic acid (4HB). Increased mitochondrial CoQ results in decreased mitochondrial superoxide, an upstream driver of insulin resistance. Data and error bars depict mean ± s.e.m. *P ≤ 0.05 as determined by Mann-Whitney (C–H) or Kruskal-Wallis test (A, B).