Extended Data Fig. 4: Ectopic expression of Aox in hepatocytes decreases mROS generation via RET and improves systemic glucose homeostasis.
From: CoQ imbalance drives reverse electron transport to disrupt liver metabolism
(A) Illustration showing mROS production in ob/ob mice ± Aox expression. Aox oxidizes excess CoQH2 and decreases mROS by RET. (B) Representative traces of cyanide-insensitive oxygen consumption in Hepa 1-6 and (C) AML12 cells expressing Aox. N-propyl gallate (n-PG) inhibits Aox activity. Top, immunoblot confirming Aox expression. Representative data from n = 3 experiments. (D) Representative traces of Amplex UltraRed oxidation to show that Aox-expressing primary hepatocytes generate less mROS by RET. Ad., adenovirus. Representative data from n = 7 experiments. (E) Immunoblot analysis and (F) quantification of 18 nM insulin action in isolated hepatocytes from 9–10-week-old obese mice incubated with ad.Aox-HA (n = 15) or ad.GFP (n = 15) for 24 h. Pooled from five independent experiments (*p = 0.0298, **p = 0.0092, ****p < 0.0001, one-tailed unpaired t-test). (G) Representative immunoblot analysis of tissue homogenates of Aox expressing mice (n = 1 mouse). (H) Immunoblot analysis of different cellular fractions from the liver of GFP or Aox mice. ndufs1 and VDAC, mitochondria; calreticulin, endoplasmic reticulum and tubulin, cytosol (n = 1 mouse per group). (I) Glucose production from primary hepatocytes isolated from obese mice expressing Aox or GFP using 20 mM lactate, 2 mM pyruvate, and 2 mM glutamine as substrates. n = 9 mice per group (*p = 0.0178, one-tailed unpaired t-test). (J) Blood glucose levels during oral glucose tolerance test (OGTT) (0.75 g • kg−1) in ob/ob mice. expressing aav.GFP (n = 7) or aav.Aox (n = 8). Inset, area under the curve. (*p = 0.0496, one-tailed unpaired t-test). (K) Liver sections from ob/ob mice expressing Aox or GFP stained with PAS, bars 200 µm. (L) Quantification of liver areas positive for PAS staining of glycogen in obese mice expressing aav.Aox (n = 7) or aav.GFP (n = 5). (*p = 0.0159, unpaired t-test). (M) Plasma insulin levels during OGTT (GFP, n = 7 vs Aox, n = 8 mice). (N) Blood glucose levels during insulin tolerance test (3.5 U of insulin • kg−1). Inset, area under the curve (GFP, n = 7 vs Aox, n = 8 mice). (O) Liver sections from ob/ob mice expressing Aox or GFP stained with H&E, bars 200 µm. (P-U) Metabolic profile of obese 10 days after Aox expression. (P) Bodyweights following aav.GFP or aav.Aox administration at 6 weeks of age (n = 8 per group). (Q) Body composition of 7-week-old ob/ob mice following AAV administration (n = 6 per group). (R) Energy expenditure (EE) as a function of bodyweight (Aox, n = 9 vs GFP, n = 7). (S) Respiratory exchange ratio measured during metabolic cage housing (Aox, n = 9 vs GFP, n = 7). (T) Analysis of RER during light and dark cycles (Aox, n = 9 vs GFP, n = 7). (U) Metabolite levels in the livers of ob/ob mice expressing Aox vs. GFP (n = 6 mice per group). Panels D and I were created with BioRender.com.Values are individual values and means ± SEM. ns, not significant.
