Age‑dependent oxidative modification of carnitine acetyltransferase limits shuttle flux despite elevated muscle carnitine

Hypothesis We hypothesize that in aging human skeletal muscle the catalytic efficiency of carnitine acetyltransferase (CAT) declines because oxidative stress induces S‑glutathionylation of active‑site cysteines, raising the Km for acetyl‑CoA and lowering Vmax. This kinetic defect persists even when total carnitine is increased by L‑carnitine supplementation, explaining why fat oxidation rises during acute exercise but resting insulin sensitivity and intramuscular lipid content won't improve.

Mechanistic rationale Aging mitochondria emit higher levels of superoxide and hydrogen peroxide, which can modify protein thiols. CAT contains a conserved cysteine (Cys‑XXX) that's essential for acetyl‑CoA binding; reversible S‑glutathionylation of this residue sterically hinders the acetyl‑CoA pocket, decreasing affinity (higher Km) and slowing turnover (lower Vmax). Because CAT also buffers acetyl‑CoA pools, its impairment limits the ability of the carnitine shuttle to match fatty‑acid uptake with oxidation, causing acetyl‑CoA accumulation, reduced pyruvate dehydrogenase activity, and persistent lipid intermediates that impair insulin signaling. Supplementation raises free carnitine but doesn't reverse the enzyme modification, so shuttle flux remains constrained.

Testable predictions

1. Permeabilized muscle fibers from older donors will show a higher apparent Km for acetyl‑CoA and a lower Vmax for CAT activity compared with young donors, when measured at identical free carnitine concentrations.
2. Oxidative inhibition assays (e.g., diamide or H2O2 pretreatment) will reproduce the aged kinetic profile in young fibers, whereas pretreatment with glutaredoxin‑1 or N-acetylcysteine will restore kinetic parameters in aged fibers.
3. In vivo, older adults receiving L‑carnitine plus a mitochondria‑targeted antioxidant (e.g., MitoQ) will exhibit a greater reduction in resting intramuscular lipid and improved insulin sensitivity than those receiving L‑carnitine alone, despite similar increases in muscle carnitine content.
4. Direct measurement of CAT S‑glutathionylation via biotin‑switch immunoblot will correlate inversely with CAT Vmax across age groups.

Experimental design

• Obtain vastus lateralis biopsies from young (20‑30 yr) and older (65‑80 yr) healthy volunteers (n = 10 per group).
• Prepare permeabilized fibers and measure CAT activity using a coupled assay that monitors acetyl‑CoA‑dependent conversion of [14C]‑acetylcarnitine to free carnitine, varying acetyl‑CoA concentrations to derive Km and Vmax.
• Parallel samples will be treated with diamide (oxidant) or glutaredoxin‑1 + GSH (reductant) to test reversibility.
• Assess CAT S‑glutathionylation by biotin‑switch followed by streptavidin pull‑down and western blot for CAT.
• For the intervention arm, recruit older adults (n = 30) randomized to L‑carnitine (2 g day⁻¹) or L‑carnitine + MitoQ (10 mg day⁻¹) for 12 weeks; pre‑ and post‑muscle carnitine content (LC‑MS), resting insulin sensitivity (hyperinsulinemic‑euglycemic clamp), IMCL (1H‑MRS), and CAT kinetics (as above) will be measured.

Potential outcomes and falsifiability If aged muscle shows unchanged CAT Km/Vmax despite elevated acylcarnitines, the hypothesis is falsified. Conversely, demonstration of age-related kinetic decline that's reversible by reducing agents and linked to S‑glutathionylation, and its partial rescue by antioxidant co‑supplementation, would support the mechanistic link between CAT oxidation and the metabolic inflexibility observed with L‑carnitine alone.