Does autophagy regulate hepatic protein acetylation through acetyl-CoA metabolism?

Acetylation falls while other modifications rise

The paper asks whether autophagy, best known for degrading proteins, also shapes how the hepatic proteome is chemically modified after translation. The authors screened five major post-translational modifications in livers of hepatocyte-specific Atg7 knockout mice (hepAtg7−/−) and found a split response: ubiquitination, SUMOylation, methylation, and tyrosine phosphorylation all increased, while global protein acetylation — the hepatic acetylome — fell markedly. The same acetylation loss appeared in Atg5-deficient livers, so the effect is not tied to one autophagy gene.

Acetylation here means the transfer of an acetyl group from acetyl-CoA onto lysine residues, detected with two independent anti-acetyl-lysine antibodies. The reduction was not confined to one compartment: subcellular fractionation showed lower acetylation in nuclear, cytosolic, and membrane fractions, and histone H3 acetylation also dropped. In other words, the change is broad rather than a single-protein event, which is why the authors frame it as a shift in the hepatic acetylome.

The acetylation decline precedes liver injury

A central confound is that autophagy deficiency in the liver causes cholestatic injury, inflammation, and fibrosis, so reduced acetylation could simply be a byproduct of damaged tissue. The authors addressed this with an inducible model, Atg7Hep-ERT2, in which Atg7 is deleted in adult hepatocytes after tamoxifen. At day 7 after induction, Atg7 was lost and P62/SQSTM1 had accumulated, confirming blocked autophagy, yet overt liver pathology in this model does not appear until about day 15. At that day-7 point, hepatic protein acetylation was already significantly reduced.

Two further checks support a direct link. Adult mice given AAV8-TBG-Cre to delete Atg7 in hepatocytes, avoiding developmental effects of embryonic deletion, also showed reduced acetylation. And when Atg7 was re-expressed via AAV8-TBG-Atg7 in Atg7Hep-ERT2 mice, autophagy function was restored and acetylation normalized. The authors therefore argue that reduced acetylation is a direct consequence of autophagy loss and may even precede injury, though the temporal ordering is an interpretation rather than a demonstrated cause of later pathology.

Partial autophagy defects are enough to lower acetylation

Complete genetic deletion is a blunt tool, and many liver diseases impair autophagic flux without eliminating autophagy. To test a milder scenario, wild-type mice received chloroquine, which blocks autophagosome-lysosome fusion, or leupeptin, which inhibits lysosomal proteases. At the doses used, chloroquine did not cause significant liver injury, yet hepatic protein acetylation fell clearly; leupeptin produced a comparable decrease. AML12 hepatocytes exposed to either agent also showed reduced acetylation.

This matters for how the result should be read: the acetylome responds to disrupted autophagic flux, not only to a complete loss of autophagy genes. The other side of the comparison is equally important. Fasting for 16–24 hours, a physiological autophagy stimulus, increased hepatic protein acetylation in wild-type mice, and Becn1F121A knock-in mice, which carry a mutation driving constitutive autophagy, showed markedly elevated acetylation relative to wild-type controls. Activation and inhibition thus move acetylation in opposite directions.

The enzyme explanation does not fit the data

The intuitive explanation for lower acetylation would be less acetyltransferase or more deacetylase activity. The authors tested this and found it wanting. mRNA levels of multiple lysine acetyltransferases, N-terminal acetyltransferases, and histone acetyltransferases were largely unchanged in autophagy-deficient livers; KAT8 showed only a modest decrease, which the authors judge insufficient to explain a global loss. Class I, IIa/b, and IV HDAC expression was also unchanged.

Sirtuins, the NAD+-dependent class III deacetylases, were actually downregulated, and SIRT6 protein was reduced. Because fewer deacetylases should raise acetylation, this runs opposite to the observed decline and further weakens the deacetylase explanation. The authors note they did not measure NAD+ abundance, NAD+/NADH ratios, or sirtuin enzymatic activity, so altered NAD+ availability remains an untested additional layer rather than an excluded one.

Acetyl-CoA supply links autophagy to acetylation

The alternative is substrate limitation. Acetylation consumes acetyl-CoA, a central metabolite produced in mitochondrial and nucleo-cytosolic compartments. Hepatic acetyl-CoA was significantly reduced in autophagy-deficient livers, and wild-type mice treated with chloroquine also showed decreased hepatic acetyl-CoA. Gene expression analysis found no significant upregulation of pathways that consume acetyl-CoA, such as fatty acid, ketone body, and steroid synthesis, which argues against increased utilization.

The decisive test was supplementation. Atg7F/F mice with AAV8-TBG-Cre-mediated deletion received acetyl-CoA at 20 mg/kg intraperitoneally for seven days, and hepatic protein acetylation was restored to near-normal; a similar rescue occurred in hepAtg5−/− mice. Acetate, an acetyl-CoA precursor, given at 500 mg/kg/day for seven days to Atg7Hep-ERT2 mice, also reversed the acetylation reduction, and acetate restored acetylation in chloroquine-treated AML12 hepatocytes. These rescue experiments are what turn a correlation between acetyl-CoA and acetylation into a supported causal chain within the models used.

NRF2 represses acetyl-CoA biosynthetic enzymes

If acetyl-CoA is limiting, why does it fall? The authors examined the enzymes that make it: Acly converts citrate to acetyl-CoA in the cytosol, Acss1 and Acss2 convert acetate in mitochondria and cytosol respectively, Mlycd generates acetyl-CoA from malonyl-CoA, and Pdha1 converts pyruvate in mitochondria. All four were significantly downregulated at both mRNA and protein levels in autophagy-deficient livers. Because nutritional state shifts which source dominates, all samples were collected in the same nutritional state and a fixed circadian window of 12:00 PM to 2:00 PM.

The proposed upstream regulator is NRF2, the antioxidant transcription factor that is activated when autophagy is impaired. In silico promoter analysis identified NRF2 binding regions in the promoters of NQO1, Acly, Acss, Mlycd, and Pdha1, and chromatin immunoprecipitation showed NRF2 occupancy at these sites in autophagy-deficient livers, with binding lost in hepAtg7/NRF2−/− mice. Co-deleting NRF2 restored expression of the acetyl-CoA biosynthetic genes and rescued hepatic protein acetylation. This is the paper's mechanistic core: autophagy loss activates NRF2, NRF2 occupancy suppresses acetyl-CoA-producing enzymes, substrate falls, and acetylation declines.

Rescue extends to injury and human liver disease

The therapeutic claim rests on what supplementation did beyond the acetylation readout. In autophagy-deficient models, acetyl-CoA or acetate supplementation restored protein acetylation and alleviated liver injury and inflammation. That combination is what makes acetyl-CoA supply a candidate intervention point rather than only a biomarker of autophagy status.

The authors also report that similar reductions in acetylation and in acetyl-CoA enzyme expression occur in preclinical models of chronic liver disease and in human liver tissue from patients with non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and cirrhosis. These human observations are descriptive comparisons of tissue, not intervention results, so they extend the relevance of the pathway without showing that restoring acetyl-CoA changes outcomes in patients. The supplementation evidence remains confined to mouse and cell models.

Where the acetyl-CoA explanation holds

The most useful way to read this paper is as a priority ordering among candidate mechanisms. Within the models tested, substrate availability outranks the classical enzyme-balance explanation: acetyltransferase and HDAC expression did not move in the direction needed, sirtuins moved the wrong way, and acetyl-CoA or acetate restored acetylation. The NRF2–Acly/Acss/Mlycd/Pdha1 axis is the specific route the authors propose, supported by promoter binding, co-deletion rescue, and enzyme downregulation at both mRNA and protein levels.

The boundaries are equally specific. The evidence comes from mouse genetic and pharmacological models plus AML12 cells, with supplementation tested over seven to eight days and acetylation measured by immunoblot and histone multiplex assays. The authors did not measure NAD+ or sirtuin activity, leaving a possible complementary layer untested. And the human data are cross-sectional tissue comparisons. The practical takeaway is therefore conditional: when autophagy is impaired, look first at acetyl-CoA supply and NRF2-driven repression of biosynthetic enzymes, and treat acetate or acetyl-CoA rescue as a validated lever in these models rather than an established human therapy.

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