Highlights d Lysine lactoylation occurs via a non-enzymatic acyl transfer from lactoylglutathione d Glycolytic enzymes are heavily modified by lactoylation d Glyoxalase 2 is the critical regulator for lactoylglutathione and lysine lactoylation d Glycolytic output is significantly repressed in glyoxalase 2 knockout cells

Non-enzymatic reactions in glycolysis lead to the accumulation of methylglyoxal (MGO), a reactive precursor to advanced glycation end-products (AGEs), which has been suggested to drive obesity- and aging-associated pathologies. We observe that a combination of nicotinamide, lipoic acid, thiamine, pyridoxamine and piperine, which were selected to lower glycation (Gly-Low), reduce toxic glycolytic byproducts, MGO and MGO-derived AGE, MG-H1. Administration of Gly-Low reduced food consumption and body weight, improving insulin sensitivity and survival in both leptin receptor deficient (Lepr db) and wildtype C57 control mouse models. Unlike calorie restriction, Gly-Low inhibited ghrelin-mediated hunger responses and upregulated Tor pathway signaling in the hypothalamus. Gly-Low also extended lifespan when administered as a late life intervention, suggesting its potential benefits in ameliorating age-associated decline by inducing voluntary calorie restriction and reducing glycation.

Post‐translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feed‐forward mechanisms of regulation. We have identified a novel PTM that is derived from the glycolytic by‐product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D‐lactate. We have identified the non‐enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a ‘LactoylLys’ modification on proteins. GLO2 knockout cells have elevated LGSH and consequently, a marked increase in LactoylLys. Using an alkyne‐tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg‐like conditions. Support or Funding Information Financial support was provided by National Institutes of Health Grants (R01 ES027593 for J.R.R.), the SENS foundation (D.A.S.), the American Diabetes Association Pathway to Stop Diabetes Grant 1‐17‐VSN‐04 (D.A.S.), and the Velux Foundations (VELUX34148, A‐M.S.O). Mass spectrometry and proteomics data were acquired by the University of Arizona Analytical and Biological Mass Spectrometry Facility supported by NIH/NCI grant CA023074 to the University of Arizona Cancer Center, UA Research Development and Innovation Office, and by the BIO5 Institute of the University of Arizona.