BACKGROUND: In animals, dietary sulfur amino acid restriction (SAAR) improves metabolic health, possibly mediated by altering sulfur amino acid metabolism and enhanced anti-obesogenic processes in adipose tissue. AIM: To assess the effects of SAAR over time on the plasma and urine SAA-related metabolites (sulfurome) in humans with overweight and obesity, and explore whether such changes were associated with body weight, body fat and adipose tissue gene expression. METHODS: Fifty-nine subjects were randomly allocated to SAAR (∼2 g SAA, n = 31) or a control diet (∼5.6 g SAA, n = 28) consisting of plant-based whole-foods and supplemented with capsules to titrate contents of SAA. Sulfurome metabolites in plasma and urine at baseline, 4 and 8 weeks were measured using HPLC and LC-MS/MS. mRNA-sequencing of subcutaneous white adipose tissue (scWAT) was performed to assess changes in gene expression. Data were analyzed with mixed model regression. Principal component analyses (PCA) were performed on the sulfurome data to identify potential signatures characterizing the response to SAAR. RESULTS: SAAR led to marked decrease of the main urinary excretion product sulfate (p < 0.001) and plasma and/or 24-h urine concentrations of cystathionine, sulfite, thiosulfate, H2S, hypotaurine and taurine. PCA revealed a distinct metabolic signature related to decreased transsulfuration and H2S catabolism that predicted greater weight loss and android fat mass loss in SAAR vs. controls (all pinteraction < 0.05). This signature correlated positively with scWAT expression of genes in the tricarboxylic acid cycle, electron transport and β-oxidation (FDR = 0.02). CONCLUSION: SAAR leads to distinct alterations of the plasma and urine sulfurome in humans, and predicted increased loss of weight and android fat mass, and adipose tissue lipolytic gene expression in scWAT. Our data suggest that SAA are linked to obesogenic processes and that SAAR may be useful for obesity and related disorders. TRIAL IDENTIFIER: https://clinicaltrials.gov/study/NCT04701346.

Department of Biostatistics Institute of Basic Medical Sciences University of Oslo Postboks 1122 Blindern 0317 Oslo Norway

Department of Endocrinology Morbid Obesity and Preventive Medicine Institute of Clinical Medicine Faculty of Medicine University of Oslo Postboks 4959 Nydalen OUS HF Aker sykehus 0424 Oslo Norway

Department of Molecular Immunology and Toxicology and the National Tumor Biology Laboratory National Institute of Oncology Ráth György u 7 9 1122 Budapest Hungary

Department of Molecular Immunology and Toxicology and the National Tumor Biology Laboratory National Institute of Oncology Ráth György u 7 9 1122 Budapest Hungary; Department of Anatomy and Histology HUN REN UVMB Laboratory of Redox Biology Research Group University of Veterinary Medicine 1078 Budapest Hungary; Chemistry Institute University of Debrecen 4012 Debrecen Hungary

Department of Nutrition Institute of Basic Medical Sciences Faculty of Medicine University of Oslo Postboks 1046 Blindern 0317 Oslo Norway

Department of Nutrition Institute of Basic Medical Sciences Faculty of Medicine University of Oslo Postboks 1046 Blindern 0317 Oslo Norway; Department of Food Safety Norwegian Institute of Public Health Postboks 222 Skøyen 0213 Oslo Norway

Department of Nutrition Institute of Basic Medical Sciences Faculty of Medicine University of Oslo Postboks 1046 Blindern 0317 Oslo Norway; Department of Pharmacology University of Oxford Mansfield Rd Oxford OX1 3QT UK

Department of Nutrition Institute of Basic Medical Sciences Faculty of Medicine University of Oslo Postboks 1046 Blindern 0317 Oslo Norway; The Lipid Clinic Department of Endocrinology Morbid Obesity and Preventive Medicine Oslo University Hospital Postboks 4959 Nydalen OUS HF Aker sykehus 0424 Oslo Norway

Department of Pathology Oslo University Hospital Rikshospitalet Postboks 45980 Nydalen OUS HF Rikshospitalet 0424 Oslo Norway

Department of Pediatrics and Inherited Metabolic Disorders Charles University 1st Faculty of Medicine and General University Hospital Ke Karlovu 2 128 00 Prague Czech Republic

Department of Physiology Faculty of Medicine University of Alexandria Chamblion street Qesm Al Attarin Alexandria 5372066 Egypt; Department of Pharmacology University of Oxford Mansfield Rd Oxford OX1 3QT UK

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