Cysteine depletion triggers adipose tissue thermogenesis and weight loss

Abstract

Caloric restriction and methionine restriction-driven enhanced lifespan and healthspan induces 'browning' of white adipose tissue, a metabolic response that increases heat production to defend core body temperature. However, how specific dietary amino acids control adipose thermogenesis is unknown. Here, we identified that weight loss induced by caloric restriction in humans reduces thiol-containing sulfur amino acid cysteine in white adipose tissue. Systemic cysteine depletion in mice causes lethal weight loss with increased fat utilization and browning of adipocytes that is rescued upon restoration of cysteine in diet. Mechanistically, cysteine-restriction-induced adipose browning and weight loss requires sympathetic nervous system-derived noradrenaline signalling via β3-adrenergic-receptors that is independent of FGF21 and UCP1. In obese mice, cysteine deprivation induced rapid adipose browning, increased energy expenditure leading to 30% weight loss and reversed metabolic inflammation. These findings establish that cysteine is essential for organismal metabolism as removal of cysteine in the host triggers adipose browning and rapid weight loss.

Fig. 1. Cysteine deficiency induces weight loss.

a, Principal-component analysis of the metabolome of subcutaneous adipose depots (SFAT) of healthy individuals at baseline and after 12 months of CR (n = 14). b, Metabolite set enrichment analysis shows that compared to baseline, 1 year of CR in humans activates TSP, with increased cysteine and taurine metabolism. c, Schematic summary of TSP and metabolites from baseline to 1 year CR, measured in human SFAT. Blue lines indicate unchanged metabolites, green and red arrows indicate significantly increased or decreased metabolites or genes respectively, via paired t-test (P < 0.05). SAM, S-adenosyl methionine. d,e, Normalized expression of changes in CTH and BHMT in human SFAT at baseline, and after 12 and 24 months of CR. Adjusted P values were calculated in the differential gene expression analysis in a separate cohort from metabolome analyses in the CALERIE-II trial (n = 8). B, baseline. f,g, Change in metabolites in human SFAT at baseline and 12 months of CR. Significance was calculated using paired t-tests (n = 14). AU, arbitrary unit. h, Mouse model used to achieve cysteine deficiency utilizing Cth^−/− mice fed a CysF diet. i, Male Cth^+/+ and Cth^−/− mice were fed control (CTRL) or CysF diets for 6 days (n = 5 Cth^+/+ CTRL, n = 12 Cth^+/+ CysF, n = 8 Cth^−/− CTRL, n = 17 Cth^−/− CysF; three experiments pooled). Per cent body weight represented over 6 days of diet. j, Cth^−/− mice were fed purified control diet (black line) or a diet containing 75% cysteine (green line) alternately switched to CysF diet (green line with red dots n = 6 per group). k, Box plots of metabolites involved in TSP in the serum of Cth^−/− mice fed CTRL or CysF diet for 6 days (n = 4 Cth^−/− CTRL, n = 5 Cth^−/− CysF). l, Schematic summary of changes in the metabolites in the serum of Cth^−/− mice fed CTRL or CysF diet for 6 days. Blue lines represent measured, but unchanged metabolites, red and green arrows indicate significantly decreased or increased metabolites, respectively (P < 0.05). See Supplementary Table 1 for the full list of metabolites. m, Total GSH content in subcutaneous (SFAT), brown (BAT) adipose depots and liver of Cth^−/− mice fed with CTRL or CysF diet for 5 days (n = 7 per group), determined by colorimetric assay. n, Box plots of GSSG and threonine quantification in the SFAT of Cth^−/− mice fed CTRL or CysF diet for 6 days (n = 6 per group). o,p, RNA-seq based expression of Gclc, Gss (o) and Bola3 (p) in the SFAT of Cth^−/− mice fed with CTRL or CysF for 6 days. FPKM, fragments per kilobase of exon model per million mapped fragments. q, Coenzyme A (CoA) content in SFAT, BAT and liver samples of Cth^−/− mice fed with CTRL or CysF diet for 5 days, determined by fluorometric assay (n = 7 per group, ND, not detectable). r, Analysis of EPR spectra of POBN-lipid radical adducts measured in Folch extracts of visceral adipose depot (VFAT), SFAT and BAT tissues from Cth^−/− mice fed with CTRL or CysF diet for 5 days, normalized to 100 mg (ND, not detectable; n = 5–6 per group). s, Aconitase activity determined in SFAT, VFAT and BAT tissues from Cth^−/− mice fed with CTRL or CysF diet for 5 days (n = 6 CTRL and 7 CysF). Data are represented as mean ± s.e.m. Box plots represent median value and extend to the 25th and 75th percentiles. Whiskers are plotted down to the minimum and up to the maximum value. Unless mentioned, differences were determined with unpaired two-tailed t-tests. Panels c, h and l created with BioRender.com. Source data

Fig. 2. Cysteine depletion induces browning of adipose tissue.

a, Representative images of subcutaneous (SFAT) and visceral (VFAT) fat sections stained for UCP1 from Cth^−/− mice fed CTRL or CysF diet for 6 days (scale bar, 100 μm). b, Representative H&E-stained sections of SFAT of Cth^−/− mice fed CTRL or CysF diet for 6 days or CysF diet followed by Cys-supplemented diet for 4 days (CysF + Cys) (scale bar, 100 μm). c, Western blot detection of ATGL and UCP1 in SFAT from Cth^−/− mice after 6 days of CTRL or CysF diet or Cys supplementation after CysF-induced weight loss. Actin is used as a loading control. d, qPCR analysis of thermogenic genes in SFAT of Cth^+/+ and Cth^−/− mice fed CysF diet for 6 days (n = 8 Cth^+/+ and n = 10 Cth^−/−). e,f, Faecal calorie content (n = 6 per group) (e) and cumulative food intake of Cth^−/− mice fed CTRL or CysF diet for 4 days (f) (n = 6 per group). g, Linear regression analysis of EE against body mass during dark cycle at 4 and 5 days of weight loss (n = 10 Cth^+/+ CysF and n = 12 Cth^−/− CysF). h, Per cent body weight change of Cth^−/− mice fed with CTRL diet or CysF diet (red line) for 5 days and then switched to Cys-containing diet (orange line) for 3 days (n = 6 per group). i, RER measured in metabolic cages, of Cth^−/− mice fed with CTRL diet (n = 6) or Cys-containing diet after CysF-induced weight loss (n = 4). j, Average food intake of Cth^−/− mice fed with CysF diet and then switched to Cys-containing diet for 2 days (n = 7 per group). Significance was measured with paired t-test. k, Linear regression analysis of EE against body mass during dark cycle of Cth^−/− mice fed with CTRL (n = 6) or Cys-supplemented diet after CysF-induced weight loss (n = 4), average values of the first two nights after diet switch. l, t-SNE plot of scRNA-seq showing cluster identities from SFAT SVF from Cth^−/− mice fed CTRL or CysF diet at day 4 of weight loss and bar chart showing population fold change (FC) in relative abundance of each cluster comparing Cth^−/− CysF versus Cth^−/− CTRL. DC, dendritic cell; APC, antigen-presenting cell. m, t-SNE plot displaying Pdgfra expression in red across all populations and Monocle analysis of clusters 0, 1 and 2, with colouring by pseudotime to show right most cluster giving rise to two separate clusters. Each cluster represented by colour in Cth^−/− CTRL and Cth^−/− CysF. Data are expressed as mean ± s.e.m. Statistical differences were calculated by two-way ANOVA with Sidak’s correction for multiple comparisons or unpaired two-tailed t-tests. NS, not significant. Source data

Fig. 3. FGF21 is partially required for cysteine-restriction-mediated weight loss.

a, Western blot detection of lipolysis regulators pHSL, HSL and ATGL in SFAT from Cth^−/− mice after 6 days of CTRL or CysF diet, actin is used as loading control. b, Volcano plot of lipid species of BAT showing FC of triglycerides in Cth^−/− mice fed CTRL or CysF diet. c,d, in vivo measurement of BAT temperature by BIRDS imaging (c) and quantification of local temperature differences in BAT (d) compared to surrounding tissue in Cth^+/+ and Cth^−/− mice on CysF diet for 6 days (n = 5 per group). e, Serum FGF21 quantification in Cth^−/− CTRL (n = 23), Cth^−/− CysF for 6 days (n = 8) and Cth^−/− CysF followed with 4 days of Cys supplementation (n = 10). f, Serum GDF15 concentrations in Cth^−/− CTRL, Cth^−/− CysF for 4 days and Cth^−/− CysF followed with 3 days of Cys supplementation (n = 6 per group). g, Immunoblot analysis of CHOP, calnexin, IRE1a, BiP in the liver of Cth^−/− mice fed with CTRL or CysF diet at day 6. Actin was used as loading control. h, Percentage body weight change of Cth^−/− and Cth^−/−CHOP^−/− mice fed with CysF diet for 5 days (n = 17 Cth^−/− and n = 15 Cth^−/−CHOP^−/−). i, Percentage body weight change of Cth^−/− and Fgf21^−/−Cth^−/− mice fed with CysF diet for 5 days (n = 13 Cth^−/− and n = 18 Fgf21^−/−Cth^−/−). j, Energy expenditure measured in metabolic cages of Cth^−/− and Cth^−/− Fgf21^−/− mice on days 3–4 of CysF diet (n = 5 per group). k, Immunoblot analysis of pHSL, HSL, ATGL and UCP1 in SFAT of Cth^+/+, Cth^−/− and Cth^−/−Fgf21^−/− mice fed CysF diet for 6 days. l, Representative H&E-stained SFAT sections of Cth^−/− and Fgf21^−/−Cth^−/− mice after 6 days of CysF diet (scale bar, 500 μm). m–p, Cth^+/+ and Cth^−/− mice were fed with CysF diet and housed at 20 °C or 30 °C for 6 days. Percentage body weight change (n = 3 Cth^+/+ 20 °C, n = 4 Cth^+/+ 30 °C, n = 4 Cth^−/− 20 °C, n = 5 Cth^−/− 30 °C) (m), representative images of H&E staining of SFAT sections (scale bar, 200 μm) (n) and qPCR analysis of thermogenic markers (n = 5 Cth^+/+ 20 °C, n = 10 Cth^+/+ 30 °C, n = 6 Cth^−/− 20 °C, n = 11 Cth^−/− 30 °C) (o,p). Data are expressed as mean ± s.e.m. Statistical differences were calculated by one-way ANOVA with Tukey’s correction for multiple comparisons or two-way ANOVA with Sidak’s correction for multiple comparisons or unpaired two-tailed t-tests. Source data

Fig. 4. Global cysteine deficiency induced adipose browning is UCP1 independent.

a, Immunoblot analyses of CTH in the liver of male and female Cth^f/f Alb:Cre⁻ or Alb:Cre⁺ mice. b, Western blot detection of CTH in the SFAT of male and female Cth^f/f Adipoq:Cre⁻ or Adipoq:Cre⁺ mice. c,d, Serum cysteine and cystine determined by LC–MS/MS in Alb:CreCth^f/f mice (n = 5 per group) (c) and Adipoq:Cre;Cth^f/f mice (n = 4 CTRL and n = 5 CysF) (d) after 6 days of CTRL or CysF diet. AU, arbitrary units. e,f, Percentage body weight changes of Alb-Cre;Cth^f/f mice (n = 5 Cth^f/f CTRL, n = 6 Cth^f/f CysF and n = 3 Alb-Cre;Cth^f/f CTRL and CysF) (e) and Adipoq-Cre;Cth^f/f mice (n = 5 per group) after 6 days of CTRL or CysF diet (f). g,h, Volcano plot of serum metabolites identified by LC–MS/MS in Alb-Cre;Cth^f/f mice (n = 5 per group) (g) and Adipoq-Cre;Cth^f/f mice (n = 4 CTRL and n = 5 CysF) (h) after 6 days of CTRL or CysF diet. Trans-sulfuration pathway related metabolites are highlighted in red. Significantly increased or decreased metabolites (−log₁₀(P) >1.3 and ∣log₂(FC)∣>1) are highlighted in blue and listed on the right. Cys, cysteine; Homocys, homocysteine; Met, methionine; SAH, S-adenosyl homocysteine. Supplementary Tables 3 and 6 provide the full list of metabolites. i–l, Cth^−/− and Cth^−/− Ucp1^−/− mice were fed a CysF diet for 6 days (n = 8 per group). Per cent body weight change over 6 days of diet (i). Representative H&E histology images of SFAT after 6 days of diet (j). Energy expenditure measured in metabolic cages on days 4 and 5 of CysF diet (k). Linear regression analysis of EE against body mass during dark and light cycles at 4 and 5 days of weight loss (when adjusted to body mass covariate, EE of Cth^−/− Ucp1^−/− is significantly decreased, during both night and day) (l). m, CBTs measured in the peritoneal cavity by implantation of Star-Oddi loggers over 6 days of diet in male Cth^−/− and Cth^−/−Ucp1^−/− mice fed CysF diet. Recordings were taken every 30 min and representative day 4 is plotted (n = 7 Cth^−/−, n = 5 Cth^−/−Ucp1^−/−). n,o, Immunoblot staining of ATGL, TH and UCP1 in BAT of Cth^−/− and Cth^−/−Ucp1^−/− fed a CysF diet for 6 days (n) and quantification using tubulin as loading control (o). p, Thermogenic markers gene expression analysis in BAT of Cth^−/− and Cth^−/−Ucp1^−/− mice fed a CysF diet for 6 days, measured by qPCR (n = 8 Cth^−/−, n = 10 Cth^−/−Ucp1^−/−). q, Gene expression of genes involved in futile creatine cycle in BAT of Cth^−/− and Cth^−/−Ucp1^−/− mice fed a CysF diet for 6 days (n = 16 Cth^−/−, n = 15 Cth^−/−Ucp1^−/−), quantified by qPCR. Data are expressed as mean ± s.e.m. Statistical differences were calculated by two-way ANOVA with Sidak’s correction for multiple comparisons or by unpaired two-tailed t-tests. Source data

Fig. 5. Cysteine-elimination-induced browning and weight loss requires noradrenergic signalling.

a, Tissue clearing and whole-brain c-Fos immunolabelling approach using iDISCO+ and CLEARMAP in Cth^−/−mice fed CTRL or CysF for 5 days. b, Scheme of the thermosensory information inflow into the brain and the thermogenic outflow to periphery, highlighting the key canonical sites responsible for a thermogenic response. LPBN, lateral parabrachial nucleus; MPOA, medial preoptic area; DMH, dorsomedial hypothalamus; PVH, paraventricular hypothalamus; RPA, raphe palladus; DRN/vlPAG, dorsal raphe nucleus/ventrolateral portion of the periaqueductal grey. c, Automated analysis of c-Fos⁺ cell distribution in Cth^−/− brains collected after 5 days CTRL (n = 6) or CysF feeding (n = 5). Panels show the reference annotation (Allen Brain Atlas; ABA) with details from the averaged density maps (5–6 brains averaged) between the two conditions, P value maps (25-μm orthogonal projection) for the canonical thermogenic regions in the brain as coronal projections. First lane shows the LPBN, the entry point of thermosensory information into the brain. Second lane shows the MPOA, the sensory integrator of thermogenic input information. Third, fourth and fifth lanes show DMH, BNST and the vlPAG, respectively; three critical sites for transmitting information received by the MPOA to the SNS-mediated thermogenic outflow. d, Quantification of c-Fos staining in the parabrachial nucleus, MPOA, DMH and BNST of Cth^−/− mice after 5 days of CTRL (n = 6) or CysF feeding (n = 5). e) Measurement of noradrenaline by orbitrap MS/MS in the SFAT of Cth^+/+ and Cth^−/− fed 6 days of CTRL or CysF diet (n = 5 Cth^+/+ CTRL, n = 5 Cth^+/+ CysF, n = 6 Cth^−/− CTRL, n = 6 Cth^−/− CysF). f, qPCR gene expression Maoa in SFAT of Cth^+/+ (n = 8) and Cth^−/− (n = 10) mice fed with CysF diet for 6 days. g,k, Cth^−/− mice were fed with CysF diet for 5 days and treated daily with a β-3 adrenergic receptor antagonist (L748337) or vehicle (PBS) (n = 7 per group). g, Percentage body weight change. h, Representative images of hematoxylin and eosin (H&E) staining of SFAT sections (scale bar, 50 μm). i, qPCR gene expression of Ucp1 in BAT depots. j, Immunoblot analysis of lipolysis regulators (pHSL, HSL and ATGL) in BAT samples. Actin is used as a loading control. k, Quantification of pHSL and ATGL signals (n = 6 per group). Data are expressed as mean ± s.e.m. Box plots represent median value and extend to the 25th and 75th percentiles. Whiskers are plotted down to the minimum and up to the maximum value. Statistical differences were calculated by two-way ANOVA with Sidak’s correction for multiple comparisons or by unpaired two-tailed t-tests. Panels a and b created with BioRender.com. Source data

Fig. 6. Cysteine deficiency reverses high-fat-diet-induced obesity in mice.

Cth^−/− mice that had been fed HFD for 12 weeks were switched to HFD-CTRL or HFD-CysF. a, Percentage body weight change after switching to HFD-CysF diet (n = 6 Cth^−/− HFD-CTRL, n = 5 Cth^−/− HFD-CysF and n = 5 Cth^+/+ HFD-CysF). b, Fasting blood glucose measured 1 week post diet switch (Cth^−/− HFD-CTRL n = 19, Cth^−/− HFD-CysF, n = 20). c, The glucose tolerance test (GTT) in Cth^−/− after diet switch from HFD-CTRL to HFD-CysF (Cth^−/− HFD-CTRL n = 19, Cth^−/− HFD-CysF, n = 20). The glucose administration is based on total body weight. d, EE of Cth^−/− mice fed with HFD-CTRL or HFD-CysF, average values of nights 4 and 5 of diet switch (n = 6 Cth^−/− HFD-CTRL, n = 5 Cth^−/− HFD-CysF). e, RER measured in metabolic chambers on days 4 and 5 of diet switch (n = 6 Cth^−/− HFD-CTRL, n = 5 Cth^−/− HFD-CysF). f, Representative histological sections of SFAT and VFAT stained for UCP1, 6 days after diet switch. g, qPCR analysis of inflammatory genes in CD11b⁺ F4/80⁺ VFAT macrophages of Cth^−/− mice after diet switch to HFD-CTRL or HFD-CysF (n = 4 per group). Data are expressed as mean ± s.e.m. Statistical differences were calculated by two-way ANOVA with Sidak’s correction for multiple comparisons or by unpaired two-tailed t-tests. Source data

Extended Data Fig. 1. Cysteine depletion induces weight-loss in mice without overt pathology.

a) Cystathionine and homocysteine measurements by MS/MS in human SFAT at baseline (B) and after 12 months of caloric restriction (n = 14). AU: arbitrary units. b) Schematic of Cth^−/− and Cth^fl/fl mice generation (KOMP construct) used to cross to either Alb:cre or Adipoq:cre. c) Body weight of Cth^+/+ and Cth^−/− mice fed with CTRL or CysF diet for 6 days (n = 5 Cth^+/+ CTRL, n = 6 Cth^+/+ CysF, n = 4 Cth^−/− CTRL, n = 5 Cth^−/− CysF). d) Fat mass and lean mass measured by EchoMRI of male Cth^+/+ and Cth^−/− after 6 days of CTRL or CysF diet (n = 5 Cth^+/+ CTRL, n = 12 Cth^+/+ CysF, n = 8 Cth^−/− CTRL, n = 17 Cth^−/− CysF). e) Cth^+/+ and Cth^−/− mice were fed ad libitum (ad lib) or pair fed CTRL or CysF diet (n = 4 Cth^+/+CysF ad lib, n = 5 Cth^+/+CysF pair fed, n = 7 Cth^−/−CTRL pair fed, n = 5 Cth^−/− CysF pair fed). Percentage body weight change over 6 days of diet. f) Accumulated food intake of Cth^+/+ and Cth^−/− mice over 6 days of CysF feeding measured in metabolic cages (n = 10 Cth^+/+ and n = 12 Cth^−/−). Cage image and video show that Cth^−/− mice on CysF diet at day 5 have normal activity. g) Qualitative assessment of nest building (score from 0 to 4) and presence (score=1) or absence (score=0) of kyphosis in WT and Cth^−/− mice (n = 12/group). h) Gait assessment, ledge test and hindlimb clasping test were performed to measure motor coordination in WT and Cth^−/− mice. Mice were scored from 0 (normal behaviour) to 1 (abnormal behaviour) (n = 12/group). i) Representative H&E-stained sections of kidney, lung, heart, and liver from female Cth^−/− mice fed control diet or cystine-deficient diet for 6 days, lack notable pathologic changes and do not differ in microscopic changes by diet in the tissues examined. C = renal cortex, M = renal medulla A = airway, P = pulmonary artery, > = central vein, and * = portal triad. Kidney scale bars=200 μm, lung, heart, liver scale bars= 100μm. Data are expressed as mean±SEM. Statistical differences were calculated by 2-way ANOVA with Sidak’s correction for multiple comparisons, or by unpaired two-tailed t-tests. Panel b created with BioRender.com. Source data

Extended Data Fig. 2. Cysteine depletion triggers metabolic rewiring without altering protein synthesis and iron-sulfur cluster maintenance.

a, b) Volcano plot of a) serum and b) subcutaneous adipose tissue (SFAT) metabolites identified by LC-MS/MS in Cth^−/− mice after 6 days of CTRL or CysF diet (n = 4 CTRL and n = 5 CysF for serum, n = 6/group for SFAT). Transsulfuration pathway related metabolites are highlighted in red. Significantly increased or decreased metabolites (-log₁₀(pvalue)>1.3 and ∣log₂(FC)∣>1) are highlighted in blue and listed on the right. Cys: cysteine. Met: methionine. SAH: S-adenosyl homocysteine. SAM: S-adenosyl methionine. Cysteine and cysteine were not detectable in SFAT samples. See Supplementary Table 1 and 2 for the full list of metabolites. c) Serum L-methionine, L-homocysteine, glutamic acid quantified by mass spectrometry in Cth^−/− mice fed with CTRL (n = 4) or CysF (n = 5) diet for 6 days. AU: arbitrary units. d) Total GSSG content in SFAT, brown adipose tissue (BAT) and liver samples of Cth^−/− mice fed CTRL or CysF diet for 5 days, determined by colorimetric assay (n = 7/group). e) Surface sensing of translation (SUnSET) assay was performed to assess protein synthesis. Immunoblot detection of puromycin incorporation in neosynthesized proteins in SFAT, visceral adipose tissue (VFAT), BAT and liver samples of Cth^−/− mice fed with CTRL or CysF diet for 5 days (n = 6/group). f) Heatmaps of RNA-seq based gene expression of genes involved in Fe-S cluster assembly in SFAT, VFAT and BAT of Cth^+/+ and Cth^−/− mice, fed with CTRL or CysF diet (n = 4/group). g) Representative EPR spectra of POBN-lipid radical adducts measured in Folch extracts of SFAT, VFAT and BAT tissues of Cth^−/− mice fed with either CTRL or CysF diet. The six-line spectrum (red arrows) is consistent with carbon-centred lipid-derived radicals, indicative of lipid peroxidation (identified through hyperfine coupling constants a^N = 15.75 ± 0.06 G and a_β^H = 2.77 ± 0.07 G). Data are expressed as mean±SEM. Box plots represent median value and extend to the 25^th and 75^th percentiles. Whiskers are plotted down to the minimum and up to the maximum value. Statistical differences were calculated by 2-way ANOVA with Sidak’s correction for multiple comparisons, or by unpaired two-tailed t-tests. Source data

Extended Data Fig. 3. Cysteine starvation induces thermogenic reprogramming of adipose tissue transcriptome.

a) Representative subcutaneous (SFAT), visceral (VFAT), and brown adipose depots (BAT), indicated with white arrows, of Cth^+/+ and Cth^−/− after 6 days of CysF diet. b) Representative H&E-stained sections of VFAT of Cth^−/− mice fed CTRL or CysF diet for 6 days or after Cys supplementation following CysF weight loss (scale bar=100 μm). c) Serum glycerol levels of Cth^−/− mice fed with CTRL (n = 20) or CysF (n = 8) or switched to Cys-containing diet after CysF feeding (n = 10). d) Ucp1, Cidea and Pparg gene expression in Cth^−/− pre-adipocytes differentiated in vitro and treated with increasing concentration of Cysteine for 48 h (n = 6/condition). e) Cumulative food intake during the initial two days of CysF feeding in Cth^+/+ and Cth^−/− mice (n = 10 Cth^+/+ and n = 12 Cth^−/−). f–j) Cth^+/+ and Cth^−/− mice were fed with CysF diet for 6 days and housed in metabolic cages (n = 10 Cth^+/+ and n = 12 Cth^−/−). f) Energy expenditure during CysF feeding. g) Linear regression analysis of unnormalized average energy expenditure measured by indirect calorimetry against body mass on days 4 and 5 of CysF diet. h) Locomotor activity. i) Respiratory exchange ratio (RER) and j) area under the curve (AUC) quantified for RER. k, l) Whole tissue RNA-seq of SFAT, VFAT, and BAT of Cth^+/+ and Cth^−/− fed 6 days of CTRL or CysF diet (n = 4/group). k) Heat map highlighting changes specifically occurring in cysteine deficiency. l) Selected top pathways being up- and down-regulated in Cth^−/− CysF vs CTRL in SFAT after gene set enrichment analysis. m) Gene expression of selected thermogenesis markers confirmed by qPCR in SFAT, in Cth^+/+ and Cth^−/− mice fed with CysF diet (n = 8 Cth^+/+ and n = 10 Cth^−/−). Data are expressed as mean±SEM. Statistical differences were calculated by one-way ANOVA, or by 2-way ANOVA with Sidak’s correction for multiple comparisons, or by unpaired two-tailed t-tests. Source data

Extended Data Fig. 4. Impact of cysteine depletion on transcriptional regulation of adipose tissue at single cell resolution.

a) Experimental design schematic of cell processing of subcutaneous adipose depot (SFAT) stromal vascular fraction (SVF) for scRNA-seq. b) t-SNE plot of scRNAseq from SFAT stromal vascular fraction with c) cluster identities. APCs: antigen presenting cells. ASCs: adipose-derived stromal cells. d) Heat map of normalized gene expression of selected markers to identify major cell lineages. e) Enrichment of CL-316,243 activated gene signature overlaid on all populations in all samples. f) t-SNE plots displaying Dpp4, Cd9, Icam1, Col5a3, F3, and Tagln expression in red across all populations in Cth^−/− CTRL and Cth^−/− CysF samples. g) Volcano plot of differentially expressed genes comparing Cth^−/− CysF and Cth^+/+ CysF in cluster 1. h) Orthogonal validation of adipocyte progenitor changes using FACS analysis of SFAT SVF in Cth^+/+ and Cth^−/− mice on CTRL and CysF diet for 4 days (n = 6 Cth^+/+ CTRL and CysF, n = 5 Cth^−/− CTRL and CysF). i) Selected top pathways from gene set enrichment comparing Cth^−/− CysF vs. Cth^+/+ CysF in cluster 1. j) Heatmap of gene expression of select stem and mature adipocyte genes in clusters 0, 1 and 2 showing the impact of cysteine depletion in mice. Data are expressed as mean±SEM. Statistical differences were calculated by 2-way ANOVA with Sidak’s correction for multiple comparisons, and by unpaired two-tailed t-tests. Panel a created with BioRender.com. Source data

Extended Data Fig. 5. Cysteine-depletion mobilizes lipids for thermogenic response independently of thermoneutrality.

a) Quantification of ATGL immunoblot shown in Fig. 3a, Actin was used as a loading control. b, c) Tissue lipidomics of brown adipose depot (BAT) from Cth^−/− mice fed CTRL (n = 4) or CysF diet (n = 5) for 6 days with b) triglycerides (TG) and c) diacylglycerol species highlighted. AU: arbitrary units. d) Core body temperature (CBT) measured in the peritoneal cavity by implantation of Star-Oddi logger of Cth^−/− mice fed with CTRL or CysF diet over 6 days and e) average day and night CBT of Cth^−/− mice fed with CTRL or CysF diet. Recordings were taken every 30 min (n = 11 Cth^−/− CTRL, n = 12 Cth^−/− CysF, 3 independent experiments pooled). f) Fgf21 gene expression in the liver of Cth^+/+ and Cth^−/− mice fed CTRL or CysF diet for 6 days (n = 8 Cth^+/+ CTRL, n = 10 Cth^+/+ CysF, n = 8 Cth^−/− CTRL, n = 12 Cth^−/− CysF). g, h) Serum levels of g) FGF21 and h) GDF15 in Cth^−/− and Cth^−/−CHOP^−/− mice after 5 days of CysF feeding, measured by ELISA (n = 9 Cth^−/− and n = 7 Cth^−/−CHOP^−/−). i) SFAT, VFAT and BAT weight normalized to body weight of Cth^−/− and Fgf21^−/−Cth^−/− mice after CysF feeding (n = 5/group). j) Respiratory exchange ratio (RER) of Cth^−/− and Fgf21^−/−Cth^−/− mice upon CysF feeding, measured at day 3 and 4 in metabolic cages (n = 5/group). k) Ucp1 gene expression in SFAT of Cth^−/− and Fgf21^−/−Cth^−/− mice after 6 days of CysF feeding (n = 11 Cth^−/− and n = 12 Fgf21^−/−Cth^−/−). l) Representative H&E histology images of SFAT showing increased browning at day 6 in Cth^+/+ and Cth^−/− mice fed CysF diet and housed at 20 °C or at 30 °C. m) Ucp1 gene expression measured by qPCR in BAT of Cth^+/+ and Cth^−/− mice fed CysF diet and housed at 20 °C or at 30 °C for 6 days (n = 3 Cth^+/+20 C, n = 4 Cth^+/+30 C and Cth^−/−20C, n = 5 Cth^−/−30C). Data are expressed as mean±SEM. Box plots represent median value and extend to the 25^th and 75^th percentiles. Whiskers are plotted down to the minimum and up to the maximum value. Statistical differences were calculated by 2-way ANOVA with Sidak’s correction for multiple comparisons, and by unpaired two-tailed t-tests. Source data

Extended Data Fig. 6. Systemic cysteine depletion-induced weight-loss is independent of microbiota and canonical thermogenic pathways.

a) Immunoblot analysis of CTH in liver, kidney, subcutaneous (SFAT), visceral (VFAT), brown (BAT) adipose depots, lung, heart, spleen, and thymus. b) Immunoblot analysis of CTH in kidney samples from male and female Cth^f/f;Alb-Cre- and Cth^f/f;Alb-Cre+ mice and in liver samples from male and female Cth^f/f;Adipoq-Cre- and Cth^f/f;Adipoq-Cre+ mice. Actin is used as a loading control. c, d) Cysteine serum levels of c) Cth^f/f and Alb-Cre;Cth^f/f mice (n = 4 Cth^f/f CysF, n = 5 Cth^f/f CTRL, Alb-Cre;Cth^f/f CTRL and CysF) and d) Cth^f/f and Adipoq-Cre;Cth^f/f (n = 4 Cth^f/f CTRL and CysF, Adipoq-Cre;Cth^f/f CTRL and n = 5 Adipoq-Cre;Cth^f/f CysF) mice after 6 days of CTRL or CysF diet. e–i) Alb-Cre;Cth^f/f mice were fed CTRL or CysF diet for 6 days. Schematic summary of changes in the metabolites in the e) serum and in the f) liver. g) Volcano plot of metabolites identified by MS/MS in the liver. h) Schematic summary of changes in the metabolites and i) volcano plot of metabolites identified by MS/MS in the SFAT. Transsulfuration pathway related metabolites are highlighted in red. Significantly increased or decreased metabolites (-log₁₀(pvalue)>1.3 and ∣log₂(FC)∣>1) are highlighted in blue and listed on the right. Cys: cysteine. Homocys: homocysteine. Met: methionine. SAM: S-adenosyl methionine. SAH: S-adenosyl homocysteine. j) Schematic summary of changes in serum metabolites of Adipoq-Cre;Cth^f/f fed with CTRL or CysF diet for 6 days. Blue lines represent measured, but unchanged metabolites, red and green arrows indicate significantly decreased or increased metabolites, respectively (p < 0.05). See Supplementary Table 6 for the full list of metabolites. k) Percentage body weight change of Cth^+/+ and Cth^−/− mice that were co-housed and fed CysF diet for 6 days (n = 4/group). l) Accumulated food intake of Cth^−/− and Cth^−/− Ucp1^−/− mice during 6 days of CysF diet (n = 7 Cth^−/− and n = 8 Cth^−/− Ucp1^−/−). m, n) RNA-seq based expression of genes associated with m) futile creatine cycle (Slc6a8, Gatm, Gamt, Ckmt2, Alpl and Ckb) and n) futile calcium cycle (Atp2a2 and Ryr2) in the SFAT of Cth^+/+ and Cth^−/− mice fed CTRL or CysF diet for 6 days (n = 4/group). o) qPCR gene expression of Sarcolipin and Atp2a2 in the soleus of Cth^+/+ and Cth^−/− mice fed CTRL or CysF diet for 6 days (n = 3 Cth^+/+CTRL, n = 6 Cth^+/+CysF, n = 3 Cth^+/+CTRL and n = 5 Cth^+/+CysF). p) RNA-seq based expression of genes associated with triglyceride and fatty acid metabolism (Dgat1, Pnpla2, Lipe, Gk) in the SFAT of Cth^+/+ and Cth^−/− mice fed CTRL or CysF diet for 6 days (n = 4/group). q–r) Heatmaps of gene expression of genes involved in creatine, calcium and lipid futile cycles in q) BAT and r) SFAT of Cth^−/− and Cth^−/−Ucp1^−/− mice fed a CysF diet for 6 days (n = 16 Cth^−/−, n = 15 Cth^−/− Ucp1^−/−), quantified by qPCR. Data are expressed as mean±SEM. Box plots represent median value and extend to the 25^th and 75^th percentiles. Whiskers are plotted down to the minimum and up to the maximum value. Statistical differences were calculated by 2-way ANOVA with Sidak’s correction for multiple comparisons, and by unpaired two-tailed t-tests. Panels e, f, h and j created with BioRender.com. Source data

Extended Data Fig. 7. Cysteine starvation induced browning requires adrenergic signalling and reverses high-fat diet induced obesity in mice.

a) Imaging mass spectrometry of noradrenaline in the BAT of Cth^+/+ and Cth^−/− fed 6 days of CTRL or CysF diet. b) qPCR gene expression of Comt in SFAT of Cth^+/+ (n = 8) and Cth^−/− (n = 10) mice fed with CysF diet for 6 days. c) Cumulative food intake during CysF feeding of Cth^−/− treated with L748337, a β-3 adrenergic receptor antagonist or vehicle (PBS) for 5 days (n = 6 PBS and n = 4 L748337). d) Body composition measured by Echo-MRI on day 6 post diet switch (n = 6 Cth^−/− HFD-CTRL and n = 4 Cth^−/− HFD-CysF). e) The glucose tolerance test (GTT) in Cth^−/− mice fed CTRL or cysF diet for 4 days (n = 5 CTRL and n = 6 CysF). Glucose dose was based on lean mass. f) Linear regression analysis of energy expenditure (EE) against body mass during dark cycle of Cth^−/− mice fed with HFD-CTRL or HFD-CysF, average values of nights 4 and 5 of diet switch. Data are expressed as mean±SEM. Statistical differences were calculated by 2-way ANOVA with Sidak’s correction for multiple comparisons, and by unpaired two-tailed t-tests. Source data