Extended Data Fig. 6: Systemic cysteine depletion-induced weight-loss is independent of microbiota and canonical thermogenic pathways.
From: Cysteine depletion triggers adipose tissue thermogenesis and weight loss
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 Cthf/f;Alb-Cre- and Cthf/f;Alb-Cre+ mice and in liver samples from male and female Cthf/f;Adipoq-Cre- and Cthf/f;Adipoq-Cre+ mice. Actin is used as a loading control. c, d) Cysteine serum levels of c) Cthf/f and Alb-Cre;Cthf/f mice (n = 4 Cthf/f CysF, n = 5 Cthf/f CTRL, Alb-Cre;Cthf/f CTRL and CysF) and d) Cthf/f and Adipoq-Cre;Cthf/f (n = 4 Cthf/f CTRL and CysF, Adipoq-Cre;Cthf/f CTRL and n = 5 Adipoq-Cre;Cthf/f CysF) mice after 6 days of CTRL or CysF diet. e–i) Alb-Cre;Cthf/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;Cthf/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 25th and 75th 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.
