Extended Data Fig. 2: TNFα promotes glutaminolysis in white adipocytes.
From: Reduced adipocyte glutaminase activity promotes energy expenditure and metabolic health
a. Pathway analysis of genes positively associated with GLS mRNA expression and negatively associated with GLUL mRNA expression. The size of the dots is proportional to the Gene Set Enrichment Analysis score. b. Correlation between GLUL/GLS mRNA expression and TNFα secretion ex vivo in human subcutaneous adipose tissue in cohort 3 (n = 45). r- and p values are shown for correlation using simple Pearson´s regression analysis. c. Freshly isolated human adipocytes were cultured with or without TNFα for 24 hrs. Analyses of gene expression by qPCR (six replicates per condition, repeated >three times) (left) and glutamine and glutamate levels including glutamine-to-glutamate ratio (six and five replicates per condition) in conditioned media (right) were performed. Data were analyzed by Student’s t-test and p values are shown. d. Analyses of gene expression by qPCR in human adipocytes incubated with vehicle (n = 8), TNFα (n = 4), isoprenaline (n = 4) or insulin (n = 4). Data were compared using one-way ANOVA with Dunnett’s post-hoc test and p values are shown (experiment repeated >three times). e. Representative examples of protein levels of GLS and the inflammatory pathways induced by TNFα (JNK, STAT3 and NFκB) in human adipocytes incubated with TNFα for four hours with or without inhibitors of each pathway (repeated two times). Proteins from the same samples were loaded on three different gels in parallel. f. Chromatin immunoprecipitation followed by qPCR analysis showing that c-Jun binds to the human GLS promoter. Complexes containing c-Jun were immunoprecipitated from cross-linked and digested chromatin isolated from human adipocytes incubated with PBS or TNFα. Following reversal of cross-links and purification of DNA, qPCR was run using primers designed to amplify a 196-bp fragment centered around the putative c-Jun binding site at position −247 to −125 bp relative to the transcriptional start site (TSS). The data presented are the relative quantity values from independent duplicate reactions. Data in panels c-d show mean ± S.E.M. Abbreviations: TNFα = Tumor necrosis factor alpha, TG = triglycerides, -insulin = cells deprived from insulin for 24 hours, +insulin = insulin treatment for six hours, isoprenaline = isoprenaline treatment for six hours.
