Extended Data Fig. 8: The inhibitory effect of TGR5 on DHHC4 is cAMP-PKA-dependent.
From: Inhibition of fatty acid uptake by TGR5 prevents diabetic cardiomyopathy
a, Representative western blot images (left) and comparisons (right) of CD36 palmitoylation in cardiac tissues. CD36 palmitoylation was determined in mice treated with NCD and HFD/STZ for 24 weeks. n = 6. b, Flow comparisons of BODIPY C-16 uptake in primary NMCMs from TGR5fl/fl and TGR5ΔCM mice transfected with siDHHC4 or siCont and treated with PA + OA for 24 h. n = 6. c, Representative western blot images of palmitoylated CD36 in cardiomyocytes. PA + OA were incubated for 24 h in primary NMCMs in the presence or absence of INT-777 and PKA inhibitor PKI. CD36 palmitoylation was determined by ABE assay. n = 6. d, HEK293A cells were transfected with the indicated vectors and treated with 1μmol/L PKI for 1 h before stimulation with INT-777 for 6 h. DHHC4 was then enriched by GST pull-down. Phosphorylation of DHHC4 was detected using p-PKA substrate antibody. n = 6. e, The graphical summary mediated by TGR5 in the regulation of DbCM. Briefly, TGR5 deficiency results in the suppression of cAMP/PKA signaling in diabetes. Inactivated PKA reduces the phosphorylation of substrate DHHC4 and activates DHHC4, resulting in increased CD36 palmitoylation. Palmitoacylated CD36 is localized to the plasma membrane, thus promoting FFA uptake and aggravating cardiolipotoxicity. Created with BioRender.com with modifications. Data are presented as mean ± SEM. Statistical significance was evaluated by two-tailed unpaired Student’s t-test (a), two-way ANOVA followed by Tukey’s post-hoc test (b,c), or one-way ANOVA followed by Tukey’s post-hoc test (d).