Fig 1: Key enzymes of ketone body oxidation are expressed in cardiac endothelial cells AOxidation of ketone bodies requires the enzymes SCOT and BDH1 and yields acetyl-CoA which enters the tricarboxylic acid (TCA) cycle.BImmunoblot of SCOT, BDH1, and the loading control VCP across various organs obtained from adult C57Bl/6J mice.C, DQuantification of SCOT and BDH1 expression levels relative to VCP in various murine organs.EImmunoblot of SCOT, BDH1, CD31, and the loading control VCP in murine endothelial cells isolated from heart, muscle, lung, and adipose tissue.FImmunoblot of SCOT, BDH1, CD31, and the loading control VCP in human cardiac ECs, HUVECs, and adipose tissue ECs.GImmunoblot of SCOT, BDH1, CD31, and VCP in cardiac endothelial cells (EC) and non-endothelial cell fraction (non-EC) isolated from adult C57Bl/6J mice.HImmunoblot of SCOT, BDH1, and ß-actin in immortalized murine cardiac endothelial cells (MCEC).I, JKetone body concentration in cell culture medium of MCEC treated with 2 mM D-ß-hydroxybutyrate (ß-OHB) or 2 mM acetoacetate (AcAc) after 24 h. Data are presented as mean ± SD. n = 3; *P < 0.05; ***P < 0.001 unpaired Student’s t-test. Source data are available online for this figure.
Fig 2: Ketone ester diet increased expression level of key ketogenic enzymes.After 4 weeks of feeding, tissue protein lysates were prepared to measure protein expression level of key ketogenic enzymes. A, A schematic representation of ketogenic pathway in the heart is shown. B, Representative blots of mitochondrial ß-hydroxybutyrate dehydrogenase (BDH1), succinyl-CoA:3-ketoacid transferase (SCOT), and mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) are shown. Summary data of (C) BDH1, (D) SCOT, and (E) ACAT1 are displayed. Data expressed as mean±SEM with n as the number of samples in the bar graphs. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001, and it is calculated using 2-way ANOVA with Tukey’s post hoc multiple comparison. Dashed line represents the basal level of protein expression in wild-type (WT) animals fed a control diet. This level was set as 100% and used for normalization of protein expression in WT animals treated with ketone ester (KE) and in diabetic animals treated with control and KE diet.
Fig 3: Cardiac endothelial cells isolated from mice fed a ketogenic diet Relative mRNA levels of cardiomyocyte, fibroblast, and smooth muscle cell marker genes in anti-CD31-isolated cells compared to levels in total heart lysates of C57Bl/6J mice.Flow cytometry analysis of anti-CD31-isolated cells from hearts of C57Bl/6J mice using the endothelial cell marker CD31 and the immune cell marker CD45.Relative fold changes of Oxct1 (Scot) and Bdh1 expression levels in mice fed a ketogenic diet relative to a control diet were obtained from RNAseq analyses.Relative mRNA levels of cardiac ECs isolated from mice fed a ketogenic or control diet confirming increased levels of pro-proliferative genes Hmgcs2 and Pdk4.Heat-map showing expression levels of genes involved in regulation of cell cycle (KEGG). Data are presented as mean ± SD. n = 3. Two-tailed unpaired Student’s t-test; **P < 0.01; ***P < 0.001.
Supplier Page from Abcam for Anti-BDH1 antibody