Fig 2: Knockdown of CPT1A inhibits fatty acid oxidation in colon cancer cells.a The expression of CPT1A protein was analyzed in stable control and CPT1A knockdown PT130 and SW480 cells using Western blotting. Two different shRNA targeting sequences (C6 and C7) were used to silence CPT1A in each cell line. β-actin was used as loading controls. b Representative OCR measurements obtained from the FAO tests performed in control (sh-Control) and CPT1A knockdown (sh-CPT1A-C6 and sh-CPT1A-C7) PT130 cells using the Seahorse XF96 Extracellular Flux analyzer. FCCP, ETO, and antimycin A (Anti-A) were added at the indicated points. c Experiments as shown in b were quantified and the relative levels of OCR associated with basal FAO, FAO capacity, and FAO reserve were calculated based on the measurements obtained from the addition of different compounds. Data represent the mean ± SD (n = 3, ¶p < 0.0001 and *p < 0.01). d Representative OCR measurements obtained from the FAO tests in sh-control, sh-CPT1A-C6, and sh-CPT1A-C7 SW480 cells using the Seahorse XF96 Extracellular Flux analyzer. e The relative levels of OCR associated with basal FAO, FAO capacity, and FAO reserve were quantified. Data represent the mean ± SD (n = 3, #p < 0.05, *p < 0.01, and §p < 0.001).

Fig 3: Metabolic reprogramming by loss of endothelial GR loss worsens diabetic kidney disease.a Radiolabeled 14C-palmitate uptake analysis in isolated endothelial cells from kidneys of nondiabetic and diabetic control, GRECKO, Apoe−/−, and DKO mice. CPM of each sample were counted. Data were normalized to μg protein. Fold activity is presented. Endothelial cells isolated and cultured from six biologically independent mice/group were performed in triplicate and analyzed for 14C-palmitate uptake analysis. Data are mean ± SEM. b Radiolabeled [14C]palmitate oxidation and [14CO2]release were measured in isolated endothelial cells from kidneys of nondiabetic and diabetic control, GRECKO, Apoe−/−, and DKO mice. CPM of each sample was counted. Data were normalized to μg protein. Fold activity is presented. Endothelial cells isolated and cultured from six biologically independent mice/group were performed in triplicate and analyzed for 14C-palmitate uptake analysis. Data are mean ± SEM. c Body weight, blood glucose, and kidney weight/body weight were measured at the end of these experiments. N = 6 biologically independent mice from two experiments. Ex vivo radiolabeled [14C]palmitate oxidation and [14CO2]release were measured in kidneys. CPM of each sample was counted. Isolated endothelial cells from five biologically independent mice/group were analyzed in triplicate. Data are mean ± SEM. d Masson trichrome, Sirius red, and PAS staining were analyzed in the kidneys of control-, etomoxir-, and C75-treated diabetic mice. Representative images are shown; original magnification ×300. Relative area of fibrosis (%) and relative collagen deposition (%) were measured using the ImageJ program. N = 8 biologically independent mice per/group from three independent experiments were analyzed. Scale bar 50 μm. Data are mean ± SEM. e Immunohistochemical analysis of CPT1a and β-catenin. Representative images are shown; original magnification ×300. Scale bar 50 μm. N = 6 biologically independent mice per group from three independent experiments. For all panels one-way ANOVA with Tukey post-test was used for the analysis of statistical significance. Significance *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided in the source data file.

Fig 4: Assessment of metabolites and key enzyme related to fatty acid oxidation in skeletal muscles. (A–D) Heatmap of relative acyl‐CoA and acyl‐carnitine levels in skeletal muscles from db/+, db/db and canagliflozin (CANA)‐treated db/db mice. Each column represents the mean metabolite from four mice, where red indicates high metabolite level and blue indicates low metabolite level within the three mouse groups. Relative acyl‐CoA (A) and acyl‐carnitine (B) levels in soleus (SOL). Relative acyl‐CoA (C) and acyl‐carnitine (D) levels in extensor digitorum longus (EDL). The scale bar indicates z‐score values, which are calculated by subtracting the mean peak intensity of metabolites across all samples from the individual peak intensity of a specific metabolite and then dividing the result by the standard deviation. (E) Schematic of key enzymatic steps in fatty acid oxidation. (F) Representative immunoblot images and quantification of carnitine palmitoyltransferase 1 (CPT1) expression in SOL and EDL. Data are expressed as the mean ± SEM. *P < 0.05 (analysis of variance [ANOVA], n = 4). ns, not significant.

Fig 5: Regulation of fatty acid metabolism-related genes by HIF-1α under hypoxia. A qRT-PCR analysis of mRNA expression levels of genes encoding fatty acid β-oxidation (FAO)-related enzymes ACSL1, CPT1, MCAD, and LCAD, as well as fatty acid transporter CD36, in HIF-1α knockdown (KD) and scramble control (SC) HepG2 and Hep3B cells. B Western blot analysis of ACSL1, CPT1, MCAD, LCAD, and CD36 protein expression in KD and SC cells. C Quantification of Western blot analysis of ACSL1, CPT1, MCAD, LCAD, and CD36 protein expression. D qRT-PCR analysis of mRNA levels of triglyceride (TG) synthesis-related genes APGAT and DGAT in KD and SC cells. E Western blot analysis of APGAT and DGAT protein expression in KD and SC cells. F Quantification of Western blot analysis of APGAT, DGAT protein expression. Data are expressed as the mean ± standard error of the mean (SEM) and are representative of three independent experiments (A, D). N.S.: not significant, *P < 0.05, **P < 0.01 versus control