FoxO1-zDHHC4-CD36 S-Acylation Axis Drives Metabolic Dysfunction in Diabetes

Abstract

Background: The fatty acid (FA) transporter CD36 (FA translocase/cluster of differentiation 36) is the gatekeeper of cardiac FA metabolism. Preferential localisation of CD36 to the sarcolemma is one of the initiating cellular responses in the development of muscle insulin resistance and in the type 2 diabetic heart. Post-translational S-acylation controls protein trafficking, and in this study we hypothesised that increased CD36 S-acylation may underpin the preferential sarcolemmal localisation of CD36, driving metabolic and contractile dysfunction in diabetes.

Methods: Type 2 diabetes was induced in the rat using high fat diet and a low dose of streptozotocin. Forkhead box O1 (FoxO1) transcriptional regulation of zDHHC4 (zinc finger DHHC-type palmitoyltransferase 4) and subsequent S-acylation of CD36 was assessed using chromatin immunoprecipitation (ChIP) sequencing, ChIP-quantitative polymerase chain reaction, luciferase assays, siRNA (small interfering RNA) and shRNA silencing.

Results: Type 2 diabetes increased cardiac CD36 S-acylation, CD36 sarcolemmal localisation, FA oxidation rates and triglyceride storage in the diabetic heart. CD36 S-acylation was increased in diabetic rats, db/db mice, diabetic pigs and insulin-resistant human iPSC-derived cardiomyocytes, demonstrating conservation between species. The enzyme responsible for S-acylating CD36, zDHHC4, was transcriptionally upregulated in the diabetic heart, and genetic silencing of zDHHC4 decreased CD36 S-acylation. We identified that zDHHC4 expression is under the regulation of the transcription factor FoxO1. Diabetic mice with cardiomyocyte-specific FoxO1 deletion had decreased cardiac zDHHC4 expression and decreased CD36 S-acylation, which was further confirmed using diabetic mice treated with the FoxO1 inhibitor AS1842856. Pharmacological inhibition of zDHHC enzymes in diabetic hearts decreased CD36 S-acylation, sarcolemmal CD36 content, FA oxidation rates and triglyceride storage, culminating in improved cardiac function in diabetes. Conversely, inhibiting the de-acylating enzymes in control hearts increased CD36 S-acylation, sarcolemmal CD36 content and FA metabolic rates in control hearts, recapitulating the metabolic phenotype seen in diabetic hearts.

Conclusions: Activation of the FoxO1-zDHHC4-CD36 S-acylation axis drives metabolic and contractile dysfunction in the type 2 diabetic heart.

Keywords: cardiovascular diseases; diabetic cardiomyopathies; heart failure; insulin resistance; myocardial infarction.

Figure 1.

Diabetes increases cardiac fatty acid metabolism associated with increased sarcolemmal CD36 (fatty acid translocase/cluster of differentiation 36). Diabetic hearts had increased fatty acid oxidation rates (A), fatty acid oxidation per unit work (B), and myocardial triglyceride concentrations (C) compared with control hearts. Total CD36 protein (D and E) was not significantly increased, but sarcolemma content of CD36 was increased in diabetic hearts compared with controls (F), with no significant differences in endosomal CD36 content (G). Data (A, B, E, and G) were compared using a 2-tailed unpaired t test and (C and F) were compared using a Mann-Whitney U test (data show the mean±SEM).

Figure 2.

Diabetes increases cardiac CD36 (fatty acid translocase/cluster of differentiation 36) S-acylation across species. Diabetic rats had increased cardiac CD36 S-acylation (A and B) compared with controls. C, CD36 is S-acylated on 4 cysteine residues in hearts from control and diabetic rats. Cardiac CD36 S-acylation is increased in diabetic pigs (D), db/db mice (E), and insulin-resistant human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) (F) compared with their respective controls. Data (B and D) were compared using a 2-tailed unpaired t test and (E and F) compared using a Mann-Whitney U test (data show the mean±SEM). +HA or CBF indicates S-acylated protein (hydroxylamine); CUF, unacylated protein; −HA, negative control (minus hydroxylamine); and IF, input fraction.

Figure 3.

Diabetes increases the expression of the S-acylating enzyme zDHHC4 (zinc finger DHHC-type palmitoyltransferase 4). A and B, Perfusion with a high-fat buffer decreased CD36 (fatty acid translocase/cluster of differentiation 36) S-acylation compared with low-fat perfused heart. C, Representative Western blot images showing zDHHC4, zDHHC5 (zinc finger DHHC-type palmitoyltransferase 5), and APT1 (Acyl protein thioesterase 1) protein levels in control and diabetic hearts. APT1 (D and E) and zDHHC5 (F and G) protein and mRNA expressions were not significantly different between control and diabetic hearts. H, In contrast, zDHHC4 total protein was significantly increased in diabetic hearts compared with controls. zDHHC4 mRNA from diabetic rats (I) and diabetic mice (J) were significantly increased compared with their respective controls. Data (B–F, H, and I) were compared using a 2-tailed unpaired t test and (G and J) were compared using a Mann-Whitney U test (data show the mean±SEM). +HA indicates S-acylated protein (hydroxylamine); and −HA, negative control (minus hydroxylamine).

Figure 4.

The transcription factor FoxO1 (forkhead box O1) drives enhanced zDHHC4 (zinc finger DHHC-type palmitoyltransferase 4) expression. A, Genomic regions around the mouse zDHHC4 gene alongside tracks showing chromatin immunoprecipitation (ChIP)-seq signal for the promoter mark H3K4Me3 in mouse cardiomyocytes and the FoxO1 transcription factor in adult mouse heart. JASPAR transcription factor track on University of California Santa Cruz (UCSC) browser identified 5 mouse-rat conserved FoxO1 binding motifs within the promoter region (core motif in bold alongside the JASPAR sequence logo for FoxO1 in both orientations; A). FoxO1 target gene enrichment scores (D) and heat map visualization of FoxO1 target genes demonstrate clustering between control and insulin-resistant human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM; B). C, Single-sample enrichment scores for the FoxO1 pathway for the genes belonging to the FoxO1 pathway were increased in insulin-resistant hiPSC-CM compared with controls. E, FoxO1 binds to the zDHHC4 promotor in H9c2 (H9c2 myoblasts) cardiomyocytes measured by chromatin immunoprecipitation/quantitative (q)PCR, which is absent in the IgG (immunoglobulin G) control group. F, Transfection of a luciferase reporter construct encoding the zDHHC4 promoter into H9c2 cardiomyocytes demonstrated increased luciferase activity in the presence of the FoxO1 wild-type (WT) plasmid relative to empty vector and FoxO4 WT plasmid. Data (C and E) were compared using a Mann-Whitney U test (data show the mean±SEM). Data (D) present the Benjamini and Hochberg false discovery rate (FDR)–corrected P value for the enrichment of FOXO (controlling the FDR for the 1115 total pathways included). Data (F) were compared using a Kruskal-Wallis test with the Dunn multiple comparison post hoc test (data show the median±95% CI).

Figure 5.

A FoxO1 (forkhead box O1)-zDHHC4 (zinc finger DHHC-type palmitoyltransferase 4)-CD36 (fatty acid translocase/cluster of differentiation 36) S-acylation axis in diabetes. In human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM; A) and endothelial sEnd.1 (skin derived endothelial cells 1) cells (B), transfection with FoxO1 siRNA (small interfering RNA) decreased the expression of zDHHC4 mRNA relative to nontarget pool (NTP) control siRNA. C, In contrast, FOXO1 siRNA did not significantly change the expression of ZDHHC5 zinc finger DHHC-type palmitoyltransferase 5) in hiPSC-CM. D and E, In hiPSC-CM, transfection with ZDHHC4 siRNA decreased CD36 S-acylation. In neonatal rat ventricular myocytes, lentivirus transduction with zDHHC4 shRNA decreased CD36 S-acylation, relative to control scramble shRNA (F and G). Expression of zDHHC4 mRNA (H) and CD36 S-acylation (I and J) were increased in diabetic αMHC^Cre (Alpha-myosin heavy chain Cre) mice compared with chow-fed αMHC^Cre controls but not in the diabetic FoxO1^{Cardiac−/−} littermates. zDHHC4 mRNA expression (K) and CD36 S-acylation (J and L) were decreased in diabetic αMHC^Cre mice pharmacologically treated with the FoxO1 inhibitor AS1842856 compared with vehicle-treated diabetic αMHC^Cre mice. Data (A–G and L) were compared using a Mann-Whitney U test and (K) compared using an unpaired t test (data show the mean±SEM). Data (H and I) were compared using aligned ranks transform-based nonparametric ANOVA, with estimated post hoc pairwise contrasts through the method described by Elkin et al and the Benjamini-Hochberg false discovery rate (FDR) correction procedure (data show the median±95% CI). +HA indicates S-acylated protein (hydroxylamine); and −HA, negative control (minus hydroxylamine).

Figure 6.

Pharmacologically inhibiting the S-acylating zDHHC (zinc finger DHHC-type palmitoyltransferase) enzymes corrects metabolism and function in the diabetic heart. The zDHHC inhibitor cyano-myracrylamide (CMA; A) decreased CD36 (fatty acid translocase/cluster of differentiation 36) S-acylation in diabetic hearts compared with untreated diabetic hearts (B and C). Sarcolemmal CD36 (D), fatty acid oxidation rates (E), and myocardial triglyceride concentrations (F) were decreased in diabetic hearts treated with CMA compared with untreated diabetic hearts. CMA treatment of diabetic hearts significantly improved cardiac function as assessed by rate pressure product (G) compared with untreated diabetic hearts. Data (C–G) were compared using a 2-tailed unpaired t test (data show the mean±SEM). +HA indicates S-acylated protein (hydroxylamine); and −HA, negative control (minus hydroxylamine).

Figure 7.

Preventing deacylation in control hearts recapitulates the excessive fatty acid metabolism seen in diabetic hearts. The APT1 (Acyl protein thioesterase 1) inhibitor ML348 (N-[2-chloro-5-(trifluoromethyl)phenyl]-4-(2-furanylcarbonyl)-1-piperazineacetamide) (A) increased CD36 (fatty acid translocase/cluster of differentiation 36) S-acylation in control hearts compared with untreated control hearts (B and C). Sarcolemmal CD36 (D), fatty acid oxidation rates (E), and myocardial triglyceride concentrations (P=0.08; F) were increased in control hearts treated with ML348 compared with untreated hearts. ML348 treatment of control hearts depressed cardiac function (P=0.05) as assessed by rate pressure product (G) compared with untreated control hearts. Data (C–G) were compared using a 2-tailed unpaired t test (data show the mean±SEM). +HA indicates S-acylated protein (hydroxylamine); and −HA, negative control (minus hydroxylamine).