Cardiovascular disease is the leading cause of mortality in type 2 diabetes (T2D) patients. Metabolic dysfunction is apparent within the T2D heart, presenting with an increased reliance on lipid metabolism and decreased glucose metabolism. Emerging work indicates that the main fatty acid (FA) transport protein, CD36, is enhanced on the sarcolemma in T2D, driving an increase in FA uptake. The lipid post translational modification S-acylation has been linked to regulating CD36 trafficking. However, it remains unknown whether CD36 is S-acylated in the heart and whether CD36 S-acylation levels are altered in T2D.
This work began through investigating the S-acylation status of substrate membrane transport proteins. CD36 was shown to be S-acylated in the heart on four cysteine residues, while the glucose transporters (i.e GLUT1 and GLUT4) and fatty acid transporter FABPpm were not S-acylated. Furthermore, in hearts from T2D rats, CD36 S-acylation was increased 2.5-fold and associated with a 2.4-fold increase in CD36 localisation at the sarcolemma. The changes in CD36 were accompanied by a 25% increase in fatty acid oxidation (FAO) rates and 65% increase in triglyceride (TG) concentrations in diabetes. The regulatory S-acylating enzyme, DHHC4, was increased 2.5-fold in diabetes compared with controls. Furthermore, DHHC4 was shown to have a binding motif for the transcription factor FoxO1, and blunting of FoxO1 decreased DHHC4 mRNA levels in T2D, indicating that ehanced DHHC4 expression in diabetes is driven by increased FoxO1 transcription.
This thesis then went on to explore the manipulation of S-acylation using increased palmiate concentrations and pharmacological compounds that inhibit the S-acylating DHHCs and de-S-acylating acyl-protein thioesterases (APTs). Acute increases in palmiate concentrations decreased CD36 S-acylation in Langendorff perfused control hearts, indicating that the abundance of palmitate does not actively drive increases in CD36 S-acylation. In T2D rat hearts, the broad spectrum DHHC inhibitor CMA decreased CD36 S-acylation by 34% with a 24% decrease in CD36 localised on the sarcolemma. Treament with CMA also reduced FAO rates by 21% with a 48% decrease myocardial TG concentrations compared to untreated T2D hearts. The decrease in lipid metabolism was accompanied by a 43% increase in cardiac function in T2D hearts perfused with CMA. In contrast, in control hearts, the inhibitor of the de-S-acylating enzyme APT1 (ML348) increased CD36 S-acylation by 32% with a 30% increase in FAO rates.
The last part of this thesis explored alterations in CD36 in human heart failure (HF) biopsies and myocardial ischemia in rodents. In HF tissue, sarcolemmal CD36 was decreased 29% and were positively correlated with left ventricle ejection fraction (LVEF). In ischemic rat hearts, CD36 S-acylation was not altered compared to non- ischemic controls, whereas the metabolic regulators AMPK and ACC were shown to have enhanced S-acylation in ischemia, suggesting S-acylation as a potential novel regulator of the metabolic response in ischemia.
In conclusion, this works provides compelling evidence that there is an increase in lipid metabolism in diabetes due to enhanced CD36 S-acylation driven by FoxO1 mediated increases in DHHC4 expression. Pharmacologically manipulating CD36 S-acylation levels has profound effects on downstream metabolism. Lastly, CD36 is altered in human HF tissue and ischemia is a stimulus for enhanced S-acylation of AMPK and ACC in rat hearts
