Cardiomyocytes function and survival during ischemic stress heavily rely on glucose uptake stimulation. However, in the diabetic myocardium chronically exposed to excess of circulating lipids, this mechanism is impaired, rendering the diabetic myocardium more vulnerable to ischemia reperfusion injury (IRI). Excessive fatty acids (FA) provision and oxidation induces a metabolic inflexibility of the diabetic myocardium, which impairs glucose uptake and utilization. Moreover, the excess of FA that are not oxidized can be either stored into lipid droplets or used to produce toxic lipid derivatives, such as diacylglycerol. On the other hand, excessive FA oxidation increases acetyl- CoA production and reduces nicotinamide adenine dinucleotide (NAD+) levels, favoring protein acetylation over deacetylation. Increased diacylglycerol (DAG) accumulation and protein acetylation levels are two mechanisms potentially involved in the FA-induced impairment of cardiac glucose uptake. Previous studies showed that inducing lipid droplets formation restored glucose uptake and protected against lipotoxicity. However, the mechanism remains elusive.

In this study, we investigated the roles of lipid droplets and NAD+-dependent deacetylation in the regulation of glucose uptake in cardiomyocytes. We induced LD with either tetradecanoyl phorbol acetate (TPA) or the AMP-activated Protein Kinase (AMPK) agonist 5-aminoimidazole-4- carboxamide-1-β-D-ribofuranoside (AICAR). Triacylglycerol biosynthesis enzymes were inhibited in cardiomyocytes exposed to FA ± LD inducers, either upstream (glycerol-3-phosphate acyltransferases; GPAT) or downstream (diacylglycerol acyltransferases; DGAT) of the diacylglycerol step. We demonstrated that chronic exposure to FA in cardiomyocytes leads to increased intracellular diacylglycerol levels, resulting in the activation of protein kinase C d (PKCd) and impaired glucose uptake during metabolic stress. However, GPAT inhibition, by reducing intracellular diacylglycerol levels, prevented PKCd activation and improved metabolic stress-stimulated glucose uptake in FA-exposed cardiomyocytes. Interestingly, we observed that promoting lipid droplets biogenesis protected glucose uptake in FA-exposed cardiomyocytes through a mechanism unrelated to intracellular diacylglycerol levels. This protection involved a distinct management of exogenous FAs within the cardiomyocytes, as assessed by reduced exogenous diacylglycerol/ triacylglycerol ratio.

We further investigate the role of FA-induced NAD+ reduction in myocardial lysine acetylation and glucose transport. We showed that restoring NAD+ levels resulted in reduced protein lysine acetylation and improved metabolic stress-stimulated glucose uptake in FA-exposed cardiomyocytes. Furthermore, we found that FA-induced NAD+ reduction led to increased acetylation of the hydroxyacyl-CoA dehydrogenase trifunctional enzyme subunit a (HADHA), which enhanced FA uptake and oxidation. However, replenishing NAD+ levels prevented HADHA acetylation and reduced FA utilization in FA-exposed cardiomyocytes. Moreover, changes in acetylation state of HADHA were associated with changes in pyruvate dehydrogenase (PDH) phosphorylation, suggesting a potential link to glucose uptake during metabolic stress. In conclusion, this study highlights the critical role of lipid droplets formation in the management of exogenous FA, which impact on the glucose uptake during metabolic stress