Diabetes is a chronic metabolic disease characterized by the deregulation of glucose and lipid metabolism, a process that aggravates myocardial damage. However, hyperglycemia and glycemic variability are not the only determinants of heightened cardiovascular risk in diabetes. Indeed, research suggests that aggressive glycemic control does not significantly reduce major cardiovascular events (Nusca et al., 2018). Other metabolic disorders such as ectopic lipid accumulation, impaired lipid metabolism, and chronic inflammation are associated with increased cardiovascular risk in diabetic and prediabetic states (Cai et al., 2021).

According to a recent meta-analysis of 10 randomized clinical trials (71,553 participants), novel antidiabetic drugs, including sodium–glucose cotransporter 2 (SGLT2) inhibitors like empagliflozin, reduce total and cardiovascular mortality in diabetic and nondiabetic individuals (Deshpande et al., 2023). Although the exact mechanism behind the cardioprotective effects of SGLT2 inhibitors is not clear, they are understood to positively affect myocardial energetics, including the metabolism of ketone bodies, lipids, and fatty acids. These effects may occur via several independent mechanisms, many of which are not yet fully understood. The beneficial cardioprotective effects of SGLT2 inhibitors are linked not only to their impacts on left ventricular remodeling (Chen and Peng, 2023) and hemodynamics (Gronda et al., 2022), but also to their systemic metabolic effects and direct effects on the heart itself.

Several studies suggest that SGLT2 inhibitors may modulate lipid metabolism independently of their glucose-lowering effects (Szekeres et al., 2021; Yaribeygi et al., 2022). Based on our previous study, which employed a prediabetic model (Hüttl et al., 2021), and other studies performed using models of diet-induced obesity followed by the onset of type 2 diabetes mellitus (Petito-da-Silva et al., 2019; Nasiri-Ansari et al., 2021), empagliflozin treatment decreases hepatic lipid accumulation and improves markers of nonalcoholic fatty liver disease (NAFLD). Clinical studies have also reported improvements in hepatic fat accumulation in diabetic subjects after empagliflozin treatment (Kahl et al., 2020; Chehrehgosha et al., 2021). However, little is known about the effect of SGLT2 inhibitors on lipid accumulation and metabolism in the heart that can be directly associated with the cardiovascular benefits of SGLT2 inhibitors. One in vitro study focusing on cardiomyocytes reported that canagliflozin may attenuate myocardial lipotoxicity and inhibit inflammatory signaling pathways in the heart (Zhang et al., 2023).

Recent research suggests that SGLT2 inhibitors may improve cardiac metabolism by shifting myocardial substrate utilization from glucose toward to lipids and ketone body oxidation (Xi et al., 2022). In our previous study, performed using a prediabetic model with serious vascular complications (Hüttl et al., 2020), we confirmed alterations in substrate utilization in the heart after empagliflozin administration. However, the utilization of ketone bodies was not affected.

We hypothesize that lipids, particularly their metabolism in the heart, contribute to the cardioprotective effects of SGLT2 inhibitors, not only by supplying an important source of energy, but also by interfering with various signaling pathways, including pathways involved in the inflammatory response.

In this study, we explore the possible effect of empagliflozin, an SGLT2 inhibitor, on the metabolism of lipids, fatty acids and ketone bodies in the heart, and compare these effects with lipid metabolism in the liver using a prediabetic model, the hereditary hypertriglyceridemic (HHTg) rat (Vrana and Kazdova, 1990; Zicha et al., 2006). This rat strain exhibits genetically determined hypertriglyceridemia, insulin resistance in peripheral tissues, fatty liver, and ectopic lipid accumulation in the absence of obesity, fasting hyperglycemia, or essential hypertension. In our previous study of this rat strain (Miklankova et al., 2021), atherogenic dyslipidemia and myocardial lipotoxicity was associated with impaired glucose oxidation and increased lipogenesis in the heart.

It is becoming increasingly evident that the cardiovascular benefits observed after treatment with SGLT2 inhibitors are not exclusively related to glycemic control and reduced glucotoxicity. Although the mechanism underlying these benefits is poorly understood, a mounting body of research suggests that modulations in inflammation and energy metabolism play a key role.

A reduction in glucose calories induced by SGLT2 inhibitors such as empagliflozin is consistent with increased lipid and ketone body mobilization (Szekeres et al., 2021). In our study, empagliflozin treatment improved serum dyslipidemia, first of all decreased serum triglycerides levels, but had no effect on circulating NEFAs or serum ketone bodies. HHTg rats typically exhibit chronically elevated levels of circulating NEFAs. However, in our study, treatment with empagliflozin did not further affect these NEFA values. Despite no change in NEFA concentration, empagliflozin significantly altered the FA profile of the NEFA lipid class, which is likely to have had a major impact on the transport and uptake of fatty acids in the myocardium. Qualitative changes in the FA profiles of NEFAs are believed to contribute to the effect of empagliflozin on the metabolism of adipose tissue. Although empagliflozin only slightly affected lipolysis in our study, it may have altered the composition of FAs released from adipose tissue. Cardiac energy metabolism depends on the availability, concentration, and FA profiles of circulating NEFAs. As previously described (Markova, et al., 2019), our prediabetic rat strain exhibits chronically elevated NEFA levels accompanied by qualitative changes in the NEFA lipid class, which may be partially attributed to a reduction in the n-3 PUFA profile. In humans, according to meta-analysis with type 2 diabetes patients SGLT2 inhibitors as monotherapy given only minor effect on serum lipid profile (Lazarte et al., 2021). In type 2 diabetes patients, significant increases in total, LDL and HDL cholesterol were observed in SGLT2 inhibitors treated-patients. Moreover, a reduction of serum triglycerides is described in both clinical and preclinical studies with SGLT2 inhibitors (Salvatore et al., 2022). The increase in HDL cholesterol and decreased in triglycerides is mediated partly through improved insulin secretion and sensitivity.

Ectopic lipid deposition and myocardial lipotoxicity play important roles in the development of myocardial damage, both structurally and functionally, and have been shown to affect substrate utilization and the development of cardiomyopathies (Xi et al., 2022). Although HHTg rats do not exhibit left ventricular hypertrophy, they do tend to accumulate lipid and lipotoxic intermediates in the heart, which is associated with accelerated lipogenesis. A previous study of ours demonstrated that this rat strain is characterized by an imbalance in substrate utilization in the myocardium, reflected in reduced glucose oxidation and increased FA oxidation (Miklankova et al., 2021). This observation tallies with a human study of diabetic patients, which found that increased intracardiac TAG concentration is associated with left ventricular remodeling and reduced cardiac energy (Levelt, et al., 2016).

In our study, empagliflozin treatment led to a reduction in the accumulation of myocardial lipids, which is a predictor of improved myocardial energy metabolism. This reduction was also observed in neutral TAGs as well in lipotoxic DAGs and LPCs. These lipotoxic metabolites interfere with intracellular signaling pathways, resulting in the impairment of cell function, partial impairment of insulin signaling, and enhanced inflammation (Ormazabal, et al., 2018). It has even been suggested that DAGs are an independent predictor of cardiac fibrosis (Marin-Royo, et al., 2018). Thus, a reduction in lipotoxic intermediates likely contributes to the anti-inflammatory effect of empagliflozin in the heart and consequent improvements in myocardial insulin resistance.

Our results indicate that a reduction in the lipogenic enzyme Scd-1 along with changes in the FA profile of NEFAs may contribute to decreased accumulation of myocardial lipids. Scd-1 is an important factor in maintaining efficient cardiac lipid metabolism through the lipogenesis of cardiomyocytes and can also improve cardiac function by shifting substrate utilization in the myocardium (Balatskyi and Dobrzyn, 2023). Therefore, decreased gene expression of Scd-1 after empagliflozin treatment not only contributes to a reduction in the accumulation of myocardial lipids, but can also optimize the utilization of glucose and lipids in the myocardium. Based on our previous study and others (Ormazabal et al., 2018; Miklankova et al., 2021), metabolic flexibility in the myocardium is impaired in an insulin-resistant state, which is associated with an imbalance between lipid and glucose uptake by cardiomyocytes, leading to intramyocardial accumulation. SGLT2 inhibitors reduce lipogenesis via several cellular pathways, including Scd-1 and SREBP-1c. They are also understood to stimulate FA oxidation. However, the effect of empagliflozin on gene expression involved in FA oxidation in the myocardium is unclear.

Composition of the phospholipids PC and PE may contribute to a reduction in lipid accumulation in the myocardium after empagliflozin treatment. The PC/PE ratio influences mitochondrial energy metabolism (van der Veen, et al., 2017) and can modulate the organization of lipid rafts in membrane regions (Gholam, et al., 2023). By altering the PC/PE ratio, empagliflozin can modulate membrane fluidity and inflammatory responses while mediating their antifibrotic effects. Precisely how SGLT2 inhibitors affect phospholipids in the myocardium is not known. However, the authors of one study of diabetic mice found that dapagliflozin altered the density of lipid rafts in the membranes of proximal tubular cells (Gholam et al., 2023).

According to our results, the effect of empagliflozin treatment on the myocardium is likely associated with the metabolism of n-3 PUFA and arachidonic acid and the generation of bioactive metabolites originating from these metabolic lipid pathways. We observed that empagliflozin influenced inflammatory metabolites generated by lipid metabolism, which is associated with altered arachidonic acid and n-3 PUFA metabolism. Interestingly, the EPA profiles of myocardial phospholipids decreased, which could be related to the generation of anti-inflammatory EET metabolites. However, the EPA profile in circulating NEFA increased. Our results support the premise that EPA is more likely to be transported into the myocardium, where it is then metabolized into anti-inflammatory metabolites. For FAs to be metabolized, they must be released from the membrane. This means that an increase in the profile of membrane-bound arachidonic acid might not necessarily lead to the formation of proinflammatory metabolites. Our results are in line with previous studies of diabetic rats, where arachidonic acid metabolites were shown to worsen cardiac function (Yousif, et al., 2009) and contribute to the development and progression of cardiac hypertrophy (Zordoky and El-Kadi, 2008). In our study, an increase in the arachidonic acid profile and a decrease in the EPA profile in myocardial phospholipids resulted in a decrease in HETEs and an increase in EET concentrations. The two eicosanoids 20-HETE and 14,15-EET play an important role in the pathogenesis of myocardial damage, modulating inflammation and oxidative stress during the development of diabetic complications and in the heart. In addition, EETs help to regulate the cardiovascular system by exerting a vasodilatory and natriuretic effect. For instance, 20-HETE can affect insulin signaling. The potent effect of 20-HETE on the cardiovascular system involves stimulating the contractility and proliferation of smooth muscle cells, as well as activating endothelial dysfunction and inflammation. Dietary DHA can reduce the plasma level of 20-HETE, highlighting the interaction between DHA metabolites and HETEs (Xi et al., 2022). The mechanism by which empagliflozin affects 20-HETE in the myocardium is not yet understood. However, multiomics analysis of a rat model with diabetic cardiomyopathy indicates that the perturbation of PUFA biosynthesis and arachidonic acid metabolism are responsible for the effects of empagliflozin (Xi et al., 2022). In our study, the decreased profile of oleic acid in myocardial phospholipids is consistent with the theory that it serves as a preferential substrate in the regulation of FA oxidation. Although the mechanism is poorly understood, it may be potentially linked to PPARα activation (Finck, et al., 2003).

In addition to affecting inflammatory metabolites generated by lipids, empagliflozin treatment also has a positive effect on the myocardial levels of the inflammatory cytokines TNFα and IL-10. In our study, the decrease in myocardial TNFα was not associated with a decrease in myocardial TNFα gene expression or with alterations in the circulating levels of TNFα. On the other hand, the increase in anti-inflammatory IL-10 in the myocardium was also evident in serum. According to one study, a significant interrelationship exists between IL-10, TNFα, and heart failure (Kaur, et al., 2009). IL-10 suppresses the production of TNFα and other proinflammatory cytokines such as IL-1, IL-6, and IL-8. However, TNFα is a mediator of cardiac pathology via inflammation pathways and the activation of apoptosis. Cardiac myocytes isolated from rat and mouse hearts were shown to contain TNFα, which suggests that heart cells are capable of producing TNFα (Kaur et al., 2009). Thus, the effect of empagliflozin may be directly attributed either to TNFα production in the heart or to the release of TNFα from adipose tissue, in particle epicardial tissue. Consistent with our results, some studies have drawn attention to the possible anti-inflammatory properties of SGLT2 inhibitors. A reduction of gene expression in proinflammatory cytokines, including IL-6 and TNFα, after empagliflozin treatment was demonstrated in the hearts of obese diabetic rats (Aragon-Herrera, et al., 2019). In studies involving type 2 diabetic patients, treatment with canagliflozin has lowered the serum levels of leptin and IL-6 (Garvey, et al., 2018), while an increase in the serum level of anti-inflammatory IL-10 has been observed after empagliflozin treatment (Iannantuoni, et al., 2019). The modulation of inflammatory signaling pathways involving NFκB inhibition and activation of AMPK signalization are understood to contribute to the anti-inflammatory properties of empagliflozin (Hawley, et al., 2016). In addition, several studies have shown that SGLT2 inhibitors can affect macrophage polarization from the M1 to the M2 subtype (Koyani, et al., 2020), albeit not in the context of cardiovascular and myocardial injury. In agreement with our results, changes in the FA profiles of myocardial phospholipids, particularly an increase in the n-3 PUFA profile, has been linked to the influence of macrophage polarization. Thus, it is possible that reduced TNFα in the heart after empagliflozin treatment is associated with decreased M1 macrophages. According to this theory, the effect of macrophage membrane polarization is to reduce infiltration of inflammatory macrophages in the heart. In summary, the anti-inflammatory effect of empagliflozin is probably either reflected in indirect improvements in metabolism or direct modulation of inflammatory signaling pathways in the heart along with inflammatory cytokine release.

Empagliflozin may also have a cardioprotective effect on ketone bodies in the myocardium (Packer, 2023). In our study, empagliflozin treatment had no significant effect on either BHB concentration or utilization in the myocardium. However, empagliflozin administration did lead to a partial normalization of the reduction in myocardial BHB levels. That empagliflozin did not affect circulating concentrations of BHB, which are determinants of myocardial concentrations, may partly explain this effect. However, our results are also consistent with a general trend observed in human studies, which is that circulating ketone bodies do not increase after the administration of SGLT2 inhibitors in nondiabetics (Marilly, et al., 2022). Ketone bodies are believed to have antioxidant, anti-inflammatory, and antifibrotic effects on the heart and may also function as important signaling molecules. These effects are probably used more than as an alternative energy sources. In our previous study, which employed a prediabetic model with serious vascular complications (Huttl et al., 2020), we noted alterations in substrate utilization in the myocardium after empagliflozin administration; however, we observed no effect on the utilization of ketone bodies in the myocardium. According to the authors of one study involving mice with heart failure, ketone bodies may contribute to the anti-inflammatory effect of empagliflozin in the heart (Byrne, et al., 2020). However, in our study, we found that the possible anti-inflammatory effects of empagliflozin related more to the generation of anti-inflammatory lipid metabolites and anti-inflammatory cytokines in cardiac tissue.

Moderate weight loss may also indirectly contribute to a reduction in cardiovascular impairment after empagliflozin treatment. In our study, empagliflozin reduced both total body weight and visceral adipose tissue weight. Interestingly, whole body weight and visceral adipose tissue weight loss occurred despite a slight increase in food intake after empagliflozin administration, a finding supported by other studies (Petito-da-Silva et al., 2019; Hojna, et al., 2021). Reduced visceral adipose tissue is probably the main mechanism involved in body weight loss. Nevertheless, other mechanisms such as increased basal metabolism (Kim, et al., 2018) and increased body temperature (Xu et al., 2017) may also contribute to weight loss following treatment with SGLT2 inhibitors. According to a recent study, empagliflozin may reduce epicardial adipose tissue mass (Sato et al., 2018), indirectly contributing to a reduction in myocardial inflammation. Our study excluded measurements of subcutaneous and epicardial adipose tissue mass. However, adipose tissue mass and metabolic activity may have an impact on cardiovascular pathology.

According to recent studies (Petito-da-Silva et al., 2019; Nasiri-Ansari et al., 2021) and our research (Huttl et al., 2021), empagliflozin can ameliorate NAFLD development, which is one of several independent cardiovascular risk factors. In one experimental model exhibiting fatty liver, empagliflozin treatment reduced hepatic lipogenesis and the accumulation of ectopic lipids and lipotoxic intermediates. In our study, although these effects were roughly comparable in the liver and in the heart, the effect of empagliflozin on lipid metabolism, reflected in the production of lipid metabolites, was more pronounced in the heart than in the liver. It may be the case that empagliflozin primarily influences lipogenesis in the liver, even though its effect on this organ was less potent than its effect on the heart in our study. In our previous study (Huttl et al., 2020), empagliflozin modulated the expression of genes involved in lipogenesis and lipid storage, including Scd-1, Fas, Srebp1, and Pparγ, in the liver. However, genes involved in lipid oxidation were not affected. To our knowledge, no study has compared the effects of empagliflozin on lipid metabolism in the heart and in the liver, and moreover, myocardial lipid metabolism was monitored in relation to myocardial ketone body metabolism. On the other hand, cardiac functions were not directly measured in our study, nor were changes in myocardial structure directly monitored. Furthermore, it is important to note that the alterations in lipid metabolism are part of the pleiotropic effects of SGLT2 inhibitors, and other mechanisms such as affecting hemodynamic parameters, including decrease in blood pressure and extracellular volume, have a significant cardioprotective effect associated with the SGLT2 inhibitors treatment (Abdul-Ghani et al., 2016).

In summary, empagliflozin may exert cardioprotective effects by positively influencing both myocardial and hepatic lipid metabolism and reducing inflammation in the heart. Empagliflozin decreased the accumulation of ectopic lipids and lipotoxic intermediates and reduced lipogenesis. We speculate that the anti-inflammatory and antifibrotic effects of empagliflozin are attributed to its influence on the metabolism of n-3 PUFA and arachidonic acid in the heart. The cardioprotective effects of empagliflozin might be connected with alterations in myocardial and hepatic lipid metabolism in prediabetic states with severe dyslipidemia.