For the first time, our study has shown the relationship between myocardial sphingolipid metabolism and CBD treatment in high-fat diet-induced obesity in rats. We found that this phytocannabinoid in such conditions is a modulator of the de novo ceramide synthesis pathway along with the salvage route, however, to a different extent in the plasma and cardiac muscle. We noticed a remarkable decrease in both SFA and SFA1P myocardial contents after CBD treatment during a high-fat feeding regime (Scheme 1) [16]. Possibly due to the extracellular sphingolipid efflux, e.g., from the liver, significantly increased concentrations of these two sphingolipids were observed in Wistar rats’ blood plasma. We believe that this might be considered as a cell protection mechanism against ceramide synthesis since SFA is a primary component of the ceramide de novo synthesis pathway. Three different metabolic pathways of CER generation are defined: (1) de novo synthesis pathway via serine palmitoyltransferase and sphinganine formation, (2) sphingomyelin hydrolysis via sphingomyelinase, and (3) the salvage pathway—a synthesis of ceramide from sphingosine via ceramide synthase (Scheme 1). Although our study did not demonstrate significant differences in CER content in the cardiac tissue after 2-week CBD treatment in a high-fat-diet group in comparison with HFD alone, we noticed a considerable elevation in blood plasma CER concentration in the same experimental group. An extracellular rise of CER content might be correlated with its enhanced cell efflux, for instance, from the liver, to reduce the intracellular CER accumulation, especially when there is FAs oversupply [17]. This sphingolipid, when accumulated, is known to demonstrate numerous destructive cell effects, including the development of metabolic disorders such as impairments of insulin signaling pathway and related insulin resistance [18,19]. Parra et al. demonstrated that CER affecting mitochondrial dynamics might be involved in the apoptosis of cardiomyocytes [20]. With regard to the above-mentioned matter, Law et al. showed that very-long-chain ceramides can develop lipotoxic effects, e.g., mitochondrial dysfunction, oxidative stress, and cell apoptosis in cardiomyocytes [21]. Interestingly, a study conducted by Bielawska et al. demonstrated that CER affects signal transduction leading to cardiomyocytes’ apoptosis during heart ischemia and reperfusion injury [22]. Considering the enzymes involved in the sphingolipid pathway, we observed a remarkable decrease in the expression of enzymes controlling the de novo ceramide synthesis pathway, namely SPTLC1 and 2, as well as LASS5 and 6 after 2-week CBD treatment in HFD fed Wistar rats, which is in line with the above-mentioned downregulation. Our study also indicated that CBD treatment in high-fat feeding lowered the expression of proteins from the salvage pathway, i.e., SPHK1 and 2, with simultaneous augmentation of the expression of neutral ceramidase in comparison with the high-fat diet-fed rats (Scheme 1). The above-mentioned alterations in the myocardial expression of enzymes are in accordance with the decreased intracellular concentration of two sphingolipid fractions, which are ceramide precursors—SFA in the de novo synthesis pathway and SFO in the salvage pathway (a trend toward reduction). This might lead to the presumption that CBD in conditions of increased FAs consumption inhibits two different ceramide synthesis pathways. We are aware of the fact that this should be confirmed in isolated primary cardiomyocytes with the additional presence of a specific inhibitor (e.g., myriocin—a serine palmitoyltransferase inhibitor); therefore, it can be considered as a limitation of this study. Nonetheless, we do believe that the obtained data undoubtedly revealed a causal relationship between downregulation of the de novo ceramide synthesis pathway and 2-week CBD administration in FAs oversupplied rats, which in the long-term may prevent negative consequences of CER accumulation in the heart. Although we did not observe a significant difference in myocardial tissue S1P content between examined groups, there was a trend toward its enhancement in the HFD+CBD group, whereas plasma concentration of this sphingolipid derivative was reduced after CBD introduction irrespective of applied diet type. Some researchers emphasize that signaling of S1P may exhibit both anti- and pro-fibrotic effects in cardiac fibrosis [23]. Furthermore, studies conducted on mice demonstrated that S1PR1 (sphingosine-1-phosphate receptor 1) signaling might be considered as a novel therapeutic option for hypertension via vascular relaxation [24]. Other studies carried out by Jin et al. showed that S1P exhibits a cardioprotective role since it protects the ex vivo heart from ischemia–reperfusion injury [25]. It corresponds with the recent study revealing vasoprotective effects of chronic CBD treatment in hypertensive rats, such as a reduction of hypertrophy and improvement of the endothelium-dependent vasodilation [26]. Simultaneously, we have shown in the previous research that chronic administration of CBD (10 mg/kg) was not successful in reducing both blood pressure value and heart rate in an animal model of primary and secondary hypertension (spontaneously and deoxycorticosterone hypertensive rats) [27]. Taking into account the above and present data, it is probable that CBD indirectly, through alternations in sphingolipid metabolism, triggers vaso- and cardioprotective outcomes. Considering the concentration of sphingomyelin, we observed a trend toward a decrease in its content in the high-fat-diet group exposed to CBD, which means that CBD probably leads to an activation of the SM breakdown. Our data are consistent with studies conducted by Burstein et al., who evaluated the effect of CBD on SM content in fibroblasts and demonstrated a considerable decrease in SM concentration after exposure to CBD treatment [28]. It is known that sphingomyelinases are widely related to cardiovascular diseases [29]. Although our Western blot analysis after treatment with CBD indicated decreased expression of both enzymes regulating the metabolism of SM—alkaline and neutral sphingomyelinase, where a significant difference in comparison to the HFD group was observed in N-SMase expression. Interestingly, Klevstig et al. indicated that inhibition of acid sphingomyelinase might result in the reduction of CER accumulation in the post-ischemic cardiac tissue. However, the same study also revealed that inhibition of SMase in mice did not improve heart function after induced myocardial infarction despite decreasing CER storage [30]. Moreover, a study conducted by Hernandez et al. on rats cardiac myocytes indicated that neutral SMase and the subsequent signaling of CER might be one of the earliest cardiac tissue responses to oxidative stress in a model of myocardial ischemia [31].

It is known that a higher intake of fat-rich food leads to increased circulating levels of LCFAs in the coronary circulation, which promotes their uptake across the plasma membrane to the cardiomyocytes. Three membrane-associated fatty acid protein transporters are known to greatly facilitate LCFAs uptake in cardiomyocytes (up to 80% of the total FAs transmembrane transport), i.e., CD36, FABPpm, and FATP1, 4, and 6 [32]. A number of animal studies and human clinical trials based on models of obesity or type 2 diabetes mellitus revealed that accumulation of different lipid fractions, including CER, is correlated with an increased expression of these protein transporters, mainly CD36 and FABPpm, in metabolically active tissues, i.e., the heart, adipose tissue, skeletal muscles, and liver [33,34]. To our knowledge, this study is the first in which it is demonstrated a remarkable decline in the expression of all five examined transporters, i.e., CD36, FABPpm, FATP1, 4, and 6, after 2-week CBD treatment under conditions of high-fat diet-induced obesity in Wistar rats (Scheme 1). Therefore, based on the above data, we can assume that CBD via its influence on LCFA transporters possibly leads to a diminished accumulation of lipids and their derivatives in the cardiomyocytes also through the de novo CER synthesis pathway, which is consistent with lower heart mass in HFD fed rats after CBD treatment even though the total body mass was not altered. This, in turn, can be considered as a cardioprotective mechanism during increased dietary fatty acid intake. However, the exact mechanism by which CBD decreases the total myocardial expression of LCFAs protein transporters should also be examined in the in vitro model. One of the hypotheses, which should be further investigated, includes peroxisome proliferator-activated receptors (PPARα and PPARβ/δ) since they serve as a link between LCFA protein transporters and the ECS [35,36]. Nevertheless, it should be underlined that in our research, a reduced expression of fatty acid transporters (a protective mechanism against lipotoxicity) is closely related with downregulated de novo ceramide synthesis pathway in the cardiac muscle of obese rats as the consequence of lower LCFAs influx. Our observations are in line with other studies in which CBD treatment resulted in a significant reduction of intracellular lipid droplets (e.g., TAGs fraction) as well as lipogenesis in hepatocytes [37].

The expanded endocannabinoid system includes elementarily G-protein-coupled receptors, known as CB₁ and CB₂, which mediate the effects of cannabinoids. The CB₁ receptor is principally expressed in the central nervous system, whereas CB₂ is the predominant cannabinoid receptor within the immune system’s cells [38]. Cannabidiol is known to interact with various complex signaling systems involved in eCBome; however, it has a very low affinity to cannabinoid receptors [39]. We can distinguish two endocannabinoids (eCBs) that are endogenous agonists of CB₁ and CB₂ receptors—anandamide (AEA) and 2-arachidonoylglycerol (2-AG), [40]. Our study indicated a significant decrease in the myocardial CB₂ receptor expression in the HFD+CBD group in comparison with both control and HFD groups. This downregulatory effect of the CBD treatment on CB₂ receptor expression was causally related with lowered expression of both fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL; a trend toward a decrease) in the HFD group after 2-week CBD application. Since FAAH catalyzes the hydrolysis of AEA to arachidonic acid and ethanolamine [41], its inhibition by CBD in high-fat diet-fed rats probably resulted in an increased level of eCBs, which in turn provoked downregulation of the myocardial expression of the CB₂ receptor. Even though CBD was successful in diminishing FAAH expression, it did not affect augmented by high-fat-diet expression of CB₁, presumably due to the fact that CBD itself is known to be a negative allosteric modulator of this cannabinoid receptor. Additionally, FAAH, apart from AEA and 2-AG, degrades endocannabinoid-like molecules, namely N-oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA) [42]. It was shown that OEA binds PPARα (with high affinity) and PPARβ/δ, whereas PEA is an agonist of only PPARα. Interestingly, Fu et al. displayed that OEA triggers satiety and reduces body weight gain in wild-type mice, but not in PPARα knockout animals [43]. Moreover, PPARα and β/δ are regulators of lipid metabolism, also through modulation of the expression of LCFAs protein transporters (e.g., CD36, FABPpm, or FATP1) in different tissues, including cardiac muscle [36,43]. Since in our study, we observed a considerable decline in the FAAH expression in the HFD+CBD group (vs. HFD-fed rats), it is probable that via OEA and/or PEA PPARs activation was affected, which in turn produced changes in de novo CER synthesis pathway and the expression of LCFAs protein transporters. Therefore, we can hypothesize that PPARs link sphingolipid metabolism, the ECS, and fatty acid transporters, which should be confirmed in in vitro experiments. Moreover, endocannabinoids are considered to be very promising cardioprotective molecules. A number of studies indicated their significant role in both physiological conditions [44] and various cardiac pathologies, e.g., myocardial infarction, hypertrophy, or hypertension affecting inflammatory response, infarct size, or blood pressure value [13,45]. A study conducted by Bátkai et al. demonstrated on FAAH knockout mice a reduced inflammation, oxidative/nitrative stress, and cardiomyocytes apoptosis in age-related cardiac dysfunctions, compared to the wild types of mice [46].