Distinct Effects of Cannabidiol on Sphingolipid Metabolism in Subcutaneous and Visceral Adipose Tissues Derived from High-Fat-Diet-Fed Male Wistar Rats

Abstract

1. Introduction

In the 21st century, obesity and type 2 diabetes mellitus (T2DM) have become a global health problem, and the need for the development of novel and safer therapeutic interventions is great. Obesity-related comorbidities generate high costs for health systems and diminish patients’ quality of life. One of these comorbidities is insulin resistance (IR), which is a pathophysiological state where vital metabolic organs (liver, muscle, adipose tissues) demonstrate reduced sensitivity to the activity of insulin [1,2,3]. IR may be distinguished as a risk factor for the occurrence of T2DM together with chronic inflammation and excessive visceral fat deposition [4].

Human adipose tissue is commonly divided, according to the regional fat distribution, into subcutaneous (SAT) and visceral adipose tissue (VAT) [5]. These two classes of fat deposits have intrinsically distinct gene expression profiles and different lipid metabolisms [6,7]. VAT exerts higher lipolytic activity and lower insulin sensitivity than SAT [8]. The accumulation of lipids in VAT impairs insulin sensitivity in metabolically obese, normal-weight individuals [9]. On the other hand, preferential deposition of fat in subcutaneous deposits, such as those detected in most women, positively correlates with metabolically healthy obesity. It is a condition in which, despite the occurrence of obesity, individuals may be protected from T2DM and the development of cardiovascular diseases [10].

Undoubtedly, adipocytes’ insulin sensitivity indirectly regulates liver and muscle function by modulating systemic glucose and fatty acid homeostasis [11]. A large lipid pool in adipose tissue caused by excessive non-esterified fatty acids (NEFAs) influx provokes the hypertrophy of adipocytes and a hyper-lipolytic phenotype that is resistant to insulin [12]. Furthermore, the disruption of lipid metabolism pathways results in elevated plasma levels of triacylglycerols (TAGs) [13], ceramide (CER) [14], and sphingosino-1-phosphate (S1P) [15] in obese individuals [13]. CER and S1P are sphingolipids that are key bioactive signaling molecules in adipose tissue [16]. Altered sphingolipid metabolism induces a dysfunctional adipose tissue phenotype by blocking insulin signaling and adipogenesis and promoting chronic inflammation [17]. Thus, a new therapeutic strategy focused on the modulation of sphingolipid metabolism is needed in order to restore the regular function and insulin sensitivity of adipose tissue [18].

The lack of an effective insulin resistance treatment has led to extensive pharmacological and physiological research, particularly on non-euphoric phytocannabinoids. One of them is CBD, which is found in medicinal and fiber-type Cannabis sativa plants (hemp) and affects lipid metabolism, but, unlike Δ⁹-THC, it lacks abuse potential [19,20]. Many research works have proved that CBD modulates both lipid and glucose homeostasis by targeting various receptors, as well as regulating endocannabinoid levels [21,22,23]. A growing body of studies suggest that CBD may be a pharmacological tool in ameliorating symptoms of insulin resistance, T2DM, and metabolic syndrome [20]. Now, we know that CBD reduces diabetes incidence in non-obese diabetic mice, has beneficial effects on complications from diabetes, and prevents palmitate-induced insulin sensitivity impairment when differentiating bone-marrow-derived mesenchymal stem cells by recovering Akt activation and increasing GLUT4 mRNA expression [24,25]. Nonetheless, there are still no data describing the possible impacts of CBD on insulin signaling in the two deposits of adipose tissue under normal and obese conditions. Similarly, it is unknown if CBD affects the metabolism of sphingolipids in adipocytes. Thus, we performed our study to delineate the novel effect of CBD on sphingolipid metabolism pathways and, as a result, on the development of insulin resistance in subcutaneous and visceral adipose tissue.

2. Results

3. Discussion

Over the last decade, phytocannabinoids, including CBD, have become emerging players in ameliorating the symptoms of insulin resistance, T2DM, and metabolic syndrome [26]. The most common features of the above-mentioned metabolic diseases are disturbances in lipid metabolism and subsequent insulin resistance. This problem is even more alarming because effective treatment of such diseases does not exist.

The effects of CBD on adipocyte lipid metabolism were first explored by Silvestri et al. in an in vitro study, demonstrating that CBD applied simultaneously with oleic acid (OA) reduced triacylglycerol accumulation in 3T3-L1 adipocytes [21]. Because the deposited TAGs may be esterified into various fractions, we resolved to evaluate the influence of CBD on a class of lipids that are more biologically active compared with other lipid fractions, namely, sphingolipids. Thus, in our experiment, which was performed on a rat model, the effects of CBD on sphingolipid concentration and their metabolism, e.g., for ceramide biosynthesis pathway enzymes were assessed. Furthermore, we formulated a hypothesis that CBD, by interfering with the sphingolipid metabolism pathway, may modulate adipocyte insulin sensitivity. Our previous study showed that chronic CBD treatment during excessive fat intake led to the alleviation of de novo CER synthesis, restored content of S1P, and an enhanced insulin signaling pathway and glycogen recovery in rat skeletal muscles [27].

The sphingolipid metabolic pathway includes simple molecules, such as ceramide, and a plurality of more complex sphingolipids, in which ceramide represents the central node in the biosynthesis and catabolism of sphingolipids [28]. The main route of ceramide formation is the de novo synthesis pathway, where the condensation of serine and palmitoyl-CoA by serine palmitoyl transferases (SPTLC1, SPTLC2) takes place [24]. It is well known that nutrient oversupply specifically, with saturated fatty acids intensifies the de novo ceramide synthesis pathway, which generates increased levels of CER in several tissues, including adipose tissue [17,29]. This phenomenon was also demonstrated in the present study in both types of adipose tissue derived from rats fed with a high-fat diet. Importantly, the higher CER level induced by the HFD was reduced in response to CBD injections. Interestingly, the decreased CER content in fat deposits after CBD administration was in line with the study conducted by Łuczaj et al., who indicated a decrease in CER levels in keratinocytes after topical application of CBD in UVA-irradiated rats [30].

The beneficial impact of CBD on subcutaneous adipose tissue observed in our experiment on insulin-resistant rats was mainly achieved through the inhibition of de novo ceramide synthesis, which manifested as a considerable decrease in the ceramide concentration of the precursor sphinganine (SFA). Moreover, this is consistent with the decreased expression of enzymes involved in sphingolipid synthesis, SPTLC2 and CerS6, after the CBD and HFD treatment. It is known that high CerS6 expression in the adipose tissue of obese humans is essential for the formation of unfavorable C16:0 ceramide, which is correlated with the development of insulin resistance in adipocytes, but not the whole body [18,31,32]. As we showed, CBD administration during the HFD feeding attenuated the CerS6 expression induced by lipid oversupply, which strongly suggests its positive metabolic effect. It is worth emphasizing that, in VAT, the inhibition of the de novo ceramide synthesis route was much more pronounced than in subcutaneous fat deposits. In this type of tissue, the reduction in SFA concentration was more visible, and a decreased expression of key de novo ceramide synthesis enzymes SPTLC1, SPTLC2, and CerS2/5 was observed. It is worth highlighting that CBD may act similarly to myriocin, a selective inhibitor of SPTLC that, through the inhibition of ceramide biosynthesis, induces profound changes in skeletal muscle ceramide content with an improvement in systemic insulin action [33]. However, due to the fact that myriocin exerts adverse effects, such as hepatotoxicity [34], CBD appears to be a more beneficial alternative in the treatment of IR.

In addition to the de novo synthesis and sphingomyelin hydrolysis, CER can also be produced from the breakdown of sphingosine through the action of ceramide synthases (CerS1-6) in the salvage pathway, which is even responsible for 90% of total sphingolipid biosynthesis [28,35]. Once formed, CER can undergo degradation because of the actions of ceramidases (ASAH1, ASAH2) toward sphingosine, which can return to the sphingolipid pathway or be transformed by sphingosine kinases (SPHK1, SPHK2) into sphingosine-1 phosphate (S1P) [36,37]. In SAT, the substantial decrease in the intracellular SFO content with the simultaneously lowered expression of enzymes involved in the salvage pathway (CerS6) and CER catabolism (ASAH1) proved the inhibition of both routes after CBD injections in IR rats. On the other hand, in VAT, under conditions of high fatty acid availability, cannabidiol favored ceramide catabolism more than its synthesis in the salvage pathway, which was reflected by the higher SFO level with increased CerS6 and ASAH1 expression. This effect seems to be very important because SFO, as a ceramide derivative with proapoptotic properties, modulates key cellular processes, has thereby been linked with metabolic disorders [38,39]. However, a further step of sphingolipid catabolism in VAT was inhibited, as evidenced by the lower SPHK1 expression with a diminished intracellular S1P content. We hypothesized that the pronounced decline in S1P concentration may also have resulted from the intensified transport of this molecule outside the cells or the redirection of S1P to further degradation into hexadecenal and ethanolamide-1-phosphate. However, at this stage of research, these are just assumptions that should be clarified in the future. Our findings are in accordance with those of the study conducted by Wang et al., where, in mice with diet-induced obesity, SPHK1 deficiency was associated with reduced S1P concentration and enhanced insulin sensitivity in epididymal adipose tissue [40]. Moreover, numerous studies have shown that obese subjects had increased SPHK1 expression accompanied by raised deposition of S1P not only in adipose tissue, but also several other tissues [40,41]. Although the specific role played by S1P in metabolic maladies remains unclear [32], some authors suggested that genetic disturbance of the SPHK1/S1P signaling pathway in HFD-fed mice led to improved systemic insulin sensitivity [40]. Based on this, we suspect that inhibition of SPHK1 by CBD may be considered as a potential therapeutic approach to restoring normal insulin activity in obesity.

Among all of the sphingolipids, there is a particular interest in ceramide because it promotes the development of adipocyte insulin resistance to the greatest extent [42]. Ceramide disrupts transport of glucose into the cells through the dephosphorylation of protein kinase B (Akt/PKB), by blocking the transfer of Akt/PKB to the plasma membrane, or by targeting protein phosphatase 2A (PP2A). Moreover, accumulation ceramide in VAT more than in SAT is associated with obesity and metabolic syndrome [43,44]. Many recent publications have summarized that the diminution of ceramide deposition in adipocytes improved their insulin sensitivity. As a consequence, it contributed to the normalization of glucose homeostasis in the whole body, which also resolved the progression of hepatic steatosis [45,46,47]. In the present study, CBD administration in HFD-fed rats elevated the pAkt (Ser473)/Akt and pGSK3β (Ser9)/GSK3β expression ratios, but only in VAT. These findings corresponded with the reduced ceramide content, which proved the restoration of insulin sensitivity through the action of the examined phytocannabinoid in visceral fat deposits. However, we did not observe any crucial changes in the expression of insulin pathway proteins in SAT between the groups of rats that were fed with a high-fat chow and were untreated or treated with CBD. In this fat deposit, the only change that was observed was an increase in the pGSK3β (Ser9)/GSK3β ratio and the trend toward an increase in the p/Akt (Ser473)/Akt ratio only after CBD treatment. This may have resulted from the reduced lipolytic activity and higher insulin sensitivity of the subcutaneous fat regions, which are specialized to provide long-term storage of excessive fatty acids, thus protecting other tissues from lipotoxic effects [8,48]. These CBD-dependent changes in insulin signaling pathway are consistent with our previous study where two-week CBD administration caused a substantial reduction in insulin plasma concentration and the trend toward a decrease in HOMA-IR value in the HFD-fed rats [27].

Our observations suggest the distinct effects of CBD on two types of fat deposits in the context of intracellular insulin signaling, and they complement the reports on the anti-diabetic properties of CBD [49,50].

4. Materials and Methods

5. Conclusions

Our data indicated that, under conditions of overnutrition, CBD injections modulated sphingolipid metabolism, leading finally to a reduction in the ceramide content in two types of adipose tissues. In visceral adipose tissue, the action of CBD was more directed toward the inhibition of the de novo ceramide synthesis pathway and the intensification of its catabolism. On the other hand, in subcutaneous fat depots, CBD restrained the de novo ceramide synthesis and salvage synthesis routes. All of these changes ultimately sensitized adipose tissue to insulin, especially in the visceral deposits. Therefore, CBD use may be considered as a potential therapeutic strategy for treating or reducing insulin resistance, T2DM, and metabolic syndrome.

Funding Statement

This work was funded by the National Science Center [grant number 2017/26/D/NZ3/01119] and the Medical University of Bialystok [grant number SUB/1/DN/21/007/1118].

Author Contributions

Conceptualization, E.H.-S. and K.B.; methodology, K.K.-N.; software, T.C.; validation, K.B. and K.K.-N.; formal analysis, A.C.; investigation, K.K.-N.; resources, E.H.-S.; data curation, K.B.; writing—original draft preparation, K.B.; writing—review and editing, K.K.-N. and A.C.; visualization, K.B.; supervision, A.C.; project administration, T.C.; funding acquisition, K.B. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the animal Ethics Committee in Olsztyn (No. 71/2018).

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

References