In this study, we demonstrated for the first time that CBD, a non-psychotropic plant-origin cannabinoid, reduces the expression of LCFA transport proteins and intramuscular lipid accumulation with simultaneous inhibition of de novo lipogenesis in skeletal muscle of rats in the course of obesity induced by a high-fat diet. Moreover, we have shown that CBD under these conditions is a modulator of the elongation and desaturation processes, which positively influenced FAs metabolism.

In our study, as we expected, the induction of obesity by feeding rats a HFD significantly increased the expression of all examined LCFAs transporters (CD36, FABPpm, FATP-1, and FATP-4) in the red gastrocnemius muscle, while in the white skeletal muscle only expression of FABPpm and FATP-1 was enhanced. These differences are probably related to the metabolism presented by these types of muscles, where white skeletal muscle exhibits glycolytic metabolism and red skeletal muscle exhibits oxidative metabolism³¹. In addition, we found that CBD appears to function as a regulator of the expression of LCFAs transport proteins, as its two-week administration markedly attenuated the total expression of specific LCFAs transporters, i.e., CD36, FABPpm, FATP-1, and FATP-4 in both types of skeletal muscles. In line with that, we observed similar effects of CBD action on the expression of fatty acid protein carriers in the myocardial tissue, which we described in our previous work³². It is worth emphasizing, that CBD had the greatest effect on the muscular expression of CD36, which is believed to play a pivotal role in maintaining cellular lipid homeostasis^12,33. Numerous studies have shown that complete deletion or downregulation of CD36 expression significantly reduces the uptake of LCFAs, thereby causing changes in fatty acid metabolism, especially concerning fatty acid oxidation, which is important in the working heart and skeletal muscles³⁴. Other researchers have also shown that the overexpression of CD36 caused by lipid oversupply, increases the influx of LCFAs into the cell, exceeding the oxidative capacity of mitochondria, which in turn results in the storage of excessive amounts of FAs in the TAG fraction and its further conversion to more metabolically active lipid metabolites (i.e., DAG, CER), which are known to inhibit the insulin signaling pathway, promoting the development of IR³⁵. Furthermore, recent studies have shown the unexpected properties of CD36 to promote de novo lipogenesis in hepatocytes by regulating SREBP1 processing³⁶. Zeng et al. found that CD36 directly interacted with an insulin-induced gene (INSIG) to attenuate its inhibitory effects on SREBP1 proteolysis. Increased processing of SREBP1 upregulated the expression of de novo lipogenesis enzymes such as acetyl-CoA carboxylase α (ACCα) and FAS, which resulted in elevated hepatic lipid accumulation³⁶. Considering the above, CBD downregulates protein-mediated LCFAs transport in myocytes of obese rats, which is desired therapeutic effect in future clinical studies.

It is well known that in the course of obesity there is the deposition of lipids in non-adipose tissues such as skeletal muscle⁹. In our research, we confirmed it by noting a significant increase in intramuscular accumulation of FAs in various lipid fractions including FFA, DAG, TAG, and PL in both muscle types in fatty acids oversupply conditions, which is in line with a significant upregulation of LCFAs transport proteins expression in either white and red skeletal muscle. However, we have observed a much greater FAs accumulation in the red gastrocnemius muscle in all examined lipid fractions. It is consistent with the metabolism shown by this type of muscle tissue, since the red muscle fibers use FAs as the primary source of energy in the oxidation process, while white skeletal muscles obtain energy through the glycolytic pathway³¹. Moreover, it is also associated with a more pronounced expression of membrane LCFAs transport proteins in the red skeletal muscle³¹. Particular attention should be paid to the increased accumulation of lipids not only in the TAG fraction but also in the fraction of DAG, which was observed in our study in the red skeletal muscle. DAG is one of the prime candidates among the lipid derivatives responsible for the development of IR. Many studies have shown that it disrupts the insulin signaling pathway by activating both classic and atypical protein kinase C (PKC), which dephosphorylates insulin receptor substrate 1 (IRS1) and thus blocks further downstream insulin signaling, thereby reducing insulin-stimulated glucose uptake^37,38. In contrast, TAG is considered to be a relatively safe fraction that does not exhibit intracellular lipotoxic activity, because both the activation and inhibition of TAG intramuscular hydrolysis do not significantly affect the mitochondrial function and cellular insulin sensitivity³⁹. Accordingly, the herein study demonstrated that in animals receiving a HFD after a two-week administration of CBD, we noticed a remarkable decrease in TAG content in the white gastrocnemius muscle, whereas in the red skeletal muscle CBD injections significantly reduced lipid accumulation in FFA, DAG, and TAG fractions. This is in line with the results obtained by Silvestri et al.²² where they showed that CBD dose-dependently inhibits TAG accumulation in hepatocytes and 3T3-L1 adipocytes treated with oleic acid (OA). Interestingly, only in the case of the PL fraction, we observed that chronic CBD treatment caused a pronounced increase in the content of accumulated lipids in the rats fed a HFD in both white and red skeletal muscle. We can hypothesize that CBD enhances the incorporation of FAs into the PL fraction and significantly influences the composition of the above-mentioned fraction in favor of n-3 PUFA, as we demonstrated in our previous work⁴⁰. Concomitantly, in our experiment, we reported intensified muscular de novo lipogenesis in rats subjected to high-fat feeding in both white and red gastrocnemius muscle as indicated by increased lipogenic indexes. De novo lipogenesis is a complex metabolic pathway in which excess carbohydrates are converted into FAs, which further are esterified and stored as TAG fraction⁴¹.This also underlines the importance of de novo lipogenesis process in the skeletal muscle, especially in the state of IR related to obesity⁴².

Furthermore, recent research has shown the relationship between ECS and FAs metabolism. Osei-Hyiaman et al.⁴³ demonstrated that hepatic CB₁ activation results in enhanced de novo fatty acid synthesis via the induction of the lipogenic transcription factor SREBP-1c and its target enzymes including FAS. Interestingly, in our previous research⁶, we revealed that CBD downregulated muscle CB₁ receptor expression, however, we can assume that CBD in skeletal muscle affects FAS expression through a different molecular mechanism than in the liver, due to the fact that both tissues differ significantly in terms of metabolic processes occurring in them. In addition, CB₁ has also been shown to be associated with SCD1 activity. A study conducted by Liu et al. revealed that various overlapping mechanisms contribute to the development of IR, more specifically stimulation of the ECS by CB₁ receptor activation in the course of high-fat diet-induced obesity promotes the activity of SCD1, an enzyme that catalyzes the biosynthesis of MUFA^44,45. The same study also revealed that endogenous MUFAs (palmitoleic and oleic acid) act as fatty acid amide hydrolase (FAAH) inhibitors leading to a reduction in AEA degradation and an elevation in its concentration, which stimulates the ECS. Furthermore, elevated levels of MUFA activate CB₁ receptors that promote de novo lipogenesis by inducing the expression of lipogenic genes, including SCD1, forming a positive feedback loop⁴⁵. This is in accordance with our studies, which also showed a pronounced upregulation of SCD1 activity in rats fed a HFD in all examined lipid fractions in the red and white gastrocnemius muscle, only with the exception of the PL fraction in the glycolytic type of muscle. The two-week administration of CBD significantly reduced the level of SCD1 in the DAG and TAG fractions in the white muscle, again affecting the red muscle to a greater extent, where it decreased SCD1 activity in the FFA, DAG, and TAG lipid pools. Based on the above data, we may suspect that the effects of CBD are related to the inhibition of the endocannabinoids action at the CB₁ receptor level since CBD has been well-described as a negative allosteric modulator of this cannabinoid receptor⁴⁶. Additionally, in our experimental model, we examined the muscle expression of fatty acid desaturase 1 (FADS1; also known as delta-5 desaturase (∆5D)) and fatty acid desaturase 2 (FADS2; referred as delta-6 desaturase (∆6D)), enzymes involved in the double bond introduction at delta ∆5 and ∆6 positions of PUFA chain, respectively¹⁹. Our analysis, after treatment of HFD-fed rats with CBD, indicated substantially decreased expression of FADS1 in the red gastrocnemius muscle and FADS2 in the white skeletal muscle. We believe that this may be considered as a cell protection mechanism since Yashiro et al. reported that FADS1 inhibition alleviates IR and reduce body weight in a HFD-induced obese (DIO) mouse model⁴⁷. Moreover, FADS1 catalyzes the production of arachidonic acid (20:4, AA), which is a precursor of pro-inflammatory eicosanoids, therefore, its accumulation promotes the synthesis of AA-derived mediators that contribute to the development of chronic inflammation related to obesity⁴⁸. Similarly, a growing number of studies demonstrated a positive correlation between increased FADS2 expression and IR as well as TD2M occurrence, whereas FADS2 inhibition resulted in resistance to HFD-induced obesity in a mouse model^49,50. Intriguingly, in the white gastrocnemius muscle we also noticed a substantial rise in the FADS1 expression in the HFD + CBD group. This is in line with the results from our previous publication, where we discovered that the most pronounced changes considering the AA and eicosapentaenoic acid (EPA) content (the synthesis of which is controlled by FADS1), were observed in the same experimental group in different lipid fractions in the white skeletal muscle⁴⁰. However, a stronger protective effect of CBD treatment was observed in the red gastrocnemius muscle, where it caused a significant shift in the n-6/n-3 PUFA balance towards the anti-inflammatory n-3 PUFA in examined lipid fractions, which we can associate with the metabolism exhibited by this muscle type. This taken altogether, can lead to the presumption that CBD in conditions of elevated FAs consumption inhibits de novo fatty acids synthesis and reduces their intracellular accumulation in the skeletal muscle in obesity.

In addition, we examined for the first time the effect of CBD action on the expression of the elongase family members, where we revealed substantial alternations in the elongation process in both white and red skeletal muscle. Overall, LCFAs supplied with the diet and formed during de novo lipogenesis (mainly 16:0; palmitic acid) can be elongated by adding two carbon units to the carboxyl end of LCFAs, using malonyl-CoA and fatty acyl-CoA as substrates. This process is catalyzed by the ELOVLs (Fig. 9). Our findings indicate that a HFD increases elongation of 16∶0 to stearic acid (18∶0) in the DAG, TAG, and PL lipid fractions in the skeletal muscle of rats regardless of the “presented metabolism”. Intriguingly, CBD treatment in high-fat feeding markedly elevated the 18:0/16:0 FA elongation ratio in the following fractions: FFA, DAG, and TAG in both muscle types. We also observed similar effects of CBD administration in rats subjected to the standard diet, where it enhanced a ratio of 18:0/16:0 elongation in the DAG, TAG, and PL fractions in the white gastrocnemius muscle, and in all examined lipid fractions in the red gastrocnemius muscle. Possibly, increased elongation of 16:0 by treatment with CBD can help prevent a buildup of 16∶0 in favor of 18∶0 in the skeletal muscle of obese rats, whichcan be considered as a cell protection mechanism. Accordingly, the increased elongation of palmitic acid to stearic acid reflects a significant rise in the expression of the ELOVL6 enzyme in rats fed a HFD in both muscle types, which was subsequently diminished by CBD. This is worth emphasizing since Matsuzaka et al.⁵¹ showed that mice with Elovl6 gene deletion were resistant to the development of diet-induced IR despite the presence of obesity, suggesting that inhibition of elongase 6 may represent a novel approach to treat IR. In the case of further elongation of the FAs chain, our results show that the supply of a HFD to rats significantly increased the elongation ratio of stearic acid (18:0) to arachidic acid (20:0), arachidic acid to behenic acid (22:0) and behenic acid to lignoceric acid (24:0) in the FFA, DAG and PL lipid pools in either muscle with glycolytic and oxidative metabolism, whereas injections with CBD caused a significant reduction of the above-mentioned indexes. In addition, our research showed that the TAG fraction was affected to a lesser extent, and surprisingly chronic exposure to a HFD resulted in a decrease in the 20:0/18:0 and 24:0/22:0 indexes in the white gastrocnemius muscle, while in the red skeletal muscle, 20:0/18:0 and 22:0/20:0 ratios were reduced. However, the implementation of CBD treatment significantly diminished the elongation of 22:0 to 24:0 in both muscle types as well. Moreover, in our study, we investigated the muscular expressions of ELOVL1 and 3, the enzymes that elongate mostly SFA and MUFA providing precursors for the synthesis of sphingolipids⁵². The total expression of ELOVL1 and ELOVL3 was significantly enhanced in both white and red gastrocnemius muscles of the rats subjected to a HFD, which is consistent with results obtained by Kozawa et al.⁵³, and coincides with the increase in elongation ratios determined in our study (18:0/16:0, 20:0/18:0, 22:0/20:0 and 24:0/22:0). Although, in the oxidative fibers, we did observe only a trend towards downregulation in the muscular expression of elongase 1 and 3 in the HFD + CBD group, while in the case of anaerobic fibers, two-week CBD administration significantly reduced total ELOVL3 expression and surprisingly increased the expression of ELOVL1 in the same group of animals. Collectively, these findings implicate that lipid oversupply to skeletal muscle, results in increased elongation of SFA chains, which, take part in further metabolic transformations, and intensify the accumulation of compounds such as CER and DAG, since most SFAs in mammals are found to be components of sphingolipids family⁵². These active biomolecules, as indicated by a substantial body of evidence, impair the insulin-signaling cascade by inhibiting insulin-stimulated phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3 (GSK3β), and subsequent GLUT4 translocation from the intracellular compartments to the plasma membrane, which partially was also showed in our previous work^6,54. Collectively, the interactions between the above processes are quite complex, and examined enzymes are involved in different synthetic pathways that can alter the levels of various LCFAs pools in response to distinct factors, which is the basis for further research examining the exact mechanisms of CBD action.