To the best of our knowledge, the following study is the first to propose new areas of interest for CBG’s medical use in obesity-related complications therapy, demonstrating that CBG treatment has a positive effect on the intramuscular phospholipids fraction by changing the content of individual PLs subclasses and modifying their FAs composition, which is associated with the attenuation of local inflammatory response in the skeletal muscle of insulin-resistant rats subjected to a high-fat, high-sucrose diet.

It is well-established, that obesity is associated with alterations in FAs metabolism, including partitioning between oxidation, storage, and changes in membrane structure, which in turn may affect the metabolic rate and initiate inflammatory processes (Zhang et al., 2010). Not surprisingly, in the present study, we noticed significant alterations in the composition of muscular PLs fraction under the FAs oversupply conditions. Specifically, the HFHS diet resulted in a marked elevation in the content of different PLs subclasses, e.g., PC and PS, which translates into the observed increase in the total intramuscular PLs pool. This is in accordance with a study of Funai et al., which demonstrated an increased PC concentration in the skeletal muscle of high-fat diet-fed mice (Funai et al., 2016). As they determined, it resulted from enhanced muscular expression of choline/ethanolamine phosphotransferase 1 (CEPT1), that is the step-limiting enzyme in the Kennedy synthesis pathway, through which PC as well as PE, are synthesized de novo (Funai et al., 2016; Tracey et al., 2018). In the same study, the authors also showed that skeletal muscle-specific CEPT1 knockout resulted in increased muscle sensitivity to insulin, which was linked to an altered composition of phospholipids in the sarcoplasmic reticulum and reduced sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity (Funai et al., 2016). Interestingly, in the case of PE, we did not observe any significant changes under conditions of excess lipids, even though it shares the same Kennedy synthesis pathway as PC. This suggests increased interconversion of PE in favor of PC or PS since PS is only formed as a result of the de novo-produced PLs species remodeling by diverse deacylation/reacylation processes (Scheme 1) (Tracey et al., 2018). Importantly, the above-mentioned compositional changes in muscular PLs fraction during the increased consumption of FAs and sucrose were substantially improved by the 2-week CBG application. Our results show that the introduction of CBG in a group of insulin-resistant and obese Wistar rats significantly reduced the content of PC and PS. One of the possible explanations for the decrease in muscular PC content is the inhibitory effect of CBG on the activity of enzymes involved in the de novo PLs synthesis, e.g., CEPT1, which could result in improved muscular insulin sensitivity, however, this hypothesis requires further detailed research. In turn, in the case of PS, CBG probably intensifies its catabolism into PE, which corresponds to the elevated PE content in the group of animals subjected to the HFHS diet and CBG treatment. Additionally, we observed a considerable enhancement of intramuscular PE amount after CBG application not only in the group of HFHS diet-fed rats but also in rats fed the standard chow. It seems likely that this is another CBG’s cell protective mechanism as numerous studies have shown that PE serves as a key player in maintaining membrane integrity, cell cycle signaling as well as glucose and lipid metabolism pathways (Funai et al., 2016; Lee et al., 2018). This assumption is supported by a study conducted by Grapentine et al. in which they demonstrated that deficiency of PE synthesizing enzyme (CTP:phosphoethanolamine cytidylyltransferase - Pcyt2) in Pcyt2 ^+/− mice results in disrupted PE milieu with subsequent deleterious metabolic effects including enhanced lipogenesis, greater intramuscular accumulation of DAGs and triacylglycerols (TAGs), impaired insulin signaling and mounting inflammatory state in skeletal muscle, which is linked to occurrence of obesity and IR (Grapentine et al., 2023). Moreover, the changes in muscle PC and PE content observed in our study are reflected in the PC:PE index, which was significantly reduced after the implementation of CBG treatment in both rats fed the basal and HFHS diets (data not shown). The above effects of CBG action should be emphasized since mounting evidence indicates that disruptions in the PC:PE tissue ratio caused by obesity are directly correlated with impaired glucose homeostasis, while correction of this ratio is associated with insulin sensitivity improvement (Fu et al., 2011; Bzdęga et al., 2023). This is in line with our previous publication, where we reported that the introduction of CBG to obese and insulin-resistant rats improved hepatic insulin sensitivity, which was reflected in increased Akt phosphorylation (in Ser and Thr phosphorylation sites), and hence, its activation, as well as in reduced phosphorylation of glycogen synthase kinase 3β (GSK-3β) in Tyr 216, resulting in decreased glycogen deposition (Bzdęga et al., 2023). Another important finding from our study is the discovery that 2-week CBG administration significantly increased intramyocellular amounts of PI after feeding rats both standard chow and HFHS diet. It is well established that PI is an essential component of the cell membrane that functions as a lipid anchor of numerous membrane proteins (Derkaczew et al., 2023). Furthermore, it also constitutes a precursor for many derivatives, such as phosphatidylinositol-3,4,5-trisphosphate (PIP₃), that activates downstream signaling proteins, the most notable of which is the protein kinase B (PKB/Akt), which plays a pivotal role in the insulin signal transduction pathway (Derkaczew et al., 2023; Khezri et al., 2023). Therefore, taking into account the above-mentioned effects caused by the CBG treatment, it seems reasonable to conclude that this phytocannabinoid may prevent the development of IR in the skeletal muscle under the increased dietary FAs bioavailability conditions through improving the cellular phospholipids milieu.

Additionally, at the same time, we observed that in our study not only the intramuscular amounts of individual PLs subclasses changed but also the fatty-acyl chain composition differed significantly in these fractions. This seems to be extremely important since the results obtained in the studies conducted on both experimental animals and humans indicate that the FAs composition of the PLs fraction in the cell lipid bilayer is related to membrane fluidity as well as to insulin responsiveness of the skeletal muscle (Kriketos et al., 1996; Clore et al., 1998). However, most of the studies were carried out in the total membrane phospholipids, therefore we decided to evaluate the composition of individual FAs considering distinct PLs species. Our experimental model showed that under the conditions of the HFHS diet feeding, the content of SFAs, MUFAs, as well as n-3 and n-6 PUFAs in the PC pool increased substantially. Noteworthy, the largest increment concerned the content of n-6 PUFAs, which resulted from the significant enrichment of this fraction in arachidonic acid (AA; C20:4n-6). The large majority of studies describe the biological effects of AA as pro-inflammatory, demonstrating that it serves as a precursor of intermediates (termed eicosanoids) whose action orchestrates many detrimental processes involved in the pathogenesis of a wide range of metabolic disorders, including IR (Wang et al., 2021). The above statement corresponds with the research conducted by Koeberle et al. (Koeberle et al., 2013), which showed that supplementation of NIH 3T3 murine fibroblasts plasma membranes with AA-rich phosphatidylcholine species (20:4-PC) impaired downstream insulin-mediated signaling apparently by inhibiting the interaction of Akt with its PIP₃ binding sites. Concomitantly, in the same study researchers showed that phosphatidylcholines enriched in SFAs and MUFAs did not affect the Akt signaling, suggesting that the ability of PC species to suppress the Akt pathway decreases with the unsaturation index of the forming FAs (Koeberle et al., 2013). Moreover, we should also bear in mind that AA constitutes a biosynthetic precursor of endocannabinoids, of which the best-studied are AEA and 2-AG (Di Marzo et al., 2008). These AA-derived metabolites are endogenous ligands of CB₁ and CB₂ receptors, and according to the current state of knowledge, overactivation of the CB₁ receptor is closely related to the induction of the inflammatory response and occurrence of obesity (Silvestri and Di Marzo, 2013; Bielawiec et al., 2020). Additionally, our research also showed that the oversupply of FAs in the diet caused similar changes in the composition of the PS fraction as in PC, including a significant enhancement in the proportions of MUFAs, n-3, and n-6 ​​PUFAs. Despite a significant elevation in the AA content of the PS fraction, the most noticeable change concerned the amount of n-3 PUFAs, which resulted from a drastic increase in the content of docosahexaenoic acid (DHA; C22:6n-3), that has been linked to broad effects in promoting metabolic health (Hishikawa et al., 2017). One of the possible explanations for the high DHA content in the PS fraction is the significant preference for DHA-rich substrates by PS synthesizing enzymes, e.g., phosphatidylserine synthase 2 (PSS2) (Kimura and Kim, 2013). Meanwhile, both in the case of the PC and PS subclasses, 2-week CBG supplementation in animals during the HFHS feeding period resulted in a significant decrease in the content of all classes of FAs, as follows SFAs, MUFAs, and PUFAs, however, the largest diminishment among these fractions concerned the amount of AA, which confirms the anti-inflammatory properties of CBG. In contrast, the alternations in the PE and PI fractions observed in our experiment are completely different from those regarding the PC and PS subclasses. Specifically, our research has shown that either in the PE or PI species, the content of individual FAs in the group of HFHS-fed rats changed only slightly in relation to rats receiving a basal diet. In turn, chronic administration of CBG to rats during both a standard and HFHS feeding course resulted in prominent fatty-acyl chain modifications, which indicates an interesting phenomenon regarding the increased incorporation of PUFAs from the n-3 pathway into these fractions. The most probable explanation seems to be the activation of desaturation and elongation processes by CBG treatment, leading to the shift of the long-chain PUFAs equilibrium in favor of n-3, well-known as anti-inflammatory precursors. Thus, the above effects of CBG can be considered beneficial since it enhances the anti-inflammatory and antioxidant potential of PE and PI subclasses, which are abundantly localized on the inner leaflet of the cell membranes (Patel et al., 2017). Collectively, the present observations strongly suggest that the lipid bilayer phospholipids remodeling, including complex interconversions of PLs species and alternations in their FAs composition, is closely associated with myocyte insulin sensitivity. Therefore, restoring the lipid homeostasis of the myocyte membrane by CBG in conditions of the overabundance of FAs and carbohydrates in the diet seems to be extremely important and indicates a new potential medical use of CBG in the treatment of obesity-related metabolic disorders.

In accordance with the above findings is the enhanced muscular FADS1 and FADS2 expression in the group of animals fed the HFHS diet, which was subsequently reduced by following CBG treatment. FADS1 and FADS2 are the crucial step-limiting enzymes involved in the PUFAs metabolism, in particular, responsible for the multistep synthesis of AA as well as eicosapentaenoic acid (EPA; C20:5n-3) and DHA from the diet-derived essential FAs: LA and ALA, respectively (Scheme 1) (Glaser et al., 2010). Downstream conversions of FADS family products result in the formation of various lipid signaling mediators that participate in both the initiation and alleviation of inflammatory processes, which might modify whole body metabolic rate (Miyazaki et al., 2008). Furthermore, recent studies have indicated a positive interrelationship between enhanced FADS1 activity and obesity state (Powell et al., 2016; Yashiro et al., 2016). Consistently, Yashiro’s research group demonstrated that the use of a selective FADS1 inhibitor prevented obesity development in HFD-fed mice (Yashiro et al., 2016), whereas, another study showed that FADS1 gene knockout (fads ^−/−) mice exhibited improved glycemic parameters and resistance against obesity induction under HFD-feeding conditions (Powell et al., 2016). Similarly, evidence accumulating during the last decades has shown that obesity, type 2 diabetes mellitus (T2DM), and distinct inflammatory diseases are also correlated with elevated FADS2 activity (Warensjö et al., 2006; Arshad et al., 2019). Wang et al. reported that male C57BL/6 J FADS2 knockout mice (fads2 ^−/−) were characterized by lower body weight, reduced adipocyte size, and enhanced lipolysis markers in comparison with wildtype mice regardless of the feeding regime (diet enriched with Lard or Flax) (Wang et al., 2023). Thus, data from our experiment support the hypothesis whereby upregulated FADS1 and FADS2 expression is associated with the occurrence of obesity and IR (insulin sensitivity results were demonstrated in our recently published paper (Bzdęga et al., 2023)). Therefore, we can assume that the observed reduction in intramuscular FADS1 and FADS2 expression is in line with the diminished AA concentration following the administration of CBG in obese rats points out the high therapeutic potential of CBG as a new class of drug useful in combating obesity-related complications. Intriguingly, we should also note that the most pronounced changes caused by CBG administration across all examined PLs fractions concern AA and DHA with no significant changes in EPA content. Hence, it seems likely that CBG promotes the process of fatty-acyl chain elongation, leading to the formation of very-long-chain PUFAs from 18- and 20-carbon substrates. Indeed, in the present study, we revealed that implementation of CBG in rats fed a high-fat, high-sucrose diet substantially elevated the intramuscular expression of ELOVL5 responsible for PUFAs chain elongation, which reflects the above-mentioned changes. This gains significant importance as recent research has shown that ELOVL5 knockout (Elovl5 ^−/−) mice developed hepatic steatosis via overactivation of sterol regulatory element-binding protein 1c (SREBP-1c) and its target genes (e.g., acetyl-CoA carboxylase–ACC and fatty acid synthase–FAS) involved in intracellular FAs synthesis (Moon et al., 2009). The underlying mechanism can be explained most likely due to a deficiency of endogenous AA and DHA, which are recognized to inhibit the activity of SREBP-1c, forming a negative feedback loop (Moon et al., 2009). Our results also demonstrated that the muscular expression of ELOVL3 and ELOVL6 in rats fed the HFHS diet was significantly upregulated compared to the control groups. These elongases preferentially utilize SFAs and MUFAs as substrates in the two-carbon units addition cycle (Guillou et al., 2010), specifically, ELOVL6 is involved in the formation of C18 long-chain SFAs and MUFAs, while ELOVL3 is responsible for C20 - C24 FAs production (Matsuzaka et al., 2007; Zach-Avec et al., 2010). In recent years, interest in the role of these enzymes has increased significantly, as a growing body of evidence has pointed to their direct connection with obesity, where they demonstrate intensified activity patterns. In accordance, the mice lacking ELOVL3 (Elovl3 ^−/− ) developed by Zach-Avec and coworkers (Zach-Avec et al., 2010) were resistant to the induction of obesity by an HFD, which was associated with a reduced FAs uptake and inhibited de novo synthesis. In a similar manner, research on ELOVL6 identified that gene deletion in mice (Elovl6 ^−/− ) ensured resistance against diet-induced IR, although the mice were characterized by obesity and hepatic steatosis (Matsuzaka et al., 2007). Noteworthy, the herein study has shown that treatment of obese and insulin-resistant rats with CBG caused a pronounced decrease in either ELOVL3 or ELOVL6 intramuscular expression. Additionally, when analyzing FAs metabolic pathways, we should not forget about another important enzyme, SCD1. It is responsible for catalyzing the MUFAs formation (mainly palmitoeleic acid (C16:1n-7) and oleic acid (C18:1n-9)) and appears to be a pivotal orchestrator of a wide range of metabolic processes including lipid synthesis and oxidation (Dobrzyn and Ntambi, 2004). Due to the multifaceted roles of MUFAs, alternations in SCD1 activity are expected to impact a variety of metabolic pathways, including those involved in insulin signal transduction and obesity (Sampath and Ntambi, 2011). Indeed, recently it has been proven that SCD1 deficient mice (Scd1 ^−/− ) exhibit enhanced energy expenditure, improved insulin sensitivity of skeletal muscle, and high resistance to diet-induced weight gain, which collectively highlight the close link between SCD1 overactivity and obesity occurrence (Dobrzyn et al., 2005; Miyazaki et al., 2007). Thus, targeted SCD1 inhibition has the potential to protect against multiple aspects of obesity-associated metabolic derangements. Given the variety of beneficial effects of CBG action, our findings revealed that it additionally possesses the capability to significantly reduce the total intramuscular SCD1 expression in the HFHS diet-fed rats, which corresponds with the inhibition of the SCD1 activity in the total PLs and PS fraction in the same experimental group. One possible mechanism for diminished SCD1 activity after CBG implementation is the ability of CBG to effectively modulate endocannabinoid signaling since the latest research has shown a functional connection between the eCBome and the lipogenesis process (Liu et al., 2013). Over the past decade, increased CB₁ receptor activation has been attributed to the promotion of lipogenic enzyme expression, such as SCD1 (Liu et al., 2013). Furthermore, it has been shown that products of SCD1, endogenous MUFAs downregulates the fatty acid amide hydrolase (FAAH) activity, resulting in impaired AEA degradation and subsequent CB₁ overactivation, which constitutes a positive feedback loop (Liu et al., 2013). Therefore, we can assume that CBG interrupts this vicious cycle that occurs during obesity since CBG has been demonstrated to be a partial agonist of the CB₁ receptor with low affinity and is considered as a strong competitive inhibitor of AEA (Nachnani et al., 2021). However, in the case of PC, PE, and PI species, we observed an increase in SCD1 activity index compared to animals receiving only the high-fat, high-sucrose diet. This reverse effect of CBG action in PC, PE, and PI subclasses is puzzling, and more experiments are needed to elucidate the exact mechanism. Moreover, it is also worth adding that in our previous research we showed results, that are consistent with the herein data and demonstrate that another phytocannabinoid, namely, cannabidiol (CBD), substantially decreased the ELOVL3 and ELOVL6 expression, as well as reduced the SCD1 activity ratio in different lipid pools in the skeletal muscle of rats with HFD-induced obesity (Bielawiec et al., 2023). Thus, this collectively suggests that phytocannabinoids are emerging as compounds that may represent a novel therapeutic approach for the treatment of obesity and accompanying IR through the modification of FAs-metabolizing enzymes activity involved in elongation and desaturation processes.

It has been generally accepted that the overabundance of FAs and carbohydrates in the diet is closely related to the occurrence of obesity, which is accompanied by inflammatory modifications resulting from the cell membrane PLs remodeling (Wu and Ballantyne, 2017). In the present study, analogously to the increased AA content in all examined PLs fractions during the course of obesity, we also reported a markedly elevated n-6 PUFAs activity pathway. Intriguingly, 2-week CBG administration in rats under the lipid overload conditions also promotes the upregulation of n-6 PUFAs activity in all PLs species in comparison with the control subjects, although compared to the rats fed HFHS diet alone, we noted profoundly decreased n-6 activity pathway only in the case of the PC and PI subclasses. Presumably, the possible explanatory mechanism could be an increase in the content of γ-linoleic acid (GLA; C18:3n-6), which serves as a precursor for AA formation. This notion is supported by the study of Vodolazska et al., where they demonstrated that a diet enriched with hemp seed oil caused a marked elevation in the GLA concentration, whose interconversion to AA may intensify the activity of the n-6 PUFAs pathway (Vodolazska and Lauridsen, 2020). Moreover, in our study, significant modifications of PUFAs activity also concerned the anti-inflammatory n-3 series. In particular, HFHS diet-fed rats (untreated and treated with CBG) have shown a substantial rise in intramuscular n-3 activity pathway in all PLs fractions except for PS. However, CBG application elevated the activity of n-3 PUFAs in the PC, PE, and PI classes to a greater extent compared to obese rats, indicating increased incorporation of FAs from the anti-inflammatory n-3 pathway. Thus, we strongly believe that CBG markedly improved the anti-inflammatory and anti-oxidative potential of membrane PLs by modifying the composition and activity of PUFAs, which is also reflected in alternations in the expression of enzymes involved in inflammatory pathways that we observed in our research. In accordance, during the high-fat, high-sucrose feeding course, we found significantly elevated intramuscular expression of cytosolic phospholipase A₂ (cPLA2), an enzyme that plays a key role in PLs hydrolysis to AA (Leslie, 2015). Increased cPLA2 activity along with a relative excess of AA in the lipid bilayer may lead to its continuous release from membrane deposits and make it available for the generation of eicosanoids with the participation of enzymes from the COX or LOX family (Leslie, 2015). Not surprisingly, in the herein study, we noted that rats with HFHS diet-induced obesity have shown profoundly enhanced enzyme expression of COX-1, COX-2, 5-LOX, and 12/15-LOX, which are engaged in the production of various cytokines with high pro-inflammatory effects attributed to the development of local and systemic inflammatory response. This suggests the occurrence of inflammation in the skeletal muscle, which may be partially linked to the onset of diet-induced IR. It is worth emphasizing that, CBG implementation in the same group of animals substantially inhibits all AA-metabolizing enzymes, such as cPLA₂, COX-1, COX-2, 5-LOX, and 12/15-LOX, which is in line with the study conducted by Ruhaak et al., demonstrating the inhibitory activity of CBG against both COX-1 and COX-2 enzymes with high effectiveness (Ruhaak et al., 2011). Presumably, one of the mechanisms that could account for the immunomodulatory properties of CBG is its ability to bind to the CB₂ receptor and PPARγ (Jastrząb et al., 2022). Specifically, Giacoppo et al. in their study suggest that CBG through the CB₂ receptor activation led to an increase in the cellular level of NF-κB inhibitor - IκB-α and, consequently, a decrease in the NF-κB activity (Giacoppo et al., 2017). Another possibility is the potent stimulation of PPARγ by CBG since it has been proven that upregulation of this transcription factor induces termination of the NF-κB signaling pathway, which leads to the suppression of genes involved in the initiation of the inflammatory response, including COX-2 (Hou et al., 2012). In line with the above hypothesis, our data showed significantly enhanced muscular PPARγ expression in HFHS diet-fed rats after CBG application in comparison with the untreated obese rats, which in turn corresponds to a significant reduction in the total NF-κB and COX-2 expression in the same group of experimental animals. In light of these findings, CBG may hold great promise for the treatment of chronic low-grade inflammatory states potentially via modulation of the activity of enzymes participating in the AA cascade, which most likely results in reduced generation of lipid pro-inflammatory mediators. Simultaneously, our immunoblotting analyses also revealed that lipid overabundance conditions substantially reduced the level of nuclear factor erythroid 2-related factor 2 (Nrf2), which is the central regulator of cellular resistance to oxidative stress (Ma, 2013). Importantly, 2-week CBG administration restored the Nrf2 activity in rats subjected to HFHS feeding, indicating the beneficial antioxidant properties of CBG, which, in addition to its anti-inflammatory activity, protects cells against obesity-induced damage. Moreover, we also have found that CBG inhibits the fibrotic muscular remodeling during the obesity state, which was revealed by a significant upregulation of secreted matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) as well as collagen type 1, and type 3 (COL-1A1 and COL-3A1) expression, which contributes to the regulation of extracellular matrix turnover and probably may increase the regenerative capacity of the skeletal muscles. Collectively, CBG favorably impacts the composition and metabolism of muscle PLs, which is associated with the inhibition of the AA-derived bioactive metabolites formation accounting for the beneficial effects of CBG therapy. Thus, our experimental study is the first step toward understanding the modulatory action of CBG on membrane PLs, however, further research should be introduced to determine the exact mechanism of CBG due to the considerable complexity of the above processes.