Cannabidiolic acid as a modulator of lipid metabolism in the liver of rats with metabolic-associated steatotic liver disease

Abstract

This study investigated the effects of cannabidiolic acid (CBDA) on hepatic lipid metabolism in a rat model of metabolic dysfunction-associated steatotic liver disease (MASLD), addressing the need for natural therapeutic compounds targeting lipid metabolism disorders. Male Wistar rats were fed a standard diet or a high-fat diet (HFD) for 8 weeks. During the last 14 days, half of the rats received CBDA intragastrically (0.1 mg/kg BW). The hepatic lipid fractions were analyzed via gas–liquid chromatography, and protein expression was assessed via Western blotting and immunohistochemistry. Compared with the control diet, the HFD significantly increased the expression of fatty acid transporters CD36, FATP5, and FABPpm and elevated the levels of free fatty acids (FFAs), triacylglycerols, diacylglycerols, and phospholipids compared with controls. CBDA treatment in HFD-fed rats significantly decreased CD36, FABPpm, and FATP5 expression as well as total diacylglycerol and phospholipid concentrations. CBDA also decreased the saturated fatty acid content in the FFA and phospholipid fractions while increasing omega-3 polyunsaturated fatty acids in the diacylglycerol and triacylglycerol fractions. CBDA ameliorated HFD-induced hepatic steatosis by modulating fatty acid transporter expression, reducing harmful lipid accumulation and improving fatty acid composition. These findings suggest the potential of CBDA as a therapeutic agent for MASLD through the targeting of multiple dysregulated pathways in hepatic lipid metabolism, potentially limiting disease progression.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-41130-0.

Introduction

With changing dietary habits, fast-paced lifestyles, a lack of physical activity, and increasing stress, global society has become more prone to civilization-related diseases worldwide. One of these diseases is metabolic dysfunction-associated steatotic liver disease (MASLD), which replaced the previously used term nonalcoholic fatty liver disease (NAFLD). MASLD is characterized by hepatic steatosis, identified through imaging or biopsy, in the presence of at least one of the five cardiometabolic criteria¹. It is the most common liver disease in the world, with a prevalence of more than 30% of the population, and is a significant problem in both developing and developed countries^2,3. In one out of every four cases, MASLD progresses to metabolic dysfunction-associated steatohepatitis, which can lead to irreversible fibrosis and even hepatocellular carcinoma⁴. The pathophysiology of MASLD is complex and involves a disorder in lipid metabolism characterized by the impairment of several processes.

The first one is the excessive influx of lipids into the liver facilitated by fatty acid transporters, including fatty acid translocase (FAT/CD36), fatty acid binding protein (FABPpm), and fatty acid transport proteins (FATP2 and FATP5)⁵. An increased amount of lipid uptake results in increased storage of triacylglycerols (TAGs). Excessive TAG accumulation is considered one of the predominant causes of hepatic steatosis leading to MASLD development⁶. Although evidence indicates that certain TAG species are less metabolically harmful than other lipid fractions are, excessive TAGs may undergo esterification to diacylglycerols (DAGs), which constitute lipotoxic lipid species⁷. Studies conducted on liver biopsies from obese nondiabetic patients revealed that excessive hepatic DAG accumulation was strongly correlated with the activation of protein kinase C in hepatic lipid droplets and was associated with impaired insulin sensitivity. Thus, observed in that study, increased deposition of DAGs in the liver, leading to the development of insulin resistance, is a key pathophysiological mechanism in MASLD deterioration⁸. The second process contributing to excessive hepatic lipid deposition is β-oxidation⁹. In in vitro and in vivo models of obesity induced by palmitate or a high-fat diet (HFD), respectively, the overexpression of the rate-limiting enzyme in β-oxidation, carnitine palmitoyltransferase 1 (CPT1), significantly reduces the amount of deposited TAGs, indicating improved steatosis¹⁰. De novo lipogenesis (DNL) is another process that substantially contributes to the amount of excessively deposited lipids in the liver, leading to the production of palmitic acid (16:0), which then undergoes elongation and desaturation¹¹. Although in healthy individuals DNL produces up to 5% of fatty acids in the liver, in patients suffering from MASLD, it increases up to 26%. Intensified DNL with concomitant increased fatty acid uptake results in excessive accumulation of toxic saturated fatty acids (SFAs) and subsequent lipotoxicity¹². However, some studies have shown decreased DNL in high-fat diet-induced animal models of MASLD¹³. The discrepancy between various studies analyzing DNL may depend on the presence of other nutrients (such as fructose) used in the diet or various dietary durations, as it was shown in studies conducted by Kowalski et al., where a changed dietary pattern resulted in various effects on liver DNL¹⁴. Studies in a mouse model of obesity induced by a HFD showed that, together with changes in the DNL, elongation was elevated, catalyzed by the fatty acid elongase family (ELOVLs), which extends the carbon chain length of fatty acids¹³. In human liver biopsies and in the liver of an animal with diet-induced MASLD, in different stages of disease, hepatic elongase activity is significantly impaired, which is correlated with increased hepatic triglyceride accumulation and altered fatty acid composition¹⁵. The process that converts SFAs to monounsaturated fatty acids (MUFAs) and polyunsaturatined fatty acids (PUFAs) is desaturation, which is mediated by stearoyl-CoA desaturase 1 (SCD1) and fatty acid desaturases 1 and 2 (FADS1 and FADS2)¹⁶. In the murine models and patients with nonalcoholic steatohepatitis, hepatic desaturase activities are dysregulated, with increased SCD1 activity promoting MUFA synthesis, whereas simultaneously reduced FADS1 expression results in a hepatic lipid composition enriched in SFA and MUFA species and depleted in protective PUFAs¹⁵. The interrelationships among these processes: fatty acid uptake, β-oxidation, DNL, elongation, and desaturation, determine the balance between hepatic lipid acquisition and disposal. Dysregulation of any of these pathways may contribute to increased lipid accumulation in the liver and play a key role in the pathogenesis of MASLD⁵.

Owing to the significant impact of lipid metabolism on the pathogenesis of MASLD, the search for natural compounds that improve this metabolism is desirable¹⁷. Recently, compounds found in Cannabis sativa have gained attention because of their pleiotropic properties and natural origin. One such substance is cannabidiolic acid (CBDA), a 22-carbon compound and the primary phytocannabinoid found in hemp and certain cannabis oil varieties¹⁸. Its biosynthesis in plants is catalyzed by cannabidiolic acid synthase, which selectively converts cannabigerolic acid into CBDA¹⁹. Compared with its decarboxylated form, cannabidiol (CBD), CBDA is much better absorbed when it is administered orally in an extract, which highlights its potential as a therapeutic agent^20,21. CBDA has no intoxicating effects, and animal studies have confirmed that it does not cause significant side effects²². Recent studies have also demonstrated the antioxidant, antiemetic, and anticancer properties of CBDA²³. However, there is a gap in understanding the role of CBDA in lipid metabolism and associated pathways, and to date, no research has discussed its exact impact on these metabolic routes. Its closest derivative, CBD, exerts beneficial effects on lipid metabolism. In Silvestri et al.’s study on ob/ob mice, CBD administered at a 3 mg/kg dose for 4 weeks markedly decreased the hepatic TAG content²⁴. Similarly, rats fed a sucrose-rich diet for 3 weeks and simultaneously treated with cannabis oil at a ratio of 2:1 CBD:tetrahydrocannabinol (THC) presented a significant decrease in liver TAG accumulation²⁵. Owing to the lack of studies on the use of CBDA for the treatment of MASLD, we aimed to investigate the impact of this phytocannabinoid on lipid metabolism and accumulation in the liver, which could serve as a basis for further human studies on the effects of CBDA on metabolic diseases.

Results

Effects of CBDA administration to the liver tissue and plasma on total lipid fraction accumulation in selected lipid fractions in rats subjected to standard and high-fat diets.

The administration of CBDA to rats fed a standard chow resulted in elevated total lipid contents of both the FFA and the DAG fractions compared with the control group (Fig. 1A, B). Compared with the control group, the high-fat diet group presented increased total lipid accumulation in all the lipid fractions relative to the control group (Fig. 1A–D). Similarly, when CBDA was co-administered with a high-fat diet, a substantial increase in the FFA, DAG, PL and TAG fractions was observed in comparison with the control group (Fig. 1A–D). Notably, compared with the high-fat diet, rats fed with both a high-fat diet and CBDA administration showed decreased levels of total DAG and PL (Fig. 1B, C).

Fig. 1.

Total lipid accumulation in the free fatty acid (FFA), diacylglycerol (DAG), phospholipid (PL) and triacylglycerol (TAG) fractions in the liver tissue. The results are shown as mean ± standard deviation and are expressed in nmol per mg of tissue, n = 10 in each group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group.

The plasma of rats subjected to a high-fat diet showed total lipid content augmented in the FFA and PL fractions, while simultaneously decreased in the TAG fraction when compared with the control group (Supplementary Fig. 1A, C, D). Notably, in relation to the control group, when a high-fat diet was combined with CBDA, the plasma FFA and PL fractions presented increased total lipid content (Supplementary Fig. 1A, D). Furthermore, compared with the high-fat diet group the combination mentioned above also resulted in an elevation in the TAG and PL fractions (Supplementary Fig. 1C, D).

Effects of CBDA administration on body weight and liver function under standard and high-fat diet conditions.

In histological images of livers from rats fed a high-fat diet, increased macrovesicular steatosis was observed compared with the control liver. However, the condition of hepatocytes after simultaneous treatment with a high-fat diet and CBDA was improved, as indicated by a decrease in the number of large, spherical lipid vacuoles (Supplementary Fig. 1E).

The effect of a high-fat diet on ALT and body weight of rats was prominent, as both were increased in comparison with the group fed a control diet (Supplementary Fig.1F, G). Moreover, in relation to the control group, the co-administration of CBDA and high-fat diet resulted in a significant increase in ALT and body weight (Supplementary Fig. 1F, G). Finally, when the aforementioned rats were contrasted with animals from the high-fat diet group, a significant decrease in ALT was noted (Supplementary Fig. 1F, G).

Effects of CBDA administration in the liver tissue and plasma on SFA, MUFA, and PUFA profiles in selected lipid fractions under standard and high-fat diet conditions.

The group of rats fed a standard chow and subjected to CBDA showed an increase in SFA content in the FFA, DAG, and PL with a decrease in TAG lipid fractions relative to the control group (Fig. 2A–D). Moreover, when only a high-fat diet was introduced to the rat group, the SFA concentrations in the FFA, DAG, PL, and TAG lipid fractions increased in reference to the control group (Fig. 2A–D). Additionally, the combination of CBDA and a high-fat diet resulted in an increase in SFA deposition in all assessed lipid fractions in comparison with the animals fed a standard diet (Fig. 2A–D). Lastly, compared with the high-fat diet group, the high-fat diet group with CBDA addition exerted decreased content of SFAs in the FFA and PL lipid fractions (Fig. 2A, C).

Fig. 2.

The saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acid (PUFA) concentrations in the free fatty acid (FFA), diacylglycerol (DAG), phospholipid (PL) and triacylglycerol (TAG) fractions in the liver tissue. The results are shown as mean ± standard deviation and are expressed in nmol per mg of tissue, n = 10 in each group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group.

In the group of animals fed a standard chow diet, CBDA administration led to an increase in the MUFA content within the FFA and DAG lipid fractions, whereas a decrease was observed in the TAG fraction compared with the control group (Fig. 2E, F, H). Furthermore, in rats fed a high-fat diet alone, MUFA levels were elevated in the FFA, DAG, and TAG fractions but decreased in the PL fraction relative to the rats from the control group (Fig. 2E–H). The combination of CBDA treatment and high-fat diet resulted in a decrease in MUFA accumulation in the PL fraction, as well as in an increase in all three other lipid fractions, namely, FFA, DAG, and TAG compared with the control group (Fig. 2E–H). However, when comparing the high-fat diet group to the group receiving both CBDA and a high-fat diet, the latter showed an increase in MUFA content in the TAG and PL fractions, and a decrease in the DAG fraction (Fig. 2F–H).

When animals fed a standard diet received CBDA, it led to an increase in the PUFA content within the FFA and DAG lipid fractions, whereas a decrease was observed in the TAG fraction relative to the group fed a control diet (Fig. 2I, J, L). Furthermore, the PUFA levels were elevated in all the assessed lipid fractions in rats fed a high-fat diet alone as well as in the animals simultaneously treated with CBDA and a high-fat diet in reference to the control group (Fig. 2I–L). Similarly, higher PUFA levels were observed only in the TAG fraction in the group receiving both CBDA and a high-fat diet compared with the high-fat diet group (Fig. 2. K).

When the levels of SFAs, MUFAs and PUFAs in the plasma of high-fat-fed rats were analyzed, the increased availability of fatty acids in the diet led to an increase in SFA levels in the FFA and PL fractions and a decrease in the DAG and TAG fractions in comparison with the control group (Supplementary Fig. 2A, C, B, D). Moreover, relative to the animals from the control group, introduction of CBDA to the standard rodent diet group resulted in a decrease in the SFA in DAG lipid fraction (Supplementary Fig. 2B). Furthermore, the CBDA and high-fat diet combination resulted in an elevation in SFA in FFA and PL fractions, with a simultaneous decrease in DAG lipid fraction when contrasted against the control group (Supplementary Fig. 2A, C, B). Lastly, the previously mentioned HFD + CBDA group also showed increased SFA levels in PL and TAG fractions in relation to the high-fat diet group (Supplementary Fig. 2C, D).

Subsequently, high-fat diet feeding resulted in a significantly increased MUFA level in the FFA fraction and a decreased level in the TAG fraction when compared with the control group (Supplementary Fig. 2E, H). Simultaneously, CBDA administration to animals fed a standard rodent diet caused a rise in the MUFA level in the FFA fraction only (Supplementary Fig. 2E). Furthermore, when coupled with CBDA, the high-fat diet group presented increased MUFA levels in the FFA, PL and TAG fractions in relation to the control group, and a rise in the PL and TAG fractions when contrasted against the high-fat diet group (Supplementary Fig. 2E, G, H).

Compared with the animals from the control group, PUFA levels were increased by the high-fat diet in DAG and decreased in TAG lipid fractions (Supplementary Fig. 2J, L). Compared with the control group, the high-fat diet consumption with CBDA administration resulted in elevated PUFA levels in the FFA, DAG and PL lipid fractions and decreased levels in TAG (Supplementary Fig. 2I, J, K, L). The previously mentioned combination led to an increased levels of PUFAs in DAG and PL lipid fractions compared with the high-fat diet group (Supplementary Fig. 2J, K).

Impact of CBDA administration on hepatic n-3 and n-6 fatty acid profiles in selected lipid fractions in rats subjected to standard and high-fat diets.

Compared with the control group, the total n-3 content in the FFA and DAG lipid fractions was increased in the standard diet-fed animals treated with CBDA, whereas a decrease in the total n-3 content was observed only in the TAG fraction (Fig. 3A, B, D). The high-fat diet group showed elevated total n-3 level in the FFA, DAG, TAG, and PL fractions relative to the control group (Fig. 3A–D). Furthermore, across all four assessed lipid fractions, the total n-3 content was increased when both CBDA and a high-fat diet were given simultaneously in comparison with the control group (Fig. 3A–D). Lastly, when compared with the high-fat diet group, the animals receiving both CBD and a high-fat diet displayed higher total n-3 content in the DAG and TAG fractions (Fig. 3B, D).

Fig. 3.

The total n-3 and n-6 contents in the free fatty acid (FFA), diacylglycerol (DAG), phospholipid (PL) and triacylglycerol (TAG) fractions in the liver tissue. The results are shown as mean ± standard deviation and are expressed in nmol per mg of tissue, n = 10 in each group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group.

Standard chow diet combined with CBDA administration led to an increase in the total n-6 content in the FFA and DAG lipid fractions, whereas a decrease was observed in the TAG fractions when contrasted with the control group (Fig. 3E, F, H). Moreover, total n-6 levels were elevated in all the lipid fractions relative to the control group in rats fed a high-fat diet alone (Fig. 3E–H). Similarly, in the group of animals in which CBDA and a high-fat diet were combined, increased total n-6 levels were detected in the FFA, DAG, and TAG fractions compared with the control group (Fig. 3E, F, H). In comparison with the high-fat diet group, lower levels of total n-6 were observed in the FFA, DAG, and PL fractions in the group receiving both CBDA and a high-fat diet (Fig. 3E, F, G).

Effects of CBDA administration on hepatic de novo lipogenesis ratio in selected lipid fractions in rats subjected to standard and high-fat diets.

To assess the de novo lipogenesis (DNL) process, the de novo lipogenesis ratio was calculated, which was decreased in the FFA and DAG fractions in reference to the control, in the standard chow diet-fed rat group that was administered CBDA (Fig. 4A, B). In animals fed a high-fat diet alone and in the group in which CBDA was co-administered with a high-fat diet, the DNL ratio was decreased in the FFA and DAG fractions relative to the control group (Fig. 4A, B). Furthermore, compared with the high-fat diet group, rats receiving both CBDA and a high-fat diet presented a lower DNL ratio only in FFAs (Fig. 4A).

Fig. 4.

The de novo lipogenesis ratio index of the free fatty acid (FFA), diacylglycerol (DAG), triacylglycerol (TAG), and phospholipid (PL) fractions in the liver tissue. The results are shown as mean ± standard deviation and are expressed in nmol per mg of tissue, n = 10 in each group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group.

Effects of CBDA administration in liver tissue on desaturation indices 16:1/16:0 and 18:1/18:0 in selected lipid fractions in rats subjected to standard and high-fat diets.

The desaturation index, calculated as a 16:1/16:0 ratio, was increased in the group of rats treated with CBDA and fed a standard rodent diet in the FFA, DAG, and TAG fractions, whereas this index was reduced in the PL lipid fraction relative to the control group (Fig. 5A–D). In animals subjected to a high-fat diet alone, the desaturation index 16:1/16:0 ratio increased in the FFA fraction but decreased in the DAG, PL and TAG fractions compared with the control group (Fig. 5A–D). Furthermore, a higher desaturation index 16:1/16:0 ratio was also present in rats simultaneously receiving CBDA with a high-fat diet in the FFA fraction, while a decrease was observed in the DAG, PL and TAG fractions compared with the control group (Fig. 5A–D). Moreover, compared with the high-fat diet group, rats receiving both CBDA and a high-fat diet exhibited a decreased desaturation index 16:1/16:0 only in the PL fraction (Fig. 5C).

Fig. 5.

The desaturation 16:1/16:0 and 18:1/18:0 indices in the free fatty acid (FFA), diacylglycerol (DAG), phospholipid (PL) and triacylglycerol (TAG) fractions in the liver tissue. The results are shown as mean ± standard deviation and are expressed in nmol per mg of tissue, n = 10 in each group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group.

An elevated desaturation index 18:1/18:0 ratio was noted in the FFA fraction in rats fed a standard chow diet subjected to CBDA administration in comparison with the control group (Fig. 5E). Furthermore, in animals receiving a high-fat diet alone, the 18:1/18:0 ratio was increased in the FFA and DAG fractions but decreased in the PL and TAG fractions relative to the control group (Fig. 5E–H). When CBDA was co-administered with a high-fat diet, the desaturation index 18:1/18:0 ratio rose in the FFA and DAG fractions, whereas decreases were noted in the PL and TAG fractions in comparison with the control group (Fig. 5E–H). Additionally, in reference to the high-fat diet group, rats treated with both CBDA and a high-fat diet displayed an increased desaturation index 18:1/18:0 ratio in the FFA fraction, whereas the DAG fraction showed reduced values (Fig. 5E, F).

Effects of CBDA administration on hepatic elongation 18:0/16:0, 20:0/18:0, 22:0/20:0, and 24:0/22:0 indices in selected lipid fractions in rats subjected to standard and high-fat diets.

In rats fed a standard chow diet, CBDA administration led to a reduction in the elongation index (18:0/16:0 ratio) in the FFA lipid fraction, whereas an increase was observed in the DAG fraction compared with the control group (Fig. 6A, B). Moreover, the 18:0/16:0 elongation index was elevated in the PL and TAG fractions relative to controls in animals maintained on a high-fat diet alone (Fig. 6C, D). Co-administration of CBDA with a high-fat diet resulted in greater elongation index values for all the lipid fractions except for FFAs when compared with the control group (Fig. 6B–D). Furthermore, in comparison with the high-fat diet group, rats receiving both CBDA and a high-fat diet presented an increased elongation index only in the DAG fraction (Fig. 6B).

Fig. 6.

The elongation 18:0/16:0, 20:0/18:0, 22:0/20:0, and 24:0/22:0 indices of the free fatty acid (FFA), diacylglycerol (DAG), phospholipid (PL) and triacylglycerol (TAG) fractions in the liver tissue. The results are shown as mean ± standard deviation and are expressed in nmol per mg of tissue, n = 10 in each group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group.

The elongation index (20:0/18:0 ratio) was increased in the rats fed a standard chow diet coupled with CBDA in the PL fraction compared with the control group (Fig. 6G). In animals subjected to a high-fat diet alone, the 20:0/18:0 elongation index increased in the FFA fraction, whereas a reduction was noted in the TAG fraction relative to the control (Fig. 6E, H). Moreover, the same elongation index in the group of rats treated simultaneously with a high-fat diet and administered CBDA, was enhanced in the FFA fraction, with a corresponding decline in the TAG fraction compared with the control group (Fig. 6E, H).

In rats that consumed a standard chow diet, CBDA increased the elongation index (22:0/20:0 ratio) in the FFA and DAG fractions relative to the control group (Fig. 6I, J). Moreover, in subjects fed a high-fat diet alone, the elongation index was elevated in the FFA and PL fractions but decreased in the TAG fraction compared with the controls (Fig. 6I, K, L). The higher 22:0/20:0 elongation index was observed in the experimental group, where animals received combined treatment with CBDA alongside a high-fat diet in the PL fraction, accompanied by a reduction in the TAG fraction when compared with the controls (Fig. 6K, L). Moreover, relative to the high-fat diet-only group, animals receiving both CBDA and a high-fat diet exhibited a diminished elongation index in the FFA fraction (Fig. 6I).

Based on the 24:0/22:0 ratio calculation, the elongation index was increased in the FFA lipid fraction of the rats fed a standard chow diet and administered with CBDA, whereas decreased values were observed in the DAG and PL fractions in the same group compared with the control group (Fig. 6M–O). Moreover, in animals receiving a high-fat diet alone, the elongation index was elevated in the TAG fraction but decreased in the FFA and DAG fractions relative to the control (Fig. 6P, M, N). Compared with the control group, the combined administration of CBDA with a high-fat diet resulted in higher elongation index values in the DAG fraction, whereas the PL fractions exhibited reduced levels (Fig. 6N, O). Furthermore, compared with the high-fat diet group, the 24:0/22:0 elongation index in the FFA and DAG fractions was increased in rats treated with both CBDA and a high-fat diet, whereas in the PL fraction it presented lower values (Fig. 6M–O).

Effects of CBDA administration on the expression of proteins being molecular targets of CBDA in rats subjected to standard and high-fat diets.

Compared with the control group, CB1 protein expression was increased when a high-fat diet was introduced to the rats (Supplementary Fig. 3A). Simultaneously, the CB2 protein expression level was notably decreased when CBDA was given to the animals, relative to the control group (Supplementary Fig. 3B). Furthermore, a high-fat diet had also affected the PPARα and PPARγ protein expression, which resulted in a decrease in their expression compared with the group fed a control diet (Supplementary Fig. 3D, E). Lastly, relative to the high-fat diet group, the combined treatment with CBDA and a high-fat diet led to increased PPARγ protein expression (Supplementary Fig. 3E).

Effects of CBDA administration on the expression of proteins responsible for fatty acid transport in rats subjected to standard and high-fat diets assessed with Western blot.

Compared with the control group, FAT/CD36 protein expression was significantly increased in the high-fat diet-fed group (Supplementary Fig. 4A). Additionally, when CBDA was combined with a high-fat diet, FAT/CD36 protein expression was decreased in comparison with the high-fat diet group (Supplementary Fig. 4A).

FABPpm protein expression was decreased in the rat group fed a standard rodent diet when CBDA was administered, compared with the control group (Supplementary Fig. 4B). In relation to the control group, the high-fat diet group presented increased FABPpm protein expression (Supplementary Fig. 4B). Moreover, when CBDA was combined with a high-fat diet, the expression of the previously mentioned protein was decreased in comparison with the high-fat diet group (Supplementary Fig. 4B).

Compared with the control group, rats fed a high-fat diet showed notably increased FATP5 protein expression (Supplementary Fig. 4D). Furthermore, simultaneous CBDA and a high-fat diet administration resulted in further increased FATP5 protein expression in comparison with the control and HFD groups (Supplementary Fig. 4D).

In liver tissue, in the group subjected to a high-fat diet as well as in animals simultaneously treated with a high-fat diet and CBDA, the expression of the ABCA1 transporter was increased in relation to the control group (Supplementary Fig. 4E).

Effects of CBDA administration on proteins involved in β-oxidation, de novo synthesis, and metabolism of lipids in rats subjected to standard and high-fat diets.

The only statistically significant change observed in the protein expression of CPT1 was an increase in the group fed a high-fat diet when compared with the control group (Fig. 7A). The expression of another protein involved in β-oxidation, namely, β-HAD, was decreased relative to the control group, in the rat group fed a standard rodent diet combined with CBDA administration (Fig. 7B). Moreover, in the group treated with CBDA but fed a high-fat diet, there was an increase in the protein expression of β-HAD protein compared with the high-fat diet group (Fig. 7B).

Fig. 7.

The expression levels of proteins associated with β-oxidation, de novo synthesis, and lipid metabolism, including carnitine palmitoyltransferase 1 (CPT1), β-hydroxyacyl-CoA dehydratase (β-HAD), fatty acid synthase (FAS), glycerol-3-phosphate acyltransferase (GPAT), adipose triglyceride lipase (ATGL) and diacylglycerol O-acyltransferase 2 (DGAT2), were assessed. The expression levels are presented as percentages relative to the control group, which was set at 100%. The data are expressed as mean ± SD, n = 6 per group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group. Original blots are presented in Supplementary Fig.5.

Compared with the control group, the protein expression of FAS was lower in the high-fat diet group (Fig. 7C). When CBDA and a high-fat diet were combined, the protein expression of FAS decreased in relation to the control group (Fig. 7C) but was greater than in the high-fat diet group (Fig. 7C).

The expression of the GPAT enzyme was significantly increased in the high-fat diet group in comparison with the control group (Fig. 7D). In contrast, a decrease in the expression of GPAT in the CBDA and high-fat diet combined groups was observed relative to the high-fat diet group (Fig. 7D).

Compared with the control group, ATGL protein expression was lower in the rat group fed a standard rodent diet coupled with CBDA administration (Fig. 7E) as well as in rats fed a high-fat diet (Fig. 7E). Rats subjected simultaneously to a high-fat diet and CBDA administration noted a significantly increased expression of ATGL in comparison with the high-fat diet group as well (Fig. 7E).

Effects of CBDA administration on the expression of proteins involved in desaturation and elongation in rats subjected to standard and high-fat diets.

The expression of the first isoform of the enzyme involved in the desaturation process, FADS1, was decreased in the group of rats fed a standard rodent diet with CBDA administration and in the group of rats fed a high-fat diet in comparison with the control group (Fig. 8A). Additionally, when CBDA was combined with a high-fat diet, FADS1 protein expression was increased compared with the high-fat diet group (Fig. 8A).

Fig. 8.

The expression levels of proteins associated with desaturation and elongation of fatty acids, including fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2), and elongation of very long-chain fatty acid proteins 3, 5, and 6 (ELOVL3, ELOVL5, and ELOVL6), were evaluated. The expression levels are presented as percentages relative to the control group, which was set at 100%. The data are expressed as mean ± SD, n = 6 per group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group. Original blots are presented in Supplementary Fig. 5.

Compared with the control group, the expression of the second isoform of the enzyme—FADS2 was greater in the two experimental groups, namely, the rat group fed a high-fat diet and in the group where CBDA was coupled with a high-fat diet (Fig. 8B).

Rats fed a high-fat diet had the expression of ELOVL6 protein substantially decreased in comparison with the control group (Fig. 8C). Moreover, when CBDA was combined with a high-fat diet, the expression of ELOVL6 was increased relative to the high-fat diet group (Fig. 8C).

Compared with the control group, the expression of ELOVL 5 was increased in the group fed a standard diet coupled with CBDA administration (Fig. 8D). Furthermore, the expression of the previously mentioned protein increased in the group treated simultaneously with CBDA and a high-fat diet in relation to both the control and high-fat diet groups (Fig. 8D).

Effects of CBDA administration on the immunohistochemical expression of fatty acid transporters in rats subjected to standard and high-fat diets.

CBDA administration in rats fed a standard chow led to enhanced FABPpm expression (Fig. 9B). Moreover, a high-fat diet also increased the FABPpm intensity density (Fig. 9B), and the combination of CBDA administration with high-fat diet resulted in elevated FABPpm expression (Fig. 9B). High-fat diet feeding suppressed FATP2 expression alone (Fig. 9C) as well as with CBDA co-administration (Fig. 9C). Similarly, rats fed solely a high-fat diet presented an increase in FATP5 expression (Fig. 9D). CBDA supplementation in the high-fat diet group resulted in an elevation of FATP5 intensity density compared with the control group and a decrease in FATP5 intensity density in comparison with that the HFD group (Fig. 9D). FAT/CD36 expression was decreased in the CBDA group (Fig. 9E) and increased in rats fed with a high-fat diet without (Fig. 9E) and with CBDA administration in comparison to the control (Fig. 9E). Compared with high-fat diet-fed rats, the HFD + CBDA group presented increased FAT/CD36 expression (Fig. 9E). CBDA administration in standard chow diet group resulted in significantly reduced ABCA1 expression (Fig. 9F), whereas high-fat diet consumption led to increased levels (Fig. 9F), and combined supplementation with CBDA and a high-fat diet also increased ABCA1 expression (Fig. 9F). Finally, CBDA administration in the standard diet group resulted in decreased MTP expression in all the experimental groups compared with the control group (Fig. 9G).

Fig. 9.

Representative images of IHC staining for selected fatty acid transporters (400x, bar : 50 μm) and quantified intensity density of fatty acid binding protein (FABPpm), fatty acid transport proteins 2 and 5 (FATP2 and FATP5), fatty acid translocase (FAT/CD36), ATP-binding cassette transporter (ABCA1), and microsomal triglyceride transfer protein (MTP). The data are expressed as mean ± SD, n = 6 per group. *p < 0.05—significant difference compared with the control group; #p < 0.05—significant difference compared with the high-fat diet group.

Discussion

Hepatic steatosis, the hallmark of MASLD, occurs when lipid acquisition exceeds lipid disposal. While multiple pathways contribute to this imbalance, research has shown that increased uptake of circulating fatty acids plays a critical role¹². According to the literature, rats fed a high-fat and high-fructose diet to induce NAFLD presented increased expression of FAT/CD36 and FATP5 compared with the control group²⁶. Similarly, in our study, HFD consumption significantly increased FAT/CD36 and FATP5 expression. However, in the study conducted by Zhou et al., the expression of FATP2 increased after HFD, and we did not observe any statistically significant changes in FATP2 expression, as determined by Western blotting, in comparison with the control group. However, the expression of FATP2 analyzed via IHC was significantly lower in the HFD group and the HFD + CBDA than in the control group. The differences in FATP2 expression between our studies and those presented in the literature may be due to the different feeding periods or diet compositions used²⁷. However, a study conducted by Eithan et al., which is in line with our findings, revealed that feeding mice a HFD for 19 weeks did not affect FATP2 expression²⁸. Moreover, we observed significantly increased FABPpm expression in HFD-fed rats compared with control rats. This finding is in line with the findings of McIntosh et al.’s study on pair-fed high glucose mice, where hepatic FABPpm expression was significantly increased. Similarly, Cao et al., however, studied different tissues than the liver and reported elevated FABPpm expression in the soleus muscle of rats after 8 weeks of HFD consumption^29,30. Compared with HFD treatment, CBDA treatment in rats with HFD-induced MASLD significantly decreased the expression of FAT/CD36 and FABPpm, suggesting that CBDA ameliorated hepatic fatty acid influx through the modulation of the expression of these transporters. However, FAT/CD36 and FABPpm expression measured via IHC staining revealed different results. FAT/CD36 expression was further increased, and FABPpm expression did not differ between the HFD + CBDA group and the HFD group. As shown by the IHC images of FAT/CD36 in the HFD + CBDA group, although a larger group of cells had higher protein intensity staining, there is a greater contrast between the group of cells and surrounding cells, whose intensity is much lower than that of the surrounding cells in the HFD group. Compared with other studies on cannabis extracts, CBDA exhibited distinct effects on FAT/CD36 expression. In the study by Assa-Glazer et al., none of the three cannabis extracts (predominantly rich in CBD or THC) administered orally significantly altered liver FAT/CD36 mRNA expression in mice fed a HFD for six weeks. We suspect that although two of the three extracts contained CBDA, its low percentage and the mixed composition of various cannabinoids in the extracts might have accounted for the difference in results between our study and the cited studies³¹. Interestingly, our study revealed that FATP5 expression, as measured by IHC, was markedly lower in the HFD + CBDA group than in the HFD group. Ran et al., in their study performed on FATP5 knockout mice, demonstrated that deletion of FATP5 reduced triglyceride and diglyceride levels in the liver and ameliorated diet-induced steatosis in mice³². Similarly, Doege et al. reported that FATP5 deletion in hepatocytes isolated from FATP5 knockout mice resulted in decreased hepatic TAG and FFA levels³³. However, research by Enooku et al. has also demonstrated that decreased hepatic FATP5 expression in patients with NAFLD is associated with the histological progression of this disease and may correlate with hepatic fat loss during the transition from nonalcoholic steatohepatitis to cirrhosis³⁴. While the Enooku et al. reported that decreased FATP5 expression is correlated with disease progression, this correlation may be related to the natural disease course being affected by factors other than therapeutic intervention. The reduction in FATP5 expression through CBDA treatment should lead to a reduction in excessive hepatic fatty acid uptake, which is fundamentally different from the pathological changes obseerved in advanced NAFLD progression and therefore might be beneficial. Moreover, our findings revealed that CBDA did not affect fatty acid efflux transporters such as ABCA1 and MTP, thereby limiting the role of CBDA to the modulation of only the previously mentioned fatty acid influx transporters.

In our study, compared with the control group altered fatty acid transporter expression in the HFD group resulted in a significant increase in the deposition of all the main lipid fractions, namely, FFAs, TAGs, DAGs, and PLs. CBDA treatment during the last 2 weeks resulted in a significant decrease in total DAG and PL concentrations compared with the HFD group. Excessively deposited DAGs are responsible for insulin signaling impairment and MASLD progression, as Magkos et al. reported a direct correlation between DAG levels and hepatic steatosis as well as decreased insulin sensitivity in patients³⁵. In addition, Li et al. reported that PLs, especially phosphatidylcholine/phosphatidylethanolamine, are important in MASLD progression to metabolic dysfunction-associated steatohepatitis and act as regulators of cell membrane integrity³⁶. Although not all assessed lipid fractions were affected in our experiment and animal body weight did not change in the HFD group after CBDA treatment, decreased total lipid accumulation was observed in histological images, which shows that the accumulation of lipid droplets in the liver was significantly lower in the HFD + CBDA group than in the HFD group. Compared with the HFD group, alanine aminotransferase (ALT) levels, which is considered a marker of liver damage, were lower in the plasma of rats treated simultaneously with a HFD and CBDA. We suspect that changes in total DAG and PL could have been caused by the previously mentioned decreased expression of FAT/CD36 and FAPBpm, which lowered lipid influx into hepatocytes. Another reason for the changes in DAG and PL deposition was the decreased expression of GPAT, which is the rate-limiting initial enzyme in the glycerolipid synthesis pathway, leading to the formation of PL and DAG³⁷. In Linden et al.’s research, adenoviral-mediated GPAT overexpression in primary cultures of rat hepatocytes led to an increase in DAG and phosphatidylcholine levels without changes in TAG content or to decreased β-oxidation³⁸. Therefore, we assume that the decreased expression of GPAT in HFD + CBDA rats compared with HFD rats may have resulted in decreased accumulation of total DAG and PL, which was observed in our study. DAG content in the liver was also decreased in the research of Wendel et al. on ob/ob mice with deficient of GPAT1, compared with ob/ob mice, which is in line with our study³⁷. Moreover, we revealed that ATGL expression, which was suppressed by HFD feeding, was increased in the HFD + CBDA group compared with the HFD group. Products from TAG hydrolysis, facilitated by increased ATGL expression, could have been redirected into β-oxidation. While CPT1 was not significantly impaired, increased β-HAD expression in the HFD + CBDA group compared with the HFD group supports our assumption. Another study confirming our findings was conducted by Rein et al. on McA-RH7777 cells, where adenoviral ATGL overexpression resulted in promoted fatty acid oxidation via increased expression of CPT1 and acyl-CoA oxidase³⁹. Similarly, Ong et al. studied primary hepatocytes from chow-fed mice and reported that increased hepatic ATGL expression promoted fatty acid oxidation. ATGL overexpression induced in these cells by adenovirus resulted in increased fatty acid oxidation to acid-soluble metabolites and CO₂⁴⁰.

In addition to changes in hepatic lipid accumulation, we observed compelling changes in circulating lipid levels. While in several studies HFD feeding resulted in increased plasma TAG levels, in our study, rats fed only a HFD presented decreased levels of TAG⁴¹. This discrepancy may be explained by the diet composition. As described by Rey et al., in their study, rats fed three different HFDs presented increased TAG plasma levels in two groups, whereas in the third group, decreased TAG plasma levels were observed⁴². Interestingly, compared with HFD consumption, CBDA administration to the HFD group increased plasma TAG levels. Owing to a lack of changes in previously mentioned ABCA1 and MTP transporters as well as no difference in the total hepatic TAG levels in the HFD + CBDA group compared with the HFD group, we suspect that CBDA could increase the secretion of TAG from tissues other than the liver. Moreover, as total plasma TAG is the sum of the TAG content across all the lipoprotein species, we may suspect that also chylomicrons, which are the largest triglyceride-rich lipoproteins carrying exogenous (dietary) triacylglycerols, may also be affected by CBDA and result in increased levels of plasma TAGs. In Zhu et al.’s study, rats treated with CBD at high doses for one week presented increased TG-rich lipoprotein transport in the lymphatic system, which may partially confirm the effect of phytocannabinoids on TAG output⁴³.

Another important process impaired in MASLD is de novo lipogenesis, whose activity can be represented by the de novo lipogenesis ratio^44,45. In our study, DNL was lower in all the experimental groups than in the control group in terms of the FFA and DAG fractions. This finding is in accordance with studies conducted on high-fat diet-fed mice, where this diet significantly decreased de novo lipogenesis in the liver and adipose tissue¹³. The high availability of dietary fatty acids may inhibit the expression of enzymes and genes involved in this pathway, which is supposedly an adaptive mechanism in which the organism prioritizes the utilization or storage of dietary lipids rather than the synthesis of new lipids⁴⁶. Moreover, in measured de novo lipogenesis ratios, we spotted a significant decrease in the FFA fraction in rats from the HFD + CBDA group compared with the HFD group. This selective effect on FFA synthesis suggests that CBDA may specifically interfere with pathways governing the production of FFAs without significantly altering the metabolism of other lipid fractions. Interestingly, CBDA administered to rats fed standard chow diminished the de novo lipogenesis ratio in comparison with the control group, which shows that CBDA’s de novo lipogenesis decreasing properties not only in pathological states such as MASLD but also under physiological conditions, which may preserve the organism from excessive production of lipids. Conversely to de novo lipogenesis ratio, we noticed that CBDA administration to the rats fed a HFD resulted in increased expression of the rate-limiting fatty acid synthesis enzyme, namely, FAS, in comparison with the HFD group. However, because of the differences between expression and activity of the protein, a more authoritative indicator is the de novo lipogenesis ratio, which determines lipogenesis based on substrates and products of this reaction; therefore, although FAS expression could have been increased, the total de novo lipogenesis activity might have been decreased. A similar difference in SCD protein expression and activity was observed in Li et al.’s study, in which 6-gingerol did not change SCD expression, but inhibited SCD activity by diminishing the desaturation of fatty acids in HFD-fed rats⁴⁷.

Not only does total lipid accumulation play an important role in MASLD pathogenesis, but so does its composition⁴⁸. Our research demonstrated that HFD feeding significantly increased total SFAs across all the lipid fractions (FFA, DAG, TAG, and PL) in the rat liver, which is consistent with the findings of Liu et al., in which rats fed a HFD for 3 weeks presented markedly increased levels of SFAs in the DAG, TAG and PL in the liver⁴¹. Notably, in our study, CBDA administration for two weeks partially reversed this trend, with significant reductions in SFA content observed in the FFA and PL fractions. These changes may be considered beneficial in MASLD development and progression since Friden et al., in their study of human patients, reported a positive correlation between PL SFAs and liver fibrosis; therefore, decreasing PL SFA accumulation should be considered beneficial⁴⁹. As confirmed by Malhi et al. in mouse hepatocytes, FFAs and SFAs are responsible for hepatic lipoapoptosis, which is a key feature of MASLD; therefore, the lowering effect of CBDA on their deposition might also be promising⁵⁰.

With respect to MUFAs, we observed a complex pattern of alterations. While a HFD increased the MUFA content in the FFA, DAG, and TAG fractions, CBDA differentially affected these lipid pools. Most notably, CBDA decreased the MUFA content in the DAG fraction compared with the HFD group. Conversely, compared with the HFD, CBDA increased the MUFA content in the PL and TAG fractions. This redistribution of MUFAs from potentially harmful lipid intermediates (DAG) to storage (TAG) and structural lipids (PL) may represent a protective adaptation, especially because hepatic TAG MUFAs demonstrated an inverse association with liver fibrosis in Friden et al.’s study in humans⁴⁹. PUFA supplementation, especially with n-3 PUFAs, was confirmed to lower hepatic inflammation, oxidative stress, and fibrosis in mice fed a Western diet for 16 weeks by Depner et al.⁵¹. Therefore, the CBDA effect in our experiment on HFD-fed rats, which resulted in a significant increase in total n-3 PUFAs in the DAG and TAG fractions compared with the HFD group, may be considered beneficial for MASLD development. Interestingly, we observed a significant decrease in total n-6 PUFAs in the FFA, DAG and PL fractions in HFD + CBDA rats compared with HFD rats. Hence, it is important for liver homeostasis, as Jeyapal et al. reported that decreased n-6 PUFA supplementation in favor of n-3 PUFAs prevented hepatic steatosis induced by high-fat, high-fructose diet feeding for 24 weeks in rats⁵².

To provide more information on the influence of CBDA on lipid metabolism, we investigated desaturation indices, which revealed significant alterations in the activity of SCD1, the enzyme responsible for converting SFAs to MUFAs⁵³. The 16:1/16:0 desaturation index, reflecting SCD1 activity, was markedly elevated in the FFA fraction but diminished in the DAG, TAG, and PL fractions in both the HFD and HFD + CBDA groups compared with the control. Compared with those of the control group, the 18:1/18:0 desaturation indices of HFD-fed rats were significantly greater in the FFA and DAG fractions and lower in the TAG and PL fractions. These findings align with a study conducted on rats by Liu et al., which also reported notable decreases in the 16:1/16:0 and 18:1/18:0 desaturation indices of the DAG, TAG and PL fractions after HFD feeding⁴¹.

Interestingly, compared with HFD alone, CBDA treatment further decreased the 16:1/16:0 index in the PL fraction, suggesting a potential suppression of desaturation in membrane phospholipids. Conversely, compared with HFD alone, CBDA increased the 18:1/18:0 index in the FFA fraction but decreased it in the DAG fraction. A further increase in the 18:1/18:0 index in the FFA fraction seems to be a compensatory mechanism of CBDA, which prevents excessive accumulation of the lipotoxic SFA – stearic acid and thus, redirects it to the less harmful MUFA – oleic acid.

The fatty acid desaturase expression data further support a regulatory role for CBDA in desaturation processes. The significant increase in FADS2 expression in both the HFD and HFD + CBDA groups indicates an increased desaturation capacity for PUFAs⁵⁴. Moreover, in our study, CBDA reversed the HFD-induced decrease in FADS1 expression, which was similar to the findings of the study conducted by Ghooray et al. In their study, the overexpression of FADS1, which was achieved via the injection of a recombinant AAV8 vector containing a promoter with a FADS1 sequence in rats fed with a high fat and high-fructose (HFHFr) diet for 8 weeks, also led to the reversal of FADS1 expression. Further in their study, FADS1 overexpression did not change total TAG accumulation in HFHFr rats, which is consistent CBDA impact, but it did not significantly change total SFA, MUFA and PUFA accumulation, which is different from the findings of our study and confirms CBDA’s broader impact on liver fatty acid metabolism than only on FADS1 expression⁵⁵.

Other findings in our study are related to the effect of CBDA on fatty acid elongation processes. The elongation index 18:0/16:0, reflecting primarily ELOVL6 activity, was significantly greater in the DAG fraction of the HFD + CBDA group than in both the control and HFD groups. This finding aligns with our observation of increased ELOVL6 expression in the HFD + CBDA group compared with the HFD group, where ELOVL6 was initially suppressed by HFD feeding. This finding is particularly intriguing considering the findings of previous studies highlighting the complex role of ELOVL6 in metabolic health. While Matsuzaka et al. demonstrated that Elovl6 gene deletion protected mice from diet-induced insulin resistance despite obesity, suggesting that the inhibition of ELOVL6 might be metabolically beneficial, their other study, which was performed on transgenic mice expressing a human ELOVL6 model, revealed that ELOVL6 overexpression leads to inflammation and liver injury^56,57. However, our findings suggest that CBDA might restore ELOVL6 expression suppressed by a HFD, potentially as part of a compensatory mechanism to maintain lipid homeostasis.

The elongation indices for longer-chain SFAs (20:0/18:0, 22:0/20:0, and 24:0/22:0) showed complex, fraction-specific changes in response to both HFD and CBDA treatment. Notably, CBDA reversed several HFD-induced alterations in these elongation indices, particularly in the FFA and DAG fractions. For instance, compared with the HFD group, CBDA decreased the 22:0/20:0 index in the FFA fraction and increased the 24:0/22:0 index in both the FFA and the DAG fractions. While there is no research discussing the mentioned changes in the liver, Martinez-Sanz et al., in their study on patients with NAFLD and infected with HIV, reported a decreased total 24:0/22:0 ratio in plasma compared with patients with NAFLD without the virus⁵⁸. On the other hand, decreased elongation index 24:0/22:0 in the PL fraction in HFD + CBDA rats compared with the HFD rats may also be considered as an adverse effect because PL behenic acid (22:0) was directly associated with liver fibrosis in the study performed by Friden et al. on human liver tissue samples⁴⁹.

Our study also revealed that CBDA significantly increased ELOVL5 expression in rats fed with HFD compared with both the control and HFD groups. ELOVL5 primarily elongates C16-C22 PUFAs and is essential for the synthesis of long-chain PUFAs⁵⁹. The upregulation of ELOVL5 by CBDA could enhance the synthesis of anti-inflammatory long-chain PUFAs, potentially contributing to its hepatoprotective effects. It would be a similar effect to that observed in a study by Tripathy et al. on obese C57BL/6 J mice with increased activity of ELOVL5 induced by infection with an adenovirus expressing ELOVL5. In their experiment, they revealed that ELOVL5 elevated activity resulted in increased TAG catabolism as well as reversed altered by HFD administration, n-3 and n-6 PUFA content. Moreover, they linked ELOVL5 upregulation with increased ATGL expression, which we also demonstrated as an effect of CBDA administration in our study⁶⁰.

CBDA exerts its effects through interactions with many receptors, including a wide system called the endocannabinoidome²³. Although CBDA has low direct binding affinity for the cannabinoid receptors, its interaction with various isoforms of PPAR receptors has been shown in breast cancer cells, hepatocellular carcinoma cells and adipocyte cell line^61,62. This finding seems to be of prime importance since, in recent years, dual agonists of PPAR-α and PPAR-γ have attracted significant interest as potential therapeutic agents for MASLD^63,64. In our study, HFD feeding resulted in a decrease in PPAR-α and PPAR-γ expression compared with that of the control, which is in line with other animal studies^65,66. CBDA treatment of rats fed a HFD resulted in significantly increased PPAR-γ expression compared with the HFD group. In D’Aniello et al.’s study performed on HepG2 and 3T3L1 cell cultures, CBDA was shown to be a dual PPAR-α and PPAR-γ agonist, which partially corresponds with the results from our animal model⁶². Therefore, we may suspect that the changes in lipid metabolism mentioned before could have been a result of a PPAR-γ modulation; however, to elucidate the exact mechanism of CBDA through PPAR regulation, more research, especially in knockout models, should be performed.

While in our study we concentrated on the effects of CBDA on MASLD animals, notably CBDA itself also afffected chow-fed animals. The most prominent changes were visible in the FFA and DAG fractions, where accumulation increased. Moreover, in control animals, CBDA treatment decreased β-oxidation, de novo lipogenesis and the expression of fatty acid exporters (ABCA1 and MTP), which diminishment may significantly contribute to increased lipid deposition in FFAs and DAGs. These changes visible in the liver are associated with a lack of changes in the plasma lipids after CBDA treatment in standard diet-fed animals. We suspect that the observed changes in the liver may be exerted via the interaction of CBDA with CB2 receptors, as its expression was significantly decreased in the control group. Although such changes may be quite surprising since CBDA promoted enhanced lipid deposition in the liver, the lack of changes in plasma lipid levels indicates that CBDA promoted liver lipid-accumulating capacity rather than adding lipids to the circulation, which is a main factor in arteriosclerosis development. The involvement of CB2 receptors in these effects seems to be reasonable, as studies on CB2-deficient macrophages revealed decreased macrophage apoptosis-induced arteriosclerosis development⁶⁷. Moreover, increased CB2 expression in the liver was observed and associated with the progression of steatosis to steatohepatitis⁶⁸. Thus, as observed in our study, decreased expression of this receptor exerted by CBDA in control animals seems to be beneficial for the whole organism. However, many gaps related to the roles of CBDA and CB2 roles in MASLD development still exist, and progression and more studies are needed.

In conclusion, this study provides compelling evidence that CBDA exerts multifaceted effects on hepatic lipid metabolism in rats with MASLD. Our findings demonstrate that CBDA administration significantly changed lipid metabolism through several complementary mechanisms. First, CBDA modulated fatty acid influx by downregulating FAT/CD36 and FABPpm transporter expression while upregulating FATP5, effectively rebalancing fatty acid trafficking into the liver. Second, CBDA promoted lipid catabolism by enhancing β-oxidation through increased β-HAD expression and stimulating TAG lipolysis via the upregulation of ATGL, collectively improving lipid disposal mechanisms. Third, CBDA reduced total DAG and PL levels, potentially mitigating lipotoxicity and inflammation associated with these lipid intermediates. Fourth, CBDA substantially remodeled the hepatic lipid composition by decreasing saturated fatty acids in the FFA and PL fractions, redistributing MUFAs from harmful DAGs to more benign TAG and structural PL pools, and increasing the PUFA content in the TAG fractions. Fifth, CBDA significantly altered fatty acid modification pathways by reversing HFD-induced suppression of FADS1 and increasing ELOVL5 and ELOVL6 expression, which may enhance the synthesis of beneficial long-chain PUFAs and reduce lipotoxicity. These comprehensive metabolic effects, coupled with decreased GPAT expression and reduced de novo lipogenesis in the FFA fraction, suggest that CBDA represents an interesting therapeutic candidate for MASLD by targeting multiple dysregulated pathways in hepatic lipid metabolism, potentially limiting disease progression by improving both the quantity and quality of the hepatic lipid content. However, still more research needs to be performed, especially with radiolabeled fatty acid precursors, to elucidate the dose-dependent effects of CBDA on lipid metabolism as well as its impact on other tissues and their metabolic pathways.

Materials and methods

Animal model

Our experimental procedures were carried out on male Wistar rats, which initially weighed between 70 and 100 g (6 weeks old). Rodents were obtained from the Centre for Experimental Medicine of the Medical University of Bialystok, Poland. The rats were housed in accredited animal care facilities (22 ± 2℃) coupled with a reverse light–dark cycle of 12/12 h and provided with unlimited access to water and standard rodent feed (Labofeed B, Animal Feed Manufacturer “Morawski”, Kcynia, Poland). Following a seven-day acclimatization period, the rats were randomly divided into four experimental groups, each consisting of 10 animals. The groups were as follows: (1) the control group, which was fed a basal rodent diet (Labofeed B, Animal Feed Manufacturer “Morawski”, Kcynia, Poland) (12.4 kcal% fat, 57.1 kcal% carbohydrates, and 30.5 kcal% protein)^69,70; (2) the CBDA group, which was fed a basal rodent diet and treated with CBDA; (3) the HFD group, which was fed a rodent diet rich in fatty acids (60 kcal% fat, 20 kcal% carbohydrates, and 20 kcal% protein (cat. no.: D12492, Research Diets Inc., New Brunswick, NJ, USA)^71,72; and the HFD + CBDA group, which was fed a rodent diet rich in fatty acids and treated with CBDA. The feeding phase lasted for eight weeks, and beginning in the seventh week, the rats received synthetic CBDA (0.1 mg/kg body mass, purity ≥ 99%; THC Pharm GmbH, Frankfurt, Germany) in a single dose intragastrically dissolved in sesame oil or only oil administered for two weeks at the same time of the day. The CBDA dose was selected on the basis of the available literature, as CBDA is the most effective when it is administered intragastrically^73,74. Thus, throughout the entire experiment, the animals received 14 doses of CBDA. In the 8th week of the experiment and 24 h after the last dose of CBDA or its solvent, animals were anaesthetized intraperitoneally with ketamine: xylazine (80 mg/kg: 5 mg/kg body mass). Deeply anaesthetised animals were exsanguinated to harvest large volumes of blood, and immediately afterwards, the heart was removed to ensure death. Liver samples were excised, immediately frozen with precooled aluminum tongs and stored at -80℃ for further analysis. All the experimental procedures were performed in accordance with the ARRIVE guidelines and were approved by the Animal Ethics Committee in Olsztyn (Poland) under license number 35/2023.

Lipid analysis

Gas–liquid chromatography (GLC) was employed to analyze the lipid content in the liver. Lipid fractions were extracted using a chloroform–methanol mixture (2:1, vol/vol) following the Folch method⁷⁵. Subsequently, the samples were centrifuged, and the lower phase was collected for further analysis. The selected lipid fractions, namely, FFA, DAG, TAG, and PL, were separated by thin-layer chromatography (TLC) on silica gel plates (silica Plate 60, 0.25 mm; Merck, Darmstadt, Germany), and heptane/isopropyl ether/acetic acid (60:40:3, vol/vol/vol) was used as the resolving solution. The dried silica plates were examined under ultraviolet light, allowing for the identification of the lipid fractions based on standard lines of authentic FFA, DAG, TAG and PL placed at the beginning and end of the TLC plate. The gel bands containing the targeted lipid fractions were subsequently scraped off and eluted in suitable solvents, followed by transmethylation of the organic phase using a 14% boron trifluoride-methanol solution. A Hewlett-Packard 5890 Series II Gas Chromatograph (Agilent Technologies, CA, USA) containing a capillary column (HP-INNOWax 50mx0.25 mm inner diameter) and a flame ionization detector was used to examine the samples with the addition of hexane. The identification and quantification of individual fatty acids in the selected fractions were performed by comparing their retention times to those of known fatty acid standards (Lanodan Research grade lipids, Stockholm, Sweden). Additionally, heptadecanoic acid (C17:0) was used as an internal standard at the start of the procedure, and the calibration curves prepared for each fatty acid were used to calculate the concentration of fatty acids. The concentrations of FFA, DAG, TAG and PL were calculated based on the sum of the specific fatty acid species content in each target fraction and expressed in nanomoles per milligram of tissue. All the measurements listed below were taken from liver tissue, from all the targeted lipid fractions.

The de novo lipogenesis ratio was measured as the palmitic/linoleic acid (16:0/18:2n-6) ratio. The elongation indices were calculated as the stearic/palmitic acid (18:0/16:0) ratio, arachidic/stearic acid (20:0/18:0) ratio, behenic/arachidic acid (22:0/20:0) ratio and lignoceric/behenic acid (24:0/22:0) ratio. The desaturation index was estimated as palmitoleic/palmitic acid (16:1n7/18:0) and oleic/stearic acid (18:1n9/18:0).

The total SFA, PUFA, MUFA, n-3, and n-6 contents were calculated as a sum of the following lipids: SFA: myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), arachidic acid (20:0), behenic acid (22:0), lignoceric acid (24:0), MUFA: palmitoleic acid (16:1), oleic acid (18:1), nervonic acid (24:1), PUFA: linoleic acid (18:2n6), ALA (18:3n3), AA (20:4n6), EPA (20:5n3), DHA (22:6n3), n-3: ALA (18:3n3), EPA (20:5n3), DHA (22:6n3), n-6: linoleic acid (18:2n6), and AA (20:4n6).

Western blotting

Protein expression was examined using the Western blotting technique, as previously described⁷⁶. Briefly, the total protein concentration in all the samples was determined using the bicinchoninic acid assay with bovine serum albumin (BSA) established as a standard. Next, the homogenates were reconstituted in Laemmli buffer (Bio-Rad, Hercules, CA, USA) and applied to commercially available CriterionTM TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA). After electrophoresis, the separated proteins were transferred onto polyvinylidene fluoride or nitrocellulose membranes, depending on the type of transfer: semi-dry or wet, respectively. Next, the nitrocellulose membranes were blocked with Tris-buffered saline containing Tween 20 supplemented with 5% nonfat dry milk or BSA. The membranes were then incubated overnight with selected primary antibodies, including FAT/CD36, FATP2, FATP5, MTP, GPAT, DGAT2, CPT1, MAGL, ATGL, β-HAD, FAS, Elovl3, Elovl6, Elovl5, PPARα, PPARγ, CB2 (Santa Cruz Biotechnology, Dallas, TX, USA), FABPpm, FADS1, FADS2, CB1 (Abcam Cambridge, UK), ABCA1, and HTR1A (Invitrogen Biotechnology, Waltham, MA, USA). The membranes were incubated with horseradish peroxidase conjugated secondary antibodies. Finally, the protein bands were visualized and quantified densitometrically using a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Protein expression levels were quantified using stain-free gels, which enabled total protein normalization with the control group set as 100%.

Immunohistochemical staining and histochemistry

Immunohistochemical (IHC) staining was performed on 4 µm-thick paraffin sections of freshly picked samples from the same fragment of the liver lobe from each rat. The immunohistochemical reaction was performed using antibodies against proteins of interest, namely, FABPpm (Abcam, Cambridge, UK), FAT/CD36, MTP, FATP2 (Santa Cruz Biotechnology, Dallas, TX, USA), FATP5, ABCA1 (Thermo Fisher Scientific, Waltham, MA, USA). The EnVision™ FLEX (Dako Agilent Technologies, Santa Clara, CA, USA) visualization kit was used for the staining procedure on formalin-fixed, paraffin-embedded tissue sections. The antibodies were diluted 1:250 in Dako Antibody Diluent (Dako Agilent Technologies, Santa Clara, CA, USA).

Antigen retrieval was conducted using the PT Link device (Dako Agilent Technologies, Santa Clara, CA, USA). After 10 min of washing, the slides were placed in EnVision™ FLEX Target Retrieval Solution, High pH (50x) buffer (Dako Agilent Technologies, Santa Clara, CA, USA), which was diluted according to the manufacturer’s instructions and incubated for 73 min, followed by incubation with EnVision™ FLEX Peroxidase Blocking Reagent for 10 min, and incubation with the primary antibody for 30 min each. Next, the slides were incubated with the secondary antibody EnVision™ FLEX /HRP for 30 min. and EnVision™ FLEX DAB + Chromogen with EnVision™ FLEX Substrate Buffer for 10 min with 10 min washes between.

The substrate system reaction product was intensely brown and localized at the site of antigen detection. For counterstaining, standard hematoxylin staining was performed. Moreover, to assess liver morphology, the standard hematoxylin–eosin staining was conducted on the same fragment of the liver lobe from each rat. After the immunohistochemical reaction and hematoxylin–eosin staining, the slides were evaluated using an Olympus BX 41 microscope, an SC50 camera, and CellSens morphometric software. Moreover, the images were quantified using ImageJ software and are presented as the intensity density of the stained area.

Enzyme-linked immunosorbent assay

The concentration of ALT in the plasma samples was assessed with the use of a commercially available enzyme-linked immunosorbent assay kit (Biorbyt, Cambridge, UK). Before the procedure, the plasma samples were centrifuged and diluted twofold. The next steps were conducted in accordance with the manufacturer’s protocol. The absorbance was measured at a wavelength of 450 nm in a hybrid multimode microplate reader Synergy H1TM (BioTek Instruments, Winooski, VT, USA), and the calculated values were based on the obtained standard curve. The ALT concentration was expressed in nanograms per milliliter of plasma.

Statistical analysis

The experimental results are based on ten independent measurements (GLC) or six independent measurements (Western blot, IHC) and are given as mean ± standard deviation (SD). The analysis of the statistical data was conducted with the use of the GraphPad Prism for macOS Version 10.2.1 Software (San Diego, CA, USA). The Shapiro–Wilk test and Bartlett’s test were used to determine whether the distribution was normal and whether the variance was homogeneous. Two-way ANOVA supported by Tukey’s test, the parametric t-test, or the non-parametric Mann–Whitney U test for differences with normal and abnormal distributions, respectively, was used to evaluate the statistical comparisons between the experimental groups. For statistical differences, a p-value of less than 0.05 was used.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization, P.F.K. and K.K.-N; methodology: P.C., P.F.K. and K.K.-N.; software P.C.; validation, K.K.-N., E.H.-S. and P.F.K.; formal analysis, P.C. and J.D.; investigation, K.K.-N., E.H.-S., J.K. and P.F.K.; resources, K.K.-N., E.H.-S. and P.R.; data curation, K.K.-N., M.Z. and P.F.K.; writing, P.C. and P.F.K.; review and editing, K.K.-N.; visualization, P.F.K.; supervision K.K.-N. and A.C.; project administration, K.K.-N. and P.F.K;

funding acquisition, K.K.-N. and P.F.K.; All authors have read and agreed to the published version of the manuscript.

Funding

Funded from the state budget underthe program of the Minister of Education and Science in Poland “Pearls of Science”, project number PN/01/0003/2022, amount of co-financing 240 000 PLN, total value of theproject 240 000 PLN.

Data availability

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

Declarations

Competing interests

The authors declare no competing interests.

Institutional review board statement

The study was conducted according to the EU Directive 2010/63/EU for animal experiments and approved by the Ethical Committee for Animal Testing in Olsztyn, Poland (permission no. 35/2023, date of approval: 19 April 2023).