Cannabinoid receptor type 1 (CB1) has been found to be involved in intrahepatocyte lipid accumulation (1,2). CB1 overexpression in hepatocytes has been hypothesized to contribute to the formation of hepatic steatosis and inflammation (3–5). Steatogenic agents, such as ethanol, and a high-fat diet can upregulate the activity of CB1 via the increased synthesis of endocannabinoids, 2-arachidonoylglycerol, and anandamide (4). CB1 receptor activation upregulates the lipogenic transcription factor, sterol regulatory element-binding protein 1c and its target enzymes, acetyl-CoA carboxylase-1 and fatty acid synthase. Furthermore, CB1 receptor activation downregulates carnitine palmitoyltransferase-1, which is the rate-limiting enzyme associated with fatty acid oxidation. Therefore, CB1 activation increases fatty acid synthesis and decreases fatty acid oxidation, which results in a fatty liver (4). Previously published images of hepatic CB1 overexpression in human diseases have predominantly been obtained from patients suffering from serious liver diseases, such as severe non-alcoholic fatty liver disease found in morbid obesity (6), stage IV primary biliary cirrhosis (7) and hepatitis C (8). However, it remains unclear whether CB1 overexpression additionally contributes to milder, more common liver diseases, such as fatty liver disease without or with mild inflammation.

The primary aim of the present exploratory study was to determine the extent of CB1 expression in steatotic rat livers. A secondary aim was to clarify whether steatosis and inflammation are more extensive in areas of increased CB1 overexpression.

Previously published images of CB1 expression have predominantly been captured from cases with severe liver diseases, including severe non-alcoholic fatty liver disease found in morbid obesity (6), stage IV primary biliary cirrhosis (7) and hepatitis C (8). Liu et al (6) have previously captured immunohistochemistry images of human livers stained with CB1 antibodies. In these figures, the nuances of the hepatocytes in the liver with no obvious pathology appear identical to the negative control, which had not been stained with CB1. Therefore, the immunohistochemistry images captured in the present study and those attained by Liu et al (6) suggested that the visible CB1 expression levels exhibited in the present study constitute overexpression. The present study used similar CB1 antibodies to those that generated the aforementioned images (6–8). The present results indicated that CB1 was overexpressed in the hepatocytes of rats with steatosis with mild or without any inflammatory activity in two strains of rats (Figs. 1 and 2). In these images, steatosis was most marked in areas exhibiting the overexpression of CB1. This is consistent with previous studies, which have reported that CB1 contributes to the formation of steatosis (4,12). In addition, lymphocyte infiltrations were more commonly observed in areas where CB1 was highly overexpressed, which is consistent with studies that have suggested that CB1 contributes to inflammation (12) and plays a role in inflammatory cells (13). In particular, a CB1 antagonist, rimonabant, has been demonstrated to suppress the lipopolysaccharide-induced production of the pro-inflammatory interleukins (IL), IL-6 and IL-8 in human macrophages (13).

The livers extracted from the 2 rats lacking normal leptin receptors had a total CB1 score of 6, and macrovesicular grade 4 steatosis. By contrast, the livers from the 36 Sprague Dawley rats exhibited a reduced CB1 score and/or less severe steatosis. These results may have been due to the increased age of the rats lacking normal leptin receptors, which allowed more time for disease progression. High CB1 activity has previously been associated with obesity (14) and hepatic steatosis.

The present results suggested that hepatic CB1 contributes to steatosis in rats, even prior to its progression to steatohepatitis. Although the present results are preliminary and require further confirmation, it is to be expected that a hepatic CB1 antagonist that is actively transported into hepatocytes may exert a protective effect against steatosis and inflammatory complications. Previous studies have suggested that a hepatic CB1 antagonist may be designed that would be actively transported into hepatocytes (12,15). For example, a glucokinase activator has been optimized for active liver uptake, leading to a >50-fold increase in free concentration in the liver compared with that in the pancreas (15). Furthermore, a hepatoselective CB1 antagonist may be capable of ameliorating steatosis, insulin resistance, dyslipidemia and inflammatory liver disease (12). The present results suggest that a hepatoselective CB1 antagonist could ameliorate steatosis and steatohepatitis in rodents, even prior to the occurrence of serious complications. Centrally acting CB1 antagonists are known to produce notable psychiatric side effects in certain patients (16); however a hepatoselective CB1 antagonist may have the potential to be effective with negligible psychiatric side effects (12).

The present study investigated tissue from spare, archived liver blocks, which were obtained from 38 rats that had been euthanized during the course of previous research at the Karolinska Institute of the Karolinska University Hospital and Lund University in order to keep the number of animals used for research to a minimum. Therefore, among the limitations of this study is the lack of individual data for the food and fructose intake levels of the rats per day, in addition to the final body weight of the rats. However, these variables do not appear to be necessary for the present investigation. In the livers of 7-week-old Sprague-Dawley rats in a previous study (17), the triglyceride content did not differ significantly between males and females, which supports the generalization of the present findings to female rats. In addition, to the best of our knowledge, no previous results have suggested that the hepatic molecular pathways associated with steatosis differ markedly between males and females. Another limitation of the present study is that the descriptive statics are not supplemented with inferential statistics. The scoring system used assigned identical scores to tissues with steatosis ranging from 26 to 75%; however, these tissues may not have had identical disease severity. In future studies, actual percentages, rather than scores, should be recorded as a basis for calculating correlations between CB1 upregulation, steatosis and inflammation

On the basis of the present histological findings and the results of previous studies on CB1 overexpression in more serious inflammatory liver diseases, we have come to the conclusion that it is likely that CB1 overexpression contributes to increased steatosis and complications across several stages of the disease, including the early stages.