The Atypical Cannabinoid Abn-CBD Reduces Inflammation and Protects Liver, Pancreas, and Adipose Tissue in a Mouse Model of Prediabetes and Non-alcoholic Fatty Liver Disease

Abstract

Background and Aims: The synthetic atypical cannabinoid Abn-CBD, a cannabidiol (CBD) derivative, has been recently shown to modulate the immune system in different organs, but its impact in obesity-related meta-inflammation remains unstudied. We investigated the effects of Abn-CBD on metabolic and inflammatory parameters utilizing a diet-induced obese (DIO) mouse model of prediabetes and non-alcoholic fatty liver disease (NAFLD). Materials and Methods: Ten-week-old C57Bl/6J mice were fed a high-fat diet for 15 weeks, following a 2-week treatment of daily intraperitoneal injections with Abn-CBD or vehicle. At week 15 mice were obese, prediabetic and developed NAFLD. Body weight and glucose homeostasis were monitored. Mice were euthanized and blood, liver, adipose tissue and pancreas were collected and processed for metabolic and inflammatory analysis. Results: Body weight and triglycerides profiles in blood and liver were comparable between vehicle- and Abn-CBD-treated DIO mice. However, treatment with Abn-CBD reduced hyperinsulinemia and markers of systemic low-grade inflammation in plasma and fat, also promoting white adipose tissue browning. Pancreatic islets from Abn-CBD-treated mice showed lower apoptosis, inflammation and oxidative stress than vehicle-treated DIO mice, and beta cell proliferation was induced. Furthermore, Abn-CBD lowered hepatic fibrosis, inflammation and macrophage infiltration in the liver when compared to vehicle-treated DIO mice. Importantly, the balance between hepatocyte proliferation and apoptosis was improved in Abn-CBD-treated compared to vehicle-treated DIO mice. Conclusions: These results suggest that Abn-CBD exerts beneficial immunomodulatory actions in the liver, pancreas and adipose tissue of DIO prediabetic mice with NAFLD, thus protecting tissues. Therefore, Abn-CBD and related compounds could represent novel pharmacological strategies for managing obesity-related metabolic disorders.

Keywords: NAFLD; adipose tissue; cannabinoids; inflammation; islets of Langerhans; liver; obesity; prediabetes.

Figure 1

Effect of Abn-CBD on body weight and food intake. Mice fed a standard diet (SD) or high fat diet (HFD) for 15 weeks were randomized to vehicle or Abn-CBD for further 2 weeks (A). Body weight was measured weekly during the first 15 weeks and daily during treatment (B). (C) Body weight after the 2-week treatment with vehicle or Abn-CBD. (D) Average of daily calorie intake per mouse and (E) per Kg of weight during the 2-week treatment and (F) food efficiency. Leptin (G) and adiponectin (H) plasma levels at the end of the study. Data show mean ± S.E.M except (C) that shows median ± min to max. n = 6 SD-Vehicle, n = 7 HFD-Vehicle and n = 8 HFD-Abn-CBD. ^*p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001 compared to SD-Vehicle; ^#p ≤ 0.05 compared to HFD-Vehicle.

Figure 2

Effect of Abn-CBD on glucose homeostasis, islet morphology and functionality. (A) Fasted (overnight) plasma insulin and (B) blood glucose levels at the end of the study. (C) Representative photomicrographs of islets. (D,E) Morphometric analysis of islets. Data show mean ± S.E.M. except (D,E) that show median ± min to max. n = 6 SD-Vehicle, n = 7 HFD-Vehicle and n = 8 HFD-Abn-CBD except (D), n = 51–66 islets from 4 mice each group, (E) n = 52–81 islets from 4 mice each group. ^*p ≤ 0.05 and ^**p ≤ 0.01 compared to SD-Vehicle. (F) Static glucose-stimulated insulin secretion in HFD-Vehicle and HFD-Abn-CBD isolated islets. N = 11–12 wells each condition, islets from 2 mice each group.

Figure 3

Effect of Abn-CBD on pancreatic beta-cell viability and intra-islet inflammation. Representative photomicrographs and quantification of islets immunostained for apoptosis (A) and p-NFκB (B); arrows indicate apoptotic cells; p-NFκB immunostaining was counterstained with haematoxylin. (C) F4/80 staining; arrows indicate macrophages. (D) Quantification of lipid peroxidation by TBARS production. (E) Representative photomicrographs of double insulin (green) and BrdU (red) immunostaining in islets and quantification of BrdU+/Insulin+ cells; arrows indicate proliferative beta cells; scale bar is 20 μm. n = 6 SD-Vehicle, n = 7 HFD-Vehicle and n = 8 HFD-Abn-CBD. ^*p ≤ 0.05 and ^***p ≤ 0.001 compared to SD-Vehicle; ^#p ≤ 0.05, ^##p ≤ 0.01, and ^###p ≤ 0.001 compared to HFD-Vehicle.

Figure 4

Effect of Abn-CBD on circulating inflammatory cytokines. Plasma levels of IL-6 (A), CXCL1 (B), IL-5 (C), IFN-γ (D), and IL-10 (E) after the 2-week treatment of mice. n = 6 SD-Vehicle, n = 7 HFD-Vehicle and n = 8 HFD-Abn-CBD. ^*p ≤ 0.05, compared to SD-Vehicle; ^#p ≤ 0.05 and ^##p ≤ 0.01 compared to HFD-Vehicle.

Figure 5

Effect of Abn-CBD on inflammation in white adipose tissue. Representative photomicrographs and quantification of (A) adipocyte size and (B) crown-like structures in white adipose tissue. Relative expression of (C) Il10 and (D) Cxcl1 in visceral adipose tissue and of (E) Cxcl1 in subcutaneous adipose tissue. Relative expression of Ucp-1 in (F) brown adipose tissue, (G) visceral adipose tissue, and (H) subcutaneous adipose tissue. β-actin was used as reference gene. ^*p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001 compared to SD-Vehicle; ^#p ≤ 0.05 and ^##p ≤ 0.01 compared to HFD-Vehicle.

Figure 6

Effect of Abn-CBD on liver structure and function. Representative photomicrographs and quantification of livers stained for lipid droplets (A) and collagen fibers (B), scale bar is 100 μm. Representative photomicrographs of immunofluorescence in liver for α-SMA (red), counterstained with DAPI (in blue) (C). Biochemical determination of hepatic glycogen content (D) and circulating levels of alanine aminotransferase (E). n = 7–8 SD-Vehicle, n = 5–8 HFD-Vehicle and n = 6–8 HFD-Abn-CBD. ^*p ≤ 0.05 and ^***p ≤ 0.001 compared to SD-Vehicle; ^#p ≤ 0.05 and ^###p ≤ 0.001 compared to HFD-Vehicle.

Figure 7

Effect of Abn-CBD on hepatocyte viability and liver inflammation. (A) Representative photomicrographs and quantification of livers stained for the inflammatory marker p-NFκB (A), the macrophage marker F4/80 (B) and apoptotic cells (C); arrows indicate stained cells; scale bar is 100 μm (A), 20 μm (B), and 200 μm (C). (D) Representative photomicrographs and quantification of BrdU immunofluorescence (in red) in the liver, counterstained with DAPI (in blue). Arrows indicate proliferating cells. n = 7–8 SD-Vehicle, n = 5–8 HFD-Vehicle and n = 6–8 HFD-Abn-CBD. ^*p ≤ 0.05, ^**p ≤ 0.01, and ^***p ≤ 0.001 compared to SD-Vehicle; ^#p ≤ 0.05, ^##p ≤ 0.01, and ^###p ≤ 0.001 compared to HFD-Vehicle.