Arachidyl amido cholanoic acid improves liver glucose and lipid homeostasis in nonalcoholic steatohepatitis via AMPK and mTOR regulation

Figure 1 Arachidyl amido cholanoic acid improves glycemic control in nonalcoholic steatohepatitis patients. ARREST is a multicenter, placebo-controlled study designed to evaluate the efficacy and safety of two arachidyl amido cholanoic acid (Aramchol) doses (400 and 600 mg tablets/d) and placebo tablets in subjects with nonalcoholic steatohepatitis, confirmed by liver biopsy, who were overweight or obese, and had prediabetes or type II diabetes. Blood hemoglobin A1c (HbA1c) content was determined at baseline and weeks 8, 24, 40, and 52. At week 52, both Aramchol doses resulted in a decrease in HbA1c, while NASH patients on the placebo arm showed an increase. The differences from placebo were statistically significant (^aP = 0.0061 and ^bP = 0.0008 for Aramchol 400 mg vs placebo and 600 mg vs placebo, respectively).

Figure 2 Arachidyl amido cholanoic acid differentially regulates AMPK and mTORC1 in cultured hepatocytes. Mouse primary hepatocytes were isolated and allowed to attach for 3 h, after which culture medium was replaced by serum-free MEM with DMSO (vehicle) or Arachidyl amido cholanoic acid (Aramchol) (20 µM), and the cells were cultured for an additional 48 h. A: Effect of Aramchol treatment on SCD1, CPT1A/B, AMPK, P-AMPKα (Thr172), ACCα/β, P-ACCα/β (Ser79), p70S6K, P-p70S6K (T389), S6, and P-S6 (Ser235/236) protein levels in cultured mouse hepatocytes by Western blot analysis. β-actin was used as a loading control; B: Densitometric analysis of the Western blot protein levels. Effect of Aramchol treatment on the ratios of phosphorylated/total protein levels of AMPK, ACC and p70S6K is shown. In the case of SCD1 and CPT1A/B, the protein level is normalized against β-actin (loading control). Results are expressed as fold of total specific protein levels. At least triplicates were used per experimental condition. Data is shown as the mean ± SEM. ^aP < 0.05.

Figure 3 Effect of Arachidyl amido cholanoic acid on the proteome of cultured mouse hepatocytes. A total of 3220 proteins were identified by proteomics analysis, of which the contents of 219 changed significantly (6.80%, P < 0.05) between the two experimental groups [vehicle vs arachidyl amido cholanoic acid (Aramchol) 20 µmol/L]. A: Hierarchical clustering showing classification of the samples into two differentiated groups: Control and Aramchol treated; B: The differentially expressed proteins according to the up- or down-regulation were classified with colors in a volcano plot depending on their biological functions; C: Classification of the selected differentially expressed proteins in key biological functions. STRING software was used for the analysis.

Figure 4 Arachidyl amido cholanoic acid regulates tricarboxylic acid cycle activity. Glucose flux in the tricarboxylic acid (TCA) cycle was investigated in order to determine glucose utilization in the liver after arachidyl amido cholanoic acid (Aramchol) treatment (20 µmol/L). Murine hepatocytes were cultured with vehicle or Aramchol (20 µmol/L) for 48 h, after which uniformly labeled ¹³C₆-glucose was added. A: Diagram of the label dispersion of fully ¹³C-labeled glucose for the initial cycle of the TCA cycle. Carbon atoms are either white, grey-shaded, or black depending on the origin of labelled and non-labelled carbons. Grey-shaded carbons originate directly from pyruvate (Pyr). Pyr is converted in oxaloacetate by pyruvate carboxylase after introduction of a carboxyl group coming from a carboxylate ion. Black coloured carbons originate from acetyl-CoA and are introduced via citrate synthase. White carbons are not labeled. The mass increases of the TCA cycle metabolites due to the incorporation of ¹³C are indicated and follow the same colour coding. The numbers indicated in blue are the total mass increase due to incorporation of labels via both pathways. Note that the formation of isocitrate (iCit) from citrate causes the formation of two distinct isotopomers because the hydroxyl group can shift equally likely to both sides. The formation of malate (Mal) from fumarate again doubles the amount of isotopomers for the same reason. In subsequent cycles, the isotopomer patterns take forms that are more complex. The transformation from iCit to succinate causes the loss of two carbon dioxide molecules; B: The ¹³C-label dispersion in the TCA cycle was determined by measuring the ratios of the different isotopomers of Mal at steady-state by mass spectrometry. After Aramchol treatment, an upregulation of the +4 labeled Mal species was found compared to control. ^aP < 0.002; ^bP < 0.0002 Aramchol treated vs control. TCA: Tricarboxylic acid; Pyr: Pyruvate; OA: Oxaloacetate; PC: Pyruvate carboxylase; CO₃-: Carboxylate ion; Ac-CoA: Acetyl-CoA; CS: Citrate synthase; Cit: Citrate; iCit: Isocitrate; Mal: Malate; Fum: Fumarate; Suc: Succinate; CO_2: Carbon dioxide.

Figure 5 Arachidyl amido cholanoic acid treatment improves glucose homeostasis in mice fed a 0. 1MCD diet. C57BL6/J mice were fed a 0.1MCD diet for 2 wk. After this, mice were divided into groups of 10 and treated by intragastric gavage with 1 or 5 mg/kg/d of Arachidyl amido cholanoic acid (Aramchol) or vehicle alone. Animals kept on a normal diet were also provided with the vehicle preparation. A: Effect of 5 mg/kg/d Aramchol treatment on AMPK, P-AMPKα (Thr172), p70S6K, and P-p70S6K (T389) protein levels in the livers by Western blot analysis. β-actin was used as loading control. Densitometric analysis of phosphorylated/total AMPK and p70S6K ratio was represented. Results are expressed as fold of total specific protein levels. Experiment was performed in cuadruplicates per experimental condition. Data is shown as the mean ± SEM. ^aP < 0.05 vs MCDD; B: Metabolomic analysis of livers from the 0.1MCD fed mice showed a reduction of glucose, glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, ribulose 5-phosphate, fructose 1, 6-bisphosphate, and pyruvate, as compared to mice fed a normal diet. The treatment with Aramchol tended to normalize the concentration of these metabolites in a dose dependent manner. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.