The capacity of bile acids to protect or aggravate fatty liver disease is not clear. In terms of liver damage CA is considered a food toxin. Feeding rodents with bile acids has been used to study bile acid toxicity in vivo. The ability of the bile acids to produce hepatotoxicity is considered to be: UDCA < CA < CDCA < DCA < LCA . Feeding rats a high cholesterol diet in the absence of bile acids resulted in relatively moderate liver damage compared to feeding of cholesterol in the presence of CA. In contrast to CA, CDCArg did not potentiate cholesterol toxicity (Additional file 4: Figure S4).
We have used bile acids in combination with saturated fatty acids from a plant source (palm stearin, containing around 50 percent of saturated fatty acids) that is cholesterol free. To the best of our knowledge, we are the first to report that CA administrated together with HFD rich in saturated fat in the absence of cholesterol potentiates liver damage. This was demonstrated by an elevation of liver enzymes in the blood. HFD, while contains fat of plants origin, has only a small capacity to induce liver damage. However, toxic bile acids may cause inflammation, apoptosis, and cell death . Currently it is not clear if the deleterious effect of CA to the liver was synergistic or simply additive to the effect of the fatty acid rich diet. However, in contrast to CA, CDCArg did not generate toxic effects in the liver and did not induce general toxicity. Electrolyte imbalance and kidney damage were not observed during the in vivo treatments indicating no acute toxicity of the diets. CDCArg ameliorated HFD-induced liver steatosis. Also blood liver enzymes levels were lower normalizing the long term effect of feeding with HFD (treatment study). Hepatomegaly was corrected by CDCArg. All these parameters indicate that CDCArg may be used with efficacy and safety for NAFLD and NASH treatment.
In the clinical setting both CDCA and urosodeoxycholic acid (UDCA) have failed as an approved treatment for NASH . The results of a randomized controlled trial of UDCA in NASH patients were disappointing, and no measurable benefit could be attributed to UDCA. Likewise, in a smaller, randomized, controlled study carried out in Switzerland no benefit of UDCA over placebo was found when looking at liver histology in NASH patients. However, there was a beneficial effect of UDCA on ALT levels in this trial.
Additionally, studies with another bile acid, the FXR activator obeticholic acid, that is under clinical investigation, have demonstrated adverse side effects of elevated LDL cholesterol .
Bile acids are used in an animal models of an atherogenic diet supplementation to potentiate the effect of cholesterol. It has been noted that the atherogenic diet can lead to the development of NASH. Atherogenic diets have been show to induce dyslipidemia, lipid peroxidation, stellate cell activation, precirrhotic steatohepatitis and cellular ballooning. Atherogenic diets in contrast to other animal models of NASH, such as the methionine and choline deficiency diet, induce the necessary histological features similar to those found in human NASH. It also generates a phenotype of insulin resistance. The addition of a high-fat component to the atherogenic diet exacerbated hepatic insulin resistance and further accelerated the pathology of steatohepatitis . Therefore, it is extremely important to develop bile acid based therapy that is free of such deleterious effects.
Bile acid sequestrates have been used for many years to treat hypercholesterolemia and dyslipidemia. On the other hand, bile acid-activated nuclear and GPCR signaling pathways may protect against inflammation in the liver. In the current study, treatment with CDCArg improved fasting glucose levels, insulin and leptin sensitivity and reduced testicular fat accumulation induced by HFD in the mouse model.
Bile acids can enter the hepatocytes in several ways. The entry of bile acids into hepatocytes at the sinusoidal surface takes place by NTCP, Na+-dependent pathway that is driven by the transmembrane Na+-gradient . NTCP is exclusively expressed in hepatocytes and localized at the sinusoidal membrane. In addition there is a less efficient system for uptake Na+-independent and involves several members of the organic anion transporting polypeptide family . Another transporter, the apical sodium-dependent bile salt transporter is expressed at high levels in the terminal ileum, renal proximal tubules, and biliary epithelium. CDCArg, due to its net positive charge, most probably cannot or is not expected to be absorbed efficiently back into the liver and therefore is probably translocated into the colon dragging along fat and preventing fat absorption. However, evaluation of plasma albumin levels indicated that CDCArg did not impair protein absorption in mice.
In the current study we investigated a primary bile acid (chenodeoxycolic acid) that was conjugated to L-arginine. The arginine guanidino group of its side chain provides a constant net positive charge to the molecule throughout the range of physiological pH range. The pKa of guanidine is 12.5, indicating that this compound will exist almost entirely as a cation in the in vivo environment. According to our results such a bile acid did not potentiate cholesterol-induced liver damage and protected against deleterious effects of HFD composed of high amounts of saturated fat.
The effect of CDCArg in HFD-induced obesity in mice was tested. Two main effects were noted, increased consumption and decreased body weight gain. In general bile acids may possess an anti-obesity properties by activation of the TGR5 receptor. TGR5 is a novel pharmacological target in the metabolic syndrome and related disorders, such as diabetes, obesity, atherosclerosis, liver diseases and cancer . However, its clinical effect is yet to be elucidated. Activation of TGR5 by bile acids releases incretins and improves glycemic control in a rodent nutritional model of HFD . However, bile acids do not appear to be key mediators of the early increase in GLP-1 and PYY response in post-bariatric patients . Therefore, the role of bile acids in obesity treatment is still unclear. Recently it has been shown that dietary fats, by promoting changes in host bile acid composition, can markedly alter conditions of the gut microbial environment due to sulfur containing bile acids, resulting in perturbation of immune homeostasis. A diet high in saturated fat promoted taurine conjugation of hepatic bile acids and promoted induction of colitis . CDCArg, due to its different chemical properties, may offer a possible treatment for obesity as it directly affects weight gain. Its specific effects on gut polypeptide secretion and its anti-obesity mechanism still need to be clarified.
CDCArg + HFD in oppose to HFD decreased blood leptin levels, which could indicate the amelioration of obesity related leptin resistance. The effect of selective leptin resistance on disease progression and the metabolic syndrome is complicated [24, 25]. Whether or not the effect of CDCArg is via leptin still needs to be clarified. CDCArg increased the expression of PGC1α and its related gene PPARα. This may accelerate mitochondrial oxidation of fat. Previously it has been reported that bile acids suppress PGC1 activity via the FXR and SHP pathway . Since CDCArg is a different chemical structure compared natural bile acids, and was unable to activate the FXR pathway it does not suppose to suppress the PGC pathway and may regulate its expression by cAMP dependent pathway. Surprisingly, CDCArg also suppressed SREBP-1 expression probably in a mechanism that may be independent of SHP expression. All of these effects culminate in improvement of liver steatosis in the mice model.
In conclusion: conjugate of the bile acid chenodeoxycholic acid and the positively charged amino acid arginine was found to be not toxic in mice. The compound was evaluated as a liver protective agent in a model of NAFLD. These results indicate that the compound did not potentiate cholesterol accumulation in the liver and protected the liver from damage caused by a HFD rich in saturated fat. This compound was also effective in improving the metabolic condition of rodents treated with HFD and was found to be effective as anti-obesity agent in a mouse model of diet-induced obesity.