L-Carnitine attenuates fibrosis and inflammation in C57BL/6 mice with dietary-induced steatohepatitis

Nonalcoholic steatohepatitis (NASH) represents an advanced stage of fatty liver disease developed in the absence of alcohol abuse and its prevalence is increasing in western countries in parallel with Type 2 Diabetes and metabolic syndrome incidence. NASH is a strong marker of cardiovascular risk and in the last few decades it has become evident that there is a mutual interaction between heart and liver influencing their individual functions. In effect, NASH characterized by excess of intracellular fatty acids, severe inflammatory and fibrotic state, plays a critical role in two principal tissues: liver and heart. Several studies have examined the effectiveness of L-Carnitine (LC) in liver function and have recognized LC as a nutritional supplementation in cardiovascular disease. The present study was designed to investigate the effects of LC administration on liver and heart function and morphology in mice models of steatohepatitis, induced by a methionine-choline deficient diet (MCD). C57BL/6 mice male (n=18, age:12 weeks) were divided in 3 different groups: control group (CONTR) were fed for 6 weeks with a normal diet while both MCD and LC groups received MCD diet. From the 4th week LC group received MCD diet enriched with 200mg/kg/die LC (drinking water). The results showed that there are no significant differences in body weight between MCD and LC mice groups, while, as expected, there is a significant weight loss in MCD and LC groups in respect with CONTR. Furthermore, insulin sensitivity (IPGTT test) and GLUT4 protein content showed no differences between groups. Tissues fat deposition, inflammatory infiltration and fibrosis were, then, investigated. Different histopathology staining methods showed that LC significantly reduces fat accumulation. Immunofluorescence assay revealed an important downregulation of the two markers of tissue fibrosis: alpha smooth muscle actin protein level and Kruppel-like factor (KLF15). To clarify LC cellular mechanisms in counteracting inflammatory liver state, we evaluated NFκB pathway and PPARγ: our results indicated that NFkB increase, caused by MCD diet, was markedly attenuated by LC. In addition, LC significantly stimulates Ca2+/calmodulin-dependent kinases II activity, suggesting that LC could ameliorate mitochondrial function. Noteworthy, we observed LC actives ERKs pathway, which is correlated with a reduction of oxidative stress and hepatotoxicity. In parallel, to investigate LC anti-inflammatory and fibrotic role in heart, we studied STAT-3 activation: LC significantly decreases STAT 3 activation observed in MCD heart. This data is in accordance with the reduction of Calcium signaling in LC heart compared to MCD. In conclusion, LC appear to exert different beneficial actions on the liver-heart axis counteracting steatohepa¬titis. LC supplementation may be used in order to prevent disease progression in these analyzed tissues, inhibiting the inflammation and fibrotic pathways.