Amelioration of nonalcoholic fatty liver disease by hepatic stimulator substance via preservation of carnitine palmitoyl transferase-1 activity

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease and so far is supposed to be related with mitochondrial impairment. Hepatic stimulator substance (HSS) has been defined as a liver-protective factor promoting hepatocyte DNA synthesis and hepatic proliferation after liver intoxication. We previously reported that HSS ameliorated hepatocyte death, probably because of its preservation of mitochondria. This study aims to explore whether HSS could protect carnitine palmitoyl transferase-1 (CPT-1), an essential enzyme responsible for β-oxidation of free fatty acids in mitochondria, from lipotoxicity, thus alleviating hepatic lipid deposition. To test this, the HSS gene was delivered into C57BL/6J mice and efficiently expressed in the liver. NASH mice were prepared with high-fat diet or methionine-choline-deficient diet. The results showed that hepatic inflammation and liver functions were alleviated in the HSS-transfected mice; meanwhile, the activity of CPT-1 was obviously protected. Moreover, oleic acid (OA) treatment resulted in remarkable lipid accumulation in HepG2 cells; this deposition was improved by HSS transfection. Simultaneously, the CPT-1 activity, which was impaired by OA treatment, was profoundly rescued in the HSS-expressing cells. CPT-1 activity was more severely impaired if the OA treatment was combined with S15176, a CPT-1 inhibitor. However, this impairment was effectively reduced by the HSS transfection, and the effect was enhanced by C75, a CPT-1 activator. Interestingly, if the cells were transfected with HSS-siRNA, the preservation of CPT-1 provided by HSS was again diminished. In conclusion, HSS reduces lipotoxicity to mitochondria most likely via preservation of CPT-1.

Deceleration of liver regeneration by knockdown of augmenter of liver regeneration gene is associated with impairment of mitochondrial DNA synthesis in mice

Hepatic stimulator substance, also known as augmenter of liver regeneration (ALR), is a novel hepatic mitogen that stimulates liver regeneration after partial hepatectomy (PH). Recent work has indicated that a lack of ALR expression inhibited liver regeneration in rats, and the mechanism seems to be related to increased cell apoptosis. The mitochondria play an important role during liver regeneration. Adequate ATP supply, which is largely dependent on effective mitochondrial biogenesis, is essential for progress of liver regeneration. However, ALR gene expression during liver regeneration, particularly its function with mitochondrial DNA synthesis, remains poorly understood. In this study, ALR expression in hepatocytes of mice was suppressed with ALR short-hairpin RNA interference or ALR deletion (knockout, KO). The ALR-defective mice underwent PH, and the liver was allowed to regenerate for 1 wk. Analysis of liver growth and its correlation with mitochondrial biogenesis showed that both ALR mRNA and protein levels increased robustly in control mice with a maximum at days 3 and 4 post-PH. However, ALR knockdown inhibited hepatic DNA synthesis and decelerated liver regeneration after PH. Furthermore, both in the ALR-knockdown and ALR-KO mice, expression of mitochondrial transcription factor A and peroxisome proliferator-activated receptor-γ coactivator-1α were reduced, resulting in impaired mitochondrial biogenesis. In conclusion, ALR is apparently required to ensure appropriate liver regeneration following PH in mice, and deletion of the ALR gene may delay liver regeneration in part due to impaired mitochondrial biogenesis.

Enhanced endoplasmic reticulum SERCA activity by overexpression of hepatic stimulator substance gene prevents hepatic cells from ER stress-induced apoptosis

Although the potential pathogenesis of nonalcoholic fatty liver disease (NAFLD) is unclear, increasing evidence indicates that endoplasmic reticulum (ER) stress may link free fatty acids to NAFLD. Since we previously reported that hepatic stimulator substance (HSS) could protect the liver from steatosis, this study is aimed to investigate whether HSS protection could be related with its inhibition on ER stress. The HSS gene was stably transfected into BEL-7402 hepatoma cells and effectively expressed in ER. The palmitic acid (PA)-induced heptocyte lipotoxicity was reproduced in the HSS-transfected cells, and HSS alleviation of the ER stress and apoptosis were subsequently examined. The results showed that PA treatment led to a heavy accumulation of fatty acids within the cells and a remarkable increase in reactive oxygen species (ROS). However, in the HSS-expressing cells, production of ROS was inhibited and ER stress-related marker glucose-regulated protein 78 (GRP-78), sterol regulatory element-binding protein (SREBP), anti-phospho-PRK-1ike ER kinase (p-PERK), anti-phospho-eukaryotic initiation factor 2α (p-eIF2α), and anti-C/EBP homologous protein (CHOP) were downregulated compared with the wild-type or mutant HSS-transfected cells. Furthermore, PA treatment severely impaired the activity of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), leading to imbalanced calcium homeostasis during ER stress, which could be rescued in the HSS-trasfected cells. The protection provided by HSS to the SERCA is identical to that observed with N-acetyl-l-cysteine (NAC) and sodium dimercaptopropane sulfonate (Na-DMPS), which are two typical free radical scavengers. As a consequence, the rate of ER stress-mediated apoptosis in the HSS-expressing cells was significantly reduced. In conclusion, the protective effect of HSS against ER stress may be associated with the removal of ROS to restore the activity of the SERCA.