| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 106 === Non-alcoholic fatty liver disease (NAFLD) is a chronic disease that accumulates excess fat in hepatocytes, resulting in increased oxidative stress and mitochondrial dysfunction. PTEN-induced kinase 1 (PINK1) is a key protein that activates mitophagy to degrade the impaired mitochondria and avoid the production of reactive oxides. Our previous studies showed that full-length PINK1 (full length-PINK1, f-PINK1) is cleaved by presenilins-associated rhomboid-like protein (PARL) in the mitochondrial inner membrane to become small PINK1 fragment (s-PINK1), then released to the cytoplasm during adipocyte differentiation. Intriguingly, adipocyte differentiation is almost aborted by PARL knockdown. In addition, expression of hepatic enzymes critical for lipogenesis is altered by PARL knockdown, suggesting PINK1 and PARL mediate lipid metabolism in hepatocytes and adipocytes. Therefore, we speculated that PINK1 may have potential to become a therapeutic target for NAFLD by regulating hepatic lipid metabolism and mitophagy. In the present study, expression of PINK1 and critical enzymes controlling lipid metabolism were examined in palmitic acid- (PA) treated BNL hepatocytes mimicking NAFLD condition. Our results showed that in PA-treated BNL cells, lipid droplets were significantly increased. Expression of f-PINK1, microtubule-associated protein 1 light chain 3 beta (LC3B), fatty acid synthase (FAS) and acetyl coenzyme A carboxylase (ACC) were decreased. Although f-PINK1 protein was reduced in the whole cell lysate, f-PINK1 and LC3B in isolated mitochondrial fraction were increased, suggesting the mitophagy activity was enhanced. In addition, copy number of mitochondrial cytochrome c oxidase I (COX1) gene was significantly reduced while mitochondrial biogenesis-promoting peroxisome proliferator-activated receptor gamma coactivator (PGC1α) remained unaltered in PA-treated hepatocytes. Immunofluorescence staining images regarding decreased mitochondrial mass and LC3B levels, and the co-localization of LC3B with mitochondria further supported the above observations. These results indicated that the decreased mitochondrial DNA was resulted from the up-regulated mitophagy rather than the change of mitochondrial biogenesis. Meanwhile, C57BL/6 mice were fed with high fat diet (HFD) for 8-, 12- and 16 weeks (wk) respectively for examining the temporal mitochondrial-related alterations during the process of NAFLD. Hematoxylin-eosin staining (H&E staining) results showed that mice had liver steatosis after 12 wk of HFD feeding. FAS and ACC were decreased after 12- and 16- wk- of HFD feeding but carnitine palmitoyl transferase 1 A (CPT1A) was significantly increased after 16- wk with impaired glucose tolerance. The above results suggest that accompanied with NAFLD progression, using glucose as the major energy source is gradually replaced by consuming fat. Although f-PINK1 and s-PINK1 showed no difference in 12- and 16- wk HFD-feeding mice, Parkin and LC3B were significantly increased after 16 wk consumption of HFD. These results indicate that mitophagy activity is evolving during NAFLD pathogenesis. The above results suggest that glucose utilization is reduced while lipid metabolism and PINK1-mediated mitophagy are enhanced during the process leading to NAFLD. Collectively, PINK1 might play an important role in energy metabolism through regulating mitophagy, and thus involved in NAFLD pathogenesis.
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