Acetyl-CoA Carboxylase 2 Suppression Rescues Human Proximal Tubular Cells From Palmitic Acid Induced Lipotoxicity Via Regulating Autophagy

Objective Diabetic nephropathy (DN) is one of the most serious complications of diabetes mellitus, and has become the leading cause of end-stage renal disease (ESRD).the ectopic accumulation of lipids may occur in non-adipose tissues when the lipids are overloaded and the balance of circular and cytosolic lipids is broken, which is termed as lipotoxicity, and is consumed to be relevant to the physiopathology of many metabolic syndromes, particularly to type 2 diabetes and its complications. The (?)-oxidation is a main pathway of FA catabolism and is a major source for renal ATP production. Acetyl-CoA carboxylase 2 (ACC2) is a key enzyme in lipid metabolism and catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which is a crucial metabolite for FA oxidation by inhibiting carnitine palmitoyltransferase I (CPTI), a rate-limiting enzyme of FA oxidation. Autophagy is known to control the cellular energy balance. The interrelationship of these two events remains unclear. Here we aim to investigate the effects of ACC2 on palmitic acid (PA) induced lipotoxicity in human proximal tubular cells and the putative role of autophagy in this process.Methods1. The cells were treated with 300 uM PA (coupled to BSA) for indicated time points, and BSA alone was used as a control and the lipid accumulation was revealed by Oil Red O staining at indicated time of points. The cell viability was measured by CCK-8 assay.2. The mRNA level of ACC2 was evaluated by qCR in vitro by PA treatment compared to BSA treated HK-2 cells. The expression of pACC2 and ACC2 were evaluated by immunohistochemical on normal diet rats and high diet rats3. HK-2 cells were transfected by scramble shRNA (CTR) or specific shRNA targeting ACC2 (ACC2 kd) and were treated by either BSA or 300 uM PA for 72h. The knockdown efficiency was evaluated by Western Blot. Oil red O staining was performed to investigate the lipid deposition in the cells. Cell viability was measured by CCK8 assay.4. Autophagy was observed in PA stimulated HK-2 cells.Western blotting for LC3, Beclin-1 and p62 in cell lysates treated with different amount of PA for 2 h. BSA alone was used as control. Autophagy was also observed in HK-2 cells with PA treatment by transmission electron microscopy (TEM) observation and mGFP-RFP-LC3 adenovirus.5. Autophagy was observed in rats with HFD by mRNA, Western Blot and immunohistochemical.6. Regulation of FA (?)-oxidation on autophagy. HK2 cells, ACC2 shRNA knockdown cells and control cells were respectively treated with PA or PA with 80 uM Etomoxir and their expression of p62, Beclinl and LC3 evaluated by Western Blot and immunostaining.7. HK-2 cells were transfected by siRNA targeting Atg5 and were treated by either BSA or PA. The knockdown efficiency was evaluated by Western Blot. Cell viability was measured by CCK8 assay. Results1. The intracellular lipid accumulation could be observed as early as at 24 h after PA treatment and increased with the time duration, indicating a time-dependent intracellular accumulation of neutral lipid. The cell viability was significantly decreased at 48 h after PA treatment, and was further reduced at 72 h (P<0.05).2. The mRNA level of ACC2 was evaluated by qPCR in vitro, and it was obviously increased (P<0.05) by PA treatment compared to BSA treated HK-2 cells. The expression of ACC2 was increased while the pACC2 level was dramatically decreased in the kidney from rats with high-fat diet treatment (P<0.05).3. ACC2 knockdown cells had obviously reduced lipid accumulation after treated by PA for 72 h. The reduced cell viability (P<0.05) by PA treatment was statistically ameliorated by ACC2 knockdown compared to PA treated CTR cells.4. Autophagy was initiated shown by increased LC3II/I ratio and Beclin-1 expression, as well as the enhanced p62 degradation. The initiation started from the lowest concentration of PA we used, which was 100 uM and showed a concentration-dependent manner (P<0.05).5. In consistent with the results in vitro, we also observed the changes of autophagy related proteins by mRNA, Western blot and immunnohistochemical analyses (in rats with HFD (P<0.05).6. The knockdown of ACC2 restrained the PA induced autophagy (P<0.05), evidenced by increased p62 accumulation and decreased Beclin-1 and LC3 expression compared to scramble shRNA transfected cells. HK-2 cells treated with PA followed by Etomoxir, which can restrain FA β-oxidation, the autophagy level was more elevated compared to PA treated alone. However, combined these two opposite steps of FA β-oxidation regulation, there was no significant difference on the levels of autophagy (PA+Et+shRNA-ACC2 vs. PA). The findings was further confirmed by immunostaining for LC3 puncta formation (P<0.05).7. Gene silencing of Atg5 partially rescued PA reduced cell viability treatment (P<0.05), which was further increased by additional silence of ACC2, although no statistic difference.Conclusion Autophagy was induced by dysregulated lipid metabolism in vitro. The downregulation of ACC2 protected cells from PA-induced lipotoxicity by suppressing autophagy. The present study demonstrates that ACC2 mediates autophagic activity, which provides a novel molecular mechanism of PA-induced renal lipotoxicity. Pharmacological targeting of these signaling pathways may help design a new approach to develop therapeutic strategies for prevention of deterioration of kidney function in metabolic syndrome.