Carnitine palmitoyl transferase 1C regulates tumor cell senescence

OBJECTIVE Passage-dependent cel ular senescence is a complex process limiting the proliferative lifespan of tumor cells but the mechanism of this process is not understood.METHODS Replicative senescenceof pancreatic carcinoma-derived PANC-1 cells wasanalyzed.Metabolomics and transcriptomic analyses were performed to find endogenous metabolites changed andassociated genes.Mitochondrial function,cell survival andtumorigenesis of replicative senescent PANC-1 cellswere analyzed.PANC-1 cells were transfected with RNAi CPT1C to specifically knockdown CPT1C expressions,then mitochondrial function,cellular senescence,cell survival and tumorigenesis were investigated.MDA-MB-231,HCT116,A549,MCF7,and He Lacells werealso transfected with si RNA CPT1C and cellular senescence were monitored.RESULTS Replicative senescenceof PANC-1 cells was confirmed.Metabolomic and transcriptomicanalyses revealed that acylcarnitines and their upstream regulator carnitine palmitoyltransferase 1C(CPT1C),an enzyme that catalyzes the initiating step of fatty acidβ-oxidation,were markedly decreased in senescent PANC-1 cells.Furthermore,low CPT1C expression caused abnormal energy metabolism and mitochondrial dysfunction of PANC-1 cells,resulting in decreased cell survival and a suppressed tumorigenesis.Most importantly,loss of CPT1C triggered mitochondrial dysfunction,leading to senescence-like growth suppression and cellular senescence,suppressed cell survival under metabolic stress,and lower tumorigenesis in a mouse xenograft model.Silencing of CPT1C also induced cellular senescence in five other tumor cell lines.CONCLUSION Low CPT1C expression is a novel biomarker and key regulator of cellular senescence in tumor cell lines.Inhibition of CPT1C may be a new cancer therapeutic target impacting cellular senescence and tumorigenesis through modulation of mitochondrial function.

The effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on fatty acid oxidation in hepatocytes isolated from neonatal piglets

In the present study, the effect of 5-aminoimidazole-4-carboxamide ribonucleoside （AICAR） on long-chain fatty acid oxidation by hepatocytes isolated from suckled neonatal pig liver （a low ketogenic and lipogenic tissue） was tested Incubation of hepatocytes with AICAR （0.5 raM） in the presence of ] mM of carnitine and 10 mM of glucose for 1 hour at 37℃ had no significant effect on total [1-14C]-palrnitate （0.5 mM） oxidation （14CO2 and 14C-Acid soluble products （ASP））. Consistent with the fatty acid oxidation, carnitine palmitoyltransferase I activity and inhibition of its activity by malonyI-CoA （10 MM） assayed in cell homogenate also remained constant. However, addition of AICAR to the hepatocytes decreased 14CO2 production by 18% compared to control （p 〈 0.06）. The reduction of labeled carboxylic carbon accumulated in C02 caused a significant difference in distribution of oxidative products between 14C02 and 14C-ASP （p 〈 0.03） compared with the control. It was also noticed that acetyI-CoA carboxylase （ACC） was increased by AICAR （p 〈 0.03）, indicating that ACC might drive acetyI-CoA toward fatty acid synthesis pathway and induce an increase in distribution of fatty acid carbon to 14C-ASP. Addition of insulin to hepatocyte incubations with AICAR did not change the oxidative product distribution between CO2 and ASP, but further promoted ACC activity. The increased ACC activity was 70% higher than in the control group when citrate was absent in the reaction medium and was 30% higher when citrate was present in the medium. Our results suggest that AICAR may affect the distribution of metabolic products from fatty acid oxidation by changing ACC activity in hepatocyte isolated from suckled neonatal piglets; however, the basis for the increase in ACC activity elicited by AICAR is not apparent.

Berberine alleviates non-alcoholic hepatic steatosis partially by promoting SIRT1 deacetylation of CPT1A in mice

Background:Berberine effectively alleviates non-alcoholic fatty liver disease(NAFLD).Nevertheless,the mechanism is incompletely comprehended.It has been reported that SIRT1 mediates lipid metabolism in liver and berberine promotes the expression of SIRT1 in hepatocytes.We hypothesized that SIRT1 mediated the effect of berberine on NAFLD.Methods:The effects of berberine on NAFLD were evaluated in C57BL/6J mice fed a high-fat diet(HFD)and in mouse primary hepatocytes and cell lines exposed to palmitate.The change of fatty acid oxidation(FAO)and the activity of CPT1A were observed in HepG2 cells.Quantitative real-time polymerase chain reaction and Western blot were employed to observe the expression of SIRT1 and lipid metabolism-related molecules.The interaction between SIRT1 and CPT1A was investigated by using co-immunoprecipitation assay in HEK293T cells.Results:Berberine treatment attenuated hepatic steatosis,reduced triglyceride(190.1611.2 lmol/g liver vs 113.667.6 lmol/g liver,P<0.001)and cholesterol(11.362.5 lmol/g liver vs 6.360.4 lmol/g liver,P<0.001)concentration in the liver,and improved lipid and glucose metabolism disorders compared with the HFD group.The expression of SIRT1 was reduced in the liver of NAFLD patients and mouse models.Berberine increased the expression of SIRT1 and promoted the protein level of CPT1A and its activity in HepG2 cells.SIRT1 overexpression mimicked the effect of berberine on reducing triglyceride levels in HepG2 cells,whereas SIRT1 knock-down attenuated the effect of berberine.Mechanistically,berberine increased the expression of SIRT1.SIRT1 deacetylated CPT1A at the Lys675 site,which suppressed its ubiquitin-dependent degradation,thereby promoting FAO and alleviating non-alcoholic liver steatosis.Conclusions:Berberine promoted SIRT1 deacetylation of CPT1A at the Lys675 site,which reduced the ubiquitin-dependent degradation of CPT1A and ameliorated non-alcoholic liver steatosis.