The Role Of CPL-1-mediated Autophagy In Diet-induced Fat Deposition In Caenorhabditis Elegans

Obesity and related metabolic diseases were a greatly threaten to human health.Fat accumulated excesssly was the key incentive of obesity.Cathepsin L(CPL)is involved in diet-induced fat deposition through degrading extracellular matrix,but the molecular mechanism of whether CPL-mediated autophagy involve in fat deposition remains unclear.As a lysosomal cysteine protease,CPL plays its major role in lysosome.Lysosome,which is an acidic organelle fused with autophgosome to form autolysosme in cell,participates in the process of autophage.Recent researches revealed that autophage plays an important function in fat deposition.Whereas,it needs to confirm whether CPL participate in the process of fat deposition through regulating cell autophagy.To study the mechanism in Caenorhabditis elegans,we first amplified Cpl-1 gene(Cathepsin L-like proteases,CPL-1,Cathepsin L homologous gene in C.elegans)and then constructed recombinant prokaryotic expression plasmid pET28a::CPL-1.The plasmid was transferred into E.coli BL21 and was induced expression.CPL-1 fusion protein was subsequently purified and yielded polyclonal antibody for CPL�1.ELISA and Western blot showed the antibody for CPL-1 had better titer and specificity.On this basis,we further study the effects of CPL-1-mediated autophage in the process of fat deposition in C.elegans.Using LGG-1: : GFP nematodes model(GFP refers to autophage level),we found autophagy-related gene expression were elevated in high glucose-induced fat deposition and autophagy level reached its maximum in 4mM glucose induced.Meanwhile,we found that CPL-1 as a lysosomal cathepsin had a very strong relationship with cell autophagy.Accordingly,autophagy related gene expression level was significantly reduced in cpl-1 mutant yq89 and qx304 which induced by high-carbohydrate diet.These results indicated that CPL-1-mediated autophage participated in fat deposition in C.elegans.All together,we prepared polyclonal antibody for CPL-1 and confirmed that cell autophage involved in fat deposition induced by high glucose in C.elegans.Importantly,we proposed a new celluar mechanism of that cathepsins regulate fat deposition.