Recombinant Buckwheat Trypsin Inhibitor Reduces Fataccumulation In C.elegans

Disorders of lipid metabolism can cause much harm to the human body,such as hyperlipidemia and fatty liver.Buckwheat is a traditional crop,which has a good preventive effect on metabolic-related disease including obesity,hyperglycemia and hyperlipidemia.Buckwheat trypsin inhibitor(BTI)is a low molecular weight protein extracted from buckwheat seeds.In this study,Caenorhabditis elegans(C.elegans)was used as a model to investigate the effect of recombinant buckwheat trypsin inhibitor(r BTI)on fat accumulation and its mechanism was explored initially.The main research contents and results are as follows:1.Effect of r BTI on fat accumulation in Caenorhabditis elegans.Nile red staining and triglyceride were used to detect fat changes in C.elegans.The results showed that in the concentration range of 2.5-10 ?M,r BTI could reduce fat accumulation in a concentration dependent manner.Compared with the control,after 10 ?M r BTI treatment,the fat accumulation of the nematodes was reduced by 37.82%,and the triglycerides were decreased by 47.51%.0.75% glucose was used to build a high-fat model of C.elegans,which was used to study the effect of r BTI on the fat accumulation.It was found that after the r BTI treatment,the fat accumulation was reduced by 38.6% and triglycerides by 38.57% in high-fat model nematodes,indicating that r BTI can reduce fat accumulation under normal and high-fat conditions.In addition,r BTI could significantly reduce the body width of worms,but it did not show a significant effect on worm body length,motility and reproduction ability,indicating that r BTI's role in reducing fat accumulation has nothing to do with movement and reproduction behaviors in C.elegans.2.Mechanism of r BTI to reduce fat accumulation in Caenorhabditis elegans.To study the mechanism of r BTI reducing fat accumulation,the fat level of mutant C.elegans was examined.The results showed that r BTI can reduce fat accumulation in the eat-2 mutant(diet restriction model),However,r BTI didn't show significant effect on reducing fat accumulation in daf-2 and daf-16 mutants.daf-2 and daf-16 are key factors in the insulin signaling pathway(IIS),hence,this result indicated that the pathway that r BTI depending on to reduce fat accumulation is the IIS pathway rather on the dietary restriction signaling pathway.The transgenic C.elegans was used to detect the expression level of lipase gene lipl-4.The results showed that r BTI increased the expression level of lipl-4,indicating that lipolysis was enhanced in C.elegans.Transcription factor DAF-16 regulates the expression of lipl-4 in the small intestine,indicating that r BTI regulates the expression of lipl-4 through the IIS pathway,thereby enhancing lipolysis.Enzyme activity detection,q RT-PCR and mutant nematode fat accumulation detection were used to further study the effect of r BTI on fat synthesis.Enzyme activity assay showed that r BTI reduced the activity of two key enzymes involved in fat synthesis,namely ACC and FAS.q RT-PCR results showed that r BTI down-regulated the expression of key genes involved in fat synthesis including sbp-1,pod-2,and fasn-1.sbp-1 is the upstream gene of pod-2 and fasn-1.At the same time,the fat accumulation test results of three nematode mutants,sbp-1,pod-2 and fasn-1,showed that the role of r BTI in reducing fat accumulation disappeared in these three mutants,indicating that sbp-1,pod-2 and fasn-1 were essential for r BTI to reduce fat accumulation.rBTI mutants with different trypsin inhibitory activity were obtained by using site-directed mutagenesis technology.And the effects of each mutant on the fat accumulation in C.elegans were examined.The results showed that when trypsin inhibitory activity was lost(r BTI-R45F),lower(r BTI-W53R),and significantly increased(r BTI-P44T),the function of reducing fat accumulation was lost to some extent.The results indicate that r BTI's reduction of fat accumulation is related to its certain trypsin inhibitory activity.When the activity is changed,the effect of reducing fat accumulation is weakened or lost.However,the relationship between the lipid-lowering effect of r BTI and its molecular structure remains to be further studied.In summary,r BTI increased the transcription and expression of lipolytic enzymes by down-regulating the IIS pathway.At the same time,r BTI reduced the activity of fat synthesis-related enzymes by regulating sbp-1,thereby reducing fat synthesis.3.Sequencing analysis of C.elegans transcriptome data after r BTI treatment.The high-throughput sequencing method was used to sequence the transcriptome of the control group and the 10 ?M r BTI-treated worms.We obtained the differentially expressed genes between the two groups.We performed GO function annotation and enrichment on differential genes,and analyzed the effect of r BTI on nematodes at the micro level.The results showed that compared with the control,there were 283 differentially expressed genes after treatment with r BTI,of which 57 genes were significantly up-regulated and 226 genes were significantly down-regulated.According to GO functional annotation,under r BTI treatment,the genes that were significantly up-regulated were cyp-14A1,acdh-1,hsp-16.1,hsp-16.2,and hsp-16.70,and the genes that were significantly down-regulated were scp-1,sbp-1,and so on.Both scp-1 and sbp-1 are related to fatty acid synthesis,and this is consistent with the experiment results.The down-regulation of scp-1 and sbp-1 indicates that r BTI can reduce the fat synthesis in C.elegans.The down-regulation of cyp-14A1 and acdh-1 indicates that the fatty acid oxidative decomposition is accelerated;further confirming that r BTI can reduce fat accumulation.Factors such as hsp-16.1,hsp-16.2,and hsp-16.70 belong to heat shock protein family factors and are related to heat stress.The expression of these genes is up-regulated,suggesting that r BTI may enhance the resistance to heat stress in nematodes through the IIS signaling pathway.In addition,r BTI may affect other physiological processes such as the immune system.These results provide support that r BTI may be an effective ingredient in buckwheat to prevent obesity and lay a basis for further research on the function of buckwheat.