| After long-term natural selection,Bactrian camel has many special biological characteristics,such as strong thirst tolerance,hunger tolerance and high blood glucose level.Nesfatin-1 is hydrolyzed from its precursor NUCB2,which can regulate the efficiency of energy metabolism and inhibit appetite.In order to study the distribution and expression of Nesfatin-1 in Bactrian camels and explore its effect on energy metabolism of Bactrian camels,the hapten polypeptide of specific epitope of Bactrian camel NUCB2/Nesfatin-1 protein was synthesized and coupled with hemocyanin(KLH)by maleimide method to prepare polyclonal antibody against single epitope of NUCB2/Nesfatin-1 protein.The expression of Nesfatin-1 in hypothalamus,anterior and posterior fat,stomach,duodenum,jejunum,ileum,cecum,colon,rectum,pancreas,liver and abdominal adipose tissue of Bactrian camel was detected by Western blot and RT-q PCR.In addition,3T3-L1 preadipocytes induced to differentiate were treated with Nesfatin-1(100 n M)for 1 h under high glucose condition,and then the glucose uptake level,pyruvate content,hexokinase and phosphofructokinase activity of adipocytes were detected by non radioactive fluorescence method,At the same time,the expression of autophagy related genes in adipocytes was detected by RT-q PCR to evaluate the effect of Nesfatin-1 on glucose metabolism and autophagy in adipocytes.The experimental results are as follows:(1)the synthesized Bactrian camel NUCB2/Nesfatin-1 protein specific epitope polypeptide has less heteropeaks,the purity is more than 95%,and the mass charge ratio [M + 4H] 4 + and [M + 3H] 3 + meet the expectation,(2)The titer of Nesfatin-1 polyclonal antibody was 1:512000,Nesfatin-1 was distributed in the hypothalamus(arcuate nucleus,solitary tract nucleus,ventromedial nucleus),anterior and posterior peak fat,gastric fundus gland,duodenum,jejunum,ileum,cecum,colon,rectum,pancreas,liver and abdominal fat of Bactrian camel,but it was significantly expressed in adipose tissue and pancreas,(3)RT-q PCR showed that the expression of Nesfatin-1 m RNA was high in abdominal fat and liver of Bactrian camel,(4)After treatment with Nesfatin-1,the glucose uptake level,hexokinase and phosphofructokinase activities of 3T3-L1 cells decreased significantly(P < 0.05),while the pyruvate content increased significantly(P < 0.05),(5)The expression of p62 m RNA in 3T3-L1 cells treated with Nesfatin-1 decreased significantly(P < 0.05),but there was no significant difference between LC3 and beclin-1 m RNA.In this study,Bactrian camel NUCB2/Nesfatin-1 antibody was successfully prepared by analyzing antigen epitopes and synthetic peptides,and the distribution and expression of Nesfatin-1 in Bactrian camel and its effects on adipocyte glucose metabolism and autophagy were analyzed.These results suggest that nesfatin-1 may promote the hydrolysis of lipid droplets in Bactrian camels by inhibiting the differentiation of adipocytes. |
