Insulin Secretion In Small Intestine Of Mice And The Effects Of Cold Stimulation And Exercise On The Nonalcoholic Fatty Liver Disease

OBJECTIVE:Studies have shown that tissues other than the pancreas also play a role in the physiological and pathological processes of diabetes.Intestinal hormones have a great influence on islet cells and have a certain role in the pathogenesis of diabetes.Cold stimulation and exercise interventions can alter the gene expression in the liver of mice,which is beneficial for accelerating fat and carbohydrate metabolism.In the previous study,we found that the small intestine of the mouse has the expression of the proinsulin gene.The muscle tremor caused by cold stimulation and the skeletal muscle contraction during exercise can mediate the browning of white fat.In the first part of the study,we determined the specific part of insulin synthesis in the small intestine by morphological methods,and demonstrated the expression of insulin in the small intestine from the level of genes and proteins,and whether there was a difference in insulin expression levels in different intestinal segments.To study the effects of intestinal insulin on pancreatic cells and the role of diabetes in the pathogenesis of diabetes.In the second part we will explore the effects of cold stimulation and exercise on which genes in the liver.METHODS:We studied the small intestine of healthy 6-week-old C57BL/6J mice,and fasted for 8 hours before sacrifice.The blood glucose level of the mice was measured.After the mice were removed from the neck,the small intestine of the mice was followed according to the duodenum.The jejunum and ileum were sampled separately.The length of the small intestine tissue of the mouse was about 24-30 cm,and the duodenum,jejunum and ileum accounted for about one-third of the total length.The cut tissue samples were made into paraffin-embedded sections,and the presence of insulin was detected by immunofluorescence assay.Total RNA was extracted from tissue samples and reverse transcribed into c DNA.The relative expression of insulin m RNA in different intestinal tissues was detected by real-time fluorescent quantitative PCR.Perform western sampling on protein samples extracted from tissues in the blot experiment;it was detected whether there was insulin protein in the small intestine and whether the expression of each intestinal segment was different.The second part is selected 6 weeks of age C57BL/6J male mice 24,6 weeks of age KKay Spontaneous male obese mice 24,divided into two Group 4:normal temperature group,normal temperature exercise group,cold stimulation group,cold stimulation exercise group.C57BL/6J mice are given a normal diet,KKay Spontaneous obese mice KKay a high-fat diet in mice.High-throughput sequencing and GEO database analysis were used to determine the differential genes to be detected,and the changes in liver gene expression in different groups were detected by real-time PCR and western blot.RESULTS : Immunofluorescence assay showed that insulin,PDX1 and ICA-512 were expressed in the intestinal villi and intestinal crypts of the small intestine of mice,and there was no significant difference in the location and expression of insulin in the same intestinal segment.Electron scanning microscopy revealed that there were insulin-like secretory granules in the cells near the small intestine crypt.Real-time fluorescent quantitative PCR showed m RNA expression ofinsulin in the small intestine of mice,and the expression of jejunum was slightly higher.Western protein level The blot experiment also confirmed the presence of insulin protein in the small intestine cells,and the difference in expression level was consistent with the difference in m RNA expression levels.Both RT-PCR and western blot showed that under the intervention of cold stimulation and exercise,the liver gene expression of mice changed,and the expression of genes related to fat metabolism was significantly up-regulated.Conclusion:There is insulin gene expression in the small intestine,and insulin protein is also found.Since insulin does not enter the cell membrane during its normal physiological process,we can think that the insulin protein found in the small intestine cells is synthesized by the small intestine cells rather than transported from the pancreas.We also found that in the small intestine,the insulin synthesis in the jejunum was the highest,the duodenum and ileum were slightly lower than the jejunum,and at normal blood glucose levels,all three were much lower than the insulin expression in the pancreas;our previous studies also found The secretion of intestinal insulin is different from that of pancreatic insulin,so we suspect that intestinal insulin may have a different effect than insulin in the pancreas.Cold stimulation and exercise play an important role in promoting fat metabolism and controlling body weight.The combination of cold exposure and exercise also provides a new way to control body weight and prevent obesity and related metabolic diseases.