The Study On Short-term High Fat Diet Induced Islets ? Cells Replication And The Regulatory Effect Of ? Cells On ? Cell Function

Diabetes Mellitus?DM?,is a group of metabolic disorder in which there are high blood sugar levels over a prolonged period.Diabetes is due to either the pancreas not producing enough insulin,or the cells of the body not responding properly to the insu-lin produced.Type 2 diabetes mellitus?T2DM?is the most commonly diabetes type,with type 2 DM making up about 90%of the cases.Diabetes can strike anyone,from any walk of life.With the continuous development of social economy,the continuous improvement of people living standard,and people's dietary structure and living hab-its change,the obesity rate increases,accompanied by the aging of the population,the incidence of diabetes is increasing rapidly with each passing year.In the last decade,the cases of people living with diabetes jumped almost 50 percent.Worldwide,it af-flicts more than 422 million people,threatening to the human life and healthy serious-ly.In China,the prevalence rate of diabetes in adults is 9.7%,and the proportion of pre-diabetes is as high as 15.5%.Therefore,looking for the risk factors of diabetes,and early intervention and prevention has become a major public safety problem in China.Studies have shown that the occurrence and development of diabetes has a very close relationship with eating habits,unhealthy diet,such as high-fat diet,is con-sidered to be the main risk factors for diabetes,obesity and other diseases.Excessive intake of fat,represented by saturated fatty acids,has been proved to be closely related to the occurrence of diabetes.Most of the type 2 Diabetes Mellitus is characterized by insulin resistance and obesity.Islets?cell mass adaptation to obe-sity has been observed in both rodent and human,with human autopsies showing in-creases of up to 50%in obese patients.The growth of?cells under high-fat diet?HFD?conditions represents a natural adaptation process of the cells.The compensatory?-cell proliferation following long-term HFD is believed to be secondary to the devel-opment of insulin resistance in response to liver-derived systemic factors.It has been revealed recently,however,that increases in?cell proliferation are initiated within days after the start of high-fat feeding in mice,with or without the development of insulin resistance,suggesting potential alternate mechanisms involved.Macrophage?M??is a type of cell of the immune system.Macrophages are de-rived from monocytes in the blood,which are released into the blood through the bone marrow.After 3 to 4 days in the blood,they enter the tissues and chorion cavi-ties and then transform into macrophages.Under the physiological conditions,there are a small number of macrophages in organs and tissues,which play specific and non-specific immune roles.In recent years,studies on macrophages have shown that macrophages are a class of cells with plasticity and versatility.Under the influence of microenvironment in vivo and in vitro,macrophages show obvious functional differ-ences.At the same time,some studies have also shown that the intrinsic macrophages in the islets can secrete a variety of factors involved in islet development and tissue remodeling process.At present,macrophages can be divided into Classical Activated Macrophage?M1?and Alternatively Activated Macrophage?M2?according to their activation status and functions.In the immune inflammatory response,the pro-inflammatory M1 macrophages are in an anti-inflammatory and pro-inflammatory balance with the anti-inflammatory M2 macrophages.In many chronic inflammatory diseases,such as diabetes and atherosclerosis,this balance is broken,macrophages will continue to infiltrate,activate and may destroy the mechanism of inhibiting in-flammation,making the disease further developed.There have been various reports describing increased inflammation in pancreas and islets of humans with type 2 diabe-tes?T2D?and in a non-obese rodent model of T2D.Non-diabetic juvenile nonhuman primates?NHPs?in the early stages of insulin resistance after chronic?12 months?HFD also showed the presence of increased numbers of islet-associated macrophages along with increased cytokine expression in the pancreatic tissue.All these findings suggest a role for local inflammation in the impairment of islet function in metabolic diseases.According to their staining and morphological characteristics,human islet cells are mainly divided into islet?cells,?cells,delta cells and PP cells.There are about20%of the islets cells are?cells.When blood glucose decreases,pancreatic?cells can secrete glucagon to raise blood glucose,when blood glucose increases,secretion decreases,and?cells secrete insulin to work together to maintain blood glucose level in vivo.In the process of exploring the pathophysiological mechanism of early diabe-tes mellitus,people often focus on the insulin-secreting islet?cells,but ignore the glucagon-secreting islet?cells.We hypothesized that short-term high-fat diet?HFD?promotes macrophage infil-tration in pancreatic islets and that macrophage serve as a regulator of?cell replica-tion.To test these hypotheses and dissect mechanisms involved in HFD-induced?cell replication,adult C57BL/6J mice were fed a HFD for 7 days with or without ad-ministration of clodronate-containing liposomes,a macrophage-depleting agent.Mouse body and epididymal fat pad weights,and non-fasting blood glucose and fast-ing serum insulin levels were measured by Elisa,and pancreatic islet?cell replication,oxidative stress,and macrophage infiltration were examined by Immunohistochemistry fluorescence staining.We argue that carefully unraveling the processes underlying HFD-induced changes in islet micro-and macro-environments will illuminate the potential role of inflammation in?cell replication,and advancing our ability to preserve functional?cell mass in persons with obesity and T2D.The experimental results are as follows.1.After a short-term high-fat diet?7 days?,the mice in the high-fat diet group showed significant macrophage infiltration and?cell replication compared with the control group.2.In mice fed a high-fat diet with clodronate containing liposomes-a macro-phage depleting agent,there was no significant increase in macrophages in the islets of pancreas compared with the normal diet group,and the replication of?cells was significantly lower than that of mice fed a high-fat diet without macrophage depletor,and there was no significant difference between the two groups.3.Compared with the control group,the fasting serum insulin level of mice in the high-fat diet group and the high-fat diet treated with macrophages depleting agent group is significantly increased,indicating that the insulin sensitivity of mice was de-creased.And the fasting serum insulin level of mice in the high-fat diet group was slightly higher than that in the high-fat diet treated with Pioglitazone?an insulin sensi-tizer?group.There was no significant difference from the control group.4.After a short-term high-fat diet,the weight of mice,epididymal fat pad weight and blood glucose levels were significantly higher than those before the experiment.The body weight and blood glucose level of mice fed with high fat diet treated with piglitazone did not increase significantly.The body weight of mice fed with high fat diet along with clodronate containing liposomes did not change significantly,but the blood sugar of this group increased significantly.5.There was no significant difference in 8-oxo-2'-deoxyguanosine and activated caspase-3 staining between the control group and the high-fat diet group,indicating that short-term high-fat diet did not significantly induce DNA damage or apoptosis.6.SDF1 in pancreas tissue of mice fed with high fat diet was not significantly different from that of mice fed with normal diet.7.After macrophage infiltration,the proportion of inducible nitric oxide synthase?iNOS?positive macrophages,M1,increased significantly in the high fat diet group.Short-term HFD promoted an increase in body and epididymal fat pad weight and blood glucose levels,along with an increased fasting serum insulin concentration.?-Cell replication,islet macrophages infiltration,and the percentage of inducible NO synthase positive macrophages in the islets increased significantly in mice fed the HFD.Immunofluorescence staining for 8-oxo-2-deoxyguanosine or activated caspase-3 revealed no significant induction of DNA damage or apoptosis,respectively.In ad-dition,no change in stromal derived factor 1-expressing cells was found induced by HFD.Despite continuous elevation of non-fasting blood glucose and fasting serum insulin levels,depletion of Ms through treatments of clodronate abrogated HFD-induced-cell replication.These findings demonstrated that HFD-induced macrophag-es infiltration is responsible for?cell replication.This study suggests the existence of macrophage-mediated mechanisms in?-cell replication that are independent of insulin resistance.Through the Fluorescence-Acticated Cell-Sorting?FACS?,we got the pure?cells group and pure?cells group.The purities of both groups are both high than 95%.We cultured the cells into a 37^oC,CO₂ incubator for three days,and then measured the glucagon secretion by?cell to determine the response of?cell to different glucose concentrations in the presence or absence of?cells and insulin to explore the regula-tion function of?cells on?cell.Following results:1.When glucose concentration was low?2 mmM?,the pure?cell group,?and?mixed cell group could secrete higher glucagon,and there was no significant differ-ence between them.2.When glucose concentration was high?16.8 mM?,the inhibitory effect on glu-cagon release from?-?mixed cell group was significantly higher than that of pure?cell group.3.The inhibitory effect of high glucose concentration and insulin on glucagon re-lease from pure?cells was significantly enhanced.Through the above studies,we established the model of human islet?cells in vitro,which provides a certain basis and new ideas for the further study of?cell.At the same time,by adding?cells and insulin to?cell,we determined the response of?cells to glucose concentration,and determined the regulation of?cells to glucagon release from?cell,providing a scientific basis for further study of its related mecha-nism.