A Pilot Study On The Role And Mechanism Of LEAP2 In The Development Of Type 2 Diabetes Mellitus

Type 2 diabetes mellitus(T2DM)is a common clinical disease.Its etiology and pathogenesis are complex,including early insulin resistance and hyperinsulinemia,and progressively deteriorated islets βcells dysfunction,leading to a series of metabolic disorder syndromes dominated by hyperglycemia.Further investigation of the pathogenesis and exploration of new targets are urgently needed in the prevention and treatment of T2 DM.Liver expressed antimicrobial peptide 2(LEAP2)is expressed and secreted by hepatocytes and first isolated from human blood in 2003.It was named for its antibacterial activity in vitro.In subsequent studies,LEAP2 was identified as an endogenous antagonist of acyl-ghrelin,through competitively binding to growth hormone secretagogue’s receptor(GHSR)with acyl-ghrelin.Acyl-ghrelin stimulates growth hormone secretion,increase appetite and food intake,regulates glucose metabolism and cardiovascular function.Recent studies have reported that plasma LEAP2 levels of obese individuals has changed,LEAP2 may be involved in the regulation of blood glucose homeostasis.However,the role of LEAP2 in the occurrence and development of T2 DM has not been investigated,which is the aim of this current research.Objective:(1)To observe the difference of serum LEAP2 and ghrelin between T2 DM patients and healthy controls in clinical.(2)To observe the m RNA expression of tissue LEAP2 and ghrelin and the effects of exogenous LEAP2 on islet function in T2 DM mice.(3)To investigate the effects of LEAP2 on insulin secretion from MIN6 cells in vitro and the possible molecular mechanism of LEAP2 action.Methods:1.The blood samples were collected from T2 DM patients and healthy individuals matched for age and body mass index(BMI).The serum was separated for ELISA detection of LEAP2 and ghrelin levels.The clinical laboratory test parameters and anthropometric parameters were collected for correlation analysis.2.High glucose and fat diet combined with low-dose multiple streptozotocin injection were used to induce a T2 DM mouse model.The m RNA expressions of LEAP2,ghrelin and GHSR were measured by q RTPCR in liver,small intestine,stomach,and pancreas of T2 DM and control mice.3.Mice were randomly divided into four groups: control,LEAP2,T2 DM and T2DM+LEAP2 groups.After successfully established T2 DM model,mice were intraperitoneal injected with LEAP2(30 μg/kg)daily for14 consecutive days.Control intraperitoneal injection was with the same amount of saline solution.During the period,the weight,food intake and fasting blood glucose of each group were recorded.The changes of islet function in mice were analyzed by intraperitoneal glucose tolerance test(IPGTT)and glucose stimulated insulin secretion test(GSIS).The histological changes of pancreas were observed by hematoxylin & eosin(H&E)staining and immunohistochemistry.4.MIN6 cells were treated in vitro with LEAP2.ELISA was used to detect the effect of LEAP2 on insulin secretion.q RT-PCR and WB were used to detect expression changes of glucokinase(Gck)and peroxidase proliferator activated receptor γ(PPARγ)at m RNA and protein levels.ATP detection kit and fluo-3 AM calcium fluorescence probe were used to detect the intracellular contents of ATP and free calcium in MIN6 cells.Results:1.The serum ghrelin levels were decreased,while the LEAP2 levels were increased in T2 DM patients.Ghrelin levels were positively correlated with insulin levels and HOMA-IR in healthy adults.LEAP2 levels were positively correlated with age and Hb A1 c in all tested samples.Ghrelin/LEAP2 ratio was negatively correlated with age,fasting blood glucose and Hb A1 c.2.Compared to normal mice,the m RNA expression of LEAP2 was decreased in the liver and increased in the small intestine of T2 DM mice.The expression of ghrelin m RNA in the liver was up-regulated and no significant change in stomach in T2 DM mice.There were reductions in GHSR m RNA expression in liver,pancreas and small intestine in T2 DM mice.3.The long-term one injection per day treatment of mice for 14 days with LEAP2 had no significant effects on body weight,food intake and fasting blood glucose in T2 DM mice.However,the treatment alleviated impaired glucose tolerance and improve the abnormal histological changes of pancreatic islets in T2 DM mice.No significant effects of the LEAP2 treatment were found on insulin sensitivity and basal insulin secretion.4.MIN6 cells treated with LEAP2 in vitro significantly increased insulin secretion,intracellular ATP contents and intracellular free calcium levels.LEAP2 increased intracellular Gck and PPARγ expression in MIN6 cells.Conclusion:1.The results showed that serum LEAP2 levels were increased in T2 DM patients and positively correlated with Hb A1 c.Ghrelin/LEAP2 ratio was significantly reduced and negatively correlated with blood glucose levels and Hb A1 c,suggesting that LEAP2 and ghrelin were associated with glycemic control in T2 DM patients.2.The m RNA expression of LEAP2 and GHSR in liver,small intestine and pancreas tissue of T2 DM mice were altered,suggesting that LEAP2 and GHSR were associated to the occurrence and progress of T2 DM in mice.3.Long term(14 days)treatment with LEAP2 injection alleviated glucose tolerance and pancreatic islet abnormalities in T2 DM mice,suggesting that the beneficial effects of LEAP2 in protecting islet β cells from dysfunction.4.LEAP2 promoted MIN6 cells to secrete insulin through an underlying mechanism involving LEAP2-GHSR-PPARγ-Gck axis.