Metabolomics Study Of Calorie Restriction In The Treatment Of Type 2 Diabetes Mellitus And The Molecular Mechanism Of Protecting Pancreatic Islet Beta Cell Function By Calorie Restriction

Study oneMetabolomic study on the mechanism of calorie restriction in Type 2 diabetes millitusObjective:To study the changes of general glucose and lipid metabolism index,safety index and plasma metabolome of patients with type 2 diabetes mellitus(T2DM)after very low calorie restriction(VLCR)therapy,to screen out specific markers that cause metabolome changes in diabetic patients after VLCR therapy,and explain the underlying mechanism of VLCR in the treatment of type 2 diabetes based on metabonomics.Methods:Ten patients with type 2 diabetes mellitus who met the inclusion criteria were selected for a 9-day VLCR,and fasting blood was collected before and after VLCR therapy.The glucose and lipid metabolism index and safety index were detected,and the changes of these indexes before and after VLCR were observed.Ultra-performance Liquid Chromatography Tandem Mass Spectrometry(UPLC-Q/TOF-MS/MS)was used to collect plasma metabolite data before and after calorie restriction in 10 diabetic patients.Partial least square analysis of variance(PLS-DA)and orthogonal partial least squares-discriminate analysis(OPLS-DA)models were used to compare plasma metabolome of diabetic patients before and after calorie restriction.Results:Glucose and lipid metabolism was improved after VLCR in patients with type 2 diabetes mellitus,and safety indicators did not change.The PLS-DA method can successfully distinguish the pre-caloric restriction group and the post-caloric restriction group in patients with type 2 diabetes.Five potential biomarkers,lysophosphatidylcholine(16:0),lysophosphatidylcholine(18:2(9z,12z)),lysophosphatidylcholine(20:4(7z,8z,11z,14z)),valerylcarnitine,and leucine were identified using the OPLS-DA method.After calorie restriction,all these five biomarkers increased in type 2 diabetics.Conclusions:VLCR can improve the glucose and lipid metabolism of patients with type 2 diabetes and partially restore islet function.Short-term VLCR is a safe and effective method for patients with type 2 diabetes.There is a significant difference between the plasma metabolome in patients before and after calorie restriction therapy.VLCR may regulate metabolic disturbances in type 2 diabetic patients by elevating lysophosphatidylcholine(16:0),lysophosphatidylcholine(1 8:2(9z,12z)),lysophosphatidylcholine(20:4(7z,8z,11z,14z)),valerylcarmitine,and leucine.Study two Calorie restriction reverses high-fat-induced pancreatic islet beta cell apoptosis and dedifferentiation Objective:To investigate the effects of calorie restriction on palmitic acid(PA)-induced apoptosis and dedifferentiation of mouse insulinoma MIN6 cells,and to explore its possible molecular mechanism.Methods:MIN6 cells were treated by PA with different concentrations(0?0.1?0.2?0.3?0.4?0.5?0.6mmol/L)for 24 hours,the thiazolyl blue method(MTT)was used to test the effect of PA on the proliferation of MIN6 cells.MIN6 cells were divided into four groups: normal culture(Control)group,Earle!s balanced salt solution(EBSS)group(simulated in vitro CR),palmitic acid(PA)group,and PA combined with EBSS group.After 5 hours of intervention,the morphology of MIN6 cells was observed by inverted microscope,MTT was used to test the proliferation of MIN6 cells.Western Blot was used to measure the protein levels of Bax and,Bcl-2,Caspase 3,p-AMPK and SIRT1.And finally,with the help of qPCR,the mRNA expressions of Insulin,Glut2,Pdxl,MafA,Nkx-6.1,NeuroDl and FOXOlwere to be detected.Results:MTT showed that PA played a significant role in inhibiting the growth of MIN6 cells,with the increasing of concentration of PA,the inhibition of proliferation of MIN6 cells became stronger and stronger,the cell viability decreased gradually and the inhibition was concentrationdependent.The morphology of the cells was observed under an inverted microscope,which showed that,compared with control group,cell density coefficient and adherent cells decreased,and the cell shape changed from fusiform to fragment and pieces in the PA group,compared with the PA group,cell density coefficient and adherent cells increased,and the cell shape tended to be normal in PA+EBSS group.MTT showed that PA+EBSS can improve the inhibition of cell proliferation and enhance viability of MIN6 cell compared with PA group(P<0.05);Western Blot showed that PA increased Bax expression,decreased Bcl-2 expression,increased Bax/Bcl-2 ratio,upregulated the expression of Cleaved Caspase3,and after the intervention of the combination of PA and EBSS,Bax expression decreased,Bcl-2 expression increased,Bax/Bcl-2 ratio was significantly reduced,and Cleaved Caspase3 expression significantly decreased(P<0.05).Meanwhile,the protein levels of p-AMPK and SIRT1 weren't different between the control group and PA group,after the intervention of combination,compared with PA group,the protein levels of p-AMPK and SIRT1 significantly increased in the PA+EBSS(P<0.05).qPCR showed that the mRNA expressions of Pdxl,MafA,FOXOl and Glut2 in PA group were lower than that in control group,and the mRNA expressions of Insulin,NeuroDl and NKX6.1 had no difference between the control group and PA group.After the combined intervention(PA+EBSS),compared with PA group,the mRNA expressions of Pdxl and MafA increased,the change were not significant(P>0.05).Insulin,NKX6.1,NeuroDl,FOXOl mRNA expression significantly increased,and Glut2 mRNA expression significantly decreased in PA+EBSS group(P<0.05).Conclusions:(1)PA can inhibit the growth of MIN6 cells in a concentration-dependent manner,induce apoptosis and dedifferentiation of pancreatic islet beta cell.(2)Calorie restriction can promote cell growth and inhibit PA-induced apoptosis and dedilferentiation of MIN6 cells.The molecular mechanism of this process may be associated with the activation of AMPK-SIRT1 pathway.