The Immunometabolism Studies in Diabetic Subjects and Diet Induced Obese Mice
|School||Central South University|
|Keywords||Autoimmune diabetes LADA Obese Immunemetabolism inflammationtory cytokines adipokines autoimmune diabetes High fat diet obesity B6 mice Myd88-/- B6 mice CD11cMyD88+/+MyD88-/-mice IPGTT ITT TG MyD88-/-B6 mice CD11cMyD88+/+MyD88-/-B6 mice immunology mechanism regulatory T cells BMDC gut immunology system inflammatory cytokines|
Part 1 Prevalence of LADA in phenotypic Type 2 Diabetes and Prevalence of obesity in autoimmune diabetes in ChinaObject:To investigate prevalence of LADA in phenotypic Type 2 diabetic patients and prevalence of obesity in autoimmune diabetic patients in China.Design:Multicenter -cross-sectional and case control study.Methods:By LADA China multicenter study, we included newly diagnosed type 2 diabetes patients 5128, within them 316 LADA patients were diagnosed after centrally GADA assay. By Hunan Type 1 diabetes collaboration net, we included newly diagnosed Type 1 diabetes patients 106. By community based investigation, we screened 216 healthy controls with OGTT. Serum samples were shipped to Diabetes Center, Central South University with a centrally GADA testing. Other information was collected locally.Results:①among 5128 newly diagnosed type 2 diabetes patients,316 [6.2%(5.5-6.8)] are GADA positive subjects and diagnosed LADA finally.②Frequency of LADA in northern China is higher than southern China (6.9%(156/2256) v.s.5.6%(160/2872), P=0.018 after adjustment).③In different age subgroups, the frequency of GADA positivity is increased with aging (P=0.004 after adjustment).④In BMI subgroups, leaner subjects have a higher frequency of positive GADA (P<0.001 after adjustment).⑤In differentβcell function group, the worse group (FCP<0.3ng/ml) has a higher GADA positivity [12.6%(39/309) vs. 5.4%(80/1479), P= 0.001 after adjustment].⑥The overweight and obese subjects in T2DM subjects are 2148(44.99%), which is much higher (P <0.001) than in T1DM subjects 8(8.25%). There are 108(34.50%) subjects having overweight and obese in LAD A, which is similar as in control group (33.18%).⑦No gender difference affected the prevalence of overweight and obese in autoimmune diabetes.⑧Among different age group, prevalence of overweight and obese subjects increased with age in T1DM subjects but not in LADA and T2DM.⑨Overweight and obese subjects in LADA and T2DM group have a better (3 cell function than leaner group.Conclusion:Type 2 diabetes related inflammation (obese) and Type 1 diabetes related autoimmunity (islet autoantibodies) exist in the same subtype of diabetes patients. The two immune mechanisms may crosstalk in the pathogenesis of diabetes. Part 2 Heterogeneity of altered cytokine levels across the clinical spectrum of diabetes in ChinaObject:Altered inflammatory markers are a feature of obesity and type 2 diabetes, but have not been extensively studied in latent adult-onset autoimmune diabetes (LADA). We determined selected cytokines in Chinese diabetes patients to determine their relationship to diabetes in general, and autoimmune diabetes in particular.Design:Multicenter-cross-sectional and case control study.Methods:Adult patients with type 1 diabetes (n=53), LADA (n=250) and type 2 diabetes (n=285), diagnosed for less than one year and recruited from multiple Chinese centers, were compared with population-based normal subjects (n=196). Serum glutamate decarboxylase autoantibodies (GADA), interlukin-6 (IL-6), lipocalin 2 (LCN2), highly sensitive-C reactive protein (hs-CRP) and adiponectin were tested centrally and clinical data collected locally.Results:①After age, gender and BMI adjustment, all three types of diabetes showed increased IL-6 and LCN2 (P<0.01), and all four cytokines (IL-6, LCN2, hs-CRP and adiponectin) were increased in LADA (P<0.01).②In type 1 diabetes, adiponectin, but not hs-CRP, was increased (P<0.01); while in type 2 diabetes, hs-CRP, but not adiponectin, was increased (P<0.01).③Adiponectin was positively correlated with GADA titer and negatively correlated with hs-CRP (p<0.01 for both),④IL-6, LCN2 and hs-CRP, were related to glucose (fasting), obesity (waist-hip ratio) and insulin secretion (HOMA-(3) (p<0.001 for all).Conclusions:In China, inflammatory markers are increased in all three major types of diabetes, but the cause is likely heterogeneous, even in autoimmune diabetes. Part 3 CD11c+cells expressing MyD88 promotes obesity in high fat diet mice.Object:Compare the phenotype of MyD88 knockout and wild type mice under high fat diet.Design:B6 wild type mice, MyD88-/-B6 mice, and B6CD11cMyd88+/ Myd88-/- mice, were divided into high fat diet group and normal chow group(n≥10 per group). The high fat diet was set up from 6 weeks old lasting for 4 months.Methods:Body weight and food consumption were monitered every week. The glucose tolerance and insulin tolerance of the mice were determined by IPGTT and ITT test. TG and HDL-ch in plasma of the mice were measured by HPLC methods. Bone Density and Fat tissue ratio was determined by scanning.Results:1) After 4 months high fat diet, the wild type mice average body weight was 47.35g. The normal chow group was only 29.60g. (P<0.001).2) The MyD88-/- mice had lower average body weight than B6 mice. (40.64g vs 47.35g, P<0.001).3) After 4 months high fat diet, the body weight of CD 11 cMyd88+/+ Myd88-/- B6 mice was similar as wild type B6 which was heavier than Myd88-/- mice.4) B6 mice had higher average food calorie intake than Myd88-/- mice. CD11cMyd88+ Myd88-/-B6 mice was in the between。5) In accordance with body weight, epidemical fat pat, the whole body adipose tissue ratio results showed that MyD88-/- mice were lower than B6 mice, and CD11cMyd88+ Myd88-/-mice were in the between and close to wild type B6。6) IPGTT results, including the area under curve (ROC) results showed MyD88-/- mice had better glucose tolerance than CD11cMyd88+ Myd88-/- mice and B6 mice (overall P<0.001).7) ITT results, including AUC results showed the trend that MyD88-/- mice also had a better insulin tolerance ability than other two mice but without statistic significance.8) TG level was dramatically elevated in B6 mice compared with MyD88-/- mice. TG of CD11cMyd88+/+ Myd88-/- mice level was in the between。(overall P<0.001).9) We didn’t find differences of Bone Density between groups.Conclusion:Knocking out MyD88 protein can positively regulate glucose and lipid metabolism dysfunction and protect from obesity under high fat diet. It is CD11c cells expressing MyD88 playing this important role in obesity. Part 4 The immunology study in high fat diet B6 wild type, MyD88-/- and CD11cMYD88+/+ MYD88-/-obese mice.Object:Compare the immunology changes in spleen, adipose tissue, bone marrow dendritic cells (BMDC), and gut related lymph node in three different mice under high fat diet.Design:B6, Myd88-/- and CD11cMyd88+/+Myd88-/- mice, were divided into high fat diet and normal chow groups (n=6 per group). High fat diet was from 6 weeks old and lasting for 4 months.Methods:Harvest spleen cells, adipose stromal cells, BMDCs, and MLN and PP lymphocytes. Stain monoclonal florescence antibodies, flow cytometry determined final results to study the phenotype of lymphocytes. Intracellular staining to determine Foxp3 regulatory T cells frequency and T cells cytokine releasing. Flow cytometry determined final results. 3H-DTR methods measure the proliferation ability of spleen lymphocytes under the stimulation by CD3, CD40 and LPS. Beads purified CD11c cells APC function was determined by co culture with BDC2.5 T cells and final determined by 3H-DTR methods. Luminex platform assayed the plasma cytokines.Results:1) Under high fat diet, the CD4+and CD8+T cells ratio in adipose tissue cells reduced, but frequency of CD 11 b+ and CD11c+ cells were increased. The CDllc+ and CDllb+ cells of MyD88-/- mice were increased in spleen but reduced in adipose tissue if compared with other two groups.2) Majority of Foxp3+ regulatory T cells were CD25-T cells, the frequency of Foxp3+CD25+CD4+ was less than CD25- one. No matter high fat diet or normal chow group, in spleen, the Foxp3+CD25+CD4+T cells frequency in MyD88-/-mice were higher than other two groups. But for CD25-regulatory T cells, they were lower than other two groups.3) High fat induced obese mice inflammatory cytokines increased greatly than normal chow group.4) MyD88-/-mice under high fat diet didn’t show difference of proliferation under LPS stimulation.5) Purified CD11c cells showed different APC function with different concentration of antigen stimulation. In a low concentration, B6 mice had better APC function than knock out mice. But in high concentration of the antigen, the APC function of B6 mice was lower than MyD88-/-mice and CD11cMyD88+/+MyD88-/-mice.6) The phenotype of BMDC didn’t show differences in different mice. However, the APC function of MyD88-/-BMDC was better than B6 and CD11cMyD88+/+MyD88-/-mice.7) High fat diet affected the phenotype of gut immune system. In MLN and PP, all the lymphocytes subgroups elevated. CD 103 gut homing marker expressing on spleen CD4+ and CD11c+ cells increased in MyD88-/-mice. The MLN and PP releasing TNF a and IL10 were changed according to High fat diet and normal chow feeding. Conclusions:High fat diet inducing obese mice showed a different immunology phenotype. MyD88-/- mice had an immunology difference with wild type B6 mice under high fat diet. CD11c cells expressing MyD88 could reverse this difference.