The Effect And Mechanisms Of Adipose-derived Mesenchymal Stem Cell On Islet β Cells And Insulin Resistance

Obesity and insulin resistance are important elements in the pathogenesis of type 2 diabetes. Stem cells are an attractive option to ameliorate diabetes for its abundant source and potential to acquire glucose-dependent insulin secretory function. Adipose-derived stem cells(ASC) have been studied over the past decade as an alternative adult stem cell source for autologous cell replacement.Objective: we induced type 2 diabetes(T2DM) in a rat model by a high-fat diet combined with streptozotocin(STZ) administration, and performed Adipose-derived stem cells(ASCs) infusion at 28 days after STZ injection, in order to investigate the therapeutic effect and possible mechanisms of ASCs infusion on type 2 diabetes of longer history.Methods: we induced T2 DM in a SD rat model by a high-fat diet combined with streptozotocin(STZ) administration. ASCs were prepared with collagenase digestion and then tissue adherence method. Forty rats fed with a high-fat diet and STZ intraperitoneal injection were confirmed as diabetic and randomly divided into two groups(n=20), including ASCs group and diabetic control group. Rats in ASCs group were treated with an intravenous infusion of ASCs through vena caudalis at the 28 th day after STZ injection, the cell number was 2×106/rat and cells were suspended in 0.5 ml physiological saline. Rats in diabetic control group were as diabetic controls and infused with 0.5 ml physiological saline through vena caudalis. Glucometer tail-vein blood glucose levels were measured bi-weekly. Glycosylated hemoglobin(Hb A1c), serum insulin and C-peptide were measured by ELISA. Homeostatic model assessment(HOMA) was used to assess changes in insulin resistance(HOMA-IR) and pancreatic β-cell function(HOMA-β) in different groups. Hyperinsulinemic- euglycemic clamp studies were used to assess insulin sensitivity at 2 weeks after ASCs infusion. Following immunofluorescence staining of the islets, the numbers of different cell types(α, β cells) and total islet cells in each islet were manually counted from the islet microscopy images. Caspase-3 activity in lysates from pancreatic islets was determined using the Caspase-3 Colorimetric Assay Kit. Levels of proinflammatory cytokines TNF-α, IL-1β, IL-6 and von Willebrand factor(v WF) in the pancreatic tissue homogenates were determined by a quantitative sandwich enzyme technique using Quantikine ELISA kits. Total proteins were extracted from samples of skeletal muscle, adipose tissue and liver by PRO-PREP Protein Extraction Kit. Detection of INSR, GLUT4, IRS-1, PDK1 and PKCζby Western blot analysis.Results: 1. Characterization of ASCs and animals ASCs isolated from inguinal adipose tissue of diabetic rats were adhered to the plastic surface of culture flasks and had a spindle-shaped morphology, and expressed CD44+(75.34%), CD105+(79.26%), CD90+(85.77%), CD73+(89.63%), CD34-(0.34%) and CD45-(0.14%). Their differentiation potential was characterized through the successful differentiation into osteoblasts and adipocytes. Messy fur, lags in response, polydipsia, polyphagia, urorrhagia, nastiness, and hypoergy were observed as expected among rats in diabetic groups. There were no significant differences in FPG and body weight of rats between diabetic control group and ASCs group at the beginning of therapy. 2. Infusion of ASCs relieved the hyperglycemia of diabetic rats As for rats in ASC group, after ASCs infusion, their body weight increased gradually. Moreover, their FPG began to decrease, reached the lowest level in two weeks, and was at that level sustained for six weeks. Also, the level of Hb A1 c of rats in ASCs group was statistically lower compared to diabetic control group(P<0.05) but higher than normal control group(P<0.05). After STZ injection, concentrations of serum insulin and C-peptide of rats in ASCs group and diabetic control group were lower than normal control group(P< 0.05), while after ASCs infusion, concentrations of serum insulin and C-peptide of rats in ASCs group were slightly higher compared to diabetic control group(P<0.05). 3. Infusion of ASCs repaired islet cells by reducing the apoptosis of islet cells and promoting islet revascularization A small increase of islet β cells in ASCs group was observed as expected when compared with diabetic control group. The average number of β cells in ASCs group was more than that in diabetic control group(P=0.032), but lower than normal control group(P=0.01). The average number of α cells in ASCs group increased after therapy and was higher compared to normal control group(P= 0.019). Eight weeks after therapy, the caspase-3 activity of pancreatic islet in diabetic control group was significantly higher than normal control group(P=0.03), while activity of caspase-3 in ASC group was much lower compared to diabetic control group(P=0.038), but still higher compared to normal control group(P= 0.031). The concentrations of v WF of pancreatic tissue in ASCs group were higher than normal control group and diabetic control group after therapy(P=0.034 and P=0.016). 4. Infusion of ASCs modulated the inflammation in pancreatic tissue Concentrations of TNF-α, IL-1β, and IL-6 in diabetic control group were higher compared to normal control group(P= 0.013, 0.022, and 0.03, respectively). After ASCs infusion, concentrations of TNF-α, IL-1β and IL-6 in ASCs group were lower compared to diabetic control group(P=0.032, 0.027, and 0.028), but higher compared to normal control group(P=0.04, 0.038, and 0.031). 5. ASCs infusion improved insulin sensitivity in diabetic rats GDR and GIR were significantly increased in rats in ASCs group. There was no significant difference in basal HGP between rats in ASCs group and diabetic control group, but clamp HGP was significantly lower in rats in ASCs group. Moreover, the levels of the IR index and HBCI in rats in ASCs group indicated their reduced insulin resistance and improved β-cell function. 6. Infusion of ASCs influenced the expression of GLUT4 and INSR in insulin target tissues Total membrane fractions of GLUT4 and INSR in skeletal muscle and liver and adipose tissues were dramatically decreased in diabetic control group compared to normal control group(P<0.01). However, after ASCs infusion, both fractions were increased in ASCs group. 7. Infusion of ASCs enhanced the phosphorylation of IRS-1, PDK1, and PKC ζ in diabetic rats Phosphorylated levels of IRS-1, PDK1 and PKC ζ in skeletal muscle and liver and adipose tissues in diabetic control group were significantly lower compared to normal control group(P<0.01), while marked increase of phosphorylated IRS-1, PDK1, and PKC ζ in rats in ASCs group was observed as expected after ASCs infusion(P<0.05).Conclusions: ASCs infusion could effectively ameliorate hyperglycemia, restore islet β cells by reducing cell apoptosis and promoting islet angiogenesis, and improve insulin resistance of T2 DM. These findings provide an important basis for exploring ASCs infusion in T2 DM therapy. Autologous ASCs infusion might be an effective method for T2 DM.