Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitro

Bckground Stem cells, which have the ability to differentiate into insulin-producing cells （IPCs）, would provide a potentially unlimited source of islet cells for transplantation and alleviate the major limitations of availability and allogeneic rejection. Therefore, the utilization of stem cells is becoming the most promising therapy for diabetes mellitus （DM）. Here, we studied the differentiation capacity of the diabetic patient＇s bone marrow-derived mesenchymal stem cells （MSCs） and tested the feasibility of using MSCs for β-cell replacement. Methods Bone marrow-derived MSCs were obtained from 10 DM patients （5 type 1 DM and 5 type 2 DM） and induced to IPCs under a three-stage protocol. Representative cell surface antigen expression profiles of MSCs were analysed by flow cytometric analysis. Reverse transcription-polymerase chain reaction （RT-PCR） was performed to detect multiple genes related to pancreatic β-cell development and function. The identity of the IPCs was illustrated by the analysis of morphology, ditizone staining and immunocytochemistry. Release of insulin by these cells was confirmed by immunoradioassay. Results Flow cytometric analysis of MSCs at passage 3 showed that these cells expressed high levels of CD29 （98.28%）, CD44 （99.56%） and CD106 （98.34%）. Typical islet-like cell clusters were observed at the end of the protocol （18 days）. Ditizone staining and immunohistochemistry for insulin were both positive. These differentiated cells at stage 2 （10 days） expressed nestin, pancreatic duodenal homeobox-1 （PDX-1）, Neurogenin3, Pax4, insulin, glucagon, but at stage 3 （18 days） we observed the high expression of PDX-1, insulin, glucagon. Insulin was secreted by these cells in response to different concentrations of glucose stimulation in a regulated manner （P〈0.05）. Conclusions Bone marrow-derived MSCs from DM patients can differentiate into functional IPCs under certain conditions in vitro. Using diabetic patient＇s own bone marrow-derived MSCs as a source of autologous IPCs for β-cell reDlacement would be feasible.

Chemerin and apelin are positively correlated with inflammation in obese type 2 diabetic patients

Background As two novel adipocytokines, chemerin and apelin play a key role in the pathological process of insulin resistance （IR）, glucose metabolism and obesity, researchers have found that the levels of chemerin and apelin changed significantly in type 2 diabetic patients with obesity, however, the underlying mechanism involved remains unclear. The aim of this study was to investigate whether chemerin and apelin play an important role in the pathophysiologic proceeding of diabetes. Methods This study enrolled 81 newly diagnosed obese type 2 diabetes mellitus （T2DM） patients （T2DM group, n=81）. All the patients were randomly assigned to DM1 group treated with metformin （n=41） and DM2 group treated with pioglitazone （n=40）. After hypoglycemic agents treatment, patients under better blood glucose control were chosen to be given antioxidant treatment. Another 79 subjects without T2DM were recruited as normal control group （NC group）, including 40 subjects （NC1 group） with normal body mass index （BMI） and 39 obese subjects （NC2 group）. Levels of chemerin, apelin, BMI, tumor necrosis factor-α（TNF-α）, homeostasis model assessment of IR （HOMA-IR） and 8-isoprotaglandim F2α（8-iso-PGF2α） were examined at baseline and post-treatment. The relationship between chemerin, apelin and BMI, TNF-α, HOMA-IR, 8-iso-PGF2α was analyzed. Results The baseline levels of chemerin, apelin, TNF-α, HOMA-IR and 8-iso-PGF2α in T2DM group were significantly higher than normal control group （P 〈0.001）. All indices mentioned above were significantly decreased after treatment （P 〈0.05）. In T2DM patients treated with pioglitazone, indices mentioned above except for HOMA-IR, were decreased significantly compared with patients treated with mefformin （P〈0.05）. After antioxidant treatment using lipoic acid, levels of chemerin, apelin, TNF-α and 8-iso-PGF2α were further significantly decreased （P 〈0.05）. Correlation analysis showed that the levels of chemerin and apelin correlated positively with BMI, TNF-α, HOMA-IR and 8-iso-PGF2α before and after treatment with hypoglycemic agents （P〈0.01）. The levels of chemerin and apelin also had positive correlation with TNF-a and 8-iso-PGF2α after antioxidant treatment （P〈0.05）. Conclusions The levels of chemerin and apelin in obese T2DM patients are closely related to IR. The increased levels may be a result of compensatory response to IR, and also may be the causative factor of IR. The levels of chemerin and apelin correlate closely with oxidative stress and inflammation. The two adipokines may be inflammatory factors playing important roles in the initiation and development of obese T2DM. Chemerin and apelin are related to the pathophysiology of IR, oxidative stress and inflammation.

Influence of blood glucose on the expression of glucose transporter proteins 1 and 3 in the brain of diabetic rats

Background The delivery of glucose from the blood to the brain involves its passage across the endothelial cells of the blood-brain barrier （BBB）, which is mediated by the facilitative glucose transporter protein 1 （GLUT1）, end then across the neural cell membranes, which is mediated by GLUT3. This study aimed to evaluate the dynamic influence of hyperglycemia on the expression of these GLUTs by measuring their expression in the brain at different blood glucose levels in e rat model of diabetes. This might help to determine the proper blood glucose threshold level in the treatment of diabetic apoplexy. Methods Diabetes mellitus was induced with streptozotocin （STZ） in 30 rats. The rats were randomly divided into 3 groups： diabetic group without blood glucose control （group DM1）, diabetic rats treated with low dose insulin （group DM2） end diabetic rats treated with high dose insulin （group DM3）. The mRNA end protein levels of GLUT1 end GLUT3 were essayed by reverse trenscriptese-polymerese chain reaction （RT-PCR） end immunohistochemistry, respectively. Results Compared with normal control rats, the G/UT1 mRNA was reduced by 46.08%, 29.80%, 19.22% （P〈0.01） in DM1, DM2, end DM3 group, respectively; end the GLUT3 mRNA was reduced by 75.00%, 46.75%, end 17.89% （P〈0.01） in DM1, DM2, end DM3 group, respectively. The abundance of GLUT1 end GLUT3 proteins had negative correlation with the blood glucose level （P〈0.01）. The density of microvessels in the brain of diabetic rats did not change significantly compared with normal rats. Conclusions Chronic hyperglycemia downreguletes G/UT1 end GLUT3 expression at both mRNA end protein levels in the rat brain, which is not due to the decrease of the density of microvessels. The downreguletion of G/UT1 end GLUT3 expression might be the adaptive reaction of the body to prevent excessive glucose entering the cell that may lead to cell damage.

Adipocytokines and breast cancer risk

Background Many researches suggested that obesity increased the risk of breast cancer, but the mechanism was currently unknown. Adipocytokines might mediate the relationship. Our study was aimed to investigate the relationship between serum levels of resistin, adiponectin and leptin and the onset, invasion and metastasis of breast cancer. Methods Blood samples were collected from 80 newly diagnosed, histologically confirmed breast cancer patients and 50 age-matched healthy controls. Serum levels of resistin, adiponectin and leptin were determined by enzyme-linked immunosorbent assays （ELISA）; fasting blood glucose （FBG）, lipids, body mass index （BMI）, and waist circumference （WC） were assayed simultaneously. Results Serum levels of adiponectin （（8.60±2.92） mg/L vs （10.37±2.81） mg/L, P=-0.001） and HDL-c were significantly decreased in breast cancer patients in comparison to controls. Serum levels of resistin （（26.35±5.36） μg/L vs （23.32±4.75） μg/L, P=-0.000）, leptin （（1.35±0.42） μg/L vs （1.06±0.39） μg/L, P=0.003）, FBG and triglyceride （TG） in breast cancer patients were increased in contrast to controls, respectively. However, we did not find the significant difference of the serum levels of resistin, adiponectin and leptin between premenopausal breast cancer patients and healthy controls （P= 0.091, 0.109 and 0.084, respectively）. The serum levels of resistin, adiponectin and leptin were significantly different between patients with lymph node metastasis （LNM） and those without LNM （P=-0.001, 0.000 and 0.006, respectively）. The stepwise regression analysis indicated that the tumor size had the close correlation with leptin （R^2=0.414, P=-0.000） and FBG （R^2=0.602, P=0.000）. Logistic regression analysis showed that reduced serum levels of adiponectin （OR：. 0.805; 95%CI： 0.704--0.921; P=0.001）, HDL （OR： 0.087; 95%C/： 0.011-0.691, P=0.021）, elevated leptin （OFt： 2.235; 95%C/：1.898--4.526; P=0.004） and resistin （OR：. 1.335; 95%C/： 1.114-2.354; P=0.012） increased the risk for breast cancer; Reduced serum levels of adiponectin （OR： 0.742; 95%C/： 0.504-0.921; P=-0.003） and elevated leptin （OR： 2.134; 95%C/：1.725-3.921; P= 0.001） were associated with lymph node metastasis of breast cancer. Conclusions The decreased serum adiponectin levels and increased serum resistin and leptin levels are risk factors of breast cancer. The low serum adiponectin levels and high serum leptin levels are independent risk factors for metastasis of cancer. The association between obesity and breast cancer risk might be explained by adipocytokines.

Dynamic expression of glucose transporters 1 and 3 in the brain of diabetic rats with cerebral ischemia reperfusion

Background Blood glucose control improves the outcome of diabetic patients with stroke, but the target range of blood glucose control remains controversial. The functional recruitment of ischemia penumbra is extremely important to the recovery after stroke. The present study aimed to explore the expression of brain-type glucose transporters （GLUT1 and GLUT3） in cerebral ischemic penumbra at different blood glucose levels and different ischemic-reperfusion time in diabetic hypoxia-ischemia rats. The results might provide an experimental basis for clinical treatment of diabetic patients with stroke. Methods The Wistar rats included in this study were randomly assigned to 4 groups （50 rats each）： normal control group （NC）, uncontrolled diabetic group （DM1）, poorly-controlled diabetic group （DM2）, and well-controlled diabetic group （DM3）. Diabetic rats were induced by single intraperitoneal injection of streptozotocin, and the focal ischemic rat model of middle artery occlusion （MCAO） was made by insertion of fishing thread in 6 weeks after the establishment of the diabetic model. Each group was divided into 5 subgroups （10 rats each）： four focal ischemic subgroups at different ischemic-reperfusion time （at 3,12, 24 and 72 hours after reperfusion, respectively） and one sham-operated subgroup. The mRNA and protein expression of GLUT1 and GLUT3 was assessed by RT-PCR and Western blotting, respectively. Results There was significant difference in the mRNA expression of GLUT1 and GLUT3 between the four focal ischemic subgroups and the sham-operated subgroup at different reperfusion time in each group. The mRNA expression of GLUT1 and GLUT3 in the 4 ischemic groups began to increase at 3 hours, peaked at 24 hours after reperfusion and maintained at a higher level even at 72 hours compared with that of the sham-operated subgroup. The mRNA expression of GLUT1 increased more significantly than that of GLUT3. The mRNA expression of GLUT1 and GLUT3 was significantly different between the diabetic groups and normal control group. The mRNA expression of GLUT1 and GLUT3 was increased more significantty in the diabetic groups than that in the normal control group. There was a significant difference in the mRNA expression in the groups with different blood glucose levels. The mRNA expression tended to decrease with increased blood glucose levels. The expression trend of GLUT1 and GLUT3 protein was similar to that of GLUT1 and GLUT3 mRNA. Conclusions GLUT1 and GLUT3 expression was notably up-regulated in the penumbra region after cerebral ischemia in this study. But the up-regulated amplitude of GLUT1 and GLUT3 in the diabetic rats with cerebral ischemic injury became smaller than that of the normal controls. In the treatment of diabetic patients with cerebral embolism, blood glucose control should not be too strict, otherwise the up-regulation of GLUT1 and GLUT3 induced by cerebral ischemic injury might not be able to meet the needs of energy metabolism in cells. Chin Med J 2009; 122（ 17）： 1996-2001

Effects of glucose and insulin on the H9c2 （2-1） cell proliferation may be mediated through regulating glucose transporter 4 expression

Background The change of glucose transporter 4 （GLUT4） expression could influence glucose uptake in the myocardial cells and then effect myocardial metabolism, which maybe one of the factor for the diabetes cardiovascular disease. This study aimed to explore the influence of glucose and insulin at different concentrations on H9c2 （2-1） cell proliferation and its GLUT4 expression in vitro, and evaluate the correlation between myocardial cells proliferation and GLUT4 expression. This might be helpful for understanding the relationship between glucose metabolism and cardiovascular disease. Methods According to glucose concentrations in culture medium, cultured H9c2 rat myocardial cells were divided into five groups： control group （NC, glucose concentration 5.0 mmol/L）, low glucose group （LG, glucose concentration 0.1 mmol/L）, high glucose group 1 （HG1, glucose concentration 10 mmol/L）, high glucose group 2 （HG2, glucose concentration 15 mmol/L）, high glucose group 3 （HG3, glucose concentration 20 mmol/L）. Then according to different insulin concentrations in culture medium, each group was further divided into two subgroups： normal insulin subgroup （INSc, insulin concentration 3.8 mU/L）, high insulin subgroup （INSh, insulin concentration 7.6 mU/L）. H9c2 （2-1） cells were cultured for 1, 2, 3 days, the proliferation of cells were assayed by cell counting Kit-8 assay, the expressions of GLUT4 mRNA and protein were detected with RT-PCR and Western Blotting technique, and the relation between myocardial cells proliferation and GLUT4 expression was evaluated.

Basal insulin therapy strategy is superior to premixed insulin therapy in the perioperative period blood glucose management

Background The probability and risk of operations increase in patients with type 2 diabetes mellitus. For diabetic patients, blood glucose control is a key factor to improving the prognosis of surgery. During perioperative period, insulin therapy is usually advised to be used for surgical patients with type 2 diabetes. However, the insulin regimen which one is better remains controversial. In this study, we estimated the efficacy, safety and advantage of different insulin therapy strategy during perioperative period.