Glucose-induced heat production, insulin secretion and lactate production in isolated Wistar rat pancreatic islets

Transplantation of pancreatic islets is efficient in improving the metabolic control and quality of life and in preventing severe hypoglycemia in patients with brittle type 1 diabetes mellitus. More accurate methods to assess islet viability would be extremely useful in designing target interventions for islet cytoprotection and in reducing the number of islets required to achieve insulin independence. Here we report on an application of calorimetry to evaluate the metabolic response of pancreatic islets to glucose stimulation. A significant increase in metabolic heat was produced by islet samples when consecutively subjected to 2.8 and 16.3 mmol L⁻¹ glucose. Under these glucose concentrations, 1000 islets released average heat values of 9.16 ± 0.71 mJ and 14.90 ± 1.21 mJ over 50 min, respectively. Additionally, the glucose stimulation indexes were 1.67 ± 0.30 for insulin, 1.72 ± 0.13 for heat and 2.91 ± 0.50 for lactate, raising the important possibility of substituting the secreted insulin index/ratio by the index/ratio of the heat released in the evaluation of Langerhans islets viability for transplantation. Altogether, our results demonstrate the applicability of calorimetry to assess the quality of isolated pancreatic islets and to study vital islet functions.     

Fabrication of Xanthan stabilized green gold nanoparticles based tolbutamide delivery system for enhanced insulin secretion in mice pancreatic islets

Abstract

Tolbutamide is an oral anti-diabetic agent for the treatment of type 2 diabetic patients. Its lower absorption results in its inferior therapeutic efficacy. Nano-carrier systems have the subject of greater interests for enhancing efficacy of such drugs. Current study was designed to improve the tolbutamide therapeutic efficacy through its delivery in Gum Xanthan (GX) stabilized green gold nanoparticles (AuNPs). GX stabilized AuNPs were characterized for surface plasmon resonance (SPR), morphology, size, polydisoersity index (PDI) and zeta potential through UV spectrophotometer, atomic force microscope (AFM) and zetasizer respectively. They were used for loading tolbutamide and loaded nanoparticles were investigated for morphology, size, PDI, zeta potential and drug loading efficiency. FT-IR analysis was used for conforming GX functional groups involvement in AuNPs stabilization and drug-excepients interactions. Tolbutamide loaded in the synthesized nanoparticles was investigated for its insulin secretion potentials in isolated mice islets. Synthesized AuNPs were found in nano-size range with spherical morphology, increased surface negativity and loaded increased concentration of drug without changing its chemical nature. They markedly enhanced the tolbutamide insulin secretion potentials as compared to simple drug solution. Results confirm that the developed nano-carrier system is highly efficient in achieving higher therapeutic efficacy of drugs like tolbutamide.     

Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice

Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microg/microgL cDNA and 25 microgL effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.     

Prolonged survival of islet allografts in mice treated with rosmarinic acid and anti-CD154 antibody

Pancreatic islet transplantation can correct the abnormal glucose metabolism of Type 1 diabetes. Although immunosuppressants greatly reduce the acute rejection rate in transplant patients, the long-term side effects can be debilitating. Therefore, researchers are seeking to develop new immunosuppressive regimens that induce maximal levels of immunosuppression with minor side effects. Rosmarinic acid (Ros A) is a secondary metabolite of certain herbs and has multiple biological activities, including anti-inflammatory effects. Here, we have investigated whether treatment of mice with a combination of Ros A and anti-CD154 monoclonal antibody (MR1) improves islet allograft survival in a murine model. After transplantation, the mice were treated with either Ros A, MR1, or both (the "double" treatment). Allograft survival was prolonged in the double-treated animals compared to animals that received only Ros A or MR1. As is the case with the single-treated animals at 15 days after transplantation, the double-treated recipients did not display a significant decrease in the expression of cytokines or the population of activated T cells. Infiltrating CD3(+) T cells were reduced in the MR1- or double therapy relative to control or RosA group. However, at the same time point, double-treated graft showed fewer apoptotic cells and increased expression of insulin and glucagons, compared to the single-treatment groups. Furthermore, long-term (>150 days) allografts that were received with double therapy exhibited larger islet clusters and contained more insulin- and glucagon-positive cells, relative to the MR1-treated grafts. In conclusion, treatment with both Ros A and MR1 has a synergistic effect in murine islet allotransplantation.     

Functional improvement of porcine neonatal pancreatic cell clusters via conformal encapsulation using an air-driven encapsulator

Transplantation of islet cells into diabetic patients is a promising therapy, provided that the islet cells are able to evade host immune rejection. With improved islet viability, this strategy may effectively reverse diabetes. We applied 2% calcium alginate to generate small and large capsules to encapsulate porcine neonatal pancreatic cell clusters (NPCCs) using an air-driven encapsulator. After encapsulation, the viability was assessed at 1, 4, 7, 14 and 28 days and secretion of functional insulin in response to glucose stimulation were tested at days 14 and 28. Selective permeability of the small alginate capsules was confirmed using various sizes of isothiocyanate-labeled dextran (FITC-dextran). Encapsulation of NPCCs was performed without islet protrusion in the small and large capsules. The viability of NPCCs in all experimental groups was greater than 90% at day 1 and then gradually decreased after day 7. The NPCCs encapsulated in large capsules showed significantly lower viability (79.50 ± 2.88%) than that of naïve NPCCs and NPCCs in small capsule (86.83 ± 2.32%, 87.67 ± 2.07%, respectively) at day 7. The viability of naïve NPCCs decreased rapidly at day 14 (75.67 ± 1.75%), whereas the NPCCs encapsulated in small capsules maintained (82.0 ± 2.19%). After 14 and 28 days NPCCs' function in small capsules (2.67 ± 0.09 and 2.13 ± 0.09) was conserved better compared to that of naïve NPCCs (2.04 ± 0.25 and 1.53 ± 0.32, respectively) and NPCCs in large capsules (2.04 ± 0.34 and 1.13 ± 0.10, respectively), as assessed by a stimulation index. The small capsules also demonstrated selective permeability. With this encapsulation technique, small capsules improved the viability and insulin secretion of NPCCs without islet protrusion.     

Effect of nicotinamide on early graft failure following intraportal islet transplantation

Intraportal islet transplantation (IPIT) may potentially cure Type 1 diabetes mellitus; however, graft failure in the early post-transplantation period presents a major obstacle. In this study, we tested the ability of nicotinamide to prevent early islet destruction in a syngeneic mouse model. Mice (C57BL/6) with chemically-induced diabetes received intraportal transplants of syngeneic islet tissue in various doses. Islets were cultured for 24 h in medium with or without 10 mM nicotinamide supplementation. Following IPIT, islet function was confirmed by an intraperitoneal glucose tolerance test (IPGTT) and hepatectomy. The effects of nicotinamide were evaluated by blood glucose concentration, serum monocyte chemoattractant protein-1 (MCP-1) concentration, and immunohistology at 3 h and 24 h after IPIT. Among the various islet doses, an infusion of 300 syngeneic islets treated with nicotinamide exhibited the greatest differences in glucose tolerance between recipients of treated and untreated (i.e., control) islets. One day after 300 islet equivalent (IEQ) transplantation, islets treated with nicotinamide were better granulated than the untreated islets (P = 0.01), and the recipients displayed a slight decrease in serum MCP-1 concentration, as compared to controls. After 15 days, recipients of nicotinamide-pretreated islets showed higher levels of graft function (as measured by IPGTT) than controls. The pretreatment also prolonged graft survival (> 100 days) and function; these were confirmed by partial hepatectomy, which led to the recurrence of diabetes. Pretreatment of islet grafts with nicotinamide may prevent their deterioration on the early period following IPIT in a syngeneic mouse model.     

胰岛封装技术及其在胰岛移植中的应用

随着全球糖尿病患者人数逐年增长,临床上亟需一种行之有效的糖尿病治疗方案。胰岛移植可通过植入具有正常功能的胰岛以替代患者体内功能受损的胰岛,从而维持正常血糖水平,有望发展为理想的糖尿病治疗方法。然而,胰岛供体资源短缺,且移植后长期服用免疫抑制剂不仅产生诸多不良反应甚至会有致癌风险。以上瓶颈极大限制了胰岛移植的临床应用。采用天然高分子、合成高分子、无机化合物等生物材料对胰岛进行封装,营造免疫隔离微环境,避免宿主免疫细胞与植入胰岛直接接触,可有效抑制免疫排斥反应。免疫隔离的同时,胰岛的封装不影响胰岛素、葡萄糖、氧气等胰岛必需或输出关键分子的交换,同时确保移植胰岛的正常生理活性及精准控制血糖水平的能力。本文综述了胰岛封装的研究现状,常用材料及设计策略,并初步展望其应用前景。     

Stromal Vascular Fraction Loaded Silk Fibroin Mats Effectively Support the Survival of Diabetic Mice after Pancreatic Islet Transplantation

The aim of this study is to assess whether stromal vascular fraction (SVF)‐soaked silk fibroin nonwoven mats (silk‐SVF) can preserve the functionality of encapsulated pancreatic endocrine cells (alginate‐PECs) after transplantation in the subcutaneous tissue of diabetic mice. Silk scaffolds are selected to create an effective 3D microenvironment for SVF delivery in the subcutaneous tissue before diabetes induction: silk‐SVF is subcutaneously implanted in the dorsal area of five healthy animals; after 15 d, mice are treated with streptozotocin to induce diabetes and then alginate‐PECs are implanted on the silk‐SVF. All animals appear in good health, increasing weight during time, and among them, one presents euglycemia until the end of experiments. On the contrary, when PECs are simultaneously implanted with SVF after diabetes induction, mice are euthanized due to suffering. This work clearly demonstrates that silk‐SVF creates a functional niche in subcutaneous tissue and preserves endocrine cell survival and engraftment.     

Fibrin glue increases the cell survival and the transduced gene product secretion of the ceiling culture-derived adipocytes transplanted in mice

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 μg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene- transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The immunodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.     

Individualized Dosage of Tacrolimus for Renal Transplantation Patients Based on Pharmacometabonomics

The clinical pharmacodynamics of tacrolimus in renal transplant patients has significant interindividual variability. T lymphocytes were selected to study the pharmacodynamic response of tacrolimus, which was significantly correlated with renal function and the outcome of renal transplant patients. Ultra-performance liquid chromatography–quadrupole time-of-flight mass spectroscopy (UPLC/Q-TOF-MS) was performed to obtain the metabolic profiles of 109 renal transplant patients. A partial least squares (PLS) model was constructed to screen potential biomarkers that could predict the efficacy of tacrolimus. Multinomial logistic regression analysis established a bridge that could quantify the relationship between the efficacy of tacrolimus and biomarkers. The results showed a good correlation between endogenous molecules and the efficacy of tacrolimus. Metabolites such as serum creatinine, mesobilirubinogen, L-isoleucine, 5-methoxyindoleacetate, eicosapentaenoic acid, N₂-succinoylarginine, tryptophyl-arginine, and butyric acid were indicated as candidate biomarkers. In addition, the key biomarkers could correctly predict the efficacy of tacrolimus with an accuracy of 82.5%. Finally, we explored the mechanism of individual variation by pathway analysis, which showed that amino acid metabolism was significantly related to the efficacy of tacrolimus. Moreover, orthogonal partial least squares discriminant analysis (OPLS-DA) showed that there was no difference in key metabolites among different pharmacodynamic groups at 1 month and 3 months after dose adjustment, suggesting that pharmacometabonomics is a useful tool to predict individual differences in pharmacodynamics and thus to facilitate individualized drug therapy.     

85例脏器移植患者手术前后血清镁、锌含量的变化

对85例脏器移植患者（45例肝移植,40例肾移植）手术前、后进行了血清Mg、Zn含量的检测。结果表明：肝、肾移植患者术后血Mg水平较术前正常水平均明显降低（P＜0．01）。肝移植患者术后血Zn水平有一过性降低现象,肾移植患者术前、术后血Zn水平均明显低于正常对照组（P＜0．001）。研究结果提示,应重视肝、肾移植患者手术后出现的低镁血症和低锌血症,并做适当的补充。     

Altered expression of insulins I and II and their mRNAs in the islets of Langerhans in dexamethasone-induced diabetic rats

Rats have two isomeric insulins (insulins I and II). There have been no reports on the expression of the isomeric insulins in glucocorticoid-induced diabetic rats. To clarify the relation of the expression of each insulin and its mRNAs in dexamethasone-induced diabetic rats, the amounts of the isomeric insulins and mRNAs in the islets of Langerhans were determined in vivo and in vitro. A sensitive and selective HPLC-fluorescence determination method for the isomeric insulins and a newly developed real-time quantitative RT-PCR method for their mRNAs were used. There was a greater reduction of insulin II than insulin I in the islets of Langerhans in dexamethasone-induced diabetic rats. This alteration may be caused by a disproportionate expression of the respective mRNA for the isomeric insulins that resulted from the direct effect of dexamethasone. In addition, continuous hyperglycemia may also suppress the expression of the insulin II mRNA. The overall effects of dexamethasone and hyperglycemia may cause a greater reduction of insulin II than insulin I in the dexamethasone-induced diabetic rat. Conversely, an elevated ratio of insulin I to II in the islets could suggest a diabetic condition.     

A dual enzymatic-biosensor for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages of diabetic mice: evaluation of the diabetes-accelerated atherosclerosis risk

In this paper, a novel dual enzymatic-biosensor is described for simultaneous determination of glucose and cholesterol in serum and peritoneal macrophages (PMs) of diabetic mice to evaluate the risk of diabetes-accelerated atherosclerosis. The biosensor was constructed by a three-step method. First, a poly-thionine (PTH) film was assembled on the surface of glassy carbon electrode by cyclic voltammetric electropolymerization of thionine, which serves as an electron transfer mediator (ETM). Second, gold nanoparticles (GNPs) were covered on the surface of PTH facilitating the electron transfer between glucose oxidase (GOx), cholesterol oxidase (ChOx) and electrode. Finally, the enzymes, GOx, cholesterol esterase (ChE), and ChOx, were covalently attached to the PTH layer through a chitosan (CH) linker. The PTH coupled with GNPs provides good selectivity, high sensitivity and little crosstalk for the dual enzymatic-biosensor. The developed biosensor had good electrocatalytic activity toward the oxidations of glucose and cholesterol, exhibiting a linear range from 0.008 mM to 6.0 mM for glucose with a detection limit of 2.0 μM, and a linear range from 0.002 mM to 1.0 mM for cholesterol with a detection limit of 0.6 μM. The results of the diabetic mice demonstrated that the cholesterol level did not change obviously with the increase of glucose level in serum, while the cholesterol level was induced with the increase of the glucose level in PMs. Previous studies have shown that the large accumulation of cholesterol in macrophage could lead to macrophage foam cell formation, which is the hallmark of early atherosclerosis. This study provides useful further evidences for the development of diabetes-accelerated atherosclerosis.     

Bioartificial Organs I: Silica Gel Encapsulated Pancreatic Islets for the Treatment of Diabetes Mellitus

The primary objective of this work is to evaluate the potential of silica gel encapsulated pancreatic islets of Langerhans, or islet tissue, as a means by which insulin secretory capacity might be restored to individuals with insulin dependent, type 1, diabetes mellitus. The encapsulation material under investigation is comprised of sol-gel derived silica ceramic that hardens under conditions of pH, salinity, and temperature that are not harmful to living cells and organisms. Preliminary efficacy has been demonstrated by measurement of insulin secretory response of silica gel encapsulated pancreatic isletsin vitro and blood sugar levels of nonobese diabetic micein vivo.     

Effect of the combination of metformin and fenofibrate on glucose homeostasis in diabetic Goto-Kakizaki rats

Metformin has been reported to increase the expression of the glucagon-like peptide-1 (GLP-1) receptor in pancreatic beta cells in a peroxisome proliferator-activated receptor (PPAR)-α-dependent manner. We investigated whether a PPARα agonist, fenofibrate, exhibits an additive or synergistic effect on glucose metabolism, independent of its lipid-lowering effect, when added to metformin. Non-obese diabetic Goto-Kakizaki (GK) rats were divided into four groups and treated for 28 days with metformin, fenofibrate, metformin plus fenofibrate or vehicle. The random blood glucose levels, body weights, food intake and serum lipid profiles were not significantly different among the groups. After 4 weeks, metformin, but not fenofibrate, markedly reduced the blood glucose levels during oral glucose tolerance tests, and this effect was attenuated by adding fenofibrate. Metformin increased the expression of the GLP-1 receptor in pancreatic islets, whereas fenofibrate did not. During the intraperitoneal glucose tolerance tests with the injection of a GLP-1 analog, metformin and/or fenofibrate did not alter the insulin secretory responses. In conclusion, fenofibrate did not confer any beneficial effect on glucose homeostasis but reduced metformin''s glucose-lowering activity in GK rats, thus discouraging the addition of fenofibrate to metformin to improve glycemic control.     

Topical propolis improves wound healing in patients with diabetic foot ulcer: a randomized controlled trial

In this randomized controlled trial, diabetic patients with foot ulcers (Wagner grades 1 and 2) were randomly assigned to conventional therapies for diabetic foot ulcer plus topical propolis ointment (5%; twice daily) or conventional therapies alone. The process of ulcer healing was observed during 4 weeks and compared between the two groups regarding the size, erythema, exudates, white blood cell (WBC) count and erythrocyte sedimentation rate (ESR). The process of ulcer size reduction during the four-week period of study was significantly different between the groups. However, this difference was not significant between the third and fourth weeks. There was no significant difference between two groups regarding erythema and exudate reduction as well as WBC count and ESR. Administration of topical propolis ointment in addition to the conventional treatments of diabetic foot ulcer could reduce the size of ulcers with Wagner grades 1 and 2.     

白血病骨髓移植病人全血微量元素铜的变化及其意义

为了研究白血病骨髓移植病人全血微量元素铜的变化及其意义 ,用原子吸收光谱法检测了正常对照组与白血病骨髓移植患者预处理前及移植后骨髓空虚期全血微量元素铜的含量。结果表明 ,正常对照组全血铜浓度与白血病骨髓移植患者预处理前全血铜浓度差异无显著性 (P >0 0 5 ) ,而与骨髓空虚期全血铜浓度有显著性差异 (P <0 0 5 ) ,说明白血病缓解后血铜接近正常对照组水平 ,而白血病骨髓移植患者预处理后骨髓空虚期全血铜含量高于正常对照组 ,说明预处理影响微量元素铜的代谢 ,本文就其影响机理进行了研究     

聚电解质复合物在药物控释中的应用

近年来聚电解质复合物在药物控释领域受到重视。对聚电解质复合物在响应型药物控释、细胞免疫隔离移植、多肽蛋白质药物的缓释及其基因治疗等领域的应用进行了综述。