Mannogalactoglucan from mushrooms protects pancreatic islets via restoring UPR and promotes insulin secretion in TIDM mice

Type 1 diabetes mellitus(T1DM) lacks insulin secretion due to autoimmune deficiency of pancreaticβ-cells.Protecting pancreatic islets and enhancing insulin secretion has been therapeutic approaches.Mannogalactoglucan is the main type of polysaccharide from natural mushroom,which has potential medicinal prospects.Nevertheless,the antidiabetic property of mannogalactoglucan in T1DM has not been fully elucidated.In this study,we obtained the neutral fraction of alkali-soluble Armillaria mellea polysaccharide(AAMP-N) with the structure of mannogalactoglucan from the fruiting body of A.mellea and investigated the potential therapeutic value of AAMP-N in T1DM.We demonstrated that AAMP-N lowered blood glucose and improved diabetes symptoms in T1DM mice.AAMP-N activated unfolded protein response(UPR) signaling pathway to maintain ER protein folding homeostasis and promote insulin secretion in vivo.Besides that,AAMP-N promoted insulin synthesis via upregulating the expression of transcription factors,increased Ca^(2+) signals to stimulate intracellular insulin secretory vesicle transport via activating calcium/calmodulin-dependent kinase Ⅱ(CamkⅡ) and cAMP/PKA signals,and enhanced insulin secretory vesicle fusion with the plasma membrane via vesicle-associated membrane protein 2(VAMP2).Collectively,these studies demonstrated that the therapeutic potential of AAMP-N on pancreatic islets function,indicating that mannogalactoglucan could be natural nutraceutical used for the treatment of T1DM.

Small intestinal submucosa improves islet survival and function during in vitro culture

AIM： To evaluate the recovery and function of isolated rat pancreatic islets during in vitro culture with small intestinal submucosa （SIS）. METHODS： Pancreatic islets were isolated from Wistar rats by standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and Euroficoll purification. Purified islets were cultured in plates coated with multilayer SIS （SIS-treated group） or without multilayer SIS （standard cultured group） for 7 and 14 d in standard islet culture media of RPMI 1640. After isolation and culture, islets from both experimental groups were stained with dithizone and counted. Recovery of islets was determined by the ratio of counts after the culture to the yield of islets immediately following islet isolation. Viability of islets after the culture was assessed by the glucose challenge best with low （2.7 mmol/L） and high glucose （16.7 mmol/L） solution supplemented with 50 mmol/L 3-isobutyl-1- methylxanthine （IBMX） solution. Apoptosis of islet cells after the culture was measured by relative quantification of histone-complexed DNA fragments using ELISA. RESULTS： After 7 or 14 d of in vitro tissue culture, the recovery of islets in SIS-treated group was significantly higher than that cultured in plates without SIS coating. The recovery of islets in SIS-treated group was about twice more than that of in the control group. In SIS treated group, there was no significant difference in the recovery of islets between short- and long-term periods of culture （95.8±1.0% vs 90.8±1.5%, P〉0.05）. When incubated with high glucose （16.7 mmol/L） solution, insulin secretion in SIS-treated group showed a higher increase than that in control group after 14 d of culture （20.7±1.1 mU/L vs 11.8±1.1 mU/L, P〈0.05）. When islets were placed in high glucose solution containing IBMX, stimulated insulin secretion was higher in SlS-treated group than in control group. Calculated stimulation index of SlS-treated group was about 23 times of control group. In addition, the stimulation index of SlS-treated group remained constant regardless of short- and long- term periods of culture （9.5±0.2 vs 10.2±1.2, P〉0.05）. Much less apoptosis of islet cells occurred in SlS-treated group than in control group after the culture. CONCLUSION： Co-culture of isolated rat islets with native sheet-like SIS might build an extracellular matrix for islets and provide possible biotrophic and growth factors that promote the recovery and subsequent function of islets.

Review of experimental attempts of islet allotransplantation in rodents:Parameters involved and viability of the procedure

The purpose of the present study was to organize the parameters involved in experimental allotransplantation in rodents to elaborate the most suitable model to supply the scarcity of islet donors. We used the PubMed database to systematically search for published articles containing the keywords “rodent islet transplantation” to review. We included studies that involved allotransplantation experiments with rodents’ islets, and we reviewed the reference lists from the eligible publications that were retrieved. We excluded articles related to isotransplantation, autotransplantation and xenotransplantation, i.e., transplantation in other species. A total of 25 studies related to allotransplantation were selected for systematic review based on their relevance and updated data. Allotransplantation in rodents is promising and continues to develop. Survival rates of allografts have increased with the discovery of new immunosuppressive drugs and the use of different graft sites. These successes suggest that islet transplantation is a promising method to overcome the scarcity of islet donors and advance the treatment options for type 1 diabetes.

小鼠Islet-1基因慢病毒表达载体的构建及其诱导C_3H_(10)T1/2细胞向心肌样细胞特异性分化

目的研究Islet-1对干细胞分化的影响。方法用PCR钓取目的基因,将目的基因与pLenO-WPI载体连接,选取阳性质粒,与辅助质粒共同感染293T细胞生产出慢病毒载体。感染C3H10T1/2细胞,实时荧光定量PCR及Western blot检测Islet-1和心肌、肝脏、骨骼及神经各系统相关标志物的表达,免疫荧光检测心肌肌钙蛋白T(cTnT)表达部位。结果 PCR及测序显示目的片段正确插入,实验组有Islet-1表达;心肌早期发育相关基因GATA-4、MEF2C、NKx2.5在检测到荧光蛋白1周后升高,2周到达高峰,3周后可检测到心肌特异性蛋白cTnT(0.582±0.0576),其时序性表达呈随时间增强趋势;cTnT表达于胞质;肝脏系统特异性标志AFP及ALB、骨骼系统特异性标志BGP及BALP、神经系统特异性标志Nestin及GFAP均未表达。结论 Islet-1具有特异性促进干细胞向心肌样细胞分化的作用。

小鼠Islet-1基因RNAi慢病毒载体的构建

目的构建高效沉默小鼠Islet-1基因的慢病毒载体。方法针对小鼠Islet-1基因设计3个RNAi靶序列,合成相应的短发卡RNA(shRNA)寡核苷酸序列(oligo):Sh1、Sh2、Sh3,分别插入经酶切后的PLVTHM载体。经PCR和测序方法筛选阳性克隆,抽提阳性克隆质粒经大肠埃希菌扩增后,与其辅助包装质粒共同感染293T细胞制备慢病毒载体,利用斑形成试验测定病毒滴度。感染C3H10T1/2细胞株,以流式细胞仪检测其感染效率、荧光定量PCR检测其干扰效率。结果与正常C3H10T1/2细胞比较,测序及PCR结果显示目的片段插入正确;病毒滴度值为3.87×108TU/ml;慢病毒载体对C3H10T1/2细胞感染效率达90.36%;3个靶点(抑制效率分别为76.8%5、5.1%和11.7%)均有干扰效果,与正常细胞比较,Sh1靶点干扰效果最为显著(76.8%,P<0.05)。结论成功构建高效沉默Islet-1基因慢病毒载体。

Islet-1在乙酰化调控网络中特异性辅助C3H10T1/2细胞向心肌样细胞分化

目的筛选并分析转染Islet-1慢病毒载体的C3H10T1/2细胞转化为心肌样细胞过程中与Islet-1相互作用的组蛋白乙酰化酶(HATs)和组蛋白去乙酰化酶(HDACs),明确Islet-1在C3H10T1/2细胞分化为心肌样细胞乙酰化调控网络中的关键枢纽作用。方法培养转染Islet-1慢病毒载体的C3H10T1/2细胞,观察细胞形态。免疫荧光和免疫印迹检测Islet-1的表达部位和最高表达时间点。免疫共沉淀与Islet-1结合的蛋白。免疫印迹验证Islet-1相互作用的HATs和HDACs。结果诱导组细胞形态出现心肌样细胞改变。各组Islet-1主要在胞质表达。诱导组Islet-1表达量在诱导后3周最高(0.782±0.015)。诱导组Islet-1表达量显著高于空白对照组和C3H10组(分别为0.819±0.026,0.127±0.006和0.126±0.001)(P<0.05),免疫共沉淀技术可行。与Islet-1相互作用的HATs和HDACs有GCN5、P300/CBP和HDAC4。结论 Islet-1与GCN5、P300/CBP和HDAC4相互作用特异性辅助C3H10T1/2细胞向心肌样细胞分化。

Encapsulated islets transplantation: Past, present and future

Islet transplantation could become an ideal treatment for severe diabetes to prevent hypoglycemia shock and irreversible diabetic complications, once some of the major and unresolved obstacles are overcome, including limited donor supplies and side effects caused by permanent immunosuppressant use. Approximately 30 years ago, some groups succeeded in improving the blood glucose of diabetic animals by transplanting encapsulated islets with semi-permeable membranes consisting of polymer. A semi-permeable membrane protects both the inner islets from mechanical stress and the recipient’s immune system (both cellular and humoral immunities), while allowing bidirectional diffusion of nutrients, oxygen, glucose, hormones and wastes, i.e., immune-isolation. This device, which enables immune-isolation, is called encapsulated islets or bio-artificial pancreas. Encapsulation with a semipermeable membrane can provide some advantages: (1) this device protects transplanted cells from the recipient’s immunity even if the xenogeneic islets (from large animals such as pig) or insulin-producing cells are derived from cells that have the potential for differentiation (some kinds of stem cells). In other words, the encapsulation technique can resolve the problem of limited donor supplies; and (2) encapsulation can reduce or prevent chronic administration of immunosuppressants and, therefore, important side effects otherwise induced by immunosuppressants. And now, many novel encapsulated islet systems have been developed and are being prepared for testing in a clinical setting.

Effects of human umbilical cord serum on proliferation and insulin content of human fetal islet-like cell clusters

BACKGROUND: Type 1 diabets is an autoimmune disease caused by the destruction of pancreatic β-cell with an in- creased incidence worldwide in the closing decades of the 20th century. This study was to investigate the effects of human umbilical cord serum (UCS) on the proliferation and function of human fetal islet-like cell clusters (ICCs) in vitro. METHODS: Eight fresh pancreatic glands obtained after in- duction of labor with water bag were mildly exposed to col- lagenase V, and the digested cells were cultured in a RPMI- 1640 medium plus 10% pooled UCS or fetal calf serum (FCS) to permit cells attachment and outgrowth of ICCs. RESULTS: In 8 consecutively explanted glands, develop- ment and proliferation of ICCs were observed. In the pre- sence of FCS, the outgrowth of ICC took place on the top of a flbroblast monocellular layer. UCS affected less growth of fibroblasts and increased the formation of ICCs about four-fold compared with explants from the same glands maintained in FCS. In both UCS and FCS, the insulin con- tent of the medium was variable to a certain extent and progressively declined from day 2 to day 6. Dithizone- stained ICCs in UCS suggested that most cell clusters were islet cells ( β-cells), and the purity of islets was estimated 80%-90%. The ultrastructure of the cultured cells showed a large number of granule-containing cells, most of which were identified as β-cells. CONCLUSION: We conclude that in comparison with ex- plants with FCS, the yield of ICCs and purification of islet cells are markedly increased by UCS and may facilitate the proliferation of pancreatic β-cells intended for islet trans- plantation.

Preparation of Recombinant Islet Cell Autoantigen 69 kD Fusion Protein

To get recombinant antigen (Is/et Cell Autoantigen 69)ICA69 which was expressed in Escherichia coli strains (E.coli) by means of the gene engineering technique so that it can be used for early diagnosis of and screening in type Ⅰ diabetes mellitus, the cDNA fragment of human ICA69 was amplified by PCR, and then cloned into pSPORT 1 vector. After DNA sequencing, it was inserted into pGEX-2T between the sites of EcoR Ⅰ and Sma Ⅰ, then recombinant plasmid p2T-ICA69 was constructed and introduced into E.coli. The GST-ICA69 fusion protein was expressed by the induction of IPTG. The recombinant ICA69 proteins were used to detect the antibodies against hICA69 in 100 healthy subjects and type Ⅰ diabetic serum by the use of indirect ELISA. The sequence analysis showed that the amplified fragments contained 1449 bp, encoded 483 amino acids, and had been correctly inserted into pGEX-2T vector. The recombinant proteins expressed in the prokaryotic cells had immunogenicity and could be used to detect antibodies against ICA69 in type Ⅰ diabetic serum. Finally it can be concluded in this paper that the expression products obtained by the method of gene engineering are recombinant ICA69 antigen and may be used to improve the forecast rate and the diagnostic rate of type Ⅰ diabetes in combination with other tests.

Intra-islet endothelial cell and β-cell crosstalk: Implication for islet cell transplantation

The intra-islet microvasculature is a critical interface between the blood and islet endocrine cells governing a number of cellular and pathophysiological processes associated with the pancreatic tissue. A growing body of evidence indicates a strong functional and physical interdependency of β-cells with endothelial cells(ECs), the building blocks of islet microvasculature. Intra-islet ECs, actively regulate vascular permeability and appear to play a role in fine-tuning blood glucose sensing and regulation. These cells also tend to behave as "guardians", controlling the expression and movement of a number of important immune mediators, thereby strongly contributing to the physiology of islets. This review will focus on the molecular signalling and crosstalk between the intra-islet ECs and β-cells and how their relationship can be a potential target for intervention strategies in islet pathology and islet transplantation.

Islet autotransplantation in a patient with hypercoagulable disorder

Total pancreatectomy and islet auto transplantation is a good option for chronic pancreatitis patients who suffer from significant pain, poor quality of life, and the potential of type 3C diabetes and pancreatic cancer. Portal vein thrombosis is the most feared complication of the surgery and chances are increased if the patient has a hypercoagulable disorder. We present a challenging case of islet auto transplantation from our institution. A 29-year-old woman with plasminogen activator inhibitor-4G/4G variant and a clinical history of venous thrombosis was successfully managed with a precise peri- and postoperative anticoagulation protocol. In this paper we discuss the anti-coagulation protocol for safely and successfully caring out islet transplantation and associated risks and benefits.

Islet-1基因变异体的发现及其在神经干细胞内的表达

目的:构建大鼠Islet-1基因逆转录病毒表达载体,利用此载体将Islet-1基因转导入神经干细胞(NSC)内。方法:利用RT-PCR技术钓取大鼠Islet-1基因,将其插入到逆转录病毒载体plEGFP-C1中,利用包装细胞PA317将Islet-1基因转导入NSC内,观察Islet-1基因在NSC内的表达。结果:经PCR、酶切及荧光检测等证实,成功构建了plEGFP-C1-Islet-1表达载体,并应用免疫组化技术证明Islet-1在NSC内有表达。在实验中首次发现了Islet-1基因的变异体。结论:重组Islet-1基因逆转录病毒载体的构建为进一步探讨Islet-1基因是否参与NSC向运动神经元分化及其可能的作用机制奠定了坚实的实验基础。

Islet1基因与先天性心脏病的关系

先天性心脏病是最常见的出生缺陷性疾病,严重危害婴幼儿健康。心脏发育过程中控制发育的基因突变是先天性心脏病的常见原因。近年来,越来越多的研究表明Islet1基因在心脏早期发育过程中起着至关重要的作用,该基因的突变或多态性可导致先天性心脏病的发生,现就近年来Islet1基因与先天性心脏病关系的研究进展做一综述。

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by Clostridium histolyticum

Clostridium histolyticum is used for production of several proteolytic enzymes such as elastase, neutral proteases, clostripain and in particular collagenase. Besides industrial applications, collagenase has been indispensable for medical purposes including isolation of pancreatic islets for diabetes treatment. The aim of this study was to optimize the method for production and partial purification of a new collagenase blend and to test its suitability for successful pancreatic islets isolation in a rat model. Bacterial strain of C. histolyticum was sequenced for presence of the collagenase genes. Different fermentation conditions were tested and the process of collagenase extraction was modified and optimized. Samples of collagenases were taken for western blot detection, activity assessment, and ability for dissociation of pancreatic tissue. Findings indicate that concentrated trypton growth medium with pepton was the most suitable for Clostridium growth and collagenase production. Whole genome sequencing revealed two genes for collagenase and also gene for clostripain. Western blot specific detection helped to select useful production modifications. Following these modifications was also improved the yield, morphology and in vitro function of intact pancreatic islets which were finally comparable or better than those achieved using standard blends of collagenase. The results support the use of the new collagenase blend for islet isolation giving thus the opportunity to choose an alternative product. Our next steps would lead to further enzyme purification through scaling up of the production method for a wider use.

A pancreatic player in dementia:pathological role for islet amyloid polypeptide accumulation in the brain

Type 2 diabetes mellitus patients have a markedly higher risk of developing dementia.While multiple factors contribute to this predisposition,one of these involves the increased secretion of amylin,or islet amyloid polypeptide,that accompanies the pathophysiology of type 2 diabetes mellitus.Islet amyloid polypeptide accumulation has undoubtedly been implicated in various forms of dementia,including Alzheimer’s disease and vascular dementia,but the exact mechanisms underlying islet amyloid polypeptide’s causative role in dementia are unclear.In this review,we have summarized the literature supporting the various mechanisms by which islet amyloid polypeptide accumulation may cause neuronal damage,ultimately leading to the clinical symptoms of dementia.We discuss the evidence for islet amyloid polypeptide deposition in the brain,islet amyloid polypeptide interaction with other amyloids implicated in neurodegeneration,neuroinflammation caused by islet amyloid polypeptide deposition,vascular damage induced by islet amyloid polypeptide accumulation,and islet amyloid polypeptide-induced cytotoxicity.There are very few therapies approved for the treatment of dementia,and of these,clinical responses have been controversial at best.Therefore,investigating new,targetable pathways is vital for identifying novel therapeutic strategies for treating dementia.As such,we conclude this review by discussing islet amyloid polypeptide accumulation as a potential therapeutic target not only in treating type 2 diabetes mellitus but as a future target in treating or even preventing dementia associated with type 2 diabetes mellitus.

25-Hydroxyvitamin D Is Associated with Islet Homeostasis in Type-2 Diabetic Patients with Abdominal Obesity

Objective Isletαcells input is essential for insulin secretion fromβcells.The present study aims to investigate the association between 25-hydroxyvitamin D[25(OH)D]and islet function homeostasis in type-2 diabetes(T2D)patients.Methods A total of 4670 T2D patients from seven communities in Shanghai,China were enrolled.The anthropometric indices,biochemical parameters,serum 25(OH)D,and islet function[including C-peptide(C-p)and glucagon]were measured.Results The fasting plasma glucose(FPG),glycated hemoglobin(HbA1c),glucagon,and C-p levels exhibited a significantly decreasing trend in T2D patients as the 25(OH)D levels increased.Next,the population was divided into two groups:abdominal obesity and non-abdominal obesity groups.After adjustment,the 25(OH)D level was found to be associated with HbA1c,glucagon,and homeostasis model assessment ofβ(HOMA-β)in the non-abdominal obesity group.There was a significant relationship between 25(OH)D and HbA1c,glucagon,HOMA-IR,baseline insulin or C-p in the abdominal obesity group.In the abdominal obesity group,the ordinary least squares(OLS)regression and quantile regression revealed that 25(OH)D was obviously associated with glucagon and fasting C-p levels.In the abdominal obesity group,the moderate analysis revealed a significant interaction effect of 25(OH)D and glucagon on C-p(P=0.0124).Furthermore,the conditional indirect effect of 25(OH)D on the glucagon/C-p ratio was significantly lower at 1 standard deviation(SD)below the mean(P=0.0002),and lower at the mean of the course of diabetes(P=0.0007).Conclusion 25(OH)D was found to be negatively correlated to glucagon and C-p in T2D patients with abdominal obesity.The 25(OH)D influenced C-p in part by influencing glucagon.The effect of 25(OH)D on the glucagon/C-p ratio in T2D patients with abdominal obesity,in terms of islet homeostasis,is influenced by the course of diabetes.

Glucose-stimulated insulin secretion in isolated pancreatic islets: Multiphysics FEM model calculations compared to results of perifusion experiments with human islets

Because insulin released by the β-cells of pancreatic islets is the main regulator of glucose levels, the quantitative modeling of their glucose-stimulated insulin secretion is of obvious interest not only to improve our understanding of the processes involved, but also to allow better assessment of β -cell function in diabetic patients or islet transplant recipients as well as the development of improved artificial or bioartificial pancreas devices. We have recently developed a general, local concentrations-based multiphysics computational model of insulin secretion in avascular pancreatic islets that can be used to calculate insulin secretion for arbitrary geometries of cultured, perifused, transplanted, or encapsulated islets in response to various glucose profiles. Here, experimental results obtained from two different dynamic glucose-stimulated insulin release (GSIR) perifusion studies performed by us following standard procedures are compared to those calculated by the model. Such perifusion studies allow the quantitative assessment of insulin release kinetics under fully controllable experimental conditions of varying external concentrations of glucose, oxygen, or other compounds of interest, and can provide an informative assessment of islet quality and function. The time-profile of the insulin secretion calculated by the model was in good agree- ment with the experimental results obtained with isolated human islets. Detailed spatial distributions of glucose, oxygen, and insulin were calculated and are presented to provide a quantitative visualization of various important aspects of the insulin secretion dynamics in perifused islets.

Islet-1诱导C_3H_(10)T1/2细胞向心肌样细胞分化过程中对心肌特异蛋白基因上组蛋白乙酰化的促进作用

目的研究Islet-1诱导C3H10T1/2细胞定向分化为心肌样细胞过程中表达增加的心肌特异蛋白基因GATA4、NKX2.5、MEF2c上组蛋白乙酰化水平的变化情况。方法 Islet-1基因慢病毒感染小鼠骨髓间充质干细胞C3H10,Western blot检测转染后乙酰化的组蛋白H3表达情况,逆转录及实时荧光定量PCR时序性检测出心肌特异性蛋白基因GATA4、NKX2.5、MEF2c的mRNA表达达高峰的时间点,采用染色质免疫共沉淀技术(chromatin immunoprecipitation,CHIP),用CHIP级乙酰化H3抗体免疫沉淀与其所结合的DNA,采用实时荧光定量PCR扩增抗体所富集的心肌特异性蛋白基因GATA4、NKX2.5、MEF2c启动子区域的DNA量,比较感染组和未感染组与抗体所结合的上述3种心肌特异蛋白基因的表达情况。结果感染高表达Islet-1的C3H10T1/2细胞组蛋白H3的乙酰化水平较未感染组升高3.04倍(P<0.05),在心肌特异性蛋白基因GATA4、NKX2.5、MEF2C的mRNA表达达高峰的2周时间点,乙酰化的H3抗体所结合的心肌特异性蛋白基因GATA4、NKX2.5、MEF2c与未转染Islet-1的细胞组相比较,表达增加(P<0.05)。结论高表达Is-let-1之后C3H10T1/2细胞定向分化为心肌样细胞过程中表达增加的心肌特异蛋白基因上组蛋白乙酰化水平增高。

Characterization of Islets from Chronic Calcific Pancreatitis Patients of Tropical Region with Distinct Phenotype

Background and Objective: Islet autotransplantation is performed to preserve endocrine function in patients undergoing pancreatic resections for painful chronic pancreatitis. We characterized islets isolated from chronic pancreatitis patients (CP) of tropical region. Patients and Methods: Pancreatic tissues were obtained from CP patients with and without diabetes undergoing pancreatic resections (n = 35) and brain-dead multi organ donors (n = 6;considered as controls). Islets isolated were assessed for yield, purity, viability and in vitro islet function (Glucose stimulated insulin release, GSIR) as per standard protocols. Results: Islets from CP patients without diabetes were similar to controls in yield (control 4120 - 6100 IE/g, CP 3550 - 5660 IE/g), purity (control 78% ± 12%, CP 70% ± 8.2%) and viability (control 85% ± 8%, CP 81% ± 10%) and islets from CP patients with diabetes showed decreases in yield (3002 - 2300 IE/g), purity (61% ± 16%) and viability (62% ± 21%). Islets measuring 50 - 200 μ were similar in abundance in controls (94.74% ± 3.2%) and CP patients with and without diabetes, 86.31% ± 4.9%, 91.03% ± 3.8%. GSIR of islets from CP patients and controls were similar at 5.5 mM glucose (2.8 - 3.1 μU/ml). However, GSIR at 16.5 mM glucose was decreased in CP patients (control 18.5 ± 0.6, CP without diabetes 11.8 ± 0.3, CP with diabetes 4.3 ± 0.3 μU/ml). Conclusion: Our results demonstrate suitability of islets isolated from CP patients of tropical region for autotransplantation.