Glucose-stimulated Insulin Secretion Test Research Articles

The effect of diabetes mellitus on differentiation of mesenchymal stem cells into insulin-producing cells

BackgroundDiabetes mellitus (DM) is a global epidemic with increasing incidences. DM is a metabolic disease associated with chronic hyperglycemia. Aside from conventional treatments, there is no clinically approved cure for DM up till now. Differentiating mesenchymal stem cells (MSCs) into insulin-producing cells (IPCs) is a promising approach for curing DM. Our study was conducted to investigate the effect of DM on MSCs differentiation into IPCs in vivo and in vitro.MethodsWe isolated adipose-derived mesenchymal stem cells (Ad-MSCs) from the epididymal fat of normal and STZ-induced diabetic Sprague–Dawley male rats. Afterwards, the in vitro differentiation of normal-Ad-MSCs (N-Ad-MSCs) and diabetic-Ad-MSCs (DM-Ad-MSCs) into IPCs was compared morphologically then through determining the gene expression of β-cell markers including neurogenin-3 (Ngn-3), homeobox protein (Nkx6.1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin-1 (Ins-1) and eventually, through performing glucose-stimulated insulin secretion test (GSIS). Finally, the therapeutic potential of N-Ad-MSCs and DM-Ad-MSCs transplantation was compared in vivo in STZ-induced diabetic animals.ResultsOur results showed no significant difference in the characteristics of N-Ad-MSCs and DM-Ad-MSCs. However, we demonstrated a significant difference in their abilities to differentiate into IPCs in vitro morphologically in addition to β-cell markers expression, and functional assessment via GSIS test. Furthermore, the abilities of both Ad-MSCs to control hyperglycemia in diabetic rats in vivo was assessed through measuring fasting blood glucose (FBGs), body weight (BW), histopathological examination of both pancreas and liver and immunoexpression of insulin in pancreata of study groups.ConclusionOur findings reveal the effectiveness of N-Ad-MSCs in differentiating into IPCs in vitro and controlling the hyperglycemia of STZ-induced diabetic rats in vivo compared to DM-Ad-MSCs.Graphical

Yiqi Huazhuo decoction increases insulin secretion in type 2 diabetic rats by regulating the pancreatic GPR40-IP3R-1 signaling pathway.

Objective: Yiqi Huazhuo Decoction (YD) reduces blood glucose, glycated hemoglobin, body weight, and insulin resistance in patients with type 2 diabetes mellitus (T2DM), but its exact mechanisms are unknown. This study investigated the therapeutic effects and mechanisms of YD on impaired insulin secretion in T2DM rats. Methods: T2DM rats were randomized to the model, YD-lo (15mg/kg/d YD, 10weeks), YD-hi (30mg/kg/d YD, 10 weeks), positive drug (TAK-875), and healthy control groups. The rats underwent an oral glucose tolerance test (OGTT), glucose-stimulated insulin secretion (GSIS) test, and serum lipid measurements. High-fat and high-glucose-injured RIN-m5f cells were treated with YD (30 or 150mg/mL) for 48h. GPR40 and IP3R-1 expression levels were determined by immunofluorescence, qRT-PCR, and western blot. Results: Compared with the model group, the OGTT area under the curve (AUC) in the YD-hi group was decreased by 26.7%, the insulin release test (IRT) AUC in the YD-hi group was increased by 45.9%, and the GSIS AUC was increased by 33.9% (p < 0.05). Compared with the model cells, the insulin secretion after glucose stimulation in the YD-hi group was increased by 24.5%, similar to the TAK-875 group (23.1%) (p > 0.05). GPR40 and IP3R-1 mRNA in the model cells were decreased by 49.5% and 51.2% compared with the control cells (p < 0.05). In the YD-hi group, GPR40 and IP3R-1 mRNA levels were increased by 58.1% and 39.3% (p < 0.05), similar to the TAK-875 group. The changes in protein expression were similar to mRNA. Conclusion: YD promotes insulin secretion from pancreatic islet β-cell in T2DM rats by regulating the GPR40-IP3R-1 pathway, thereby reducing blood glucose.

Isolation and Purification of Human Pancreatic Islets.

Successful islet isolation is the key to islet transplantation in diabetic patients. However, islet isolation is a technically complex and time-consuming manual process. Optimizing the islet isolation process can improve islet yield and quality, reduce operators, and thus reduce costs.The isolation and purification of human islets include pancreas acquisition and preservation, pancreas digestion, islet purification, islet culture, and islet quality identification. Briefly, after the duodenum was removed, the pancreas was trimmed, the main pancreatic duct was intubated at the distal end of the pancreatic head, collagenase was injected into the pancreatic duct, and the perfused pancreatic tissue was cut and then digested in a Ricordi chamber. A digestion temperature of 37°C was continuously used to assess the number of samples and the integrity of the lysed and released islets. At the end of the digestion process, collect the digested tissue in a 500 mL centrifuge tube prefilled with 25mL of cold (4°C) human serum albumin and centrifuge twice at 150g for 3min. After mixing with UW solution as islet storage solution, put it on ice (shake occasionally to prevent clumping) after 30min. Digested pancreatic tissue was centrifuged at 2200rpm for 5min in a COBE 2991 cell processor to isolate islets from exocrine tissue using a continuous density gradient. The purified islet fractions were washed twice in HBSS supplemented with 10% human serum albumin and finally collected in CMRL1066 medium supplemented with the corresponding liquid. The purity of purified islets was calculated by DTZ staining, the survival rate of islets was calculated by FDA/PI staining, and islet function was determined by in vitro glucose-stimulated insulin secretion test.

Mutated lncRNA increase the risk of type 2 diabetes by promoting β cell dysfunction and insulin resistance

Islet β cell dysfunction and insulin resistance are the main pathogenesis of type 2 diabetes (T2D), but the mechanism remains unclear. Here we identify a rs3819316 C > T mutation in lncRNA Reg1cp mainly expressed in islets associated with an increased risk of T2D. Analyses in 16,113 Chinese adults reveal that Mut-Reg1cp individuals had higher incidence of T2D and presented impaired insulin secretion as well as increased insulin resistance. Mice with islet β cell specific Mut-Reg1cp knock-in have more severe β cell dysfunction and insulin resistance. Mass spectrometry assay of proteins after RNA pulldown demonstrate that Mut-Reg1cp directly binds to polypyrimidine tract binding protein 1 (PTBP1), further immunofluorescence staining, western blot analysis, qPCR analysis and glucose stimulated insulin secretion test reveal that Mut-Reg1cp disrupts the stabilization of insulin mRNA by inhibiting the phosphorylation of PTBP1 in β cells. Furthermore, islet derived exosomes transfer Mut-Reg1cp into peripheral tissue, which then promote insulin resistance by inhibiting AdipoR1 translation and adiponectin signaling. Our findings identify a novel mutation in lncRNA involved in the pathogenesis of T2D, and reveal a new mechanism for the development of T2D.

Targeting ferroptosis with miR-144-3p to attenuate pancreatic β cells dysfunction via regulating USP22/SIRT1 in type 2 diabetes

BackgroundRecently, ferroptosis has been implicated in the pathologic process of several diseases including type 2 diabetes mellitus (T2DM). However, molecular mechanisms underlying ferroptosis in T2DM remain obscure.MethodsTwenty four mice were included in this study. T2DM model mice were established by a high-fat diet combined with streptozotocin injection. INS-1 cells were stimulated with high glucose (HG). Cell viability was detected by CCK-8 kit. The levels of GSH, MDA, iron, and lipid ROS, and SOD activity, were detected by the corresponding kits. The interaction between miR-144-3p and USP22 was validated by dual-luciferase reporter assay. The relationship between USP22 and its substrate was verified using Co-IP and ubiquitination assays. The mRNA and protein expressions were examined by RT-qPCR and western blot, respectively. The functions of β cells in vitro and in vivo were evaluated glucose-stimulated insulin secretion test and HOMA-β, respectively.ResultsFerroptosis occurred in the pancreas of T2DM mice and HG-induced INS-1 cells. Silencing miR-144-3p blocked the effect of HG on the cell viability and accumulation of lipid peroxides, thereby improving the insulin secretion in INS-1 cells. Mechanistically, USP22 is a direct target of miR-144-3p, which could stabilize SIRT1 expression, thereby suppressing ferroptosis. Overexpressing USP22 attenuated deleterious roles of HG in INS-1 cells; but its roles were reversed by up-regulating miR-144-3p. In vivo study demonstrated that miR-144-3p antagomir exerted an anti-hyperglycemic effect and regulated the ferroptosis-related proteins in the pancreas.ConclusionThe up-regulation of miR-144-3p suppressed USP22/SIRT1 to induce ferroptosis, which causes pancreatic β cells dysfunction, thereby promoting T2DM development.

Effects of Zinc Deficiency in Male Mice on Glucose Metabolism of Male Offspring.

As an essential metal, zinc is central to insulin biosynthesis and energy metabolism. Zinc can not only maintain the activity of insulin, but also has insulin-like effect. When zinc is sufficient, the body needs less insulin. Zinc can correct abnormal glucose tolerance and even replace insulin to improve glucose metabolism disorder in diabetic rats. However, the effect of paternal zinc deficiency on glucose metabolism of offspring is still unclear. In the present study, sixteen 8-week-old male mice were randomly allocated into low-zinc group and control group (8 mice in each group), which were fed with low zinc and standard diet for 6 weeks, respectively. The mice were mated with female mice fed with standard diet to get the first generation of mice (F1) to explore the effect zinc deficiency on the glucose metabolism of offspring. Glucose tolerance, insulin sensitivity and insulin secretion in mice were determined by oral glucose tolerance test (OGTT), insulin tolerance test (ITT) and glucose stimulated insulin secretion test (GSIS), respectively. Compared with the control group, the fasting blood glucose levels of F1 generation male mice in the low zinc group increased at 15 and 30 min after glucose injection. The blood glucose of F1 generation male mice in the low zinc group of mice decreased at 60, 90, 120, 180, 240 min after insulin injection. Compared with the control group, serum insulin of F1 generation male mice in the low zinc group decreased at 15 min after glucose injection (all p values <0.05). However F1 female mice in the low zinc group did not show abnormal glucose metabolism. Marginal zinc deficiency in male mice can cause abnormal glucose homeostasis in male offspring, but not in female offspring.

High Fat Diet and High Fructose Diet‐fed Male and Female Liv‐ARKO Mice Displayed Impair Glucose‐Stimulated Insulin Secretion

Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome (PCOS) affects up to 10% of reproductive‐age adult women. The liver plays an essential component in the metabolism of insulin and androgen signaling. Hyperandrogenism in females can also increase predisposition to insulin resistance. In our study, we placed female and male liver androgen receptor knock‐out mice (Liv‐ARKO) on high fructose diets (HFrD) and high fat diets (HFD) to determine if Liv‐ARKO mice demonstrated altered glucose‐stimulated insulin secretion on special diets compared to a control diet.In this study, female and male Liv‐ARKO mice were placed on three distinct diets: Control (Research Diets Inc), High Fat (HFD), and High Fructose (HFrD). After one month and two months on these diets, Glucose‐Stimulated Insulin Secretion tests were performed: each group was injected with a 20% glucose solution at 2g of glucose/kg body mass and blood samples were taken via tail milking at 0 minutes, 15 minutes, and 30 minutes. Blood samples were immediately centrifuged and serum supernatant was extracted for further analysis via Sigma Insulin Assay.In the current study, after two months, female mice on HFrD demonstrated lower basal insulin levels compared to female mice fed a control diet. Interestingly, mice on a HFD showed insulin levels that were lower than the basal levels of both control mice and HFrD mice. Furthermore, compared to the control mice, the HFD mice demonstrated lower insulin levels at both basal level and 30 minutes post‐glucose administration.It was hypothesized that HFD and HFrD‐fed Liv‐ARKO mice would impair glucose‐stimulated insulin secretion, which our current study supports. These data suggest that diets high in fructose and fat both blunt insulin secretion following a meal, and to a greater extent in high fat diets. This dysfunction may ultimately play a role in the development of insulin resistance. More research is necessary in order to further investigate the effects of high fat and high fructose diets on glucose‐stimulated insulin secretion and consequent development of insulin resistance.

The 26RFa (QRFP)/GPR103 neuropeptidergic system in mice relays insulin signalling into the brain to regulate glucose homeostasis.

26RFa (pyroglutamilated RFamide peptide [QRFP]) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localised in the hypothalamus. In this study we investigated whether 26RFa neurons are involved in the hypothalamic regulation of glucose homeostasis. 26Rfa+/+, 26Rfa-/- and insulin-deficient male C57Bl/6J mice were used in this study. Mice received an acute intracerebroventricular (i.c.v.) injection of 26RFa, insulin or the 26RFa receptor (GPR103) antagonist 25e and were subjected to IPGTTs, insulin tolerance tests, acute glucose-stimulated insulin secretion tests and pyruvate tolerance tests (PTTs). Secretion of 26RFa by hypothalamic explants after incubation with glucose, leptin or insulin was assessed. Expression and quantification of the genes encoding 26RFa, agouti-related protein, the insulin receptor and GPR103 were evaluated by quantitative reverse transcription PCR and RNAscope in situ hybridisation. Our data indicate that i.c.v.-injected 26RFa induces a robust antihyperglycaemic effect associated with an increase in insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26Rfa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26Rfa are mainly localised in the lateral hypothalamic area, that they co-express the gene encoding the insulin receptor and that insulin induces the expression of 26Rfa in these neurons. Concurrently, the central antihyperglycaemic effect of insulin is abolished in the presence of a GPR103 antagonist and in 26RFa-deficient mice. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production. We have identified a novel mechanism in the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system, and we provide evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.

Downregulation of Kcnq1ot1 attenuates β-cell proliferation and insulin secretion via the miR-15b-5p/Ccnd1 and Ccnd2 axis.

To examine the effect of lncRNA Kcnq1ot1 on pancreatic β cells in the development of diabetes. The expression levels of Kcnq1ot1 were detected in the islets of diabetes mouse models and the serum of patients with type 2 diabetes by qRT-PCR. CCK8, Ki67 staining, immunohistochemical analyses, glucose-stimulated insulin secretion and intraperitoneal glucose tolerance test were performed to detect the effect of Kcnq1ot1 on β-cell proliferation and insulin secretion in vitro and in vivo. The relationship between Kcnq1ot1 and miR-15b-5p was predicted by bioinformatics prediction, which was confirmed by luciferase reporter assay. Kcnq1ot1 was more abundant in the pancreas. The expression of Kcnq1ot1 was decreased in the islets of db/db mice and diet-induced obese mice and in the serum of patients with type 2 diabetes. Silencing Kcnq1ot1 inhibited the β-cell proliferation concomitant with a reduction in the levels of Ccnd1 and Ccnd2. Insulin synthesis and secretion were impaired, along with the decreased expression of Ins1, Ins2, and insulin-related transcription factors. Moreover, Kcnq1ot1 knockdown in vivo reduced glucose tolerance and decreased insulin secretion, consistent with the reduction in the relative islet area and Ki67-positive β-cells detected by immunochemistry and immunofluorescence staining, respectively. Mechanistically, Kcnq1ot1 directly targeted miR-15b-5p which regulated β-cell proliferation and insulin secretion through Ccnd1 and Ccnd2. Notably, the suppression of miR-15b-5p attenuated the inhibition of Min6 proliferation and insulin production induced by Kcnq1ot1 knockdown. Kcnq1ot1 regulated β-cell proliferation and insulin secretion via the miR-15b-5p/Ccnd1 and Ccnd2 axis, which is worthy of further investigation considering its potential in diabetes treatment.

HMSCs Migrate under the Chemotaxis of CXCL-13 and Enhance Islet B Cell Activity through p-AKT Signaling Pathway in High-Glucose Environment.

As a common clinical chronic disease, the incidence of diabetes is increasing year by year. According to the latest statistics from the International Diabetes Federation, as of 2019, the global prevalence of diabetes has reached 8.3%. This study aims to investigate the effect of CXCL-13 on the migration ability of human mesenchymal stem cells (hMSCs) and to clarify the specific molecular mechanism of the protective effect of hMSCs on islet B cells. The hMSCs were cultured in high-glucose environment, and the effect of CXCL-13 on the migration ability of hMSCs was determined by Transwell experiment. After coculture of hMSCs and islet B cells, the activity of cells was detected by CCK8 assay, the expression of Ki-67 in cells was detected by RT-PCR, and the expression of P53 was detected by Western blot to investigate the effect of hMSCs on the proliferation and apoptosis of islet B cells. The effect of hMSCs on the function of islet B cells was determined by glucose stimulated insulin secretion experiment. Transwell experiment results showed that CXCL-13 could promote the migration of hMSCs to islet B cells in high-glucose environment. The results of CCK-8 showed that the cell activity in the coculture group was significantly higher than that of the other groups, and RT-PCR showed that the expression of Ki-67 was significantly increased in the coculture group of hMSCs and islet B cells. The results of Western blot showed that the expression of P53 was significantly decreased in the coculture group, and the glucose stimulated insulin secretion test showed that insulin secretion was significantly increased. It was found that after the inhibition of ATK, cell activity was significantly reduced, and apoptosis was significantly increased. Meanwhile, the expression of Ki-67 was inhibited, the expression of P-53 was significantly increased, and insulin secretion was significantly reduced. To sum up, in a high-glucose environment, CXCL-13 effectively promoted the migration of hMSCs, and hMSCs protected the activity and function of islet B cells through Akt signaling pathway.

P.169: Decellularized Cotyledons Isolated From Human Placenta Used Like a Scaffold for the Generation of a Human Bioartificial Pancreas

Background: Replacing damaged organs with biological substitutes capable of protecting the islets and facilitating vascularization is a great objective in the field of islet transplantation. A decellularized placenta includes a large number of cotyledons with a conserved vessel structure of the native organ. Our goal is to obtain a perfect decellularization protocol to generate pre-vascularized organoids by recellularizing this ECM with HUVECs and pancreatic islets. Methods: After blood removal, cotyledons were dissected from the placentas and decellularized using a bioreactor. Cell removal was assessed by histology and quantification of residual DNA. Presence of structural proteins and ECM structure were analyzed using SEM, CT scan and mass spectrometry. Recellularization protocols were conducted, with HUVECs or BOECs as endothelial cell sources, and with Ins-1E cells or rat islets as insulin secreting cell sources. Function of recellularized cotyledons was assessed in vitro with glucose stimulated insulin secretion tests (GSIS). To assess in vivo biocompatibility and function of the scaffolds, we transplanted in diabetic NSG mice. Glycaemia was measured every day to monitor normalization of blood glucose levels. Results: Our protocol led to successful decellularization, as evidenced by the absence of cells and the preserved ECM structure. Moreover, DNA quantification did not reveal any residual DNA. Quantification of GAG and hydroxyproline, and mass spectrometry analysis show that structural proteins are conserved. SEM and CT scan images revealed that the ECM structure was preserved after the decellularization protocol. Cells after recellularization showed a good vascularization after 7 days. The GSIS test shows a perfect organ response in the production of insulin. Conclusions: The decellularized cotyledon is the perfect scaffold to reproduce a prevascularized insulin-producing organ, which allows transplanted cells to survive during the peri-transplantation period.

β Cell GHS-R Regulates Insulin Secretion and Sensitivity.

Growth hormone secretagogue receptor (GHS-R) is widely known to regulate food intake and adiposity, but its role in glucose homeostasis is unclear. In this study, we investigated the expression of GHS-R in mouse pancreatic islets and its role in glycemic regulation. We used Ghsr-IRES-tauGFP mice, with Green Fluorescent Protein (GFP) as a surrogate for GHS-R, to demonstrate the GFP co-localization with insulin and glucagon expression in pancreatic islets, confirming GHS-R expression in β and α cells. We then generated β-cell-specific GHSR-deleted mice with MIP-Cre/ERT and validated that GHS-R suppression was restricted to the pancreatic islets. MIP-Cre/ERT;Ghsrf/f mice showed normal energy homeostasis with similar body weight, body composition, and indirect calorimetry profile. Interestingly, MIP-Cre/ERT;Ghsrf/f mice exhibited an impressive phenotype in glucose homeostasis. Compared to controls, MIP-Cre/ERT;Ghsrf/f mice showed lower fasting blood glucose and insulin; reduced first-phase insulin secretion during a glucose tolerance test (GTT) and glucose-stimulated insulin secretion (GSIS) test in vivo. The isolated pancreatic islets of MIP-Cre/ERT;Ghsrf/f mice also showed reduced insulin secretion during GSIS ex vivo. Further, MIP-Cre/ERT;Ghsrf/f mice exhibited improved insulin sensitivity during insulin tolerance tests (ITT). Overall, our results confirmed GHS-R expression in pancreatic β and α cells; GHS-R cell-autonomously regulated GSIS and modulated systemic insulin sensitivity. In conclusion, β cell GHS-R was an important regulator of glucose homeostasis, and GHS-R antagonists may have therapeutic potential for Type 2 Diabetes.

Long-term reversal of diabetes by subcutaneous transplantation of pancreatic islet cells and adipose-derived stem cell sheet using surface-immobilized heparin and engineered collagen scaffold

ObjectiveEsterified collagen (EC) can be functionalized with heparin to enhance islet graft stability. Growth factors secreted by human adipose-derived stem cells (hADSCs) can bind efficiently to EC-heparin (EC-Hep), which enhances...

Pancreatic Islets Aging in Old Rhesus Monkey

BackgroundType 2 diabetes is an age associated disease which characterized by insulin resistance and loss of β cell mass and function. Accumulation of senescent β cells with age probably lead to compromised capacity of insulin synthesis and secretion. Nevertheless, other studies also suggested systemic aging may impair glucose tolerance via compromised peripheral insulin sensitivity and microcirculation. So, the pathophysiological process of pancreatic β cell senescence, and the relationship between senescent β cells and diabetes are not clarified. Rhesus monkey is one of the most ideal non‐human primate models of human aging. To date, the researches on islets function of rhesus monkeys, particularly aged monkeys are so limited. So, in this study, we are aiming to explore the link between islets senescence and diabetes in aged rhesus monkeys of approximately 20 years old that may reflect the characters of humans around 80 years old.MethodsSix middle age rhesus monkeys (average 10 years old) and six old rhesus monkeys (average 20 years old) were used in this study. Blood chemistry was detected and the intravenous glucose tolerance tests (IVGTT) were carried out. After sacrifice, the pancreatic islets were isolated to evaluate β cell function, and the histopathological analysis was done on the pancreatic tissues.ResultsIn the blood chemistry, no significant differences in kidney and liver functions were observed between the two groups. The old monkeys show slight increase on the fasting blood glucose, while their fasting insulin levels were increased distinctly. In addition, the IVGTT results showed that the glucose tolerance of the old group was slightly impaired, indicating the possible occurrence of peripheral insulin resistance. In respect to pancreatic histology, the immunohistochemistry staining with anti‐insulin antibody showed that both the total area of insulin producing β cells in pancreatic sections and the single islet mass decreased a lot in old rhesus monkeys, but interestingly, a notable elevated expression of pancreatic/duodenal homeobox‐1(PDX1), an important biomarker for β cell function, was observed. In respect to isolated islets, no significant difference was found in either glucose‐stimulated insulin secretion test(GSIS) or β‐Gal staining, suggesting the islets may function well in these old monkeys.ConclusionIn our 20 years old rhesus monkeys, the insulin resistance has appeared, and their islets area decreased obviously but not accompany significantly compromise of islets function, suggesting that the islets of old rhesus monkey may still have a strong compensatory ability.Support or Funding InformationThis study was supported by the Program of National Natural Science Foundation of China (81870609, 31571474, and 81571808), and the National Key Clinical Project.A, B, C) The fasting blood glucose, fasting blood insulin and intravenous glucose tolerance tests of middle age and old rhesus monkey. D) The glucose‐stimulated insulin secretion test (GSIS) of isolated isletsFigure 1E) The pancreatic sections immunohistochemistry staining with anti‐insulin antibody of middle age(left) and old(right) rhesus monkeyFigure 2

Heterogeneity of Human Pancreatic Islet Isolation Around Europe: Results of a Survey Study

Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries. A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan. Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities. The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria.

Islet-specific Prmt5 excision leads to reduced insulin expression and glucose intolerance in mice.

Protein arginine methyltransferase 5 (PRMT5), a symmetric arginine methyltransferase, regulates cell functions by influencing gene transcription through posttranslational modification of histones and non-histone proteins. PRMT5 interacts with multiple partners including menin, which controls beta cell homeostasis. However, the role of Prmt5 in pancreatic islets, particularly in beta cells, remains unclear. A mouse model with an islet-specific knockout (KO) of the Prmt5 gene was generated, and the influence of the Prmt5 excision on beta cells was investigated via morphologic and functional studies. Beta cell function was evaluated by glucose tolerance test (GTT) and glucose-stimulated insulin secretion (GSIS) test. Beta cell proliferation was evaluated by immunostaining. Gene expression change was determined by real-time qPCR. Molecular mechanisms were investigated in beta cells in vitro and in vivo in Prmt5 KO mice. The results show that islet-specific KO of Prmt5 reduced expression of the insulin gene and impaired glucose tolerance and GSIS in vivo. The mechanistic study indicated that PRMT5 is involved in the regulation of insulin gene transcription, likely via histone methylation-related chromatin remodeling. The reduced expression of insulin in beta cells in the Prmt5 KO mice may contribute to impaired glucose tolerance (IGT) and deficient GSIS in the mouse model. These results will provide new insights into exploring novel strategies to treat diabetes caused by insulin insufficiency.

Circadian misalignment alters insulin sensitivity during the light phase and shifts glucose tolerance rhythms in female mice.

Shift work and jet lag, characterized by circadian misalignment, can disrupt several physiological activities, but whether they affect the rhythm of glucose uptake and insulin sensitivity remain unclear. In the present study, female C57BL/6J mice were maintained for four weeks under the condition of 8-hour phase advance and delay every 3–4 days to mimic shift work. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed repeatedly at Zeitgeber time (ZT) 0, ZT6, ZT12, and ZT18. Glucose-stimulated insulin secretion (GSIS) test was performed at ZT6. We found that the average level of daily glucose tolerance did not decrease but the phase of glucose tolerance advanced by 2.27 hours and the amplitude attenuated by 20.4% in shift work mice. At ZT6, IPITT showed blood glucose at 30 min after insulin injection decreased faster in shift work mice (−3.50±0.74mmol/L, −61.58±7.89%) than that in control mice (−2.11±1.10mmol/L, −33.72±17.24%), but IPGTT and GSIS test showed no significant difference between the two groups. Food intake monitor showed that the feeding time of shift work mice continued to advance. Restricting feed to a fixed 12-hour period alleviated the increase of insulin sensitivity induced by shift-work. We also observed that an increase of blood glucose and liver glycogen at ZT0, as well as a phase advance of liver clock genes and some glucose metabolism-related genes such as forkhead box O1 (Foxo1) and peroxisome proliferator activated receptor alpha (Pparα) in shift work mice. Our results showed that light change-simulated shift work altered insulin sensitivity during the light phase and shifted glucose tolerance rhythms in female mice, suggesting a causal association between long-term shift work and type 2 diabetes.

Age-Related Alteration in Serum Adiponectin in Rats and Its Association with Insulin Resistance Markers

Introduction: Adiponectin is one of the most important adipose derived hormone that conflicting data are available about serum changes of adiponectin at different ages.The present study was done to determine the age related changes in serum adiponectin and its association with insulin resistance (IR) indices in aging in male rats.&#x0D; Methods: In this study, serum samples were obtained from male rats at different ages, including 2, 5, 10, 18, 52 and 72 weeks age (n=10 in each age group). Oral glucose tolerance and glucose stimulated insulin secretion tests were measured using determination of glucose concentrations at 15, 30, 45 and 60 min after oral ingestion of glucose (1 mg/kg body weight) for each animal. Serum adiponectin and insulin levels were determined using species specific ELISA kits. HOMA-IR was calculated based on glucose and insulin concentrations. Data were analyzed using SPSS version 16 software (SPSS Inc., Chicago, IL) also using one way analysis of variance and LSD posthoc tests.&#x0D; Results: Our results showed an age dependent decrease in serum adiponectin concentration, and 72-week old rats had the lowest level of adiponectin compared with those in other ages (p&lt;0.05). IR indices, including fasting glucose, insulin, HOMA-IR and response to oral glucose ingestion was increased in an age dependent manner and 72-week old rats showed the highest levels of the IR indices.&#x0D; Conclusion: Regarding the role of adiponectin in glucose homeostasis and insulin sensitization, it seems that reduction of serum adiponectin with age progression may be an important mechanism of insulin resistance in aging.

Compact fluidic system for functional assessment of pancreatic islets.

Transplantation of pancreatic islets is becoming a promising therapy for people with type I diabetes. In this study, we present a compact fluidic system that enables assessment of islet functionality ex vivo for efficient islet transplantation. The fluidic system includes a micromesh sheet-embedded chip. Islets can be loaded easily on the micromesh sheet and observed clearly by microscopy. Islets on the mesh sheet mainly remained in place during perfusion and did not get damaged by hydraulic pressure because of high porosity of the micromesh sheet. The fluidic system was assembled with a sample fraction chip of polydimethylsiloxane. The chip includes a channel and columns, both having surfaces that were super-hydrophilized so that solutions could flow smoothly within the chip by gravity. Using mouse pancreatic islets, a dynamic glucose-stimulated insulin secretion test was performed to examine the performance of the fluidic system. The system successfully analyzed levels and patterns of insulin secretion upon exposure of the islets to low- and high-glucose solutions in turns, thus demonstrating its capacity to assess islet functions more easily and cost-effectively.

47: Bioartificial Endocrine Pancreas Generated from Decellularized Human Placenta for Type-1 Diabetes Treatment

Background Development of vascularized biological scaffolds providing mechanical protection for islets is a challenging objective of modern regenerative medicine. The aim of our study was to develop vascularized endocrine pancreas using decellularized placental cotyledon. Methods/ Materials Human placentas were decellularized by perfusion, using 0.5% sodium dodecyl sulfate. Decellularization was assessed by histological analyses, scanning electron microscopy (SEM) and residual DNA quantification. Glucosaminoglycans (GAG) and hydroxyproline were analyzed. Placental cotyledons were recellularized by a intravascular perfusion of human umbilical vein endothelial cells (HUVECs) during the 24h, followed by intraparenchimal injection of INS-1E cells. Recellularization was assessed by hystological and immunohistochemical methods, and endocrine function was confirmed by glucose stimulated insulin secretion tests performed at different time points. Results Histological staining and SEM showed complete decellularization and well preserved ECM structure. This was confirmed by the absence of residual DNA. Immunostainings of recellularized cotyedons showed insulin expressing islet like structures with intense vascularization confirmed by CD31 staining. Adequate insulin secretion in response to high glucose stimulation was also observed, confirming the functional activity of INS-1E cells. Conclusion These data demonstrate that acellular placental cotyledons seeded with endocrine pancreatic tissue and endothelial cells could be used for functional pancreas bioengineering.