Islets最新文献

Inhibiting Class IIa Histone Deacetylase (HDAC) function is a promising approach to therapeutically enhance skeletal and cardiac muscle metabolic health in several chronic diseases including type 2 diabetes. However, the importance of Class IIa HDACs in the beta-cell remains unknown. As beta-cell function is vital to maintaining glycaemia it is essential that the importance of Class IIa HDACs in the beta-cell is determined. Here we used the INS-1E cell line cultured in normal glucose (11.1 mM) or hyperglycaemic (20 mM) conditions for 48 hrs to represent cells in a normal and diabetic environment respectively. Cells cultured in high glucose showed significantly reduced insulin secretory function and increased apoptotic signalling compared to cells cultured in normal glucose. Class IIa HDACS, HDAC-4 and -5, were not regulated at the transcript or protein level under normal or hyperglycaemic conditions suggesting that they may not play a role in beta-cell dysfunction. Furthermore, overexpression of wild-type HDAC-4 and -5 or dominant negative HDAC-4 and -5 did not alter insulin secretion, insulin mRNA expression or apoptotic signalling under normal or hyperglycaemic conditions. This suggests that Class IIa Histone Deacetylases do not play an important physiological role in the beta-cell under normal or diabetic conditions. Thus, Class IIa Histone Deacetylase inhibitors are not likely to have a detrimental effect on beta-cells supporting the use of these inhibitors to treat metabolic diseases such as type 2 diabetes.

Background: We previously reported the utility of engineered cell sheets composed of human islets and supporting cells in vitro and in vivo. It is unclear which type of supporting cell is most suitable for constructing cell sheets with human islets. The present study aimed to compare human fibroblasts, bone marrow-derived mesenchymal stem cells (BM-MSCs), and adipose-derived mesenchymal stem cells (ADSCs) as a supporting source for cell sheets.

Methods: Engineered cell sheets were fabricated with human islets using human fibroblasts, BM-MSCs, or ADSCs as supporting cells. The islet viability, recovery rate, glucose-stimulated insulin release (determined by the stimulation index), and cytokine secretion (TGF-β1, IL-6, and VEGF) of groups-including an islet-alone group as a control-were compared.

Results: All three sheet groups consistently exhibited higher viability, recovery rate, and stimulation index values than the islet-alone group. The ADSC group showed the highest viability and recovery rate among the three sheet groups. There were no discernible differences in the stimulation index values of the groups. The fibroblast group exhibited significantly higher TGF-β1 values in comparison to the other groups. The IL-6 level of the ADSC group was more than five times higher than that of the other groups. The ADSC group showed the VEGF level; however, it did not differ from that of the BM-MSC group to a statistically significant extent.

Conclusion: Engineered cell sheets composed of islets and supporting cells had a cytoprotective effect on islets. These results suggest that individual cell types could be a more attractive source for crafting engineered cell sheets in comparison to islets alone.

Since the report of the Edmonton protocol in 2000, islet transplantation has been implemented worldwide, and xenotransplantation using porcine islets has also been reported. In addition, many basic experiments using pancreatic islets and exocrine tissue after isolation have been reported. Recently, exocrine cells have been found to be essential for inducing the differentiation of pancreatic islets. Therefore, the importance of the culture conditions for pancreatic tissue when conducting experiments using pancreatic tissue is also increasing. In this study, we focused on the coat material and examined the adhesive properties of porcine pancreatic islets and exocrine tissue after isolation. Porcine islet isolation was performed, and isolated islets (purity ≥95%) and exocrine tissue (purity ≥99%) were used to achieve adhesion to several extracellular matrixes, fibronectin, collagen type I, collagen type IV, laminin I, fibrinogen, and bovine serum albumin (BSA). DMEM with 0.5% FBS was used as the assay medium. For exocrine tissue, the adhesion was promoted in fibronectin, collagen type I, laminin I, and fibrinogen. The adhesive ability to fibronectin was more than twice that to BSA, while the adhesive ability to collagen type I, laminin I, and fibrinogen was less than twice that to BSA. For islets, the adhesive ability to fibronectin was weaker than that of exocrine tissue. Furthermore, the adhesion effect in fibronectin was obtained within 30 minutes and in medium containing little serum for both islets and exocrine tissues. These data suggest that fibronectin may be useful for the adhesion of pancreatic tissue.

Glucose metabolism is regulated by insulin, which is produced from β-cells in the pancreas. Because insulin is secreted into vessels in response to blood glucose, vascular structures of the pancreas, especially the relationship between vessels and β-cells, are important for physiological and pathological glucose metabolism. Here, we developed a system to visualize vessels surrounding mature β-cells expressing transcription factor MafA in a three-dimensional manner. Optical clearing of the pancreas prevented light scattering of fluorescence driven by the bacterial artificial chromosome (BAC)-mafA promoter in β-cells. Reconstruction of confocal images demonstrated mature β-cells and the glomerular-like structures of β-cell vasculatures labeled with DyLight 488-conjugated lectin in normal mice as well as in low-dose streptozotocin-injected diabetes model mice with reduced β-cell mass. This technological innovation of organ imaging can be used to investigate morphological changes in vascular structures during transplantation, regeneration and diabetes development.

The endocrine pancreas expands markedly in the first postnatal days and the insulin producing β-cells initiate a functional maturation preceded by a morphological change of the islets of Langerhans. Trefoil factor 3 (TFF3) is a secreted peptide expressed in intestinal epithelia, where it promotes migration, but its role in the pancreas is not characterized. The aim of this study was to examine the expression and function of TFF3 in perinatal rat pancreas, ex vivo cultured fetal rat pancreas and in the rat β-cell line INS-1E. Control or gestational low-protein diet perinatal rat pancreas was harvested at embryonic day 20 (E20), day of birth (P0) and postnatal day 2 (P2). TFF3 mRNA was upregulated 4.5-fold at P0 vs. E20 and downregulated again at P2. In protein-undernourished pups induction of TFF3 at P0 was further increased to 9.7-fold and was increased at P2. TFF3 caused tyrosine phosphorylation of EGFR in INS-1E β-cells, and purified recombinant TFF3 increased both attachment and spreading of INS-1E β-cells. In ex vivo cultures of collagenase digested fetal rat pancreas, a model of perinatal β-cell maturation, TFF3 increased cellular spreading as well as insulin mRNA levels. TFF3 also increased the expression of Pref1/Dlk1 that shares similarities in expression and regulation with TFF3. These results suggest that TFF3 may promote adhesion and spreading of cells to accelerate β-cell maturation. This study indicates a functional role for TFF3 in pancreatic β-cell maturation in the perinatal period, which is altered by low protein diet during gestation.

Recently, we showed that pancreatitis in the context of profound β-cell deficiency was sufficient to induce islet cell transdifferentiation. In some circumstances, this effect was sufficient to result in recovery from severe diabetes. More recently, we showed that the molecular mechanism by which pancreatitis induced β-cell neogenesis by transdifferentiation was activation of an atypical GPCR called Protease-Activated Receptor 2 (PAR2). However, the ability of PAR2 to induce transdifferentiation occurred only in the setting of profound β-cell deficiency, implying the existence of a repressive factor from those cells. Here we show that the repressor from β-cells is insulin. Treatment of primary islets with a PAR2 agonist (2fLI) in combination with inhibitors of insulin secretion and signaling was sufficient to induce insulin and PAX4 gene expression. Moreover, in primary human islets, this treatment also led to the induction of bihormonal islet cells coexpressing glucagon and insulin, a hallmark of islet cell transdifferentiation. Mechanistically, insulin inhibited the positive effect of a PAR2 agonist on insulin gene expression and also led to an increase in PAX4, which plays an important role in islet cell transdifferentiation. The studies presented here demonstrate that insulin represses transdifferentiation of α- to β-cells induced by activation of PAR2. This provides a mechanistic explanation for the observation that α- to β-cell transdifferentiation occurs only in the setting of severe β-cell ablation. The mechanistic understanding of islet cell transdifferentiation and the ability to modulate that process using available pharmacological reagents represents an important step along the path towards harnessing this novel mechanism of β-cell neogenesis as a therapy for diabetes.

It is currently unknown how the islet transcriptional pattern changes as glucose metabolism deteriorates and progresses to fulminant type 2 diabetes (T2D). In this study, we hypothesized that islets from donors with elevated HbA1c levels, but not yet diagnosed with T2D, would show signs of cell stress on a transcriptional level. Laser capture microdissection and qPCR arrays including 330 genes related to mitochondria, oxidative stress, or the unfolded protein response were used to extract and analyze islets from organ donors with HbA1c <5.5% (37 mmol/mol), elevated HbA1c (6.0-6.5% (42-48 mmol/mol)), high HbA1c (>6.5% (48 mmol/mol)) or established T2D. Principal component analysis and hierarchical clustering based on the expression of all 330 genes displayed no obvious separation of the four different donor groups, indicating that the inter-donor variations were larger than the differences between groups. However, 44 genes were differentially expressed (P < 0.05, false discovery rate <30%) between islets from donors with HbA1c <5.5% (37 mmol/mol) compared with islets from T2D subjects. Twelve genes were differentially expressed compared to control islets in both donors with established T2D and donors with elevated HbA1c (6.0-6.5% (42-48 mmol/mol)). Overexpressed genes were related mainly to the unfolded protein response, whereas underexpressed genes were related to mitochondria. Our data on transcriptional changes in human islets retrieved by LCM from high-quality biopsies, as pre-diabetes progresses to established T2D, increase our understanding on how islet stress contributes to the disease development.

Intraportal islet transplantation has proven to be efficacious in preventing severe hypoglycemia and restoring insulin independence in selected patients with type 1 diabetes. Multiple islet infusions are often required to achieve and maintain insulin independence. Many challenges remain in clinical islet transplantation, including substantial islet cell loss early and late after islet infusion. Contributions to graft loss include the instant blood-mediated inflammatory reaction, potent host auto- and alloimmune responses, and beta cell toxicity from immunosuppressive agents. Protective strategies are being tested to circumvent several of these events including exploration of alternative transplantation sites, stem cell-derived insulin producing cell therapies, co-transplantation with mesenchymal stem cells or exploration of novel immune protective agents. Herein, we provide a brief introduction and history of islet cell transplantation, limitations associated with this procedure and methods to alleviate islet cell loss as a means to improve engraftment outcomes.

Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.

Collagens are the most abundant fibrous protein in the human body and constitute the main structural element of the extracellular matrix. It provides mechanical and physiological support for cells. In the pancreas, collagen VI content is more than double that of collagen I or IV. It is a major component of the islet-exocrine interface and could be involved in islet-cell survival. To test the impact of collagen VI on human encapsulated pancreatic islets-cells, we tested the effects of exogenous collagen type VI on in vitro functional survival of alginate encapsulated human islet-cells. Concentrations tested ranged from 0.1 to 50 µg/ml. Islets in capsules without collagen type VI served as control. Islet-cell interaction with collagen type VI at concentrations of 0.1 and 10 µg/ml, promoted islet-cell viability (p<0.05). Although no improvement in glucose induced insulin secretion (GSIS) was observed, islets in capsules without incorporation of collagen type VI showed more dysfunction and oxygen consumption rates was improved by inclusion of collagen type VI. Our results demonstrate that incorporation of collagen type VI in immunoisolated human islets supports in vitro viability and survival of human pancreatic islets.