Implanted islets in the anterior chamber of the eye are prone to autoimmune attack in a mouse model of diabetes

Aims/hypothesis

Type 1 diabetes is an autoimmune disease resulting from the destruction of insulin-producing beta cells. Along with advances in generating replacement beta cells for treating diabetes, there is also increasing demand for non-invasive tools to evaluate the recurrence of autoimmune attack on transplanted tissue. Here, we examined the anterior chamber of the eye as a potential islet transplant site, and also evaluated whether in vivo imaging of the islets transplanted in the eye could enable real-time visualisation of autoimmune processes underway in the pancreas.

Methods

Syngeneic islet equivalents were transplanted into the eye or kidney capsule of streptozotocin-induced diabetic C57BL/6 mice to compare islet dose (25–125 islet equivalents) and function across transplant sites. Autoimmune attack of syngeneic islets was evaluated in the pancreas and eye tissues of NOD and NOD-severe combined immunodeficient (SCID) mice given diabetogenic splenocytes.

Results

Islet transplantation in the eye decreased fasting plasma glucose levels and increased weight gain and survival in an islet-dose-dependent manner. Even 50 islets in the eye reduced blood glucose levels, whereas ≥200 islets were required in the kidney for a similar effect. Autoimmune destruction of pancreatic islets in the eye mirrored that in the pancreas and could be visualised in real time by non-invasive imaging.

Conclusions/interpretation

We found that far fewer islets were required to restore normoglycaemia when transplanted into the anterior chamber of the eye vs the kidney capsule. However, our results suggest that islets are not protected against autoimmune attack in the eye, making this a suitable site for visualising autoimmune processes against transplanted tissue.     

Association of proinflammatory cytokines and islet resident leucocytes with islet dysfunction in type 2 diabetes

Aims/hypothesis

Chronic inflammation in type 2 diabetes is proposed to affect islets as well as insulin target organs. However, the nature of islet inflammation and its effects on islet function in type 2 diabetes remain unclear. Moreover, the immune cell profiles of human islets in healthy and type 2 diabetic conditions are undefined. We aimed to investigate the correlation between proinflammatory cytokine expression, islet leucocyte composition and insulin secretion in type 2 diabetic human islets.

Methods

Human islets from organ donors with or without type 2 diabetes were studied. First and second phases of glucose-stimulated insulin secretion were determined by perifusion. The expression of inflammatory markers was obtained by quantitative PCR. Immune cells within human islets were analysed by FACS.

Results

Type 2 diabetic islets, especially those without first-phase insulin secretion, displayed higher CCL2 and TNFa expression than healthy islets. CD45⁺ leucocytes were elevated in type 2 diabetic islets, to a greater extent in moderately functional type 2 diabetic islets compared with poorly functional ones, and corresponded with elevated ALOX12 but not with CCL2 or TNFa expression. T and B lymphocytes and CD11c⁺ cells were detectable within both non-diabetic and type 2 diabetic islet leucocytes. Importantly, the proportion of B cells was significantly elevated within type 2 diabetic islets.

Conclusions/interpretation

Elevated total islet leucocyte content and proinflammatory mediators correlated with islet dysfunction, suggesting that heterogeneous insulitis occurs during the development of islet dysfunction in type 2 diabetes. In addition, the altered B cell content highlights a potential role for the adaptive immune response in islet dysfunction.     

Insulitis in human diabetes: a histological evaluation of donor pancreases

Aims/hypothesis

According to the consensus criteria developed for type 1 diabetes, an individual can be diagnosed with insulitis when ≥ 15 CD45⁺ cells are found within the parenchyma or in the islet–exocrine interface in ≥ 3 islets. The aim of this study was to determine the frequency of individuals with type 2 diabetes fulfilling these criteria with reference to non-diabetic and type 1 diabetic individuals.

Methods

Insulitis was determined by examining CD45⁺ cells in the pancreases of 50, 13 and 44 organ donors with type 2 diabetes, type 1 diabetes and no diabetes, respectively. CD3⁺ cells (T cells) infiltrating the islets were evaluated in insulitic donors. In insulitic donors with type 2 diabetes, the pancreases were characterised according to the presence of CD68 (macrophages), myeloperoxidase (MPO; neutrophils), CD3, CD20 (B cells) and HLA class I hyperstained islets. In all type 2 diabetic donors, potential correlations of insulitis with dynamic glucose-stimulated insulin secretion in vitro or age, BMI, HbA_1c or autoantibody positivity were examined.

Results

Overall, 28% of the type 2 diabetic donors fulfilled the consensus criteria for insulitis developed for type 1 diabetes. Of the type 1 diabetic donors, 31% fulfilled the criteria. None of the non-diabetic donors met the criteria. Only type 1 diabetic donors had ≥ 15 CD3⁺ cells in ≥ 3 islets. Type 2 diabetic donors with insulitis also had a substantial number of CD45⁺ cells in the exocrine parenchyma. Macrophages constituted the largest fraction of CD45⁺ cells, followed by neutrophils and T cells. Of type 2 diabetic pancreases with insulitis, 36% contained islets that hyperstained for HLA class I. Isolated islets from type 2 diabetic donors secreted less insulin than controls, although with preserved dynamics. Insulitis in the type 2 diabetic donors did not correlate with glucose-stimulated insulin secretion, the presence of autoantibodies, BMI or HbA_1c.

Conclusions/interpretation

The current definition of insulitis cannot be used to distinguish pancreases retrieved from individuals with type 1 diabetes from those with type 2 diabetes. On the basis of our findings, we propose a revised definition of insulitis, with a positive diagnosis when ≥ 15 CD3⁺ cells, not CD45⁺ cells, are found in ≥ 3 islets.

     

Islet infiltration,cytokine expression and beta cell death in the NOD mouse,BB rat,Komeda rat,LEW.1AR1-iddm rat and humans with type 1 diabetes

Aims/hypothesis

Research on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation.

Methods

We compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data.

Results

After type 1 diabetes manifestation CD8⁺ T cells, CD68⁺ macrophages and CD4⁺ T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1β and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed.

Conclusions/interpretation

With the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.     

SMAD2 disruption in mouse pancreatic beta cells leads to islet hyperplasia and impaired insulin secretion due to the attenuation of ATP-sensitive K+ channel activity

Aims/hypothesis

The TGF-β superfamily of ligands provides important signals for the development of pancreas islets. However, it is not yet known whether the TGF-β family signalling pathway is required for essential islet functions in the adult pancreas.

Methods

To identify distinct roles for the downstream components of the canonical TGF-β signalling pathway, a Cre-loxP system was used to disrupt SMAD2, an intracellular transducer of TGF-β signals, in pancreatic beta cells (i.e. Smad2β knockout [KO] mice). The activity of ATP-sensitive K⁺ channels (K_ATP channels) was recorded in mutant beta cells using patch-clamp techniques.

Results

The Smad2βKO mice exhibited defective insulin secretion in response to glucose and overt diabetes. Interestingly, disruption of SMAD2 in beta cells was associated with a striking islet hyperplasia and increased pancreatic insulin content, together with defective glucose-responsive insulin secretion. The activity of K_ATP channels was decreased in mutant beta cells.

Conclusions/interpretation

These results suggest that in the adult pancreas, TGF-β signalling through SMAD2 is crucial for not only the determination of beta cell mass but also the maintenance of defining features of mature pancreatic beta cells, and that this involves modulation of K_ATP channel activity.     

Early deficits in insulin secretion,beta cell mass and islet blood perfusion precede onset of autoimmune type 1 diabetes in BioBreeding rats

Aims/hypothesis

Genetic studies show coupling of genes affecting beta cell function to type 1 diabetes, but hitherto no studies on whether beta cell dysfunction could precede insulitis and clinical onset of type 1 diabetes are available.

Methods

We used 40-day-old BioBreeding (BB) DRLyp/Lyp rats (a model of spontaneous autoimmune type 1 diabetes) and diabetes-resistant DRLyp/+ and DR+/+ littermates (controls) to investigate beta cell function in vivo, and insulin and glucagon secretion in vitro. Beta cell mass was assessed by optical projection tomography (OPT) and morphometry. Additionally, measurements of intra-islet blood flow were performed using microsphere injections. We also assessed immune cell infiltration, cytokine expression in islets (by immunohistochemistry and qPCR), as well as islet Glut2 expression and ATP/ADP ratio to determine effects on glucose uptake and metabolism in beta cells.

Results

DRLyp/Lyp rats were normoglycaemic and without traces of immune cell infiltrates. However, IVGTTs revealed a significant decrease in the acute insulin response to glucose compared with control rats (1685.3?±?121.3 vs 633.3?±?148.7; p?<?0.0001). In agreement, insulin secretion was severely perturbed in isolated islets, and both first- and second-phase insulin release were lowered compared with control rats, while glucagon secretion was similar in both groups. Interestingly, after 5–7 days of culture of islets from DRLyp/Lyp rats in normal media, glucose-stimulated insulin secretion (GSIS) was improved; although, a significant decrease in GSIS was still evident compared with islets from control rats at this time (7393.9?±?1593.7 vs 4416.8?±?1230.5 pg islet^?1 h^?1; p?<?0.0001). Compared with controls, OPT of whole pancreas from DRLyp/Lyp rats revealed significant reductions in medium (4.1?×?10⁹?±?9.5?×?10⁷ vs 3.8?×?10⁹?±?5.8?×?10⁷ μm³; p?=?0.044) and small sized islets (1.6?×?10⁹?±?5.1?×?10⁷ vs 1.4?×?10⁹?±?4.5?×?10⁷ μm³; p?=?0.035). Finally, we found lower intra-islet blood perfusion in vivo (113.1?±?16.8 vs 76.9?±?11.8 μl min^?1 [g pancreas]^?1; p?=?0.023) and alterations in the beta cell ATP/ADP ratio in DRLyp/Lyp rats vs control rats.

Conclusions/interpretation

The present study identifies a deterioration of beta cell function and mass, and intra-islet blood flow that precedes insulitis and diabetes development in animals prone to autoimmune type 1 diabetes. These underlying changes in islet function may be previously unrecognised factors of importance in type 1 diabetes development.

     

B lymphocyte-induced maturation protein 1 (BLIMP-1) attenuates autoimmune diabetes in NOD mice by suppressing Th1 and Th17 cells

Aims/hypothesis

Recent reports indicate that B lymphocyte-induced maturation protein 1 (BLIMP-1), encoded by the Prdm1 gene, expands its control over T cells and is associated with susceptibility to colitis in mice with T cell-specific BLIMP-1 deficiency. In this study, we aimed to investigate the potential role of BLIMP-1 in regulating autoimmune diabetes and T helper type 17 (Th17) cells.

Methods

We generated T cell-specific Blimp1 (also known as Prdm1) transgenic (Tg) or conditional knockout (CKO) NOD mice, in which Blimp1 is overexpressed or deleted in T cells, respectively. By side-by-side analysing these Tg or CKO mice, we further dissected the potential mechanisms of BLIMP-1-mediated modulation on autoimmune diabetes.

Results

Overproduction of BLIMP-1 in T cells significantly attenuated insulitis and the incidence of diabetes in NOD mice. Consistent with these results, the diabetogenic effect of splenocytes was remarkably impaired in Blimp1 Tg mice. Moreover, overproduction of BLIMP-1 repressed the proliferation and activation of lymphocytes and enhanced the function of regulatory T cells (Tregs) in NOD mice. In contrast, mice lacking BLIMP-1 in T cells markedly increased Th1 and Th17 cells, and developed highly proliferative and activated lymphocytes. Strikingly, overexpansion of Th1 and Th17 cells in CKO mice was significantly reduced by introducing a Blimp1 transgene, reinforcing the emerging role of BLIMP-1 in autoimmunity.

Conclusions/interpretation

We conclude that BLIMP-1 orchestrates a T cell-specific modulation of autoimmunity by affecting lymphocyte proliferation and activation, Th1 and Th17 cell differentiation, and Treg function. Our results provide a theoretical basis for developing BLIMP-1-manipulated therapies for autoimmune diabetes.     

The anterior chamber of the eye as a clinical transplantation site for the treatment of diabetes: a study in a baboon model of diabetes

Aims/hypothesis

The aim of this study was to provide evidence that the anterior chamber of the eye serves as a novel clinical islet implantation site.

Methods

In a preclinical model, allogeneic pancreatic islets were transplanted into the anterior chamber of the eye of a baboon model for diabetes, and metabolic and ophthalmological outcomes were assessed.

Results

Islets readily engrafted on the iris and there was a decrease in exogenous insulin requirements due to insulin secretion from the intraocular grafts. No major adverse effects on eye structure and function could be observed during the transplantation period.

Conclusions/interpretation

Our study demonstrates the long-term survival and function of allogeneic islets after transplantation into the anterior chamber of the eye. The safety and simplicity of this procedure provides support for further studies aimed at translating this technology into the clinic.     

Increased islet beta cell replication adjacent to intrapancreatic gastrinomas in humans

Aims/hypothesis

Type 1 and type 2 diabetes are characterised by a beta cell deficit. Islet hyperplasia has been described in patients with Zollinger–Ellison syndrome secondary to gastrin-producing tumours (gastrinomas), and gastrin therapy has increased beta cell mass in rodents and human islets in vitro. In the present studies we addressed the following questions: (1) In pancreas specimens from gastrinoma cases, is the fractional beta cell area increased? (2) If so, is this restricted to tumour-adjacent islets or also present in tumour-distant islets? (3) Is new beta cell formation (beta cell replication and islet neogenesis) increased and beta cell apoptosis decreased in pancreas specimens from gastrinoma cases?

Methods

Pancreas was obtained at surgery from four patients with Zollinger–Ellison syndrome caused by pancreatic gastrinomas and 15 control subjects at autopsy.

Results

Islet fractional beta cell area (p<0.001), islet size (p<0.001) and beta cell replication (Ki67 staining) (p<0.05) were increased in islets adjacent to the tumours, but not in tumour-distant pancreas, compared with control subjects. We did not observe any differences in beta cell apoptosis or in the number of insulin-positive cells in ducts either adjacent to or distant from the tumour.

Conclusions/interpretation

One or more factors released by human gastrinomas increase beta cell replication in islets immediately adjacent to the tumour, but not in tumour-distant islets. While these findings demonstrate that adult human beta cells can be driven into the cell cycle, they caution against the therapeutic usefulness of gastrin, since islets located >1 cm away from the gastrinomas did not exhibit changes in beta cell turnover, despite markedly elevated systemic gastrin levels sufficient to cause severe gastrointestinal symptoms.     

Production and function of IL-12 in islets and beta cells

Aims/hypothesis

IL-12 is an important cytokine in early inflammatory responses and is implicated in the immune-mediated pathogenesis of pancreatic islets in diabetes. However, little is known about the direct effects of IL-12 on islets and beta cells.

Methods

In this study, beta cell function, gene expression and protein production were assessed in primary human donor islets and murine beta cell lines in response to stimulation with IL-12 or a pro-inflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ).

Results

The pro-inflammatory cytokine cocktail induced islet dysfunction and potently increased the expression and production of IL-12 ligand and IL-12 receptor in human islets. In human islets, the receptor for IL-12 co-localised to the cell surface of insulin-producing cells. Both IL-12 ligand and IL-12 receptor are expressed in the homogeneous beta cell line INS-1. IL-12 induced changes in gene expression, including a dose-dependent upregulation of IFNγ (also known as IFNG), in INS-1 cells. A neutralising antibody to IL-12 directly inhibited IFNγ gene expression in human donor islets induced by either IL-12 or pro-inflammatory cytokine stimulation. Functionally, IL-12 impaired glucose-stimulated insulin secretion (GSIS) in INS-1 cells and human donor islets. A neutralising antibody to IL-12 reversed the beta cell dysfunction (uncoupling of GSIS or induction of caspase-3 activity) induced by pro-inflammatory cytokines.

Conclusions/interpretation

These data identify beta cells as a local source of IL-12 ligand and suggest a direct role of IL-12 in mediating beta cell pathology.     

Mesenchymal stromal cells improve transplanted islet survival and islet function in a syngeneic mouse model

Aims/hypothesis

Islet transplantation is used therapeutically in a minority of patients with type 1 diabetes. Successful outcomes are hampered by early islet beta cell loss. The adjuvant co-transplantation of mesenchymal stromal cells (MSCs) has the promise to improve islet transplant outcome.

Methods

We used a syngeneic marginal islet mass transplantation model in a mouse model of diabetes. Mice received islets or islets plus 250,000 MSCs. Kidney subcapsule, intra-hepatic and intra-ocular islet transplantation sites were used. Apoptosis, vascularisation, beta cell proliferation, MSC differentiation and laminin levels were determined by immunohistochemical analysis and image quantification post-transplant.

Results

Glucose homeostasis after the transplantation of syngeneic islets was improved by the co-transplantation of MSCs together with islets under the kidney capsule (p?=?0.01) and by intravenous infusion of MSCs after intra-hepatic islet transplantation (p?=?0.05). MSC co-transplantation resulted in reduced islet apoptosis, with reduced numbers of islet cells positive for cleaved caspase 3 being observed 14 days post-transplant. In kidney subcapsule, but not in intra-ocular islet transplant models, we observed increased re-vascularisation rates, but not increased blood vessel density in and around islets co-transplanted with MSCs compared with islets that were transplanted alone. Co-transplantation of MSCs did not increase beta cell proliferation, extracellular matrix protein laminin production or alpha cell numbers, and there was negligible MSC transdifferentiation into beta cells.

Conclusions/interpretation

Co-transplantation of MSCs may lead to improved islet function and survival in the early post-transplantation period in humans receiving islet transplantation.     

Population dynamics of islet-infiltrating cells in autoimmune diabetes

Type-1 diabetes in the nonobese diabetic (NOD) mouse starts with an insulitis stage, wherein a mixed population of leukocytes invades the pancreas, followed by overt diabetes once enough insulin-producing β-cells are destroyed by invading immunocytes. Little is known of the dynamics of lymphocyte movement into the pancreas during disease progression. We used the Kaede transgenic mouse, whose photoconvertible fluorescent reporter permits noninvasive labeling and subsequent tracking of immunocytes, to investigate pancreatic infiltrate dynamics and the requirement for antigen specificity during progression of autoimmune diabetes in the unmanipulated NOD mouse. Our results indicate that the insulitic lesion is very open with constant cell influx and active turnover, predominantly of B and T lymphocytes, but also CD11b⁺c⁺ myeloid cells. Both naïve- and memory-phenotype lymphocytes trafficked to the insulitis, but Foxp3⁺ regulatory T cells circulated less than their conventional CD4⁺ counterparts. Receptor specificity for pancreatic antigens seemed irrelevant for this homing, because similar kinetics were observed in polyclonal and antigen-specific transgenic contexts. This “open” configuration was also observed after reversal of overt diabetes by anti-CD3 treatment. These results portray insulitis as a dynamic lesion at all stages of disease, continuously fed by a mixed influx of immunocytes, and thus susceptible to evolve over time in response to immunologic or environmental influences.Type-1 diabetes (T1D) is an organ-specific autoimmune disease initiated by a breakdown in T lymphocyte tolerance to islet-cell antigens; it comprises two stages: an occult phase of pancreatic inflammation, which reduces the number and function of insulin-producing β-cells and eventually provokes sufficient damage to result in the overt phase of diabetes, when insulin production is insufficient for proper glucose homeostasis. The genetics of T1D in mice and humans point primarily to a dysfunction of CD4⁺ T cells, because class II genes of the MHC, and several other loci that modify T-cell activation and regulation, are linked to T1D susceptibility (1).In nonobese diabetic (NOD) mice and other animal models (2), this initial inflammatory phase takes the form of insulitis, wherein a mixed population of leukocytes invades the islets of Langerhans. Insulitis starts around 3–4 wk of age and amplifies progressively until the onset of clinically overt diabetes (predominantly between 14 and 25 wk of age); it involves a wide array of cell types—T and B lymphocytes as well as myeloid cells—macrophages and dendritic cells (3), which can take on the organization of typical tertiary lymphoid structures (4). Importantly, insulitis variably affects different islets in a given animal, heavily infiltrated islets coexisting with fully intact and functional ones, even in advanced prediabetic mice.Inflammation in islets of human patients has been harder to evaluate, because access to material is obviously more difficult. As cogently reviewed by In’t Veld (5), few individuals have actually been analyzed, often in conditions of uncertain diagnosis, with missing genetic or biochemical data, and the histological evaluation complicated by ketoacidosis and variable handling of cadaveric or surgical samples. Insulitis seems more frequent in individuals presenting with T1D at a young age and assessed soon after onset (6, 7); further, it has been observed in two of three high-risk prediabetic individuals analyzed (8). The proportion of infiltrated islets and the extent of infiltration appear generally lower in human patients than in NOD mice, and a dominance of CD8⁺ over CD4⁺ T cells seems frequent (7, 9, 10), with a variable frequency of B lymphocytes (10). These differences may reflect fluctuations over time, or merely the much faster progression of diabetes in the NOD mouse (weeks rather than years).Two related questions concerning the progression of insulitis arise. First, what are its population dynamics? Is insulitis isolated from the remainder of the immune system and growing locally in response to autoantigens presented there, or is it continuously replenished, and thus possibly modified, by a regular influx of freshly recruited cells? Second, are all of the lymphocytes in the pancreatic infiltrate antigen-specific, or is there also a significant bystander population? The latter point has been debated: using double-retrogenic mice, in which specificity is controlled, Lennon et al. (11) showed that only antigen-specific cells accumulate in the insulitis after transfer into NOD.scid mice, and only T cells specific for β-cell antigens localized to islets in the first hours after transfer (12, 13) in other studies. However, antigen-specific cells identified by tetramer staining are only modestly enriched in the pancreas relative to irrelevant lymphoid organs, and there is still ample participation by other cells (14, 15). Polyclonal populations, unlikely to be antigen specific, accompanied cells from specific clones injected into NOD mice (16). Calderon et al. (17) showed that, in transgenic models, the initial entry of antigen-specific cells modifies local conditions, allowing the recruitment of nonspecific T cells. It should also be noted that chemokine expression resulting from inflammation has the potential to attract T cells to the islets (17, 18).To avoid the potential caveats of transfer systems, we set out to investigate population dynamics of the pancreatic infiltrate during disease progression in NOD mice, using a noninvasive labeling and tracking approach. We used the Kaede/NOD transgenic (tg) mouse, which expresses ubiquitously the photoconvertible fluorescent protein Kaede (19–22). We tracked cell movement from a known point of “labeling”—namely, s.c. lymph nodes (LNs), into the pancreas tissue itself, which allowed us to define the kinetics of entry for lymphoid and myeloid cells, much of which turned out to be independent of antigenic specificity.     

Hydrogen sulfide-induced inhibition of L-type Ca2+ channels and insulin secretion in mouse pancreatic beta cells

Aims/hypothesis

L-type voltage-dependent Ca²⁺ channels (VDCCs) in pancreatic beta cells play a critical role in regulating insulin secretion. The gasotransmitter H₂S is mostly generated from l-cysteine in pancreatic beta cells by cystathionine γ-lyase (CSE) and has been reported to inhibit insulin release by opening ATP-sensitive K⁺ channels. However, whether and how H₂S affects VDCCs in beta cells is unknown.

Methods

The whole-cell patch-clamp technique was used to record VDCCs in beta cells from Cse (also known as Cth)-knockout (KO) and wild-type (WT) mice. Insulin secretion from pancreatic islets and endogenous H₂S production in pancreas were measured.

Results

The H₂S donor NaHS reversibly decreased L-type VDCC current density in a concentration-dependent fashion in WT pancreatic beta cells, and the current density was further inhibited by nifedipine. Furthermore, NaHS inhibited the channel recovery from depolarisation-induced inactivation, but did not shift the current–voltage (I–V) relationship. ACS67, another H₂S donor, also inhibited L-type VDCCs in beta cells. Inhibiting CSE activity with dl-propargylglycine increased the basal L-channel activity of beta cells from WT mice, but not that of beta cells from Cse-KO mice. Beta cells from Cse-KO mice displayed higher L-type VDCC density than those from WT mice. Insulin secretion from pancreatic islets was elevated in Cse-KO mice compared with WT mice. NaHS dose-dependently inhibited glucose-stimulated insulin secretion, which was further inhibited by nifedipine. Bay K-8644 increased glucose-stimulated insulin secretion, but this was counteracted by NaHS and nifedipine.

Conclusions/interpretation

Exogenous and endogenous H₂S inhibit L-type VDCC activity and pancreatic insulin secretion, constituting a novel mechanism for the regulation of insulin secretion by the CSE/H₂S system.     

Histone deacetylases 1 and 3 but not 2 mediate cytokine-induced beta cell apoptosis in INS-1 cells and dispersed primary islets from rats and are differentially regulated in the islets of type 1 diabetic children

Aims/hypothesis

Histone deacetylases (HDACs) are promising pharmacological targets in cancer and autoimmune diseases. All 11 classical HDACs (HDAC1–11) are found in the pancreatic beta cell, and HDAC inhibitors (HDACi) protect beta cells from inflammatory insults. We investigated which HDACs mediate inflammatory beta cell damage and how the islet content of these HDACs is regulated in recent-onset type 1 diabetes.

Methods

The rat beta cell line INS-1 and dispersed primary islets from rats, either wild type or HDAC1–3 deficient, were exposed to cytokines and HDACi. Molecular mechanisms were investigated using real-time PCR, chromatin immunoprecipitation and ELISA assays. Pancreases from healthy children and children with type 1 diabetes were assessed using immunohistochemistry and immunofluorescence.

Results

Screening of 19 compounds with different HDAC selectivity revealed that inhibitors of HDAC1, -2 and -3 rescued INS-1 cells from inflammatory damage. Small hairpin RNAs against HDAC1 and -3, but not HDAC2, reduced pro-inflammatory cytokine-induced beta cell apoptosis in INS-1 and primary rat islets. The protective properties of specific HDAC knock-down correlated with attenuated cytokine-induced iNos expression but not with altered expression of the pro-inflammatory mediators Il1α, Il1β, Tnfα or Cxcl2. HDAC3 knock-down reduced nuclear factor κB binding to the iNos promoter and HDAC1 knock-down restored insulin secretion. In pancreatic sections from children with type 1 diabetes of recent onset, HDAC1 was upregulated in beta cells whereas HDAC2 and -3 were downregulated in comparison with five paediatric controls.

Conclusions/interpretation

These data demonstrate non-redundant functions of islet class I HDACs and suggest that targeting HDAC1 and HDAC3 would provide optimal protection of beta cell mass and function in clinical islet transplantation and recent-onset type 1 diabetic patients.