Noel G. Morgan

Analysis of islet inflammation in human type 1 diabetes

The immunopathology of type 1 diabetes (T1D) has proved difficult to study in man because of the limited availability of appropriate samples, but we now report a detailed study charting the evolution of insulitis in human T1D. Pancreas samples removed post‐mortem from 29 patients (mean age 11·7 years) with recent‐onset T1D were analysed by immunohistochemistry. The cell types constituting the inflammatory infiltrate within islets (insulitis) were determined in parallel with islet insulin content. CD8+ cytotoxic T cells were the most abundant population during insulitis. Macrophages (CD68+) were also present during both early and later insulitis, although in fewer numbers. CD20+ cells were present in only small numbers in early insulitis but were recruited to islets as beta cell death progressed. CD138+ plasma cells were infrequent at all stages of insulitis. CD4+ cells were present in the islet infiltrate in all patients but were less abundant than CD8+ or CD68+ cells. Forkhead box protein P3+ regulatory T cells were detected in the islets of only a single patient. Natural killer cells were detected rarely, even in heavily inflamed islets. The results suggest a defined sequence of immune cell recruitment in human T1D. They imply that both CD8+ cytotoxic cells and macrophages may contribute to beta cell death during early insulitis. CD20+ cells are recruited in greatest numbers during late insulitis, suggesting an increasing role for these cells as insulitis develops. Natural killer cells and forkhead box protein P3+ T cells do not appear to be required for beta cell death.

The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes

Aims/hypothesisEvidence that the beta cells of human patients with type 1 diabetes can be infected with enterovirus is accumulating, but it remains unclear whether such infections occur at high frequency and are important in the disease process. We have now assessed the prevalence of enteroviral capsid protein vp1 (vp1) staining in a large cohort of autopsy pancreases of recent-onset type 1 diabetic patients and a range of controls.MethodsSerial sections of paraffin-embedded pancreatic autopsy samples from 72 recent-onset type 1 diabetes patients and up to 161 controls were immunostained for insulin, glucagon, vp1, double-stranded RNA activated protein kinase R (PKR) and MHC class I.Resultsvp1-immunopositive cells were detected in multiple islets of 44 out of 72 young recent-onset type 1 diabetic patients, compared with a total of only three islets in three out of 50 neonatal and paediatric normal controls. vp1 staining was restricted to insulin-containing beta cells. Among the control pancreases, vp1 immunopositivity was also observed in some islets from ten out of 25 type 2 diabetic patients. A strong correlation was established between islet cell vp1 positivity and PKR production in insulin-containing islets of both type 1 and type 2 diabetic patients, consistent with a persistent viral infection of the islets.Conclusions/interpretationImmunoreactive vp1 is commonly found in the islets of recent-onset type 1 diabetes patients, but only rarely in normal paediatric controls. vp1 immunostaining was also observed in some islets of type 2 diabetes patients, suggesting that the phenomenon is not restricted to type 1 diabetes patients.

Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.

Monogenic causes of autoimmunity provide key insights into the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in five individuals with a spectrum of early-onset autoimmune disease, including type 1 diabetes. These findings emphasize the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in hyper IgE syndrome.

Life and death decisions of the pancreatic β-cell: the role of fatty acids

Both stimulatory and detrimental effects of NEFAs (non-esterified fatty acids) on pancreatic β-cells have been recognized. Acute exposure of the pancreatic β-cell to high glucose concentrations and/or saturated NEFAs results in a substantial increase in insulin release, whereas chronic exposure results in desensitization and suppression of secretion, followed by induction of apoptosis. Some unsaturated NEFAs also promote insulin release acutely, but they are less toxic to β-cells during chronic exposure and can even exert positive protective effects. Therefore changes in the levels of NEFAs are likely to be important for the regulation of β-cell function and viability under physiological conditions. In addition, the switching between endogenous fatty acid synthesis or oxidation in the β-cell, together with alterations in neutral lipid accumulation, may have critical implications for β-cell function and integrity. Long-chain acyl-CoA (formed from either endogenously synthesized or exogenous fatty acids) controls several aspects of β-cell function, including activation of specific isoenzymes of PKC (protein kinase C), modulation of ion channels, protein acylation, ceramide formation and/or NO-mediated apoptosis, and transcription factor activity. In this review, we describe the effects of exogenous and endogenous fatty acids on β-cell metabolism and gene and protein expression, and have explored the outcomes with respect to insulin secretion and β-cell integrity.

Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.

Aims/hypothesisEndoplasmic reticulum (ER) stress may play a role in cytokine-mediated beta cell death in type 1 diabetes, but it remains controversial whether ER stress markers are present in islets from type 1 diabetic individuals. Therefore, we evaluated by immunostaining the expression of markers of the three main branches of the ER stress response in islets from 13 individuals with and 15 controls without type 1 diabetes (eight adults and seven children).MethodsAntibodies against the ER stress markers C/EBP homologous protein (CHOP), immunoglobulin heavy chain (BIP) and X-box binding protein 1 (XBP-1) were validated using HeLa cells treated with the ER stressor thapsigargin. These antibodies were then used to stain serial sections of paraffin-embedded pancreas from type 1 diabetic and non-diabetic individuals; samples were also immunostained for CD45, insulin and glucagon. Immunostaining intensities of the ER stress markers were quantified using a software-based, unbiased quantitative approach.ResultsIslets from individuals with type 1 diabetes showed increased levels of CHOP and, at least for insulitis-positive and beta cell-containing islets, BIP. XBP-1 expression was not, however, increased.Conclusions/interpretationIslet cells from individuals with type 1 diabetes display a partial ER stress response, with evidence of the induction of some, but not all, components of the unfolded protein response.

Islet-associated macrophages in type 2 diabetes

To the Editor: Recently evidence has emerged that supports a role for islet inflammation in the development of type 2 diabetes in man, suggesting that there may be certain common features underlying the pathology of beta cell loss in both type 1 and type 2 diabetes. In particular, data have recently been presented revealing an increased number of macrophages infiltrating the islets of nine type 2 diabetic patients, as well as in several animal models of type 2 diabetes (including high-fat-fed C57BL6/6J mice, GK rats and the db/db mouse) when compared with relevant controls [1]. Those authors argued that this evidence implies that macrophage infiltration could be involved in mediating beta cell dysfunction and loss in type 2 diabetes. In view of these conclusions, we considered it important to verify whether increased macrophage infiltration is also observed in a different and larger cohort of human patients with type 2 diabetes and to assess the magnitude of this response. Serial sections of paraffin-embedded pancreas recovered at autopsy from 15 type 2 diabetic patients (mean age [±SEM] 69.2±1.8 years) and 16 non-diabetic controls (age 52.9±3.9 years) were processed and stained with antiinsulin and anti-CD68 antibodies (DAKO, Ely, UK) using a standard immunoperoxidase technique. The use of all tissue was undertaken with full ethical permission. A quantitative analysis of up to 50 randomly selected islets per individual was carried out and the number of CD68 cells (taken to indicate the presence of macrophages) either within the islets or in the peri-islet area was counted. Statistical comparisons were performed by χ analysis. Within the control group, we did not observe any tendency for the number of macrophages present within islets to change with age. Therefore we consider that, although the mean age of the type 2 diabetic patients was slightly lower than the controls, this difference per se is unlikely to account for variations in macrophage infiltration. In order to confirm that the number of CD68 cells counted per islet was not distorted by a change in islet size or area in the patients vs controls, random images were examined from slides stained for insulin from six cases of type 2 diabetes (mean age 62.7±2.3 years) and four controls (mean age 64.0± 2.6 years). This revealed that the percentage of pancreatic tissue occupied by endocrine cells was similar in the two groups (1.99±0.23% in type 2 diabetes; 2.17±0.32% in controls). In addition, the mean endocrine cell area within the islets was also unchanged in the sections studied, implying that the overall size of the islets was not decreased in the cohort of patients with type 2 diabetes compared with the controls. A total of 545 and 564 islets were analysed Diabetologia (2009) 52:1686–1688 DOI 10.1007/s00125-009-1410-z

The α2-adrenoceptor antagonist efaroxan modulates K+ATP channels in insulin-secreting cells

Abstract The actions of efaroxan, a highly selective and potent α 2 -adrenoceptor antagonist, on insulin secretion, cAMP levels, 86 Rb4 + efflux and ATP-regulated potassium (K + ATP ) channels have been studied using isolated pancreatic islets of Langerhans and RINm5F cells. In the absence of an adrenoceptor agonist, efaroxan (1–100 μM) potentiated glucose-induced secretion over the range 4–10 mM glucose, but was without effect upon the maximal rate of secretion induced by 20 mM glucose. Efaroxan did not affect cAMP levels. Suppression of insulin release by the potassium channel opener diazoxide, was partially alleviated by efaroxan and was associated with an inhibition of the diazoxide-induced increase in the rate of 86 Rb + efflux. Using isolated patches of membrane we found efaroxan to be an effective blocker of k + ATP channels, with a K 1 value of 12 μm and a Hill coefficient of approximately 1. These data indicate that efaroxan promotes insulin secretion, in the absence of exogenous agonists, by a mechanism that involves inhibition of ATP-regulated K + channels.

The imidazoline site involved in control of insulin secretion : characteristics that distinguish it from I1- and I2-sites

1 The nature of the binding site mediating the insulin secretagogue activity of certain imidazoline compounds remains unclear and the pharmacology of the I1‐ and I2‐imidazoline sites, described in many tissues, does not correlate with the observed responses to imidazolines in islets. In the present paper, we describe further results which support the concept that the islet imidazoline site may represent a novel subtype of imidazoline receptor. 2 Culture of rat isolated islets in the presence of imidazoline secretagogues (either efaroxan or phentolamine) resulted in loss of responsiveness on subsequent re‐exposure to these agents. However, culture of islets with either idazoxan or UK 14,304 (imidazoline ligands that do not stimulate insulin secretion) did not lead to any loss of response when the islets were subsequently exposed to efaroxan. By contrast, islets cultured with UK 14,304 (a potent α2‐adrenoceptor agonist), displayed loss of sensitivity to noradrenaline, consistent with down‐regulation of α2‐adrenoceptors. 3 In order to characterize the imidazoline site further, radioligand binding studies were performed in membranes from RINm5F insulinoma cells using [3H]‐RX821002, an imidazoline insulin secretagogue that does not interact significantly with imidazoline sites in other tissues. [3H]‐RX821002 labelled α2‐adrenoceptors with high affinity (2.01 ± 0.7 nM) but also labelled a second, non‐adrenoceptor site with much lower affinity. 4 Under conditions of α2‐adrenoceptor blockade (in the presence of adrenaline), efaroxan displaced [3H]‐RX821002 binding to the low affinity site, in a dose‐dependent manner. Competition studies employing additional imidazoline compounds of varying secretagogue activity revealed that the pharmacological profile of the low affinity site correlates well with that observed in secretion experiments. 5 The results obtained from the down‐regulation experiments with isolated islets and from the radioligand binding studies suggest that the low affinity [3H]‐RX821002 binding site may represent the functional receptor responsible for the secretagogue activity of imidazoline compounds in the endocrine pancreas and that it has a pharmacological profile distinct from those of I1‐ and I2‐sites.

Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B‐cell‐directed cytotoxicity of cytokines in insulin‐dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B‐cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B‐cell line HIT‐T15 in a dose‐ and time‐dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8‐bromo cGMP (100 μM) and dibutyryl cGMP (300 μM) were able to fully relieve this inhibition. In addition, incubation of HIT‐T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP‐dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose‐dependent over the concentration range of 10–250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT‐T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.

Differential regulation of the endoplasmic reticulum stress response in pancreatic β-cells exposed to long-chain saturated and monounsaturated fatty acids

Exposure of pancreatic beta-cells to long-chain fatty acids leads to the activation of some components of the endoplasmic reticulum (ER) stress pathway and this mechanism may underlie the ability of certain fatty acids to promote beta-cell death. We have studied ER stress in BRIN-BD11 beta-cells exposed to either the saturated fatty acid palmitate (C16:0) or the monounsaturated palmitoleate (C16:1). Palmitate (0.025-0.25 mM) induced the expression of various markers of the RNA-dependent protein kinase-like ER eukaryotic initiation factor 2 alpha (eIF2 alpha) kinase (PERK)-dependent pathway of ER stress (phospho-eIF2 alpha; ATF4, activating transcription factor 4 and C/EBP homologous protein (CHOP-10)) although it failed to promote the expression of the ER chaperone GRP78. By contrast, palmitoleate did not induce any markers of the ER stress pathway even at concentrations as high as 1 mM. When palmitate and palmitoleate were added in combination, a marked attenuation of the ER stress response occurred. Under these conditions, the levels of phospho-eIF2 alpha, ATF4 and CHOP-10 were reduced to less than those found in control cells. Palmitoleate also attenuated the ER stress response to the protein glycosylation inhibitor, tunicamycin, and improved the viability of the cells exposed to this agent. Exposure of the BRIN-BD11 cells to the protein phosphatase inhibitor, salubrinal, in the absence of fatty acids resulted in increased eIF2 alpha phosphorylation but this was abolished by co-incubation with palmitoleate. We conclude that saturated fatty acids activate components of the PERK-dependent ER stress pathway in beta-cells, ultimately leading to increased apoptosis. This effect is antagonised by monounsaturates that may exert their anti-apoptotic actions by regulating the activity of one or more kinase enzymes involved in mediating the phosphorylation of eIF2 alpha.