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ABSTRACT: The beta cell transcriptional factor musculoaponeurotic fibrosarcoma oncogene family A (MafA) regulates genes important for beta cell function. Loss of nuclear MafA has been implicated in beta cell dysfunction in animal models of type 2 diabetes. We sought to establish if nuclear MafA is less abundant in beta cell nuclei in humans with type 2 diabetes.
Pancreas obtained at surgery from five non-diabetic individuals and six individuals with type 2 diabetes was immunostained for insulin, glucagon and MafA.
Beta cell nuclear MafA was markedly decreased in type 2 diabetes (1.6 ± 1.2% vs 46.3 ± 8.3%, p < 0.001).
Beta cell nuclear MafA is markedly decreased in humans with type 2 diabetes, which may contribute to impaired beta cell dysfunction.
Diabetologia 07/2012; 55(11):2985-8. · 6.81 Impact Factor
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Y. Saisho,
A. E. Butler,
E. Manesso,
R. Galasso,
L. Zhang, T. Gurlo,
G. M. Toffolo,
C. Cobelli,
K. Kavanagh,
J. D. Wagner,
P. C. Butler
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ABSTRACT: Aims/hypothesisWe sought to establish the relationship between fasting plasma glucose concentrations and pancreatic fractional beta cell
area in adult cynomolgus monkeys (Macaca fascicularis).
MethodsFasting plasma glucose and pancreatic fractional beta cell area were measured in 18 control and 17 streptozotocin-treated
adult primates (17.0 ± 1.2 vs 15.4 ± 1.2years old).
ResultsFasting plasma glucose was increased (12.0 ± 2.0 vs 3.4 ± 0.1mmol/l, p < 0.01) and fractional beta cell area was decreased (0.62 ± 0.13% vs 2.49 ± 0.35%, p < 0.01) in streptozotocin-treated monkeys. The relationship between fasting plasma glucose and pancreatic fractional beta
cell area was described by a wide range of beta cell areas in controls. In streptozotocin-treated monkeys there was an inflection
of fasting blood glucose at ∼50% of the mean beta cell area in controls with a steep increase in blood glucose for each further
decrement in beta cell area.
Conclusions/interpretationIn adult non-human primates a decrement in fractional beta cell area of ∼50% or more leads to loss of glycaemic control.
KeywordsBeta cell mass-Cynomolgus monkey-Streptozotocin-Type 1 diabetes-Type 2 diabetes
Diabetologia 04/2012; 53(1):111-114. · 6.81 Impact Factor
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ABSTRACT: In type II diabetes (T2DM), there is a deficit in β-cells, increased β-cell apoptosis and formation of intracellular membrane-permeant oligomers of islet amyloid polypeptide (IAPP). Human-IAPP (h-IAPP) is an amyloidogenic protein co-expressed with insulin by β-cells. IAPP expression is increased with obesity, the major risk factor for T2DM. In this study we report that increased expression of human-IAPP led to impaired autophagy, due at least in part to the disruption of lysosome-dependent degradation. This action of IAPP to alter lysosomal clearance in vivo depends on its propensity to form toxic oligomers and is independent of the confounding effect of hyperglycemia. We report that the scaffold protein p62 that delivers polyubiquitinated proteins to autophagy may have a protective role against human-IAPP-induced apoptosis, apparently by sequestrating protein targets for degradation. Finally, we found that inhibition of lysosomal degradation increases vulnerability of β-cells to h-IAPP-induced toxicity and, conversely, stimulation of autophagy protects β-cells from h-IAPP-induced apoptosis. Collectively, these data imply an important role for the p62/autophagy/lysosomal degradation system in protection against toxic oligomer-induced apoptosis.
Cell death and differentiation 03/2011; 18(3):415-26. · 8.24 Impact Factor
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Y Saisho,
A E Butler,
E Manesso,
R Galasso,
L Zhang, T Gurlo,
G M Toffolo,
C Cobelli,
K Kavanagh,
J D Wagner,
P C Butler
[show abstract]
[hide abstract]
ABSTRACT: We sought to establish the relationship between fasting plasma glucose concentrations and pancreatic fractional beta cell area in adult cynomolgus monkeys (Macaca fascicularis).
Fasting plasma glucose and pancreatic fractional beta cell area were measured in 18 control and 17 streptozotocin-treated adult primates (17.0 +/- 1.2 vs 15.4 +/- 1.2 years old).
Fasting plasma glucose was increased (12.0 +/- 2.0 vs 3.4 +/- 0.1 mmol/l, p < 0.01) and fractional beta cell area was decreased (0.62 +/- 0.13% vs 2.49 +/- 0.35%, p < 0.01) in streptozotocin-treated monkeys. The relationship between fasting plasma glucose and pancreatic fractional beta cell area was described by a wide range of beta cell areas in controls. In streptozotocin-treated monkeys there was an inflection of fasting blood glucose at approximately 50% of the mean beta cell area in controls with a steep increase in blood glucose for each further decrement in beta cell area.
In adult non-human primates a decrement in fractional beta cell area of approximately 50% or more leads to loss of glycaemic control.
Diabetologia 10/2009; 53(1):111-4. · 6.81 Impact Factor
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ABSTRACT: Beta cell destruction in type 1 diabetes is apparently mediated by the release of cytokines. We questioned whether cytokine-induced apoptosis preferentially kills replicating beta cells.
In the first experiment, rat insulinoma (RIN) cells were studied for 36 h by time-lapse video microscopy. Cells were exposed to three doses of a cytokine mixture (maximal concentration: IL-1beta 50 U/ml; TNF-alpha 1,000 U/ml; IFN-gamma 1,000 U/ml) or vehicle and analysed for the total cell number (2-h intervals) and timing of each cell death and division. In the second experiment, isolated human islets were incubated with the same cytokine mixture for 24 h and examined for replication and paired (postmitotic) apoptosis.
In the first experiment, after application of cytokines, apoptosis occurred most frequently immediately after the next or subsequent cell mitosis (p<0.05). In the second experiment, cytokines caused increased apoptosis in human islets, with an increase in the proportion of postmitotic apoptotic pairs (p<0.001).
Cytokine-induced beta cell death preferentially affects newly forming beta cells, which implies that replicating beta cells might be more vulnerable to cytokine destruction. Efforts to expand beta cell mass in type 1 diabetes by fostering beta cell replication are likely to fail unless cytokine-induced apoptosis is concurrently suppressed.
Diabetologia 02/2006; 49(1):83-9. · 6.81 Impact Factor
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ABSTRACT: Prostaglandins (PG) are released during tissue injury and inflammation, and inhibit immune responses at many points. PG may be one of several factors that protect not only against injury-induced, but also spontaneous, organ-specific autoimmune disease. Here we show that the production of PGE(2), normally produced at a very low rate in islets of Langerhans, is significantly increased in inflamed islets of non-obese diabetic (NOD) mice. We investigated a possible role of PGE(2) in controlling TCR-dependent release of IFN-gamma from islet-reactive NOD CD8(+) T cells. PGE(2) inhibited anti-TCR antibody-triggered release of IFN-gamma from CD8(+) T cell clone 8D8 and from polyclonal cytotoxic T lymphocytes (CTL). Using receptor subtype selective agonists, we present evidence that the effect of PGE(2) is mediated by EP(2) and EP(4) receptors, both of which are coupled to an increase in intracellular cAMP production. The cAMP analogs 8-Br-cAMP and Sp-cAMPS mimic the effect of EP(2)/EP(4) receptor agonists, inhibiting TCR-triggered IFN-gamma release from NOD CD8(+) T cells in a dose-dependent manner. The inhibitory effect of PGE(2) was largely reversed by IL-2 added at the time of culture initiation and decreased with increasing strength of stimulation through the TCR. Resting CTL were more sensitive to PGE(2) than recently expanded CTL and NOD CD8(+) T cells remained insensitive to PGE(2) for a longer time than BALB/c cells. Our study suggests that PGE(2) may be part of a regulatory network that controls local activation of T cells and may play a role in the balance between the development of islet autoimmunity or maintenance of tolerance.
International Immunology 07/2000; 12(6):851-60. · 3.41 Impact Factor
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ABSTRACT: Cell-enzyme-linked immunosorbent assay (cell-ELISA) is a technique for the rapid, convenient, and quantitative detection of molecules expressed on the cell surface. Here we present an evaluation of beta-galactosidase as an antibody-tag for cell-ELISA. In contrast to substrates for horseradish peroxidase (HRP) and alkaline phosphatase, murine splenocytes do not hydrolyze the beta-galactosidase substrate chlorophenolred-beta-D-galactopyranoside (CPRG). beta-Galactosidase-antibody conjugates show much lower background binding to murine T cells than conjugates with HRP or alkaline phosphatase. We describe step-by-step procedures for direct and indirect beta-galactosidase based cell-ELISA to quantitate the expression of molecules on the surface of unfixed, live cells. Variations of the basic protocol are suitable for adherent and non-adherent cells, large scale screening for expression of cell surface molecules, and the screening of hybridomas for production of antibodies to cell surface epitopes. Since relatively few beta-galactosidase conjugated antibodies are commercially available, we describe an efficient method to couple beta-galactosidase to antibodies using a novel water soluble heterobifunctional crosslinker, sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC). We demonstrate the utility of this method by conjugating F(ab')(2) fragments of an anti-B7-2 antibody, and using this conjugate to assay B7-2 on Fc-receptor bearing cells.
Journal of Immunological Methods 03/2000; 234(1-2):P153-67. · 2.20 Impact Factor
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Immunology Letters 01/2000; 70(3):139-41. · 2.53 Impact Factor
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ABSTRACT: To investigate how CD8+ T cells interact with beta cells and local inflammatory cells in islets, we have isolated CD8+ T cell clones from nonobese diabetic (NOD) spleen that recognize and destroy both islets and the NOD insulinoma cell line NIT-1. The clones destroyed NOD islets with pre-existing inflammation better than islets without signs of inflammation. Islets from NOD-scid mice were destroyed only poorly, but that could be improved by adding IL-7 to the assay. Anti-IFN-gamma Abs inhibited destruction of infiltrated islets. Single islets were effective stimulators of IFN-gamma production by cloned CD8+ T cells, which varied >50-fold depending on the degree of islet infiltration. This effect of the islet mononuclear infiltrate could be mimicked by adding spleen cells to NIT-1 cells, which augmented IFN-gamma production above the level stimulated by NIT-1 cells alone. The enhancing effect of spleen cells could be attributed to their macrophage subpopulation and was not MHC restricted, although recognition of islet Ag by cloned CD8+ T cells and subsequent islet destruction was restricted to islets expressing H-2Db molecules. An inhibitor of inducible NO synthase inhibited destruction of inflamed islets by cloned CD8+ T cells. We propose that macrophages in inflamed islets provide a form of bystander costimulation of beta cell-specific CD8+ T cells. CD8+ T cells respond to Ag and costimulation by producing IFN-gamma that activates macrophages. Activated macrophages facilitate islet destruction by CD8+ T cells through a NO synthesis-dependent pathway.
The Journal of Immunology 12/1999; 163(11):5770-80. · 5.79 Impact Factor
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ABSTRACT: The cytotoxic T cell line CTLL-2 and the T helper cell line HT2 proliferate in response to interleukin 2 (IL-2) or IL-4 without requiring stimulation by antigen through the T cell receptor and therefore lend themselves for studies of IL-2- and IL-4-dependent proliferation and signalling through their cognate receptors. Here we have used CTLL-2 and HT2 cells to investigate the effect of the inflammatory mediator prostaglandin E2 (PGE2) on IL-2- and IL-4-dependent proliferation. PGE2 inhibited IL-2- as well as IL-4-dependent proliferation of both CTLL-2 and HT2 cells, with IL-4-dependent proliferation being more sensitive than IL-2-dependent proliferation and CTLL-2 cells being more sensitive than HT2 cells. A quantitative dose-effect analysis revealed a two-step increase of inhibition (around 10(-10) M and 10(-5) M PGE2) for all combinations of cells and cytokines approaching 100% at high concentrations of PGE2. The data suggest that even in cases where synthesis of IL-2 and IL-4 is differentially affected by PGE2, IL-2- and IL-4-dependent T cells may still be similarly sensitive to PGE2 by way of their cytokine responsiveness. Furthermore, the effects of PGE2 may be mediated by more than one functional binding site or receptor subtype. PGE2 levels are an important consideration when CTLL-2 and HT2 cells are used for the measurement of IL-4 and IL-2.
Cytokine 05/1998; 10(4):265-74. · 3.02 Impact Factor
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ABSTRACT: A cloned Th1 cell line was isolated from pancreatic lymph nodes of NOD mice that carries a T-cell receptor encoding Vbeta14 and proliferates in response to NOD islets, islet supernatant, and crystalline bovine and rat insulin, specifically to a B-chain peptide bound to IA(g7). The response to islet supernatant was reduced by 75% by anti-insulin antibody treatment. The insulin-reactive clone reduced insulitis and totally blocked the development of spontaneous diabetes in NOD mice (n = 8) as well as the adoptive transfer of diabetes into irradiated NOD mice following the injection of splenocytes from diabetic mice (n = 13). Trafficking of the adoptively transferred cells was assessed by labeling the clone or diabetic splenocytes with a fluorescent marker (DiI). The labeled clone was detected in the islet periphery, whereas labeled splenocytes alone invaded the islets by 3 days. In contrast, the protective clone dramatically delayed and reduced the number of labeled diabetic splenocytes infiltrating the islet, although their appearance in the spleen was unaffected. In vitro, the clone as well as supernatant derived from the clone blocked the proliferation of diabetic NOD splenocytes to islets. This inhibitory effect was diminished by anti-transforming growth factor-beta. In conclusion, an insulin-specific Th1 cell was isolated from NOD mice that traffics to the islet and prevents the spontaneous development and the adoptive transfer of diabetes. It appears to act locally by releasing transforming growth factor-beta and/or other factors that inhibit homing to and/or proliferation of diabetic splenocytes within the islet. These findings may provide insights into and suggest mechanisms for the protective effects of insulin therapy against diabetes.
Diabetes 08/1997; 46(7):1124-32. · 8.29 Impact Factor
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Y Saisho,
A E Butler,
E Manesso,
R Galasso,
L Zhang, T Gurlo,
G. M. Toffolo,
C Cobelli,
K Kavanagh,
J D Wagner,
P C Butler
