A genomic-based approach identifies FXYD domain containing ion transport regulator 2 (FXYD2)γa as a pancreatic beta cell-specific biomarker

Aims/hypothesis

Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers.

Methods

We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays.

Results

After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na⁺–K⁺-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2γa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2γa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2γa and loss of insulin-positive cells.

Conclusions/interpretation

We propose human FXYD2γa as a novel beta cell-specific biomarker.     

The human L-type calcium channel Cav1.3 regulates insulin release and polymorphisms in CACNA1D associate with type 2 diabetes

Aims/hypothesis

Voltage-gated calcium channels of the L-type have been shown to be essential for rodent pancreatic beta cell function, but data about their presence and regulation in humans are incomplete. We therefore sought to elucidate which L-type channel isoform is functionally important and its association with inherited diabetes-related phenotypes.

Methods

Beta cells of human islets from cadaver donors were enriched using FACS to study the expression of the genes encoding voltage-gated calcium channel (Ca_v)1.2 and Ca_v1.3 by absolute quantitative PCR in whole human and rat islets, as well as in clonal cells. Single-cell exocytosis was monitored as increases in cell capacitance after treatment with small interfering (si)RNA against CACNA1D (which encodes Ca_v1.3). Three single nucleotide polymorphisms (SNPs) were genotyped in 8,987 non-diabetic and 2,830 type 2 diabetic individuals from Finland and Sweden and analysed for associations with type 2 diabetes and insulin phenotypes.

Results

In FACS-enriched human beta cells, CACNA1D mRNA expression exceeded that of CACNA1C (which encodes Ca_v1.2) by approximately 60-fold and was decreased in islets from type 2 diabetes patients. The latter coincided with diminished secretion of insulin in vitro. CACNA1D siRNA reduced glucose-stimulated insulin release in INS-1 832/13 cells and exocytosis in human beta cells. Phenotype/genotype associations of three SNPs in the CACNA1D gene revealed an association between the C allele of the SNP rs312480 and reduced mRNA expression, as well as decreased insulin secretion in vivo, whereas both rs312486/G and rs9841978/G were associated with type 2 diabetes.

Conclusion/interpretation

We conclude that the L-type calcium channel Ca_v1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.     

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.     

The CD19 signalling molecule is elevated in NOD mice and controls type 1 diabetes development

Aims/hypothesis

Type 1 diabetes is characterised by early peri-islet insulitis and insulin autoantibodies, followed by invasive insulitis and beta cell destruction. The immunological events that precipitate invasive insulitis are not well understood. We tested the hypothesis that B cells in diabetes-prone NOD mice drive invasive insulitis through elevated expression of CD19 and consequent enhanced uptake and presentation of beta cell membrane-bound antigens to islet invasive T cells.

Methods

CD19 expression and signalling pathways in B cells from NOD and control mice were compared. Expansion of CD8⁺ T cells specific for insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were compared in CD19-deficient and wild-type NOD mice and this was correlated with insulitis severity. The therapeutic potential of anti-CD19 treatment during the period of T cell activation was assessed for its ability to block invasive insulitis.

Results

CD19 expression and signalling in B cells was increased in NOD mice. CD19 deficiency significantly diminished the expansion of CD8⁺ T cells with specificity for the membrane-bound beta cell antigen, IGRP. Conversely the reduction in CD8⁺ T cells with specificity for the soluble beta cell antigen, insulin, was relatively small and not significant.

Conclusions/interpretation

Elevated CD19 on NOD B cells promotes presentation of the membrane-bound antigen, IGRP, mediating the expansion of autoreactive T cells specific for antigens integral to beta cells, which are critical for invasive insulitis and diabetes. Downregulating the CD19 signalling pathway in insulin autoantibody-positive individuals before the development of type 1 diabetes may prevent expansion of islet-invasive T cells and preserve beta cell mass.     

Replication of genome-wide association studies (GWAS) loci for fasting plasma glucose in African-Americans

Aims/hypothesis

Chronically elevated blood glucose (hyperglycaemia) is the primary indicator of type 2 diabetes, which has a prevalence that varies considerably by ethnicity in the USA, with African-Americans disproportionately affected. Genome-wide association studies (GWASs) have significantly enhanced our understanding of the genetic basis of diabetes and related traits, including fasting plasma glucose (FPG). However, the majority of GWASs have been conducted in populations of European ancestry. Thus, it is important to conduct replication analyses in populations with non-European ancestry to identify shared loci associated with FPG across populations.

Methods

We used data collected from non-diabetic unrelated African-American individuals (n?=?927) who participated in the Howard University Family Study to attempt to replicate previously published GWASs of FPG. Of the 29 single nucleotide polymorphisms (SNPs) previously reported, we directly tested 20 in this study. In addition to the direct test, we queried a 500?kb window centred on all 29 reported SNPs for local replication of additional markers in linkage disequilibrium (LD).

Results

Using direct SNP and LD-based comparisons, we replicated multiple SNPs previously associated with FPG and strongly associated with type 2 diabetes in populations with European ancestry. The replicated SNPs included those in or near TCF7L2, SLC30A8, G6PC2, MTNR1B, DGKB-TMEM195 and GCKR. We also replicated additional variants in LD with the reported SNPs in ZMAT4 and adjacent to IRS1.

Conclusions/interpretation

We identified multiple GWAS variants for FPG in our cohort of African-Americans. Using an LD-based strategy we also identified SNPs not previously reported, demonstrating the utility of using diverse populations for replication analysis.     

Genetic variants at CDC123/CAMK1D and SPRY2 are associated with susceptibility to type 2 diabetes in the Japanese population

Aims/hypothesis

Recently, rs10906115 in CDC123/CAMK1D, rs1359790 near SPRY2, rs1436955 in C2CD4A/C2CD4B and rs10751301 in ODZ4 were identified as genetic risk variants for type 2 diabetes by a genome-wide association study in a Chinese population. The aim of the present study was to ascertain the role of these four variants in conferring susceptibility to type 2 diabetes in the Japanese population.

Methods

We genotyped 11,530 Japanese individuals (8,552 type 2 diabetes cases, 2,978 controls) for the above single nucleotide polymorphisms (SNPs) and used logistic regression analysis to determine whether they were associated with type 2 diabetes.

Results

In accordance with the findings in a Chinese population, rs10906115 A, rs1359790 C and rs1436955 G were found to be risk alleles. Both rs10906115 and rs1359790 were significantly associated with susceptibility to type 2 diabetes in our study (rs10906115 OR 1.15, 95% CI 1.08, 1.22; p?=?6.10?×?10^?6; rs1359790 OR 1.14, 95% CI 1.06, 1.21; p?=?2.24?×?10^?4). Adjustment for age, sex and BMI had no significant effects on the association between these variants and the disease. We did not observe any significant associations between the SNPs and any metabolic traits, e.g. BMI, fasting plasma glucose (determined for 1,332 controls), HOMA of beta cell function (900 controls) and HOMA of insulin resistance (900 controls; p?>?0.05).

Conclusions/interpretation

The SNPs rs10906115 A and rs1359790 C are significantly associated with susceptibility to type 2 diabetes in the Japanese population, confirming that these alleles are common susceptibility variants for type 2 diabetes in East Asian populations.     

Epidemiology and genetic determinants of progressive deterioration of glycaemia in American Indians: the Strong Heart Family Study

Aims/hypothesis

Type 2 diabetes is a chronic, heterogeneous disease and a major risk factor for cardiovascular diseases. The underlying mechanisms leading to progression to type 2 diabetes are not fully understood and genetic tools may help to identify important pathways of glycaemic deterioration.

Methods

Using prospective data on American Indians from the Strong Heart Family Study, we identified 373 individuals defined as progressors (diabetes incident cases), 566 individuals with transitory impaired fasting glucose (IFG) and 1,011 controls (normal fasting glycaemia at all visits). We estimated the heritability (h²) of the traits and the evidence for association with 16 known variants identified in type 2 diabetes genome-wide association studies.

Results

We noted high h² for diabetes progression (h²?=?0.65??±??0.16, p?=?2.7?×?10^?6) but little contribution of genetic factors to transitory IFG (h²?=?0.09??±??0.10, p?=?0.19) for models adjusted for multiple risk factors. At least three variants (in WFS1, TSPAN8 and THADA) were nominally associated with diabetes progression in age- and sex-adjusted analyses with estimates showing the same direction of effects as reported in the discovery European ancestry studies.

Conclusions/interpretation

Our findings do not exclude these loci for diabetes susceptibility in American Indians and suggest phenotypic heterogeneity of the IFG trait, which may have implications for genetic studies when diagnosis is based on a single time-point measure.     

Thiopurine S-methyltransferase (TPMT) Activity Is Better Determined by Biochemical Assay Versus Genotyping in the Jewish Population

Background

Thiopurine S-methyltransferase (TPMT) is a key enzyme that deactivates thiopurines, into their inactive metabolite, 6-methylmercaptopurine. Intermediate and low TPMT activity may lead to leukopenia following thiopurine treatment. The aim of this study was to determine TPMT activity and TPMT alleles (genotype–phenotype correlation) in Jews, aiming to develop an evidence-based pharmacogenetic assay for this population.

Methods

TPMT activity was determined in 228 Jewish volunteers by high performance liquid chromatography. Common allelic variants in the Caucasian population [TPMT*2 (G238C), TPMT *3A (G460A and A719G), TPMT* 3B (G460A) and TPMT*3C (A719G)] were tested. Phenotype–genotype correlation was examined and discordant cases were fully sequenced to identify novel genetic variants.

Results

Mean TPMT activity was 15.4 ± 4 U/ml red blood cells (range 1–34). Intermediate activity was found in 33/228 (14 %) subjects and absent activity was found in one sample (0.4 %). Only eight individuals (3.5 % of the entire cohort and 24 % of those with intermediate/low activity) were identified as carriers of a TPMT genetic variant, all of whom had the TPMT*3A allele. Sequencing the entire TPMT coding region and splice junctions in the remainder of the discordant cases did not reveal any novel variants.

Conclusion

Genotyping TPMT in Jews yields a much lower rate of variants than identified in the general Caucasian population. We conclude that a biochemical assay to determine TPMT enzymatic activity should be performed in Jews before starting thiopurine treatment in order to identify low activity subjects.     

Common variation in oxidative phosphorylation genes is not a major cause of insulin resistance or type 2 diabetes

Aims/hypothesis

There is substantial evidence that mitochondrial dysfunction is linked to insulin resistance and is present in several tissues relevant to the pathogenesis of type 2 diabetes. Here, we examined whether common variation in genes involved in oxidative phosphorylation (OxPhos) contributes to type 2 diabetes susceptibility or influences diabetes-related metabolic traits.

Methods

OxPhos gene variants (n?=?10) that had been nominally associated (p?n?=?10,108) were selected for follow-up in 3,599 type 2 diabetic and 4,956 glucose-tolerant Danish individuals. A meta-analysis of these variants was performed in 11,729 type 2 diabetic patients and 43,943 non-diabetic individuals. The impact on OGTT-derived metabolic traits was evaluated in 5,869 treatment-naive individuals from the Danish Inter99 study.

Results

The minor alleles of COX10 rs9915302 (p?=?0.02) and COX5B rs1466100 (p?=?0.005) showed nominal association with type 2 diabetes in our Danish cohort. However, in the meta-analysis, none of the investigated variants showed a robust association with type 2 diabetes after correction for multiple testing. Among the alleles potentially associated with type 2 diabetes, none negatively influenced surrogate markers of insulin sensitivity in non-diabetic participants, while the minor alleles of UQCRC1 rs2228561 and COX10 rs10521253 showed a weak (p?p?Conclusions/interpretation We cannot rule out the possibility that common variants in or near OxPhos genes may influence beta cell function in non-diabetic individuals. However, our quantitative trait studies and a sufficiently large meta-analysis indicate that common variation in proximity to the examined OxPhos genes is not a major cause of insulin resistance or type 2 diabetes.     

A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets

Aims/hypothesis

Glucagon-like peptide 1 (GLP-1) is a major incretin, mainly produced by the intestinal L cells, with beneficial actions on pancreatic beta cells. However, while in vivo only very small amounts of GLP-1 reach the pancreas in bioactive form, some observations indicate that GLP-1 may also be produced in the islets. We performed comprehensive morphological, functional and molecular studies to evaluate the presence and various features of a local GLP-1 system in human pancreatic islet cells, including those from type 2 diabetic patients.

Methods

The presence of insulin, glucagon, GLP-1, proconvertase (PC) 1/3 and PC2 was determined in human pancreas by immunohistochemistry with confocal microscopy. Islets were isolated from non-diabetic and type 2 diabetic donors. GLP-1 protein abundance was evaluated by immunoblotting and matrix-assisted laser desorption–ionisation-time of flight (MALDI–TOF) mass spectrometry. Single alpha and beta cell suspensions were obtained by enzymatic dissociation and FACS sorting. Glucagon and GLP-1 release were measured in response to nutrients.

Results

Confocal microscopy showed the presence of GLP-1-like and PC1/3 immunoreactivity in subsets of alpha cells, whereas GLP-1 was not observed in beta cells. The presence of GLP-1 in isolated islets was confirmed by immunoblotting, followed by mass spectrometry. Isolated islets and alpha (but not beta) cell fractions released GLP-1, which was regulated by glucose and arginine. PC1/3 (also known as PCSK1) gene expression was shown in alpha cells. GLP-1 release was significantly higher from type 2 diabetic than from non-diabetic isolated islets.

Conclusions/interpretation

We have shown the presence of a functionally competent GLP-1 system in human pancreatic islets, which resides in alpha cells and might be modulated by type 2 diabetes.     

TLR2/6 and TLR4-activated macrophages contribute to islet inflammation and impair beta cell insulin gene expression via IL-1 and IL-6

Aims/hypothesis

Inflammation contributes to pancreatic beta cell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in recently diagnosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequences of a high-fat diet. Here we investigated the role of macrophages in TLR2/6- and TLR4-mediated effects on islet inflammation and beta cell function.

Methods

Genetic and pharmacological approaches were used to determine the effects of TLR2/6 and TLR4 ligands on mouse islets, human islets and purified rat beta cells. Islet macrophages were depleted and sorted by flow cytometry and the effects of TLR2/6- and TLR4-activated bone-marrow-derived macrophages (BMDMs) on beta cell function were assessed.

Results

Macrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expression in mouse and human islets and IL-1β secretion from human islets. TLR2/6 and TLR4 ligands also reduced insulin gene expression; however, this occurred in a non-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretion partly via reducing Ins1, Ins2, and Pdx1 mRNA expression. Antagonism of the IL-1 receptor and neutralisation of IL-6 completely reversed the effects of activated macrophages on beta cell gene expression.

Conclusions/interpretation

We conclude that islet macrophages are major contributors to islet IL-1β secretion in response to TLR2/6 and TLR4 ligands. BMDMs stimulated with TLR2/6 and TLR4 ligands reduce insulin secretion from pancreatic beta cells, partly via IL-1β- and IL-6-mediated decreased insulin gene expression.     

Sleep duration does not mediate or modify association of common genetic variants with type 2 diabetes

Aims/hypothesis

Short and long sleep duration are associated with increased risk of type 2 diabetes. We aimed to investigate whether genetic variants for fasting glucose or type 2 diabetes associate with short or long sleep duration and whether sleep duration modifies the association of genetic variants with these traits.

Methods

We examined the cross-sectional relationship between self-reported habitual sleep duration and prevalence of type 2 diabetes in individuals of European descent participating in five studies included in the Candidate Gene Association Resource (CARe), totalling 1,474 cases and 8,323 controls. We tested for association of 16 fasting glucose-associated variants, 27 type 2 diabetes-associated variants and aggregate genetic risk scores with continuous and dichotomised (≤5 h or ≥9 h) sleep duration using regression models adjusted for age, sex and BMI. Finally, we tested whether a gene × behaviour interaction of variants with sleep duration had an impact on fasting glucose or type 2 diabetes risk.

Results

Short sleep duration was significantly associated with type 2 diabetes in CARe (OR 1.32; 95% CI 1.08, 1.61; p?=?0.008). Variants previously associated with fasting glucose or type 2 diabetes and genetic risk scores were not associated with sleep duration. Furthermore, no study-wide significant interaction was observed between sleep duration and these variants on glycaemic traits. Nominal interactions were observed for sleep duration and PPARG rs1801282, CRY2 rs7943320 and HNF1B rs4430796 in influencing risk of type 2 diabetes (p?<?0.05).

Conclusions/interpretation

Our findings suggest that differences in habitual sleep duration do not mediate or modify the relationship between common variants underlying glycaemic traits (including in circadian rhythm genes) and diabetes.     

Imaging dynamics of CD11c+ cells and Foxp3+ cells in progressive autoimmune insulitis in the NOD mouse model of type 1 diabetes

Aims/hypothesis

The aim of this study was to visualise the dynamics and interactions of the cells involved in autoimmune-driven inflammation in type 1 diabetes.

Methods

We adopted the anterior chamber of the eye (ACE) transplantation model to perform non-invasive imaging of leucocytes infiltrating the endocrine pancreas during initiation and progression of insulitis in the NOD mouse. Individual, ACE-transplanted islets of Langerhans were longitudinally and repetitively imaged by stereomicroscopy and two-photon microscopy to follow fluorescently labelled leucocyte subsets.

Results

We demonstrate that, in spite of the immune privileged status of the eye, the ACE-transplanted islets develop infiltration and beta cell destruction, recapitulating the autoimmune insulitis of the pancreas, and exemplify this by analysing reporter cell populations expressing green fluorescent protein under the Cd11c or Foxp3 promoters. We also provide evidence that differences in morphological appearance of subpopulations of infiltrating leucocytes can be correlated to their distinct dynamic behaviour.

Conclusions/interpretation

Together, these findings demonstrate that the kinetics and dynamics of these key cellular components of autoimmune diabetes can be elucidated using this imaging platform for single cell resolution, non-invasive and repetitive monitoring of the individual islets of Langerhans during the natural development of autoimmune diabetes.     

A variant in the G6PC2/ABCB11 locus is associated with increased fasting plasma glucose, increased basal hepatic glucose production and increased insulin release after oral and intravenous glucose loads

Aims/hypothesis

An association between elevated fasting plasma glucose and the common rs560887 G allele in the G6PC2/ABCB11 locus has been reported. In Danes we aimed to examine rs560887 in relation to plasma glucose and serum insulin responses following oral and i.v. glucose loads and in relation to hepatic glucose production during a hyperinsulinaemic–euglycaemic clamp. Furthermore, we examined rs560887 for association with impaired fasting glycaemia (IFG), impaired glucose tolerance (IGT), type 2 diabetes and components of the metabolic syndrome.

Methods

rs560887 was genotyped in the Inter99 cohort (n?=?5,899), in 366 young, healthy Danes, in non-diabetic relatives of type 2 diabetic patients (n?=?196), and in young and elderly twins (n?=?159). Participants underwent an OGTT, an IVGTT or a 2 h hyperinsulinaemic–euglycaemic clamp.

Results

The rs560887 G allele associated with elevated fasting plasma glucose (p?=?2?×?10^?14) but not with plasma glucose levels at 30 min (p?=?0.9) or 120 min (p?=?0.9) during an OGTT. G allele carriers had elevated levels of serum insulin at 30 min during an OGTT (p?=?1?×?10^?4) and relatives of type 2 diabetes patients carrying the G allele had an increased acute insulin response (p?=?4?×?10^?4) during an IVGTT. Among elderly twins, G allele carriers had higher basal hepatic glucose production (p?=?0.04). Finally, the G allele associated with the risk of having IFG (OR 1.26, 95% CI 1.08–1.47, p?=?0.002), but not with IGT (OR 0.94, 95% CI 0.82–1.08, p?=?0.4) or type 2 diabetes (OR 0.93, 95% CI 0.84–1.04, p?=?0.2).

Conclusions/interpretation

The common rs560887 G allele in the G6PC2/ABCB11 locus is associated with increased fasting glycaemia and increased risk of IFG, associations that may be partly related to an increased basal hepatic glucose production rate.     

Common genetic variants differentially influence the transition from clinically defined states of fasting glucose metabolism

Aims/hypothesis

Common genetic variants have been associated with type 2 diabetes. We hypothesised that a subset of these variants may have different effects on the transition from normal fasting glucose (NFG) to impaired fasting glucose (IFG) than on that from IFG to diabetes.

Methods

We identified 16 type 2 diabetes risk variants from the Illumina Broad Candidate-gene Association Resource (CARe) array genotyped in 26,576 CARe participants. Participants were categorised at baseline as NFG, IFG or type 2 diabetic (n?=?16,465, 8,017 or 2,291, respectively). Using Cox proportional hazards and likelihood ratio tests (LRTs), we compared rates of progression by genotype for 4,909 (NFG to IFG) and 1,518 (IFG to type 2 diabetes) individuals, respectively. We then performed multinomial regression analyses at baseline, comparing the risk of assignment to the NFG, IFG or diabetes groups by genotype.

Results

The rate of progression from NFG to IFG was significantly greater in participants carrying the risk allele at MTNR1B (p?=?1?×?10^?4), nominally greater at GCK and SLC30A8 (p?IGF2BP2 (p?=?0.01) than the rate of progression from IFG to diabetes by the LRT. Results of the baseline, multinomial regression model were consistent with these findings.

Conclusions/interpretation

Common genetic risk variants at GCK, SLC30A8, IGF2BP2 and MTNR1B influence to different extents the development of IFG and the transition from IFG to type 2 diabetes. Our findings may have implications for understanding the genetic contribution of these variants to the development of IFG and type 2 diabetes.     

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.     

Cardiotrophin 1 protects beta cells from apoptosis and prevents streptozotocin-induced diabetes in a mouse model

Aims/hypothesis

Cardiotrophin 1 (CT-1) is a recently described cytokine originally isolated from the heart where it has been shown to play an important role in apoptotic protection of cardiomyocytes and heart hypertrophy. Its beneficial properties have also been described in other organs such as liver and neuromuscular tissue. In the present study, we investigated whether CT-1 can confer protection against pro-apoptotic stimuli in pancreatic beta cells, and its role in insulin secretion and diabetes development.

Methods

The effects of CT-1 on apoptosis and function were studied using MIN6B1 cells and freshly isolated murine pancreatic islets. The impact on the development of diabetes was evaluated in Ct1-null (Ct1 ^?/?) mice (the gene Ct1 is also known as Ctf1) using two streptozotocin (STZ)-induced models of diabetes.

Results

CT-1 has a protective effect in MIN6B1 cells and murine islets under the pro-apoptotic stimulus of serum deprivation, which correlates with the expression of B cell lymphoma-extra large, or following exposure to a mixture of cytokines. In addition, CT-1 enhances glucose-stimulated insulin secretion in MIN6B1 cells and this was repressed by inhibitors of phospholipase C. Furthermore, Ct1 ^?/? mice were more prone to develop diabetes, and their glucose tolerance test showed impaired plasma glucose clearance which correlated with decreased pancreatic insulin secretion.

Conclusions/interpretation

The results obtained from both in vitro and in vivo experiments show that CT-1 improves beta cell function and survival, and protects mice against STZ-induced diabetes.