Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes

Human and rodent islets differ substantially in several features, including architecture, cell composition, gene expression and some aspects of insulin secretion. Mouse pancreatic islets are highly vascularized with interactions between islet endothelial and endocrine cells being important for islet cell differentiation and function. To determine whether human islets have a similar high degree of vascularization and whether this is altered with diabetes, we examined the vascularization of islets from normal human subjects, subjects with type 2 diabetes (T2D), and normal mice. Using an integrated morphometry approach to quantify intra-islet capillary density in human and mouse pancreatic sections, we found that human islets have five-fold fewer vessels per islet area than mouse islets. Islets in pancreatic sections from T2D subjects showed capillary thickening, some capillary fragmentation and had increased vessel density as compared with non-diabetic controls. These changes in islet vasculature in T2D islets appeared to be associated with amyloid deposition, which was noted in islets from 8/9 T2D subjects (and occupied 14% ± 4% of islet area), especially around the intra-islet capillaries. The physiological implications of the differences in the angioarchitecture of mouse and human islets are not known. Islet vascular changes in T2D may exacerbate β cell/islet dysfunction and β cell loss.     

Altered islet composition and disproportionate loss of large islets in patients with type 2 diabetes

Human islets exhibit distinct islet architecture with intermingled alpha- and beta-cells particularly in large islets. In this study, we quantitatively examined pathological changes of the pancreas in patients with type 2 diabetes (T2D). Specifically, we tested a hypothesis that changes in endocrine cell mass and composition are islet-size dependent. A large-scale analysis of cadaveric pancreatic sections from T2D patients (n = 12) and non-diabetic subjects (n = 14) was carried out combined with semi-automated analysis to quantify changes in islet architecture. The method provided the representative islet distribution in the whole pancreas section that allowed us to examine details of endocrine cell composition in individual islets. We observed a preferential loss of large islets (>60 µm in diameter) in T2D patients compared to non-diabetic subjects. Analysis of islet cell composition revealed that the beta-cell fraction in large islets was decreased in T2D patients. This change was accompanied by a reciprocal increase in alpha-cell fraction, however total alpha-cell area was decreased along with beta-cells in T2D. Delta-cell fraction and area remained unchanged. The computer-assisted quantification of morphological changes in islet structure minimizes sampling bias. Significant beta-cell loss was observed in large islets in T2D, in which alpha-cell ratio reciprocally increased. However, there was no alpha-cell expansion and the total alpha-cell area was also decreased. Changes in islet architecture were marked in large islets. Our method is widely applicable to various specimens using standard immunohistochemical analysis that may be particularly useful to study large animals including humans where large organ size precludes manual quantitation of organ morphology.     

Gender differences in the relationship of ENPP1/PC-1 variants to obesity in a Turkish population

The ectoenzyme ENPP1 (also termed membrane glycoprotein PC-1 or ENPP1/PC-1) is an inhibitor of insulin-induced activation of the insulin receptor. There is evidence from previous studies that coding variants of ENPP1/PC-1 (K121Q) are associated with type 2 diabetes (T2D) and obesity. Studies in the general Turkish population have demonstrated: unique plasma lipid characteristics, a high prevalence of cardiovascular risk factors, and an increased prevalence of obesity and T2D. We investigated, therefore, the association of ENPP1/PC-1 variants with obesity and T2D in Turkish individuals. The TaqMan allelic discrimination assay was used for genotyping the relationship of ENPP1/PC-1 variants to obesity and T2D in a genetic association study of 1,553 genotyped, randomly selected subjects from the Turkish Heart Study. The K121Q (rs1044498) variant and other previously reported variants (rs997509, rs1799774, rs1044548, rs11964389, rs7754561) were analyzed. In this cohort, the minor allele frequency (MAF) of the K121Q variant was associated with obesity in male, but not in female subjects (male, odds ratio 1.64, 95% confidence interval 1.004-2.698, P = 0.048; female, odds ratio 1.003, 95% confidence interval 0.684-1.471, P = ns). In addition, the previously reported ENPP1/PC-1 "risk haplotype" (Q (rs1044498), delT (rs1799774), and G (rs7754561) alleles) was found to be associated with obesity in male, but not in female, subjects (P = 0.035). In contrast, there was no association of either the K121Q variant or the ENPP1/PC-1 haplotype with T2D. We find evidence that variants of ENPP1/PC-1 are associated with obesity in the male Turkish population; thus, these variants may contribute to the development of the obesity in these individuals.     

Replication of association between polymorphisms of the pancreatic ATP-sensitive potassium channel and susceptibility to type 2 diabetes in two Russian urban populations

The KCNJ11 and ABCC8 genes encode components of the pancreatic ATP-sensitive potassium (KATP) channel. Previously, we reported association of the KCNJ11 E23K and ABCC8 R1273R G/A variants with type 2 diabetes (T2D) in a small Russian population sample (n=244). Here we replicated association between these genetic variants and T2D in a larger cohort (588 diabetic and 597 non-diabetic subjects). Using the ANCOVA analysis, Odds Ratios (ORs) and relationships between the carriage of a genotype and biochemical parameters of the patients were assessed and then adjusted for confounders (age, gender, HbA1c, hypertension, and obesity). The KCNJ11 K23 variant and the ABCC8 R1273R allele A showed association with higher risk of T2D (adjusted OR of 1.41 and 2.03, P<0.0001, respectively). Diabetic patients homozygous for K/K had lower 2h insulin (P_adjusted=0.044). The ABCC8 A/A variant was associated with increased 2h serum insulin in diabetic and non-diabetic subjects (P_adjusted=0.027 and 0.033, respectively). The carriage of the risk variant K/K of KCNJ11 E23K or A/A of ABCC8 G/A R1273R was associated with reduced response to nonsulfonylurea and sulfonylurea blockers of the pancreatic KATP channel. Adjusted attributable population risk was 3.0% (KCNJ11 E23K) and 4.8% (ABCC8 G/A) suggesting for the modest effects of these genetic variants on diabetes susceptibility.     

An Exploratory Study of the Association between KCNB1 rs1051295 and Type 2 Diabetes and Its Related Traits in Chinese Han Population

Since the KCNB1 encoding Kv2.1 channel accounts for the majority of Kv currents modulating insulin secretion by pancreatic islet beta-cells, we postulated that KCNB1 is a plausible candidate gene for genetic variation contributing to the variable compensatory secretory function of beta-cells in type-2 diabetes (T2D). We conducted two studies, a case-control study and a cross-section study, to investigate the association of common single-nucleotide polymorphisms (SNPs) in KCNB1 with T2D and its linking traits. In the case-control study, we first examined the association of 20 tag SNPs of KCNB1 with T2D in a population with 226 T2D patients and non-diabetic subjects (screening study). We then identified the association in an enlarged population of 412 T2D patients and non-diabetic subjects (replication study). In the cross-sectional study, we investigated the linkage between the candidate SNP rs1051295 and T2D by comparing beta-cell function and insulin sensitivity among rs1051295 genotypes in a general population of 1051 subjects at fasting and after glucose loading (oral glucose tolerance tests, OGTT) in 84 fasting glucose impaired subjects, and several T2D-related traits. We found that among the 19 available tag SNPs, only the KCNB1 rs1051295 was associated with T2D (P = 0.027), with the rs1051295 TT genotype associated with an increased risk of T2D compared with genotypes CC (P = 0.009). At fasting, rs1051295 genotype TT was associated with a 9.8% reduction in insulin sensitivity compared to CC (P = 0.008); along with increased plasma triglycerides (TG) levels (TT/CC: P = 0.046) and increased waist/hip (W/H) ratio (TT/CC: P = 0.013; TT/TC: P = 0.002). OGTT confirmed that genotype TT exhibited reduced insulin sensitivity by 16.3% (P = 0.030) compared with genotype TC+CC in a fasting glucose impaired population. The KCNB1 rs1051295 genotype TT in the Chinese Han population is associated with decreased insulin sensitivity and increased plasma TG and W/H ratio, which together contribute to an increased risk for T2D.     

Polymorphic variant at the IL2 region is associated with type 1 diabetes and may affect serum levels of interleukin-2

Polymorphic variants at the interleukin-2 (IL2) locus affect the risk of several autoimmune disorders. Our aim was to evaluate the association of the four IL2 polymorphisms (rs6822844, rs6534349, rs2069762 and rs3136534) with type 1 diabetes (T1D) in the Polish population, and to correlate them with the serum interleukin-2 levels. 543 unrelated T1D patients and 706 healthy control subjects were enrolled. The minor T allele at rs6822844 was significantly less frequent in T1D compared to controls (p = 0.002; OR 0.71; 95 % CI 0.571–0.880). Likewise, the frequency of the TT genotype was decreased among the affected individuals (p = 0.007). In healthy subjects, stratification according to the rs6822844 genotype revealed significant differences in circulating interleukin-2 (p = 0.037) with the highest levels in TT protective genotypes. Three other IL2 polymorphisms did not display significant differences in allele and genotype distribution. In conclusion, the rs6822844 variant is associated with T1D and may play a functional role, or reflect the influence of another causative genetic variant in linkage disequilibrium.     

Regional Differences in Islet Distribution in the Human Pancreas - Preferential Beta-Cell Loss in the Head Region in Patients with Type 2 Diabetes

While regional heterogeneity in islet distribution has been well studied in rodents, less is known about human pancreatic histology. To fill gaps in our understanding, regional differences in the adult human pancreas were quantitatively analyzed including the pathogenesis of type 2 diabetes (T2D). Cadaveric pancreas specimens were collected from the head, body and tail regions of each donor, including subjects with no history of diabetes or pancreatic diseases (n = 23) as well as patients with T2D (n = 12). The study further included individuals from whom islets were isolated (n = 7) to study islet yield and function in a clinical setting of islet transplantation. The whole pancreatic sections were examined using an innovative large-scale image capture and unbiased detailed quantitative analyses of the characteristics of islets from each individual (architecture, size, shape and distribution). Islet distribution/density is similar between the head and body regions, but is >2-fold higher in the tail region. In contrast to rodents, islet cellular composition and architecture were similar throughout the pancreas and there was no difference in glucose-stimulated insulin secretion in islets isolated from different regions of the pancreas. Further studies revealed preferential loss of large islets in the head region in patients with T2D. The present study has demonstrated distinct characteristics of the human pancreas, which should provide a baseline for the future studies integrating existing research in the field and helping to advance bi-directional research between humans and preclinical models.     

Association analysis of v-AKT murine thymoma viral oncogene homolog 1 (AKT1) polymorphisms and type 2 diabetes mellitus in the Korean population

V-AKT murine thymoma viral oncogene homolog 1 (AKT1) is an important downstream target of the insulin-signaling pathway and may be an important regulator of pancreatic beta cell growth. This study investigated the association of theAKT1 gene with susceptibility to type 2 diabetes mellitus and its related traits. By sequencing theAKT1 gene in 24 unrelated individuals, we iden-tified 32 genetic variations including 30 single nucleotide polymorphisms and 2 deletions. For the association analysis, we selected seven single nucleotide polymorphisms (rs10138227, ?726G>A; rs3730358, +12574C>T; rs2494737, +12656T>A; rs2498796, +15761T>C; rs2498799, +19087 A>G; rs2494732, +19789G>A; rs3803304, +19835G>C) based on minor allele frequency (>0.05) and linkage disequilibrium status. The study included 483 type 2 diabetes patients (206 men and 277 women with mean age 64±2.8 years and mean age at onset 56 ± 8.1 years) and 1,138 non-diabetic control subjects (516 men and 622 women with mean age 64 ±2.9 years). Two single nucleotide polymorphisms (rs2498796, +15761T>C and rs2494732, +19789G>A) were found to be associated with risk of type 2 diabetes mellitus, and showed an increased risk of type 2 diabetes mellitus in a recessive model (OR=1.343, 95% CI 1.021–1.765,p=0.035 and OR=1.534, 95% CI 1.058–2.225,p=0.024, respectively). These SNPs were also associated with diabetes-related traits such as levels of fasting blood glucose and hemoglobin A1c. In addition, type 2 diabetes mellitus patients who also have dyslipidemia or high blood pressure showed significant association with single nucleotide polymorphisms in AKT1 when compared with healthy controls. These results indicate that genetic variation in AKT1 influences the development of type 2 diabetes mellitus in the Korean population.     

Localisation of islet amyloid peptide in lipofuscin bodies and secretory granules of human B-cells and in islets of type-2 diabetic subjects

Summary Islet amyloid peptide (or diabetes-associated peptide), the major component of pancreatic islet amyloid found in type-2 diabetes, has been identified by electronmicroscopic immunocytochemistry in pancreatic B-cells from five non-diabetic human subjects, and in islets from five type-2 diabetic patients. The greatest density of immunoreactivity for islet amyloid peptide was found in electrondense regions of some lysosomal or lipofuscin bodies. The peptide was also localised by quantification of immunogold in the secretory granules of B-cells, and was present in cytoplasmic lamellar bodies. Acid phosphatase activity was also demonstrated in these organelles. Immunoreactivity for insulin was found in some lysosomes. These results suggest that islet amyloid peptide is a constituent of normal pancreatic B-cells, and accumulates in lipofuscin bodies where it is presumably partially degraded. In islets from type-2 diabetic subjects, amyloid fibrils and lipofuscin bodies in B-cells showed immunoreactivity for the amyloid peptide. Abnormal processing of the peptide within B-cells could lead to the formation of islet amyloid in type-2 diabetes.     

Genetic polymorphisms at the human liver/islet glucose transporter (GLUT2) gene locus in Caucasian and West Indian subjects with type 2 (non-insulin-dependent) diabetes mellitus.

The liver/islet glucose transporter (GLUT2) is mainly expressed in the hepatocytes of the liver and the beta-cells of the pancreatic islets and a defect in this transporter could lead to diabetic phenotypes, such as relative hypoinsulinaemia and reduced uptake and metabolism of glucose in the liver. DNA from unrelated individuals was digested with the restriction endonucleases Bgl-I and Taq-I followed by blotting and hybridisation with a 32P-labelled GLUT2 cDNA which revealed three restriction fragment length polymorphisms (RFLPs) (B1, T1 and T2) in both Caucasian and West Indian populations. Linkage analysis between these variant sites demonstrated that the alleles of these polymorphisms were in strong linkage disequilibrium. Disease association of genetic variants at the GLUT2 locus with type 2 diabetes was examined with these RFLPs in both Caucasian (n = 54) and West Indian (n = 46) populations with type 2 diabetes. There were no significant differences in the frequency of alleles, genotypes or haplotypes between diabetic patients and non-diabetic controls. However, there were significant differences in the allele frequencies of all these three polymorphisms between Caucasian and West Indian populations.     

Vitamin D receptor gene polymorphisms and HLA DRB1*04 cosegregation in Saudi type 2 diabetes patients

The vitamin D receptor (VDR) gene has been involved in the modulation of susceptibility to inflammatory and autoimmune conditions, and could play a role in the pathogenesis of type 2 diabetes mellitus (T2DM). Susceptibility to T2DM was recently also suggested to associate with HLA alleles. We evaluated possible correlations between VDR polymorphisms, HLA alleles, and risk for development of T2DM by analyzing 627 individuals (368 T2DM patients and 259 healthy control subjects) part of a well-characterized cohort followed in Riyadh, Kingdom of Saudi Arabia. Genomic DNA was genotyped for the VDR gene single nucleotide polymorphisms of Fok-1, Taq-1, ApaI, and Bsm-I. Analyses were run by allelic discrimination real-time PCR. HLA genotyping was performed as well by PCR using sequence-specific primers, whereas cytokine production was evaluated by FACS. Results showed T2DM to be significantly associated with the VDR Taq1 (rs731236-AG) and Bsm-I (rs1544410-CT) genotypes, and the VDR rs1544410-T allele. Cosegregations resulting in significant increases of T2DM odds ratio were detected between Taq1 and Bsm-I VDR polymorphisms and HLA DRB1*04. Notably, the VDR polymorphisms observed to be more frequent in T2DM patients correlated with increased VDR expression and IL-12 production, as well as with metabolic parameters of susceptibility to T2DM, including serum cholesterol and high-density lipoprotein levels. VDR polymorphisms are present in T2DM, and correlate with HLA DRB1*04 and with immunologic and metabolic parameters; results from this study add T2DM to the list of diseases that are likely modulated by an HLA/VDR interaction.     

Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.