Diabetes

Published by American Diabetes Association
Online ISSN: 0012-1797
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Primer sequences 
Article
Currently, 16 loci that contribute to the development of IDDM in the NOD mouse have been mapped by linkage analysis. To fine map these loci, we used congenic mapping. Using this approach, we localized the Idd3 locus to a 0.35-cM interval on chromosome 3 containing the Il2 gene. Segregation analysis of the known variations within this interval indicated that only one variant, a serine-to-proline substitution at position 6 of the mature interleukin-2 (IL-2) protein, consistently segregates with IDDM in crosses between NOD and a series of nondiabetic mouse strains. These data, taken together with the immunomodulatory role of IL-2, provide circumstantial evidence in support of the hypothesis that Idd3 is an allelic variation of the Il2 gene, or a variant in strong linkage disequilibrium.
 
Article
The primary associations of the HLA class II genes, HLA-DRB1 and HLA-DQB1, and the class I genes, HLA-A and HLA-B, with type 1 diabetes (T1D) are well established. However, the role of polymorphism at the HLA-DRB3, HLA-DRB4, and HLA-DRB5 loci remains unclear. In two separate studies, one of 500 cases and 500 controls and one of 366 DRB1*03:01-positive samples from selected multiplex T1D families, we used Roche 454 sequencing with Conexio Genomics ASSIGN™ATF HLA genotyping software analysis to analyze sequence variation at these three HLA-DRB loci. Association analyses were performed on the two HLA-DRB locus (DRB1-DRB3, -DRB4, or -DRB5) haplotypes. Three common HLA-DRB3 alleles (*01:01, *02:02, *03:01) were observed. DRB1*03:01 haplotypes carrying DRB3*02:02 conferred a higher (p=0.033) T1D risk than did DRB1*03:01 haplotypes carrying DRB3*01:01, primarily in DRB1*03:01/*03:01 homozygotes with two DRB3*01:01 alleles (OR = 3.4 95% CI= 1.46-8.09), compared with those carrying one or two DRB3*02:02 alleles (OR = 25.5; 95% CI - 3.43-189.2). For DRB1*03:01/*04:01 heterozygotes, however, the HLA-DRB3 allele did not significantly modify the T1D risk of the DRB1*03:01 haplotype (OR = 7.7 for *02:02; OR = 6.8 for *01:01). These observations were confirmed by sequence analysis of HLA-DRB3 exon 2 in a targeted replication study of 280 informative T1D family members and 86 Affected Family Based Control (AFBAC) haplotypes. The frequency of DRB3*02:02 was 42.9% in the DRB1*03:01/*03:01 patients and 27.6% in the DRB1*03:01/*04 (p = 0.005), compared to 22.6% in AFBAC DRB1*03:01 chromosomes (p = 0.001). Analysis of T1D associated alleles at other HLA loci (HLA-A, HLA-B and HLA-DPB1) on DRB1*03:01 haplotypes suggests that DRB3*02:02 on the DRB1*03:01 haplotype can contribute to T1D risk.
 
Article
In an attempt to elucidate the basic genetic defect (s) in diabetes, the acute effect of insulin on the conversion of glucose-C-14 to C-14-02 in cultured human diploid fibroblasts was measured under various conditions. Freshly trypsinized cells did not respond to insulin, but after more than sixteen hours in growth medium, a small but significant stimulation, requiring high concentrations of insulin (0.26 U. per ml.) was observed. Stimulation by insulin in various normal strains ranged from 5 to 45 per cent with a mean of 18 per cent (p < .001). C-14-O2 production was approximately linear with time after an initial lag phase and proportional to the number of cells inoculated. Increasing the concentration of glucose in the medium increased C-14-O2 production, with stimulation by insulin apparent at only very low concentrations and at 250 mg. per 100 ml., the highest concentration studied. Experiments comparing the oxidation of glucose-l-C-14 versus glucose-6-C-14 showed that substantial C-14-O2 production occurred via the hexose monophosphate shunt, but that most of the increase in C-14-O2 production in the presence of insulin occurred via the Krebs cycle. A comparison of basal and insulin-stimulated C-14-O2 production in normal and diabetic fibroblasts revealed no significant differences. Possible explanations for the failure to distinguish between the two groups are discussed.
 
Article
The human leukocyte antigen (HLA) class II genotype DQA1*0301-DQB1*0302/DQA1*0501-DQB1*0201 has been identified as a marker strongly predisposing to insulin-dependent diabetes mellitus (IDDM) in Caucasian populations. Its frequency in control populations (1-3%) is still, however, 1 order of magnitude higher than the prevalence of IDDM, suggesting that its penetrance can be modified by protective factors. In this study we searched for such a factor in the DRB1 locus by studying DRB1*04 polymorphism in 174 European Caucasian IDDM patients and 73 nondiabetic control subjects, all sharing the HLA-DR3/DR4 phenotype. Significant protection was encoded by the DRB1*0403 allele, which was observed in 5 of 49 control subjects (10%) and none of 171 IDDM patients (0%) with the DQA1*0301-DQB1*0302/DQA1*0501-DQB1*0201 genotype (RR = 0.02 [0.01-0.18], P < 0.0005). These data support the concept that protective HLA class II genes can overrule the risk caused by HLA-DQ susceptibility dimers. They also contribute to a possible strategy to screen for nondiabetic individuals with increased genetic risk of developing IDDM.
 
Protective effects of DPB1*0402 and DRB1*0403 in DR3-DQB1*0201/DR4-DQB1*0302 NECs and SOCs. A: Anti-islet autoantibody development in NECs (newborn/general population cohort). B: Progression to diabetes in NECs. C: Anti-islet autoantibody development in SOCs (sibling/offspring cohort). D: Progression to diabetes in SOCs. , no protective alleles (no DPB1*0402 or DRB1*0403); OE, DRB1*0403 and/or DPB1*0402. 
Article
A major goal in genetic studies of type 1A diabetes is prediction of anti-islet autoimmunity and subsequent diabetes in the general population, as >85% of patients do not have a first-degree relative with type 1A diabetes. Given prior association studies, we hypothesized that the strongest candidates for enhancing diabetes risk among DR3-DQB1*0201/DR4-DQB1*0302 individuals would be alleles of DP and DRB1*04 subtypes and, in particular, the absence of reportedly protective alleles DPB1*0402 and/or DRB1*0403. We genotyped 457 DR3-DQB1*0201/DR4-DQB1*0302 Diabetes Autoimmunity Study of the Young (DAISY) children (358 general population and 99 siblings/offspring of type 1 diabetic patients) at the DPB1, DQB1, and DRB1 loci using linear arrays of immobilized sequence-specific oligonucleotides, with direct sequencing to differentiate DRB1*04 subtypes. By survival curve analysis of DAISY children, the risk of persistently expressing anti-islet autoantibodies is approximately 55% for relatives (children with a parent or sibling with type 1 diabetes) in the absence of these two protective alleles vs. 0% (P = 0.02) with either protective allele, and the risk is 20 vs. 2% (P = 0.004) for general population children. Even when the population analyzed is limited to DR3-DQB1*0201/DR4-DQB1*0302 children with DRB1*0401 (the most common DRB1*04 subtype), DPB1*0402 influences development of anti-islet autoantibodies. The ability to identify a major group of general population newborns with a 20% risk of anti-islet autoimmunity should enhance both studies of the environmental determinants of type 1A diabetes and the design of trials for the primary prevention of anti-islet autoimmunity.
 
Article
Cytotoxic T-lymphocytes (CTLs) are considered to be essential for beta-cell destruction in type 1 diabetes. However, few islet-associated peptides have been demonstrated to activate autoreactive CTLs from type 1 diabetic subjects. In an effort to identify novel epitopes, we used matrix-assisted algorithms to predict peptides of glial fibrillary acidic protein (GFAP), prepro-islet amyloid polypeptide (ppIAPP), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) that likely bind to HLA-A*0201 with a strong affinity and contain a COOH-terminal proteasomal cleavage site. Seven peptides stabilized HLA-A*0201 expression in binding assays and were used to stimulate peripheral blood mononuclear cells and were evaluated for granzyme B secretion. We found that 5 of 13 type 1 diabetic subjects and 4 of 6 antibody-positive relatives exhibited greater numbers of granzyme B-secreting cells in response to at least one putative epitope compared with healthy control subjects. The most prevalent responses in antibody-positive and type 1 diabetic subjects were to ppIAPP(9-17). Other peptides recognized by type 1 diabetic or antibody-positive subjects included GFAP(143-151), IGRP(152-160), and GFAP(214-222). These data implicate peptides of ppIAPP, GFAP, and IGRP as CTL epitopes for a heterogenous CD8(+) T-cell response in type 1 subjects and antibody-positive relatives.
 
Article
CD8(+) T-cells specific for islet antigens are essential for the development of type 1 diabetes in the NOD mouse model of the disease. Such T-cells can also be detected in the blood of type 1 diabetic patients, suggesting their importance in the pathogenesis of the human disease as well. The development of peptide-based therapeutic reagents that target islet-reactive CD8(+) T-cells will require the identification of disease-relevant epitopes. We used islet-infiltrating CD8(+) T-cells from HLA-A*0201 transgenic NOD mice in an interferon-gamma enzyme-linked immunospot assay to identify autoantigenic peptides targeted during the spontaneous development of disease. We concentrated on insulin (Ins), which is a key target of the autoimmune response in NOD mice and patients alike. We found that HLA-A*0201-restricted T-cells isolated from the islets of the transgenic mice were specific for Ins1 L3-11, Ins1 B5-14, and Ins1/2 A2-10. Insulin-reactive T-cells were present in the islets of mice as young as 5 weeks of age, suggesting an important function for these specificities early in the pathogenic process. Although there was individual variation in peptide reactivity, Ins1 B5-14 and Ins1/2 A2-10 were the immunodominant epitopes. Notably, in vivo cytotoxicity to cells bearing these peptides was observed, further confirming them as important targets of the pathogenic process. The human versions of B5-14 and A2-10, differing from the murine peptides by only a single residue, represent excellent candidates to explore as CD8(+) T-cell targets in HLA-A*0201-positive type 1 diabetic patients.
 
Cytotoxicity of anti–2–11, anti– 6 –14, anti–14 –23, and anti–15–24 CD8 T-cell clones against P815 cells transfected with the HHD-encoding and the human preproinsulin genes. Individual CD8 T-cell clones were tested for cytolytic activity against HHD-and preproinsulin-transfected P815 targets loaded with relevant preproinsulin peptide (circles for 2–11, rectangles for 6 –14, triangles for 14 –23, squares for 15–24; open symbols) or without peptide (closed symbols, respectively), using chromium release assay. The figure represents the percentage of specific lysis (vertical axis) obtained for 1:1, 2:1, and 3:1 effector:target (E/T) ratio (horizontal axis).  
Binding of preproinsulin peptides to selected HLA class I alleles
Recognition of viral peptides by PBMCs of patients and of control individuals
Relative recognition frequencies of preproinsulin peptides in diabetic versus control subjects
Article
A restricted region of proinsulin located in the B chain and adjacent region of C-peptide has been shown to contain numerous candidate epitopes recognized by CD8(+) T-cells. Our objective is to characterize HLA class I-restricted epitopes located within the preproinsulin leader sequence. Seven 8- to 11-mer preproinsulin peptides carrying anchoring residues for HLA-A1, -A2, -A24, and -B8 were selected from databases. HLA-A2-restricted peptides were tested for immunogenicity in transgenic mice expressing a chimeric HLA-A*0201/beta2-microglobulin molecule. The peptides were studied for binding to purified HLA class I molecules, selected for carrying COOH-terminal residues generated by proteasome digestion in vitro and tested for recognition by human lymphocytes using an ex vivo interferon-gamma (IFN-gamma) ELISpot assay. Five HLA-A2-restricted peptides were immunogenic in transgenic mice. Murine T-cell clones specific for these peptides were cytotoxic against cells transfected with the preproinsulin gene. They were recognized by peripheral blood mononuclear cells (PBMCs) from 17 of 21 HLA-A2 type 1 diabetic patients. PBMCs from 25 of 38 HLA-A1, -A2, -A24, or -B8 patients produced IFN-gamma in response to six preproinsulin peptides covering residues 2-25 within the preproinsulin region. In most patients, the response was against several class I-restricted peptides. T-cells recognizing preproinsulin peptide were characterized as CD8(+) T-cells by staining with peptide/HLA-A2 tetramers. We defined class I-restricted epitopes located within the leader sequence of human preproinsulin through in vivo (transgenic mice) and ex vivo (diabetic patients) assays, illustrating the possible role of preproinsulin-specific CD8(+) T-cells in human type 1 diabetes.
 
Article
To elucidate the genetic factors contributing to heterogeneity of the rate of beta-cell destruction in type 1 diabetes, we investigated the relationship between the time course of complete beta-cell loss and HLA class I and II alleles. HLA allele frequencies were also examined among subgroups classified by the mode of onset. The subjects were 266 type 1 diabetic patients (among whom 196 patients were studied longitudinally) and 136 normal control subjects. Earlier complete loss of beta-cell function was observed in patients who possessed both HLA-A24 and HLA-DQA1*03 and in patients who had HLA-DR9, compared with those without these HLA alleles (P=0.0057 and 0.0093, respectively). Much earlier complete beta-cell loss was observed in the patients who possessed all of HLA-A24, -DQA1*03, and -DR9 compared with the remaining patients (P=0.0011). The combination of HLA-A24, -DQA1*03, and -DR9 showed a higher frequency in acute-onset than slow-onset type 1 diabetes (P=0.0002). In contrast, HLA-DR2 was associated with a slower rate of progression to complete beta-cell loss. These results indicate that the combination of HLA-A24, -DQA1*03, and -DR9 contributes to the acute-onset and early complete beta-cell destruction, whereas HLA-DR2 has a protective effect against complete beta-cell loss in type 1 diabetes.
 
Article
Susceptibility to IDDM has been associated with specific alleles at the HLA class II loci in a variety of human populations. Previous studies among Mexican-Americans, a group ancestrally derived from Native Americans and Hispanic whites, showed that the DR4 haplotypes (DRB1*0405-DQB1*0302 and DRB1*0402-DQB1*0302) and the DR3 haplotype (DRB1*0301-DQB1*0201) were increased among patients and suggested a role for both DR and DQ alleles in susceptibility and resistance. Based on the analysis of 42 Mexican-American IDDM families and ethnically matched control subjects by polymerase chain reaction/sequence-specific oligonucleotide probe typing, we report an association of IDDM with the DPB1 allele, *0301 (relative risk = 6.6; P = 0.0012) in this population. The analysis of linkage disequilibrium patterns in this population indicates that the observed increased frequency in DPB1*0301 among patients cannot be attributed simply to linkage disequilibrium with high-risk DR-DQ haplotypes. These data suggest that in addition to alleles at the DRB1 and DQB1 loci, polymorphism at the DPB1 locus may also influence IDDM risk.
 
A: DQ staining of peripheral blood lymphocytes (PBLs) from three IA n u l l F2 littermates, which are negative (mouse 3), hemizygous (mouse 11), or homozygous (mouse 10) for HLA-DQ8. B: Dot-blot of 10 µg of tail DNA from the same three mice, probed with a DQ8-spe- cific probe. C: FACScan analysis of splenocytes from NOD human C D 4 +/+ D Q 8 + / + I A n u l l transgenic mice (l e f t) compared with wild-type NOD mice (r i g h t), stained with anti-DR, anti-DQ, anti-IA, and anti-IE monoclonal antibodies. D: Expression of human CD4 in CD3 + s p l e n ocytes from the transgenic line (l e f t) compared with wild-type NOD mice (r i g h t) .  
Comparison of various populations of immune cells found in NOD human CD4 +/+ D Q 8 + / + I A n u l l transgenic mice versus IA n u l l N O D mice. Splenocytes (A – H) and peripheral blood lymphocytes (PBLs) (I – L) were purified and stained with FITC-conjugated antibodies as follows: GK1.5 (A, B, I, J), LY-2 (C, D, K, L), F4/80 (E, F), and B220 (G , H). IA n u l l NOD mice (A, C, E, G, I, K) were compared with CD4 + / + D Q 8 + / + I A n u l l NOD mice (B, D, F, H, J, L). Portions of cells staining within the M1 gate are given as percentages for each FACS plot.  
Anti-DQ antibodies block T-cell responses to GAD65 () and to the immunodominant peptide P201-220 ( ). A GAD65-specific T-cell line was established from GAD65-immunized NOD human CD4 + / + D Q 8 + / + I A n u l l transgenic mice, and T-cells tested in a 72-h proliferation assay in the presence of syngeneic irradiated APCs and GAD65 (5 µg/ml) or peptide (20 µg/ml), with or without purified monoclonal antibodies at 100 µg/ml. Antibodies tested included 4D12 (anti-DQ), 10- 3-6 (anti-IA), and H25B10 (anti-HBV surface antigen, which provides isotype control for 4D12). ND, not done.  
Antibody responses against GAD65 after immunization of NOD human CD4 +/+ D Q 8 + / + I A n u l l transgenic mice with recombinant GAD65. Serum was collected 3 weeks () and 9 weeks ( ) after immunization and was tested by ELISA against GAD65-and BSAcoated plates. Bars represent the means of six independent sera, expressed as OD ± SEM, with each serum tested at a 1:1,000 dilution. Pre-immune serum produced only background reactivity against GAD65 (data not shown).  
A: Fine specificity of anti-GAD65 T-cell responses in GAD65- immunized NOD human CD4 +/+ D Q 8 + / + I A n u l l transgenic mice. Mice were sacrificed 2–8 weeks after immunization, and splenocytes were prepared and tested against the overlapping peptide set (20 µg/ml) in a 72-h proliferation assay. Peptides are numbered consecutively from the N H 2 -terminus to the COOH-terminus (e.g., peptide 3 corresponds to residues 21–40, and peptide 10 corresponds to residues 91–110). Data are derived from five separate experiments involving a total of 30 immunized animals; splenocytes from each individual animal were tested on the entire peptide set. Asterisks indicate responses that differ significantly (P < 0.05) using analysis of variance (ANOVA) with post hoc test. Table 2 shows representative values for counts per minute obtained in these experiments. B: Fine specificity of antibody responses in GAD65-immunized NOD human CD4 +/+ D Q 8 + / + I A n u l l t r a n sgenic mice. Serum was diluted 1:1,000 and tested by ELISA against the overlapping peptide set. Data are expressed as mean OD ± SE. Data were obtained from three separate experiments, involving a total of 12 animals. Asterisks indicate responses that differ significantly (P < 0.05) using ANOVA with post hoc test.  
Article
The 65KD isoform of GAD is considered to be a major target autoantigen in many humans with autoimmune prediabetes or diabetes. The major histocompatibility complex class II allele DQA1*0301, DQB1*0302, which encodes HLA-DQ8, confers susceptibility to type 1 diabetes and occurs in up to 80% of affected individuals. To map T-cell epitopes for GAD65 restricted to the diabetes-associated DQ8 heterodimer, we generated transgenic NOD mice expressing HLA-DQ8 and human CD4 while having the mouse class II gene (IA(beta)) deleted. These mice were immunized with full-length purified recombinant GAD65, and the fine specificity of T-cell responses was mapped by examining recall responses of bulk splenocytes to an overlapping set of 20-mer peptides encompassing the entire GAD65 protein. Four different peptides (P121-140, P201-220, P231-250, and P471-490) gave significant T-cell recall responses. P201-220 and P231-250 have been shown previously to bind DQ8, whereas the other two peptides had been classified as nonbinders. Interestingly, the peptide giving the greatest response (P201-220) encompasses residues 206-220 of GAD65, a region that has been shown to be a dominant T-cell epitope in wild-type IA(g7) NOD mice. Overlap in this T-cell epitope likely reflects structural similarities between DQ8 and IA(g7). The fine specificity of antibody responses in the GAD65-immunized mice was also examined by testing the antisera by enzyme-linked immunosorbent assay (ELISA) against the same overlapping set of peptides. The two dominant B-cell epitopes were P361-380 and P381-400; P121-140 and P471-490 appeared to correspond to both B- and T-cell epitopes. Although the NOD human CD4, DQ8, IA(null) transgenic mice generated in these studies do not develop autoimmune diabetes either spontaneously or after cyclophosphamide treatment, they can be used to map DQ8-restricted T-cell epitopes for a variety of human islet autoantigens. They can also be used to test T-cell-specific reagents, such as fluorescently labeled DQ8 tetramers containing GAD65 peptides or other beta-cell peptides, which we believe will be useful in analyzing human immune responses in diabetic and prediabetic patients.
 
Article
The genes encoding the HLA-DQ heterodimer molecules, DQB1 and DQA1, have been found to have the strongest association with IDDM risk, although there is cumulative evidence for the effect of other gene loci within the major histocompatibility complex gene region. After the HLA-DQ locus, the HLA-DR locus has been suggested most often as contributing to the disease susceptibility. In this study we analyzed at the population level the effect of DR4 subtypes and class I, HLA-B alleles, on IDDM risk when the influence of the DQ locus was stratified. In all three populations studied (Estonian, Latvian, and Russian), DQB1*0302 haplotypes most frequently carried DRB1*0401 or DRB1*0404. DRB1*0401 was the most prevalent subtype in IDDM patients, whereas DRB1*0404 was decreased in frequency. DRB1*0402 was also prevalent among Russian haplotypes, but was not associated with IDDM risk. When HLA-B alleles were analyzed, strong associations between the presence of specific B alleles and DRB1*04 subtypes were detected. The HLA-B39 allele was found significantly more often in DRB1*0404-DQB1*0302-positive patients than in healthy control subjects positive for this haplotype: 27 of 54 (50%) vs. 4 of 49 (8.2%) (P < 0.0001). The results demonstrate that DQ and DR genes cannot explain all of the HLA-linked susceptibility to IDDM, and that the existence of a susceptibility locus telomeric to DR is probable.
 
Article
In January 2013, Diabetes retracted the above-listed article at the authors’ request. The original retraction statement appears below: The authors have formally requested to retract the above-titled paper, which was published online on 19 November 2012. The authors cite concerns that portions of Fig. 4 were submitted without knowledge of inherent errors or abnormalities that they recognized in retrospect after submission. Therefore, the article has been retracted so the authors can readdress the work and submit it for publication at a later time. The corresponding author requests to update …
 
Type 1 diabetes causes elevated PAI-1, suppresses MMP9 activation, and increases collagen content during early regeneration time points. Significantly elevated PAI-1 levels in Ins2 WT/C96Y mice compared with WT mice were found in blood plasma collected (A) throughout type 1 diabetes progression (main effect: P < 0.001; N = 16) and (B) after CTX injury (main effect: P < 0.001; N = 16). This led to the hypothesis that collagen would be elevated in the Ins2 WT/C96Y mice because of suppression of the fibrinolytic pathway. C: Picrosirius red staining of injured TA sections revealed increased collagen (red color), which was statistically significant (D, main effect: P = 0.001, interaction: P = 0.004; N = 16). E: MMP9, an important protease in skeletal muscle collagen cleavage, was also found to be significantly repressed in Ins2 WT/C96Y mice (labeled Ins2, E) at the 5-day time point (N = 8). F: The short-term Ins2 WT/C96Y mice also exhibited increased collagen at 10 days postinjury (t test: P = 0.047, N = 3). *Differences between groups at specific time points identified by Bonferroni post hoc analysis after 2-way ANOVA (A, B, D, E). A-E: White bars/circles represent WT, and black bars/squares represent Ins2 WT/C96Y . (A high-quality digital representation of this figure is available in the online issue.)
Pharmacologic treatment against PAI-1 improves fibrinolytic pathway activity, collagen degradation, and regeneration at 5 days post-CTX injury in type 1 diabetes. A: Treatment with PAI-039 caused an increase in free uPA in Ins2 WT/C96Y compared with untreated Ins2 WT/C96Y (t test: P = 0.004; N = 4). B: Similarly, active MMP9 was elevated in PAI-039-treated Ins2 WT/C96Y (t test: P = 0.025; N = 4). These findings are characteristic of restored fibrinolytic pathway activity, which presumably led to the (C and D) reduced collagen levels (t test: P < 0.001; N = 4) and (E and F) increased Myh3-positive area (t test: P = 0.011; N = 4) observed in PAI-039-treated Ins2 WT/C96Y compared with untreated Ins2 WT/C96Y (labeled Ins2, C and E). A-E: Black bars represent Ins2 WT/C96Y , and striped bars represent PAI-039-treated Ins2 WT/C96Y . Data are presented relative to the mean of the WT and WT + vehicle pooled data. *Differences between groups identified by t test. (A high-quality digital representation of this figure is available in the online issue.)
Article
Diabetes Mellitus impairs the ability to heal cutaneous wounds, leading to hospitalization, amputations and death. Diabetic patients suffer from elevated levels of Plasminogen Activator Inhibitor-1 (PAI-1), regardless of their glycemic control. It has been demonstrated that PAI-1 deficient mice exhibit improved cutaneous wound healing, and that PAI-1 inhibition improves skeletal muscle repair in T1DM mice, leading us to hypothesize that pharmacologically-mediated reductions in PAI-1 using PAI-039 would normalize cutaneous wound healing in streptozotocin-induced diabetic mice (STZ-diabetic). To simulate the human condition of variations in wound care, wounds were aggravated or minimally handled post-injury. Following cutaneous injury, PAI-039 was orally administered twice daily for 10 days. Compared to non-diabetics, STZ-diabetic wounds healed more slowly. Wound site aggravation exacerbated this deficit. PAI-1 inhibition had no effect on dermal collagen levels or wound bed size, but did elevate dermal macrophage content in STZ-diabetic wounds. PAI-039 treatment failed to improve angiogenesis in STZ-diabetic wounds, and blunted angiogenesis in non-diabetic wounds. Importantly, PAI-039 treatment significantly improved epidermal cellular migration and wound re-epithelialization compared to vehicle-treated STZ-diabetics. These findings support the use of PAI-039 as a novel therapeutic to improve diabetic wound closure, and demonstrates the primary mechanism of its action to be related to epidermal closure. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
 
Distribution of DRB1 alleles according to self-reported retinopathy status in case and control patients
Article
The purpose of this study was to examine whether known genetic risk factors for type 1 diabetes (HLA-DRB1, -DQA1, and -DQB1 and insulin locus) play a role in the etiology of diabetic nephropathy. RESEARCH DESIGN AND METHODS; Genetic analysis of HLA-DRB1, -DQA1, -DQB1 and the insulin gene (INS) was performed in the Genetics of Kidneys in Diabetes (GoKinD) collection of DNA (European ancestry subset), which includes case patients with type 1 diabetes and nephropathy (n = 829) and control patients with type 1 diabetes but not nephropathy (n = 904). The availability of phenotypic and genotypic data on GoKinD participants allowed a detailed analysis of the association of these genes with diabetic nephropathy. Diabetic probands who were homozygous for HLA-DRB1*04 were 50% less likely to have nephropathy than probands without any DRB1*04 alleles. In heterozygous carriers, a protective effect of this allele was not as clearly evident; the mode of inheritance therefore remains unclear. This association was seen in probands with both short (<28 years, P = 0.02) and long (>/=28 years, P = 0.0001) duration of diabetes. A1C, a marker of sustained hyperglycemia, was increased in control probands with normoalbuminuira, despite long-duration diabetes, from 7.2 to 7.3 to 7.7% with 0, 1, and 2 copies of the DRB1*04 allele, respectively. This result is consistent with a protective effect of DRB1*04 that may allow individuals to tolerate higher levels of hyperglycemia, as measured by A1C, without developing nephropathy. These data suggest that carriers of DRB1*04 are protected from some of the injurious hyperglycemic effects related to nephropathy. Interestingly, DRB1*04 appears to be both a risk allele for type 1 diabetes and a protective allele for nephropathy.
 
Article
CS-045 is a new oral antidiabetic agent that was effective in insulin-resistant diabetic animal models, including the KK mouse, the ob/ob mouse, and the Zucker fatty rat. CS-045 was not effective in the streptozocin-treated mouse, an insulin-deficient diabetic animal model. In fed KK mice, CS-045 lowered the plasma glucose levels in a dose-dependent manner after a single oral administration, and the hypoglycemic effect lasted for at least 18 h. In normal rats, however, plasma glucose levels were not changed after administration of CS-045. CS-045 when given chronically (2 wk) to diabetic KK and ob/ob mice as a 0.2% food admixture dramatically improved hyperglycemia, hyperinsulinemia, and hypertriglyceridemia to near-normal values and decreased plasma lactate, free fatty acid, and ketone body levels without reducing food intake or body weight. In the obese Zucker fatty rat, oral administration of CS-045 had a similar effect in lowering plasma glucose, insulin, triglyceride, free fatty acid, lactate, and ketone body levels. The CS-045-treated Zucker fatty rats showed increased glucose tolerance and decreased insulin secretion in response to oral glucose. After 9 days of treatment, insulin binding to adipocyte plasma membranes from both CS-045-treated Zucker fatty rats and KK mice was increased. Furthermore, 2-deoxyglucose uptake in CS-045-treated adipocytes was increased and the insulin dose-response curve was shifted to the left. These findings suggest that CS-045 increases not only insulin sensitivity but also insulin responsiveness. Based on its pharmacological profile, CS-045 is a new orally effective antidiabetic agent that may reduce abnormalities of glucose and lipid metabolism in obese and non-insulin-dependent diabetes mellitus patients with insulin resistance.
 
Article
HLA-DQB1 alleles confer susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). We investigated whether the susceptibility alleles DQB1*0302 and DQB1*0201 affect progression to diabetes among islet cell antibody-positive (ICA+) first-degree relatives of IDDM patients and whether the protective allele DQB1*0602 can be found and is still protective among such relatives. We human leukocyte antigen-typed and periodically tested beta-cell function (first-phase insulin release [FPIR] during the intravenous glucose tolerance test) in 72 ICA+ relatives, of whom 30 became diabetic on follow-up (longest follow-up 12 years); 54 (75%) relatives carried DQB1*0302 and/or DQB1*0201. The frequency of DQB1*0302 and DQB1*0201 and of the high-risk genotype DQB1*0302/DQB1*0201 did not differ significantly between diabetic relatives and those remaining nondiabetic. On follow-up, progression to IDDM was not statistically different for relatives with or without the DQB1*0302/DQB1*0201 genotype. However, those relatives with the DQB1*0302/DQB1*0201 genotype had a tendency to develop diabetes at an earlier age (log-rank P = 0.02). We found DQB1*0602 in 8 of 72 (11.1%) ICA+ relatives. Relatives with DQB1*0602 did not develop diabetes or show any decline of FPIR versus 28 of 64 DQB1*0602- relatives who developed IDDM (log-rank P = 0.006; Wilcoxon's P = 0.02). The protective allele DQB1*0602 is found in ICA+ relatives who have minimal risk of progression to IDDM. Therefore, DQB1*0602 is associated with protection from IDDM both in population studies and among relatives with evidence of autoimmunity who should not enter prevention trials.
 
Article
In the Diabetes article by Edgehill et al. (1), the authors confirm and expand the recent demonstration that proinsulin gene ( INS ) mutations may cause permanent neonatal diabetes (2). The sequence analysis (1) is resolving a 30-year-old mystery in the etiology of permanent neonatal diabetes in a sibship that we published in Diabetes 20 years ago (3). The older brother was born in 1975 with diabetes recognized at 3 days of age, and in 1978 his brother was born with diabetes, which was diagnosed on the first day of life. In an attempt to understand the etiology of neonatal diabetes, we reported the …
 
Mutations identified in the preproinsulin molecule. Black-filled circles represent amino acid changes identified in probands with diabetes diagnosed before 12 months, gray circles represent amino acid changes identified in probands with possible MODY. Where the number of probands with the mutation is greater than one, the total number is indicated in the square brackets. 
Clinical characteristics of probands diagnosed in infancy and their family members with an INS gene mutation
Article
Insulin gene (INS) mutations have recently been described as a cause of permanent neonatal diabetes (PND). We aimed to determine the prevalence, genetics, and clinical phenotype of INS mutations in large cohorts of patients with neonatal diabetes and permanent diabetes diagnosed in infancy, childhood, or adulthood. The INS gene was sequenced in 285 patients with diabetes diagnosed before 2 years of age, 296 probands with maturity-onset diabetes of the young (MODY), and 463 patients with young-onset type 2 diabetes (nonobese, diagnosed <45 years). None had a molecular genetic diagnosis of monogenic diabetes. We identified heterozygous INS mutations in 33 of 141 probands diagnosed at <6 months, 2 of 86 between 6 and 12 months, and none of 58 between 12 and 24 months of age. Three known mutations (A24D, F48C, and R89C) account for 46% of cases. There were six novel mutations: H29D, L35P, G84R, C96S, S101C, and Y103C. INS mutation carriers were all insulin treated from diagnosis and were diagnosed later than ATP-sensitive K(+) channel mutation carriers (11 vs. 8 weeks, P < 0.01). In 279 patients with PND, the frequency of KCNJ11, ABCC8, and INS gene mutations was 31, 10, and 12%, respectively. A heterozygous R6C mutation cosegregated with diabetes in a MODY family and is probably pathogenic, but the L68M substitution identified in a patient with young-onset type 2 diabetes may be a rare nonfunctional variant. We conclude that INS mutations are the second most common cause of PND and a rare cause of MODY. Insulin gene mutation screening is recommended for all diabetic patients diagnosed before 1 year of age.
 
Article
ATP-sensitive K(+) (K(ATP)) channels are activated by a diverse group of compounds known as potassium channel openers (PCOs). Here, we report functional studies of the Kir6.2/SUR1 Selective PCO 3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide (NNC 55-9216). We recorded cloned K(ATP) channel currents from inside-out patches excised from Xenopus laevis oocytes heterologously expressing Kir6.2/SUR1, Kir6.2/SUR2A, or Kir6.2/SUR2B, corresponding to the beta-cell, cardiac, and smooth muscle types of the K(ATP) channel. NNC 55-9216 reversibly activated Kir6.2/SUR1 currents (EC(50) = 16 micromol/l). This activation was dependent on intracellular MgATP and was abolished by mutation of a single residue in the Walker A motifs of either nucleotide-binding domain of SUR1. The drug had no effect on Kir6.2/SUR2A or Kir6.2/SUR2B currents. We therefore used chimeras of SUR1 and SUR2A to identify regions of SUR1 involved in the response to NNC 55-9216. Activation was completely abolished and significantly reduced by swapping transmembrane domains 8-11. The reverse chimera consisting of SUR2A with transmembrane domains 8-11 and NBD2 consisting SUR1 was activated by NNC 55-9216, indicating that these SUR1 regions are important for drug activation. [(3)H]glibenclamide binding to membranes from HEK293 cells transfected with SUR1 was displaced by NNC 55-9216 (IC(50) = 105 micromol/l), and this effect was impaired when NBD2 of SUR1 was replaced by that of SUR2A. These results suggest NNC 55-9216 is a SUR1-selective PCO that requires structural determinants, which differ from those needed for activation of the K(ATP) channel by pinacidil and cromakalim. The high selectivity of NNC 55-9216 may prove to be useful for studies of the molecular mechanism of PCO action.
 
Article
1,2-Diacylglycerol has been proposed to be a secondary messenger; therefore, in this study we evaluated the amount of 1,2-diacylglycerol in heart tissue from streptozocin-induced diabetic rats and examined the effect of insulin treatment on 1,2-diacylglycerol content. Diabetic rats had lower body and ventricular weights and higher ratios of ventricular to body weight, all of which shifted toward normal values after 4 wk of untreated diabetes followed by 4 wk of insulin treatment. The contents of major phospholipids were significantly depressed in the diabetic rat hearts. In contrast, the triglyceride and cholesterol contents in the myocardium were increased by streptozocin injection and completely normalized by insulin treatment, and glucose levels returned to normal. The 1,2-diacylglycerol content in the myocardium was also significantly elevated in the diabetic rats compared with age-matched controls. Moreover, the 1,2-diacylglycerol content was significantly higher in rats with 4 wk of diabetes than in those with 8 wk of diabetes. Insulin treatment in the diabetic rats, however, did not produce any decrease in 1,2-diacylglycerol content. The results of this study suggest that the development of cardiomyopathy induced by streptozocin injection is associated with a high 1,2-diacylglycerol level, which may result in the activation of protein kinase C. Insulin is one of the agonists that generates 1,2-diacylglycerol in myocytes; however, the relationship between the sustained 1,2-diacylglycerol level and the normalization of diabetes by insulin administration is unclear.
 
Article
These experiments were conducted to determine 1,2-diacylglycerol (DAG) in the thoracic aorta obtained from streptozocin-induced diabetic rats because 1,2-DAG is assumed to be a second messenger associated with phosphoinositide metabolism. After preincubation for a 25-min stabilization, 1,2-DAG content in isolated thoracic aortas 4 and 8 wk after streptozocin injection was significantly decreased by 42 and 31%, respectively, compared with age-matched control rats on 10-min norepinephrine stimulation (10(-5) M). However, 4 wk of daily insulin injection after 4 wk of untreated diabetes significantly shifted 1,2-DAG toward normal levels. Analysis of its fatty acid composition showed a significant difference between control and diabetic rat aortas at both 4 and 8 wk. In particular, the percentage of arachidonate, a precursor of eicosanoids, decreased. Such alteration in the fatty acid profile in diabetic rat aortas was inhibited by insulin treatment. 1,2-DAG content in the 8-wk diabetic group was also significantly decreased by 33% compared with control in the absence of norepinephrine, whereas 1,2-DAG content was lower than in the presence of norepinephrine in both the control and diabetic groups. Cholesterol, triglyceride, and phosphatidylcholine content in diabetic rat aortas was lower than control. Lower levels of 1,2-DAG in the thoracic aorta from diabetic rats were observed in the presence and absence of norepinephrine, suggesting that a defect in 1,2-DAG production may be associated with abnormalities of vascular smooth muscle responsiveness by agonists, as described previously.
 
Article
In the past, endogenous retroviral sequences have been isolated from patients suffering from different kinds of autoimmune diseases. Recently, a full length retroviral genome, termed IDDMK(1,2)22, was isolated from patients with new-onset IDDM. This genome contains a major histocompatibility complex II-dependent superantigen within its envelope gene. The viral sequence was found in ten patients with new-onset IDDM, but not in age-matched control subjects (Conrad et al. [9]). We searched for the presence of this viral genome by nested reverse transcription-polymerase chain reaction (RT-PCR) in a cohort of six patients with new-onset IDDM and six control subjects of the same age. We found all samples to be positive without any differences between patients and control subjects. The same results were obtained with supernatants of activated peripheral blood mononuclear cells. We performed isopycnic ultracentrifugation in sucrose density gradients on all samples and were unable to detect particles of the new virus in any of our samples. However, positive signals were obtained from all pellet fractions. RNase, DNase treatment and nested PCRs without reverse transcription showed that the positive signals were probably derived from intracellular RNA and DNA. In summary, no correlation between a positive nested PCR signal for IDDMK(1,2)22 and diabetes was found indicating that the new sequence represents just an additional member of the human endogenous retrovirus (HERV) family with lack of an exogenous counterpart.
 
Article
In this study we examine the hypothesis that an inositol glycan phosphate can act similarly to insulin on intact cells. The inositol glycan phosphate used in this study (glycan alpha) was isolated previously from the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase and was shown to have the structure glycine-ethanolamine-PO4-Man-Man-(N,N-dimethylethanolamine-PO4)Man- (N,N-dimethyl)GlcN-inositol-PO4. The cellular response investigated was the glucagon-stimulated activation of glycogen phosphorylase in rat hepatocytes. When hepatocytes were incubated with 20 nM glucagon for 4 min, the ratio of phosphorylase a activity to total phosphorylase increased from a basal value of 0.49 +/- 0.02 to 0.82 +/- 0.03 (mean +/- SE, n = 15). Inclusion of either 100 nM insulin or 3-10 microM glycan alpha during the glucagon incubation significantly decreased the glucagon-stimulated activity ratio to 0.74 +/- 0.03 for either agent. Furthermore, hepatocyte preparations differed in their response to insulin and were divided into insulin-responsive and -resistant groups. Glycan alpha had a significant effect only in the insulin-responsive group for which the observed activity ratio for 10 microM glycan alpha plus glucagon (0.68 +/- 0.05) compared closely with that for insulin plus glucagon (0.70 +/- 0.04). For the insulin-resistant group, the activity ratio in the presence of 10 microM glycan alpha was 0.81 +/- 0.03, unchanged from the control with glucagon alone. Because glycan alpha contains an inositol phosphate group, the effect of inositol cyclic 1,2-phosphate on the glucagon-stimulated activity ratio was determined.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
Duodenal calcium absorption and serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations are decreased and body growth is arrested in the streptozotocin-diabetic rat taking commercial chow with high (1.2-2%) calcium content. Treatment with insulin restores 1,25-(OH)2D3, calcium absorption, and body growth to normal. We hypothesized that the depressed 1,25-(OH)2D3 in diabetics is due in part to the minimal requirement for vitamin-D-mediated intestinal calcium transport under conditions of arrested growth and high calcium intake. We tested this hypothesis by comparing the response of serum 1,25-(OH)2D3 concentration to low (0.02%) and normal (0.5%) calcium diets in control and streptozotocin-diabetic rats. To evaluate response to short-term insulin treatment, serum 1,25-(OH)2D3 was measured after 12 or 36 h of treatment. Serum 1,25-(OH)2D3 concentrations in the 0.5% calcium diet groups were 175, 25, and 120 pg/ml for control, diabetic, and insulin-treated 36-h groups, respectively. Low calcium diets increased concentration to 625, 100, and 370 pg/ml for controls, diabetics, and insulin-treated 36-h groups, respectively. In conclusion, the diabetic retains the ability to respond to calcium deficiency, even in the insulin-deficient state. Low calcium intake, in addition to enhancing 1,25-(OH)2D3 formation in diabetics, also modulates the response to insulin treatment. These studies demonstrate that the regulatory factor(s) suppressing serum, 1,25-(OH)2D3 in diabetes is not simply insulin deficiency per se. Implications of these findings for diabetes are discussed.
 
Article
It has previously been shown that vitamin D deficiency impairs arginine-induced insulin secretion from the isolated, perfused rat pancreas (Science 1980; 209:823-25). Since vitamin D is known to be metabolized to 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) and 24R,25-dihydroxyvitamin D3 (24,25[OH]2D3), it is essential to clarify which vitamin D metabolite has the important role of enhancing insulin secretion. In this report, a comparison is made of the relative efficacy of 3-wk repletion with vitamin D3 (980 pmol/day), 1,25(OH)2D3 (39 pmol/day or 195 pmol/day), and 24,25(OH)2D3 (650 pmol/day) on arginine-induced insulin secretion from the isolated, perfused rat pancreas; in this experiment, the daily caloric intake of the animals receiving vitamin D or its metabolites was controlled by pair feeding to the caloric intake of the vitamin D-deficient rats. 1,25(OH)2D3 repletion was found to completely restore insulin secretion to the levels seen in vitamin D3-replete, pair-fed controls in both the first and second phases, while 24R,25(OH)2D3 only partially improved insulin secretion, and then only in the first phase. Changes of both serum calcium levels and dietary caloric intake after vitamin D metabolite administration are concluded to play a lesser role on the enhancement of insulin secretion, since, in a separate experiment, vitamin D-deficient rats with normal serum calcium levels did not show recovery of insulin secretion equivalent to the vitamin D-replete animals under conditions of dietary pair feeding. These results suggest that 1,25(OH)2D3 but not 24,25(OH)2D3 plays an essential role in the normal insulin secretion irrespective of the dietary caloric intake and prevailing serum calcium levels.
 
Article
Recent autoradiographic studies demonstrated that B-cells concentrate 1,25 (OH)2 D3 in their nuclei, suggesting a genomic action on B-cell function. This study was undertaken to investigate the effects of 1,25 (OH)2 D3 on insulin secretion in vitamin D-deficient rats. Mature vitamin D-deficient rats were injected with 1,25 (OH)2 D3 or the ethanol-isotonic saline vehicle. Administration of 1,25 (OH)2 D3 to 10 rats resulted in a 17 microunits/ml (113%) increase in insulin levels and 0.9 mg/dl (16%) increase in plasma calcium. No changes were found in insulin or calcium levels in 5 control rats given vehicle alone. A group of vitamin D-deficient rats with plasma calcium levels of 5.4 +/- 0.1 mg/dl had insulin levels that were the same as those observed in a group of vitamin D-deficient rats with plasma calcium levels of 6.3 +/- 0.1 mg/dl. The difference in calcium levels between these two groups is similar to the increase in plasma calcium found after 1,25 (OH)2 D3 administration. The results of these studies indicate that 1,25 (OH)2 D3 action on pancreatic B-cells affects insulin secretion. Since insulin increases synthesis of 1,25 (OH)2 D3, the existence of a feedback loop between B-cells and kidney proximal tubule cells is suggested.
 
Article
The NOD mouse, a model for type 1 diabetes, is characterized by resistance to apoptosis in immunocytes. The aim of this study was to investigate a link between apoptosis in NOD thymocytes and autoimmunity. First, we demonstrated that the sexual dimorphism in diabetes incidence in NOD mice (females are more diabetes-prone than males) is reflected by differences in apoptosis. Apoptosis in NOD thymocytes, 24 h after dexamethasone, was decreased in both sexes compared with C57B1/6, but it was lower in female mice (26 +/- 2%) than in male mice (50 +/- 3%, P < 0.001). Further, we demonstrated that sex hormones themselves play a central role in this difference, since castration of NOD male mice, which increases diabetes incidence, decreased apoptosis levels (32 +/- 2%), while treatment of NOD female mice with dihydrotestosterone, which protects against diabetes, restored apoptosis to male levels (42 +/- 1.5%). Finally, we demonstrated that 1,25-dihydroxyvitamin D3, a steroid hormone that prevents diabetes in NOD mice, restored apoptosis levels to C57B1/6 reference levels. This improved apoptosis was seen in male (68 +/- 1 vs. 50 +/- 3% in untreated NOD mice, P < 0.001) but especially in female NOD mice (51 +/- 5 vs. 26 +/- 2% in untreated NOD mice, P < 0.001). Fluorescence-activated cell sorter analysis of thymocyte subsets revealed marked differences, especially in CD4+CD8+ and CD4+ cells. We conclude that the sexual dimorphism in diabetes incidence in NOD mice is paralleled by a dimorphism in resistance to apoptotic signals in NOD thymocytes. This resistance to apoptosis is driven by sex hormones and is corrected by 1,25-dihydroxyvitamin D3.
 
Characterization of cell surface markers on human immature (day 6) and mature DCs (day 8) generated from isolated monocytes in the presence of GM-CSF and IL-4. DC cultures were either nontreated (dotted line) or treated with 10 8 mol/l 1,25(OH) 2 D 3 (solid thin line) or TX527 (solid thick line). DC maturation was induced by addition of LPS and IFN-.
Morphology of variously treated mature DCs. Cultures were either nontreated (NT) or continuously treated with 10 8 mol/l 1,25(OH) 2 D 3 or TX527.
1,25(OH) 2 D 3 or TX527 treatment affects DC cytokine produc- 
Effect of 1,25(OH) 2 D 3 or TX527 treatment on T-cell stimulatory capacity of mature DCs. Mature DCs were generated from isolated monocytes in the absence () or presence of 10 8 mol/l 1,25(OH) 2 D 3 (o) or TX527 (f), after which GAD65-specific T-cells were added. T-cells were cultured in the presence of 0.2 g/ml GAD (339-352) peptide for the induction of T-cell proliferation and cytokine production. A representative experiment of five is shown.
1,25(OH) 2 D 3 or TX527 treatment during maturation yields DCs that marginally affect GAD65-committed T-cells. Immature DCs were matured in the absence () or presence of 10 8 mol/l 1,25(OH) 2 D 3 (p) or TX527 (f). After 48 h, DCs were cocultured with GAD65-specific T-cells in the absence or presence of 0.2 g/ml GAD (339-352) peptide for the induction of proliferation and cytokine production. A representative experiment of three is shown.
Article
The active form of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is a potent immunomodulator known to affect T-cells through targeting antigen-presenting cells such as dendritic cells (DCs). We studied the effects of a novel nonhypercalcemic 1,25(OH)(2)D(3) analog, TX527, on DC differentiation, maturation, and function with respect to stimulation of a committed human GAD65-specific autoreactive T-cell clone. Continuous addition of TX527 impaired interleukin (IL)-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF)-driven DC differentiation as well as lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-induced maturation into Th1-promoting DC (DC1), as characterized by marked changes in DC morphology and abrogation of IL-12p70 release upon CD40 ligation. Addition of TX527 during maturation did not affect DC morphology but significantly changed DC cytokine profiles. The potential of treated DCs to alter the response pattern of committed autoreactive T-cells was found to depend on the timing of TX527 exposure. Continuously TX527-treated DCs significantly inhibited T-cell proliferation and blocked IFN-gamma, IL-10, but not IL-13 production, whereas DCs treated during maturation failed to inhibit T-cell proliferation but affected IL-10 and IFN-gamma production. Collectively, we provide evidence that nonhypercalcemic TX527 is a potent in vitro DC modulator, yielding DCs with the potential to change cytokine responses of committed autoreactive T-cells.
 
Histology of pancreatic Islets from representative 1,25(OH) 2 D 3-treated (A and 5) and control NOD mice (Cand D). Hematoxylln and eosin x 330. Biopsies were taken at 100 days of age, after 80 days of treatment. 
Article
The active form of vitamin D, 1,25(OH)2D3, can prevent various forms of experimentally induced autoimmune disorders. The aim of this study was to confirm these findings in NOD mice that spontaneously develop an autoimmune type of diabetes mellitus. Therefore, the effect of a long-term 1,25(OH)2D3 treatment on the incidence of insulitis, the histological lesion preceding diabetes, was studied. Forty-three NOD mice were treated with 1,25(OH)2D3 (5 micrograms/kg) i.p. every other day from age 21 days on, when no insulitis was present yet. At day 100, 16 control mice receiving the treatment vehicle (arachis oil) had an incidence of insulitis of 75%, whereas only 41% of the 1,25(OH)2D3-treated animals developed insulitis (P < 0.025). Calcemia, determined 24 h after the last 1,25(OH)2D3 injection was 2.5 +/- 0.1 mM, which was higher than in control animals (2.3 +/- 0.1 mM), but was well tolerated. Cellular immunity, as assessed with the mixed lymphocyte reaction performed at day 100, was not impaired significantly. This study demonstrates that long-term treatment with high doses of 1,25(OH)2D3 is able to decrease the incidence of insulitis in spontaneous autoimmune diabetes without major side effects.
 
Article
The alterations of mineral homeostasis observed in the streptozotocin-induced diabetic rat have been attributed to low circulating levels of 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2-D 3], the biologically active metabolite of vitamin D 3. However, the effect of diabetes and subsequent insulin repletion on production of 1,25-(OH) 2-D 3 and 24,25-(OH) 2-D 3 by the kidney has not been investigated. We studied the renal production of 1,25-(OH) 2-D 3 and 24,25-(OH) 2-D 3 in diabetic and insulin-treated diabetic rats using the renal slice technique. In rats made diabetic with streptozotocin, the renal production of 1,25-(OH) 2-D 3 was markedly reduced (0.195 ± 0.011 pg/min/mg) compared with controls (0.685 ± 0.107 pg/min/mg, P < 0.05). The renal production of 24,25-(OH) 2-D 3 in diabetic rats was markedly increased (0.529 ± 0.052 pg/min/mg) compared with controls (0.233 ± 0.035 pg/min/mg, P < 0.05). Treatment of diabetic rats with insulin for 12 days resulted in a significant increase in 1,25-(OH) 2-D 3 production (0.331 ± 0.053 pg/min/mg) and decrease in 24,25-(OH) 2-D 3 production (0.329 ± 0.054 pg/min/mg). The possibility that diabetes decreased renal 1,25-(OH) 2-D 3 production by decreasing parathyroid hormone (PTH) secretion or depressing the action of PTH on the kidney was also studied. Diabetes caused no decrease in serum PTH levels relative to the control group and urinary cyclic AMP excretion in the diabetic group was not depressed. The cyclic AMP content of diabetic rat kidney slices in response to PTH was similar to controls (39.8 ± 3.3 versus 39.7 ± 4.6 pmol/mg wet weight). Serum immunoreactive calcitonin (iCT) was significantly depressed in diabetic rats compared with controls. Insulin treatment of diabetic rats resulted in a marked increase in serum iCT. These data suggest the impairment of renal 1,25-(OH) 2-D 3 production was not related to impairment of the PTH activation of adenylate cyclase in the diabetic state.
 
Light micrographs of native neonatal porcine pancreas (A), freshly digested porcine NIC aggregates (B), 9-day culture islets (C), and 9-day culture dissociated islet cells (D). Sections were double immunohistochemically stained for Gal epitope (brown) and insulin (red). Arrows point to some double-positive cells. 
FACS analysis of PAECs (A) and dissociated 9-day culture porcine NIC aggregates (B) stained with FITC-conjugated IB4 lectin.-, negative controls not stained with IB4. 
Binding of human xenoreactive antibodies to porcine NICs. Single-cell suspensions of neonatal porcine islet cells were incubated with 1:32 dilution of heat-inactivated pooled human AB serum for 1 h, then incubated with 1:10 dilution of FITC-conjugated rabbit anti-human IgG (A) or IgM (B) for an additional 1 h.-, negative control islet cells incubated in FITC-labeled secondary antibodies alone. Results are representative of three independent experiments. 
Article
Neonatal porcine pancreases may be a potential source of islets for transplantation into patients with type 1 diabetes; however, whether these cellular grafts will be susceptible to damage by human natural antibody-mediated rejection remains controversial. Although we and others have demonstrated that porcine islets bind human IgG and IgM, it remains unknown if they express the xenoreactive antigen Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (Gal epitope). In this study, by using the Gal-specific lectin IB4 for immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis, we determined which cell types present in porcine neonatal islet cell (NIC) aggregates express the Gal epitope and which ones are susceptible to lysis by activation of the human complement. After FACS analysis, 30.0 +/- 3.0% of porcine NICs were shown to express Gal, whereas 70.0 +/- 2.0% did not. Histological assessment of Gal-expressing cells revealed that 54.9 +/- 8.8% stained positive for either insulin or glucagon. In contrast, 68.8 +/- 8.4% of the Gal-negative population stained positive for the pancreatic hormones insulin and glucagon. Incubation of either the Gal-positive or -negative cells with human AB serum plus complement for 1.5 h resulted in the lysis of >90% of the cells. These results demonstrate that porcine NIC aggregates are composed of Gal-expressing cells and that expression of Gal is not restricted to nonendocrine cells. Furthermore, both Gal-positive and Gal-negative cells are susceptible to human antibody/complement-mediated cytolysis, suggesting that this form of immunological destruction is an obstacle that will need to be overcome before porcine NIC aggregates can be used clinically.
 
Article
Because we were interested in assessing glucose-mediated regulation of the activity of sarcolemmal ATP-sensitive K(+) channels (K(ATP) channels) (which are closed by physiological levels of intracellular ATP and serve to couple intracellular metabolism with the membrane excitability in the heart) during ischemia, we performed experiments designed to test whether high extracellular glucose would have effects on sarcolemmal K(ATP) channels per se. Surprisingly, we found that high extracellular glucose (50 mmol/l) activates sarcolemmal K(ATP) channels in isolated guinea pig cardiomyocytes. To activate K(ATP) channels, glucose had to be transported into cardiomyocytes and subjected to glycolysis. The activation of these channels was independent of ATP production and intracellular ATP levels. The effect of glucose on sarcolemmal K(ATP) channels was mediated by the catalytic activity of glyceraldehyde-3-phosphate dehydrogenase and consequent generation of 1,3-bisphosphoglycerate. The 1,3-bisphosphoglycerate (20 mmol/l), an intermediate product of glycolysis, directly targeted and activated K(ATP) channels, despite physiological levels of intracellular ATP (5 mmol/l). We conclude that glucose, so far exclusively viewed as a metabolic fuel in the heart important only during ischemia/hypoxia, may serve a signaling role in the nonstressed myocardium by producing an agent that regulates cardiac membrane excitability independently of high-energy phosphates.
 
Article
In a series with 1,332 pregnancies in women with diabetes, perinatal fetal mortality varied in a statistically significant degree, with maternal factors and pregnancy complications expressed respectively by the White and Prognostically Bad Signs in Pregnancy (PBSP) classifications. Mortality declined steadily over the years 1946 to 1972, with fatal congenital malformation being the most important single cause of perinatal death in recent years. A controlled trial must take into account the year of admission as well as the White and PBSP classifications. With the prevailing low and decreasing mortality, even large centers may be unable to fulfil the requirements as to sample size within a few years. Therefore, criteria other than perinatal mortality may be needed to assess the value of changes in treatment.
 
Article
The pseudotetrasaccharide acarbose, previously known as a potent inhibitor of intestinal alpha-glucoside hydrolases, was investigated with regard to its influence on islet lysosomal enzyme activities and the insulin secretory processes. We observed that acarbose was a potent inhibitor of mouse islet lysosomal acid glucan-1,4-alpha-glucosidase activity, EC50 approximately 5 mumol/l, as well as of acid alpha-glucosidase activity. In contrast, acarbose did not influence other lysosomal enzyme activities such as acid phosphatase and N-acetyl-beta-D-glucosaminidase. Neutral alpha-glucosidase (endoplasmic reticulum) was only moderately inhibited in homogenate and was unaffected in intact islets. Incubation of isolated mouse islets with acarbose revealed that the pseudotetrasaccharide was a strong inhibitor of glucose-induced insulin secretion, EC50 approximately 500 nmol/l, and a significant inhibition was already observed at a concentration of acarbose as low as 100 nmol/l. The acarbose analogue maltotetrose did not influence either glucose-induced insulin release or islet lysosomal enzyme activities. Further, acarbose as well as two other alpha-glucoside hydrolase inhibitors, the deoxynojirimycin derivatives miglitol and emiglitate, did not affect islet glucose oxidation at low or high glucose levels. Acarbose also inhibited insulin release induced by the sulfonylurea glibenclamide, whereas insulin secretion stimulated by the cholinergic muscarinic agonist carbachol or the phosphodiesterase inhibitor isobutylmethylxanthine was unaffected by the drug. Moreover, complementary in vivo experiments showed that pretreatment of mice with acarbose to allow for endocytosis of the compound markedly suppressed the insulin secretory response to an intravenous glucose load.(ABSTRACT TRUNCATED AT 250 WORDS)
 
Article
To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [125I-Tyr8]bradykinin or [3H]bradykinin in three types of skeletal muscle preparations: membrane fractions from guinea pig hindlimb quadriceps, dog semimembranosus and semitendinosus muscles, and L8 rat skeletal muscle myoblasts. Scatchard analysis of [125I-Tyr8]bradykinin x bradykinin competition binding demonstrated specific bradykinin binding of 4.9 and 3.2 fmol/mg protein in dog and guinea pig skeletal muscle preparations, respectively. Unlabeled bradykinin specifically displaced [125I-Tyr8]bradykinin with IC50 values of 36.5 +/- 6 and 118.0 +/- 16.0 pmol/l from dog and guinea pig muscle membranes, respectively. The B2 bradykinin receptor antagonist HOE 140 and the B1 bradykinin receptor antagonist des-Arg9[Leu8]bradykinin displaced the binding of [3H]bradykinin from dog membranes with IC50 values of 0.38 and 217.3 nmol/l, respectively, suggesting that bradykinin binds to a B2-type receptor. In addition, unlabeled bradykinin competed with [3H]bradykinin for binding to dog skeletal muscle membrane preparations in a biphasic manner. To assess whether this represents multiple bradykinin receptor subtypes present in skeletal muscle homogenates or several affinity states of a single binding site, we examined bradykinin receptors on a pure skeletal muscle system, the L8 neonatal rat skeletal muscle myoblast cell line. These myoblasts also contain specific [3H]bradykinin-binding sites with a Bmax of 271 fmol/mg protein and a Kd of 0.83 nmol/l. Competitive agonist binding curves were biphasic (high-affinity IC50 = 3.9 pmol/l, low-affinity IC50 = 22.6 nmol/l) in the absence of guanosine 5'-O-(3-thio-trisphosphate) (GTP gamma S); they shifted to a model of one affinity (8.1 nmol/l) in the presence of GTP gamma S. Because the enzyme neutral endopeptidase 24.11 is an important kininase in skeletal muscle, we examined the effect of the neutral endopeptidase inhibitor phosphoramidon on the binding of bradykinin to dog skeletal muscle membranes. We found that phosphoramidon decreased the apparent Bmax from 7.3 to 5.8 fmol/mg protein. In addition, in this cell line we investigated the action of bradykinin on phosphoinositide hydrolysis. Inositol 1,4,5-trisphosphate (IP3) was measured with a radioreceptor assay. Bradykinin (0.1 nmol/l to 1 mumol/l) induced IP3 formation in a dose-dependent manner (EC50 = 1.42 nmol/l) from a basal level of 72.8 +/- 16 pmol/mg protein to 433 +/- 35.5 at the highest (1 mumol/l) concentration. We conclude that bradykinin B2 receptors are expressed in skeletal muscle. Phosphoinositide hydrolysis upon stimulation of this receptor is an indicator of intracellular signal transduction. Part of the bradykinin binding in skeletal muscle is due to interaction with the enzyme neutral endopeptidase.
 
Article
Glucose-stimulated insulin secretion is associated with transients of intracellular calcium concentration ([Ca2+]i) in the pancreatic beta-cell. We tested the hypothesis that inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] [Ca2+]i release is incorporated in glucose-induced [Ca2+]i oscillations in mouse islets and MIN6 cells. We found that depletion of intracellular Ca2+ stores with thapsigargin increased the oscillation frequency by twofold and inhibited the slow recovery phase of [Ca2+]i oscillations. We employed a pleckstrin homology domain-containing fluorescent biosensor, phospholipase C partial differential pleckstrin homology domain-enhanced green fluorescent protein, to visualize Ins(1,4,5)P3 dynamics in insulin-secreting MIN6 cells and mouse islets in real time using a video-rate confocal system. In both types of cells, stimulation with carbamoylcholine (CCh) and depolarization with KCl results in an increase in Ins(1,4,5)P3 accumulation in the cytoplasm. When stimulated with glucose, the Ins(1,4,5)P3 concentration in the cytoplasm oscillates in parallel with oscillations of [Ca2+]i. Maximal accumulation of Ins(1,4,5)P3 in these oscillations coincides with the peak of [Ca2+]i and tracks changes in frequencies induced by the voltage-gated K+ channel blockade. We show that Ins(1,4,5)P3 release in insulin-secreting cells can be stimulated by depolarization-induced Ca2+ flux. We conclude that Ins(1,4,5)P3 concentration oscillates in parallel with [Ca2+]i in response to glucose stimulation, but it is not the driving force for [Ca2+]i oscillations.
 
Electrically evoked Ca 2 ؉ transients in ventricular cardiomyocytes from wild-type and ob / ob mice. Confocal line-scan images from a 
Spatially averaged Ca 2 ؉ transients (expressed as normalized 
Spatially averaged Ca 2 ؉ transients from an ob / ob cell recorded 
Mitochondrial Ca 2 ؉ measured with rhod-2 in isolated wild-type 
Article
Obesity, insulin resistance, and type 2 diabetes are leading causes of heart failure, and defective cellular Ca2+ handling seems to be a fundamental problem in diabetes. Therefore, we studied the effect of insulin on Ca2+ homeostasis in normal, freshly isolated mouse ventricular cardiomyocytes and whether Ca2+ handling was changed in an animal model of obesity and type 2 diabetes, ob/ob mice. Electrically evoked Ca2+ transients were smaller and slower in ob/ob compared with wild-type cardiomyocytes. Application of insulin (6 or 60 nmol/l) increased the amplitude of Ca2+ transients in wild-type cells by approximately 30%, whereas it broadened the transients and triggered extra Ca2+ transients in ob/ob cells. The effects of insulin in ob/ob cells could be reproduced by application of a membrane-permeant inositol trisphosphate (IP3) analog and blocked by a frequently used IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate. In ob/ob cardiomyocytes, insulin increased the IP3 concentration and mitochondrial Ca2+ handling was impaired. In conclusion, we propose a model where insulin increases IP3 in ob/ob cardiomyocytes, which prolongs the electrically evoked Ca2+ release. This, together with an impaired mitochondrial Ca2+ handling, results in insulin-mediated extra Ca2+ transients in ob/ob cardiomyocytes that may predispose for arrhythmias in vivo.
 
Article
To elucidate the value of using plasma 1,5-anhydro-D-glucitol (AG) as a marker of glycemic control in diabetic patients, the relationship between the plasma concentration of AG and glucosuria was examined in 152 patients with non-insulin-dependent diabetes mellitus (NIDDM). After recovery from the deterioration of glycemic control in NIDDM patients had started, AG began to increase day by day. The recovery of plasma AG showed a constant linear increase curve when excellent glycemic control was attained. The ordinary daily recovery rate of plasma AG was estimated to be 0.3 microgram/ml, which was independent of body weight, sex, age, the difference in treatment, the duration of diabetes, or the level of plasma AG among NIDDM patients. This rate decreased according to the increase in urinary glucose. When we calculated the decrease rate of plasma AG (delta AG), assuming 0.3 microgram/day to be the maximum increase rate in a day, we found a high correlation between delta AG and urinary glucose at almost all AG levels except the normal range and observed that plasma AG (A) times urinary glucose (G) was relatively constant. The formula A x G = 16 is a simple equation for rough estimation of urinary glucose from the plasma AG concentration in a stable glycemic-controlled NIDDM patient, and we call it the A.G index. The plasma AG also correlated significantly with fasting plasma glucose (r = -.810) and glycosylated hemoglobin (r = -.856) in the same stable glycemic-controlled NIDDM patients. Based on these observations, we propose that plasma AG can serve as a new marker that may provide sensitive and analytical information about glycemic control.
 
Article
To evaluate the use of serum 1,5-anhydroglucitol (AG) levels in screening for diabetes mellitus, we compared the sensitivity and specificity of HbA1c, fructosamine (FA), and AG in 1620 randomly selected subjects in 11 institutions throughout Japan. Most individuals were receiving diet and/or drug therapy for diabetes. Subjects were separated into four groups based on World Health Organization criteria: nondiabetic control subjects, subjects with impaired glucose tolerance (IGT), patients with diabetes, and patients with other disorders without IGT. The overlap of AG values between each group was less than that of HbA1c or FA values. AG levels were significantly correlated with fasting plasma glucose (r = -0.627), HbA1c (r = -0.629), and FA (r = -0.590) levels. If we took 14 micrograms/ml as the normal lower limit, AG level was highly specific (93.1%), and a decreased AG level indicated diabetes mellitus (84.2% sensitivity). According to the selectivity index (sensitivity value times specificity value), AG determinations were superior to both HbA1c and FA measurements for diabetes screening. When combinations of these tests were used, only AG and HbA1c together were slightly better than AG alone. Thus, together with other advantages of AG, e.g., its wide variance with relatively fair glycemic control and the negligible influence of the sampling conditions, AG level has more potential than HbA1c or FA level as a screening criterion for diabetes.
 
Article
The plasma concentration of 1,5-anhydro-D-glucitol (AG) was measured in 135 newly diagnosed patients who were referred for oral glucose tolerance tests. AG concentrations in the nondiabetic patients indicated that the mean value of normal AG concentration was 21.8 micrograms/ml (SD = 5.9 micrograms/ml, range 9.6-38.8 micrograms/ml). This distribution of AG concentration was significantly different from that in patients with impaired glucose tolerance (IGT) (13.3 +/- 5.4 micrograms/ml) and definitely different from that in diabetic patients (2.1 +/- 1.8 micrograms/ml). In a standard glucagon test, it was suggested that the decrease of plasma AG was affected not only by glycemic control of the patients but also by pancreatic cell secretory activity. The reduction of AG concentration was more marked in IDDM patients than in NIDDM patients. In longitudinal studies, AG concentration was shown to be sensitive to glycemic control. However, its recovery showed a tendency toward much delay after the improvement of fasting blood glucose or HbA1 concentrations. On the other hand, AG concentration showed negligible diurnal change and no immediate change as a result of diet, oral glucose load, or acute shift of the insulin level in both normal and diabetic subjects.
 
Article
Plasma levels of 1,5-anhydroglucitol (1,5AG), a major polyol resembling glucose in structure, fell rapidly and dramatically in streptozocin (STZ)-treated rats. 1,5AG fell immediately after STZ injection, reaching a plasma level 6 h after administration of the drug that was one-third that in the plasma of control rats. Reduction of 1,5AG was independent of the profile of blood glucose induced by STZ. After intravenous injection of [14C]-1,5AG, its plasma half-life was determined to be between 120 and 180 min. After a phase of acute decrease, the reduction of 1,5AG became gradual, stopping within 6 days after treatment. However, in some cases, the drop in 1,5AG was partially reversible by insulin treatment. The extent to which 1,5AG fell did not strictly correspond to the dose of STZ. The particular organ(s) consuming or accumulating 1,5AG was not identified. However, aside from the large amount of 1,5AG in plasma and the small amount of 1,5AG in the urine, the liver appears to be a significant organ for metabolism of 1,5AG.
 
Article
We thank Masotti (1) for his interest and comment in this issue of Diabetes on our recently published article (2). In his letter, Masotti proposed, on the basis of our study findings and those from his own laboratory, that the glycolytic/gluconeogenic pathways may play an integral role in body weight regulation. Fructose-1,6-bisphosphate is not only the key substrate in gluconeogenesis for fructose-6-phosphate production but is also the substrate for the production of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate via the enzyme aldolase A ( ALDOA )—a glycolytic-pathway activated reaction. Masotti and colleagues (3) recently demonstrated that increased expression of microRNA-122 was associated with the development of nonalcoholic fatty liver disease in rats fed a …
 
FBPase upregulation in mouse models of obesity and diabetes
Generation of MIN6 cells overexpressing huFBPase. A: MIN6 cells were cotransfected with 1-2 g transgene construct shown in Fig. 1A and 0.1-0.2 g pPGK-puro selection plasmid. Eighteen puromycinresistant colonies were isolated, of which three colonies were selected to be extensively studied with no (pPGK-puro), low, and high levels of the FBPase protein, respectively, as assessed by immunoblotting. Lane 1, parental MIN6 cells; lane 2, low FBPase; lane 3, high FBPase; lane 4, pGK-puro (no FBPase); lane 5, mouse liver control. B: FBPase enzyme activity in the cell lines depicted in A, showing increased activity in the cells with the highest FBPase protein levels, whereas the parental MIN6 and pPGK-puro cells had no detectable (ND) enzyme activity. Results are presented as means SE (n 3). *P < 0.05 compared with low-expressing FBPase transgenic cells.
Glucose-and non– glucose-induced insulin secretion in FBPase transgenic MIN6 cells. A: The cells were incubated in KRBB containing 2.8 mmol/l glucose, 16.8 mmol/l glucose, or 16.8 mmol/l glucose plus a cocktail of secretagogues, including 10 mmol/l arginine, 0.1 mmol/l IBMX, and 5 mol/l carbachol for 1 h. Medium was taken to determine levels of insulin secretion. B: Total cellular insulin content was determined by lysing the cells with 0.18 mol/l HCl/95% ethanol, followed by sonification. Insulin secretion was then expressed as relative to total cellular insulin content of cells. Results are presented as means SE of three independent experiments. *P < 0.05 compared with parental MIN6 cells.  
Glucose utilization, glucose oxidation, glycolytic intermediate, and cellular ATP levels in MIN6 cells overexpressing FBPase. A: Glucose utilization was measured by the conversion of [5-3 H]glucose into 3 H 2 O as described in RESEARCH DESIGN AND METHODS. Results are expressed as means SE of three independent experiments. *P < 0.05 compared with parental MIN6 cells. B: Glucose oxidation was measured by the conversion of [U-14 C]glucose into 14 CO 2 as described in RESEARCH DESIGN AND METHODS. Results are expressed as means SE of five independent experiments. *P < 0.05 compared with parental MIN6 cells. C: Fructose-1,6-bisphosphate and fructose-6-phosphate levels in parental, pGK-puro (no FBPase), low-FBPase, and high-FBPase MIN6 cells. Results are expressed as means SE of three independent experiments. *P < 0.05 compared with control MIN6 cells. D: Cellular ATP content was measured in parental, low-FBPase, and high-FBPase MIN6 cells. Results are expressed as means SE of three independent experiments. *P < 0.05 compared with parental cells.  
Cell proliferation rates in MIN6 cells overexpressing FBPase. A: Cells were counted on days 1, 3, 5, and 7 after seeding. Results are expressed as means SE of three independent experiments. *P < 0.05 compared with parental cells. B: The cell culture medium was supplemented so that the final concentration of pyruvate was 2.5 mmol/l, and cells were counted on days 1, 3, 5, and 7 after seeding. Results are expressed as means SE of three independent experiments. *P < 0.05 compared with parental cells.  
Article
Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme that is upregulated in islets or pancreatic beta-cell lines exposed to high fat. However, whether specific beta-cell upregulation of FBPase can impair insulin secretory function is not known. The objective of this study therefore is to determine whether a specific increase in islet beta-cell FBPase can result in reduced glucose-mediated insulin secretion. To test this hypothesis, we have generated three transgenic mouse lines overexpressing the human FBPase (huFBPase) gene specifically in pancreatic islet beta-cells. In addition, to investigate the biochemical mechanism by which elevated FBPase affects insulin secretion, we made two pancreatic beta-cell lines (MIN6) stably overexpressing huFBPase. FBPase transgenic mice showed reduced insulin secretion in response to an intravenous glucose bolus. Compared with the untransfected parental MIN6, FBPase-overexpressing cells showed a decreased cell proliferation rate and significantly depressed glucose-induced insulin secretion. These defects were associated with a decrease in the rate of glucose utilization, resulting in reduced cellular ATP levels. Taken together, these results suggest that upregulation of FBPase in pancreatic islet beta-cells, as occurs in states of lipid oversupply and type 2 diabetes, contributes to insulin secretory dysfunction.
 
Article
The effect of dehydroepiandrosterone (DHEA) on the hepatic and muscle glucose metabolizing enzymes and on blood glucose were investigated in insulin-resistant diabetic C57BL/KsJ-db/db mice and their heterozygote littermates (db/+m). The results were compared with those after troglitazone administration under the same conditions. Despite hyperinsulinemia, hepatic glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FBPase) activities are higher in db/db than in db/+m mice. Dietary administration of DHEA and that of troglitazone for 15 days to respective groups of five mice each significantly decreased blood glucose in db/db mice and hepatic G6Pase and FBPase activities in both db/db and db/+m mice. Hepatic G6Pase and FBPase activities showed a linear relationship with blood glucose in all groups of mice, suggesting that the activities of G6Pase and FBPase are closely related to blood glucose levels. Because androstenedione, a DHEA metabolite, barely affected either of these enzyme activities or blood glucose in db/db mice, the actions of DHEA, which are similar to those of troglitazone, are presumed to be caused by DHEA itself. DHEA is considered to be a modulating agent for the activities of hepatic gluconeogenic enzymes in db/db mice.
 
Structures of MB06322 and MB05032. MB06322 is an oral prodrug of MB05032, a potent FBPase inhibitor (IC 50 [half-maximal inhibitory concentration] 16 nmol/l) (26) that binds to the allosteric AMP-binding site of the enzyme. MB06322 is converted to MB05032 in vivo via the sequential actions of an esterase and phosphoramidase.
Physiological and metabolic parameters in control and MB06322-treated pre-diabetic male ZDF rats (nonfasted)*
Effect of acute MB06322 administration (300 mg/kg) on endogenous glucose production (EGP) rates ( A ), the fractional contribution of gluconeogenesis and glycogenolysis to endogenous glucose production ( B ), and gluconeogenesis and glycogenolysis rates ( C ) in fasting conscious ϳ 10-week-old male ZDF rats. Endogenous glucose production was determined by standard tracer methodology, whereas the fractional contribution of gluconeogenesis and glycogenolysis to endogenous glucose production was determined by the deuterated water method, as described in RESEARCH DESIGN AND METHODS . Statistical analysis was not applied to the gluconeogenesis and glycogenolysis rates because they are the products of parameters (endogenous glucose production and fractional contributions of gluconeogenesis and glycogenolysis at 1:00 P . M .) measured in two different, but carefully matched, sets of animals ( n ؍ 6 per group). * P < 0.05 compared with vehicle (Student’s t test). 
Physiological and metabolic parameters in control and MB06322-treated high-fat diet-fed female ZDF rats (nonfasted)
Article
Gluconeogenesis is increased in type 2 diabetes and contributes significantly to fasting and postprandial hyperglycemia. We recently reported the discovery of the first potent and selective inhibitors of fructose 1,6-bisphosphatase (FBPase), a rate-controlling enzyme of gluconeogenesis. Herein we describe acute and chronic effects of the lead inhibitor, MB06322 (CS-917), in rodent models of type 2 diabetes. In fasting male ZDF rats with overt diabetes, a single dose of MB06322 inhibited gluconeogenesis by 70% and overall endogenous glucose production by 46%, leading to a reduction in blood glucose of >200 mg/dl. Chronic treatment of freely feeding 6-week-old male Zucker diabetic fatty (ZDF) rats delayed the development of hyperglycemia and preserved pancreatic function. Elevation of lactate ( approximately 1.5-fold) occurred after 4 weeks of treatment, as did the apparent shunting of precursors into triglycerides. Profound glucose lowering ( approximately 44%) and similar metabolic ramifications were associated with 2-week intervention therapy of 10-week-old male ZDF rats. In high-fat diet-fed female ZDF rats, MB06322 treatment for 2 weeks fully attenuated hyperglycemia without evidence of metabolic perturbation other than a modest reduction in glycogen stores ( approximately 20%). The studies confirm that excessive gluconeogenesis plays an integral role in the pathophysiology of type 2 diabetes and suggest that FBPase inhibitors may provide a future treatment option.
 
Top-cited authors
Ralph Defronzo
  • University of Texas Health Science Center at San Antonio
Michael Brownlee
  • Albert Einstein College of Medicine
Robert Rizza
  • Mayo Foundation for Medical Education and Research
Jens J Holst
  • University of Copenhagen
Clifton Bogardus
  • The National Institute of Diabetes and Digestive and Kidney Diseases