Published by American Diabetes Association


Not Available
  • Article

January 1959


8 Reads

Abraham M Tornow





TABLE 2 Primer sequences 
Mapping of the IDDM Locus Idd3 to a 0.35-cM Interval Containing the Interleukin-2 Gene
  • Article
  • Full-text available

May 1997


88 Reads






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.

Next Generation Sequencing Reveals the Association of DRB3*02:02 With Type 1 Diabetes

March 2013


148 Reads

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.

In vivo cytotoxicity of insulin-specific CD8(+) T-cells in HLA-A*0201 transgenic NOD mice

November 2007


64 Reads

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.

Association of HLA-DPB1(*)0301 with IDDM in Mexican-Americans

June 1996


14 Reads

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.

FIG. 1. 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) .  
FIG. 2. 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.  
FIG. 4. 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.  
FIG. 5. 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).  
FIG. 3. 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.  
Major DQ8-restricted T-cell epitopes for human GAD65 mapped using human CD4, DQA1*0301, DQB1*0302 transgenic IA(null) NOD mice

April 1999


95 Reads

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.

The effect of HLA-B allele on the IDDM risk defined by DRB1*04 subtypes and DQB1*0302

November 1997


14 Reads

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.

Update of Retraction--Blockade of Receptor for Advanced Glycation End Products in a Model of Type 1 Diabetic Leukoencephalopathy. Diabetes. 19 November 2012 [Epub ahead of print]. DOI: 10.2337/db12-0317

May 2014


19 Reads

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 …

TABLE 4 Distribution of DRB1 alleles according to self-reported retinopathy status in case and control patients
Nephropathy in Type 1 Diabetes Is Diminished in Carriers of HLA-DRB1*04: The Genetics of Kidneys in Diabetes (GoKinD) Study

February 2008


54 Reads

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.

Characterization of New Oral Antidiabetic Agent CS-045: Studies in KK and ob/ob Mice and Zucker Fatty Rats

December 1988


43 Reads

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.

HLA-DQB1*0602 Is Associated With Dominant Protection From Diabetes Even Among Islet Cell Antibody-Positive First-Degree Relatives of Patients with IDDM

July 1995


38 Reads

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.

FIG. 1. 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. 
TABLE 2 Clinical characteristics of probands diagnosed in infancy and their family members with an INS gene mutation
Insulin Mutation Screening in 1,044 Patients With Diabetes Mutations in the INS Gene Are a Common Cause of Neonatal Diabetes but a Rare Cause of Diabetes Diagnosed in Childhood or Adulthood

April 2008


370 Reads

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.

No evidence for association between IDDMK(1,2)22, a novel isolated retrovirus, and IDDM

February 1999


10 Reads

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.

1,2-Diacylglycerol Content and Its Fatty Acid Composition in Thoracic Aorta of Diabetic Rats

August 1991


6 Reads

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.

Inositol Glycan Phosphate Derived from Human Erythrocyte Acetylcholinesterase Glycolipid Anchor and Inositol Cyclic 1,2-Phosphate Antagonize Glucagon Activation of Glycogen Phosphorylase

October 1993


19 Reads

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)

Alteration of 1,2-Diacylglycerol Content in Myocardium From Diabetic Rats

October 1988


26 Reads

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.

FIG. 1. 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-.
FIG. 2. 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.
FIG. 3. 1,25(OH) 2 D 3 or TX527 treatment affects DC cytokine produc- 
FIG. 5. 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.
FIG. 7. 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.
Redirection of Human Autoreactive T-Cells Upon Interaction With Dendritic Cells Modulated by TX527, an Analog of 1,25 Dihydroxyvitamin D3

August 2002


86 Reads

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.

Effect of 1,25 Dihydroxyvitamin D3 on Insulin Secretion

June 1981


12 Reads

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.

Demonstration that the Vitamin D Metabolite 1,25(OH)2-Vitamin D3 and Not 24R,25(OH)2-Vitamin D3 Is Essential for Normal Insulin Secretion in the Perfused Rat Pancreas

May 1985


15 Reads

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)2D3but 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.

FIG. 1. 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. 
1,25-Dihydroxyvitamin D3 Prevents Insulitis in NOD Mice

November 1992


87 Reads

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.

FIG. 1. 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. 
FIG. 2. 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. 
FIG. 3. 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. 
Expression of Galα(1,3)Gal on neonatal porcine islet β-cells and susceptibility to human antibody/complement lysis

September 1998


64 Reads

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.

Assessors of Fetal Perinatal Mortality in Diabetic Pregnancy: Analysis of 1,332 Pregnancies in the Copenhagen Series, 1946-1972

May 1974


7 Reads

In a series of 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 PBSP classifications. Also, mortality declined steadily over the years 1946 to 1972. Fatal congenital malformation was 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 fulfill 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.

Bradykinin B2 Receptors on Skeletal Muscle are Coupled to Inositol 1,4,5-Trisphosphate Formation

February 1996


15 Reads

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.

FIG. 1. Electrically evoked Ca 2 ؉ transients in ventricular cardiomyocytes from wild-type and ob / ob mice. Confocal line-scan images from a 
FIG. 2. Spatially averaged Ca 2 ؉ transients (expressed as normalized 
FIG. 3. Spatially averaged Ca 2 ؉ transients from an ob / ob cell recorded 
FIG. 5. Mitochondrial Ca 2 ؉ measured with rhod-2 in isolated wild-type 
Insulin and Inositol 1,4,5-Trisphosphate Trigger Abnormal Cytosolic Ca2+ Transients and Reveal Mitochondrial Ca2+ Handling Defects in Cardiomyocytes of ob/ob Mice

September 2005


90 Reads

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.

Plasma 1,5-Anhydro-D-Glucitol as New Clinical Marker of Glycemic Control in NIDDM Patients

July 1989


36 Reads

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.

Marked Depletion of Plasma 1,5-Anhydroglucitol, a Major Polyol, in Streptozocin-induced Diabetes in Rats and the Effect of Insulin Treatment

March 1986


15 Reads

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.

Reduction and Recovery of Plasma 1,5-Anhydro-D-Glucitol Level in Diabetes Mellitus

July 1987


19 Reads

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.

Comparison of 1,5-Anhydroglucitol, HbA1c, and Fructosamine for Detection of Diabetes Mellitus

February 1991


31 Reads

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.

Dehydroepiandrosterone suppresses the elevated hepatic glucose-6-phosphatase and fructose-1,6-bisphosphatase activities in C57BL/Ksj-db/db mice - Comparison with troglitazone

September 1999


10 Reads

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.

Response to Comment on: Visinoni et al. The Role of Liver Fructose-1,6-Bisphosphatase in Regulating Appetite and Adiposity. Diabetes 2012;61:1122–1132

December 2012


58 Reads

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 …

Impaired Regulation of Hepatic Fructose-1,6-Bisphosphatase in the New Zealand Obese Mouse Model of NIDDM

January 1994


14 Reads

The New Zealand obese mouse, a model of NIDDM, is characterized by hyperglycemia, hyperinsulinemia, and hepatic and peripheral insulin resistance. The aim of this study was to investigate the biochemical basis of hepatic insulin resistance in NZO mice. Glycolytic and gluconeogenic enzyme activities were measured in fed and overnight fasted 19- to 20-wk-old NZO and control New Zealand chocolate mice. The NZO mice were twice as heavy as the NZC mice. The activity of the glycolytic enzymes glucokinase and pyruvate kinase was higher, whereas that of the gluconeogenic enzymes PEPCK and glucose-6-phosphatase was lower in fed and fasted NZO mice. These enzyme changes are consistent with a normal response to the hyperinsulinemia in NZO mice. In contrast, the activity of the third regulated gluconeogenic enzyme, fructose-1,6-bisphosphatase, was similar in fed and fasted NZO and NZC mice despite the higher insulin and glucose levels in the NZO mouse. This enzyme is primarily regulated by the powerful inhibitor fructose-2,6-bisphosphate. The levels of this metabolite were measured and found to be increased in both the fed and fasted states in the NZO mouse, suggesting that the activity of the bifunctional enzyme that regulates the level of inhibitor (6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase) is normally regulated in the NZO mouse. We conclude that most insulin-responsive gluconeogenic and glycolytic enzymes are normally regulated in the NZO mouse, but an abnormality in the regulation of fructose-1,6-bisphosphatase may contribute to the increase hepatic glucose production in these mice.

The Role of Liver Fructose-1,6-Bisphosphatase in Regulating Appetite and Adiposity

May 2012


373 Reads

Liver fructose-1,6-bisphosphatase (FBPase) is a regulatory enzyme in gluconeogenesis that is elevated by obesity and dietary fat intake. Whether FBPase functions only to regulate glucose or has other metabolic consequences is not clear; therefore, the aim of this study was to determine the importance of liver FBPase in body weight regulation. To this end we performed comprehensive physiologic and biochemical assessments of energy balance in liver-specific transgenic FBPase mice and negative control littermates of both sexes. In addition, hepatic branch vagotomies and pharmacologic inhibition studies were performed to confirm the role of FBPase. Compared with negative littermates, liver-specific FBPase transgenic mice had 50% less adiposity and ate 15% less food but did not have altered energy expenditure. The reduced food consumption was associated with increased circulating leptin and cholecystokinin, elevated fatty acid oxidation, and 3-β-hydroxybutyrate ketone levels, and reduced appetite-stimulating neuropeptides, neuropeptide Y and Agouti-related peptide. Hepatic branch vagotomy and direct pharmacologic inhibition of FBPase in transgenic mice both returned food intake and body weight to the negative littermates. This is the first study to identify liver FBPase as a previously unknown regulator of appetite and adiposity and describes a novel process by which the liver participates in body weight regulation.

FIG. 1. 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.
TABLE 1 Physiological and metabolic parameters in control and MB06322-treated pre-diabetic male ZDF rats (nonfasted)*
FIG. 2. 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). 
TABLE 3 Physiological and metabolic parameters in control and MB06322-treated high-fat diet-fed female ZDF rats (nonfasted)
Inhibition of Fructose 1,6-Bisphosphatase Reduces Excessive Endogenous Glucose Production and Attenuates Hyperglycemia in Zucker Diabetic Fatty Rats

June 2006


197 Reads

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.

Imaging Islets Labeled With Magnetic Nanoparticles at 1.5 Tesla

December 2006


277 Reads

We have developed a magnetic resonance imaging (MRI) technique for imaging Feridex (superparamagnetic iron oxide [SPIO])-labeled islets of Langerhans using a standard clinical 1.5-Tesla (T) scanner and employing steady-state acquisition imaging sequence (3DFIESTA). Both porcine and rat islets were labeled with SPIO by a transfection technique using a combination of poly-l-lysine and electroporation. Electron microscopy demonstrated presence of SPIO particles within the individual islet cells, including beta-cells and particles trapped between cell membranes. Our labeling method produced a transfection rate of 860 pg to 3.4 ng iron per islet, dependent on the size of the islet. The labeling procedure did not disrupt either the function or viability of the islets. In vitro 3DFIESTA magnetic resonance images of single-labeled islets corresponded with their optical images. In vivo T2*-weighted scan using 1.5 T detected as few as 200 SPIO-labeled islets transplanted under rat kidney capsule, which correlated with immunohistochemistry of the transplant for insulin and iron. Ex vivo 3DFIESTA images of kidneys containing 200, 800 or 2,000 SPIO-labeled islet isografts showed good correlation between signal loss and increasing numbers of islets. These data provide evidence that islets can be labeled with SPIO and imaged using clinically available 1.5- T MRI.

Diabetes Incidence: A Two-year Follow-up of 10,000 Men in a Survey of Ischemic Heart Disease in Israel

December 1970


8 Reads

In 1963, 10,059 men aged forty years and over were examined in an I.H.D. survey and 9,711 of them were re-examined two years later. At both these examinations suspect diabetics werescreened out by a casual blood glucose value and/or a positive history of diabetes. Further information was collected on all these suspect diabetics to enable their classification into diabetic categories using similar criteria on both occasions. Among 9,079 persons judged at risk of developing diabetes, 144 new cases of diabetes were discovered two years after the 1963 examination. The over-all two-year incidence rate for men over forty was 15.5 per thousand. Four regions of origin, Central Europe, Southeastern Europe, Israel and North Africa had incidence rates approximately equal to the over-all rate. The age adjusted incidence rate for men from Eastern Europe was lower (8.5 per 1,000) and from the Middle East higher (20.3 per 1,000) than the average.

Studies of Association of Variants Near the HHEX, CDKN2A/B, and IGF2BP2 Genes With Type 2 Diabetes and Impaired Insulin Release in 10,705 Danish Subjects Validation and Extension of Genome-Wide Association Studies

January 2008


241 Reads

In the present study, we aimed to validate the type 2 diabetes susceptibility alleles identified in six recent genome-wide association studies in the HHEX/KIF11/IDE (rs1111875), CDKN2A/B (rs10811661), and IGF2BP2 (rs4402960) loci, as well as the intergenic rs9300039 variant. Furthermore, we aimed to characterize quantitative metabolic risk phenotypes of the four variants. The variants were genotyped in the population-based Inter99 cohort (n = 5,970), the ADDITION Study (n = 1,626), a population-based sample of young healthy subjects (n = 377), and in additional type 2 diabetic case (n = 2,111) and glucose-tolerant (n = 521) subjects. The case-control studies involved a total of 4,089 type 2 diabetic patients and 5,043 glucose-tolerant control subjects. We validated association of variants near HHEX/KIF11/IDE, CDKN2A/B, and IGF2BP2 with type 2 diabetes. Interestingly, in middle-aged people, the rs1111875 C-allele of HHEX/KIF11/IDE strongly associated with lower acute insulin response during an oral glucose tolerance test (P = 6 x 10(-7)). In addition, decreased insulin release following intravenous tolbutamide injection was observed in young healthy subjects (P = 0.02). Also, a reduced insulin release was observed for the CDKN2A/B rs10811661 T-allele after both oral and intravenous glucose challenges (P = 0.001 and P = 0.009, respectively). We validate that variants in the proximity of the HHEX/KIF11/IDE, CDKN2A/B, and IFG2BP2 loci associate with type 2 diabetes. Importantly, variations within the HHEX/KIF11/IDE and CDKN2A/B loci confer impaired glucose- and tolbutamide-induced insulin release in middle-aged and young healthy subjects, suggesting a role for these variants in the pathogenesis of pancreatic beta-cell dysfunction.

Activation of Aldose Reductase by Interaction With Tubulin and Involvement of This Mechanism in Diabetic Cataract Formation. Diabetes. 10 April 2014 [Epub ahead of print]. DOI: 10.2337/db13-1265

April 2014


99 Reads

Our previous studies have shown that high levels of glucose induce inhibition of Na(+),K(+)-ATPase (NKA) via stimulation of aldose reductase (AR), polymerisation of microtubules, and formation of an acetylated tubulin/NKA complex. Inhibition of AR eliminated the effect of high glucose on NKA activity. In this study, we investigated the mechanism of regulation of AR activity by tubulin. Purified tubulin and AR were used. The results indicate that: (i) tubulin and AR interact with each other directly; (ii) tubulin/AR interaction results in a 6-fold increase of AR activity under microtubule growing conditions; (iii) AR interacts preferentially with tubulin that contains 3-nitro-L-tyrosine (3-NTyr); (iv) free tyrosine and 3-nitro-tyrsine are able to block tubulin/AR interaction and thereby prevent AR activation; (v) exposure of cultured COS cells to high glucose concentrations promotes microtubule polymerisation and NKA inhibition, and both these promoting effects are inhibited by addition of free Tyr or 3-NTyr; (vi) treatment of experimental (STZ-induced) diabetic rats with 3-NTyr prevented cataract formation, suggesting that this complication of diabetes involves tubulin/AR interaction and AR activity. Taken together, these findings indicate that AR activity is controlled by association/dissociation of the tubulin/AR complex, that Tyr and 3-NTyr block such effect by preventing tubulin/AR complex formation, and that AR activity can be reduced by 3-NTyr or other compounds that inhibit tubulin/AR interaction.

Adenovirus Early Region 3 Antiapoptotic 10.4K, 14.5K, and 14.7K Genes Decrease the Incidence of Autoimmune Diabetes in NOD Mice

June 2003


40 Reads

Genes in the early region 3 (E3) of the adenovirus genome allow the virus to evade host immune responses by interfering with major histocompatibility (MHC) class I-mediated antigen presentation and tumor necrosis factor-alpha (TNF-alpha)- or Fas-induced apoptosis of infected cells. Autoimmune type 1 diabetes (T1D) is inhibited in NOD mice transgenically expressing all E3 genes under control of a rat insulin promoter (RIPE3/NOD). For dissecting the protective mechanisms afforded by various E3 genes, they were subdivided into RIP-driven transgene constructs. Strong T1D protection mediated at the beta-cell level characterized DL704/NOD mice lacking the E3 gp19K gene suppressing MHC class I expression but retaining the 10.4K, 14.5K, and 14.7K genes inhibiting Fas- or TNF-alpha-induced apoptosis and TNF-alpha-induced NF-kB activation. Much weaker protection characterized DL309/NOD mice expressing the gp19K but not the 10.4K, 14.5K, and 14.7K genes. While RIPE3/NOD splenocytes had an unexpected decrease in ability to adoptively transfer T1D, splenocytes from both the DL704 and DL309 stocks efficiently did so. These findings indicate that all E3 genes must be expressed to inhibit the diabetogenic potential of NOD immune cells. They also demonstrate that the antiapoptotic E3 genes most effectively protect pancreatic beta-cells from diabetogenic immune responses.

A Search for Variants Associated With Young-Onset Type 2 Diabetes in American Indians in a 100K Genotyping Array

December 2007


81 Reads

To identify genetic variants in linkage disequilibrium with those conferring diabetes susceptibility, a genome-wide association study for young-onset diabetes was conducted in an American-Indian population. Data come from 300 case subjects with type 2 diabetes with age of onset <25 years and 334 nondiabetic control subjects aged >or=45 years. To provide for tests of within-family association, 121 nondiabetic siblings of case subjects were included along with 140 diabetic siblings of control subjects (172 sibships). Individuals were genotyped on the Affymetrix 100K array, resulting in 80,044 usable single nucleotide polymorphisms (SNPs). SNPs were analyzed for within-family association and for general association in case and control subjects, and these tests were combined by Fisher's method, with priority given to the within-family test. There were more SNPs with low P values than expected theoretically under the global null hypothesis of no association, and 128 SNPs had evidence for association at P < 0.001. The association of these SNPs with diabetes was further investigated in 1,207 diabetic and 1,627 nondiabetic individuals from the population study who were not included in the genome-wide study. SNPs from 10 genomic regions showed evidence for replication at P < 0.05. These included SNPs on chromosome 3 near ZNF659, chromosome 11 near FANCF, chromosome 11 near ZBTB15, and chromosome 12 near SENP1. These studies suggest several regions where marker alleles are potentially in linkage disequilibrium with variants that confer susceptibility to young-onset type 2 diabetes in American Indians.

A 100K Genome-Wide Association Scan for Diabetes and Related Traits in the Framingham Heart Study Replication and Integration With Other Genome-Wide Datasets

January 2008


71 Reads

To use genome-wide fixed marker arrays and improved analytical tools to detect genetic associations with type 2 diabetes in a carefully phenotyped human sample. A total of 1,087 Framingham Heart Study (FHS) family members were genotyped on the Affymetrix 100K single nucleotide polymorphism (SNP) array and examined for association with incident diabetes and six diabetes-related quantitative traits. Quality control filters yielded 66,543 SNPs for association testing. We used two complementary SNP selection strategies (a "lowest P value" strategy and a "multiple related trait" strategy) to prioritize 763 SNPs for replication. We genotyped a subset of 150 SNPs in a nonoverlapping sample of 1,465 FHS unrelated subjects and examined all 763 SNPs for in silico replication in three other 100K and one 500K genome-wide association (GWA) datasets. We replicated associations of 13 SNPs with one or more traits in the FHS unrelated sample (16 expected under the null); none of them showed convincing in silico replication in 100K scans. Seventy-eight SNPs were nominally associated with diabetes in one other 100K GWA scan, and two (rs2863389 and rs7935082) in more than one. Twenty-five SNPs showed promising associations with diabetes-related traits in 500K GWA data; one of them (rs952635) replicated in FHS. Five previously reported associations were confirmed in our initial dataset. The FHS 100K GWA resource is useful for follow-up of genetic associations with diabetes-related quantitative traits. Discovery of new diabetes genes will require larger samples and a denser array combined with well-powered replication strategies.

Response to Comment on: Padmavathi et al. (2010) Chronic Maternal Dietary Chromium Restriction Modulates Visceral Adiposity: Probable Underlying Mechanisms. Diabetes;59:98–104

April 2010


28 Reads

We thank Vincent and Rasco (1) for showing interest in our work. In this present study (2), diets were prepared as per AIN 93G formulations, which use chromium (Cr) at a level of 1 mg/kg diet as recommended earlier for rodents (3). Despite this, the finding that the control diet had 1.56 mg Cr/kg diet suggests that this could be due to contributions from other dietary ingredients. This is corroborated by our observation that Cr-restricted diet, in which Cr salt was deleted, had 0.51 mg Cr/kg diet. As indicated by Vincent …

Salicylate Downregulates 11 -HSD1 Expression in Adipose Tissue in Obese Mice and in Humans, Mediating Insulin Sensitization

February 2012


194 Reads

Recent trials show salicylates improve glycemic control in type 2 diabetes, but the mechanism is poorly understood. Expression of the glucocorticoid-generating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in adipose tissue is increased in vitro by proinflammatory cytokines and upregulated in obesity. 11β-HSD1 inhibition enhances insulin sensitivity. We hypothesized that salicylates downregulate 11β-HSD1 expression, contributing to their metabolic efficacy. We treated diet-induced obese (DIO) 11β-HSD1-deficient mice and C57Bl/6 mice with sodium salicylate for 4 weeks. Glucose tolerance was assessed in vivo. Tissue transcript levels were assessed by quantitative PCR and enzyme activity by incubation with (3)H-steroid. Two weeks' administration of salsalate was also investigated in a randomized double-blind placebo-controlled crossover study in 16 men, with measurement of liver 11β-HSD1 activity in vivo and adipose tissue 11β-HSD1 transcript levels ex vivo. In C57Bl/6 DIO mice, salicylate improved glucose tolerance and downregulated 11β-HSD1 mRNA and activity selectively in visceral adipose. DIO 11β-HSD1-deficient mice were resistant to these metabolic effects of salicylate. In men, salsalate reduced 11β-HSD1 expression in subcutaneous adipose, and in vitro salicylate treatment reduced adipocyte 11β-HSD1 expression and induced adiponectin expression only in the presence of 11β-HSD1 substrate. Reduced intra-adipose glucocorticoid regeneration by 11β-HSD1 is a novel mechanism that contributes to the metabolic efficacy of salicylates.

FIG. 2. Effect of prenatal dexamethasone on 11-HSD1 expression in the liver. Hepatic 11-HSD1 mRNA expression was measured by realtime PCR in 4-month-old (A) and 24-month-old (B) offspring of mothers that received vehicle (Control) or dexamethasone during early (Early- DEX) or late (Late-DEX) pregnancy. 11-HSD1 enzyme activity was assessed in 24-month-old livers (C). Results represent mRNA expression relative to control animals. *P < 0.05 compared with control animals.  
FIG. 5. Effect of dexamethasone on glucocorticoid receptor expression. Tissue glucocorticoid receptor mRNA was measured in liver (A), subcutaneous fat (B), peritoneal fat (C), and pancreas (D) by real-time PCR in 24-month-old offspring of mothers that received vehicle (Control) or dexamethasone during early (Early-DEX) or late (Late-DEX) pregnancy. Results are expressed as mRNA expression relative to control animals.  
Prenatal Programming of Metabolic Syndrome in the Common Marmoset Is Associated With Increased Expression of 11 -Hydroxysteroid Dehydrogenase Type 1

September 2009


58 Reads

Recent studies in humans and animal models of obesity have shown increased adipose tissue activity of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which amplifies local tissue glucocorticoid concentrations. The reasons for this 11beta-HSD1 dysregulation are unknown. Here, we tested whether 11beta-HSD1 expression, like the metabolic syndrome, is "programmed" by prenatal environmental events in a nonhuman primate model, the common marmoset monkey. We used a "fetal programming" paradigm where brief antenatal exposure to glucocorticoids leads to the metabolic syndrome in the offspring. Pregnant marmosets were given the synthetic glucocorticoid dexamethasone orally for 1 week in either early or late gestation, or they were given vehicle. Tissue 11beta-HSD1 and glucocorticoid receptor mRNA expression were examined in the offspring at 4 and 24 months of age. Prenatal dexamethasone administration, selectively during late gestation, resulted in early and persistent elevations in 11beta-HSD1 mRNA expression and activity in the liver, pancreas, and subcutaneous-but not visceral-fat. The increase in 11beta-HSD1 occurred before animals developed obesity or overt features of the metabolic syndrome. In contrast to rodents, in utero dexamethasone exposure did not alter glucocorticoid receptor expression in metabolic tissues in marmosets. These data suggest that long-term upregulation of 11beta-HSD1 in metabolically active tissues may follow prenatal "stress" hormone exposure and indicates a novel mechanism for fetal origins of adult obesity and the metabolic syndrome.

FIG. 1. Association analysis of 52 SNPs in ARHGEF11 with type 2 diabetes cases (DM; n ؍ 145) and combined type 2 diabetes/IGT cases (DMIGT; n ؍ 293) compared with NGT control subjects ( n ؍ 358). ARHGEF11 gene structure is shown above the graph, which plots the P values as a function of physical location on the chromosome. Below the graph is an LD plot of the 52 SNPs in ARHGEF11. D ؅ values are depicted as a color scale with darker shades of orange representing higher values. LD plot was generated using Haploview version 3.2. 
TABLE 1 ARHGEF11 SNPs significantly associated with type 2 diabetes and type 2 diabetes/IGT in the Amish
FIG. 2. A : Examples of two ARHGEF11 SNPs associated with glucose levels during a 3-h oral glucose tolerance test in nondiabetic subjects. B : Eighteen type 2 diabetes–associated SNPs were significantly associated with oral glucose tolerance test glucose AUC (GAUC) and post-challenge glucose levels in nondiabetic subjects. * P < 0.05 (1, common allele; 2, minor allele). 
TABLE 2 Association analysis of ARHGEF11 haplotypes with type 2 diabetes and type 2 diabetes/IGT and glucose AUC during an oral glucose tolerance test
Evidence That Rho Guanine Nucleotide Exchange Factor 11 (ARHGEF11) on 1q21 is a Type 2 Diabetes Susceptibility Gene in the Old Order Amish

June 2007


97 Reads

Rho guanine nucleotide exchange factor 11 (ARHGEF11), located on chromosome 1q21, is involved in G protein signaling and is a pathway known to play a role in both insulin secretion and action. We genotyped 52 single nucleotide polymorphims (SNPs) in ARHGEF11 and compared the genotype frequencies of subjects with type 2 diabetes (n = 145) or type 2 diabetes/impaired glucose tolerance (IGT) (n = 293) with those of control subjects with normal glucose tolerance (NGT) (n = 358). Thirty SNPs, spanning the entire gene, were significantly associated with type 2 diabetes or type 2 diabetes/IGT. The most significantly associated SNP was rs6427340 (intron 2), in which the less common allele was the risk allele (odds ratio [OR] 1.82 [95% CI 1.20-2.70], P = 0.005 for type 2 diabetes vs. NGT and 1.79 [1.27-2.50], P = 0.0008 for type 2 diabetes/IGT vs. NGT). In an expanded set of nondiabetic subjects (n = 754), most of the type 2 diabetes-and IGT-associated SNPs were significantly associated with glucose levels during an oral glucose tolerance test, with the same SNP (rs6427340) showing the most significant associations (P = 0.007). All type 2 diabetes-and IGT-associated SNPs were in high linkage disequilibrium and constitute a single 133-kb haplotype block. These results, coupled with similar findings in Pima Indians, suggest that sequence variation in ARHGEF11 may influence risk of type 2 diabetes.

The Kruppel-Like Factor 11 (KLF11) Q62R Polymorphism Is Not Associated With Type 2 Diabetes in 8,676 People

January 2007


169 Reads

Krüppel-like factor 11 is a pancreatic transcription factor whose activity induces the insulin gene. A common glutamine-to-arginine change at codon 62 (Q62R) in its gene KLF11 has been recently associated with type 2 diabetes in two independent samples. Q62R and two other rare missense variants (A347S and T220M) were also shown to affect the function of KLF11 in vitro, and insulin levels were lower in carriers of the minor allele at Q62R. We therefore examined their impact on common type 2 diabetes in several family-based and case-control samples of northern-European ancestry, totaling 8,676 individuals. We did not detect the rare A347S and T220M variants in our samples. With respect to Q62R, despite >99% power to detect an association of the previously published magnitude, Q62R was not associated with type 2 diabetes (pooled odds ratio 0.97 [95% CI 0.88-1.08], P = 0.63). In a subset of normoglycemic individuals, we did not observe significant differences in various insulin traits according to genotype at KLF11 Q62R. We conclude that the KLF11 A347S and T220M mutations do not contribute to increased risk of diabetes in European-derived populations and that the Q62R polymorphism has, at best, a minor effect on diabetes risk.

FIG. 1. Plasma membrane GLUT4 ( A ), insulin receptor ( B ), and actin ( C ) expression in adipose tissue extracts from control (linoleic acid), c9,t11-CLA (CLA) dietary treatments, and TZD-treated C57Bl-6 ob / ob mice fed specialized diets for 4 weeks ( n ؍ 3 per group). Representative blot is displayed. Histogram from densitometric analysis expressed as arbitrary units are means ؎ SE, normalized to the actin control. Significantly different from control (linoleic acid), * P < 0.05. D : The effect of feeding a control (linoleic acid) or c9,t11-CLA– enriched diet for 4 weeks ( n ؍ 8 per group) on mRNA expression of molecular markers of insulin sensitivity and adipocytokine expression in white adipose tissue of fasting male ob / ob mice. Target gene mRNA levels were normalized to GAPDH and are expressed relative to the control group. Values represent group means ؎ SE. Significantly different from the control diet, * P ؍ 0.05. 
FIG. 2. Immunostaining of F4/80 in white adipose tissue sections from ob / ob mice fed control (linoleic acid) ( n ؍ 10) ( A ) and c9,t11-CLA– enriched diet ( n ؍ 10) ( B ). Staining is representative of three experiments at 40 ؋ magnification. C : F4/80 immunohistochemistry quantification representing macrophage surface area/total surface area in each dietary group ( n ؍ 10 per group). Data are means ؎ SE. * P < 0.05 vs. control diet. 
FIG. 3. Nuclear and cytosolic NF- ␬ B p65 and I ␬ B ␣ expression and NF- ␬ B:DNA binding in white adipose tissue after a control (linoleic acid) or c9,t11-CLA– enriched diet ( n ؍ 3 per group). NF- ␬ B p65 expression ( A ), cytosolic NF- ␬ B p65 expression ( B ), nuclear I ␬ B ␣ expression ( C ), cytosolic I ␬ B ␣ expression ( D ), nuclear actin expression ( E ), cytosolic actin expression ( F ), and NF- ␬ B:DNA binding ( G ). Representative blots are displayed. Histograms from densitometric analysis expressed as arbitrary units are group means ؎ SE. Western blots are normalized to the actin control. Significantly different from the control diet, ** P < 0.01, † P < 0.001. 
FIG. 5. Effect of linoleic acid and c9,t11-CLA on GLUT4, IRS-1, and TNF-mRNA expression in 3T3-L1 adipocytes (A) and TNF-induced insulin-resistant 3T3-L1 adipocytes (B). C: TNF-concentration (pg/ ml) in the culture media of TNF-induced insulin-resistant 3T3-L1 adipocytes. Target gene mRNA levels were normalized to GAPDH and expressed relative to DMSO. Values represent group means SE of six independent experiments (n 6 per group). In normal adipocytes, GLUT4 significantly increased (*P < 0.001) by c9,t11-CLA compared with DMSO and linoleic acid. In TNF-treated insulin-resistant adipocytes, c9,t11-CLA significantly increased GLUT4 and IRS-1 mRNA (*P < 0.05) and decreased TNF-mRNA expression ( †P < 0.05) and TNF-levels in the cell culture media compared with DMSO and linoleic acid-treated cells (B).
Antidiabetic Effects of cis-9, trans-11–Conjugated Linoleic Acid May Be Mediated via Anti-Inflammatory Effects in White Adipose Tissue

March 2007


237 Reads

Adipose tissue may be the source of insulin desensitizing proinflammatory molecules that predispose to insulin resistance. This study investigated whether dietary fatty acids could attenuate the proinflammatory insulin-resistant state in obese adipose tissue. The potential antidiabetic effect of cis-9, trans-11-conjugated linoleic acid (c9,t11-CLA) was determined, focusing on the molecular markers of insulin sensitivity and inflammation in adipose tissue of ob/ob C57BL-6 mice. Feeding a c9,t11-CLA-enriched diet reduced fasting glucose (P < 0.05), insulin (P < 0.05), and triacylglycerol concentrations (P < 0.01) and increased adipose tissue plasma membrane GLUT4 (P < 0.05) and insulin receptor (P < 0.05) expression compared with the control linoleic acid-enriched diet. Interestingly, after the c9,t11-CLA diet, adipose tissue macrophage infiltration was less, with marked downregulation of several inflammatory markers in adipose tissue, including reduced tumor necrosis factor-alpha and CD68 mRNA (P < 0.05), nuclear factor-kappaB (NF-kappaB) p65 expression (P < 0.01), NF-kappaB DNA binding (P < 0.01), and NF-kappaB p65, p50, c-Rel, p52, and RelB transcriptional activity (P < 0.01). To define whether these observations were direct effects of the nutrient intervention, complimentary cell culture studies showed that c9,t11-CLA inhibited tumor necrosis factor-alpha-induced downregulation of insulin receptor substrate 1 and GLUT4 mRNA expression and promoted insulin-stimulated glucose transport in 3T3-L1 adipocytes compared with linoleic acid. This study suggests that altering fatty acid composition may attenuate the proinflammatory state in adipose tissue that predisposes to obesity-induced insulin resistance.

FIG. 1. Effects of 11-HSD-1 deficiency and high-fat feeding on fat distribution and glucose homeostasis in MF-1 mice. A: Mesenteric visceral (MES) and epididymal (EPI) fat pad weights in MF-1 (f) or MF-1-11HSD-1 / () mice fed a control (C) or high-fat (HF) diet. *P < 0.05, effect of high fat within genotype; †P < 0.05 between genotypes. B: Glucose tolerance in MF-1 (F) and MF-1-11-HSD-1 / () mice on a high-fat diet. The horizontal broken line represents the mean peak glucose value for MF-1 mice on the control diet (omitted for clarity). *P < 0.01 for effect of high fat in MF-1 mice; †P < 0.01 for effect of genotype. 
Novel Adipose Tissue-Mediated Resistance to Diet-Induced Visceral Obesity in 11 -Hydroxysteroid Dehydrogenase Type 1-Deficient Mice

May 2004


104 Reads

The metabolic syndrome (visceral obesity, insulin resistance, type 2 diabetes, and dyslipidemia) resembles Cushing's Syndrome, but without elevated circulating glucocorticoid levels. An emerging concept suggests that the aberrantly elevated levels of the intracellular glucocorticoid reamplifying enzyme 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD-1) found in adipose tissue of obese humans and rodents underlies the phenotypic similarities between idiopathic and "Cushingoid" obesity. Transgenic overexpression of 11 beta-HSD-1 in adipose tissue reproduces a metabolic syndrome in mice, whereas 11 beta-HSD-1 deficiency or inhibition has beneficial metabolic effects, at least on liver metabolism. Here we report novel protective effects of 11 beta-HSD-1 deficiency on adipose function, distribution, and gene expression in vivo in 11 beta-HSD-1 nullizygous (11 beta-HSD-1(-/-)) mice. 11 beta-HSD-1(-/-) mice expressed lower resistin and tumor necrosis factor-alpha, but higher peroxisome proliferator-activated receptor-gamma, adiponectin, and uncoupling protein-2 mRNA levels in adipose, indicating insulin sensitization. Isolated 11 beta-HSD-1(-/-) adipocytes exhibited higher basal and insulin-stimulated glucose uptake. 11 beta-HSD-1(-/-) mice also exhibited reduced visceral fat accumulation upon high-fat feeding. High-fat-fed 11 beta-HSD-1(-/-) mice rederived onto the C57BL/6J strain resisted diabetes and weight gain despite consuming more calories. These data provide the first in vivo evidence that adipose 11 beta-HSD-1 deficiency beneficially alters adipose tissue distribution and function, complementing the reported effects of hepatic 11 beta-HSD-1 deficiency or inhibition.

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