bluuebunny/crossref_metadata_2025 huggingface dataset:
Diabetes-Induced, Progressive Endometrial Involution Characterization of Periluminal Epithelial Lipoatrophy
Authors: Garris David R. , Garris Bryan L.
Published: March 2007

The present studies detail the cytopathological alterations in uterine epithelial, basal lamina, and stromal endometrial subregions, and associated endocrine parameters that occur during the progressive exacerbation of the diabetes syndrome in this species of mouse. These alterations result in a cellular lipoatrophic condition that compromises uterine tissue integrity and promotes reproductive involution. Uterine tissue samples were obtained from litter-matched control (+/?) and diabetic (db/db) C57BL/KsJ mice at four designated stages of the progressive expression of the diabetes mutation. In db/db mice between the ages of 4 and 12 weeks, the uterine epithelial cellular architecture exhibited progressive deterioration, characterized by cytoplasmic lipid imbibition (accumulation), organelle disintegration, apical membrane ciliary regression, and peristromal lamina separation from basal membrane surfaces, as compared with control indexes. The cytoplasmic volume occupied by lipid inclusions dominated the epithelial cells in diabetic mice, presenting dense basal pole lipid vacuoles, with perinuclear-intracytoplasmic migration of the inclusions promoting an apical cytoplasmic lipid condensation of increasing volume 8–12 weeks after mutation expression. These cytoplasmic lipid accumulations occurred under altered metabolic and endocrine conditions characterized by hyperglycemic, hyperinsulinemic, hypertriglyceridemic, and enhanced noradrenergic indexes, which were exacerbated between 4- and 12-week stages. These structural changes were accompanied by enhanced adrenergic counterregulatory metabolic responses as well as elevated lipoprotein and triacylglycerol lipase activities. These data indicate that diabetes-associated uterine involution is characterized by a progressive cellular and peristromal lipoatrophy of epithelial cell cytology and metabolic parameters, promoting stromal separation and ultimate endometrial involution.
Survival and Maturation of Microencapsulated Porcine Neonatal Pancreatic Cell Clusters Transplanted into Immunocompetent Diabetic Mice
Authors: Omer Abdulkadir , Duvivier-Kali Valérie F. , Trivedi Nitin , Wilmot Karen , Bonner-Weir Susan , Weir Gordon C.
Published: March 2007

Differentiation and maturation of porcine neonatal pancreatic cell clusters (NPCCs) microencapsulated in barium alginate were assessed after transplantation into immunocompetent mice. Microencapsulated NPCCs were transplanted into the peritoneal cavity of streptozocin-induced diabetic B6AF1 mice (n = 32). The microcapsules were removed at 2, 6, and 20 weeks and examined for cellular overgrowth, insulin content, and insulin secretory responses to glucose and glucose with theophylline. The differentiation, maturation, and proliferation of the β-cells in the NPCCs were assessed by immunohistochemistry. Blood glucose levels were normalized in 81% of the animals that received a transplant and remained normal until termination of the experiments at 20 weeks. Hyperglycemic blood glucose levels after explantation of the capsules confirmed the function of the encapsulated NPCCs. Insulin content of the encapsulated NPCCs was increased 10-fold at 20 weeks after transplantation compared with pretransplantation levels. A 3.2-fold increase of the ratio of the β-cell area to the total cellular area was observed at 20 weeks, demonstrating the maturation of NPCCs into β-cells. In conclusion, NPCCs encapsulated with simple barium alginate can differentiate into β-cells and reverse high blood glucose levels in immunocompetent mice without immunosuppression for >20 weeks.
Inhibition of Net HepG2 Cell Apolipoprotein B Secretion by the Citrus Flavonoid Naringenin Involves Activation of Phosphatidylinositol 3-Kinase, Independent of Insulin Receptor Substrate-1 Phosphorylation
Authors: Borradaile Nica M. , de Dreu Linda E. , Huff Murray W.
Published: March 2007

The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes. Thus, we determined whether naringenin activates this pathway. Insulin and naringenin induced PI3K-dependent increases in cytosolic and nuclear SREBP-1 and LDLr expression. Similar PI3K-mediated increases in SREBP-1 were observed in McA-RH7777 rat hepatoma cells, which express predominantly SREBP-1c. Reductions in HepG2 cell media apoB with naringenin were partially attenuated by wortmannin, whereas the effect of insulin was completely blocked. Both treatments reduced apoB100 secretion in wild-type and LDLr−/− mouse hepatocytes to the same extent. Insulin and naringenin increased HepG2 cell PI3K activity and decreased insulin receptor substrate (IRS)-2 levels. In sharp contrast to insulin, naringenin did not induce tyrosine phosphorylation of IRS-1. We conclude that naringenin increases LDLr expression in HepG2 cells via PI3K-mediated upregulation of SREBP-1, independent of IRS-1 phosphorylation. Although this pathway may not regulate apoB secretion in primary hepatocytes, PI3K activation by this novel mechanism may explain the insulin-like effects of naringenin in vivo.
Intercellular Adhesion Molecule-1–Deficient Mice Are Resistant Against Renal Injury After Induction of Diabetes
Authors: Okada Shinichi , Shikata Kenichi , Matsuda Mitsuhiro , Ogawa Daisuke , Usui Hitomi , Kido Yuichi , Nagase Ryo , Wada Jun , Shikata Yasushi , Makino Hirofumi
Published: March 2007

Diabetic nephropathy is a leading cause of end-stage renal failure. Several mechanisms, including activation of protein kinase C, advanced glycation end products, and overexpression of transforming growth factor (TGF)-β, are believed to be involved in the pathogenesis of diabetic nephropathy. However, the significance of inflammatory processes in the pathogenesis of diabetic microvascular complications is poorly understood. Accumulation of macrophages and overexpression of leukocyte adhesion molecules and chemokines are prominent in diabetic human kidney tissues. We previously demonstrated that intercellular adhesion molecule (ICAM)-1 mediates macrophage infiltration into the diabetic kidney. In the present study, to investigate the role of ICAM-1 in diabetic nephropathy, we induced diabetes in ICAM-1–deficient (ICAM-1−/−) mice and ICAM-1+/+ mice with streptozotocin and examined the renal pathology over a period of 6 months. The infiltration of macrophages was markedly suppressed in diabetic ICAM-1−/− mice compared with that of ICAM-1+/+ mice. Urinary albumin excretion, glomerular hypertrophy, and mesangial matrix expansion were significantly lower in diabetic ICAM-1−/− mice than in diabetic ICAM-1+/+ mice. Moreover, expressions of TGF-β and type IV collagen in glomeruli were also suppressed in diabetic ICAM-1−/− mice. These results suggest that ICAM-1 is critically involved in the pathogenesis of diabetic nephropathy.
Endoneurial Capillary Abnormalities Presage Deterioration of Glucose Tolerance and Accompany Peripheral Neuropathy in Man
Authors: Thrainsdottir Soley , Malik Rayez A. , Dahlin Lars B. , Wiksell Peter , Eriksson Karl F. , Rosén Ingmar , Petersson Jesper , Greene Douglas A. , Sundkvist Göran
Published: March 2007

To explore whether microangiopathy is associated with disturbed glucose tolerance and peripheral neuropathy, we assessed endoneurial capillary morphology in sural nerve biopsies from men with diabetes, impaired glucose tolerance (IGT), and normal glucose tolerance (NGT). Baseline morphology was related to glucose tolerance and neuropathy at baseline and at follow-up 6 years later. Capillary density (in number per millimeters squared) at baseline was higher in subjects with diabetes (n = 10) compared with those with NGT (n = 5) at follow-up (median [interquartile range]) (86.0 [24.3] vs. 54.9 [17.1]; P = 0.0200) and in those progressing from IGT to diabetes (n = 4) compared with those with persistent IGT (n = 4) (86.7 [25.2] vs. 54.1 [14.6]; P = 0.0433). The capillary luminal area (in micrometers squared) was lower in subjects with NGT progressing to IGT (n = 2) or subjects with IGT progressing to diabetes (n = 3) compared with subjects with constant NGT (n = 6) or constant IGT (n = 4) (11.9 [2.4] vs. 20.8 [7.8]; P = 0.0201). The capillary basement membrane area (in micrometers squared) was increased in patients with peripheral neuropathy (n = 10) compared with those without (n = 7) (114.6 [68.8] vs. 75.3 [28.7]; P = 0.0084). In conclusion, increased capillary density was associated with current or future diabetes, decreased capillary luminal area with future deterioration in glucose tolerance, and increased basement membrane area with peripheral neuropathy.
A Novel Gene for Neonatal Diabetes Maps to Chromosome 10p12.1-p13
Authors: Sellick Gabrielle S. , Garrett Christine , Houlston Richard S.
Published: March 2007

We report a genomewide linkage analysis of a large consanguineous family segregating autosomal recessively inherited neonatal diabetes and the identification of a novel neonatal diabetes locus. Neonatal diabetes was characterized by low levels of circulating C-peptide with very low to undetectable levels of insulin in the presence of severe hyperglycemia unresponsive to insulin infusion. A dense genomewide linkage search of the family was undertaken using a first generation 10K single nucleotide polymorphism chip containing 10,044 markers. A region of homozygosity harboring the neonatal diabetes disease gene on chromosome 10p12.1-p13 was identified (multipoint logarithm of odds score 3.25). There is a strong history of type 2 diabetes in carriers of the disease gene. It is likely that chromosome 10p12.1-p13 may harbor a maturity-onset diabetes of the young or type 2 diabetes gene.
Identification of a β-Cell-Specific HLA Class I Restricted Epitope in Type 1 Diabetes
Authors: Panagiotopoulos Constadina , Qin Huilian , Tan Rusung , Verchere C. Bruce
Published: March 2007

Type 1 diabetes is an autoimmune disease in which pancreatic β-cells are destroyed by cytotoxic T-cells that recognize peptide epitopes presented by HLA class I molecules. The identification of human β-cell epitopes may significantly improve the prospects for immunodiagnosis and immunotherapy in type 1 diabetes. Using algorithms to predict nonameric β-cell peptides that would bind to the common HLA allele, HLA-A*0201, we identified a potential epitope from the leader sequence of islet amyloid polypeptide (human islet amyloid polypeptide [IAPP] precursor protein [preproIAPP] 5-13: KLQVFLIVL). Peripheral blood mononuclear cells (PBMCs) were isolated from 18 HLA-A*0201 patients with type 1 diabetes (9 with recent-onset [<180 days; range, 1–120 days] and 9 with long-standing diabetes [>180 days; range, 183–3,273 days]) and 9 healthy, nondiabetic control subjects. PBMCs were screened for peptide recognition using interferon-γ enzyme-linked immunospot (ELISpot) assays. Of the nine patients with recent-onset type 1 diabetes, six had ELISpot responses to preproIAPP 5-13 that were >3 SDs above the mean of the nondiabetic control subjects (P = 0.002). In contrast, no patients with type 1 diabetes for >180 days had a response above this threshold. In summary, preproIAPP 5-13 is a novel HLA class I epitope recognized by a significant proportion of cytotoxic T-cells from HLA-A*0201 patients with recent-onset type 1 diabetes and may prove to be a useful tool for the prediction and/or prevention of this disease.
Parathyroid Hormone–Related Protein Induces Insulin Expression Through Activation of MAP Kinase–Specific Phosphatase-1 That Dephosphorylates c-Jun NH2-Terminal Kinase in Pancreatic β-Cells
Authors: Zhang Bin , Hosaka Masahiro , Sawada Yoshie , Torii Seiji , Mizutani Shin , Ogata Masato , Izumi Tetsuro , Takeuchi Toshiyuki
Published: March 2007

Parathyroid hormone–related protein (PTHrP) increases the content and mRNA level of insulin in a mouse β-cell line, MIN6, and primary-cultured mouse islets. We examined the mechanism of PTHrP-induced insulin expression. The PTHrP effect was markedly augmented by SB203580, a mitogen-activated protein (MAP) kinase inhibitor, and SB203580 itself increased insulin expression extensively, even without PTHrP. Because SB203580 inhibits both p38 and c-jun NH2-terminal kinases (JNKs), we investigated the JNK-specific inhibitor SP600125. SP600125 also increased insulin content and its mRNA level. PTHrP induced dephosphorylation of JNK1/2, and PTHrP-induced insulin expression was blocked by a dominant-negative type JNK-APF. We suspected that dual specificity MAP kinase phosphatases (MKPs) may be involved in the PTHrP-induced insulin expression by inactivating JNK1/2. MIN6 cells contained at least five MKPs, among which only MKP-1 was inducible by PTHrP. PTHrP-induced insulin expression was blocked by the MKP-1 expression inhibitor Ro-31-8220, indicating that the PTHrP effect is mediated by MKP-1. Indeed, adenoviral MKP-1 expression increased insulin expression by decreasing a phosphorylation form of JNKs and a resulting phosphorylated form of c-jun in MIN6 cells. The phosphorylated form of c-jun is known to repress cAMP-dependent insulin gene promoter activity. Thus, MKP-1 controls the insulin expression by downregulating a JNK/c-jun pathway.
Rapamycin Has a Deleterious Effect on MIN-6 Cells and Rat and Human Islets
Authors: Bell Ewan , Cao Xiaopei , Moibi Jacob A. , Greene Scott R. , Young Robert , Trucco Matteo , Gao Zhiyong , Matschinsky Franz M. , Deng Shaoping , Markman James F. , Naji Ali , Wolf Bryan A.
Published: March 2007

Rapamycin (sirolimus) is a macrolide fungicide with immunosuppressant properties that is used in human islet transplantation. Little is known about the effects of rapamycin on MIN-6 cells and islets. Rapamycin had a dose-dependent, time-dependent, and glucose-independent deleterious effect on MIN-6 cell viability. At day 1, using the MTT method, 0.01 nmol/l rapamycin reduced cell viability to 83 ± 6% of control (P < 0.05). Using the calcein AM method, at day 2, 10 nmol/l rapamycin caused a reduction in cell viability to 73 ± 5% of control (P < 0.001). Furthermore, 10 and 100 nmol/l rapamycin caused apoptosis in MIN-6 cells as assessed by the transferase-mediated dUTP nick-end labeling assay. Compared with control, there was a 3.1 ± 0.6-fold increase (P < 0.01) in apoptosis in MIN-6 cells treated with 10 nmol/l rapamycin. A supra-therapeutic rapamycin concentration of 100 nmol/l significantly impaired glucose- and carbachol-stimulated insulin secretion in rat islets and had a deleterious effect on the viability of rat and human islets, causing apoptosis of both α- and β-cells.
Counterregulatory Hormone and Symptom Responses to Insulin-Induced Hypoglycemia in the Postprandial State in Humans
Authors: Porcellati Francesca , Pampanelli Simone , Rossetti Paolo , Cordoni Cristina , Marzotti Stefania , Scionti Luciano , Bolli Geremia B. , Fanelli Carmine G.
Published: March 2007

Plasma counterregulatory hormones and symptoms were measured during hypoglycemia in the postprandial and in the fasting state in humans to establish differences in physiological responses. We studied 8 nondiabetic subjects and 10 subjects with type 1 diabetes on two different occasions during clamped insulin-induced hypoglycemia (2.4 mmol/l) in the sitting position. On one occasion, subjects ate a standard mixed meal, and on the other they remained fasting. In response to postprandial as compared with fasting hypoglycemia, nondiabetic subjects exhibited lower total symptom scores (6.6 ± 0.4 vs. 11.5 ± 0.8, P = 0.001), which was due to less hunger (1.1 ± 0.1 vs. 4.2 ± 0.2), lower suppression of plasma C-peptide (0.23 ± 0.1 vs. 0.08 ± 0.07 nmol/l, P = 0.032), and greater responses of plasma glucagon (248 ± 29 vs. 163 ± 25 ng · l−1 · min−1, P = 0.018), plasma adrenaline (4.5 ± 0.6 vs. 3.1 ± 0.4 nmol · l−1 · min−1, P = 0.037), norepinephrine (3.8 ± 0.3 vs. 3.2 ± 0.2 nmol · l−1 · min−1, P = 0.037), and pancreatic polypeptide (217 ± 12 vs. 159 ± 22 pmol · l−1 · min−1, P = 0.08). Except for plasma C-peptide, responses in diabetic subjects were similarly affected. Notably, in diabetic subjects responses of glucagon, which were absent in the fasting state, nearly normalized after a meal. In conclusion, in the postprandial compared with the fasting hypoglycemic state, total symptoms are less, but counterregulatory hormones are greater and responses of glucagon nearly normalize in type 1 diabetic subjects.
Intermittent High Glucose Enhances Apoptosis Related to Oxidative Stress in Human Umbilical Vein Endothelial Cells
Authors: Quagliaro Lisa , Piconi Ludovica , Assaloni Roberta , Martinelli Lucia , Motz Enrico , Ceriello Antonio
Published: March 2007

The effects of intermittent and constant high glucose in the formation of nitrotyrosine and 8-hydroxydeoxyguanosine (markers of oxidative stress), as well as the possible linkage between oxidative stress and apoptosis in endothelial cells, have been evaluated. Stable high glucose increased nitrotyrosine, 8-hydroxydeoxyguanosine (8-OHdG), and apoptosis levels. However, these effects were more pronounced in intermittent high glucose. Protein kinase C (PKC) was elevated in both such conditions, particularly in intermittent glucose. The adding of the PKC inhibitors bisindolylmaleimide-I and LY379196, a specific inhibitor of PKC-β isoforms, normalized nitrotyrosine and reduced 8-OHdG concentration and cell apoptosis in both stable and intermittent high glucose. Similar results were obtained with the MnSOD mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride that normalized nitrotyrosine, 8-OHdG, and apoptosis and inhibited PKC activation. NAD(P)H oxidase was also measured. NAD(P)H oxidase components p47phox, p67phox, and p22phox was overexpressed during both stable and intermittent high glucose. PKC inhibition and MnSOD mimetic normalized this phenomenon. In conclusion, our study shows that the exposure of endothelial cells to both stable and intermittent high glucose stimulates reactive oxygen species overproduction also through PKC-dependent activation of NAD(P)H oxidase, leading to increased cellular apoptosis. Our data suggest that glucose fluctuations may also be involved in the development of vascular injury in diabetes.
Endurance Training in Humans Leads to Fiber Type-Specific Increases in Levels of Peroxisome Proliferator-Activated Receptor-γ Coactivator-1 and Peroxisome Proliferator-Activated Receptor-α in Skeletal Muscle
Authors: Russell Aaron P. , Feilchenfeldt Jonas , Schreiber Sylvia , Praz Manu , Crettenand Antoinette , Gobelet Charles , Meier Christoph A. , Bell David R. , Kralli Anastasia , Giacobino Jean-Paul , Dériaz Olivier
Published: March 2007

The peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1 (PGC-1) can induce mitochondria biogenesis and has been implicated in the development of oxidative type I muscle fibers. The PPAR isoforms α, β/δ, and γ control the transcription of genes involved in fatty acid and glucose metabolism. As endurance training increases skeletal muscle mitochondria and type I fiber content and fatty acid oxidative capacity, our aim was to determine whether these increases could be mediated by possible effects on PGC-1 or PPAR-α, -β/δ, and -γ. Seven healthy men performed 6 weeks of endurance training and the expression levels of PGC-1 and PPAR-α, -β/δ, and -γ mRNA as well as the fiber type distribution of the PGC-1 and PPAR-α proteins were measured in biopsies from their vastus lateralis muscle. PGC-1 and PPAR-α mRNA expression increased by 2.7- and 2.2-fold (P < 0.01), respectively, after endurance training. PGC-1 expression was 2.2- and 6-fold greater in the type IIa than in the type I and IIx fibers, respectively. It increased by 2.8-fold in the type IIa fibers and by 1.5-fold in both the type I and IIx fibers after endurance training (P < 0.015). PPAR-α was 1.9-fold greater in type I than in the II fibers and increased by 3.0-fold and 1.5-fold in these respective fibers after endurance training (P < 0.001). The increases in PGC-1 and PPAR-α levels reported in this study may play an important role in the changes in muscle mitochondria content, oxidative phenotype, and sensitivity to insulin known to be induced by endurance training.
Elevation of Free Fatty Acids Induces Inflammation and Impairs Vascular Reactivity in Healthy Subjects
Authors: Tripathy Devjit , Mohanty Priya , Dhindsa Sandeep , Syed Tufail , Ghanim Husam , Aljada Ahmad , Dandona Paresh
Published: March 2007

To test the possible acute proinflammatory effects of fatty acids, we induced an increase in plasma free fatty acid (FFA) concentrations after a lipid and heparin infusion for 4 h in 10 healthy subjects. We determined the nuclear factor-κB (NF-κB) binding activity in mononuclear cells (MNCs), the p65 subunit of NF-κB, reactive oxygen species (ROS) generation by MNC, and polymorphonuclear leukocytes (PMN). Brachial artery reactivity, using postischemic flow-mediated dilation, was also measured. NF-κB binding activity in the MNC nuclear extracts increased to 163 ± 17% and 144 ± 14% as compared with basal levels at 2 and 4 h (P < 0.005) and remained elevated (P < 0.05) at 6 h (2 h after cessation of lipid infusion). NF-κB p65 subunit protein expression in MNC homogenates also increased at 2, 4, and 6 h (P < 0.05). ROS generation by PMNs increased significantly at 2 and 4 h (P < 0.005), whereas that by MNCs increased at 4 h (P < 0.05). Plasma macrophage migration inhibitory factor increased at 2 (P < 0.05) and 4 h (P < 0.005), respectively, and declined to baseline at 6 h. The postischemic flow-mediated dilation of brachial artery decreased from 6.3 ± 1.1% at baseline to 4.3 ± 1.9% and 2.7 ± 2.1% (P < 0.01) at 2, 4, and 6 h, respectively. We conclude that an increase in FFA concentration induces oxidative stress and has a proinflammatory effect; it also impairs postischemic flow-mediated vasodilation of the brachial artery.
Gestational Diabetes Induces Placental Genes for Chronic Stress and Inflammatory Pathways
Authors: Radaelli Tatjana , Varastehpour Ali , Catalano Patrick , Hauguel-de Mouzon Sylvie
Published: March 2007

A physiological state of insulin resistance is required to preferentially direct maternal nutrients toward the feto-placental unit, allowing adequate growth of the fetus. When women develop gestational diabetes mellitus (GDM), insulin resistance is more severe and disrupts the intrauterine milieu, resulting in accelerated fetal development with increased risk of macrosomia. As a natural interface between mother and fetus, the placenta is the obligatory target of such environmental changes. However, the molecular basis for the imbalance that leads to fetal, neonatal, and adult metabolic compromises is not well understood. We report that GDM elicits major changes in the expression profile of placental genes with a prominent increase in markers and mediators of inflammation. Within the 435 transcripts reproducibly modified, genes for stress-activated and inflammatory responses represented the largest functional cluster (18.5% of regulated genes). Upregulation of interleukins, leptin, and tumor necrosis factor-α receptors and their downstream molecular adaptors indicated an activation of pathways recruiting stress-activated protein/c-Jun NH2-terminal kinases. Transcriptional activation of extracellular matrix components and angiogenic activators pointed to a major structural reorganization of the placenta. Thus, placental transcriptome emerges as a primary target of the altered environment of diabetic pregnancy. The genes identified provide the basis to elucidate links between inflammatory pathways and GDM-associated insulin resistance.
Nephrinuria in Diabetic Nephropathy of Type 1 Diabetes
Authors: Pätäri Anu , Forsblom Carol , Havana Marika , Taipale Heidi , Groop Per-Henrik , Holthöfer Harry ,   the FinnDiane Study Group
Published: March 2007

Diabetic nephropathy is the leading cause of end-stage renal disease. Because early diagnosis and treatment may prevent the complication, new tools for an early detection are needed. One of the key components of the glomerular filtration slit spanning between neighboring podocytes is nephrin. Its expression is altered in experimental models of diabetes and also in various human proteinuric diseases, including diabetes. We studied whether type 1 diabetic patients with or without nephropathy exhibit immunoreactive nephrin in the urine, reflecting early damage of the filtration barrier. Diabetic patients with normoalbuminuria (n = 40), with microalbuminuria (n = 41), and with macroalbuminuria (n = 39) and patients previously normoalbuminuric but now testing positive for microalbuminuria (newMicro, n = 39) were screened for nephrinuria with Western blotting using two affinity-purified anti-nephrin antibodies. Nondiabetic healthy subjects (n = 29) were also studied. Nephrinuria was present in 30% of normoalbuminuric, 17% of microalbuminuric, 28% of macroalbuminuric, and 28% of newMicro patients. Of female patients, 35% were nephrinuric compared with only 19% of male patients (P = 0.02). None of the control subjects was nephrinuric. In conclusion, glomerular filtration barrier may be affected in one-third of diabetic patients manifesting as early nephrinuria. Nephrinuria may have prognostic value and become a marker of susceptibility for kidney complications in diabetes.
The Human MC4R Promoter
Authors: Lubrano-Berthelier Cecile , Cavazos Martha , Le Stunff Catherine , Haas Kurt , Shapiro Astrid , Zhang Sumei , Bougneres Pierre , Vaisse Christian
Published: March 2007

Heterozygous mutations in the coding sequence of the serpentine melanocortin 4 receptor (MC4R) are the most frequent genetic cause of severe human obesity. Since haploinsufficiency has been proposed as a causal mechanism of obesity associated with these mutations, reduction in gene transcription caused by mutations in the transcriptionally essential regions of the MC4R promoter may also be a cause of severe obesity in humans. To test this hypothesis we defined the minimal promoter region of the human MC4R and evaluated the extent of genetic variation in this region compared with the coding region in two cohorts of severely obese subjects. 5′RACE followed by functional promoter analysis in multiple cell lines indicates that an 80-bp region is essential for the transcriptional activity of the MC4R promoter. Systematic screening of 431 obese children and adults for mutations in the coding sequence and the minimal core promoter of MC4R reveals that genetic variation in the transcriptionally essential region of the MC4R promoter is not a significant cause of severe obesity in humans.
The Role of Insulin Receptor Substrate-1 Gene (IRS1) in Type 2 Diabetes in Pima Indians
Authors: Kovacs Peter , Hanson Robert L. , Lee Yong-Ho , Yang Xiaolin , Kobes Sayuko , Permana Paska A. , Bogardus Clifton , Baier Leslie J.
Published: March 2007

The insulin receptor substrate-1 (IRS1) is a critical element in insulin-signaling pathways, and mutations in the IRS1 gene have been reported to have a role in determining susceptibility to traits related to type 2 diabetes. In gene expression studies of tissue biopsies from nondiabetic Pima Indians, IRS1 mRNA levels were reduced in adipocytes from obese subjects compared with lean subjects, and IRS1 mRNA levels were also reduced in skeletal muscle from insulin-resistant subjects compared with insulin-sensitive subjects (all P < 0.05). Based on these expression differences and the known physiologic role of IRS1, this gene was investigated as a candidate gene for susceptibility to type 2 diabetes in Pima Indians, a population with an extremely high incidence and prevalence of type 2 diabetes. Thirteen variants were identified, and among these variants, several were in complete linkage disequilibrium. Four genotypically unique variants were further genotyped in 937 DNA samples from full-heritage Pima Indians. Three of the variants were modestly associated with type 2 diabetes (P < 0.05), one of which was additionally associated with 2-h plasma insulin and glucose as well as insulin action at physiologic and maximally stimulating insulin concentrations (all P < 0.05). The association of variants in IRS1 with type 2 diabetes and type 2 diabetes-related phenotypes and the differential expression of IRS1 in adipocytes and skeletal muscle suggest a role of this gene in the pathogenesis of type 2 diabetes in Pima Indians.
The Human Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) Pro12Ala Polymorphism Is Associated With Decreased Risk of Diabetic Nephropathy in Patients With Type 2 Diabetes
Authors: Caramori Maria Luiza , Canani Luis Henrique , Costa Luciana A. , Gross Jorge Luiz
Published: March 2007

The peroxisome proliferator-activated receptor γ2 (PPARγ2) Pro12Ala polymorphism has been associated with a decreased risk of type 2 diabetes and a lower albumin excretion rate (AER) in patients with established diabetes. We performed a case-control study aiming to evaluate the association between the Pro12Ala polymorphism and diabetic nephropathy. Genomic DNA was obtained from 104 type 2 diabetic patients (case subjects) with chronic renal insufficiency (78 on dialysis and 26 with proteinuria [AER ≥200 μg/min] and serum creatinine ≥2.0 mg/dl) and 212 normoalbuminuric patients (AER <20 μg/min) with known diabetes duration ≥10 years (control subjects). The genotypic distribution of the PPARγ2 Pro12Ala polymorphism in these diabetic patients was in Hardy-Weinberg equilibrium, and the Ala allele frequency was 9%. The frequency of Ala carriers (Ala/Ala or Ala/Pro) was 20.3% in control subjects and 10.6% in case subjects (P = 0.031). The odds ratio of having diabetic nephropathy for Ala carriers was 0.465 (95% CI 0.229–0.945; P = 0.034). Carriers of the Ala allele were not different from noncarriers (Pro/Pro) regarding sex (38.9 vs. 44.1% males) or ethnicity (77.4 vs. 71.7% white) distribution, age (61 ± 10 vs. 61 ± 10 years), known diabetes duration (17 ± 7 vs. 16 ± 7 years), BMI (27 ± 4 vs. 28 ± 5 kg/m2), fasting plasma glucose (184 ± 81 vs. 176 ± 72 mg/dl), HbA1c (6.7 ± 2.3 vs. 6.9 ± 2.4%; high-performance liquid chromatography reference range: 2.7–4.3%), and systolic (145 ± 27 vs. 0.144 ± 24 mmHg) or diastolic (87 ± 14 vs. 85 ± 14 mmHg) blood pressure, respectively. In conclusion, the presence of the Ala allele may confer protection from diabetic nephropathy in patients with type 2 diabetes.
Plasma Adiponectin Concentrations Predict Insulin Sensitivity of Both Glucose and Lipid Metabolism
Authors: Tschritter Otto , Fritsche Andreas , Thamer Claus , Haap Michael , Shirkavand Fatemeh , Rahe Stefanie , Staiger Harald , Maerker Elke , Häring Hans , Stumvoll Michael
Published: March 2007

In animals, the adipocyte-derived hormone adiponectin has been shown to improve insulin sensitivity, a key factor in the pathogenesis of type 2 diabetes. In Pima Indians, high plasma adiponectin levels are associated with increased insulin sensitivity and reduced risk of type 2 diabetes. It is unclear whether this is also the case in white individuals and whether an additional beneficial effect on lipid metabolism exists. We therefore analyzed in nondiabetic individuals the associations between plasma adiponectin concentrations and insulin sensitivity measured by a euglycemic-hyperinsulinemic clamp (n = 262) and estimated by an oral glucose tolerance test (OGTT; n = 636) and serum lipid parameters using correlational analysis. Plasma adiponectin concentrations were positively correlated with insulin sensitivity, both measured with the clamp (r = 0.28, P = 0.0015 in women; r = 0.42, P < 0.0001 in men) and estimated from the OGTT (r = 0.37, P < 0.0001 in women; r = 0.41, P < 0.0001 in men) before and after adjusting for sex and percentage of body fat (all P < 0.001). Fasting triglycerides and the free fatty acid (FFA) concentrations during the OGTT (area under the curve) and at 120 min were negatively correlated in both women and men, whereas HDL was positively correlated with plasma adiponectin concentrations (all P < 0.004). Most notable, these relationships remained significant after adjusting for insulin sensitivity of glucose disposal in addition to sex and percentage of body fat (all P < 0.05). In conclusion, high adiponectin predicts increased insulin sensitivity. This relationship is independent of low body fat mass and affects not only insulin-stimulated glucose disposal but also lipoprotein metabolism and insulin-mediated suppression of postprandial FFA release. This suggests pleiotropic insulin sensitizing effects of adiponectin in humans.
Leptin Receptor−Deficient Obese Zucker Rats Reduce Their Food Intake in Response to a Systemic Supply of Calories From Glucose
Authors: Gilbert Marc , Magnan Christophe , Turban Sophie , André Jocelyne , Guerre-Millo Michèle
Published: March 2007

It has been established that leptin exerts a negative control on food intake, allowing one to maintain stable caloric intake over time. The aim of the present study was to investigate whether leptin regulates food intake when a supply of calories is provided by the systemic route. Experiments were carried out in leptin receptor−deficient obese fa/fa rats and lean Fa/fa controls. In both groups, 48 h of glucose infusion reduced food intake in proportion to caloric supply, resulting in virtually no change in total caloric intake as compared to before the infusion. This hypophagic response was reproduced without adding systemic calories, but by increasing glucose and insulin concentrations specifically in the brain through carotid artery infusion. Concomitant intracerebroventricular administration of 5-(tetradecyloxy)-2-furoic acid, an acetyl CoA carboxylase inhibitor that precludes malonyl-CoA synthesis, abolished the restriction of feeding in carotid-infused lean and obese rats. These data indicate that a supply of calories via glucose infusion induces a hypophagic response independent of leptin signaling in the rat, and support the hypothesis that a rise in central malonyl-CoA, triggered by increased glucose and insulin concentrations, participates in this adaptation. This process could contribute to the limiting of hyperphagia, primarily when leptin signaling is altered, as in the obese state.
Acute Shock Induced by Antigen Vaccination in NOD Mice
Authors: Overbergh Lut , Decallonne Brigitte , Branisteanu Dumitru D. , Valckx Dirk , Kasran Ahmad , Bouillon Roger , Mathieu Chantal
Published: March 2007

Type 1 diabetes in NOD mice can be prevented through autoantigen vaccination by shifting lymphocyte differentiation toward a T-helper 2 (Th2) response. However, in other models of autoimmunity, this approach may be accompanied by unexpected triggering of Th2-dependent anaphylactic shock. To test the safety of vaccination therapy in the NOD mouse model, we evaluated the effects of immunization with a wide battery of antigens in NOD, BALB/c, and C57BL/6 mice. Surprisingly, a nondiabetogenic antigen, hen egg white lysozyme, induced severe shock exclusively in NOD mice (shock in 11 of 11 mice, lethal in 3 mice). Shock severity was further increased by a more pronounced Th2 setting generated by 1α,25(OH)2D3 administration (17 of 17 mice, lethal in 14 mice, P < 0.0001). Pretreatment with dexamethasone resulted in full rescue, indicating an immune-mediated mechanism. Serum IgE levels and Th1/Th2 cytokine profile analysis showed that the shock phenomenon was paralleled by a Th2 response. mRNA expression of platelet-activating factor receptor (PAF-R) was significantly higher in NOD mice (P < 0.01) and was further increased by 1α,25(OH)2D3. Pretreatment with WEB2086 (PAF-R antagonist) again protected all mice from lethal shock, indicating PAF as an anaphylaxis effector. In conclusion, in NOD mice, vaccination leading to a Th2 immune shift can result in a lethal anaphylactic reaction.
Stimulation of Insulin Secretion by Denatonium, One of the Most Bitter-Tasting Substances Known
Authors: Straub Susanne G. , Mulvaney-Musa Jennifer , Yajima Hiroki , Weiland Gregory A. , Sharp Geoffrey W.G.
Published: March 2007

Denatonium, one of the most bitter-tasting substances known, stimulated insulin secretion in clonal HIT-T15 β-cells and rat pancreatic islets. Stimulation of release began promptly after exposure of the β-cells to denatonium, reached peak rates after 4–5 min, and then declined to near basal values after 20–30 min. In islets, no effect was observed at 2.8 mmol/;l glucose, whereas a marked stimulation was observed at 8.3 mmol/;l glucose. No stimulation occurred in the absence of extracellular Ca2+ or in the presence of the Ca2+-channel blocker nitrendipine. Stimulated release was inhibited by α2-adrenergic agonists. Denatonium had no direct effect on voltage-gated calcium channels or on cyclic AMP levels. There was no evidence for the activation of gustducin or transducin in the β-cell. The results indicate that denatonium stimulates insulin secretion by decreasing KATP channel activity, depolarizing the β-cell, and increasing Ca2+ influx. Denatonium did not displace glybenclamide from its binding sites on the sulfonylurea receptor (SUR). Strikingly, it increased glybenclamide binding by decreasing the Kd. It is concluded that denatonium, which interacts with K+ channels in taste cells, most likely binds to and blocks Kir6.2. A consequence of this is a conformational change in SUR to increase the SUR/;glybenclamide binding affinity.
The Influence of GLP-1 on Glucose-Stimulated Insulin Secretion
Authors: Kjems Lise L. , Holst Jens J. , Vølund Aage , Madsbad Sten
Published: March 2007

The intestinally derived hormone glucagon-like peptide 1 (GLP-1) (7-36 amide) has potent effects on glucose-mediated insulin secretion, insulin gene expression, and β-cell growth and differentiation. It is, therefore, considered a potential therapeutic agent for the treatment of type 2 diabetes. However, the dose-response relationship between GLP-1 and basal and glucose-stimulated prehepatic insulin secretion rate (ISR) is currently not known. Seven patients with type 2 diabetes and seven matched nondiabetic control subjects were studied. ISR was determined during a graded glucose infusion of 2, 4, 6, 8, and 12 mg · kg−1 · min−1 over 150 min on four occasions with infusion of saline or GLP-1 at 0.5, 1.0, and 2.0 pmol · kg−1 · min−1. GLP-1 enhanced ISR in a dose-dependent manner during the graded glucose infusion from 332 ± 51 to 975 ± 198 pmol/kg in the patients with type 2 diabetes and from 711 ± 123 to 2,415 ± 243 pmol/kg in the control subjects. The β-cell responsiveness to glucose, expressed as the slope of the linear relation between ISR and the glucose concentration, increased in proportion to the GLP-1 dose to 6 times relative to saline at the highest GLP-1 dose in the patients and 11 times in the control subjects, but it was 3 to 5 times lower in the patients with type 2 diabetes compared with healthy subjects at the same GLP-1 dose. During infusion of GLP-1 at 0.5 pmol · kg−1 · min−1 in the patients, the slope of ISR versus glucose became indistinguishable from that of the control subjects without GLP-1. Our results show that GLP-1 increases insulin secretion in patients with type 2 diabetes and control subjects in a dose-dependent manner and that the β-cell responsiveness to glucose may be increased to normal levels with a low dose of GLP-1 infusion. Nevertheless, the results also indicate that the dose-response relation between β-cell responsiveness to glucose and GLP-1 is severely impaired in patients with type 2 diabetes.
Dominant Negative Pathogenesis by Mutant Proinsulin in the Akita Diabetic Mouse
Authors: Izumi Tetsuro , Yokota-Hashimoto Hiromi , Zhao Shengli , Wang Jie , Halban Philippe A. , Takeuchi Toshiyuki
Published: March 2007

Autosomal dominant diabetes in the Akita mouse is caused by mutation of the insulin 2 gene, whose product replaces a cysteine residue that is engaged in the formation of an intramolecular disulfide bond. These heterozygous mice exhibit severe insulin deficiency despite coexpression of normal insulin molecules derived from three other wild-type alleles of the insulin 1 and 2 genes. Although the results of our previous study suggested that the mutant proinsulin 2 is misfolded and blocked in the transport from the endoplasmic reticulum to the Golgi apparatus, its dominant negative nature has not been fully characterized. In the present study, we investigated the possible pathogenic mechanisms induced by the mutant proinsulin 2. There is no evidence that the mutant proinsulin 2 attenuates the overall protein synthesis rate or promotes the formation of aberrant disulfide bonds. The trafficking of constitutively secreted alkaline phosphatase, however, is significantly decreased in the islets of Akita mice, indicating that the function of early secretory pathways is nonspecifically impaired. Morphological analysis has revealed that secretory pathway organelle architecture is progressively devastated in the β-cells of Akita mice. These findings suggest that the organelle dysfunction resulting from the intracellular accumulation of misfolded proinsulin 2 is primarily responsible for the defect of coexisting wild-type insulin secretion in Akita β-cells.
Connexin 36 Controls Synchronization of Ca2+ Oscillations and Insulin Secretion in MIN6 Cells
Authors: Calabrese Alessandra , Zhang Min , Serre-Beinier Véronique , Caton David , Mas Christophe , Satin Leslie S. , Meda Paolo
Published: March 2007

Cx36 is the predominant connexin isoform expressed by pancreatic β-cells. However, little is known about the role of this protein in the functioning of insulin-secreting cells. To address this question, we searched for a cell line expressing Cx36 and having glucose-induced insulin secretion comparable to that of primary β-cells. By evaluating Cx36 expression in MIN6, βTC3, RIN2A, INS1, and HIT cell lines, which differ in their sensitivity to glucose, we found that wild-type MIN6 cells fit these requirements. Therefore, we stably transfected MIN6 cells with a cDNA coding for a Cx36 antisense sequence to study the role of Cx36 in these cells. Independent clones of MIN6 cells were obtained that had a markedly reduced Cx36 expression. Loss of Cx36 decreased functional gap junctional conductance in these clones. This alteration impaired the synchronization of glucose-induced [Ca2+]i oscillations and insulin secretion in response to glucose, to secretagogues that increase [cAMP]i, and to depolarizing conditions. These data provide the first evidence that Cx36-made channels 1) mediate functional coupling in MIN6 cells, 2) provide for synchronous [Ca2+]i oscillations, and 3) are necessary for proper insulin secretion in response to metabolizable and nonmetabolizable secretagogues.
Pax-6 Activates Endogenous Proglucagon Gene Expression in the Rodent Gastrointestinal Epithelium
Authors: Trinh Denny K.Y. , Zhang Kai , Hossain Moazzem , Brubaker Patricia L. , Drucker Daniel J.
Published: March 2007

The proglucagon gene encodes pancreatic glucagon and the glucagon-like peptides, which exert diverse effects on nutrient absorption and assimilation. The therapeutic potential of glucagon-like peptide-1 (GLP-1) has fostered interest in development of cellular engineering approaches to augment endogenous intestinal-derived GLP-1 for the treatment of type 2 diabetes. We have used adenovirus technology to examine the potential roles of the transcription factors Cdx-2/3 and Pax-6 as activators of endogenous proglucagon gene expression in enteroendocrine cell lines and in nontransformed rat intestinal cells. Adenoviral-expressed Cdx-2/3 and Pax-6 activated proglucagon promoter-luciferase activity in baby hamster kidney (BHK) fibroblasts, HEK 293 cells, and enteroendocrine cell lines. Pax-6, but not Cdx-2/3, induced expression of the endogenous proglucagon gene in enteroendocrine cell lines, but not in heterologous fibroblasts. Furthermore, transduction of primary rat intestinal cell cultures in vitro, or the rat colonic epithelium in vivo, with Ad-Pax-6 activated endogenous proglucagon gene expression. These data demonstrate that Pax-6, but not Cdx-2/3, is capable of activating the endogenous proglucagon gene in both immortalized enteroendocrine cells and the nontransformed intestinal epithelium in vivo.
Demonstration of Glycated Insulin in Human Diabetic Plasma and Decreased Biological Activity Assessed by Euglycemic-Hyperinsulinemic Clamp Technique in Humans
Authors: Hunter Steven J. , Boyd Alison C. , O’Harte Finbarr P.M. , McKillop Aine M. , Wiggam M. Ivan , Mooney Mark H. , McCluskey Jane T. , Lindsay John R. , Ennis Cieran N. , Gamble Raymond , Sheridan Brian , Barnett Christopher R. , McNulty Helene , Bell Patrick M. , Flatt Peter R.
Published: March 2007

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA1c 8.1 ± 0.2%, plasma glucose 8.7 ± 1.3 mmol/l [means ± SE]) revealed two major insulin-like peaks with retention times of 14–16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)3+ species at 1,991.1 m/z, representing monoglycated insulin with an intact Mr of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (Mr 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 ± 2.3 pmol/l, corresponding to ∼9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe1-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 μg · kg−1 · min−1, followed by 2 h at 83.0 μg · kg−1 · min−1; corresponding to 0.4 and 2.0 mU · kg−1 · min−1). At the lower dose, the exogenous glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P < 0.01) and ∼70% more glycated insulin was required to induce a similar rate of insulin-mediated glucose uptake. Maximal responses at the higher rates of infusion were similar for glycated and control insulin. Inhibitory effects on endogenous glucose production, insulin secretion, and lipolysis, as indicated by measurements of C-peptide, nonesterified free fatty acids, and glycerol, were also similar. Receptor binding to CHO-T cells transfected with human insulin receptor and in vivo metabolic clearance revealed no differences between glycated and native insulin, suggesting that impaired biological activity is due to a postreceptor effect. The present demonstration of glycated insulin in human plasma and related impairment of physiological insulin-mediated glucose uptake suggests a role for glycated insulin in glucose toxicity and impaired insulin action in type 2 diabetes.