bluuebunny/crossref_metadata_2025 huggingface dataset:
Impaired Molecular Regenerative Responses in Sensory Neurones of Diabetic Rats: Gene Expression Changes in Dorsal Root Ganglia After Sciatic Nerve Crush
Authors: Mohiuddin L , Tomlinson D R
Published: September 2013

This study investigated changes in gene expression in lumbar dorsal root ganglia (DRG), contralateral and ipsilateral to a sciatic nerve crush in control and streptozotocin (STZ)-induced diabetic rats. After 10 weeks of diabetes, the left sciatic nerves of all rats were crushed at mid-thigh level, and the rats were maintained for a further 2 weeks. Northern blots, with internal standards, were made from L4 and L5 (pooled) DRG on each side to compare RNA hybrids from ganglia attached to crushed, nerves with those attached to intact nerves. The expression of growth-associated proteins, GAP-43 and Tα1 α-tubulin mRNA in DRG, was stimulated (all P < 0.05) by crush injury in control and diabetic rats. Steady-state expression of transcripts for neurofilament (NF) proteins (NF-L, NF-H) and the high-affinity NGF receptor, trkA was decreased by diabetes in the contralateral ganglia to the crush (all P < 0.05). Crush injury further decreased expression of these transcripts in both control and diabetic rats (all P < 0.05). This reduced expression of mRNA coding for both growth-associated proteins, and neurofilament proteins in ganglia of diabetic rats could participate in the reduced competence of the regenerative response to nerve crush.
High-Fat Feeding Impairs Insulin-Stimulated GLUT4 Recruitment via an Early Insulin-Signaling Defect
Authors: Zierath Juleen R , Houseknecht Karen L , Gnudi Luigi , Kahn Barbara B
Published: September 2013

Glucose transport in skeletal muscle can be mediated by two separate pathways, one stimulated by insulin and the other by muscle contraction. High-fat feeding impairs glucose transport in muscle, but the mechanism remains unclear. FVB mice (3 weeks old) were fed a high-fat diet (55% fat, 24% carbohydrate, 21% protein) or standard chow for 3–4 weeks or 8 weeks. Insulin-stimulated glucose transport, assessed with either 2-deoxyglucose or 3-O-methylglucose was decreased 35–45% (P < 0.001) in isolated soleus muscle, regardless of diet duration. Similarly, glucose transport stimulated by okadaic acid, a serine/threonine phosphatase inhibitor, was also 45% lower with high-fat feeding, but the glucose transport response to hypoxia or N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7) (which are stimulators of the “contraction pathway”) was intact. Hexokinase I, II, and total activity were normal in soleus muscle from highfat–fed mice. GLUT4 expression in soleus muscle from the high-fat–fed mice was also normal, but the insulinstimulated cell surface recruitment of GLUT4 assessed by exofacial photolabeling with [3H]-ATB bismannose was reduced by 50% (P < 0.001). Insulinreceptor substrate 1 (IRS-1) associated phosphatidylinositol (PI) 3-kinase activity stimulated by insulin was also reduced by 36% (P < 0.001), and expression of p85 and p110b subunits of PI 3-kinase was normal. In conclusion, high-fat feeding selectively impairs insulin-stimulated, but not contraction-pathway–mediated, glucose transport by reducing GLUT4 translocation to the plasma membrane. This appears to result from an acquired defect in insulin activation of PI 3-kinase. Since effects of okadaic acid on glucose transport are independent of PI 3-kinase, a second signaling defect may also be induced.
Systemic Bias of Cytokine Production Toward Cell-Mediated Immune Regulation in IDDM and Toward Humoral Immunity in Graves' Disease
Authors: Kallmann Boris A , Hüther Manfred , Tubes Monika , Feldkamp Joachim , Bertrams Jörg , Gries Friedrich A , Lampeter Eberhard F , Kolb Hubert
Published: September 2013

Disturbed immune regulation has been postulated to be crucial in the pathogenesis of IDDM and other autoimmune or allergic diseases. We therefore tested the hypothesis of a general bias in the peripheral immune system in patients with recent-onset IDDM or Graves' disease in comparison to healthy control subjects by studying whole blood cultures stimulated with phytohemagglutinin. Cells from IDDM patients (n = 53) produced significantly higher amounts of Thl cytokines γ-interferon (IFN-γ) (P = 0.028) and tumor necrosis factor α (TNF-α) (P = 0.007) than normal control subjects (n = 56), while Th2 cytokine levels (interleukin [IL]-4, IL-10) were similar. Low levels of islet cell antibodies (ICAs) in IDDM patients were associated with high levels of Thl and Th2 cytokines. Antibodies to GAD, ICA512, or insulin did not correlate with individual cytokine profiles. Also, HLA-DQ types did not significantly correlate with either Thl or Th2 cytokine production. Conversely, whole blood cultures from patients with Graves' disease (n = 18) produced significantly less TNF-α and IL-4 than normal subjects (P = 0.001–0.006). However, when the balance between Thl and Th2 cytokine production was analyzed in individuals, the ratio between IFN-γ or TNF-α and IL-4 or IL-10 was clearly biased toward Thl reactivity in patients with IDDM (P = 0.0001), while a dominance of Th2 cytokine production was seen in Graves' disease (P = 0.0001). The ratio of counterregulatory cytokines appeared to be the most reliable marker of the individual disease process. This study provides first evidence of a systemic bias in the immune regulation of humans, which might be either toward cell-mediated immunity (Thl) in IDDM or humoral immunity (Th2) in Graves' disease.
Antioxidant Defense: Vitamins E and C and Carotenoids
Authors: Stahl Wilhelm , Sies Helmut
Published: September 2013

Reactive oxygen species are thought to be implicated in the pathogenesis of various human diseases. They are generated endogenously under physiological and pathological conditions but also upon exposure to exogenous challenge. The organism maintains defense systems against reactive oxygen species, including enzymes and low-molecular-weight antioxidants. Important antioxidants such as vitamins E and C and carotenoids are provided from the diet, vitamin E, as the major chain-breaking antioxidant, inhibits lipid perox-idation, thus preventing membrane damage and modification of low-density lipoproteins. It is regenerated by the water-soluble vitamin C. Carotenoids efficiently scavenge singlet molecular oxygen and peroxyl radicals. There is increasing evidence from epidemiological studies, animal experiments, and in vitro investigations that an increased intake of antioxidants is associated with a diminished risk for several diseases.
Role of Neurotrophins in Diabetic Neuropathy and Treatment with Nerve Growth Factors
Authors: Tomlinson DR , Fernyhough P , Diemel LT
Published: September 2013

In rodent models of diabetes, there are expression deficits in nerve growth factor (NGF) and in mRNA for its high-affinity receptor, trkA, leading to decreased retrograde axonal transport of NGF and decreased support of NGF-dependent sensory neurons, with reduced expression of their neuropeptides, substance P and calcitonin gene-related peptide (CGRP). Treatment of diabetic rats with intensive insulin normalized these deficits, and treatment with exogenous NGF caused dose-related increases, giving levels of NGF and neuropeptides that were greater than those of controls. Neurotrophin-3 (NT-3) mRNA was also deficient in leg muscle from diabetic rats, and administration of recombinant NT-3 to diabetic rats increased the conduction velocity of sensory nerves without affecting motor conduction velocity. In regenerating nerves after experimental crush injury, expression of NGF in the nerve trunk is increased in diabetes to a greater extent than in controls, but this is offset by a greater reduction in the neuronal expression of trkA in dorsal root ganglia of diabetic rats. Nonetheless, targeted administration of exogenous NGF via impregnated conduits stimulated regeneration in both control and diabetic rats. These findings implicate deficient neurotrophic support in diabetic neuropathy and suggest that its correction should be a paramount therapeutic target.
The Pathogenesis of Diabetic Foot Problems: An Overview
Authors: Shaw Jonathan E , Boulton Andrew JM
Published: September 2013

Foot ulceration and lower limb amputation are still common complications of diabetes. Diabetic peripheral neuropathy and peripheral vascular disease are the most important etiologic factors, but there is a complex interplay between these abnormalities and a number of other contributory factors, such as altered foot pressures, limited joint mobility, glycemic control, ethnic background, and cardiovascular parameters. Identification of patients at high risk of ulceration is nevertheless simple, and education of such patients can achieve a major reduction in amputation and ulceration rates.
α-Lipoic Acid in the Treatment of Diabetic Peripheral and Cardiac Autonomic Neuropathy
Authors: Ziegler Dan , Gries F Arnold
Published: September 2013

Antioxidant treatment has been shown to prevent nerve dysfunction in experimental diabetes, providing a rationale for a potential therapeutic value in diabetic patients. The effects of the antioxidant α-lipoic acid (thioctic acid) were studied in two multicenter, randomized, double-blind placebo-controlled trials. In the Alpha-Lipoic Acid in Diabetic Neuropathy Study, 328 patients with NIDDM and symptomatic peripheral neuropathy were randomly assigned to treatment with intravenous infusion of α-lipoic acid using three doses (ALA 1,200 mg; 600 mg; 100 mg) or placebo (PLAC) over 3 weeks. The total symptom score (TSS) (pain, burning, paresthesia, and numbness) in the feet decreased significantly from baseline to day 19 in ALA 1,200 and ALA 600 vs. PLAC. Each of the four individual symptom scores was significantly lower in ALA 600 than in PLAC after 19 days (all P < 0.05). The total scale of the Hamburg Pain Adjective List (HPAL) was significantly reduced in ALA 1,200 and ALA 600 compared with PLAC after 19 days (both P < 0.05). In the Deutsche Kardiale Autonome Neuropathie Studie, patients with NIDDM and cardiac autonomic neuropathy diagnosed by reduced heart rate variability were randomly assigned to treatment with a daily oral dose of 800 mg α-lipoic acid (ALA) (n = 39) or placebo (n = 34) for 4 months. Two out of four parameters of heart rate variability at rest were significantly improved in ALA compared with placebo. A trend toward a favorable effect of ALA was noted for the remaining two indexes. In both studies, no significant adverse events were observed. In conclusion, intravenous treatment with α-lipoic acid (600 mg/day) over 3 weeks is safe and effective in reducing symptoms of diabetic peripheral neuropathy, and oral treatment with 800 mg/day for 4 months may improve cardiac autonomic dysfunction in NIDDM.
The Mystery of Diabetes and Atherosclerosis: Time for a New Plot
Authors: Semenkovich Clay F , Heinecke Jay W
Published: September 2013

Most patients with diabetes die from macrovascular complications. Little is known about the pathogenesis of diabetic vascular disease, but recent advances in molecular genetics and oxidation chemistry provide clues to the mystery of diabetes and atherosclerosis. Genetic variants of well-known proteins such as lipoprotein lipase and apolipoprotein E are common. These proteins are suitable candidates for mediating diabetic vascular risk because their variants can produce hypertriglyceridemia, a risk factor for atherosclerosis in diabetes. However, mutations could have different effects on lipoprotein flux across arteries depending on whether expression is dominant in the vascular space or the vascular wall. Lipoproteins retained in the arterial wall are subject to oxidative modification, which could be dependent on glycoxidation, the enzyme myeloperoxidase, or reactive nitrogen species derived from nitric oxide. Accelerated vascular disease in diabetes is likely the result of complex interactions between metabolic derangements such as hyperglycemia, mutations in genes controlling lipid metabolism, and antioxidant defense mechanisms.
Interactions Between Leptin and Hypothalamic Neuropeptide Y Neurons in the Control of Food Intake and Energy Homeostasis in the Rat
Authors: Wang Qiong , Bing Chen , Al-Barazanji Kamal , Mossakowaska Danuta E , Wang Xin-Min , McBay Diane L , Neville William A , Taddayon Mohammad , Pickavance Lucy , Dryden Simon , Thomas Moira E A , McHale Mark T , Gloyer Israel S , Wilson Shelagh , Buckingham Robin , Arch Jonathan R S , Trayhurn Paul , Williams Gareth
Published: September 2013

Leptin acts on the brain to inhibit feeding, increase thermogenesis, and decrease body weight. Neuropeptide Y (NPY)-ergic neurons of the hypothalamic arcuate nucleus (ARC) that project to the paraventricular nuclei (PVN) and dorsomedial nuclei (DMH) are postulated to control energy balance by stimulating feeding and inhibiting thermogenesis, especially under conditions of energy deficit. We investigated whether leptin's short-term effects on energy balance are mediated by inhibition of the NPY neurons. Recombinant murine leptin (11 μg) injected into the lateral ventricle of fasted adult Wistar rats inhibited food intake by 20–25% between 2 and 6 h after administration, compared with saline-treated controls (P < 0.05). Uncoupling protein mRNA levels in brown adipose tissue (BAT) rose by 70% (P < 0.01). Leptin treatment significantly reduced NPY concentrations by 20–50% (P < 0.05) in the ARC, PVN, and DMH and significantly decreased hypothalamic NPY mRNA levels (0.61 ± 0.02 vs. 0.78 ± 0.03 arbitrary units; P < 0.01). A second study examined changes in leptin during 5 days' intracerebroventricular NPY administration (10 μg/day), which induced sustained hyperphagia and excessive weight gain. In NPY-treated rats, leptin mRNA levels in epididymal fat were comparable to those in saline-treated controls (0.94 ± 0.17 vs. 1.0 ± 0.28 arbitrary units; P > 0.1), but plasma leptin levels were significantly higher (4.88 ± 0.66 vs. 2.85 ± 0.20 ng/ml; P < 0.01). Leptin therefore acts centrally to decrease NPY synthesis and NPY levels in the ARC-PVN projection; reduced NPY release in the PVN may mediate leptin's hypophagic and thermogenic actions. Conversely, NPY-induced obesity results in raised circulating leptin concentrations. Leptin and the NPY-ergic ARC-PVN neurons may interact in a homeostatic loop to regulate body fat mass and energy balance.
MHC Class II–Dependent Abnormal Reactivity Toward Bacterial Superantigens in Immune Cells of NOD Mice
Authors: Radons J , Burkart V , Kolb H
Published: September 2013

Superantigens have been implicated in the pathogenesis of type I diabetes and other immune-mediated diseases. We therefore tested the hypothesis of an abnormal reactivity of the immune system toward bacterial superantigens during the prediabetic phase. For this purpose, splenocytes from NOD (H-2g7) mice were exposed to two well-characterized superantigens: Staphylococcal aureus enterotoxin-B (SEB) and toxic shock syndrome toxin-1 (TSST-1). Cells from BALB/c (H-2d) and C57BL/6 (H-2b) mice as well as those from NON (H-2non) and NOR (H-2g7) mice were used as controls. After 72 h of co-culture with the superantigens or the mitogen concanavalin A (Con A), proliferative response and mitochondrial activity were determined. In the culture supernatants, the cytokines γ-interferon (IFN-γ) and interleukin 10 (IL-10) were measured. Striking similarities between NOD cells and major histocompatiblity complex (MHC)-identical NOR cells could be observed with regard to a low proliferative and mitochondrial response to SEB, accompanied by a normal response to TSST-1 and Con A, respectively. In addition, only NOD and NOR spleen cells were low producers of the T-helper 1 (Th1) cytokine IFN-γ in response to SEB. Conversely, abnormally high IFN-γ levels were induced by TSST-1 in NOD and NOR spleen cells. The cytokine response to Con A was also biased toward IFN-γ in both NOD and NOR. Since IFN-γ and IL-10 are crucial disease-promoting or -protecting mediators in prediabetic NOD mice, superantigens may affect pathogenesis by acting on the Th1/Th2 cytokine balance. The low responder status toward SEB in NOD spleen cells may be of pathogenetic relevance in view of recent findings that the insulin B-chain also interacts with the SEB binding site on MHC class II molecules. In conclusion, we show here that immune cells from mice with a diabetes-associated MHC type respond differently to common environmental superantigens than do immune cells from control strains.
Insulin Lispro in CSII: Results of a Double-Blind Crossover Study
Authors: Zinman Bernard , Tildesley Hugh , Chiasson Jean-Louis , Tsui Elaine , Strack Thomas
Published: September 2013

Insulin lispro is a human insulin analog that dissociates more rapidly than human regular insulin after subcutaneous injection, resulting in higher insulin levels at an earlier point in time and a shorter duration of action. The aim of the study was to evaluate if this pharmacokinetic difference would translate into better postprandial and overall control in 30 IDDM patients (age, 35.1 ± 1.5 years; male-female ratio, 17:13; BMI, 24.8 ± 0.5 kg/m2; HbA1c, 8.03 ± 0.13% at baseline) treated with continuous subcutaneous insulin infusion (CSII; Disetronic H-TRON V100) in a double-blind crossover clinical study. Patients were randomized to insulin lispro or human regular insulin for 3 months before crossing over to the other insulin for another 3 months. All meal boluses were given immediately before breakfast, lunch, and supper. An eight-point blood glucose profile was measured once weekly, and HbA1c levels were measured monthly. At the end of the 3-month treatment period, HbA1c levels were significantly lower with insulin lispro, compared with human regular insulin: 7.66 ± 0.13 vs. 8.00 ± 0.16% (P = 0.0041). While preprandial, bedtime, and 2:00 A.M. values for blood glucose were not significantly different, 1-h postprandial blood glucose was significantly improved after breakfast, lunch, and dinner with insulin lispro, compared with human regular insulin: 8.35 vs. 9.79 mmol/l (P = 0.006), 7.58 vs. 8.74 mmol/l (P = 0.049), and 7.85 vs. 9.01 mmol/l (P = 0.03). The incidence of hypoglycemia per 30 days (blood glucose levels, <3.0 mmol/l) was 8.4 ± 1.3 before randomization, decreasing to 6.0 ± 0.9 for insulin lispro and to 7.6 ± 1.3 for regular insulin during the last month of the study. Two patients in each group reported insulin precipitation. We conclude that insulin lispro improves glycemic control in CSII without increasing the risk of hypoglycemia.
Expression of Transforming Growth Factor-β and Type IV Collagen in Early Streptozotocin-Induced Diabetes
Authors: Park In-Seok , Kiyomoto Hideyasu , Abboud Sherryd L , Abboud Hanna E
Published: September 2013

The earliest manifestations of type I diabetic nephropathy include mesangial matrix expansion, basement membrane thickening, and renal hypertrophy. Transforming growth factor (TGF)-β, a potent inducer of matrix protein synthesis, is a prime candidate to mediate the glomerular changes observed in diabetes. However, the temporal expression of TGF-β and matrix proteins during the early stage of diabetic nephropathy has not been clearly defined. Using in situ hybridization and immunohistochemistry, we determined the expression of TGF-β and type IV collagen mRNAs and proteins in glomeruli and interstitium of diabetic rats 3, 7, and 14 days after streptozotocin (STZ) administration. There was a marked increase in the expression of TGF-β and α1(IV) procollagen mRNAs in glomerular and tubulointerstitial cells as early as 3 days after induction of diabetes, an effect that persisted for 14 days. A concomitant increase in TGF-α and type IV collagen proteins was also observed at each time point. Insulin treatment substantially inhibited the increased expression of TGF-α and collagen type IV mRNAs and proteins. We conclude that TGF-β is increased in glomeruli during the early phase of rapid renal growth in diabetes. These findings suggest that TGF-β may be a key factor involved in the pathogenesis of basement membrane thickening and extracellular matrix accumulation. Inhibition of TGF-P and type IV collagen expression by insulin treatment suggests that they may be useful structural markers for determining the efficacy of therapeutic intervention during early diabetic nephropathy.
Phosphorylation of Myosin Light Chain in Resting Platelets From NIDDM Patients Is Enhanced: Correlation With Spontaneous Aggregation
Authors: Fukuda Koichiro , Ozaki Yukio , Satoh Kaneo , Kume Shoji , Tawata Masato , Onaya Toshimasa , Sakurada Katsuhiko , Seto Minoru , Sasaki Yasuharu
Published: September 2013

Platelet function in patients with NIDDM is enhanced. We have found that spontaneous aggregation (i.e., the formation of small-sized aggregates in the absence of agonist stimulation) occurs at a high rate in platelets from NIDDM patients. We then investigated basal myosin light chain 20 (MLC) phosphorylation, which plays a key role in platelet shape change and aggregation, using a monoclonal antibody against a phosphorylation site (serine 19 residue) in the MLC molecule in platelets from these patients. Standard calibration curves obtained from purified MLC or the phosphorylated form of myosin light chain 20 (MLC-P) were linear within the range of 0–150 ng for MLC and 0–3 ng for MLC-P. The amount of MLC or MLC-P in platelets was estimated, and basal MLC phosphorylation was calculated. Platelets were obtained from 9 young healthy control subjects, 13 age- and sex-matched nondiabetic control subjects, and 13 patients with NIDDM. The basal MLC phosphorylation in platelets was significantly higher in the NIDDM patients than in the control subjects, irrespective of age. These findings suggest that platelets from NIDDM patients are activated in vivo. Platelets obtained from NIDDM patients generated spontaneous aggregation, the degree of which was significantly higher than that in control subjects. Platelet spontaneous aggregation correlated well with basal MLC phosphorylation. These findings suggest that increases in basal MLC in platelets may be one factor leading to hyperaggregability of platelets in these patients.
Sequence Variants in the Pancreatic Islet β-Cell Inwardly Rectifying K+ Channel Kir6.2 (Bir) Gene: Identification and Lack of Role in Caucasian Patients with NIDDM
Authors: Inoue Hiroshi , Ferrer Jorge , Warren-Perry Margaret , Zhang Yun , Millns Helen , Turner Robert C , Elbein Steven C , Hampe Carol L , Suarez Brian K , Inagaki Nobuya , Seino Susumu , Permutt M Alan
Published: September 2013

Signals derived from the metabolism of glucose in pancreatic β-cells lead to insulin secretion via the closure of ATP-sensitive K+ channels (KATP). The cloning of the gene encoding the (β-cell inward rectifier Kir6.2 (Bir), a subunit of the β-cell KATP channel, provided the opportunity to look for mutations in this gene that might contribute to the impaired insulin secretion of NIDDM. By single-strand conformational polymorphism (SSCP) analysis on 35 Northern-European Caucasian patients with NIDDM, six sequence variants were detected: Glu10gag→Lys10aag (E10K), Glu23gag→Lys23aag (E23K), Leu270ctg→Val270gtg (L270V), Ile337atc→Val337gtc (I337V), and two silent mutations. Allelic frequencies for the missense variants were compared between the NIDDM group (n = 306) and nondiabetic control subjects (n = 175) and did not differ between the two groups. Pairwise allelic associations indicated significant linkage disequilibrium between the variants in Kir6.2 and between them and a nearby pancreatic β-cell sulfonylurea receptor (SUR1) missense variant (S1370A), but these linkage disequilibria did not differ between the NIDDM and control groups. The results of these studies thus revealed that mutations in the coding region of Kir6.2 1) were not responsible for the previously noted association of the SUR1 variants with NIDDM (Inoue H et al., Diabetes 45:825–831, 1996) and 2) did not contribute to the impaired insulin secretion characteristic of NIDDM in Caucasian patients.
Mutations in the Hepatocyte Nuclear Factor-1α Gene in MODY and Early-Onset NIDDM: Evidence for a Mutational Hotspot in Exon 4
Authors: Kaisaki Pamela J , Menzel Stephan , Lindner Tom , Oda Naohisa , Rjasanowski Ilona , Sahm Jürgen , Meincke Gustav , Schulze Jan , Schmechel Harald , Petzold Cornelia , Ledermann Hellmuth M , Sachse Günther , Boriraj V Vicky , Menzel Ruth , Kerner Wolfgang , Turner Robert C , Yamagata Kazuya , Bell Graeme I
Published: September 2013

We have recently shown that mutations in the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1α are the cause of one form of maturity-onset diabetes of the young (MODY3). Here, we report the exon-intron organization and partial sequence of the human HNF-1α gene. In addition, we have screened the ten exons and flanking introns of this gene for mutations in a group of 25 unrelated white subjects from Germany who presented with NIDDM before 35 years of age and had a first-degree relative with NIDDM. Mutations were identified in nine of these individuals, suggesting that mutations in the HNF-1α gene are a common cause of diabetes in German subjects with early-onset NIDDM and a family history of diabetes. Thus, screening for mutations in this gene may be indicated in subjects with early-onset NIDDM. Interestingly, three of the nine mutations occurred at the same site in exon 4 with insertion of a C in a polyCtract, centered around codon 290 (designated Pro291fsinsC), thereby resulting in a frameshift during translation and premature termination. Analyses of linked DNA polymorphisms in the HNF-1α gene indicated that the Pro291fsinsC mutation was present on a different haplotype in each subject, implying that the polyC tract represents a mutational hot spot. We have also identified the mutation in the HNF-1α gene in the Jutland pedigree, one of the original MODY pedigrees reported in the literature, as being a T→G substitution in codon 241, resulting in the replacement of a conserved Cys by Gly (C241G). The information on the sequence of the HNF-1α gene and its promoter region will facilitate the search for mutations in other subjects and studies of the role of the gene in determining normal β-cell function.
Combined Analysis and Single-Step Detection of GAD65 and IA2 Autoantibodies in IDDM Can Replace the Histochemical Islet Cell Antibody Test
Authors: Wiest-Ladenburger Uta , Hartmann Ralf , Hartmann Ulrike , Berling Kerstin , Böhm Bernhard O , Richter Wiltrud
Published: September 2013

Cytoplasmic islet cell antibodies (ICAs) are the classical serological markers for diagnosis and prediction of IDDM, but high technical demands have limited the widespread use of the histochemical ICA test. To investigate whether combined analysis of autoantibodies to two defined islet antigens can replace the histochemical ICA test, we established quantitative radioimmunoassays for autoantibodies to glutamate decarboxylase (GAD65-A), the tyrosine phosphatase IA2/ICA512 (IA2-A), and the cytoplasmic part of IA2 (IA2C-A). The GAD65-A and IA2C-A profiles of 920 sera from healthy individuals and from patients with IDDM, other organ-specific autoimmune diseases, and polyendocrine autoimmune syndrome were compared with the ICA profiles from these same individuals. Combined analysis of GAD65-A and IA2C-A detected 93–100% of the ICA+ sera, and, at equal specificity, improved the diagnostic sensitivity (85%) for IDDM compared with that of ICA (74%). This effect was especially pronounced in children with disease onset before 16 years of age (91% sensitivity). To replace ICA testing in risk assessment for IDDM, we designed a strategy adapted to study groups with low antibody prevalence. A combined radioimmunoassay for single-step detection of GAD65-A and IA2C-A was developed, and positive sera were reanalyzed to define their single autoantibody specificity. We identified 93% of the ICA+ sera from 204 first-degree relatives of IDDM patients. Single-step detection reduced costs and effort by more than 40% compared with separate testing, allowing an efficient large-scale screening of sera for GAD65-A and IA2C-A in IDDM. In sum, GAD65-A and IA2C-A detected much ICA reactivity, and their combined evaluation and detection is suitable to replace the histochemical ICA test.
α-Cell Neogenesis in an Animal Model of IDDM
Authors: O'Reilly Lorraine A , Gu Danling , Sarvetnick Nora , Edlund Helena , Phillips Jenny M , Fulford Tony , Cooke Anne
Published: September 2013

Currently there is debate regarding the capacity of pancreatic islets to regenerate in adult animals. Because pancreatic endocrine cells are thought to arise from duct cells, we examined the pancreatic ductal epithelium of the diabetic NOD mouse for evidence of islet neogenesis. We have evidence of duct proliferation as well as ductal cell differentiation, as suggested by bromodeoxyuridine-labeling and the presence of glucagon-containing cells within these ducts. In addition, the ductal epithelia in diabetic NOD mice expressed the neuroendocrine markers neuropeptide Y and tyrosine hydroxylase. These ducts also expressed the homeobox gene product, insulin promoter factor 1. Ductal cell proliferation and expression of these markers was not observed in transgenic NOD mice (NOD-E), which do not develop clinical or histopathological symptoms of IDDM. This suggests that the observed ductal cell proliferation and differentiation was a direct result of β-cell destruction and insulin insufficiency in these adult diabetic mice, which further suggests that these events are recapitulating islet ontogeny observed during embryogenesis. It is possible that comparable processes occur in the human diabetic pancreas.
Effects of Pramlintide, an Analog of Human Amylin, on Plasma Glucose Profiles in Patients With IDDM: Results of a Multicenter Trial
Authors: Thomas Robert G , Peterson Janet , Gottlieb Alan , Mullane John
Published: September 2013

The effects of subcutaneous administration of 10, 30, or 100 μg q.i.d. pramlintide, an analog of human amylin, on plasma glucose regulation in patients with IDDM were evaluated in a multicenter trial. The plasma glucose response to a Sustacal test meal was significantly reduced compared with placebo both after 1 week and after 2 weeks of administration of 30 or 100 μg pramlintide. In addition, 24-h mean plasma glucose concentrations were significantly lowered in patients receiving 30 μg of pramlintide for 2 weeks compared with placebo, while the 100-μg pramlintide dose did not reach statistical significance for the 24-h glucose profiles. At 10 μg, pramlintide had no effect on the 24-h glucose profile or on the plasma glucose response to a Sustacal test meal. The reduction in 24-h glucose concentrations and glucose concentrations after the Sustacal test meal observed at the 30-μg pramlintide dose was not accompanied by an increased incidence of hypoglycemic events. The most frequent adverse events were dose-related and involved transient upper gastrointestinal symptoms. A majority (>80%) of the patients who reported these adverse events during week 1 did not report them in week 2. These data indicate that pramlintide effectively reduces plasma glucose concentrations as reflected in both a 24-h glucose profile and a Sustacal test meal while maintaining an acceptable safety profile.
Acylation of Human Insulin With Palmitic Acid Extends the Time Action of Human Insulin in Diabetic Dogs
Authors: Myers Sharon , Yakubu-Madus Fatima E , Johnson William T , Baker James E , Cusick Tania S , Williams Valerie K , Tinsley Frank C , Kriauciunas Aidas , Manetta Joseph , Chen Victor J
Published: September 2013

To test whether the binding of insulin to an endogenous serum protein can be used to extend the time action of insulin, human insulin was acylated at the epsilonamino group of Lys(B29) with palmitic acid to promote binding to serum albumin. Size-exclusion chromatography was used to demonstrate specific binding of the resulting analog, [N∈-palmitoyl Lys(B29)] human insulin, to serum albumin in vitro, and the time action and activity of the analog were determined in vivo using overnight-fasted, insulin-withdrawn diabetic dogs. In the diabetic animal model, the duration of action of [N∈-palmitoyl Lys(B29)] human insulin administered intravenously was nearly twice that of unmodified human insulin, and the plasma half-life was nearly sevenfold that of the unmodified protein. Administered subcutaneously, [N∈-palmitoyl Lys(B29)] human insulin had a longer duration of action; a flatter more basal plasma insulin profile; and a lower intersubject variability of response than the intermediate-acting insulin suspension Humulin L (Lilly, Indianapolis, IN). These studies support the concept that modification of insulin to promote binding to an existing serum protein can be used to extend the time action of human insulin. In addition, the time action, pattern, and decreased variability of response to [N∈-palmitoyl Lys(B29)] human insulin support the development and further testing of this soluble insulin analog as a basal insulin to increase the safety of intensive insulin therapy.
Aldose Reductase Inhibition Increases CNTF-Like Bioactivity and Protein in Sciatic Nerves From Galactose-Fed and Normal Rats
Authors: Mizisin Andrew P , Calcutt Nigel A , DiStefano Peter S , Acheson Ann , Longo Frank M
Published: September 2013

The impact of exaggerated polyol pathway flux on ciliary neurotrophic factor (CNTF)-like bioactivity and expression of CNTF in rat sciatic nerve was examined after 2 months of galactose intoxication. Polyol content was elevated (P < 0.001) and motor nerve conduction velocity reduced (P < 0.05) in galactose-fed rats compared with control animals or control and galactose-fed rats treated with the aldose reductase inhibitor (ARI) Ponalrestat. CNTF-like bioactivity in the galactose-fed group was reduced to 30% of that assayed in the control group (P < 0.001). ARI treatment significantly increased CNTF-like bioactivity by 60% compared with the untreated galactose group (P < 0.05) but did not restore it to control levels. Unexpectedly, bioactivity in ARI-treated control animals was increased by nearly 250% compared with untreated controls (P < 0.005). In addition to the deficit in CNTF bioactivity in untreated galactose rats, the expression of protein, but not of mRNA, was reduced (P < 0.05). In ARI-treated control and galactose-fed rats, the expression of CNTF peptide was significantly enhanced above control levels (both P < 0.05). Concomitant with the reduction in CNTF levels, there was a shift in the axonal size-frequency distribution of myelinated fibers toward smaller axons in galactose-fed rats that was prevented by ARI treatment. Since galactose feeding has little impact on levels of CNTF mRNA, these observations suggest that deficits in CNTF-like bioactivity may result from a posttranscriptional modification of neurotrophic protein expression or turnover. Unlike other functional and structural disorders in galactose neuropathy, factors other than polyol accumulation may contribute to the deficit in CNTF-like bioactivity.
Apoptosis Is the Mode of β-Cell Death Responsible for the Development of IDDM in the Nonobese Diabetic (NOD) Mouse
Authors: O'Brien Bronwyn A , Harmon Brian V , Cameron Donald P , Allan David J
Published: September 2013

The NOD/Lt mouse, a widely used model of human autoimmune IDDM, was used to establish the mode of β-cell death responsible for the development of IDDM. Apoptotic cells were present within the islets of Langerhans in hematoxylin and eosin–stained sections of pancreases harvested from 3- to 18-week-old female NOD/Lt mice (a range of 11–50 apoptotic cells per 100 islets). Immunohistochemical localization of insulin to the dying cells confirmed the β-cell origin of the apoptosis. Although some islets from age-matched control female NOD/scid mice contained apoptotic cells, virtually all of these cells were insulin negative as determined by immunohistochemistry. The small number of apoptotic insulin-positive cells identified in islets from NOD/scid mice (a range of 0–1 apoptotic cells per 100 islets) was not statistically significant, compared with the numbers recorded in NOD/Lt mice. All dying cells showed the morphological changes characteristic of cell death by apoptosis and stained positively with the TUNEL method for end-labeling DNA strand breaks. The maximum mean amount of β-cell apoptosis occurring in NOD/Lt mice was at week 15 (50 apoptotic cells per 100 islets), which coincided with the earliest onset of diabetes as determined by blood glucose, urine glucose, and pancreatic immunoreactive insulin measurements. While there was no peak incidence of β-cell apoptosis throughout the time period studied (weeks 3–18), the incidence of apoptosis decreased at week 18, by which time 50% of the animals had overt diabetes. The low levels of β-cell apoptosis observed is indicative of a gradual deletion of the β-cell population throughout the extensive preclinical period seen in this model and would be sufficient to account for the β-cell loss resulting in IDDM. Apoptosis of β-cells preceded the appearance of T-cells (CD3-positive by immunohistochemistry) in islets. Lymphocytic infiltration of islets (insulitis) was not detected until week 6. The results show that β-cell apoptosis is responsible for the development of IDDM in the NOD/Lt mouse and that its onset precedes lymphocytic infiltration of the islets.
Hsp60 Peptide Therapy of NOD Mouse Diabetes Induces a Th2 Cytokine Burst and Downregulates Autoimmunity to Various β-Cell Antigens
Authors: Elias Dana , Meilin Aviram , Ablamunits Vitaly , Birk Ohad S , Carmi Pnina , Könen-Waisman Stephanie , Cohen Irun R
Published: September 2013

A peptide of the human 60-kDa heat-shock protein (hsp60), designated p277, was found to be useful as a therapeutic agent to arrest the autoimmune process responsible for diabetes in nonobese diabetic (NOD) mice. The effectiveness of peptide treatment was associated with the induction of peptide-specific antibodies of the IgG1 but not of the IgG2a isotype, suggesting the possibility that a Th2-type response may have been induced. We now report that the effectiveness of p277 treatment is associated with the transient activation of anti-p277 splenic T-cells that produce the Th2 cytokines interleukin-4 (IL-4) and IL-10. The Th2 response to p277 was associated with reduced Thl-type autoimmunity to hsp60 and to two other target antigens associated with diabetes: GAD and insulin. The Th2 shift appeared to be relatively specific; spontaneous T-cell reactivity to a bacterial antigen peptide remained in the Th1 mode in the p277-treated mice. Moreover, treatment with the bacterial peptide did not induce a change in cytokine profile, and it did not affect progression of the disease. Thus, effective peptide treatment of the diabetogenic process associated with the induction of antibodies may be explained by selective and transient activation of Th2 autoimmune reactivity.
Interferon-γ Independently Activates the MHC Class I Antigen Processing Pathway and Diminishes Glucose Responsiveness in Pancreatic β-Cell Lines
Authors: Baldeón Manuel E , Neece David J , Nandi Dipankar , Monaco John J , Gaskins H Rex
Published: September 2013

The mouse pancreatic βTC3 and βTC6-F7 cell lines were used to characterize the effects of interferon-γ (IFN-γ) on (β-cell phenotype and function. Initially, intracellular and secreted insulin were compared in glucose-stimulated cells over time. A significant reduction in insulin content and secretion was observed on a per-cell basis in glucose-stimulated βTC3 and βTC6-F7 cells after 12 h of exposure to IFN-γ. The steadystate level of pre-proinsulin mRNA expression was not affected by IFN-γ. Thus, we postulate that IFN-γ's inhibitory actions occur after transcription of preproinsulin genes. Time-course analysis of IFN-γ–regulated mRNA expression of the two intra-MHC-encoded subunits of the proteasome (low–molecular-mass polypeptide [Lmp]-2 and Lmp-7) revealed a correlation between their induction and the inhibitory effects of IFN-γ on glucose-stimulated insulin production. Increased expression of Lmp-2 and Lmp-7 mRNA was accompanied by a corresponding induction of LMP2 and LMP7 protein expression. Subsequently, major histocompatibility complex (MHC) class I cell-surface expression was significantly increased in IFN-γ–treated βTC3 and βTC6-F7 cells. Exposure of IFN-γ-treated β-cells to a peptide aldehyde inhibitor of the proteasome (MG132) significantly attenuated MHC class I cell-surface expression but did not prevent the negative effects of IFN-γ on glucose responsiveness. Enhanced expression of the MHC class I antigen processing and presentation pathway and diminished insulin production appear to be distinct pathological alterations in β-cells exposed to the insulitic cytokine IFN-γ.
Effect of MHC Transgene Expression on Spontaneous Insulin Autoantibody Class Switch in Nonobese Diabetic Mice
Authors: Hutchings Patricia , Tonks Paul , Cooke Anne
Published: September 2013

A study of spontaneous anti-insulin autoantibodies in nonobese diabetic (NOD) mice revealed that when first detected, the antibodies are immunoglobulin M (IgM), but by age 10 weeks, immunoglobulin G (IgG) autoantibodies have appeared in many of these animals. When NOD strains, partially or completely protected from IDDM by the insertion of transgenes in the class II region, were compared, it was found that the switch to IgG autoantibodies was inhibited and the autoantibodies remained IgM indefinitely. We speculate that the switch to IgG may be a marker of events leading to IDDM in NOD mice and an indication that T-cell help has been generated for responses to β-cell antigens. Such help not only directs the development of IgG autoantibodies, but more importantly, allows the emergence of potentially pathogenic T-cell clones that are capable of infiltrating the pancreas and mediating β-cell damage.
Activation of Autonomic Nerves and the Adrenal Medulla Contributes to Increased Glucagon Secretion During Moderate Insulin-Induced Hypoglycemia in Women
Authors: Havel Peter J , Ahren Bo
Published: September 2013

Despite evidence that the autonomic nervous system (ANS) makes a significant contribution to increased glucagon secretion during insulin-induced hypoglycemia in several animal species, including a recent study in nonhuman primates, the role of the ANS in mediating this important counterregulatory response in humans remains controversial. Therefore, glucagon responses to insulin-induced hypoglycemia were examined in seven nondiabetic women (BMI, 28.0 ± 2.0 kg/m2) with and without the presence of the ganglionic nicotinic receptor antagonist trimethaphan. Trimethaphan impairs neurotransmission across parasympathetic and sympathetic autonomic ganglia and in the adrenal medulla and, therefore, markedly impairs autonomic activation during insulin-induced hypoglycemia. The studies were performed in random order at least 4 weeks apart. Trimethaphan was infused at a variable rate (0.3−0.6 mg/min) to modestly lower blood pressure (∼10 mmHg) without producing hypotension. Regular human insulin was infused (0.28 pmol · m−2 · min−1) with a variable rate glucose infusion to lower the plasma glucose from 4.9 ± 0.2 to 2.6 ± 0.2 mmol/l in the control study and from 4.9 ± 0.2 to 2.5 ± 0.2 mmol/l in the trimethaphan study. Trimethaphan impaired parasympathetic and sympathoadrenal activation during insulin-induced hypoglycemia as assessed by 70% reductions of the plasma pancreatic polypeptide response and epinephrine response (both P < 0.05 vs. control study). Glucagon secretory responses during insulin-induced hypoglycemia were assessed as peak responses and as the area under the curve (AUC) above baseline values during insulin-induced hypoglycemia. Plasma glucagon increased in the control study from 44 ± 5 ng/l to a peak of 76 ± 9 ng/l (∆ = 32 ± 8 ng/l; P < 0.005 vs. baseline) and in the trimethaphan study from 41 ± 3 to 50 ± 7 ng/l (∆ = 10 ± 5 ng/l; P < 0.02 vs. control subjects). The glucagon response to insulin-induced hypoglycemia as assessed by the AUC was 948 ± 272 ng · I−1 · 45 min−1 in the control study (P < 0.01 vs. baseline), but was reduced by 75% in the trimethaphan study (AUC = 203 ± 94 ng · 1−1 · 45 min−1; P < 0.02 vs. control subjects). Trimethaphan did not affect the glucagon response to arginine administration. These results demonstrate that the ANS mediates the majority of the glucagon response to insulininduced hypoglycemia of 2.5 mmol/l in postmenopausal nondiabetic women.
Importance of Cerebral Blood Flow to the Recognition of and Physiological Responses to Hypoglycemia
Authors: Matthew Thomas , Sherwin Robert S , Murphy June , Kerr David
Published: September 2013

During hypoglycemia, cerebral blood flow (CBF) does not increase significantly until peripheral glucose levels are very low (2.0 nmol/l), that is, well below the blood glucose threshold for impairment of cognitive function (3.0 nmol/l). Because increased rates of cerebral blood flow will increase glucose transport, a failure of flow to rise earlier, before brain function is threatened, might be considered maladaptive. To examine the influence of inducing an earlier rise in CBF during hypoglycemia, eight healthy volunteers participated in three studies using a randomized, placebo-controlled design. In all three studies, a hyperinsulinemic (60 mU · m2 · min−1) clamp was used to maintain blood glucose levels at 4.5 nmol/l for 60 min. Thereafter, for EUGACZ, blood glucose was maintained at 4.5 nmol/l from 60 to 170 min and at 90 min from the start of this study, and 1-g acetazolamide i.v. was given to induce an early rise in CBF; for HYPO-ACZ, glucose was lowered over 20 min to 2.8 mmol/l and kept at that level for 90 min, and acetazolamide was given 90 min from the start of this study; and for HYPO-CON, glucose was treated as in HYPO-ACZ, and matching placebo was given in place of acetazolamide. Injection of acetazolamide was associated with a 30% rise in right (95% CI24–34%) and left (20–32%) middle cerebral artery velocity (an index of CBF) during euglycemia without any change in hypoglycemia awareness or counterregulatory hormone levels. When glucose was lowered to 2.8 nmol/l, acetazolamide caused a similar rise in middle cerebral artery velocity in the HYPO-ACZ study. However, all subjects were less “aware” of hypoglycemia, had fewer adrenergic symptoms (sweating, palpitations, tremors; all P < 0.05), and had lower plasma epinephrine levels (1,026 vs. 1,790 pmol/l; –764 [437 to 1,097] pmol/l, point estimate of difference [95% CI]; P < 0.001), compared with the HYPO-CON study, whereas levels of other counterregulatory hormones and norepinephrine were similar. Cognitive function (latency of the P300 evoked response) was unaffected by increasing CBF. In conclusion, enhanced rates of cerebral blood flow at the onset of systemic hypoglycemia are associated with diminished perception of low blood glucose levels and attenuation of the epinephrine counterregulatory response. These findings suggest that augmenting cerebral blood flow leads to an enhanced rate of substrate delivery to the central nervous system.