bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological Concentrations of Insulin Induce Cellular Desensitization to the Mitogenic Effects of Insulin-Like Growth Factor I
Authors: Conover Cheryl A , Clarkson Jay T , Bale Laurie K
Published: September 2013

Insulin and insulin-like growth factor I (IGF-I) are structurally related peptides capable of stimulating a variety of metabolic and mitogenic processes. In this study, we investigated the interaction between these peptides and their receptor-mediated pathways in an untransformed cell line. Cultured bovine fibroblasts specifically bound IGF-I and insulin, and each peptide could stimulate DNA synthesis and cell replication through its own receptor. Preincubation of bovine fibroblasts with concentrations of insulin that did not bind to the IGF-I receptor resulted in complete but reversible cellular desensitization to IGF-I-stimulated mitogenesis. Preincubation with as little as 0.1 nM insulin was sufficient to inhibit subsequent IGF-I action. Various insulin analogs produced desensitization in direct relation to the affinity of the insulin for the insulin receptor. Desensitization required > 4 h of cell exposure to insulin and was blocked in the presence of cycloheximide. Neither serum-stimulated mitogenesis nor IGF-I-stimulated glucose uptake were affected by insulin pretreatment. 125I-labeled IGF-I affinity cross-linking experiments indicated that preincubation with insulin did not affect labeling of the 130,000-Mr α-subunit of the IGF-I receptor, but was associated with the loss of IGF-I– and insulin-inhibitive bands at Mr = 100,000, 85,000, 58,000, and 34,000. These studies suggest that insulin, via interaction with insulin receptors on bovine fibroblasts, regulates IGF-I action at a step distal to IGF-I receptor binding and are consistent with desensitization occurring at an intracellular step in the mitogenic pathway shared by insulin and IGF-I.
Induction of Insulitis in Athymic (Nude) Mice: The Effect of NOD Thymus and Pancreas Transplantation
Authors: Georgiou Harry M , Mandel Thomas E
Published: September 2013

The NOD mouse is a model for human insulin-dependent diabetes mellitus. The disease is thought to have an autoimmune etiology because it is T-cell dependent and is characterized by mononuclear cell infiltration in and around the pancreatic islets of Langerhans. The mechanism by which autoreactive T-cells are generated is not fully understood, but it has been postulated that there is a breakdown in self-tolerance induction during intrathymic T-cell maturation. The aim of these studies was to determine whether transplantation of NOD thymus into diabetes-resistant mouse strains would generate islet-reactive T-cells. Neonatal thymus was pretreated either by irradiation or culture in 2-deoxyguanosine (dGua) and then transplanted into athymic BALB/c, CBA, and C57BL/6 nude mice. Generally, insulitis was not seen in the CBA or C57BL/6 recipients, but was found in 56% of BALB/c mice transplanted with an irradiated NOD thymus and in 46% BALB/c mice with a dGua-treated thymus. Similar experiments in which a NOD fetal pancreas was transplanted into nude BALB/c mice before NOD thymus transplantation showed a similar frequency and severity of insulitis in both the host pancreas and grafted NOD pancreas. This suggests that NOD islets are no more prone than the host islets to autoimmune attack and do not exacerbate insulitis. Overall, the data suggest that a defect of thymic origin (and correlating with the thymic epithelium) in the NOD mouse can lead to the development of autoreactive T-cells and specific islet cell damage. Autoreactivity appears to be restricted to the H-2Kd allele that is shared by NOD and BALB/c mice.
A Novel Insulin Secretagogue Is a Phosphodiesterase Inhibitor
Authors: Leibowitz Mark D , Biswas Chhabi , Brady Edward J , Conti Macro , Cullinan Catherine A , Hayes Nancy S , Manganiello Vincent C , Saperstein Richard , Wang Lu-hua , Zafian Peter T , Berger Joel
Published: September 2013

The arylpiperazine L-686,398 was described as an oral hypoglycemic agent and is shown to be an insulin secretagogue in vitro. The characteristics of its activity were similar to those of the incretin glucagon-like peptide I (GLP-I). We demonstrate that both the peptide and L-686,398 increase the accumulation of cAMP in isolated ob/ob mouse pancreatic islet cells, but by different mechanisms. Although GLP-I activates adenylate cyclase, the arylpiperazine has no effect on this enzyme or on the binding of 125I-labeled GLP-I to its receptor on RINm5F rat insulinoma cell membranes. However, L-686,398 inhibits the total cAMP phosphodiesterase (PDE) activity in homogenates of ob/ob mouse pancreatic islets with an EC50 of ∼ 50 μmol/l. To determine the mechanism of PDE inhibition by the arylpiperazine and to examine its specificity, we studied the kinetics of arylpiperazine inhibition of two recombinant PDEs. The arylpiperazine is a competitive inhibitor of both a human heart type III PDE and a rat type IV-D PDE. Inhibition of the type III and IV isozymes are characterized by Ki values of 27 and 5 μmol/l, respectively. Although not extremely potent, the arylpiperazine does exhibit modest selectivity between these PDEs. The observation that L-686,398 acts as a PDE inhibitor suggests that exploration for β-cell-specific PDE isoforms may reveal novel PDEs as targets for the development of therapeutically useful glucose-dependent insulin secretagogues.
A Monitor of Secretion From Single Pancreatic β-Cells
Authors: Kuromi Hiroshi , Mizuno Nobuhisa , Seino Susumu
Published: September 2013

ATP is known to be coreleased with insulin from pancreatic β-cells. To monitor insulin secretion from single β-cells, a single β-cell was surrounded in culture by Fura 2–loaded calf pulmonary artery endothelium (CPAE) cells, which can detect the ATP. CPAE cells did not respond with an elevation in cytoplasmic calcium concentration ([Ca2+]i) to either tolbutamide (100 mumol/l) or kainate (1 mmol/l) but did respond with an elevation in [Ca2+]i to ATP (0.1–10 μmol/l) without desensitization and in a dose-dependent manner. A brief application of tolbutamide (10 μmol/l) increased [Ca2+]i in both the β-cell and the adjacent CPAE cells in co-culture. Suramin (100 μmol/l), an ATP-receptor blocker, inhibited the tolbutamide-induced elevation in [Ca2+]i in the CPAE cells but did not inhibit the elevation in [Ca2+]i in the β-cell, confirming that the insulin secretagogue-induced Ca2+ response in CPAE cells in co-culture is mediated by ATP released from the β-cell. When co-culture of the β-cell and CPAE cells was stimulated by kainate (1 μmol/l) and then tolbutamide (10 μmol/l), the CPAE cells showed elevations in [Ca2+]i in response to kainate and tolbutamide during elevation in [Ca2+]i in the β-cell. This strongly suggests that insulin secretion as well as an increase in [Ca2+]i in response to different agents, i.e., kainate and tolbutamide, occurs in a single β-cell. A long exposure of tolbutamide (100 μmol/l, 4 min) resulted in a long-lasting elevation in [Ca2+]i in the β-cell. During the elevation in [Ca2+]i, induced by tolbutamide in the β-cell, secretion was observed immediately after its application and again after some resting time. This temporal analysis shows that the secretory response of a single intact β-cell rapidly desensitizes despite the [Ca2+]i remaining elevated during the secretagogue stimulation and that it can return, after some desensitized period, in an intact cell. The present co-culture system using CPAE cells as reporter cells is, therefore, useful for monitoring insulin secretion from single intact β-cells.
Hypoglycemic Effects of Peroxovanadium Compounds in Sprague-Dawley and Diabetic BB Rats
Authors: Yale Jean-François , Lachance Danielle , Bevan A Paul , Vigeant Claire , Shaver Alan , Posner Barry I
Published: September 2013

Highly purified peroxovanadium (pV) compounds, each containing an oxo ligand, one or two peroxo anions, and an ancillary ligand in the inner coordination sphere of vanadium, were shown to decrease plasma glucose markedly in both normal Sprague-Dawley and insulin-deprived diabetic BB rats. Maximal decreases in plasma glucose were at 60–100 min after intravenous, intraperitoneal, or subcutaneous administration. Synergism between these compounds and insulin was observed. Whereas parenterally administered orthovanadate or vanadyl sulfate did not induce hypoglycemia before inducing acute mortality, pV compounds effected hypoglycemia at doses much lower than those inducing acute mortality. When administered subcutaneously over a period of 3 days to insulin-deprived diabetic BB rats, pV compounds, but not vanadate, caused a significant decrease in plasma glucose concentrations and prevented the appearance of ketosis in these animals. Thus, pV compounds are the first agents other than insulin that acutely and markedly reduce plasma glucose in hypoinsulinemic diabetic BB rats.
Insulin Gene 5′ Flanking Polymorphism: Length of Class 1 Alleles in Number of Repeat Units
Authors: McGinnis Ralph E , Spielman Richard S
Published: September 2013

The 5′ flanking polymorphism (5′FP) is a minisatellite, variable number of tandem repeat (VNTR) locus adjacent to the 5′ end of the insulin gene (INS). Alleles of the 5′FP are highly variable in length but fall into three discrete size classes. The shortest, or class 1, alleles are associated with insulin-dependent diabetes mellitus (IDDM). Here we present a polymerase chain reaction (PCR)-based technique for subtyping 5′FP class 1 alleles by determining their exact lengths in number of repeat units (RUs). The technique resolves small length differences not detectable by Southern blot and produces a frequency distribution of class 1 allele lengths, which serve as subtypes of the crude class 1 category. We have applied the technique to 132 Caucasian families with IDDM offspring and have found that the lengths of 5′FP class 1 alleles form a quasi-continuous distribution with three distinct modes. We also found precise correlation between class 1 allele length and the allele present on the same chromosome at HUMTH01, a second VNTR locus in the INS region. Specifically, each of the four common alleles of HUMTH01 exhibited near-total association with a narrow size range belonging to one of the three components of the class 1 distribution. We discuss these results in relation to the population history of the 5′FP and INS region haplotypes and in relation to IDDM susceptibility in the INS region.
Upregulated Hexokinase Activity in Isolated Islets from Diabetic 90% Pancreatectomized Rats
Authors: Hosokawa Hitoshi , Hosokawa Yuka Amino , Leahy Jack L
Published: September 2013

Glucokinase is the β-cell glucose sensor, i.e., the site in glucose metabolism that determines the glucose set-point (sensitivity) for insulin secretion. Hexokinase is also present, but it normally contributes little to glucose metabolism because of end-product inhibition by glucose 6-phosphate. There is a lowered glucose set-point for insulin secretion in 90% pancreatectomized (Px) diabetic rats. We investigated the mechanism by measuring hexokinase and glucokinase activity in islet extracts. Glucokinase activity was minimally raised in Px islets (Vmax 125% of sham-operated control rats). In contrast, hexokinase Vmax was 250% of the control value, suggesting that the increased hexokinase activity caused the β-cell glucose hypersensitivity. Additional evidence was obtained with a 40-h fast that was performed because of a previous observation that the inhibitory effect of fasting on insulin secretion was impaired in Px rats. Glucokinase activity fell normally in the Px rats (32 ± 4% reduction in sham vs. 37 ± 4% in Px rats) as opposed to hexokinase activity, which was unaffected in either group. In summary, a feature of hyperglycemia is upregulated islet hexokinase activity. The result is that hexokinase assumes partial control over the glucose set-point for insulin secretion. As such, regulatory effects on insulin secretion, such as fasting, that are mediated through glucokinase activity may be altered.
Action Profile of Cobalt(III)-Insulin: A Novel Principle of Protraction of Potential Use for Basal Insulin Delivery
Authors: Kurtzhals Peter , Ribel Ulla
Published: September 2013

The Co3+-insulin hexamer is an extraordinary stable insulin hexamer that has no affinity for the insulin receptor per se but is converted into active insulin in vivo. In the present study, we evaluated the action profile of Co3+-insulin after subcutaneous injection into nondiabetic pigs and showed that the Co3+-hexamer does not dissociate before absorption. After absorption, Co3+-insulin is accumulated in the bloodstream because the complex is distributed and eliminated more slowly than human insulin. The degree of protraction of Co3+-insulin is similar to that of NPH insulin when evaluated in an euglycemic glucose clamp. We suggest that the long plasma half-life and a gradual in vivo activation contribute to prolong the effect of Co3+-insulin. The Co3+-insulin hexamer provides a novel principle of protraction of potential use for basal insulin delivery to the diabetic patient.
Low Birth Weight: A Risk Factor for Development of Diabetic Nephropathy?
Authors: Rossing Peter , Tarnow Lise , Nielsen Flemming S , Hansen Birgitte V , Brenner Barry M , Parving Hans-Henrik
Published: September 2013

It has been demonstrated that intrauterine growth retardation, defined as birth weight below the 10th percentile, gives rise to a reduction in nephron number. Oligonephropathy has been suggested to increase the risk for systemic and glomerular hypertension in adult life as well as enhance risk for expression of renal disease after exposure to potentially injurious renal stimuli. The aimof this study was to determine if low birth weight is a risk factor for development of diabetic nephropathy. In a case-control study, we investigated 184 (110 men) insulin-dependent diabetes mellitus (IDDM) patients with diabetic nephropathy (persistent albuminuria >300 mg/24 h)(age [mean ± SD] 41.0 ± 9.3 years, duration of diabetes 26.9 ± 8.2 years) and 182 (111 men) normoalbuminuric (<30 mg/24 h) IDDM patients (age 42.1 ± 9.8 years, duration of diabetes 25.8 ± 8.6 years). Information about weight at birth was obtained from the midwife's original registrations. In women below the 10th percentile in birth weight (≤2,700 g, n = 16), 75% had nephropathycompared with only 35% among patients whose birth weights were above the 90th percentile (≥4,000 g, n = 17) (P = 0.05). In men below the 10th percentile in birth weight (≤ 2,910 g, n = 22), the prevalence of patients with nephropathy (50%) was similar to the prevalence among patients above the 90th percentile in birth weight (≥4,200 g, n = 24) (54%). Weights at birth (means ± SD) were similar in patients with and without diabetic nephropathy: men, 3,548 ± 554 and 3,555 ± 493 g; women, 3,265 ± 621 and 3,373 ± 577 g, respectively. Adult height was significantly correlated with weight at birth (r = 0.18, P = 0.008 for men; r = 0.28, P < 0.001 for women). Men with diabetic nephropathy were significantly shorter than men with normoalbuminuria (176.9 ± 7.1 vs. 179.4 ± 6.5 cm, P < 0.01). In conclusion, our study supports the hypothesis that genetic predispositionor factors operating in utero or early childhood or both contribute to the development of diabeticnephropathy.
The Pharmacokinetics of Nicotinamide in Humans and Rodents
Authors: Petley Ann , Macklin Beryl , Renwick Andrew G , Wilkin Terence J
Published: September 2013

Nicotinamide, a derivative of the B vitamin niacin, is currently under trial for the prevention of insulin-dependent diabetes mellitus after success in the NOD mouse. However, the dose, route of administration, and formulation of nicotinamide given to humans is quite different from those used successfully in animals, and the aim of this study was to investigate the plasma pharmacokinetics of oral nicotinamide in humans in two doses and in two different formulations (standard and the long-acting Enduramide). There were no significant differences in the kinetics of the low dose of standard nicotinamide (2.5 mg/kg) and low-dose Enduramide (6.7 mg/kg) in young adult men. Nonlinear kinetics were found with both formulations at higher doses, e.g., a 10-fold increase in the dose of the standard nicotinamide produced a 62-fold increase in the area under the plasma concentration-time curve (AUC). The high dose of standard nicotinamide (25 mg/kg body wt) produced a mean peak plasma concentration 75% higher than that achieved with the sustained release nicotinamide preparation given in a dose similar to that currently used in prevention trials (2 g ≡ 26.6 mg/kg body wt for a 75-kg subject). The AUC was also significantly greater with the standard formulation, indicating a higher bioavailability. Long-term plasma levels for high doses of both formulations were modeled from the single-dose kinetics by computer program. The AUC for standard nicotinamide was 1.7 times higher than that for Enduramide. We conclude that standard nicotinamide offers greater bioavailability than the long-acting formulation tested and that the metabolic clearance pathways of nicotinamide are saturated at the doses currently used in human trials.
Important Role of Insulin and Flow in Stimulating Glucose Uptake in Contracting Skeletal Muscle
Authors: Hespel Peter , Vergauwen Lieven , Vandenberghe Katleen , Richter Erik A
Published: September 2013

The relative role of contractions, insulin, and increased supply of glucose and insulin, via an increase in blood flow, in stimulating glucose uptake in skeletal muscle during contractions was studied in isolated perfused rat hindlimbs. ffindlimbs were perfused with a standard per-fusate medium containing 6 mmol/1 glucose and four different insulin concentrations (0, 100, 500, and 20,000 μU/ml). Contractions were induced by supramaximal intermittent electrical stimulation of the sciatic nerve. Three different perfusion protocols were used: 1) muscles were stimulated to contract without concomitantly increasing perfusate flow; 2) flow was increased in the absence of electrical stimulation; and 3) muscles were stimulated to contract together with a flow increase. Both contractions and increased flow of perfusate, applied as separate stimuli, increased (P < 0.05) glucose uptake in the absence of insulin. Yet when submaximal insulin concentrations were added to the perfusate, the stimulatory action of both contractions and increased blood flow on muscle glucose uptake was augmented. The higher the submaximal insulin concentration, the greater the increment (P < 0.05). This effect, however, faded at supra-maximal insulin concentration. Electrical stimulation associated with an increase in perfusion flow rate produced a greater (P < 0.05) rise in glucose uptake than did contractions alone. In fact, stimulation of muscle glucose uptake by contractions and increased flow proved to be additive at any insulin concentration. We conclude that contractions and increased blood flow act as additional stimuli to muscle glucose uptake at any insulin concentration. Furthermore, insulin appears to be a major contributor to stimulating glucose uptake rate in muscle during contractions by facilitating both the flow-induced and the contraction-induced increment in muscle glucose uptake.
Normal Effect of Insulin to Stimulate Leg Blood Flow in NIDDM
Authors: Dela Flemming , Larsen Jens J , Mikines Kári J , Galbo Henrik
Published: September 2013

In patients with non-insulin-dependent diabetes mellitus (NIDDM), a decreased effect of insulin in stimulating leg blood flow (LBF) has been reported. We reinvestigated the effect of insulin on LBF and validated our data by use of other measures. Eight healthy men (control group) and seven men with NIDDM were studied (age 59 ± 1 and 58 ± 3 years, weight 83 ± 3 and 86 ± 6 kg, fat-free mass 66 ± 1 and 64 ± 3 kg, respectively [mean ± SE, all P > 0.05]; body mass index 26 ± 1 and 29 ± 1 kg/m2, fasting plasma insulin 72 ± 7 and 187 ± 22 pmol/l, fasting plasma glucose 5.8 ± 0.2 and 10.2 ± 1.7 mmoM [all P < 0.05]). A three-step hyperinsulinemic glucose clamp (ambient glucose level) was performed, combined with catheterization of an artery and both femoral veins. Expiratory air was collected, LBF was measured by thermodilution, and blood was sampled and analyzed for oxygen content. Insulin concentration was increased to 416 ± 22 and 509 ± 43 (step I), 1,170 ± 79 and 1,299 ± 122 (step II), and 15,936 ± 1,126 and 16,524 ± 1,916 (step III) pmol/l in control and NIDDM subjects, respectively (P > 0.05). LBF increased similarly (P > 0.05) in the two groups (from 287 ± 23 and 302 ± 12 [basal] to 308 ± 31 and 362 ± 9 [I], 371 ± 29 and 409 ± 17 [II], and 434 ± 32 and 472 ± 29 [III] ml min−1 leg−1 in control and NIDDM subjects, respectively). Leg oxygen uptake always increased in the face of constant venous Po2 (JP > 0.05; 4.3 ± 0.2 and 4.5 ± 0.2 [basal], 4.3 ± 0.2 and 4.6 ± 0.2 [I], 4.8 ± 0.2 and 4.6 ± 0.2 [II], and 4.7 ± 0.1 and 4.4 ± 0.2 [III] kPa in control and NIDDM subjects, respectively). Both leg and whole body O2 uptake increased similarly in the two groups. In conclusion, at ambient glucose levels, the effect of insulin in stimulating LBF is normal in NIDDM. Moreover, insulin-mediated vasodilation is closely linked to muscle metabolic rate.
Different Roles of Class I and Class II Clostridium Histolyticum Collagenase in Rat Pancreatic Islet Isolation
Authors: Wolters Gerrit H J , Vos-Scheperkeuter Greetje H , Lin Hun-Chi , Schilfgaarde Reinout van
Published: September 2013

Crude Clostridium histolyticum collagenase was purified by gel filtration and fractionated by anion exchange chromatography into class I with high collagen digestion activity (CDA) and low FALGPA (2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine) hydrolysis activity (FHA), class II with low CDA and high FHA, and a fraction called class WI with intermediate activities. The roles of these collagenase classes in rat pancreatic islet isolation were investigated. Dissociations were carried out with 360 mg of pancreatic tissue in 10 ml of buffer containing 10% (wt/vol) albumin to suppress endogenous proteolytic activity, 100 U of C. histolyticum neutral protease, and one or two purified collagenase(s). For purified nonfraction-ated (PNF) collagenase, 2.6 mg of enzyme containing 2.4 U CDA and 38.0 U FHA was used, and for the separate classes, comparable amounts of activity were added. PNF collagenase dissociated the tissue completely in 32 min and yielded 5.0 ± 0.4 μl islet tissue/g pancreas. Class I collagenase alone dissociated pancreatic tissue extremely slowly and incompletely; only a few islets were released (0.7 ± 0.2 μl/g pancreas). Class II collagenase alone dissociated the tissue adequately in 50 min, and a high islet yield of 5.7 ± 0.6 μl/g was obtained. With class I/II, a similar dissociation time (47 min) and islet yield (5.5 ± 0.3 μl/g) were obtained. Combining class I and class II collagenase resulted in a more rapid dissociation (32 min) and a higher islet yield (7.1 ± 0.8 μl/g) than that obtained with PNF collagenase (P < 0.05). Similar results were obtained when purified class I collagenase was replaced by recombinant class I collagenase (32 min, islet yield 6.1 ± 0.3 μl/g). We conclude that class U collagenase plays a major role in pancreas dissociation and islet isolation and that class I collagenase acts synergistically with class II collagenase.
Trinucleotide Repeats at the rad Locus: Allele Distributions in NIDDM and Mapping to a 3-cM Region on Chromosome 16q
Authors: Doria Alessandro , Caldwell James S , Ji Linong , Reynet Christine , Rich Stephen S , Weremowicz Stanislawa , Morton Cynthia C , Warram James H , Kahn C Ronald , Krolewski Andrzej S
Published: September 2013

A 10-allele polymorphism was identified in rad (ras associated with diabetes), a gene that is overexpressed in non-insulin-dependent diabetes mellitus (NIDDM) muscle. The polymorphism, designated RADI, consists of a variable number of trinucleotide repeats (GTT and ATT) located in the poly(A) region of an intronic Alu sequence. Based on the number of GTT and ATT repetitions, the alleles can be grouped into four classes (I-TV). RADI allele frequencies were determined in 210 NIDDM patients and 133 nondiabetic control subjects, all Caucasians. One allele (number 8, class III) accounted for >80% of the chromosomes in both groups. However, an excess of minor alleles, all belonging to class I, II, or IV, was observed among NIDDM chromosomes (P < 0.025), suggesting a possible association between RADI and NIDDM predisposition. To promote further studies to test the hypothesis that genetic variability at the rad locus contributes to NIDDM, we mapped rad on the human genome. Using the fluorescence in situ chromosomal hybridization technique, rad was unequivocally assigned to chromosomal band 16q22. In families that were informative for RAD1, the rad locus was mapped within a 3-cM region defined by the markers D16S265, D16S186, and D16S397 (logarithm of odds scores = 10.08, 10.9, and 10.84 at recombination fractions of 0.024, 0.001, and 0.03, respectively). The high degree of heterozygosity of these markers will allow large-scale family studies to be performed to test the presence of linkage between rad and NIDDM.
Differential Effects of Human and Animal Insulin on the Responses to Hypoglycemia in Elderly Patients With NIDDM
Authors: Meneilly Graydon S , Milberg William P , Tuokko Holly
Published: September 2013

Recent studies suggest that insulin-dependent diabetes mellitus patients switched from animal to human insulin may have decreased awareness of hypoglycemic warning symptoms. The risk of severe or fatal hypoglycemia associated with the treatment of diabetes increases with age. We conducted these studies to determine if awareness of hypoglycemic warning symptoms was greater with animal than with human insulin in elderly patients with diabetes. Nonobese elderly patients with non-insulin-dependent diabetes mellitus (NIDDM) (n = 13; age, 74 ± 1 years; body mass index, 26.6 ± 0.7 kg/m2) underwent paired hyperinsulinemic glucose clamp studies (insulin infusion rate 60 mU ç m−2 ç min) in random order. In one study, regular human insulin was infused, and in the other study, regular beef/pork insulin was infused. In all studies, plasma glucose was decreased from fasting levels to 5 mmol/l during the first 60 min and was then allowed to fall to 4.4, 3.8, 3.3, and 2.8 mmol/l in each subsequent hour. Subjects were blinded as to which study they were undergoing. In each study, a hypoglycemic symptom checklist was administered, and counterregulatory hormones were measured every 15 min. Neuropsychological tests were performed every hour. Counterregulatory hormone responses to the two insulin preparations were similar. Autonomic (P < 0.05) and neuroglycopenic (P < 0.01) symptom scores were significantly higher during the beef/pork insulin studies. The responses on the neuropsychological tests were not significantly different. We conclude that beef/pork insulin results in greater awareness of hypoglycemic warning symptoms than does human insulin in elderly patients with NIDDM.
Slow [Ca2+]i Oscillations Induced by Ketoisocaproate in Single Mouse Pancreatic Islets
Authors: Martin Franz , Sanchez-Andres Juan Vicente , Soria Bernat
Published: September 2013

The effect of α-ketoisocaproate (KIC), the first catabolic metabolite of the amino acid leucine, on [Ca2+]i, insulin release, and membrane potential was measured in mouse pancreatic islets of Langerhans. Stimulatory concentrations of KIC (2.5–10 mmol/l) caused slow oscillations of [Ca2+]i and cyclic variations of the membrane potential. Slow [Ca2+]i oscillations depended on extracellular calcium. Simultaneous measurements of [Ca2+]i and insulin release resolved pulsatile insulin secretion that paralleled slow [Ca2+]i oscillations. Whereas 11 mmol/l glucose induced a significant increase in cAMP, KIC was unable to modify it. Glucagon (10 nmol/l), which significantly increased cAMP in mouse islets, also increased the frequency of glucose-induced fast [Ca2+]i oscillations. However, neither glucagon (10 nmol/l) nor dibutyryl cAMP (1 mmol/l) was able to change the slow oscillation pattern into a fast pattern. Imaging of Ca2+ showed that KIC-induced slow oscillations were synchronic throughout the whole islet. It is suggested that β-cell electrical activity plays a role in the origin of slow [Ca2+]i oscillations.
Nω-Nitro-L-Arginine Methyl Ester Reduces the Incidence of IDDM in BB/E Rats
Authors: Lindsay R Mark , Smith William , Rossiter Sharon P , McIntyre Margaret A , Williams Brent C , Baird Joyce D
Published: September 2013

Evidence that nitric oxide (NO) is involved in cytokine-mediated islet β-cell dysfunction and destruction in vitro has led to the hypothesis that increased production of NO may contribute to the pathogenesis of insulin-dependent diabetes mellitus (IDDM). This study demonstrates that oral administration of Nω-nitro-L-arginine methyl ester (an inhibitor of NO synthase) from 30 to 150 days of age significantly reduced (P < 0.05) the incidence of IDDM in diabetes-prone BB/E rats. This supports the idea that NO plays a significant role in the pathogenesis of IDDM in this animal model.
Glucose-Induced Changes in Turnover of Na+/H+ Exchanger of Immortalized Lymphoblasts From Type I Diabetic Patients With Nephropathy
Authors: Davies Joan E , Siczkowski Martin , Sweeney Frank P , Quinn Paulene A , Krolewski Bozena , Krolewski Andrzej S , Ng Leong L
Published: September 2013

Increased cellular Na+/H+ exchanger (NHE) activity has been demonstrated in type I diabetic patients with nephropathy. Such patients also have a previous history of poor glycemic control. The interaction between hyperglycemia and changes in NHE activity remains obscure. Therefore, we examined the effects of media containing 5 and 25 mmol/l glucose on the increased NHE activity and turnover number in Epstein-Barr virus-transformed lymphoblasts from patients with diabetic nephropathy compared with normoalbuminuric diabetic and nondiabetic control subjects. NHE activity was determined fluorometrically, and NHE isoform 1 (NHE-1) density was measured with specific polyclonal antibodies. In the presence of 5 mmol/l glucose, cells from patients with diabetic nephropathy exhibited higher NHE activity with intracellular pH clamped to 6.0 compared with diabetic and nondiabetic control subjects (P < 0.005 for both), due to a higher turnover number of NHE-1. Incubation in 25 mmol/l glucose for 48 h caused an increase in NHE activity (P < 0.001) and turnover number (P < 0.01) in the diabetic nephropathy group only, with no significant change in the diabetic or nondiabetic control groups. The rate constants for cell proliferation and NHE activity or turnover number were correlated when cells were cultured in 5 mmol/l glucose (r = 0.34 and 0.32, respectively; P < 0.05) or 25 mmol/l glucose media (r = 0.66 and 0.65, respectively; P < 0.001). We conclude that only lymphoblasts from the diabetic nephropathy group show an increase in NHE activity and turnover number under conditions mimicking hyperglycemia. Thus, high glucose levels exaggerate the differences in NHE activity, turnover number, and cell proliferation rate already present between cells from diabetic nephropathy patients and those from diabetic and nondiabetic control subjects.
Association of the Prohormone Convertase 2 Gene (PCSK2) on Chromosome 20 with NIDDM in Japanese Subjects
Authors: Yoshida Hitomi , Ohagi Shinya , Sanke Tokio , Furuta Hiroto , Furuta Machi , Nanjo Kishio
Published: September 2013

Proinsulin is converted to insulin by the concerted action of two sequence-specific subtilisin-like proteases termed prohormone convertase 2 (PC2) and prohormone convertase 3. PC2 is a type II proinsulin-processing enzyme, and it cleaves the proinsulin molecule on the COOH-terminal side of dibasic peptide, Lys64-Arg65, which joins the C-peptide and the A-chain domains. We have previously cloned and characterized the exon-intron organization of the human PC2 gene (gene symbol PCSK2), localized this gene to human chromosome 20 band p11.2 by fluorescence in situ hybridization, and identified a simple tandem-repeat DNA polymorphism (STRP) in intron 2 of the form (CA)n, suitable for genetic studies. Since non-insulin-dependent diabetes mellitus (NIDDM) is associated with increased secretion of proinsulin and proinsulin-like molecules, we conducted a case-control study to determine whether a genetic variation in PCSK2 might contribute to the development of NIDDM. The study population consisted of 152 Japanese NIDDM subjects and 102 normal healthy nondiabetic control subjects matched for age and body mass index. The subjects were genotyped at the STRP in intron 2, and the results indicated a significant difference (P = 0.004) in the overall allele frequency distribution between the two groups. The A1 allele was found more frequently in NIDDM than in nondiabetic subjects (11 vs. 4%, P = 0.0068). The NIDDM patients were divided into two subgroups according to the presence or absence of the A1 allele. There were no significant differences between these two subgroups with respect to age, sex, body mass index, family history of NIDDM, or current HbAlc, fasting plasma glucose, and serum proinsulin levels. The 12 exons of the PC2 gene of 60 NIDDM subjects were screened for mutations that might explain the observed association using the technique of single-strand conformational polymorphism analysis. A single variant was noted in exon 1. One NEDDM subject was heterozygous for a G→T substitution at 2 base pairs before the translational start site. This nucleotide substitution creates an in-frame methionine codon, which, if functional, would generate a protein with two methionine residues at its NH2-terminus rather than one. The functional consequences of such a change on the enzymatic properties of PC2 are unknown.
Two-Dimensional Gel Electrophoresis of Rat Islet Proteins: Interleukin 1β-Induced Changes in Protein Expression are Reduced by L-Arginine Depletion and Nicotinamide
Authors: Andersen Henrik U , Larsen Peter Mose , Fey Stephen J , Karlsen Allan E , Mandrup-Poulsen Thomas , Nerup Jørn
Published: September 2013

Interleukin (IL)-1 β-mediated damage to β-cells in isolated islets of Langerhans depends upon de novo synthesis of proteins that have not been fully identified. Further, IL-1 β-induced and tumor necrosis factor alpha-induced islet damage partly depends on the intracellular production of the nitric oxide (NO) radical. IL-1 β has also been reported to induce the synthesis of cellular defense proteins, e.g., heme-oxygenase and heat shock proteins 70 and 90. Nicotinamide, while in itself inactive, inhibited IL-1 β-induced NO production in a time- and dose-dependent manner. To enable the identification of IL-1 β-induced proteins with possible protective and deleterious effects, we characterized the effects of IL-1 β, nicotinamide, and NO synthesis inhibition by L-arginine depletion on rat islet protein expression detected by high-resolution two-dimensional gel electrophoresis. More than 1,600 proteins were reproducibly detected in control rat islets. Incubation with IL-1 β–, nicotinamide-, or L-arginine-depleted control medium upregulated 29, 3, and 1 protein, respectively, and downregulated 4, 0, and 1 protein, respectively. Addition of nicotinamide and L-arginine depletion reduced the upregulation of 16 and 20 IL-1 β-induced proteins, respectively. The identity of these proteins is under study. The demonstrated changes in protein expression caused by IL-1 β ± nicotinamide and L-arginine depletion may form the basis for identification of proteins with possible protective and deleterious roles in the initial β-cell destruction in insulin-dependent diabetes mellitus.
Prolongation of Near-Normoglycemic Remission in Black NIDDM Subjects With Chronic Low-Dose Sulfonylurea Treatment
Authors: Banerji Mary Ann , Chaiken Rochelle L , Lebovitz Harold E
Published: September 2013

Microvascular and neuropathic complications of diabetes mellitus can be significantly decreased by long-term, near-normoglycemic regulation in patients with insulin-dependent diabetes mellitus. Prevention or delay of onset of hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM) patients should reduce morbidity and mortality from these complications. NIDDM can be nearly normoglycemic when diagnosed by screening before its symptomatic stage or when clinically hyperglycemic NIDDM goes into remission. One potential strategy to delay the onset of hyperglycemia in individuals at high risk is chronic low-dose sulfonylurea therapy. Thirty black NIDDM subjects who recently had developed near-normoglycemia were followed with no treatment or were randomly assigned to a 3-year, double-blind glipizide or placebo treatment. Baseline and follow-up parameters included fasting plasma glucose (FPG), HbA1c, plasma insulin, and glucose responses to an oral glucose tolerance test and insulin action, as determined by the euglycemic insulin clamp. Baseline FPG and HbA1c for all three groups were 107 mg/dl and 4.7%, respectively. Relapse to hyperglycemia was defined as an FPG level ≥ 140 mg/dl on several consecutive visits or an FPG level ≥ 140 mg/dl and symptoms of hyperglycemia. During the course of the treatment and follow-up, hyperglycemia occurred in 6 of 10 subjects in the no treatment group, 6 of 10 in the placebo group, and 2 of 10 in the glipizide treatment group. Prolongation of near-normoglycemia was significantly (P < 0.05) increased by low-dose (2.5 mg/day) glipizide compared with placebo treatment. Low-dose sulfonylurea therapy delays the onset of hyperglycemia in NIDDM subjects in remission and may be a useful method to delay the onset of NIDDM in high-risk individuals.
Altered Glucose Dependence of Glucagon-Like Peptide I(7–36)–Induced Insulin Secretion from the Zucker (fa/fa) Rat Pancreas
Authors: Jia Xiaoyan , Elliott Ruan , Kwok Yin Nam , Pederson Raymond A , McIntosh Christopher H S
Published: September 2013

In previous studies on the enteroinsular axis in Zucker rats, it was found that glucose-dependent insulinotropic polypeptide (GIP) levels were normal in obese animals, but the glucose threshold for the insulinotropic action of GIP in the perfused rat pancreas was reduced. Glucagonlike peptide I (GLP-I)(7–36) is also an important incretin, and in the current study, glucose, insulin, and immunoreactive (IR)–COOH-terminal GLP-I responses to oral glucose were compared in lean (Fa/?) and obese (fa/fa) rats. In addition, the concentration thresholds for stimulation and glucose dependence of perfused pancreases to GLP-I(7–36) were examined. Glucose responses to oral glucose were similar in fa/fa and Fa/? rats. Obese animals were hyperinsulinemic when fasting and after oral glucose. Significant increases in IR–GLP-I levels in response to glucose were only observed in fa/fa rats. Perfused pancreases from fa/fa rats hypersecreted insulin at all glucose concentrations. In the presence of 4.4 mmol/l glucose, GLP-I(7–36) increased insulin secretion in fa/fa pancreases ∼25-fold, whereas there was only a 5-fold increase in Fa/? Pancreases. Pancreases from fa/fa rats, perfused with a glucose gradient (2.8–11 mmol/l) in the presence of GLP-I(7–36), responded with an immediate increase in insulin secretion, i.e., at a glucose concentration of 2.8 mmol/l, whereas Fa/? pancreases required a minimum of 4.22 mmol/l glucose for stimulation. With high glucose (16.7 mmol/l), both fa/fa and Fa/? rat pancreases exhibited similar responsiveness to GLP-I(7–36), having thresholds of <50 pmol/l. In conclusion, in adult obese Zucker rats, GLP-I(7–36) may contribute to the fasting hyperinsulinemia seen in vivo, and there appears to be a general reduction in the glucose dependence of responses to incretins.
Decreased Interstitial Apolipoprotein A-I Levels in IDDM Patients with Diabetic Nephropathy
Authors: Tamsma Jouke T , Beverdam Frederique H , Leuven Jan A. Gevers , Lemkes Herman H P J
Published: September 2013

The risk of cardiovascular morbidity and mortality is highly increased in patients with diabetic nephropathy. Postulating that the generalized vasculopathy observed in these patients may enhance transcapillary filtration of lipids and lipoproteins resulting in a more atherogenic interstitial lipid profile, we set out to analyze the composition of their interstitial fluid. We studied healthy control subjects (n = 9), normoalbuminuric insulin-dependent diabetes mellitus (IDDM) patients (n = 16), and IDDM patients with diabetic nephropathy (n = 11) matched for age, body mass index, smoking habits, duration of diabetes, and metabolic control. Interstitial fluid was collected after an overnight fast by applying mild suction (200 mmHg) to the skin. Interstitial apolipoprotein A-I (apoA-I) levels were significantly lower in patients with nephropathy (0.18 ± 0.10 g/l [mean ± SD]) compared with normoalbuminuric diabetic patients (0.29 ± 0.08 g/l) and healthy control subjects (0.30 ± 0.09 g/l). Interstitial apolipoprotein B:apoA-I ratios tended to be higher in patients with diabetic nephropathy. In these patients, normal interstitial low-density lipoprotein cholesterol concentrations were observed in the presence of lower apoA-I levels. Transcapillary filtration of apoA-I was significantly lower in patients with diabetic nephropathy. Furthermore, an altered multiple regression model explaining interstitial apoA-I levels was observed in diabetic nephropathy. In this model, transcapillary protein (IgG) filtration and serum apoA-I levels no longer explained interstitial apoA-I levels. If we assume that interstitial apoA-I is involved in reverse cholesterol transport, these data suggest a more atherogenic interstitial lipoprotein profile in IDDM patients with nephropathy.
Levels of Tap-1 and Tap-2 mRNA and Expression of Kd and Db on Splenic Lymphocytes Are Normal in NOD Mice
Authors: Pearce Richard B , Trigler Lucas , Svaasand Eva K , Chen Hui-Min , Peterson Charles M
Published: September 2013

It has been reported that the level of Tap-1 (transporter associated with antigen processing) mRNA and the expression of class I on splenocytes are low in NOD mice. Class I expression at 37°C depends on an adequate supply of peptides, so a decrease in Tap could lead to lower class I levels. Since hypoexpression of class I correlated uniformly with the development of diabetes, it has also been suggested that Tap-1nod is diabetogenic. However, others report normal Tap-1 and class I levels in NOD mice. We examined Tap-1 and Tap-2 mRNA levels in NOD/Smrf mice using a reverse transcriptase-polymerase chain reaction method that detects ≥25% changes in mRNA. We also assessed class I expression with three monoclonal antibodies. No difference in Tap-1 or Tap-2 mRNA levels for females of different ages or between diabetic and nondiabetic animals was observed. Tap-1 mRNA levels were identical between NOD/Smrf and BALB/cJ mice. Kd expression was significantly lower on NOD lymphocytes than in BALB/cJ cells, but the difference was due to the smaller size of the NOD splenic lymphocyte. When cells of the same size were analyzed, no difference in class I levels was observed. Class I levels were also identical in diabetic and age-matched nondiabetic NOD and BALB/c females. Both NOD Tap-1 mRNA and class I were increased by interferon-γ. We find no evidence for impaired NOD Tap gene activity or class I expression, as previously reported for this strain.
Overexpression of Apolipoprotein E Prevents Development of Diabetic Hyperlipidemia in Transgenic Mice
Authors: Yamamoto Koji , Shimano Hitoshi , Shimada Masako , Kawamura Masako , Gotoda Takanari , Harada Kenji , Ohsuga Jun-ichi , Yazaki Yoshi , Yamada Nobuhiro
Published: September 2013

To determine the role of apolipoprotein E (apoE) in diabetic hyperlipidemia, we induced diabetes in transgenic mice overexpressing apoE by intravenous injection of streptozotocin (STZ) and examined plasma lipoprotein metabolism in these mice. In STZ-induced diabetic mice, blood glucose levels were > 19 mmol/l (350 mg/dl) and plasma insulin levels were reduced to = 5 pmol/l (1 μU/ml). The diabetic nontransgenic mice developed hypercholesterolemia (plasma total cholesterol level: 4.55 ± 1.32 vs. 1.97 ± 0.13 mmol/l [176 ± 51 vs. 76 ± 5 mg/dl]) and hypertriglyceridemia (plasma triglyceride level: 0.82 ± 0.29 vx. 0.42 ± 0.11 mmol/l [73 ± 26 vs. 37 ± 10 mg/dl]) compared with values before induction of diabetes. In the diabetic nontransgenic mice, enhanced intestinal acylCoA:cholesterol acyltransferase activity was demonstrated, a factor that may contribute to the development of diabetic hyperlipidemia. Induction of apoE remarkably reduced the development of hyperlipidemia in diabetic transgenic mice compared with diabetic nontransgenic mice (plasma cholesterol level: 4.55 ± 1.32 vs. 3.31 ± 0.47 mmol/l [176 ± 51 vs. 128 ± 18 mg/dl], P < 0.01, and plasma triglyceride level: 0.82 ± 0.29 vs. 0.17 ± 0.11 mmol/l [73 ± 26 vs. 15 ± 10 mg/dl], P < 0.01). Plasma lipoprotein analysis by gel filtration chromatography showed that the reduction of plasma cholesterol and triglyceride levels was due to the disappearance of lipoproteins containing apoB. In these studies, we demonstrated the usefulness of STZ-induced diabetes in mice as an animal model for diabetic hyperlipidemia and demonstrated that endogenous induction of apoE in transgenic mice improved diabetic hyperlipidemia.
Genetic Linkage of Thymic T-Cell Proliferative Unresponsiveness to Mouse Chromosome 11 in NOD Mice: A Possible Role for Chemokine Genes
Authors: Gill Bruce M , Jaramillo Andrés , Ma Lingli , Laupland Kevin B , Delovitch Terry L
Published: September 2013

Thymic and peripheral T-cells from NOD mice display a proliferative unresponsiveness on stimulation through the T-cell receptor/CD3 complex. Interleukin 4 reverses NOD T-cell unresponsiveness in vitro and prevents the onset of diabetes in vivo, suggesting a causal relationship between the T-cell unresponsiveness and diabetes susceptibility in NOD mice. Both quantitative trait loci analysis of BXD recombinant inbred mice and linkage analysis of NOD outcross populations reveal that the control of NOD thymic T-cell proliferative unresponsiveness genetically maps to a central region on mouse chromosome 11, which includes the β-chemokine gene family. This finding raises the possibility that a β-chemokine(s) may regulate T-cell unresponsiveness as well as diabetes susceptibility in NOD mice.