bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Age on Responsiveness to Diabetogenic Action of Growth Hormone
Authors: Cameron Christopher M , Kostyo Jack L
Published: September 2013

Responsiveness to the growth-promoting action of growth hormone (GH) develops gradually during postnatal life, and as the organism ages, sensitivity to the hormone declines. Our study was undertaken to determine whether there is a similar age-related pattern of sensitivity to the diabetogenic action of purified native human GH (hGH) in the obese (ob/ob) mouse. The ob/ob mouse was used because adults of this strain respond to chronic GH treatment with increases in fasting plasma insulin and blood glucose concentrations and with glucose intolerance. When hGH was given subcutaneously to adult (4-mo-old) female mice at doses of 5, 10, or 25 μg/day for 3 days, a significant increase in fasting blood glucose concentration and animpairment in glucose tolerance were produced by the 10-μg/day dose. Larger effects were obtained with the 25-μg/day dose. A threefold increase in fasting plasma insulin concentration was also produced with this dose of the hormone. By contrast, 25 μg/day of hGH had no effect on fasting plasma insulin, blood glucose, or glucose tolerance in 1-mo-old ob/ob mice. When a dose of 100 μg/day of hGH was given to 1-mo-old animals, a significant increase in fasting plasma insulin concentration occurred, but again there were no effects on fasting blood glucose or glucose tolerance. Mice 1.5 mo old showed marginal changes in fasting blood glucose and glucose tolerance when given hGH at doses of 25 or 100 μg/day. Mice 9 or 12 mo old exhibited responsiveness similar to that of 4-mo-old animals. Thus, responsiveness to the diabetogenic action of GH develops gradually in the ob/ob mouse during postnatal growth. However, there does not appear to be a significant decline in sensitivity to the diabetogenic action of GH as the adult animal ages.
Spontaneously Diabetic BB Rats Have Age-Dependent Islet β-Cell—Specific Surface Antibodies at Clinical Onset
Authors: Pipeleers Daniel , Van De Winkel Marnix , Dyrberg Thomas , Lernmark Ånke
Published: September 2013

Diabetes-prone BB rats were examined before, during, and after clinical onset of diabetes for the occurrence of circulating islet cell surface antibodies (ICSAs) with a specific binding to islet β-cells. The presence of ICSA was assessed by incubating serum immunoglobulin fractions with normal Wistar rat islet cells and identifying cell-bound immunoglobulins by indirect immunofluorescence and by a complementinduced cell toxicity reaction. Under the selected experimental conditions, none of the diabetes-resistant rats were ICSA positive over the entire study period (45–120 days of age). In two diabetes-prone BB rat strains, 19 animals developed diabetes with onset between 50 and 120 days. At day 45, none of these animals was positive for ICSA. In rats developing diabetes between 50 and 85 days of age, 7 of 9 animals presented ICSA at clinical onset, determined by either the immunofluorescence or cytotoxicity test. The antibodies bound to insulin-containing β-cells but not to other islet cell types, and binding was not eliminated by absorption with liver powder. In animals developing diabetes between 85 and 120 days, only 1 of 10 was positive for β-cell-specific surface antibodies at onset of the disease. After 30 days of insulin treatment, β-cell-specific antibodies were detectable in 3 of 4 animals of age 50-85 days, whereas only 3 of 12 older rats presented ICSAs that were, in addition, of low titer or affinity. Our data confirm that ICSAs develop in diabetic BB rats and indicate that these antibodies can bind specifically to islet 7bgr;-cells compared with other islet cell types. Our study also suggests that the occurrence of circulating β-cell-specific surface antibodies in diabetes-prone BB rats is not only related to the time of clinical onset of the disease but also to the age of the animals. Our study illustrates the advantage of using purified normal islet cells in the analysis of the autoimmune phenomena in insulin-dependent diabetes.
Nutrition and Somatomedin: XVII. Circulating Somatomedin C During Treatment of Diabetic Ketoacidosis
Authors: Glaser Elyse W , Goldstein Steven , Phillips Lawrence S
Published: September 2013

Although somatomedin levels may be low in animals with experimental diabetes, values in humans have generally been normal. Because humans with severely decompensated diabetes have not been studied, we characterized somatomedin C responses during metabolic restoration in 21 patients with diabetic ketoacidosis. Somatomedin C values were compared with those of 25 outpatient controls. Somatomedin C in controls (mean ± SE) was 0.72 ± 0.09 U/ml, 69% of the mean sex-adjusted normal level; 28% of patients had values below the lower limit of normal. In ketoacidotic subjects, initial somatomedin C was 0.43 ± .06 U/ml, 33% of the mean normal level; 52% of subjects had somatomedin C below the lower limit of normal. Initial levels in ketoacidotic subjects were unrelated to presenting levels of glucose, bicarbonate, ketones, or blood urea nitrogen but were significantly lower in patients of less than ideal body weight (0.30 vs. 0.58 U/ml, P < .03). Presenting levels of somatomedin C in ketoacidotic subjects were significantly lower than in controls (P <.02). During insulin-infusion therapy, somatomedin C rose to a peak of 1.16 ± 0.21 U/ml after 28 h, significantlyhigher than initial levels (P <.05). With continued subcutaneous insulin, somatomedin C fell to a nadir after an additional 22 h then rose more slowly to a final value of 0.75 ± 0.12 U/ml, significantly higher than the nadir (P <.05) but lower than peak values; final values in the ketoacidotic subjects were comparable to outpatient somatomedin C levels. Although final somatomedin C exhibited no apparent relationship to percent ideal body weight or final metabolic indices, values were lower in patients who had higher initial glucose (r = .43, P <.05). During the rapid rise of somatomedin C from initial to peak levels, somatomedin C values were correlated with levels of both bicarbonate (r = .37, P <.001) and ketones (r = −.31 , P <.005). Altered somatomedin C could not be attributed to changes in serum somatomedinbinding activity. Although improvement in somatomedin C levels over several days may be due to gradual intracellular normalization in response to subcutaneous insulin, the acute rise during the 1st day of intravenous therapy probably reflects rapid anabolic flux conferred by high levels of insulin and metabolic fuels. These findings establish a strong relationship between levels of somatomedin C and metabolic status in diabetic humans.
Subunit Structure, Autophosphorylation, and Tyrosine-Specific Protein Kinase Activity of Hepatic Insulin Receptors in Fetal, Neonatal, and Adult Rats
Authors: Sinha Madhur K , Jenquin Michelle
Published: September 2013

The ontogeny of the structural and functional characteristics of insulin receptors is determined by examining insulin binding, subunit structure, autophosphorylation, and tyrosine-specific protein kinase activity in partially purified solubilized liver receptors from fetal (∼21 days postconception), neonatal (1- and 7-day-old), and adult rats. Specific 125l-labeled insulin binding to these receptor preparations in the presence of different insulin concentrations was higher in fetal and neonatal rats compared with that in the adult rats. The electrophoretic mobilities of the α- and β-subunits on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography were similar at different stages of development. Insulin-stimulated autophosphorylation of insulin receptors was similar in the different groups. With fixed amounts of protein, the tyrosine-specific protein kinase activity in the presence of different insulin concentrations (1 × 10−8 to 1 × 10−6 M) was significantly higher in the fetal and neonatal rats than in adult rats. However, when expressed as a function of insulin-binding activity, the insulin-stimulated tyrosine-specific protein kinase activity in fetal and neonatal rats appears to be similar to that in adult rats because of decreased insulin binding in the latter group. These results demonstrate the structural and functional similarities of hepatic insulin receptors in fetal, neonatal, and adult rats. The relative differences in insulin-mediated biological functions in fetal and adult rat livers as reported previously are due to alterations ina step(s) distal to activation of insulin-receptor kinase.
Calculated Pattern of Intraportal Insulin Appearance Without Independent Assessment of C-Peptide Kinetics
Authors: VøLund Aage , Polonsky Kenneth S , Bergman Richard N
Published: September 2013

Prehepatic (β-cell insulin release can be calculated with C-peptide measurements, but this requires independent determination of kinetics of C-peptide disappearance from plasma. We introduce an approach by which a prehepatic insulin release pattern is calculated from plasma insulin and C-peptide, without separate C-peptide kihetic analysis. Human insulin and C-peptide were infused intraportally into conscious dogs (n = 11) at equimolar rates; endogenous insulin and C-peptide release were suppressed with somatostatin (0.8 μg · kg−1 · min−1). Insulin and C-peptide were infused at basal and equimolar rates (range 19–72 pmol/min in dogs), and the infusions were slowly increased, in stepwise fashion, to a maximum at 60 min (range 152—613 pmol/min) and subsequently renormalized at either 85 (n = 6) or 195 (n = 5) min. Plasma insulin and C-peptide measurements were described simultaneously by a composite model of insulin and C-peptide plasma kinetics, with the molar intraportal appearance rate due to the infusion R(t) as an unknown input for both insulin and C-peptide catabolism. The model assumes one-compartment disappearance kinetics for both peptides. Fitting the model to the measured insulin and C-peptide data, we were able to compute the insulin-appearance pattern accurately for every experiment; calculated and actual secretion rates were highly correlated (r = .93−.97) and had very similar temporal patterns. Also calculated were the fractional disappearance rates for human insulin (t1/2 = 6.9 min) and C-peptide (t1/2 = 14 min) in the dog, as well as the C-peptide distribution volume (12.3 ±0.5% body wt). Islet cell insulin secretion can be determined in vivo from insulin and C-peptide measurement without separate analysis of C-peptide kinetics. This method will be applicable for quantification of β-cell secretion for diagnostic and experimental purposes.
Effect of Hypocholesterolemia on Cholesterol Synthesis in Small Intestine of Diabetic Rats
Authors: Feingold Kenneth R , Moser Arthur H
Published: September 2013

Studies by our and other laboratories have demonstrated that cholesterol synthesis is increased in the small intestine of insulinopenic diabetic animals. In normal animals, many factors have been shown to regulate cholesterol synthesis in the small intestine, including changes in plasma cholesterol levels. The purpose of this study was to determine the effect of lowering plasma cholesterol levels on small intestine cholesterol synthesis in streptozocin-induced diabetic rats. In diabetic rats, 4-aminopyrazolo[3,4-d]pyrimidine (4-APP)-induced hypocholesterolemia (plasma cholesterol levels 20 mg/dl) resulted in a 2.5-fold increase in small intestine cholesterol synthesis, which was most marked in the distal small intestine, decreasing proximally. In the distal small intestine the incorporation of 3H2O into cholesterol was 0.28 ± 0.04 μmol · h−1 · g−1 in diabetic rats versus 1.60 ± 0.38 in diabetic rats administered 4-APP (P < .01). This stimulation of cholesterol synthesis occurred in the upper villus, middle villus, and crypt cells isolated from the middle intestine of the 4-APP-treated diabetic animals. In agreement with these observations, “functional hypocholesterolemia” due to Triton WR-1339 administration also stimulated cholesterol synthesis 2.5-fold in the small intestine of normal and diabetic animals. In the distal small intestine, cholesterol synthesis was 0.43 ± 0.10 μmol · h−1 · g−1 in the diabetic rats versus 1.08 ± 0.21 in diabetic rats treated with Triton WR-1339 (P < .05). In both the 4-APP and Triton WR-1339 experiments, the response of the diabetic rats was similar to that observed in normal rats. Moreover, cholesterol administered orally could prevent the Triton WR-1339-induced increase in small intestine cholesterol synthesis, indicating that providing cholesterol via an alternate route (i.e., intraluminal) can block the increase in synthesis. The results reported in this article, together with our earlier observations demonstrating that luminal factors regulate cholesterol synthesis, indicate that small intestine cholesterol synthesis in diabetic animals is regulated by both luminal and circulating factors in the expected fashion.
Permeability Changes in Blood-Retinal Barrier of Galactosemic Rats Are Prevented by Aldose Reductase Inhibitors
Authors: Lightman Susan , Rechthand Emanuel , Terubayashi Hirofumi , Palestine Alan , Rapoport Stanley , Kador Peter
Published: September 2013

Permeability-surface-area products (PA) for sucrose at the blood-retinal barrier (BRB) were determined with quantitative in vivo techniques and compared in control rats, rats fed a 50% galactosemic diet, and rats fed a diet containing both galactose and the aldose reductase inhibitor sorbinil. The mean PA ± SE for controls was 0.656 × 10−5 ± 0.13 ml · g1 · s1 and increased by ∼500% in galactose-fed animals to 3.13 × 10−8 ± 0.32 ml · g1 · s1 . Animals fed both galactose and sorbinil showed no significant difference from control animals (P > .05), with a PA of 0.91 × 10−5 ± 0.22 ml · g1 · s1. No breach in the BRB to horseradish peroxidase was detected in any of the groups. These results demonstrate an increased permeability at the BRB to small molecules in galactosemic rats that is prevented by an aldose reductase inhibitor. This suggests that the retinal capillary basement membrane thickening seen in galactosemic rats is associated with an increased permeability of the BRB and that aldose reductase is implicated in its pathogenesis.
Competitive Insulin Autoantibody Assay: Prospective Evaluation of Subjects at High Risk for Development of Type I Diabetes Mellitus
Authors: Vardi Pnina , Dib Sergio A , Tuttleman Marilyn , Connelly Jeanne E , Grinbergs Marta , Radizabeh Albert , Riley William J , Maclaren Noel K , Eisenbarth George S , Soeldner J Stuart
Published: September 2013

A quantitative fluid-phase radioassay for autoantibodies reacting with insulin (competitive insulin autoantibody assay, CIAA) was developed. The assay's features include 1) use of a physiologic amount of 125I-labeled insulin, 2) parallel incubations with supraphysiologic cold insulin (competitive), and 3) an incubation time of 7 days and a single-step multiple-wash polyethylene glycol separation. Mean ± SE CIAA levels in 50 controls were 8 ± 1.4 nU/ml (range −16–33.3). In 36 cytoplasmic islet cell antibody (ICA)-positive nondiabetic first-degree relatives of type I (insulin-dependent) patients <30 yr of age, CIAA levels exceeded the normal range in 20 (55.6%) of 36 (mean 86.8 ± 17.1 nU/ml). In 26 ICA-positive relatives >30 yr of age, only 5 (19.2%) of 26 exceeded the normal range (mean 26.1 ± 9.4 nU/ml); P < .001 compared with younger ICA-positive relatives). Six ICA-negative HLA-identical siblings of type I diabetic patients had normal CIAA levels (mean 3.6 ± 5.8 nU/ml), and only 2 of 13 ICA-negative identical twins discordant for diabetes (mean 15.4 ± 6.6 nU/ml) exceeded the normal range. Nine (50%) of 18 ICApositive schoolchildren exceeded the normal range (mean 105.3 ± 36.7 nU/ml). Genetically susceptible subjects negative for CIAA (with only 3 exceptions) remained negative for CIAA on multiple determinations (3 conversions observed), and CIAA levels of positive subjects were relatively stable. Linear regression of the first CIAA level versus last (interval between sampling 1 mo to 10 yr) in genetically susceptible individuals showed a highly significant correlation (r = .95, P < .001). This simple radioimmunoassay appears to have requisite specificity and sensitivity to allow screening populations at risk to develop type I diabetes.
Increased Frequency of Apolipoprotein ∈4 Allele in Type II Diabetes With Hypercholesterolemia
Authors: Eto Masaaki , Watanabe Kiyoshi , Iwashima Yasunori , Morikawa Akizuki , Chonan Norihide , Oshima Eiji , Sekiguchi Masatomo , Ishii Kaneo
Published: September 2013

The apolipoprotein E (apoE) phenotype and allele frequencies were examined in type II (non-insulindependent) diabetic patients with normolipidemia (n = 134) and hypercholesterolemia (type IIa hyperlipoproteinemia, n = 35; type lib hyperlipoproteinemia, n = 42). The frequencies of apoE4-present phenotypes (apoE4/3, apoE4/4, and apoE4/2) were highest in the type IIa group (51.4%), followed by the type Mb group (38.1%) and the normolipidemic group (16.4%), respectively, whereas the frequency of the most common phenotype, apoE3/3, was lowest in the type IIa group (48.6%), followed by the type lib group (61.9%) and the normolipidemic group (79.9%), respectively. There were significant differences in the apoE phenotype frequencies between the normolipidemic group and the type IIa and Mb groups. The frequency of the ∈4 allele was significantly higher in the type IIa (28.6%) and Mb (20.2%) groups than in the normolipidemic group (8.9%), whereas the frequency of the ∈3 allele was significantly lower in the type IIa (71.4%) and Mb (78.6%) groups than in the normolipidemic group (89.2%). The frequency of the ∈2 allele tended to be lower in diabetic patients with hypercholesterolemia. In addition, these frequencies were also examined in nondiabetic subjects (n = 59). The frequency of the ∈4 allele tended to be higher in hypercholesterolemic diabetic subjects (24.1%) than in hypercholesterolemicnondiabetic subjects (15.3%). These data suggest that diabetic patients with the ∈4 allele may be more susceptible to hypercholesterolemia than diabetic patients without the ∈4 allele and possibly nondiabetic subjects with the ∈4 allele, although the underlying mechanism is unknown.
Cholecystokinin-lnduced Alterations in β-Cell Sensitivity: Duration, Specificity, and Involvement of Phosphoinositide Metabolism
Authors: Zawalich Walter S , Diaz Victoria A , Zawalich Kathleen C
Published: September 2013

Prior exposure of isolated per if used rat islets to the sulfated gut hormone cholecystokinin-8 (CCK-8S) dramatically increased their insulin secretory response to 7.5 mM glucose, 10 mM arginine, and 10 mM α-ketoisocaproate. In the case of glucose, the heightened secretory response was still apparent 60–80 min after CCK-8S removal from the perifusion medium. Prior exposure of perifused islets to arginine (10 mM), tolbutamide (25 μM), or forskolin (1.0 μM) did not sensitize them to 7.5 mM glucose. CCK-8S exposure increased 3H efflux from islets prelabeled with [3H]inositol, and the increase in 3H efflux was sustained after CCK-8S removal from the perifusion medium. The duration of this increase in 3H efflux paralleled the temporal characteristics of this sensitization process and was significantly attenuated by 25 μM asperlicin, a competitive antagonist of CCK binding to its membrane receptor. Arginine, tolbutamide, or forskolin treatment of islets did not increase 3H efflux from [3H]inositol-prelabeled islets. The results suggest that the turnover of membrane phosphoinositides induced by CCK-8S is largely responsible for this heightened state of secretory responsiveness to various stimulants. Secondmessenger molecules generated during phosphoinositide turnover may be responsible for the phenomenon of sensitization displayed by islet tissue to CCK-8S addition.
64,000-Mr Autoantigen in Type I Diabetes: Evidence Against Its Surface Location on Human Islets
Authors: Colman Peter G , Campbell Iain L , Kay Thomas W H , Harrison Leonard C
Published: September 2013

The sera of type I (insulin-dependent) diabetic subjects are reported to contain autoantibodies against a 64,000-Mr protein identified in [35S]methionine biosynthetically labeled pancreatic islet cells. We have attempted to localize this autoantigen to the surface of the β-cell and to define its properties. Sera from 10 newly diagnosed type I diabetic subjects, including five of the index sera originally used to identify the autoantigen, were shown to specifically precipitate a reduced protein of 67,000 Mr from Triton-solubilized, surface 125I-labeled cultured adult human islet and rat insulinoma (RINm5F) cells but not from fresh rat spleen cells. Further characterization revealed that this protein was bovine serum albumin (BSA) adsorbed to cells from fetal calf serum (FCS)-supplemented culture medium and precipitated by BSA antibodies present in many diabetic sera. No labeled proteins were specifically precipitated when surface 125I-labeled and solubilized human islet or RINm5F cells were precleared with anti-BSA immunoglobulins or when cells were first cultured in human serum. In contrast, a 64,000-Mr protein, clearly not BSA, was precipitated by diabetic globulins from human islets but not from RINm5F cells labeled with [35]Slmethionine. In addition, a protein of the same size as well as proteins of ∼35,000, 43,000, 140,000, and 200,000 Mr were specifically precipitated by diabetic globulins from freshly isolated human islets solubilized in Triton X- 100 and then labeled with 125I. These findings suggest that the 64,000-Mr antigen is not expressed on the surface of human islet cells, at least in culture, and therefore question its relevance as a target for islet cell surface antibodies in initiating β-cell damage. They also reveal the existence of other possible protein antigens in islet cells. Finally, our results indicate that assays for islet cell surface antibodies via immunofluorescence on cultured cells should be controlled for the presence of BSA antibodies.
Insulinotropic Effects of Vanadate
Authors: Fagin James A , Ikejiri Kenji , Levin Seymour R
Published: September 2013

Vanadium compounds are known to affect multiple membrane and cytosolic phosphoenzymes from various tissues; the most characterized effect is the inhibition of Na+-K+-ATPase. Since we previously reported that immunoreactive insulin (IRI) secretagogues tend to inhibit rat islet cationdependent ATPases, we examined the effects of sodium vanadate on rat IRI secretion from incubated and perifused rat islets. In the presence of 2.4 mM Ca2+, vanadate (10−3 M) induced biphasic IRI secretion with a background glucose of 100 mg/dl. In the absence of extracellular Ca2+, IRI released from incubated islets by vanadate at 100 and 300 mg/dl glucose was doubled and tripled, respectively. Furthermore, this stimulatory effect was completely abolished by known inhibitors of IRI release such as somatostatin, epinephrine, and diphenylhydantoin. Although we found the expected dose-dependent inhibition by vanadate of islet membrane Na+-K+- ATPase activity, the mechanism of action of vanadate on IRI secretion remains unknown. Vanadate probably interacts in a complex fashion with different islet phosphoenzymes and may prove to be a useful probe to further unravel the mechanisms leading to insulin secretion.
Regulation of Vascular Permeability in Cell Culture
Authors: King George L , Berman Audrey B , Bonner-Weir Susan , Carson Mary Pat
Published: September 2013

Although one of the earliest findings of diabetic retinopathy is altered capillary permeability, metabolic factors in diabetes that may increase the permeability of capillaries to fluorescein are unknown. We have studied the effect of a variety of vascular and retinal cells and hyperglycemia on the diffusion rate of fluorescein. These studies were performed with a cell culture system that mimics the cross-section of a capillary by having two chambers separated with a porous membrane that can support the growth of cells on either side of the membrane. The addition of a confluent layer of endothelial cells or retinal pigmented epithelial (RPE) cells inhibited fluorescein diffusion between the two chambers 20- and 300-fold, respectively, after cells were cultured for >5 days. Exposure of endothelial cells to 400 mg/dl glucose for either 3 or 100 days did not affect the barrier function of these cells. The barrier function of capillary endothelial cells isolated from BB rats with chronic diabetes and from nondiabetic animals did not differ. In contrast to endothelial cells and RPE cells, arterial smooth muscle and pericytes, which are not known to form tight junctions, did not inhibit the diffusion of fluorescein more than 2-fold. Surprisingly, the dual culture of endothelial cells with either retinal pericytes or smooth muscle cells resulted in a 50-fold increase in the rate of fluorescein diffusion, showing a disruption of the endothelial barrier. In summary, the intercellular connections between endothelial and epithelial cells that are responsible for the barrier to fluorescein diffusion are not functionally affected by chronic exposure to hyperglycemia or diabetic conditions. The interaction between endothelial cells with capillary pericytes is important in maintaining vascular permeability and may be altered in the diabetic state.
Glyburide Modulates Insulin-Mediated Locomotor Response of Confluent Cell Cultures to Wounding
Authors: Mascardo Renato N , Sherline Peter
Published: September 2013

We determined whether the sulfonylurea glyburide could modify the insulin-induced locomotor response to wounding of bovine aortic, pulmonary artery endothelial, and gerbil fibroma cells. Serum-deprived cells were preincubated in medium containing glyburide (10−4 M) or diluent, then exposed to insulin (10−6 to 10−12 M) at the time of wounding, after which the centrosomal-orientation and migratory responses of the cells bordering the experimental wound were monitored by immunofluorescence microscopy and time-lapse cinemicrophotography, respectively. Insulin (10−6 M) stimulated the centrosomal-orientation and locomotor responses, resulting in the narrowing of the linear wound. Glyburide enhanced the effect of insulin on both parameters of the locomotor response and also sensitized the cells to a previously ineffective concentration of insulin (10−12 M). The action of glyburide depended on a preincubation period of ≥60 min and was blocked by cycloheximide and actinomycin D. The relevance of this novel action of glyburide to the impaired healing process observed in diabetic patients requires further study.
Free Covalent Aggregates of Therapeutic Insulin in Blood of Insulin-Dependent Diabetics
Authors: Robbins David C , Mead Patricia M
Published: September 2013

Immunoreactive insulin (IRI) in the circulation of diabetics using insulin includes a 12,000-Mr covalent aggregate of insulin. In this study, both free and bound IRI were measured in nine type I diabetics who were treated sequentially for 3 wk with constant doses of conventional beef-pork, biosynthetic human, and beefpork insulins (phases 1, 2, and 3, respectively).

The aggregate accounted for 19 ± 5.7, 38 ± 7.7, and 26 ± 7% of bound IRI, and 19 ± 8.3, 35 ± 7.3, and 31 ± 5.0% of free IRI during phases 1, 2, and 3, respectively. Taken as concentrations (μU/ml), the absolute amounts of aggregate in the bound fraction were significantly (P < .001) greater than those that were free, whereas the ratios of aggregate to monomer within each pool were similar (P > .1). The relative and absolute amounts of the insulin aggregate during each of the three treatment phases (compared by analysis of variance) were indistinguishable (P > 0.5). However, the aggregate was overestimated by a factor of ∼2 when measurements were made on the basis of an insulin- monomer standard.

We conclude that both the bound and free fractions of IRI contain insulin aggregate. Overreactivity of the aggregate in the radioimmunoassay contributes to the so-called hyperinsulinism of type I diabetes. As with insulin monomer, most of the 12,000-Mr aggregate is bound to antibodies. Because some of the aggregate is free, its biologic consequences must be assessed.
Effects of Heparin on Insulin Binding and Biological Activity
Authors: Kriauciunas Kristina M , Grigorescu Florin , Kahn C Ronald
Published: September 2013

The effect of heparin, a potyanionic glycosaminoglycan known to alter the function of many proteins, on insulin binding and bioactivity was studied. Cultured human lymphocytes (IM-9) were incubated with varying concentrations of heparin, then extensively washed, and 125I-labeled insulin binding was measured. Heparin at concentrations used clinically for anticoagulation (1-50 U/ml) inhibited binding in a dose-dependent manner; 50% inhibition of binding occurred with 5-10 U/ml. Scatchard analysis indicated that the decrease in binding was due to a decrease in both the affinity and the apparent number of available insulin receptors. The effect occurredwithin 10 min at 22°C and persisted even after the cells were extensively washed. Inhibition of insulin binding also occurred when cells were preincubated with heparinized plasma or heparinized serum but not when cells were incubated with normal serum or plasma from blood anticoagulated with EDTA. By contrast, other polyanions and polycations, e.g., poly-L-glutamic acid, poly-L-lysine, succinylated poly-L-lysine, and histone, did not inhibit binding.

Heparin also inhibited insulin binding in Epstein-Barr (EB) virus-transformed lymphocytes buthad no effect on insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells. When isolated adipocytes were incubated with heparin, there was a dose-dependent inhibitionof insulin-stimulated glucose oxidation and, to a lesser extent, of basal glucose oxidation. Although heparin has no effect on insulin binding to intact hepatoma cells, heparin inhibited both insulin binding and insulin-stimulated autophosphorylation in receptors solubilized from these cells. These data indicate that heparin may interact with insulin receptors at concentrations normally used for anticoagulation, altering insulin binding and/or action in various cell types.
Restriction-Fragment—Length Polymorphisms of 5′-Flanking Region of Insulin I Gene in BB and Other Rat Strains: Absence of Association With IDDM
Authors: Winter William E , Beppu Hidehivo , Maclaren Noel K , Cooper David L , Bell Graeme I , Wakeland Edward K
Published: September 2013

Because the 5′-flanking hypervariable region of the human insulin gene may be associated with insulin-dependent diabetes mellitus (IDDM), we examined the spontaneously diabeticBB rat and other rat strains for polymorphisms of the two rat insulin genes, the localization of such polymorphisms, and their possible association with IDDM. By use of restriction-fragmentlength polymorphism analysis, we found that the transcribed portion of the insulin I gene, its 3′-flanking region, and the insulin II gene were not polymorphic. However, four alleles of the insulin I gene were identified, two of which (IA and IB) were found in BB rats. Alleles IA and IB varied in their 5′-flanking regions, yet neither was associated with IDDM in the BB rat.
Inhibitor of Calmodulin and cAMP Phosphodiesterase Activity in BB Rats
Authors: Solomon S S , Steiner M S , Little W L , Rao R H , Sanders L L , Palazzolo M R
Published: September 2013

Diabetes mellitus in humans is associated with increased plasma and tissue levels of cAMP and decreased cAMP phosphodiesterase (PDE) activity. Calmodulin (CM) is a low-molecular-weight protein essential for activation of cAMP PDE. The inhibitor (INH) is a low-molecular-weight substance that inhibits the activity of CM in multiple systems, including PDE. Spontaneously diabetic BB rats (SDR) and their nondiabetic littermates (NDR) were used in this study. Holtzman rats were rendered diabetic by streptozocin (STZ). STZ-induced diabetic rats (STZ-DR) and BB rats werestudied with and without the benefit of insulin therapy. Calmodulin was assayed both by bioassay and by specific radioimmunoassay. The inhibitor was bioassayed by its ability to inhibit CM-activated PDE. Results showed that both spontaneous and STZ-induced diabetes are associated with a decrease in activity of the low-Michaelis constant (Km) cAMP PDE in the liver (39%, SDR; 70% STZ-DR). Calmodulin activity was also decreased in the livers of both animals (13%, SDR; 68%, STZ-DR). Similar data were obtained for NDRs. The inhibitor, on the other hand, was increased in the livers of untreated SDRs and STZ-DRs (155%, SDR; 125%, STZ-DR). No change was noted for NDRs. All these changes were restored toward normal after treatment with insulin. These data suggest that in diabetes the defect in the cAMP PDE-CM-INH system is demonstrated in both an environmental model, as illustrated by STZ-DRs, and a genetic model, as shown by SDRs and NDRs. The inhibitor activity, however, is not changed significantly in NDRs. We speculate that the inhibitor activity plays a role in dictating whether the genetic NDR will or will not become clinically diabetic.
Studies of Midaglizole (DG-5128): A New Type of Oral Hypoglycemic Drug in Healthy Subjects
Authors: Kawazu S , Suzuki M , Negishi K , Watanabe T , Ishii J
Published: September 2013

Midaglizole (DG-5128), 2-[2-(4,5-dihydro-1H-imidazol-2- yl)-1-phenylethyl]pyridine dihydrochloride sesquihydrate, is a novel α2-adrenoceptor antagonist. Its effects on plasma glucose, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG) in healthy male volunteers were investigated.

Volunteers received single oral administrations of midaglizole (150–500 mg), multiple increasing oral administrations on 3 separate days (150–300 mg 3 times daily), or successive daily oral administration for 1 wk (200 mg 3 times daily).

The hypoglycemic action of midaglizole was observed within 0.5–1.0 h after its administration and thereafter for 5 h. The maximum hypoglycemic effect was found 1.0–1.5 h after administration. Midaglizole decreased postprandial hyperglycemia ina dose-dependent manner. In the fasting state, midaglizole significantly increased IRI secretion and suppressed IRG secretion. Midaglizole inhibited epinephrine-induced platelet aggregation after successive administration for 1 wk (200 mg 3 times daily).

The plasma half-life of midaglizole was only 3 h, and the drug was rapidly excreted into the urine and feces, with >80% in its unchanged form, within 24 h.

Midaglizole did not affect the results of any clinical or laboratory tests performed. Our data indicate that midaglizole is a possible hypoglycemic agent. Further clinical investigations are required to confirm its effects on diabetes mellitus.
Kidney Morphology in Experimental Hyperglycemia
Authors: Kern Timothy S , Engerman Ronald L
Published: September 2013

To evaluate the role of hyperglycemia in the pathogenesis of diabetic nephropathy, the kidneys from dogs experimentally galactosemic for 5 yr have been compared with the kidneys from age-matched normal dogs and dogs with alloxan-induced diabetes for 5 yr. The width of glomerular capillary basement membrane and the quantity of plasma protein immunohistochemically demonstrable in the basement membrane were supranormal in the galactosemics, as they were in the diabetics.In contrast, kidney weight, mesangial volume, and the prevalence of obliterated glomeruli, glomerular exudates, and mesangial nodules in the galactosemic animals were comparable to those of normal animals and clearly were less than observed in the insulin-deficient diabetic animals. These galactosemic dogs are known to have developed a retinopathy morphologically indistinguishable from that of diabetic patients and dogs. Thus, galactosemia sufficient to produce diabetic-like lesions in the glomerular basement membrane and retina was found to be nevertheless insufficient to elicit several renal abnormalities that are typical of diabetes. The polyol concentration in erythrocytes was greater than normal in the galactosemics and the diabetics and was greatest in the galactosemics. The absence of mesangial expansion, glomerular obliteration, and nephromegaly in galactose-fed dogs raises the possibility that these abnormalities in diabetes are not a result of excessive polyol pathway activity.
Autonomic Neuropathy in BB Rat: Assessment by Improved Method for Measuring Heart-Rate Variability
Authors: McEwen Tom A J , Sima Anders A F
Published: September 2013

Sinus arrhythmia is a normal respiration-related variation in heart rate that is diminished or absent in autonomic nerve dysfunction. The conventionally used measurements, R-R intervals and their standard deviations (RRSD), were made and compared with a computed statistic () in spontaneously diabetic BB rats and in age-matched nondiabetes-prone control rats. In addition, the effects of pentobarbital sodium anesthesia on RRSD and values in normal control rats were compared with those of nonanesthetized animals.

BB rats demonstrated a decrease in values after 8 wk of diabetes. This decrease became more marked with increased duration of diabetes. No differences in mean R-R intervals or RRSD were obtained between diabetic and nondiabetic rats until 24 wk of diabetes. Normal control rats showed a decrease in sinus arrythmia with age. Both RRSD and values were greatly decreased in anesthetized animals.
Self-Association of Insulin: Its pH Dependence and Effect of Plasma
Authors: Helmerhorst E , Stokes G B
Published: September 2013

We investigated some self-association properties of monocomponent porcine insulin with a simple and novel procedure that for the first time permits the association properties of insulin to be studied in mixed protein solutions. With this semiquantitative procedure, the monomer form of insulin was separated from aggregates during centrifuge desalting on Sephadex G-25. The proportion of monomer in solutions of insulin was estimated by monitoring [125I]monoiodoinsulin recovery off the gel. The self-association properties of insulin were studied in plasma with this procedure over the concentration range of physiologically circulating levels to storage concentrations of the hormone. We estimated the equilibrium constant for the formation of dimers to be 1.5 × 104/M in pooled human plasma at pH 7.4 and 37°C. This estimate was indistinguishable from that obtained for insulin in a defined phosphate-albumin buffer under similar conditions. This result provides direct confirmation that insulin is not associated with a circulating serum binding proteins) and that insulin is present predominantly as a monomer in plasma. We also used the method to investigate the self-association properties of insulin over a wide pH range. The equilibrium constant for the formation of dimers decreased only 6-fold over a 9.2 pH unit increase from pH 2 to 11.2 but almost 200-fold for the additional 1.5 pH unit increase from pH 11.2 to 12.7. We conclude that a residue of pK 12 is critical to the maintenance of the quaternary structure of insulin. We assign this role to the single B22-arginyl residue in insulin.
Role of Hyperglucagonemia in Maintenance of Increased Rates of Hepatic Glucose Output in Type II Diabetics
Authors: Baron Alain D , Schaeffer Linda , Shragg Paul , Kolterman Orville G
Published: September 2013

Elevated rates of basal hepatic glucose output (bHGO) are significantly correlated with the fasting serum glucose (FSG) level in subjects with non-insulin-dependent diabetes mellitus (NIDDM). This observation suggests that bHGO is a major determinant of the severity of the diabetic state in these subjects. In addition, basal glucagon levels (bGL) are higher in these diabetics than in control subjects, despite the concurrent basal hyperglycemia and hyperinsulinemia, two factors known to suppress glucagon secretion. Although bGL is responsible for sustaining two-thirds of bHGO in normal humans, its role in sustaining elevated rates of bHGO in NIDDM has not been previously defined. To this end, we have studied 13 normal and 10 NIDDM subjects; mean FSG levels were 90 ± 2 and 262 ± 21 mg/dl, respectively (P <.001). The mean fasting serum insulin and glucagon levels were higher in the diabetics than in the controls: 17 ± 2 vs. 9 ± 1 μU/ml (P < .01) and 208 ± 37 vs. 104 ± 15 pg/ ml (P < .01), respectively. On separate days, HGO was assessed isotopically (with 3-[3H]glucose) in the basal state and during infusion of somatostatin (SRIF) (600 μg/h) alone and in conjunction with replacement infusions of glucose and insulin. The results demonstrate that 1) bHGO is higher in diabetics than in controls (145 ± 12 vs. 89 ± 3 mg m2 ml−1, P < .01); 2) during infusion of SRIF alone, HGO was suppressed by 25% (P < .05) and 34% (P < .05) of the basal value in controls and diabetics, respectively; 3) when the studies were repeated with glucose levels held constant at or near the FSG level by the glucose-clamp technique, the pattern and degree of HGO suppression was similar to that obtained by infusion of SRIF alone; 4) during isolated glucagon deficiency (SRIF + insulin, 5 mU m−2 min−1 with serum glucose maintained at basal level), HGO was suppressed by 71 ± 8% of the basal value in controls (P < .001) and by 58 ± 7% in diabetics (P < .001); and 5) when isolated glucagon deficiency with similar hyperglycemia was created in control subjects, HGO was suppressed by 87% of the basal value. We conclude that 1) elevated glucagon levels contribute significantly to the elevated rates of bHGO in NIDDM subjects, 2) basal glucagon levels sustain ∼71 % of basal HGO in control and 58% in NIDDM subjects, and 3) the excess glucagon effect is largely responsible for the apparent hepatic insulin resistance seen in NIDDM subjects.
Adverse Effects of Insulin Antibodies on Postprandial Plasma Glucose and Insulin Profiles in Diabetic Patients Without Immune Insulin Resistance: Implications for Intensive Insulin Regimens
Authors: Van Haeften Timon W , Heiling Valarie J , Gerich John E
Published: September 2013

To assess the possible influence of moderate titer insulin antibodies on diabetic glycemic control, we examined insulin-antibody equilibrium binding characteristics, postprandial glucose tolerance, and plasma free-insulin profiles after subcutaneous injection of both porcine and human insulin (0.15 U/kg) in 12 patients with insulin-dependent diabetes mellitus under conditions simulating intensive insulin therapy. The patients' antibodies bound porcine and human insulin indistinguishably, and their plasma glucose and freeinsulin profiles after ingestion of a standard meal were similar with both insulins. Initial increases in plasma free-insulin levels after injection of both insulins were negatively correlated with both insulin-antibody binding (r = −.55, P < .006) and postprandial hyperglycemia (peak level r = −.56, P < .006); the latter was positively correlated with insulin-antibody binding (r = .48, P < .02). The effects of insulin antibodies on postprandial plasma free-insulin and glucose levels could be accounted for substantially by the association constant of the high-affinity insulin-antibody binding sites (K1); patients in the highest quartile for K1 had significantly slower initial increments in plasma free insulin (0.31 ± 0.04 vs. 0.46 ± 0.06 μU/min, P < .05) and greater postprandial hyperglycemia (peak value 237 ± 10 vs. 166 ± 12 mg/dl, P < .001) than patients in the lowest quartile. We conclude that moderate insulin-antibody titers commonly found in insulin-treated patients can slow the early increase in plasma free insulin after subcutaneous injection and that this impairs postprandial glucose tolerance; such an effect may Received for publication 22 April 1986 and accepted in revised form 18 September 1986. limit the effectiveness of intensive insulin therapy.
Induced Hyperglycemia Alters Antithrombin III Activity but not its Plasma Concentration in Healthy Normal Subjects
Authors: Ceriello Antonio , Giugliano Dario , Quatraro Antonio , Consoli Giuseppe , Stante Aldo , Russo Patrizia Dello , D'Onofrio Felice
Published: September 2013

The effects of induced hyperglycemia on both antithrombin III (ATIII) biologic activity and plasma concentration in normal subjects are reported. A decrease in ATIII activity parallel to hyperglycemia was observed, while ATIII concentration was unchanged. When the glycemia returned to basal values ATIII activity concomitantly showed values in the basal range. Heparin infusion was able to significantly preserve ATIII activity from glycemia-induced alterations. These data demonstrate that hyperglycemia by itself may alter ATIII biologic activity. Moreover, the effect of heparin administration suggests that both glucose and heparin compete in vivo for the same functional site. Our study, showing the possible role of hyperglycemia in altering the biologic function of some proteins, stresses the role of increased blood glucose in the development of some complications in diabetes.
New, Simple Insulin-Receptor Assay With Universal Application to Solubilized Insulin Receptors and Receptors in Broken and Intact Cells
Authors: Frandsen Erik K , Bacchus Robby A
Published: September 2013

A new, simple insulin-receptor-binding assay has been devised. The assay is based on the separation of free and receptor-bound 125I-labeled insulin in 80% ethanol. It was found that the insulin-receptor complex was fully stable at this ethanol concentration, regardless of the source of the receptor employed. The assay has been evaluated with solubilized insulin receptors and membrane-bound receptors from human placenta and porcine liver as well as intact cells with the IM-9 cell line. The assay is simple, rapid, and has large capacity. Comparisons of the ethanol-based assay to the conventionally employed assays with polyethylene glycol or microfuge centrifugation for the separation of free and bound 125I-insulin revealed large discrepancies between the assays. The ethanol-based assay always appeared to provide a better separation. Microfuge centrifugation of placental membranes precipitated ∼3% of the ethanol-precipitable insulin-receptor complex, while polyethylene glycol precipitation of solubilized insulin receptors varied between 40 and 80% of the ethanol precipitability, depending on the receptor concentration employed.