bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and Use in Immunoradiometric Assay of Monoclonal Antibodies Directed Against Human Proinsulin
Authors: Gray I Peter , Siddle Kenneth , Frank Bruce H , Hales C Nicholas
Published: September 2013

Proinsulin in human serum is heterogenous. Existing assay methods do not distinguish between the various forms intermediate on the pathway of processing from proinsulin to insulin. We report the production of mouse monoclonal antibodies against human proinsulin with biosynthetic human proinsulin (produced by recombinant DNA technology) as immunogen. Four monoclonal antibody-producing cell lines were obtained from two separate fusions. Two of the antibodies had affinity constants for intact proinsulin of 3.1 × 1010 and 5.6 × 1010 L/mol and bound all forms of proinsulin and insulin. Crossreactivity with intact proinsulin was greater than with 65-66 and 32-33 split proinsulins and des 64-65 and des 31-32 proinsulins. The third antibody had an affinity constant for intact proinsulin of 6.3 × 1010 L/mol and reacted only with intact proinsulin, 65-66 split proinsulin, and des 64-65 proinsulin; there was no reaction with 32-33 split proinsulin, des 31-32 proinsulin, or C-peptide. The fourth antibody reacted only with intact proinsulin and had an affinity constant of 4.1 × 108 L/mol. We report the use of two antibodies in a sensitive two-site immunoradiometric assay for intact proinsulin. Insulin, C-peptide, and 32-33 split proinsulin did not react in the assay up to a concentration of 100 pM. The 65-66 split proinsulin and des 64-65 proinsulin reacted with a potency of ∼55% relative to intact proinsulin. Serum proinsulin concentrations measured with this assay were compared with those determined by an alternative method that detects only split proinsulins. The results suggest that both intact and split proinsulins occur in human serum but that the forms differ in their kinetics of release and removal in response to an oral glucose load.
Prevalence of Diabetes Mellitus and Impaired Glucose Tolerance Among Second-Generation Japanese-American Men
Authors: Fujimoto Wilfred Y , Leonetti Donna L , Kinyoun James L , Newell-Morris Laura , Shuman William P , Stolov Walter C , Wahl Patricia W
Published: September 2013

We describe the initial findings from a multidisciplinary, epidemiologic study of diabetes mellitus conducted in a population of secondgeneration Japanese-American (Nisei) men born between 1910 and 1939 who reside in King County, Washington (n = 1746). From this study population, 487 volunteered, and 229 were enrolled to comprise the study sample. A random sample of Nisei men was also drawn from the population to develop a reference sample of 189 men.

All subjects participated in a 75-g oral glucose tolerance test; the National Diabetes Data Group (NDDG) and World Health Organization (WHO) diagnostic criteria as well as a modification of the WHO criteria were used to classify individuals with normal glucose tolerance, impaired glucose tolerance (IGT), or diabetes. Within the study sample, 79 men were found to have normal glucose tolerance, 72 had IGT, and 78 had type II diabetes. The mean age of the study sample was 61.4 yr. Based on comparison of the study sample to the reference sample, the study sample was ascertained to be representative of Nisei men in King County.

Extrapolating from our observations in the reference sample and in the study sample, we have estimated that ∼56% of Nisei men in the study population have abnormal glucose tolerance. Much of this is undiagnosed because only ∼13% of the reference sample of Nisei men reported a prior diagnosis of diabetes. Of the men who enrolled in the study as nondiabetic subjects, 11.1% had diabetes and 39.2% had IGT; i.e., 50.3% had previously unknown abnormalities in glucose tolerance. We estimate that ∼20% of Nisei men have diabetes (both previously diagnosed and undiagnosed) and ∼36% have IGT.
Prevalence of Complications Among Second-Generation Japanese-American Men With Diabetes, Impaired Glucose Tolerance, or Normal Glucose Tolerance
Authors: Fujimoto Wilfred Y , Leonetti Donna L , Kinyoun James L , Shuman William P , Stolov Walter C , Wahl Patricia W
Published: September 2013

In a study sample of 229 second-generation Japanese- American (Nisei) men, 79 with normal glucose tolerance, 72 with impaired glucose tolerance (IGT), and 78 with non-insulin-dependent diabetes, we have determined prevalence rates for certain conditions (ischemic heart disease, peripheral vascular disease, hypertension, retinopathy, neuropathy, and nephropathy) associated with diabetes. All subjects participated in a 75-g oral glucose tolerance test. World Health Organization (WHO) diagnostic criteria and information from the subject's medical history and personal physician were used to classify the subjects.

Retinopathy was observed only in diabetic men in the study sample (11.5% of diabetic men). Furthermore, it was observed only in men who were receiving drug treatment for diabetes—40.0% of insulin-treated and 17.2% of sulfonylurea-treated men. Electrophysiologic evidence of peripheral neuropathy was observed in 46.2% of diabetic men and in 4.0% of nondiabetic (normal and IGT) men. For diabetic men with fasting serum glucose ≥140 mg/dl, 63.8% had peripheral neuropathy and 19.1% had retinopathy, whereas for diabetic men with fasting serum glucose <140 mg/dl, 19.4% had neuropathy and none had retinopathy. For diabetic men with a diabetes duration of 2:10 yr, 72.7% had neuropathy and 31.8% had retinopathy; with a diabetes duration of 5-9 yr, 70.6% had neuropathy and 11.8% had retinopathy; and with a diabetes duration of <5 yr, 20.5% had neuropathy and none had retinopathy. Nephropathy was distinctly uncommon, and among the measurements of kidney function, only proteinuria was clearly abnormal with diabetes. Prevalence rates of hypertension, peripheral vascular disease, and ischemic heart disease were highest in Nisei men with diabetes, lowest in men with normal glucose tolerance, and intermediate in men with IGT. None of these conditions appeared to be strongly related to the degree of fasting hyperglycemia. Hypertension occurred in 53.8% of diabetic men, 51.4% of men with IGT, and 29.1% of men with normal glucose tolerance (blood pressure > 140/90 and/or treatment with medication for hypertension). Arteriosclerosis obliterans of the lower leg vessels was determined by Doppler technique to occur in 24.4% of diabetic men, 16.7% of men with IGT, and 8.9% of men with normal glucose tolerance. Ischemic heart disease, as determined from a resting electrocardiogram and the Minnesota Code criteria for ischemic heart disease, occurred in 29.5% of diabetic men, 23.6% of men with IGT, and only 6.3% of men with normal glucose tolerance.
Progression of Incipient Diabetic Retinopathy During Good Glycemic Control
Authors: Engerman Ronald L , Kern Timothy S
Published: September 2013

To assess the extent to which the progression of diabetic retinopathy can be arrested by improved glycemic control, 35 normal dogs were randomly divided into a nondiabetic and three alloxan-induced diabetic groups prospectively identified according to glycemic control: poor control for 5 yr (PC), good control for 5 yr (GC), and poor control for 2.5 yr followed by good control for 2.5 yr (PGC). To achieve good control, insulin was given twice daily together with a measured diet so that hyperglycemia and glucosuria were mild and infrequent, and HbA, was comparable to normal. Retinal capillary aneurysms and other lesions developed during 60 mo of poor control (group PC) and were inhibited if good control was begun promptly within 2 mo (group GC). In group PGC, retinopathy was absent or equivocal at 2.5 yr of poor control and, surprisingly, was found to develop subsequently despite good glycemic control. Retinopathy in group PGC was greater at autopsy than at 2.5 yr and was greater than in group GC. The results indicate that retinopathy may be preventable but tends to resist arrest even in its incipient stages, before more than the first few aneurysms have appeared.
Nonenzymatic Glycosylation of Serum IgG and Its Effect on Antibody Activity in Patients With Diabetes Mellitus
Authors: Kaneshige Hideaki
Published: September 2013

Susceptibility to infection is assumed to be increased in diabetic patients, although its mechanism is unknown. The purpose of this study was to determine whether glycosylation of circulating immunoglobulins is related to the decrease of antibody activity in diabetic patients. Thirty-five patients with type II (non-insulin-dependent) diabetes and 14 age-matched normal controls were examined. Nonenzymatic glycosylation of serum immunoglobulin G (IgG) in vivo was measured by two different techniques, colorimetry and affinity chromatography. The levels of glycosylated IgG were significantly higher in diabetic patients than in normal controls.

To evaluate the antibody activity of glycosylated IgG, anti-streptolysin O (ASO) titers after in vitro glycosylation of IgG and antibody titers before and after in vivo immunization with influenza vaccine were determined. IgG specific for streptolysin O purified by affinity chromatography decreased ASO titers after in vitro glycosylation. In diabetic patients, serum titers of hemagglutinin-inhibiting antibody against influenza viruses 4 wk after initial immunization were significantly lower than those in normal controls. These results indicate that serum IgG in diabetic patients was nonenzymaticaliy glycosylated, and this modification in vivo might be associated with its functional alteration.
Diabetes Decreases Na+-K+ Pump Concentration in Skeletal Muscles, Heart Ventricular Muscle, and Peripheral Nerves of Rat
Authors: Kjeldsen Keld , Brændgaard Hans , Sidenius Per , Larsen Jim Stenfatt , Nørgaard Aage
Published: September 2013

Na+-K+-ATPase or the Na+-K+ pump is essential for some specific properties of muscle and nerve tissue such as contractility and excitability. Previous studies have shown conflicting variations in Na+-K+-ATPase activity or Na+-K+ pump concentration of muscle cells in experimental diabetes. Our study demonstrates that early untreated diabetes in rats induced by injection of streptozocin is associated with decreases in [3H]ouabain binding-site concentration of 24-48% in various skeletal muscles and 16% in peripheral nerves as well as a decrease in K+-dependent 3-O-methylfluorescein phosphatase activity of 21% in the heart ventricle. These effects could be prevented by insulin treatment. They probably represent a decrease in the concentration of Na+-K+ pumps. There was no evidence for more than one population of Na+-K+ pumps in intact samples of skeletal muscle and nerves from normal, diabetic, and insulin-treated animals. The decrease in Na+-K+ pump concentration in nerve cells may be due to atrophy of the axons. In skeletal muscles, myocardium, and peripheral nerves, the observed decrease in Na+-K+ pump concentration may be important for the pathophysiology of diabetes. We emphasize that quantification of Na+-K+-ATPase or the Na+-K+ pump in muscle and nerve tissue from diabetic animals should preferably be performed with either intact samples or crude homogenates of whole tissue.
Reversal of Insulin Resistance in Diabetic Rat Adipocytes by Insulin Therapy: Restoration of Pool of Glucose Transporters and Enhancement of Glucose-Transport Activity
Authors: Karnieli Eddy , Armoni Michal , Cohen Pinchas , Kanter Yoram , Rafaeloff Ronit
Published: September 2013

To determine the role of insulin in reversing the insulin resistance associated with depletion of the intracellular pool of glucose transporters, streptozocin-induced diabetic rats were treated with 5 U/day s.c. of insulin for 0, 8, or 14 days. At each time point, adipose cells were isolated, and 3-O-methylglucose transport was measured in the absence and presence of 1000 μU/ml insulin. With the cytochalasin B-binding assay, concentrations of glucose transporters in the plasma and the low-density microsomal membrane fractions were determined. Eight-day insulin therapy enhanced glucose transport rate (mean ± SE) from 0.2 ± 0.0 to 1.1 ± 0.1 fmol · cell−1 · min−1 in the basal state and from 0.8 ± 0.1 to 5.5 ± 0.4 fmol · cell−1 · min−1 in the insulin-stimulated state in untreated and treated diabetic rats, respectively; this is a 3-fold increment of glucose transport rate in both states compared with control rats. After 14-day insulin therapy, glucose-transport activity declined toward normal but still remained ∼1.5- and 4-fold higher than control and diabetic rats, respectively. Despite the persistent enhancement of glucose transport rate, concentration of glucose transporters in the intracellular pool was restored only to its prediabetic state. Likewise, the increased concentration of glucose transporters in the plasma membranes after insulin stimulation was similar to that of control rats. Thus, we suggest that 8–14 days of insulin therapy reversed the insulin resistance in diabetic rat adipocytes by at least two mechanisms: restoration of the intracellular pool of glucose transporters and enhancement of glucose-transport activity. The mechanism(s) responsible for this supernormal glucose-transport activity is unknown but may be related to transient appearance of a factor coregulating cellular glucose transport and/ or modulation of the intrinsic activity of the glucose transporter.
Role of Cyclosporin A in Macromolecular Synthesis of β-Cells
Authors: Eun Hyone-Myong , Pak Chin-Yong , Kim Chang-Jin , McArthur Robert G , Yoon Ji-Won
Published: September 2013

Wistar rats developed hypoinsulinemia and hyperglycemia within 7 days when treated daily with 40 mg/kg body wt of cyclosporin A (CsA) and recovered from the metabolic alteration within 1 wk when CsA treatment was terminated. By light microscopy, there was no lymphocytic infiltration, but cytoplasmic vacuolization in the islets of Langerhans from the CsA-treated rats was seen. By electron microscopy, severe degranulation, cytoplasmic vacuolization, and dilation of endoplasmic reticulum were clearly seen in the pancreatic β-cells. Islet cells isolated from the CsA-treated rats showed >50% reduction in mRNA synthesis. A similar inhibitory pattern of mRNA synthesis was observed in in vitro CsA-treated (10 μg/ml) human pancreatic islet cells from one biopsy sample and in similarly treated rat insulinoma cells (RINm5F). The inhibitory effect of CsA on mRNA synthesis in RINm5F cells was dose dependent, with a 50%-inhibiting dose of 5 μg/ml. In addition to the inhibition of mRNA synthesis, CsA also inhibited protein and DNA syntheses, although the inhibitory effect on these macromolecular syntheses was significantly less than that on mRNA synthesis. However, there was only a minor effect of CsA on in vitro transcription and translation compared with that on RINm5F and islet cells. It is concluded that CsA-induced degranulation of the β-cells in Wistar rats, accompanied by hypoinsulinemia and hyperglycemia, may be due to indirect, reversible interference of the cellular function primarily involved in mRNA synthesis.
Alterations in Free Radical Tissue-Defense Mechanisms in Streptozocin-Induced Diabetes in Rat: Effects of Insulin Treatment
Authors: Wohaieb Saleh A , Godin David V
Published: September 2013

We investigated the possible involvement of reactive oxygen radical-related processes in chronic (12-wk) diabetes induced in rats by streptozocin (STZ). Diabetes was associated with significantly increased activities of catalase (CAT), glutathione reductase (GSSG-RD), and CuZn-superoxide dismutase (SOD) in the pancreas and of CAT and GSSG-RD in the heart. On the other hand, the liver of diabetic rats showed a generalized decrease in CAT, glutathione peroxidase (GSH-PX), and SOD as well as in the levels of reduced glutathione (GSH). Diabetic kidney also showed decreases in CAT and SOD, but the activities of GSH-PX were increased. Insulin treatment (9–12 U/kg body wt) that was started after 8 wk of diabetes and continued for 4 wk reversed all of the foregoing alterations in tissue antioxidant status.

Our results suggest the presence of increased oxidative stress in uncontrolled diabetes as manifested by the marked alterations in tissue antioxidant enzyme activities, the magnitude of which increased with the degree of emaciation. The complex patterns of changes observed in the various tissues examined are believed to be the result of compensatory increases in enzyme activities (usually involving enzymes whose activity in control tissues is low) and direct inhibitory effects, possibly resulting from an increased tissue-oxidant activity. Our findings support the view that tissue antioxidant status may be an important factor in the etiology of diabetes and its complications.
Response of Diabetic Basement Membrane—Producing Cells to Glucose and Insulin
Authors: Ledbetter Steven R , Wagner Clayton W , Martin George R , Rohrbach David H , Hassell John R
Published: September 2013

The effect of insulin and glucose on the synthesis of basement membrane components was studied in organ cultures of a basement membrane-producing tumor grown in diabetic and normal mice. Tumor tissue grown in diabetic mice produced more protein and basement membrane-specific proteoglycan in response to insulin than tissue grown in normal mice. Addition of high levels of glucose to the culture medium did not alter insulin-stimulated protein synthesis by diabetic or normal tissue but dampened insulin-stimulated production of proteoglycan. These data suggest that basement membrane-producing cells in diabetic hosts may be hypersensitive to insulin and that stimulation of protein production by insulin may play some role in the in situ hypertrophy of basement membranes.
Glomerular Morphology by Light Microscopy in Non-Insulin-Dependent Diabetes Mellitus: Lack of Glomerular Hypertrophy
Authors: Schmitz Anita , Gundersen Hans Jørgen Gottlieb , Østerby Ruth
Published: September 2013

We quantitated glomerular structure by light microscopy in 19 subjects with non-insulin-dependent diabetes mellitus (NIDDM) and studied the possible connection between morphology and urinary albumin concentration. Autopsy material was collected retrospectively from diabetic subjects in whom urinary albumin concentration had been measured within 1.5 yr. Nineteen consecutive sex- and age-matched nondiabetic subjects were controls. A quantitative study of a random sample of glomeruli was performed blindly on periodic acid–Schiff (PAS)-stained sections. The main parameters obtained were 1) mean volume of open glomeruli, 2) frequency of glomerular occlusion, and 3) volume fraction of red-stained material (PAS-positive substance) in open glomeruli [Vv(R/G)]. There was no increase in glomerular volume in these NIDDM subjects, contrary to the glomerular hypertrophy found early as well as late in insulin-dependent diabetes mellitus. An increase in Vv(R/G) was found in diabetic subjects, demonstrating the presence of glomerulopathy as it is diagnosed by light microscopy. The frequency of glomerular occlusion was not significantly different between the groups. A high urinary albumin concentration did not necessarily reflect more advanced glomerulopathy.
Mineral Metabolism and Bone Mass at Peripheral and Axial Skeleton in Diabetes Mellitus
Authors: Auwerx John , Dequeker Jan , Bouillon Roger , Geusens Piet , Nijs Jos
Published: September 2013

Bone mineral content (BMC), mineral homeostasis, and diabetes control were evaluated in 31 Caucasian insulin-dependent diabetic patients (disease duration 18.3 ± 7.7 yr, mean ± SD) with normal kidney function. To evaluate bone mass, we performed radiogrammetry and single- and dual-photon absorptiometry. In women, a significantly lower mean BMC was found in the distal radius, at a mixed trabecular-cortical (P < .01) and a cortical (P < .05) site, as well as in the lumbar spine (P < .02). In diabetic men, mean BMC was significantly reduced at the trabecularcortical (P < .01) and cortical (P < .05) sites of the radius but not in the lumbar spine. When expressed as densities (i.e., BMC/width or lumbar BMC/area), only the BMC/width at the radius cortical area was significantly reduced in women (P < .05). The results of the radiogrammetry showed a larger endosteal diameter in the diabetic women, resulting in a significantly lower cortical thickness (P < .05). Diabetic men did not show abnormalities on radiogrammetry. Diabetic patients had diminished serum calcium and phosphorus concentrations (P < .001), whereas serum parathyroid, 25-hydroxyvitamin D3, and concentrations of both total and free 1,25-dihydroxyvitamin D3 were normal. No correlation between parameters of diabetes control (HbA1, insulin dose, and triglycerides) or calcium-regulating hormones and BMC were found. These data confirm that, despite large overlap of individual values, mean bone mass at the peripheral skeleton is significantly decreased in diabetic patients. Moreover, we report that the BMC of the lumbar spine is significantly reduced in female diabetic patients.
Measurement of L-[1-14C]Leucine Kinetics in Splanchnic and Leg Tissues In Humans: Effect of Amino Acid Infusion
Authors: Gelfand Robert A , Glickman Morton G , Castellino Pietro , Louard Rita J , DeFronzo Ralph A
Published: September 2013

Although whole-body leucine flux is widely measured to study body protein turnover in humans, the contribution of specific tissues to the total-body measurement remains unknown. By combining the organ-balance technique with the systemic infusion of L-[1-14C]leucine, we quantitated leucine production and disposal by splanchnic and leg tissues and by the whole body, simultaneously, in six normal men before and during amino acid infusion. At steady state, disposal of arterial leucine by splanchnic and leg tissues was calculated from the percent extraction (E) of L-[1-14C]leucine counts: uptake = E × [Leu]a × flow. Tissue release of cold leucine (from protein turnover) into vein was calculated as the difference between leucine uptake and the net tissue leucine balance. In the postabsorptive state, despite substantial (P < .01) extraction of L-[1-14C]leucine by splanchnic (23 ± 1%) and leg (18 ± 2%) tissues, net leucine balance across both tissue beds was small, indicating active simultaneous disposal and production of leucine at nearly equivalent rates. Splanchnic tissues accounted for −50% of the measured total-body leucine flux. During amino acid infusion, the net leucine balance across splanchnic and leg tissues became positive, reflecting not only an increase in leucine uptake but also a marked suppression (by ∼50%, P < .02) of cold leucine release. This reduction in splanchnic and leg leucine release was indicated by a sharp decline in whole-body endogenous leucine flux. Conclusions: 1) combining the organ-balance method with systemic L-[1-14C]leucine infusion enables leucine kinetics to be measured simultaneously in the whole body and in specific tissues; 2) splanchnic tissues account for −50% of whole-body leucine flux in postabsorptive humans; and 3) amino acid infusion markedly suppresses leg and splanchnic tissue leucine release, which may indicate inhibition of proteolysis.
Expression of Class II MHC Antigens in Murine Pancreas After Streptozocin-Induced Insulitis
Authors: Farr Andrew G , Mannschreck John W , Anderson Susan K
Published: September 2013

In this study, most class II antigen expression during streptozocin-induced insulitis was associated with the mononuclear cells infiltrating the pancreas. During the early stages of the insulitis response, phagocytic cells expressing class II antigens and containing β-cell debris were often observed. Although there was de novo expression of class II antigens by ductular epithelium associated with the exocrine portion of the pancreas and transient expression by vascular endothelial cells, the endocrine cells of the islets remained devoid of class II antigens throughout the period of observation (42 days).
Opposite Effects of Insulin and Catecholamines on LDL-Receptor Activity in Human Mononuclear Leukocytes
Authors: Krone Wilhelm , Naegele Herbert , Behnke Bert , Greten Heiner
Published: September 2013

The mechanisms by which insulin and catecholamines affect low-density lipoprotein (LDL)-receptor activity were studied in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 24 h in a lipid-free medium resulted in an increase in the specific binding, accumulation, and degradation of 125I-labeled LDL. Insulin stimulated the ability of the cells to bind, accumulate, and degrade the lipoprotein with high affinity, which may be caused by an increase in the LDL-receptor number without altering binding affinity. (–)-Epinephrine inhibited the specific binding, accumulation, and degradation of 125I-LDL. This effect appears to be mediated by a decrease in the number of LDL receptors and not by a change in the binding affinity. (–)-Norepinephrine, the unspecific β-adrenergic agonist (–)-isoproterenol, and the β2-specific agonist terbutaline mimicked the effect of epinephrine on LDL-receptor activity. Catecholamines and β-adrenergic agonists yielded sigmoidal log-concentration effect curves. The action of epinephrine was attenuated by the β-antagonist (dl)-propranolol. These results demonstrate that insulin stimulates and catecholamines suppress the specific binding, accumulation, and degradation of 125I-LDL in human mononuclear leukocytes. The catecholamine action appears to be mediated by β2-adrenergic receptors. A suppression of LDL-receptor activity resulting from deficiency of insulin and elevated plasma catecholamine concentrations in uncontrolled insulin-dependent diabetic patients may contribute to the increased levels of LDL cholesterol observed in these patients.
Sodium Butyrate Increases Glucagon and Insulin Gene Expression by Recruiting Immunocytochemically Negative Cells to Produce Hormone
Authors: Powers Alvin C , Philippe Jacques , Hermann Heather , Habener Joel F
Published: September 2013

Current evidence suggests that a multipotential endodermal progenitor ceil may give rise to all islet cell phenotypes. We characterized two hormone-producing rat islet (RIN) cell lines derived from a radiation-induced islet tumor by immunocytochemistry, Northern blot analysis, and radioimmunoassay of secreted hormone. Using antisera to glucagon, insulin, and somatostatin, we found that <15% of the cells in any of these three islet cell lines contained immunopositive cells. The number of cells staining for the hormone correlated with mRNA levels and immunoreactive secreted hormones. Sodium butyrate, a short-chain aliphatic fatty acid, slowed cell growth and increased dramatically the percentage of cells staining for glucagon and insulin. The increase in immunopositive cells was accompanied by an increase in glucagon and insulin mRNAs and secreted glucagon and insulin. These observations indicate that sodium butyrate increases glucagon and insulin gene expression by recruiting previously immunonegative cells to produce hormone. The relationship of DNA synthesis and hormone production was assessed by pulse-labeling RIN cells with [3H]thymidine, which was followed by autoradiography and immunocytochemistry. [3H]thymidine incorporation was observed in a lower percentage of immunopositive compared with immunonegative cells. Furthermore, sodium butyrate reduced the number of [3H]thymidine-labeled cells and increased the number of immunopositive cells. These observations suggest that sodium butyrate differentiates the islet cells and thereby increases the expression of the glucagon and insulin genes.
Stimulatory Effects of Cholecystokinin on Isolated Perifused Islets Inhibited by L 364718 Potent and Specific Antagonist
Authors: Zawalich Walter S , Diaz Victoria A , Zawalich Kathleen C
Published: September 2013

The influence of L 364718 on islet responsiveness to sulfated cholecystokinin (CCK-8S) was investigated. In islets whose inositol-containing phospholipids were prelabeled during a 2-h incubation period, subsequent exposure to L 364718 (1 nM) significantly impaired the secretion of insulin usually noted in response to 200 nM CCK-8S in the simultaneous presence of 7 mM glucose. A higher level of the antagonist (10 nM) completely abolished insulin secretion. L 364718 (1-10 nM) reduced the efflux of 3H from myo-[2-3H]-inositol prelabeled islets in parallel with the reduction in secretion. L 364718 (10 nM) significantly reduced the accumulation of 3H-containing inositol phosphates usually noted with CCK-8S addition. L 364718, at levels 10- to 100-fold greater than those necessary to attenuate CCK-8S–induced insulin secretion, had no adverse effect on the insulin secretory response of freshly isolated islets to 10 mM glucose alone, 5 mM D-glyceraldehyde, 15 mM α-ketoisocaproate, or 50 ng/ml gastric inhibitory polypeptide. L 364718 (1000 nM) had no adverse influence on carbamylcholine (1 mM)–induced phosphoinositide hydrolysis. These results establish L 364718 as a potent and highly selective antagonist of cholecystokinin's stimulatory actions on βc-ells. Because of its potency, selectivity, and oral effectiveness, in vivo studies with L 364718, aimed at unraveling the pleiotropic effects of CCK-8S on glucose and insulin homeostasis, seem feasible.
Effect of Proteinuria on Mortality in NIDDM
Authors: Nelson Robert G , Pettitt David J , Carraher Michael J , Baird H Robert , Knowler William C
Published: September 2013

The effect of proteinuria (≥ ∼1 g/day) on mortality in non-insulin-dependent diabetes mellitus (NIDDM) was assessed in Pima Indians aged ≥45 yr. Among 1426 subjects, 48% with NIDDM at the beginning of followup, there were 489 deaths in 13,345 person-yr of observation. The age- and sex-adjusted mortality rate was 32.7/1000 person-yr (95% CI = 27.6, 37.8) in diabetic subjects without proteinuria, similar to the rate of 30.1/1000 person-yr (95% CI = 25.7, 34.4) in nondiabetic subjects without proteinuria. By contrast, in diabetic subjects with proteinuria the mortality rate was 121.4/1000 person-yr (95% CI = 97.5, 145.3). When controlled for age, sex, and diabetes duration, diabetic subjects with proteinuria had a death rate 3.5 times as high (95% CI = 2.8, 4.4) as those without proteinuria. Of the excess mortality associated with NIDDM in Pima Indians, 97% was found in subjects with proteinuria. The death rate in diabetic subjects without proteinuria was not appreciably greater than the rate in nondiabetic subjects. Mortality rates from uremia and cardiovascular disease were significantly higher in diabetic Pima Indians with proteinuria than in those without. These relationships are similar to observations reported in people with insulin-dependent diabetes.
Regulation of Glucose-Transporter Gene Expression by Insulin in Cultured Human Fibroblasts
Authors: Kosaki Atsushi , Kuzuya Hideshi , Yoshimasa Yasunao , Yamada Kazunori , Okamoto Motozumi , Nishimura Haruo , Kakehi Takako , Takeda Jun , Seino Yutaka , Imura Hiroo
Published: September 2013

To clarify the effect of insulin on glucose-transporter (GT) biosynthesis, we determined GT mRNA levels in human cultured skin fibroblasts, using HepG2 GT cDNA as a probe. Insulin specifically increased the GT mRNA level in a time- and dose-dependent manner. Time-course study demonstrated that the mRNA level peaked within 3 h of insulin (1 × 10−7 M) addition. After remaining elevated for several hours, mRNA decreased and returned to the basal level after 24 h. In the cell strains from seven normal subjects, the mean (±SE) GT mRNA level determined after 3 h of treatment with 1 × 10−7 M insulin was 164.3 ± 8.5% of the level found in untreated control cells. The insulin dose-response curve of GT mRNA levels showed that the maximum stimulation was elicited at 1 × 10−7 M, and the half-maximum stimulation occurred at ∼5 × 10−10 M. Degradation rates of GT mRNA determined in the presence of actinomycin D were not different between insulin-treated and untreated cells. These results suggest that insulin increases GT gene expression in cultured human fibroblasts.
Aldose Reductase Expression in Human Diabetic Retina and Retinal Pigment Epithelium
Authors: Vinores Stanley A , Campochiaro Peter A , Williams Edward H , May Estelle E , Green W Richard , Sorenson Robert L
Published: September 2013

The conversion of glucose to sorbitol by aldose reductase (AR) and its subsequent intracellular accumulation have been implicated in the pathogenesis of diabetic cataracts. There is also evidence linking AR activity with retinal capillary basement membrane thickening in galactosemic rats, suggesting a possible role in diabetic retinopathy. In this study, we explored one feature of this issue by examining diabetic and nondiabetic eyes for immunoreactive AR. AR was immunohistochemically undetectable in the retinal pigment epithelia (RPE) and neural retinas of nondiabetic human eyes. Weak, focal staining for AR was present unilaterally in the RPE of 1 of 11 diabetic patients without pathologic ocular findings and in 43% of diabetic patients with mild ocular findings. Retinal positivity was found (unilaterally) in only 2 of 19 individuals from either of these mildly affected groups. Fifty-five percent of patients with background retinopathy demonstrated AR positivity in the RPE, and half of these expressed AR in the RPE of both eyes. Of the individuals with proliferative diabetic retinopathy, 87.5% showed bilateral staining in the RPE. Retinal positivity was present in 36% of background retinopathy and 75% of proliferative retinopathy cases, demonstrating a positive correlation between AR expression and the severity of the disorder. In weakly staining retinas, only the ganglion cell bodies, nerve fibers, and Müller cells were positive, whereas in intensely staining cases, virtually the entire retina, except for the rod outer segments, was positive. Eyes from patients who had had diabetes ≤6 yr were negative for AR, but those from long-term-diabetic patients (14–45 yr)manifested positivity. These data suggest that enhanced expression of AR may play a role in the development of diabetic retinopathy.
Maternal Diabetes in Rats: I. Effects on Placental Growth and Protein Turnover
Authors: Robinson Judith , Canavan James P , El Haj Alicia J , Goldspink David F
Published: September 2013

The developmental growth of the rat placenta was investigated between days 14 and 21 of gestation in normal control, gestational-diabetic, established-diabetic, and insulin-maintained-diabetic mothers. While established–diabetic mothers were hyperglycemic for 2 wk before and throughout the pregnancy, gestational-diabetic mothers were only hyperglycemic for the second half of pregnancy. Daily insulin replacements successfully restored normoglycemia. The wet weight and protein content of control placentas increased linearly between days 14 and 21. Although placentas from diabetic animals were initially smaller, placentomegaly was found at full term. Placental glycogen concentrations were also markedly increased in all diabetic animals. These changes were largely prevented by insulin replacement. The changes in placental size during normal development and in association with the diabetic state were explained by measuring placental rates of protein turnover (in vivo). In normal placentas, protein synthetic and degradative rates progressively declined over the last week of gestation. Because synthesis rates were unchanged in placentas of diabetic mothers, it appears that the differences in placental size primarily arise from alterations in protein degradation.
Maternal Diabetes in Rats: II. Effects on Fetal Growth and Protein Turnover
Authors: Canavan James P , Goldspink David F
Published: September 2013

The developmental growth of the rat fetus was studied between days 14 and 21 of pregnancy in normal control, established-diabetic, gestational-diabetic, and insulin-maintained–diabetic mothers. Measurements of fetal body weights and protein mass revealed a suppression of growth in the diabetic pregnancies, probably arising from reduced hyperplasia. Growth of the liver and skin appeared to be suppressed in proportion to the whole fetus, whereas the lung, brain, and particularly the heart were relatively well protected from growth retardation. Fetal growth during development, and its retardation in association with the hyperglycemic state, was explained by measuring the rates of fetal protein turnover in vivo. Both the protein synthetic and degradative rates gradually declined during normal development. However, in the diabetic pregnancies, fetal protein synthesis was consistently lower than control rates, whereas protein degradation increased sharply toward the end of gestation. These changes in protein synthesis and breakdown probably combine to yield a smaller fetus in the absence of normoglycemia.
Effects of Growth Hormone and Insulin-Like Growth Factor I on Endocrine Function of Human Fetal Islet-Like Cell Clusters During Long-Term Tissue Culture
Authors: Otonkoski Timo , Knip Mikael , Wong Inés , Simell Olli
Published: September 2013

The effects of recombinant human growth hormone (GH, 1 µg/ml) and insulin-like growth factor I (IGF-I, 200 ng/ml) on the production of insulin and glucagon by human fetal islet-like cell clusters (ICCs) were studied in tissue culture. ICCs were derived after collagenase digestion and culture of pancreases from 16 fetuses (mean gestational age 15.6 wk). The ICCs were cultured with or without GH or IGF-I for 7 or 31 days. Basal rates of insulin and glucagon production were not altered by GH during the first 17 days of culture, but the release of both hormones was increasingly augmented by GH during the last 2 wk of culture (131% increase in insulin and 85% in glucagon compared with controls). ICCs cultured for 7 days in the presence of GH secreted more insulin when incubated for 120 min in 20 mM than in 2 mM glucose (2.1-fold response, P < .05), whereas ICCs mantained in basal medium did not respond to glucose. GH had no effect on DNA and insulin content or insulin biosynthesis. Exogenous IGF-I caused a 28% suppression of insulin release (P < .05) between days 4 and 10 of culture but induced a 49% increase in the mean secretion rate during the last week (days 25–31, P < .01). Glucagon release was not affected by exogenous IGF-I. In contrast to GH, exogenous IGF-I induced a twofold increase in the DNA content of the 7-day-cultured ICCs. However, insulin biosynthesis and release were markedly suppressed. We conclude that GH influences the functional maturation of human fetal islet cells in vitro. The effects of GH could not be reproduced by the addition of IGF-I, suggesting a direct rather than a somatomedin-mediated action for GH.
β → α → δ Pancreatic Islet Cellular Perfusion in Dogs
Authors: Stagner John I , Samols Ellis , Bonner-Weir Susan
Published: September 2013

Intraislet communication between β-, α-, and δ-cells and their secretory products may theoretically occur via the paracrine (interstitial) and/or vascular routes. Recently, we have shown that there is a directed microvascular circulation in the rat islet with a cellular order of perfusion of β → α → δ. The direction of microvascular perfusion of cells within the dog islet has been controversial. Anterograde (arterial) perfusion and retrograde (reversed or venous) perfusion of a segment of isolated dog pancreas with potent insulin antibodies yielded results similar to those found in the rat pancreas (anterograde, 158 ±44% increase in glucagon and 65 ± 20% increase in somatostatin; retrograde, no change in glucagon or somatostatin). Anterograde infusion of glucagon antibody (no change in insulin, −33.5 ± 3% decrease in somatostatin) or somatostatin antibody (no change in insulin or glucagon) also yielded the same results as in the rat pancreas. Anterograde infusion of 500 pg/ml glucagon caused a larger increase in insulin secretion (245 ± 10%) than retrograde infusion (45 ± 4%), whereas somatostatin was stimulated more retrogradely (339 ± 17%) than anterogradely (121 ± 9%). Anterograde infusion of somatostatin produced a larger decrease in insulin and glucagon than did retrograde perfusion (P < .0001 for both comparisons). The retrograde infusion of 0.3 mU/ml insulin caused a decrease in glucagon but was without effect anterogradely. The results from the infusion of exogenous hormones suggest that the sensitivity of the α-, β-, and δ-cells to insulin, glucagon, and somatostatin is determined by the β → α → δ order of perfusion. The antibody studies indicate that directed microvascular perfusion is central to intraislet regulation of insular secretions in dogs.
Association of β-Cell–Specific Expression of Endogenous Retrovirus With Development of Insulitis and Diabetes in NOD Mouse
Authors: Suenaga Kenji , Yoon Ji-Won
Published: September 2013

The administration of cyclophosphamide to nonobese diabetic (NOD) male mice produces a rapid progression to overt diabetes (>70%) with severe insulitis in 2–3 wk, whereas none of the untreated control NOD male mice became diabetic. When the thin sections of islets from the NOD male mice, which received silica for the preservation of islets and subsequently cyclophosphamide, were examined under the electron microscope, clusters of endogenous retrovirus-like particles (type A) were frequently found in the β-cells. In contrast, retroviruslike particles were rarely found in the β-cells from NOD male mice that received only silica. Other endocrine cells, including α-, δ-, pancreatic polypeptideproducing, and exocrine acinar cells, did not contain such viruslike particles. These viruslike particles were also not found in spleen, liver, or kidney in either cyclophosphamide-treated or untreated NOD male mice. There was a clear correlation between the presence of retrovirus-like particles in the β-cells and insulitis lesions in the cyclophosphamide-treated mice. On the basis of these observations, we conclude that the β-cell–specific expression of endogenous retrovirus (like particles) is associated with the development of insulitis and diabetes in NOD mice.
Induction of Insulitis by Adoptive Transfer With L3T4+ Lyt2− T-Lymphocytes in T-Lymphocyte–Depleted NOD Mice
Authors: Hanafusa Toshiaki , Sugihara Shigetaka , Fujino-Kurihara Hiroko , Miyagawa Jun-Ichiro , Miyazaki Atsushi , Yoshioka Takayuki , Yamada Kentaro , Nakajima Hiromu , Asakawa Hideki , Kono Norio , Fujiwara Hiromi , Hamaoka Toshiyuki , Tarui Seiichiro
Published: September 2013

To clarify the pathogenesis of insulitis in the nonobese diabetic (NOD) mouse, an animal model for human insulin-dependent diabetes mellitus, T-lymphocyte-depleted NOD mice (B mice) were adoptively transferred with spleen and lymph node cells from cyclophosphamide-treated NOD mice after separating the cells with monoclonal antibodies against various T-lymphocyte surface antigens plus complement. Light-microscopic and immunohistochemical studies were also performed to investigate the lymphocytic infiltrations. The incidence of insulitis detected in B mice was much lower when compared with that of the lesion naturally occurring in the NOD mouse. However, higher incidence of insulitis was inducible in B mice by transferring unfractionated lymphoid cells from NOD mice. When the Thy1+ cell-depleted fraction was transferred into the B mice, no increase in the incidence of insulitis was observed. The Lyt1+ or L3T4+ cell-eliminated fraction was also unable to transfer insulitis. Conversely, donor cells depleted of Lyt2+ components successfully induced insulitis in the recipient B mice. These data were consistent with the immunohistochemical study, which showed that the main phenotype of the cells infiltrating the islets was L3T4+. These results suggest the importance of L3T4+Lyt2− T-lymphocytes in the pathogenesis of insulitis in NOD mice.
Muscle Glycogen Synthesis and Disposition of Infused Glucose in Humans With Reduced Rates of Insulin-Mediated Carbohydrate Storage
Authors: Young Andrew A , Bogardus Clifton , Wolfe-Lopez Deborah , Mott David M
Published: September 2013

Six men with a low rate of insulin-stimulated, non-oxidative carbohydrate disposal (storage) and six with a high storage rate were recruited for study of the fate of insulin-stimulated glucose uptake. [3-3H]glucose was infused before and during a 4-h hyperinsulinemic euglycemic clamp procedure in a dosage regimen designed to maintain a constant specific activity. From the disposition of label, the rate of insulin-mediated glucose incorporation into glycogen in the low-storage subjects was one-fourth that of the high-storage subjects (P < .02). The insulin-mediated increase in muscle glycogen synthase activity in the low-storage subjects was one-fourth that of the high-storage subjects (P < .05), suggesting the possibility of a causal relationship. In the high-storage but not the low-storage subjects, the rate of glycolysis inferred from the appearance of metabolized tritium in body water exceeded the carbohydrate oxidation rate (P < .002). This suggests that in these subjects there is a significant fraction of glycolysis that is not oxidized and that this component of carbohydrate metabolism therefore contributes to storage.
Measurement of Rat Insulin: Enzyme-Linked Immunosorbent Assay With Increased Sensitivity, High Accuracy, and Greater Practicability Than Established Radioimmunoassay
Authors: Kekow Jörn , Ulrichs Karin , Müller-Ruchholtz Wolfgang , Gross Wolfgang L
Published: September 2013

Total immunoreactive insulin (IRI) is conventionally determined by radioimmunoassays. IRI measurement in rats can be made more sensitive, accurate, and practical, as demonstrated by a new modified enzyme-linked immunosorbent assay (ELISA). It is characterized by indirect binding of an anti-insulin antibody by an antiglobulin antibody and uses the principle of competitive saturation. In this ELISA, IRI can be determined in a wide range of concentrations, corresponding to the standards. The standard curve ranges from 100 to 0.049 ng/ml IRI (1 ng/ml ∼ 23.4 μU/ml ∼ 172 pM rat insulin). The statistical analysis shows between- and within-assay coefficients of variation of ≤ 15%.
Decreased Collagen Production in Diabetic Rats
Authors: Spanheimer Robert G , Umpierrez Guillermo E , Stumpf Victoria
Published: September 2013

Many of the chronic complications of diabetes mellitus involve defects in the connective tissue such as poor wound healing, diminished bone formation, and decreased linear growth. Because collagen is the major protein component of these connective tissues, we examined collagen production in diabetic rats as a probe of this generalized defect in connective tissue metabolism. Doses of streptozocin ranging from 35 to 300 mg/kg were used to induce diabetes of graded metabolic severity in rats. Parietal bone or articular cartilage was removed and incubated at 37°C with 5 μCi L-[5-3H]proline for 2 h, and collagen and noncollagen protein production were quantitated after separation with purified bacterial collagenase. Within 2 wk after induction of diabetes, collagen production was significantly reduced in bone and cartilage from diabetic rats to 52% (P < .01) and 51% (P < .01) of control (buffer-injected) levels, respectively. In contrast, noncollagen protein production in bone and cartilage from diabetic animals was no different from in tissue from control rats. The correlation between collagen relative to total protein production (relative rate) and the degree of hyperglycemia was highly significant for both bone (r = −.77, P < .001) and cartilage (r = −.87, P < .001). Other factors found to correlate with altered collagen production were the duration of diabetes and the amount of weight loss. Thus, diabetes is associated with a marked decrease in collagen production, which was seen early after induction of diabetes and was specific when compared with noncollagen protein production. Cumulative effects of these marked changes in collagen production may contribute to the chronic connective tissue complications in diabetes.