bluuebunny/crossref_metadata_2025 huggingface dataset:
Somatomedin Inhibitors From Diabetic Rat Serum Alter Growth and Development of Mouse Embryos in Culture
Authors: Sadler T W , Phillips L S , Balkan W , Goldstein S
Published: September 2013

Offspring of diabetic humans and laboratory animals have been shown to have a higher incidence of congenital malformations with attendant growth retardation. These defects have been attributed to alterations in the intrauterine environment and specifically to changes in maternal serum factors, e.g., glucose and ketone bodies. Our investigation examines the potential teratogenicity of a low-molecular-weight (940) serum fraction with demonstrated somatomedin inhibitory activity isolated by column chromatography from streptozocin-induced diabetic rats. Mouse embryos were exposed to control or the inhibitor fraction at concentrations of 0.25–0.6% vol/vol (0.9–3.0 μg protein/ml culture medium) in whole embryo culture and evaluated for the presence of malformations and growth retardation. Embryos exposed to inhibitor during the period of neurulation (3–5 somites) exhibited neural tube and craniofacial defects, whereas those exposed during early limb bud stages (18–19 somites) exhibited abnormalities of the forebrain and face. In addition, both stages were growth retarded. Control fractions produced no abnormalities. These results demonstrate a potential role for somatomedin inhibitors in diabetic embryopathy and suggest that factors other than hyperglycemie and hyperketonemia may contribute to the higher incidence of malformations among infants of diabetic mothers.
Reduction of Ischemia-Induced Acyl Carnitine Accumulation by TDGA and Its Influence on Lactate Dehydrogenase Release in Diabetic Rat Hearts
Authors: Hekimian G , Feuvray D
Published: September 2013

The contribution of long-chain acyi carnitine to increase enzyme release during ischemia was investigated both in normal and diabetic rat hearts. 2-Tetradecylglycidic acid (TDGA) was used to inhibit acyl carnitine formation. Isolated working-heart preparations were perfused with glucose (11 mM) and palmitate (0.1 mM) in control and mild ischemie conditions. Ischemia induced lactate dehydrogenase (LDH) release from both normal and diabetic hearts, but the release was higher from the diabetics over a 15-min ischemie period. The ischemia-induced tissue accumulation of long-chain acyl carnitine also was greater in diabetic hearts compared with normal hearts. When TDGA was provided in the perfusate 10 min before the addition of palmitate, levels of acyl carnitine were significantly reduced (by ∼80%) in the ischemie tissue of both groups of hearts. Similarly, LDH release from ischemie hearts was markedly decreased in the presence of TDGA. A positive correlation was shown between LDH release over the ischemie period and the tissue levels of acyl carnitine at the end of ischemia. Significant improvement in mechanical function with TDGA was only observed in ischemie diabetic hearts. There was absolutely no difference in high-energy compounds under a given perfusion condition, either with or without TDGA, between normal and diabetic hearts. It is concluded that lessening the accumulation of fatty acid intermediates, such as acyl carnitine, may be important to prevent or to limit the loss of sarcolemmal integrity under ischemie conditions, especially in diabetic hearts.
Insulin-Receptor Development in Normal and Diabetic Pregnancies: Role of Membrane Fluidity
Authors: Neufeld N D , Corbo L M
Published: September 2013

In the hyperinsulinemic offspring of the diabetic mother, both significant macrosomia and postnatal hypoglycemia are thought to be due to increased insulin sensitivity. The purpose of this study is to characterize changes in insulin-receptor development in fetal offspring of an experimental model of diabetic pregnancy. Two groups of Sprague-Dawley female rats were studied after timed mating. Both groups received injections of either vehicle (controls) or streptozocin (diabetic), 40 mg/100 g body wt, on day 7 of pregnancy and were killed at either 17, 20, or 21 days of gestation. Maternal and fetal blood were assayed for glucose and insulin, and fetal liver membranes were prepared for 125I-labeled insulin binding, lipid composition, and fluorescence polarization studies with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH).

Maternal and pooled fetal glucose levels were elevated in streptozocin-treated rats; however, pooled fetal insulin values were not elevated in the offspring of diabetic animals compared with controls (33 ± 1 vs. 50 ± 5 μU/ml). 125I-insulin binding was greater in fetal offspring of diabetic (FO) rat membranes at each gestational age studied [P < .001 by analysis of variance (ANOVA)] due to significantly greater numbers of both high- and low-affinity receptors. The highest insulinbinding capacity was seen on membranes obtained from FD rats at day 21 (9.92 M · L1 · 100 μg membranes1 vs. 6.38 M · L1 · 100 μg protein1 in fetal control (FC) rats. At each gestational age, membranes from FDs had lower values for fluorescence polarization (using the probe DPH) than did gestational-age-matched controls. These values suggest that membrane fluidity was lower in membranes from the FD group. Lipid analyses of plasma membranes showed significantly higher levels of phospholipid-to-protein ratios in samples from 20- and 21-day FD rats than in matched controls (0.297 ± 0.032 and 0.0547 ± 0.116 vs. 0.193 ± 0.001 and 0.261 ± 0.016, P < .05 by ANOVA). Significant correlations existed between insulin tracer binding and fluorescence polarization (FP) (r = −.82, P < .05) and between insulin-binding capacity and flow activation energy, a measurement derived from FP data obtained over a range of temperatures (r = − .95, P < .005).

In summary, experimental diabetic pregnancy was associated with increased expression of insulin receptors on hepatic plasma membranes. This could be correlated with certain physical properties of the membrane related to lipid content and composition. The data did not exclude the possibility that other factors such as different rates of receptor synthesis and/or degradation could be operative in fetal development in diabetic pregnancy.
Effects of Fetal Insulin Secretory Deficiency on Metabolism in Fetal Lamb
Authors: Philipps A F , Rosenkrantz T S , Grunnet M L , Connolly M E , Porte P J , Raye J R
Published: September 2013

Fetal insulin secretion may be of importance in determining both fetal metabolic rate and glucose homeostasis in the resting state. To investigate this question, streptozocin (STZ) was injected into 10 late-gestation fetal lambs, and the effects of STZ on fetal pancreatic insulin storage and secretion, fetal metabolic rate, and umbilical glucose uptake were then studied. Fetal STZ injection caused a relative fetal hyperglycemia by 24 h after injection. Fetal hyperglycemia reached a maximum by 72 h and persisted for at least 10 days after injection. Neonates delivered after fetal injection were frankly diabetic. Fetal STZ injection was associated with complete suppression of both glucose- and tolbutamide-stimulated insulin release, although no changes in peripheral insulin concentration were observed when compared with controls. Fetal pancreatic insulin content was only 13% of that expected on the basis of gestational age. In a subgroup of 7 STZ-treated fetal lambs, fetal hyperglycemia was related to decrements in umbilical venoarterial difference of glucose, umbilical glucose uptake, and glucose-O2 quotient. No changes in maternal glucose homeostasis or in fetal O2 consumption were noted. The data suggest that deficient fetal insulin storage and secretion are associated with a decrement in exogenous fetal glucose entry but not in fetal metabolic rate. Whether the observed fetal changes relate to enhanced endogenous fetal glucoseproduction with a passive decrease in maternofetal glucose transfer or are simply due to a decrease in overall fetal glucose utilization is not known. It is speculated that a quantitative decrease in pancreatic insulin secretion is responsible for the observed changes.
Primary Hypoandrogenism in Experimental Diabetes in the Long-Evans Rat
Authors: Anderson Judy E , Jones Don , Penner S Brian , Thliveris James A
Published: September 2013

The hormonal milieu of the testis was examined in streptozocin-induced diabetic (STZ-D) adult male Wistar and Long-Evans rats. Serum testosterone, creatinine, and urea nitrogen (BUN) levels and blood glucose concentrations were determined in diabetic and control Wistar rats (experiment 1). These parameters plus luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels were studied in experiments 2 and 3 with Long-Evans rats in untreated diabetic, control, insulin-treated diabetic, nondiabetic STZ-injected, and semistarved groups. Wistar diabetic rats had significantly decreased serum testosterone and increased blood glucose, BUN, and serum creatinine compared with controls. Several findings in Long-Evans rats suggested the existence of a primary Leydig cell defect in steroidogenesis during untreated diabetes that was completely or partially compensated for by increased pituitary gonadotropin secretion. Serum LH and FSH levels increased in Long-Evans diabetic rats. Serum testosterone was significantly reduced only in experiment 2. These hormonal alterations from control levels were not seen in insulin-treated diabetic animals. Semistarved animals, weight matched to the diabetic group in experiment 2, had significantly decreased serum testosterone and increased FSH levels. In addition, Long-Evans diabetic rat BUN and serum creatinine levels increased much less or were unchanged from control values compared with the increase noted in diabetic Wistar rats. In light of the hypogonadism that complicates clinical uremia, these findings suggest the more apt use of the Long- Evans strain rather than the Wistar strain in the study of STZ-D hypogonadal function. Indices to evaluate fertility (sperm count and motility) in diabetic rats in experiment 3 showed no change from those determined in control animals. The same diabetic animals, however, had a complete lack of reproductive success after pairing with cycling control females. These latter findings suggested that, in addition to a primary Leydig cell defect and resulting lowered testosterone, other factors may have contributed to the reproductive dysfunction of Long-Evans male rats with STZ-D.
Effects of Fasting on Plasma Glucose and Prolonged Tracer Measurement of Hepatic Glucose Output in NIDDM
Authors: Glauber Harry , Wallace Penny , Brechtel Ginger
Published: September 2013

We studied the measurement of hepatic glucose output (HGO) with prolonged [3-3H]glucose infusion in 14 patients with non-insulin-dependent diabetes mellitus (NIDDM).Over the course of 10.5 h, plasma glucose concentration fell with fasting by one-third, from 234 ± 21 to 152 ± 12 mg/dl, and HGO fell from 2.35 ± 0.18 to 1.36 ± 0.07 mg · kg−1 · min−1 (P < .001). In the basal state, HGO and glucose were significantly correlated (r = 0.68, P = .03), and in individual patients, HGO and glucose were closely correlated as both fell with fasting (mean r = 0.79, P < .01). Plasma [3-3H]glucose radioactivity approached a steady state only 5-6 h after initiation of the primed continuous infusion, and a 20% overestimate of HGO was demonstrated by not allowing sufficient time for tracer labeling of the glucose pool. Assumption of steadystate instead of non-steady-state kinetics in using Steele's equations to calculate glucose turnover resulted in a 9-24% overestimate of HGO. Stimulation of glycogenolysis by glucagon injection demonstrated no incorporation of [3-3H]glucose in hepatic glycogen during the prolonged tracer infusion. In a separate study, plasma glucose was maintained at fasting levels (207 ± 17 mg/dl) for 8 h with the glucose-clamp technique. Total glucose turnover rates remained constant during this prolonged tracer infusion. However, HGO fell to 30% of the basal value simply by maintaining fasting hyperglycemia in the presence of basal insulin levels. Inconclusion, elevated HGO is a major determinant of fasting hyperglycemia in NIDDM, and the close relationship of plasma glucose and HGO is maintained as both fall during a day-long fast. Insufficient time for tracer equilibration, and assumption of steady-state kinetics may result in significant overestimates of HGO. HGO is suppressed by 70% when the fall in glucose level is prevented by performing a glucose clamp at the fasting glucose level. This effect should be considered in studies with the isoglycemic clamp technique to study effects of interventions on HGO of NIDDM.
Interleukin 2 Activates BB/W Diabetic Rat Lymphoid Cells Cytotoxic to Islet Cells
Authors: Pukel Clifford , Baquerizo Hernan , Rabinovitch Alexander
Published: September 2013

We compared the cytotoxic effects to islet cells of lymphoid cells from diabetic and diabetes-resistant (DR) BioBreeding/Worcester (BB/W) rats with a 51Cr-release assay to detect lysis of normal rat islet cells. Splenic lymphoid cells from diabetic rats were more cytotoxic to islet cells (11.3 ± 3.8%) than were lymphoid cells from DR rats (4.0 ± 2.6%). This difference was amplified by incubating the lymphoid cells for 20 h with 5 μg/ml concanavalin A (ConA); islet cell lysis was 39.3 ± 4.5% by ConA-activated diabetic cells and 9.6 ± 2.7% by ConA-activated DR cells. The cytotoxic lymphoid cells were identified as natural killer (NK) cells, because treatment of diabetic lymphoid cells with anti-asialo GM1 serum and complement selectively removed a monoclonal antibody-defined subset of NK cells (OX8+), and the NK-depleted lymphoid cells were not cytotoxic to either islet or NK-sensitive YAC-1 cells, even after culture with ConA. Of several lymphokine products of ConA-stimulated lymphoid cells, interleukin 2 (IL-2), but not interleukin 1 or interferon-γ, significantly activated splenic lymphoid cells cytotoxic to islet cells, and the lymphoid cells from diabetic rats were more sensitive to IL-2 (3 U/ml) than were the cells from DR rats (30 U/ml). This study reveals the presence of ConA- and IL-2-responsive islet cytotoxic NK cells in the diabetic BB/W rat and suggests that IL-2 activation of NK cells may contribute to islet β-cell destruction and diabetes in this animal.
High Glucose Prolongs Cell-Cycle Traversal of Cultured Human Endothelial Cells
Authors: Lorenzi Mara , Nordberg Judith A , Toledo Silva
Published: September 2013

There is evidence suggesting that the diabetic state adversely affects replication of certain cell populations. We document that exposure to high ambient glucose (20 mM) induces delay in various phases of the cell cycle of human endothelial cells in primary culture. Cells in S phase were labeled with bromodeoxyuridine (an analogue of thymidine), and the cell-cycle position of the labeled cohort was analyzed by flow cytometry at successive time points. The movement of cells exposed to high glucose for 7–8 days was retarded both in S and G2 phases so that the increase in bromodeoxyuridine-positive cells over 24 h was 1.6-fold, versus 2.0-fold in control cultures. In experiments in which mitotic arrest with vinblastine was used to investigate the movement of cells out of G, phase without interference from reentering cells, depletion of the G, compartment was significantly inhibited in cultures grown in high glucose. The effects of chronic high glucose on cell cycle occurred while total protein synthesis was not diminished. Acute exposure to high glucose had no effect on cell-cycle traversal or cell generation time. Cell-cycle abnormalities observed in this study may relate to the DNA damage we have previously observed in endothelial cells exposed to high glucose and, if occurring in vivo, could be of pathogenetic importance for the vascular lesions and teratogenicity of diabetes.
Inhibition of Leukocyte Chemotaxis by Factor in Alloxan-Induced Diabetic Rat Plasma
Authors: Pereira Maria Adelaide A , Sannomiya Paulina , Leme João Garcia
Published: September 2013

The early local exudative cellular reaction in an inflammatory lesion was impaired in alloxan-induced diabetic rats due to reduced migration of neutrophils to the inflamed area. Neutrophils, however, were capable of moving from reserve compartments into blood in these animals. Furthermore, the functional integrity of their surface membranes, assessed by the capacity of the cells to adhere to nylon fiber, was not altered by alloxan diabetes. An intrinsic cellular defect also did not occur, because the cells were capable of responding to chemotactic stimuli in the Boyden chamber system, provided they were suspended in Eagle's medium or normal serum. Suspended in the corresponding diabetic serum, a blockade of the chemotactic response was observed. Increasing concentrations of diabetic serum, added to a suspension containing neutrophils collected from normal donors, progressively inhibited the response of the cells to a chemotactic stimulus. Hyperglycemia alone or hyperosmolality secondary to hyperglycemia, the presence of ketone bodies, or a direct effect of alloxan did not explain the results. In addition, the capacity to generate chemotactic factors remained intact in diabetic serum. Pretreatment of the diabetic animals with insulin resulted in a gradual recovery of the chemotactic response in vivo and in vitro. We conclude that alloxan-induced diabetic rat serum contains a substance that inhibits neutrophil chemotaxis and that insulin administration is essential for the clearance of this substance from plasma.
Na+-K+-ATPase Pumping Activity Is Not Directly Linked to myo-inositol Levels After Sorbinil Treatment in Lenses of Diabetic Rats
Authors: Yeh Li-An , Rafford Cindy E , Goddu Kym J , Ashton Michael A , Beyer Thomas A , Hutson Nancy J
Published: September 2013

Changes in tissue levels of sorbitol, myo-inositol, and Na+-K+-ATPase enzyme activity have been implicated in the development of diabetic complications in animal models of the disease and in humans. The ability of the aldose reductase inhibitor sorbinil to reverse the hyperglycemia-induced changes in these lenticular metabolite and enzyme-activity levels in the streptozocin-induced diabetic rat was examined to determine what, if any, relationship exists between these changes. Two weeks of untreated diabetes did not change ouabain-inhibitable ATPase enzyme activity assayed in lens homogenates but did result in a decrease in the Na+-K+-ATPase transport activity as measured by 86Rb uptake in the intact lens. This was accompanied by a 100-fold increase in the levels of sorbitol and significant decreases in the levels of myo-inositol, ATP, and glutathione in the lens. Whereas all of these changes could be reversed by sorbinil treatment, the dose required for restoration of the depleted myo-inositol level (ED50 > 20 mg · kg−1 · day−1) was much higher than the dose required to reverse the other changes (ED50 range 2-5 mg · kg−1 · day−1). These results suggest that the restoration of lenticular Na+-K+-ATPase activity is not secondary to a normalization of myo-inositol levels and may provide evidence that the two parameters are not strictly associated in diabetic tissues.
Thermic Effect of Glucose in Obese Subjects With Non-Insulin-Dependent Diabetes Mellitus
Authors: Ravussin Eric , Zawadzki Joanna K
Published: September 2013

A reduced thermic effect of glucose in non-insulindependent diabetes mellitus (NIDDM) has been previously reported. To investigate whether this defect is related to 1) a decreased rate of glucose storage, 2) a reduced energy cost of glucose storage, or 3) a defect in the sympathetically mediated component of thermogenesis, we studied the thermic effect of ingested and infused glucose in nine NIDDM obese Pima Indians [90.5 ± 3.9 kg, 39 ± 2% fat, fasting plasma glucose (FPG) 130 ± 10 mg/dl] and in nine nondiabetic obese Pima Indians (99.3 ± 7.2 kg, 38 ± 2% fat, FPG 103 ± 2 mg/dl). Energy expenditure and glucose storage were derived from indirect calorimetry measurements. The thermic effect of 100 g of glucose was found to be similar in both groups (4.0 ± 0.9 vs. 5.0 ± 1.3% of energy ingested in diabetic and nondiabetic subjects, respectively) but lower than that previously reported in nonobese subjects. The cost of glucose storage calculated after stimulating storage by constant glucose infusion (0.46 g/min) and variable insulin infusion (19.5 ± 3.8 vs. 2.9 ± 0.6 mU · kg−1 · min−1 in diabetic and nondiabetic subjects, respectively; P < .01) was similar in both groups (∼0.35 kcal/g glucose stored) and not different from that reported in lean subjects. As opposed to lean and obese Caucasian subjects, energy expenditure failed to markedly decrease during propranolol infusion in both nondiabetic and diabetic Pima Indians. We postulate that the decreased rate of tissue glucose uptake and storage associated with insulin resistance is the major cause of the lower thermic effect of ingested glucose in NIDDM. It is unlikely that a reduced thermic effect of glucose causes or perpetuates obesity in NIDDM, because a low thermic response is the consequence of the increased insulin resistance and is opposed by greater increases in resting metabolic rate.
Identification of a Major Defect in Insulin-Resistant Tissues of Genetically Obese (fa/fa) Rats: Impaired Protein Kinase C
Authors: Van De Werve Gerald , Zaninetti Daniel , Lang Ursula , Vallotton Michel B , Jeanrenaud Bernard
Published: September 2013

In perfused lean rat hearts, the activator of protein kinase C phorbol myristate acetate (PMA), when present alone, stimulates glucose transport but inhibits the insulin stimulation of this transport. PMA also inactivates glycogen synthase in hepatocytes. In contrast, none of these effects are observed in hearts and hepatocytes of obese animals, indicating an impaired protein kinase C activation in these tissues, which are insulin resistant. Direct measurements of protein kinase C activity in lean rat hearts revealed that PMA provokes a translocation of the enzyme from a soluble to a particulate fraction. In obese rat hearts, the basal distribution of protein kinase C is altered (more activity is found in the soluble and less in the particulate fraction), and the translocation induced by PMA is impaired. Pretreatment of lean rats with PMA in vivo, aimed at downregulating protein kinase C, induces the same defects (i.e., insulin resistance and unresponsiveness to PMA) as those observed in hearts of untreated obese animals. The results indicate that part of the insulin resistance might be the consequence of altered modulation of insulin action by protein kinase C.
Role of α2-Adrenergic Receptor in Platelet Activation During Insulin-Induced Hypoglycemia in Normal Subjects
Authors: Kishikawa Hideki , Takeda Haruo , Kiyota Satoshi , Sakakida Michiharu , Fukushima Hideo , Ichinose Kenshi , Matsuda Hiromi , Nakamura Nobuhisa , Uzawa Haruo
Published: September 2013

The effects of α2-adrenergic-receptor blocker mianserin on the responses of blood glucose, plasma β- thromboglobulin (β-TG), and various counterregulatory hormones to insulin-induced hypoglycemia were studied in nine healthy male subjects. The α2-adrenoceptor- blocking action of mianserin was confirmed by its inhibitory effect on platelet activation in vitro. Mianserin was given orally 90 min before insulin injection; the same study without mianserin was performed on another day as the control study. The time courses of blood glucose and serum C-peptide (0, 20, 45, and 180 min after the insulin injection) were identical in both studies, indicating that mianserin has no effect on these parameters. However, a significant increase of p-TG at 45 min after insulin injection was completely suppressed by the administration of mianserin (mean ± SE, 68.5 ± 6.0 vs. 28.8 ± 7.6 ng/ml, n = 6, P < .05). No significant differences were obtained between the two studies in the responses of plasma or serum catecholamines, cortisol, glucagon, growth hormone, thromboxane B2, and 6-ketoprostaglandin F1a. These results suggest that epinephrine is responsible for some, if not all, of the β-TG release from the platelets during insulin-induced hypoglycemia.
Insulin and Insulin-Receptor Autoantibodies in Children With Newly Diagnosed IDDM Before Insulin Therapy
Authors: Ludwig Sora M , Faiman Charles , Dean Heather J
Published: September 2013

Twenty-nine children, aged 1–15 yr, with newly diagnosed insulin-dependent diabetes mellitus (IDDM) had sera taken before insulin therapy to be examined for the presence of insulin-receptor antibodies by measuring the inhibition of binding of radiolabeled insulin to IM-9 lymphocytes in both whole serum and purified IgG fractions. Groups of children with long-standing IDDM and autoimmune endocrine disease as well as a normal control group were studied. A positive result, defined as binding ≥2 SD below the mean zero standard, was found in 3 (10.3%) of the 29 newly diagnosed diabetic patients. As a group, they showed significantly greater binding inhibition than the normal control group for both whole serum and purified IgG (onetailed t test, P < .05 and P < .002, respectively). Insulin autoantibodies were also measured by a sensitive radioimmunoassay technique. A positive result, defined as binding ≥3 SD above the normal control pooled sera, was found in 9 (37.5%) of 24 of the newly diagnosed IDDM group tested. All 3 subjects positive for insulin-receptor antibodies were also positive for insulin autoantibodies, whereas 6 of the 21 receptor-antibody—negative subjects were positive for insulin autoantibodies (Fisher's exact test, P = .0415). This suggests the possibility that the presence of insulin autoantibodies is a prerequisite for the development of insulin-receptor antibodies, i.e., as an anti-idiotypic response. Insulin-receptor antibodies and insulin autoantibodies may play a currently undefined pathophysiologic role in the development of IDDM. Conversely, these may represent epiphenomena of the disease process itself or may indicate a predilection for development of the disease. Taken together with other markers associated with IDDM, e.g., HLA haplotype and islet cell antibodies, it may prove possible in the future to define a select population at risk for the development of the disease and/or to predict the clinical course in individual cases.
Increase in cAMP Levels in β-Cell Line Potentiates Insulin Secretion Without Altering Cytosolic Free-Calcium Concentration
Authors: Hill Ronald S , Oberwetter James M , Boyd Aubrey E
Published: September 2013

With an SV40-transformed hamster β-cell line (HIT cells) as a model system, we tested the hypothesis that a rise in cAMP levels potentiates insulin release by an effect on the cytosolic free-Ca2+ concentration ([Ca2+])i. Intracellular cAMP levels were measured by radioimmunoassay, and ([Ca2+])i was monitored with the fluorescent Ca2+ indicator quin 2. Insulin secretion was followed in static incubations or perifusion of the cells. In perifusion, both high glucose and depolarization of the β-cell with 40 mM K+ trigger a monophasic pattern of insulin release without altering the HIT cell cAMP content. Addition of either the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) or the adenylate cyclase activator forskolin dramatically increased the cellular cAMP content, potentiated the burst phase of insulin release, and coupled the immediate phase of insulin secretion to a sustained secretory response. However, increases in cellular cAMP content were not associated with a change in ([Ca2+])i,. Thus, the potentiation of insulin secretion by a rise in cAMP in the HIT cell is not mediated by a release of stored Ca2+. Either a glucose-generated signal or a rise in ([Ca2+])i triggered by high K+ can synergize with a rise in cAMP to couple the burst or immediate release of insulin evoked by either secretagogue to the sustained release of insulin.
Inhibition of Glycogenolysis and Glycogen Phosphorylase by Insulin and Proinsulin in Rat Hepatocyte Cultures
Authors: Hartmann Heinz , Probst Irmelin , Jungermann Kurt , Creutzfeldt Werner
Published: September 2013

The inhibitory action of insulin and proinsuiin on basal and glucagon-activated glycogenolysis was studied in cultured rat hepatocytes containing [14C]glycogen. Insulin or proinsuiin given as sole hormones in the presence of 5 mM glucose decreased basal release of [14C]glucose from [14C]glycogen to 20%. Half-maximal effective concentration of insulin was ∼0.15 nM and of proinsuiin was ∼5 nM. Inhibition of [14C]lactate release from [14C]glycogen required slightly higher hormone concentrations with a similar difference in potency for insulin and proinsuiin.

The glucagon-stimulated release of [14C]glucose was completely blocked by insulin or proinsuiin with half-maximal effective concentrations of ∼0.2 and 8 nM, respectively. In contrast, release of [14C]lactate in the presence of glucagon was increased slightly by insulin and proinsuiin.

Basal and glucagon-activated phosphorylase activity was inhibited by ∼50% in a dose-dependent manner by both hormones, with differences in potency similar to those for the inhibition of glycogenolysis.

These data point to a direct regulatory role of insulin in the control of hepatic glycogen breakdown even when acting as sole hormone. The results do not support the notion of a preferential inhibitory potency of proinsuiin on hepatic glycogenolysis.
Therapeutic Modulation of Growth-Promoting Activity in Platelets From Diabetics
Authors: Sugimoto Hidekatsu , Franks Douglas J , Lecavalier Louise , Chiasson Jean-Louis , Hamet Pavel
Published: September 2013

Proliferation of vascular smooth muscle is thought to be involved in the major diabetic complication atherosclerosis. We have previously reported an increase of growth-promoting activity (GA) in platelets from insulin-dependent diabetics. In this study, GA was measured in the platelet extract (PE) from eight diabetic patients who had been treated by conventional insulin therapy. Vascular smooth muscle cells from rat aorta were cultured and used as an assay system for GA. Incorporation of [3H]thymidine into DNA of cultured cells was stimulated by diabetic PE significantly more (P < .05) than by normal PE. Diabetic PE incubated with cells for 4 days increased cell numbers significantly more (P < .05) than normal PE. These abnormalities were corrected by long-term intensive insulin treatments (continuous subcutaneous insulin infusion and Pen infuser). The decrease of platelet extract GA appeared to correlate with the amount of insulin administered before meals as shortacting boluses, whereas the level of basal or longacting insulin appeared to correlate with an increase of PE GA. Thus, the growth-promoting potential of platelets can be normalized by intensive insulin therapy. The relationship of insulin levels to this activity needs further evaluation.
HPLC Analysis of Insulin Degradation Products From Isolated Hepatocytes: Effects of Inhibitors Suggest Intracellular and Extracellular Pathways
Authors: Hamel Frederick G , Peavy Daniel E , Ryan Michael P , Duckworth William C
Published: September 2013

Isolated rat hepatocytes were incubated with A14-[125I]monoiodotyrosyl insulin for 30 min, and labeled material was extracted from the cells and incubation media. The medium and the cell extract were chromatographed on a Sephadex G-50 column, and radioactivity eluting in the position of intact insulin was concentrated and analyzed on HPLC. The HPLC analysis of the cell extract showed two major products eluting from the column at 19 and 23 min, whereas medium extracts showed one prominent product eluting at 14 min. Inclusion of chloroquine in the incubation blocked the formation of cellular products at 19 and 23 min and caused the accumulation of a product eluting at 41 min while not affecting the media products. After sulfitolysis all cellular products contained an intact A-chain. Dansylcadaverine increased media products and altered the cellextracted product pattern such that it had a major peak at 14 min, similar to media. These results suggest that two pathways for insulin degradation exist within hepatocytes. The extracellular process forms products that are essentially unchanged by chloroquine and dansylcadaverine. The intracellular process is altered by chloroquine and apparently inhibited by dansylcadaverine.
Spontaneous Reassociation of Dispersed Adult Rat Pancreatic Islet Cells Into Aggregates With Three-Dimensional Architecture Typical of Native Islets
Authors: Halban Philippe A , Powers Susan L , George Katherine L , Bonner-Weir Susan
Published: September 2013

Islets of Langerhans consist of four major endocrine cell types assembled in a highly organized manner critical for their function. The molecular forces governing islet cellular architecture are not understood. We determined whether adult rat islet cells carry information necessary for orderly assembly. Dispersed cells from adult rat islets were maintained in static suspension culture for 6 days. During this time the cells reassociated to form numerous aggregates. These aggregates were approximately half the size of native islets with a commensurate reduction in DNA and insulin content. However, both cellular composition and organization were remarkably similar to that of adult rat islets, in which the β-cells form a central core surrounded by a discontinuous mantle of non-β-cells. Thus, immunoperoxidase staining showed that in the aggregates, just as in intact islets cultured in parallel, 26% of the cells were non-β-cells and of these, 94% were clearly peripheral. Non-β-cells were similarly found to be peripheral, with β-cells located centrally, even when the ratio of non-β-cells to β-cells had been altered. This was achieved by sorting the two cell populations by fluorescence-activated flow cytometry, resulting in aggregates with 79% non-β-cells and 21% β-cells. Insulin release from the aggregates was stimulated approximately ninefold by raising glucose from 50 to 300 mg/dl, which was comparable to that found for intact islets. The spontaneous formation of isletlike aggregates displaying appropriate cellular architecture indicates that the signals (molecules) needed for such organization are intrinsic to islet cells and are still expressed by them in adult life.
Anterograde Fast Component of Axonal Transport During Insulin-Induced Hypoglycemia in Nondiabetic and Diabetic Rats
Authors: Sidenius Per , Jakobsen Johannes
Published: September 2013

To elucidate the pathogenesis of the peripheral neuropathy associated with hypoglycemia the anterograde fast component (aFC) of axonal transport was studied in nondiabetic rats during acute and prolonged insulin-induced hypoglycemia and in streptozocin-diabetic (STZ-D) rats with acute hypoglycemia. [35S]methionine and [3H]fucose were injected into the dorsal root ganglion (L5) to label protein and glycoprotein, respectively. During the 4 h of transport, thigh temperature was maintained constant. Acute severe hypoglycemia (1.5 ± 0.2 mM) was associated with a 36% decrease in the amount of aFC (2.3 ± 0.7% in the test group vs. 3.6 ± 0.8% in the controls), whereas transport velocity was unaffected. Prolonged hypoglycemia, obtained by pretreatment with insulin for 3 days, prevented the decrease in amount of aFC. In STZ-D rats, acute severe hypoglycemia (1.5 ± 0.6 mM) produced a similar but less-pronounced decrease of aFC. We conclude that hypoglycemia is associated with alterations in axonal transport that could play a role in development of neuropathy. Prolonged hypoglycemia protects axonal transport against the effects of glucopenia, and an untreated diabetic state maintained for several days has a partially protective effect against episodes of hypoglycemia.
Presence of Pancreatic Hormones in Islet Cells With MHC-Class II Antigen Expression
Authors: Pipeleers Daniel G , In't Veld Peter A , Pipeleers-Marichal Miriam A , Gepts Willy , Van De Winkel Marnix
Published: September 2013

In the normal rat pancreas, only few islet cells express MHC-class II antigens. Their nature and function has not yet been elucidated. We report a method for the purification of la-positive islet cells by fluorescenceactivated cell sorting. The isolated mononuclear cells appear of nonendocrine origin but contain vacuoles with partially digested secretory vesicles. Both insulinand glucagon-immunoreactive granules were identified in these vacuoles of varying size and composition. It is suggested that at least part of the rat islet cells with class II antigen expression can exhibit phagocytotic properties leading to the uptake of fragments from damaged endocrine cells. This functional characteristic may implicate this particular islet cell type in the pathology of the endocrine pancreas in type I diabetes.
Alterations in Organization of Phospholipids in Erythrocytes as Factor in Adherence to Endothelial Cells in Diabetes Mellitus
Authors: Wali Ramesh K , Jaffe Stuart , Kumar Dinesh , Kalra Vijay K
Published: September 2013

Erythrocytes from patients with diabetes mellitus exhibit increased adherence to cultured human vascular endothelial cells. We investigated the alterations in erythrocyte surface characteristics that may contribute to their abnormal adherence. The organization of phospholipids in the lipid bilayer, as determined by phospholipase A2 treatment and chemical labeling with fluorescamine and trinitrobenzene sulfonic acid (TNBS), is altered in erythrocytes from diabetic patients. Specifically, 12–18% of phosphatidylserine in diabetic erythrocytes (n = 25) is accessible to phospholipase A2 hydrolysis and TNBS labeling, compared to none in normal subjects. These results suggest either a loss in lipid asymmetry or in vivo destabilization of erythrocyte membranes in diabetic patients, causing increased accessibility to phospholipase A2 degradation. The dye merocyanine 540 (MC-540), which is sensitive to the packing of lipids in the bilayer of the membrane, revealed more binding and fluorescence in erythrocytes from diabetic patients than in those from normal subjects. On flow cytometric analysis, 64.5 ± 17.0% red blood cells (RBCs) in diabetic patients, compared to 35.1 ± 25.9% RBCs in normal subjects, showed positive MC-540 binding, indicating significant (P < .001) differences in the packing of lipids in the external leaflet of the bilayer. The results of our study suggest that a loss of lipid asymmetry and/or less ordered packing in the outer leaflet of the diabetic erythrocyte membrane may be responsible for the increased propensity of erythrocytes to adhere to vascular endothelium.
Destruction of Rat Islet Cell Monolayers by Cytokines: Synergistic Interactions of Interferon-γ, Tumor Necrosis Factor, Lymphotoxin, and Interleukin 1
Authors: Pukel Clifford , Baquerizo Hernan , Rabinovitch Alexander
Published: September 2013

An assay was developed to detect the cytotoxic effects of cytokines on rat pancreatic islet cells in monolayer culture. Cell lysis was detected by a 51Cr-release assay after 4 days of incubation with various cytokines. When tested alone, murine (rat and mouse) interferon-γ (mIFN-γ) produced a small dose-dependent lysis of islet cells; human IFN-γ, mouse IFN-α/β, interleukins 1 and 2 (IL-1 and IL-2), tumor necrosis factor (TNF), and lymphotoxin (LT) were inactive. When added together, the following combinations of cytokines showed synergistic cytotoxic effects: TNF (or LT) plus IL-1, TNF (or LT) plus mIFN-γ, and IL-1 plus mIFN-γ. These results indicate that the cytokine products of mononuclear cells of the immune system, IFN-γ, TNF, LT, and IL-1 have strong synergistic cytotoxic effects on islet cells and therefore may act as direct chemical mediators of islet β-cell destruction in type I (insulin-dependent) diabetes.
Effect of Insulin Therapy on Established Diabetic Nephropathy in Rats
Authors: Petersen Jeffrey , Ross Jon , Rabkin Ralph
Published: September 2013

The effectiveness of insulin therapy on early diabetic nephropathy has not been established. In this study we examined the influence of continuous subcutaneous insulin on the progression of established nephropathy in streptozocin-induced diabetic rats. Normal controls and diabetic rats were studied for 11 mo. During the first 6 mo, all the diabetic rats received 2 U protamine zinc insulin s.c. twice weekly. During the last 5 mo of study, diabetic rats either continued on the occasional subcutaneous insulin regimen or received regular insulin by continuous subcutaneous infusion. Six months after the initiation of the study, the diabetic rats were severely hyperglycemie, and their relative mesangial areas had increased. Continued poor glycemie control in the rats receiving occasional insulin was associated with relative increased mesangial area (25.2 ± 1.0% of glomerular area) and significant proteinuria (148 ± 17 mg/24 h) compared with normal controls. In contrast, the use of continuous subcutaneous insulin therapy with improved glycemie control arrested mesangial changes (19.5 ± 1.4% of glomerular area) and prevented the excessive proteinuria (71 ±13 mg/24 h). Indeed, these parameters did not differ from age-matched controls. We conclude that in the rat, continuous subcutaneous insulin therapy instituted after the development of early glomerular pathology is effective in arresting the disease process.
Cyclosporin-Induced Remission of IDDM After Early Intervention: Association of 1 yr of Cyclosporin Treatment With Enhanced Insulin Secretion
Authors:   The Canadian-European Randomized Control Trial Group
Published: September 2013

A randomized double-blind placebo-controlled trial was undertaken to determine whether cyclosporin enhances remission of insulin-dependent diabetes mellitus (IDDM) through the 1st yr after diagnosis. Dosage with insulin was minimized with target control of blood glucose levels ≤7.8 mM (140 mg/dl) before meals. Metabolic control was evaluated by serial determinations of glycosylated hemoglobin levels, and endogenous secretion of insulin was evaluated by determination of the levels of glucagon-stimulated insulin-connecting peptide (CP) in the plasma at 3-mo intervals. A compound definition of remission required a glucagon-stimulated CP level in plasma ≥0.6 nM or a non-insulin-receiving state (NIR) in which target control of glycemia was maintained without administration of insulin. A clinical definition of remission required only the NIR state as defined. One hundred eighty-eight patients aged 10–35 yr entered the study within 6 wk of initiation of insulin therapy and within 14 wk of onset of symptoms and were studied for 1 yr. There were no significant differences in metabolic control between the two treatment groups during the study. The anticipated adverse effects of cyclosporin were not more frequent or severe than in other experience with the drug, but histological changes attributable to cyclosporin were present in some kidney biopsies obtained from selected patients after 1 yr. At 1 yr, by the compound definition, 33% of the cyclosporin-group and 21% of the placebo-group patients were in remission, when the corresponding rates for NIR remissions were 24 and 10%. With adjustment for differences in the baseline variables, the differences in rates between the treatment groups were statistically significant in all comparisons, and the relative odds for NIR remission at 1 yr were increased approximately five times in the cyclosporin-treated group. Furthermore, a relatively short duration of diabetes (entry ≤6 wk after onset of symptoms, with ≤2 wk of insulin therapy) was associated with greater efficacy of cyclosporin. The mean glucagon-stimulated CP levels in the plasma in cyclosporin-treated patients exceeded those in placebo-treated patients after 3 mo and were maintained through 12 mo, in contrast to a decline after 3 mo in the placebo group. Thus, the results show that cyclosporin treatment initiated soon after diagnosis increases the rate of remission and enhances β-cell function during the 1st yr of IDDM. We conclude that a continuing search for clinically beneficial immunomodulatory therapies designed to prevent or ameliorate the disease is feasible and worthwhile.
NOD Marrow Stem Cells Adoptively Transfer Diabetes to Resistant (NOD × NON)F1 Mice
Authors: Serreze David V , Leiter Edward H , Worthen Sherri M , Shultz Leonard D
Published: September 2013

Autoimmune β-cell destruction occurred in otherwise diabetes-resistant F1 mice from an outcross between the nonobese diabetic (NOD) and nonobese normal (NON) inbred strains after adoptive transfer of hematopoietic stem cells from NOD donors. F1 mice were lethally irradiated and reconstituted with either NOD, NON, or F1 bone marrow. Only F1 mice reconstituted with NOD bone marrow developed hyperglycemia. The long (≥ 16-wk) prodromal period required for expression of overt diabetes contrasted with the rapidity (4–6 days) with which kidney-grafted F1 or NON islets (but not anterior pituitary) were eliminated from diabetic F1 mice. Thus, development of β-cell-specific immunologic effectors was a chronic process, but once sufficient levels of autoimmunity were achieved, implanted β-cells could be eliminated in an acute fashion. Thus, expression of NOD diabetogenic alleles in hematopoietic progenitor cells is sufficient for development of anti-β-cell immunity. The elimination of grafted NON islets shows the effectors are capable of eliminating β-cells from mice without the diabetogenic genotype.
Decreased Activation of Skeletal Muscle Glycogen Synthase by Mixed-Meal Ingestion in NIDDM
Authors: Wright Kimberly S , Beck-Nielsen Henning , Kolterman Orville G , Mandarino Lawrence J
Published: September 2013

Glycogen synthase (GS) catalyzes the formation of glycogen in human skeletal muscle, the tissue responsible for disposal of a significant portion of an oral carbohydrate load. Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by fasting and postprandial hyperglycemia in conjunction with reduced rates of insulin-stimulated glucose disposal and storage in peripheral tissues, including muscle. Our objectives in this study were to determine whether ingestion of a mixed meal activates GS in control nondiabetic subjects and whether meal-related GS activation is reduced in NIDDM. To accomplish this, mixed formula meals were administered to 11 NIDDM and 9 age- and weight-matched nondiabetic control subjects. Plasma glucose and insulin values were measured before and for 90 min after meal ingestion. Skeletal muscle biopsies were performed just before and 90 min after meal ingestion for measurement of GS activity. Compared with control subjects, NIDDM subjects had significantly higher postprandial hyperglycemia and reduced postprandial hyperinsulinemia. GS was activated by meal ingestion in control subjects to a significantly greater extent than in NIDDM subjects. In NIDDM subjects, activation of GS was inversely correlated with fasting plasma glucose (r = .69, P < .05). Therefore, NIDDM is characterized by reduced activation of a key step in the process of muscle glycogen repletion after a meal. Reduced activation of GS by a mixed meal in NIDDM may contribute to the reduced glucose disposal after a meal, thus contributing to the hyperglycemia observed in these subjects.