bluuebunny/crossref_metadata_2025 huggingface dataset:
Reversible Impairment of Glucose-Induced Insulin Secretion in SHR/N-cp Rats: Genetic Model of Type II Diabetes
Authors: Voyles Nancy R , Powell Andrea M , Timmers Kim I , Wilkins Sam D , Bhathena Sam J , Hansen Carl , Michaelis Otho E , Recant Lillian
Published: September 2013

The SHR/N-cp rat is a new genetically obese model for non-insulin-dependent diabetes mellitus. Expression of the diabetes is enhanced by a high-sucrose (54%) diet. After 4 wk on the diet, the cp/cp rats weigh significantly more than their +/? controls, have postprandial hyperglycemia (> 400 mg/dl), and are hyperinsulinemic, with immunoreactive insulin (IRI) levels 10- to 20-fold > controls. Total pancreatic IRI tends to be increased 1.6-fold in the cp/cp rats (although not significantly). There is no increase in pancreatic proinsulin content as a percent of total IRI. Studies of in vitro pancreatic function were carried out with the isolated nonrecirculating perfused pancreas method. The cp/cp rats (n = 10) showed impaired or absent IRI responses to 16.5 mM glucose, whereas +/? rats (n = 9) responded with classic biphasic curves. Comparison of insulin secreted in 20 min revealed a > 53% decrease in IRI secretion in cp/cp rats (P < .05). A paradoxical hypersecretion of IRI at glucose concentrations of 0–2.7 mM was noted in cp/cp but not lean rats, i.e., 1.8 ± 0.2 mU/min IRI in cp/cp rats vs. 0.04 ± 0.007 mU/min in +/? rats. Perfusion of pancreases for 45 min with buffers containing no glucose resulted in restoration of a normal biphasic IRI response to 16.5 mM glucose in the cp/cp rats, whereas response in the lean rats was markedly reduced. Brisk IRI responses to 10 mM arginine in buffers with no glucose also occurred in cp/cp but not +/? rats. Glucagon secretion was relatively suppressed in the cp/cp rats. These findings are similar to those reported in glucose-infused normal rats and suggest that hyperglycemia per se may be responsible for the impaired β-cell responses to glucose in cp/cp rats.
Automated Method for Isolation of Human Pancreatic Islets
Authors: Ricordi Camillo , Lacy Paul E , Finke Edward H , Olack Barbara J , Scharp David W
Published: September 2013

We describe an automated method for the isolation of human pancreatic islets. The procedure meets the following requirements: 1) minimal traumatic action on the islets, 2) continuous digestion in which the islets that are progressively liberated can be saved from further enzymatic action, 3) minimal human intervention in the digestion process, and 4) high yield and purity of the isolated islets. After purification on Ficoll gradients, an average of 164,600 islets/pancreas was obtained (2279 islets/g), with an average purity of 78.5% islets. The average volume and average insulin content of the final islet preparation were 348 mm3 and 93.4 U, respectively. The islets were morphologically intact with a normal degree of β-granulation and responded to glucose stimulation with a fivefold increase of insulin secretion over basal levels. The procedure is now being used for the initiation of the second phase of clinical trials on human islet transplants.
Congenital Susceptibility to NIDDM: Role of Intrauterine Environment
Authors: Pettitt David J , Aleck Kirk A , Baird H Robert , Carraher Michael J , Bennett Peter H , Knowler William C
Published: September 2013

Non-insulin-dependent diabetes mellitus (NIDDM) during pregnancy in Pima Indian women results in offspring who have a higher prevalence of NIDDM (45%) at age 20–24 yr than in offspring of nondiabetic women (1.4%) or offspring of prediabetic women (8.6%), i.e., women who developed diabetes only after the pregnancy. These differences persist after taking into account paternal diabetes, age at onset of diabetes in the parents, and the offspring's weight relative to height. The findings suggest that the intrauterine environment is an important determinant of the development of diabetes and that its effect is in addition to effects of genetic factors.
Developmental Regulation of Insulin and Type I Insulin-Like Growth Factor Receptors and Absence of Type II Receptors in Chicken Embryo Tissues
Authors: Bassas Lluis , Lesniak Maxine A , Serrano Jose , Roth Jesse , De Pablo Flora
Published: September 2013

Chicken embryos are a suitable model for studying the role of insulin, insulin-like growth factors I and II (IGF-I and IGF-II), and their receptors in embryogenesis. We show that plasma membranes from heart, liver, and limb buds, as reported earlier for brain, each have a distinct developmental profile for insulin receptors and type I IGF receptors. In heart and limb buds, IGF binding is higher than insulin binding, but in liver, insulin receptors dominate. Expression of these receptors is, therefore, developmentally regulated and tissue specific. The wide distribution of high-affinity receptors capable of mediating insulin and IGF actions in early organogenesis further supports the possible importance of this family of peptides for differentiation and growth in vertebrates. In all chicken embryo tissues studied, both IGF-I and IGF-II appeared to bind to a type I IGF receptor. We have not detected a receptor with the peptide binding and structural characteristics of the mammalian type II IGF receptor. The type II receptor was absent in embryos, liver from newly hatched chicks, and adipocytes from older chicks, which suggests that the chicken may lack this subtype of IGF receptor.
Circulating Levels and Bone Contents of Bone γ-Carboxyglutamic Acid-Containing Protein Are Decreased in Streptozocin-Induced Diabetes: Possible Marker for Diabetic Osteopenia
Authors: Ishida Hitoshi , Seino Yutaka , Taminato Tomohiko , Usami Masaru , Takeshita Noritaka , Seino Yoshiki , Tsutsumi Chiharu , Moriuchi Sachiko , Akiyama Yasuhiro , Hara Kuniko , Imura Hiroo
Published: September 2013

To investigate the pathophysiology of diabetic osteopenia, circulating levels and bone contents of bone γ-carboxyglutamic acid-containing protein (BGP) were measured in streptozocin-induced diabetic rats . Plasma calcium and total protein were significantly decreased (P < .01) in the diabetic group, and the plasma level of BGP in diabetic rats was 19.6 ± 2.8 (mean ± SE) ng/ml, which is significantly lower than the value of 89.2 ± 14.0 ng/ml in control rats (P < .01). Bone contents of calcium and hydroxyproline per femur were significantly decreased in the diabetic group (P < .01), and the ratios of bone calcium to hydroxyproline were not different. Bone BGP content per femur in the diabetic group was 669 ± 58 μg, which was also significantly lower compared with 1241 ± 126 μg in control rats (P < .01). The decreased bone content of BGP is consistent with the hypothesis that BGP synthesis is impaired in insulin-deficient diabetes. Because a relationship between plasma levels of BGP and bone turnover has been established, the low plasma BGP value suggests there is a decrease in bone turnover in diabetic rats. Therefore, we postulate that the low bone turnover is one of the pathological features of diabetic osteopenia and is at least partly responsible for the occurrence of this complication in diabetes mellitus.
Insulin Action and Insulin Secretion in Identical Twins With MODY: Evidence for Defects in Both Insulin Action and Secretion
Authors: Beck-Nielsen Henning , Nielsen Ole Hother , Pedersen Oluf , Bak Jens , Faber Ole , Schmitz Ole
Published: September 2013

To evaluate the pathogenetic mechanisms responsible for development of diabetes in the genetically inherited disease maturity-onset diabetes of the young (MODY), we have investigated a pair of identical twins (19 yr old) from a MODY family. One twin had nondiabetic fasting plasma glucose values but impaired glucose tolerance (IGT), whereas the other suffered from frank diabetes (fasting plasma glucose 12.5 mM). Differences in insulin secretion pattern and/or insulin action between the twins is supposed to be responsible for development of hyperglycemia in MODY. On the other hand, identical defects in insulin secretion and action in the twins may point to the primary genetic defect in MODY. Therefore, our aim was to investigate insulin secretion and insulin action in the twins to find these differences and similarities. We found that fasting plasma insulin and C-peptide values were slightly increased in the twins, whereas the responses of insulin and C-peptide to oral glucose tolerance tests (OGTT) and meals were similar in the twins and within normal range. The insulin responses to OGTT were, however, lower than expected from the glucose values, indicating a β-cell defect. Despite elevated plasma insulin levels, basal hepatic glucose output (HGO) was normal in the IGT twin but increased by 75% in the diabetic twin. The maximally inhibitory effect of insulin on HGO, when estimated at euglycemia, was normal in the IGT twin but reduced by 60% in the diabetic twin. Furthermore, the maximal insulin-mediated glucose uptake in peripheral tissues was reduced by 40% in the diabetic twin. lular defect that is responsible for the insulin resistance appears to be a reduced cellular insulin binding and, in surplus, a postreceptor defect in glucose metabolism in the diabetic twin. These data indicate that a cellular defect in insulin action at the postreceptor level may be responsible for the progression from a state of IGT to frank diabetes, whereas the genetic defect in MODY may be localized to the β-cells. A primary insulin-receptor defect, however, cannot be excluded.
Physiological Importance of Deficiency in Early Prandial Insulin Secretion in Non-Insulin-Dependent Diabetes
Authors: Bruce David G , Chisholm Donald J , Storlien Leonard H , Kraegen Edward W
Published: September 2013

Patients with non-insulin-dependent diabetes mellitus (NIDDM) have a deficiency in early prandial insulin secretion. To determine the contribution of this early deficiency to prandial hyperglycemia, exogenous intravenous insulin (1.8 U over 30 min) was delivered to eight NIDDM subjects in a profile designed to simulate the normal initial rise in insulin levels. The same dose of insulin was also administered 1) in the same profile but delayed by 30 min and 2) as a constant infusion over 180 min. Augmentation of the early insulin response caused a 33 ± 4% reduction in the glycemic response to a mixed meal (P < .005); the peak blood glucose increment above baseline was reduced by 1.4 mM (P < .005) to an increment identical to nondiabetic subjects (3.3 ± 0.3 vs. 3.2 ± 0.2 mM), and blood glucose levels were still 0.9 mM lower after 180 min (P < .05). In contrast, the delayed profile or constant infusion did not significantly alter the glycemic response to the meal. Early insulin augmentation resulted in elevated peripheral insulin levels initially (peak level 81 ± 11 mU/L), but subsequent insulin and C-peptide levels were lower than in the control study (at 180 min after the meal, 22 ± 5 vs. 33 ± 8 mU/L, P < .05, and 4.0 ± 0.5 vs. 5.3 ± 0.6 μg/L, P < .02, respectively). Early insulin delivery caused free-fatty acid (FFA) levels to fall at a faster rate after the meal and also attenuated the initial rise in glucagon levels typical of NIDDM. We conclude that the deficiency in early prandial insulin secretion contributes to prandial hyperglycemia and late hyperinsulinemia and may be partially responsible for the abnormal FFA and glucagon responses in NIDDM.
Maintenance of High-Energy Brain Phosphorous Compounds During Insulin-Induced Hypoglycemia in Men: 31P Nuclear Magnetic Resonance Spectroscopy Study
Authors: Hilsted Jannik , Jensen Karl Erik , Thomsen Carsten , Larsen Steen , Henriksen Ole
Published: September 2013

31P nuclear magnetic resonance (NMR) spectroscopy allows noninvasive studies of cerebral energy-rich phosphorous compounds in humans. In an attempt to characterize the relationship between peripheral blood glucose concentrations and whole-brain phosphate metabolism during insulin-induced hypoglycemia, 31P NMR spectra were obtained before and after intravenous injection of insulin (0.15 IU/kg body wt) in six men. Compared with prehypoglycemic measurements, no significant changes were found in brain content of Pi, sugar phosphates, phosphocreatine, phosphodiesters, and ATP, and brain pH remained constant during the experiment. These results show that the integrated brain profile of energy-rich phosphorous compounds is unaffected by experimental insulin-induced hypoglycemia in humans.
Taste-Induced Changes in Plasma Insulin and Glucose Turnover in Lean and Genetically Obese Rats
Authors: Ionescu Elisabeth , Rohner-Jeanrenaud Françoise , Proietto Joe , Rivest Robert W , Jeanrenaud Bernard
Published: September 2013

Cephalic-phase insulin release (CPIR) and the changes in glucose turnover induced by saccharin ingestion were studied in freely moving lean and genetically obese fa/fa rats equipped with chronic catheters for blood sampling. Six-hour-fasted lean and obese rats were trained to drink 1 ml sodium saccharin (0.15%) or 1 ml glucose (70%), and blood samples were taken before and after the stimuli. As early as 1–1.5 min poststimulus, there was a significant increase in CPIR in lean and obese rats. The amplitude of the CPIR induced either by saccharin or by glucose in the obese rats was significantly higher than it was in the lean rats. The effect of saccharin ingestion on the hepatic glucose production (HGP) and the rate of glucose disappearance (Rd) was studied in 6-h-fasted lean and obese rats, under non-steady-state conditions, according to a method previously validated. Saccharin ingestion produced a significant increase in HGP and Rd in lean and obese rats compared with basal values. The saccharin-induced increments in HGP and Rd were higher in the obese than in the lean animals. We conclude that saccharin (through taste) appears to elicit parasympathetic (insulin release) and sympathetic (HGP increase) reflexes in lean and obese rats. These taste-induced changes in plasma insulin and glucose turnover are exaggerated in the obese rats and may participate in obesity and in insulin resistance of the overall syndrome.
Evidence for Two Independent Pathways of Insulin-Receptor Internalization in Hepatocytes and Hepatoma Cells
Authors: McClain Donald A , Olefsky Jerrold M
Published: September 2013

A study of insulin-receptor internalization and recycling was undertaken in primary cultures of rat hepatocytes and a human hepatoma cell line (HepG2). Receptors were quantitated by measuring 125I-insulin binding to partially purified soluble receptor preparations from untreated cells (total receptors) and trypsinized cells (intracellular receptors). In resting HepG2 cells, exposure to insulin results in internalization of insulin receptors, the rate and extent of which is dependent on the insulin concentration. However, receptors do not accumulate inside the cell in proportion to the higher rates of internalization at high concentrations of insulin. This lack of accumulation is explained by much higher recycling rates after exposure to high concentrations of insulin. Similar results were noted for primary cultures of rat hepatocytes. These results imply qualitatively different fates for receptors internalized after exposure to different concentrations of insulin. To further investigate the possibility of different pathways for insulin-receptor internalization and processing, cells in low (1 ng/ml) or high (100 ng/ml) concentrations of insulin were exposed to drugs or treatments known to affect receptor metabolism. Hypotonic shock and hypokalemia, which arrest coated-pit formation, blocked internalization of insulin and insulin receptors at low concentrations of insulin but allowed internalization in response to high concentrations of insulin. The lysosomotropic drugs monensin and chloroquine caused intracellular accumulation of insulin and its receptors internalized at low concentrations of insulin but had a relatively smaller effect on receptors internalized at high concentrations of insulin. All internalization is blocked by 2,4-dinitrophenol. We conclude that high doses of insulin lead to insulin-receptor internalization and recycling through a pathway that is functionally distinct from the pathway taken by receptors internalized by low (physiologic) concentrations of insulin. The pharmacologic experiments raise the possibility that the high-dose pathway, unlike the low-dose pathway, may proceed independently of coated pits and endosomal acidification.
Induction of Memory in Rat Pancreatic Islets by Tolbutamide: Dependence on Ambient Glucose Level, Calcium, and Phosphoinositide Hydrolysis
Authors: Zawalich Walter S , Zawalich Kathleen C
Published: September 2013

The ability of the sulfonylurea tolbutamide to induce insulin output, increase phosphoinositide (PI) hydrolysis, and modulate the insulin response to other agonists was assessed. At 200 μM, tolbutamide increased both insulin release and the efflux of 3H from [3H]inositol-prelabeled islets only in the presence of 5.5 or 7 mM glucose. When the glucose level was maintained at 2.75 mM, tolbutamide (200 μM) had no positive impact on either parameter. The calcium-influx inhibitor nitrendipine (200 nM) blocked the effects of 200 μM tolbutamide (with 7 mM glucose) on 3H efflux and insulin output. Prior exposure of islets to tolbutamide (200 μM) in the presence of 7 mM glucose amplified their subsequent insulin response to 10 mM glucose and 5 mM glyceraldehyde. The effect of 200 μM tolbutamide (with 7 mM glucose) was blocked by nitrendipine. Furthermore, the effect of 200 μM tolbutamide was not observed with 2.75 mM glucose; however, if the level of tolbutamide was increased to 1 mM, both PI hydrolysis and potentiated release to subsequent stimulation with 10 mM glucose were observed. Tolbutamide (200 μM with 7 mM glucose) stimulation for 20 min resulted in an increase in 3H efflux from [3H]inositol-prelabeled islets. Despite the rapid fall in insulin secretion, elevated rates of 3H efflux persisted long after the removal of the sulfonylurea from the medium. The duration of the 3H-efflux response paralleled the duration of potentiation. Furthermore, nitrendipine (200 nM) blocked the sustained increase in 3H efflux usually noted after tolbutamide, indicating that this efflux represented the continued hydrolysis of PI and was not simply the result of the slow efflux and/or degradation of preformed labeled inositol phosphates. Direct measurements of labeled inositol phosphate accumulation substantiate all the 3H-efflux data. These results support the following conclusions: 1) tolbutamide increases PI hydrolysis in islets, an effect dependent on the level of tolbutamide, the ambient glucose concentration, and calcium influx; 2) once initiated, the increase in PI hydrolysis is sustained even after the removal of tolbutamide; and 3) biochemical events in the inositol cycle appear at least partly responsible for time-dependent potentiation (memory) to subsequent stimulation induced by prior exposure to tolbutamide.
Sulfonylurea Receptors, Ion Channels, and Fruit Flies
Authors: Boyd Aubrey E
Published: September 2013

Recent studies have identified a high-affinity receptor on the plasma membrane of the β-cell that is specific for all of the sulfonylureas. The most potent secondgeneration drugs, glyburide and glipizide, bind to the receptor and trigger insulin release at nanomolar concentrations. The affinity to the receptor-ligand interaction of all sulfonylureas correlates with their potency as insulin secretagogues, further implicating receptor occupancy with signal transduction. These drugs also inhibit the electrical activity of ATPsensitive K+ channels and K+ efflux through these channels. The channels are also closed by the metabolism of the major insulin secretagogues, glucose and the amino acids, which signal insulin release by increasing the ATP level or the [ATP]-to-[ADP] ratio on the cytoplasmic side of the channel. Based on the channel number and the amount of K+ current they pass, it is possible to calculate that these channels control the resting membrane potential of the β-cell. Inactivation of the ATP-inhibitable K+ channel results in a fall in the resting membrane potential, cell depolarization, and influx of extracellular Ca2+ through the voltage-dependent Ca2+ channel. The rise in intracellular free Ca2+ level triggers exocytosis. Thus, it is now possible to link either a stimulus from the metabolism of insulin secretagogues or the sulfonylureas to ionic and electrical events that elicit insulin release. These data also suggest that the sulfonylurea receptor or a closely associated protein is an ATP-sensitive K+ channel.
Use of In Vitro DNA Amplification to Screen Family Members for an Insulin Gene Mutation
Authors: Miyano Motoshige , Nanjo Kishio , Chan Shu Jin , Sanke Tokio , Kondo Michi , Steiner Donald F
Published: September 2013

The DNA polymerase chain reaction can be a powerful tool for amplifying selected segments of genomie DNA for investigation of point mutations that are inaccessible via classic restriction-fragment–length polymorphism analysis. We have applied this method to an analysis of the incidence of heterozygosity for the mutant insulin allele insulin Wakayama (A3 Val→Leu) in two unrelated Japanese families having the hyperinsulinemic mutant insulin syndrome. The results indicate that this method is simple, sensitive, and accurate and should be useful for screening larger (diabetic) populations to detect single-base substitutions in the insulin gene that lead to either altered (pro)insulin structure and/or insulin production.
Glucose Transport Is Rate Limiting for Skeletal Muscle Glucose Metabolism in Normal and STZ-Induced Diabetic Rats
Authors: Ziel Frederick H , Venkatesan Natarajan , Davidson Mayer B
Published: September 2013

To test the hypothesis that glucose transport is rate limiting for skeletal muscle glucose metabolism, intracellular free glucose was measured in in situ abdominal wall muscle and liver that were rapidly freeze-clamped and removed from normal and streptozocin-induced diabetic (STZ-D) Sprague-Dawley rats exposed to different concentrations of serum insulin and glucose achieved by 90-min glucose clamps. Three groups of anesthetized normal (n = 17) and three groups of STZ-D (n = 19) animals were studied. In each group, infusions were adjusted to expose rats to basal, euglycemic-hyperinsulinemic, and hyperglycemic-hyperinsulinemic conditions. Final steady-state serum glucose and insulin concentrations (mean ± SE) achieved in the six groups ranged from 112 ± 2 to 467 ± 26 mg/dl and from 5.8 ± 1.9 to 3644 ±116 μU/ml, respectively. Intracellular free-glucose content of all tissue was calculated from total tissue glucose content minus the contribution of glucose in extracellular water estimated with [14C]inulin. Intracellular free glucose was not detected in muscle of any of the experimental groups. However, intracellular free glucose was detected in liver from all normal and STZ-D rats, and the calculated intracellular concentrations of free glucose correlated with serum glucose concentrations (r = .68, P < .001). We conclude that intracellular free glucose does not accumulate, regardless of glucose or insulin concentration, in normal skeletal muscle and muscle made insulin resistant (by STZ-D), suggesting that glucose transport remains rate limiting.
Evidence for Initial Involvement of Macrophage in Development of Insulitis in NOD Mice
Authors: Lee Ki-Up , Amano Kazuhiko , Yoon Ji-Won
Published: September 2013

Macrophages have been shown to be the major population of infiltrated immunocytes at the early stage of insulitis in diabetes-prone BB rats. This study was undertaken to investigate the role of macrophages in the development of insulitis in nonobese diabetic (NOD) mice. Administration of cyclophosphamide to NOD mice resulted in a significant increase in the incidence of overt diabetes and severity of insulitis compared with that in untreated NOD mice. Intraperitoneal injections of silica completely prevented the development of diabetes and insulitis in both cyclophosphamide-treated and untreated animals. Because silica is selectively toxic to macrophages, the results suggest that macrophages play an important role in the initiation of insulitis in NOD mice.
Modeling Error and Apparent Isotope Discrimination Confound Estimation of Endogenous Glucose Production During Euglycemic Glucose Clamps
Authors: Finegood Diane T , Bergman Richard N , Vranic Mladen
Published: September 2013

We previously demonstrated that conventional tracer methods applied to euglycemic-hyperinsulinemic glucose clamps result in substantially negative estimates for the rate of endogenous glucose production, particularly during the first half of 180-min clamps. We also showed that addition of tracer to the exogenous glucose infusate resulted in nonnegative endogenous glucose production (Ra) estimates. In this study, we investigated the underlying cause of negative estimates of Ra from conventional clamp/tracer methods and the reason for the difference in estimates when tracer is added to the exogenous glucose infusate. We performed euglycemic-hyperinsulinemic (300-μU/ml) clamps in normal dogs without (cold GINF protocol, n = 6) or with (hot GINF protocol, n = 6) tracer (D-[3-3H]glucose) added to the exogenous glucose infusate. In the hot GINF protocol, sufficient tracer was added to the exogenous glucose infusate such that arterial plasma specific activity (SAa) did not change from basal through the clamp period (P > .05). In the cold GINF studies, plasma SAa fell 81 ± 2% from the basal level by the 3rd h of clamping. We observed a significant, transient, positive venous-arterial difference in specific activity (SAv-SAa difference) during the cold GINF studies. The SAv-SAa difference reached a peak of 27 ± 6% at 30 min and diminished to a plateau of 7 ± 1% between 70 and 180 min. We also observed a positive but constant SAv-SAa difference (4.6 ± 0.2% between 10 and 180 min) during the hot GINF studies. The observations of a difference between hot and cold GINF endogenous Ra estimates and a positive but transient SAv-SAa difference during the cold GINF studies are consistent with the interpretation that a portion of the underestimation of Ra is due to insufficient mixing of endogenous and exogenous glucose for the one-compartment, fixed-pool volume model to be applicable. Alternatively, our results suggest that the one-compartment, fixed-pool volume model of glucose kinetics is insufficient to account for the complex dynamics of labeled and unlabeled glucose during euglycemic-hyperinsulinemic clamps. Improved mixing through addition of tracer to the exogenous glucose infusate or improved modeling by allowing for a variable-pool volume appears to improve the accuracy of the tracer methods; however, these approaches remain to be validated. The constant positive SAv-SAa difference observed during the hot GINF studies is consistent with the interpretation that an additional contributor to underestimation of endogenous Ra is apparent isotope discrimination. Whether this apparent discrimination is due to a true isotope effect or contamination of the tracer infusate remains to be determined.
Geographic Patterns of Childhood Insulin-Dependent Diabetes Mellitus
Authors:   Diabetes Epidemiology Research International Group
Published: September 2013

Population-based registries of insulin-dependent diabetes mellitus (IDDM) worldwide have reached the critical mass needed to investigate global patterns of the disease. International collaboration of 24 registries from 15 countries resulted in the first set of standardized incidence measures among divergent areas and ethnic groups. The average annual age-adjusted incidence under age 15 yr ranged from 1.7/100,000 person-yr in Hokkaido, Japan, to 29.5/100,000 person-yr in Finland during the years 1978–1980. The geographic differences in IDDM in childhood are rarely seen among chronic diseases. It appears that the risk for IDDM is determined by factor(s) correlated to the average yearly temperature of environment and to the ethnicity of the population at risk.
Nonenzymatic Glycation* of Basement Membranes From Human Glomeruli and Bovine Sources: Effect of Diabetes and Age
Authors: Garlick Robert L , Bunn H Franklin , Spiro Robert G
Published: September 2013

The nonenzymatic glycation of glomerular basement membranes (GBMs) from 14 diabetic and 19 nondiabetic human subjects was determined after boronic acid affinity and high-performance cation-exchange chromatography of their NaB[3H]4-reduced ketoamine adducts. The glucitol-lysine (Glc-Lys) and the glucitol-hydroxylysine (Glc-Hyl) content of diabetic GBM was found to be about twofold higher than that of nondiabetic samples (P < .001). The content of these glycated amino acids did not correlate with age over the range examined (20–91 yr) or with the length of disease in diabetic subjects (2–16 yr). However, analyses of Glc-Lys and Glc-Hyl in calf and adult bovine GBM and lens capsules indicated that the levels of these glycated amino acids were several times greater in basement membranes from older animals. We also observed that guanidine-insoluble collagen of bovine GBM is more extensively glycated (∼4-fold) than primarily noncollagenous proteins that are extracted by this reagent. In all of the basement membranes examined, the percentage of glycation of lysine was > of hydroxylysine. Characterization of the components released by alkaline hydrolysis indicated that O-glycosylated hydroxylysine residues are nonenzymatically N-glycated to the same extent as those without an enzymatically attached carbohydrate unit. Our study indicates that more than a hundred times as many hydroxylysine residues are enzymatically glycosylated in human and bovine GBM as those containing the nonenzymatically formed ketoamine adduct.
Galanin—Sympathetic Neurotransmitter in Endocrine Pancreas?
Authors: Dunning Beth Elaine , Taborsky Gerald J
Published: September 2013

The effects of sympathetic neural activation on basal pancreatic hormone secretion cannot be explained solely by the actions of the classic sympathetic neurotransmitter norepinephrine. The nonadrenergic component may be mediated by the 29-amino acid peptide galanin in that this neuropeptide meets several of the criteria necessary to be considered a sympathetic neurotransmitter in the endocrine pancreas. 1) Galanin administration inhibits basal insulin and somatostatin secretion and stimulates basal glucagon secretion from the pancreas, qualitatively reproducing the effects of sympathetic nerve stimulation. These sympathomimetic effects appear to be mediated by direct actions of galanin on the islet. 2) Galanin-like immunoreactivity exists in fibers that innervate pancreatic islets. 3) Galanin is released during electrical stimulation of pancreatic nerves. The quantity released is sufficient to reproduce sympathetic nerve stimulation-induced effects on insulin secretion and to contribute to the neural effects on somatostatin and glucagon release. 4) Whether interference with galanin action or release reduces the islet response to sympathetic nerve stimulation remains to be determined. We hypothesize that galanin and norepinephrine act together to mediate the islet response to sympathetic neural activation. If galanin is a sympathetic neurotransmitter in the endocrine pancreas, it may contribute to the inhibition of insulin secretion that occurs during stress and thereby to the hyperglycemic response. Moreover, the local presence of this potent β-cell inhibitor in the islet leads to speculation on galanin's contribution to the impairment of insulin secretion that occurs in non-insulin-dependent diabetes mellitus and therefore on the potential utility of a galanin antagonist in the treatment of this disease.
Charge Selectivity of Proteinuria in Diabetic Glomerulopathy
Authors: Nakamura Yoshio , Myers Bryan D
Published: September 2013

Differential macromolecule clearances were used to elucidate the mechanism of proteinuria in patients with diabetic glomerulopathy. Uncharged dextrans of graded size, combined with albumin and IgG separated into narrow fractions of varying charge by preparative electrofocusing, were used to probe the filtration barrier. Analysis of the fractional clearance profile of dextrans in the 30- to 60-Å interval revealed a small fraction of filtrate volume (0.0023–0.0097) permeating large nonrestrictive glomerular pores and correlating strongly with the fractional clearances of albumin (r = .88, P < .001) or IgG (r = .91, P < .001). The fractional clearance of the most anionic species of albumin [isoelectric point (pl) 4.0–4.5] significantly exceeded that of less anionic species (pl 4.5–5.5) at all levels of proteinuria. A corresponding increase in fractional clearance of anionic (pl 4.5–5.0) over neutral (pl 7.0–7.5) IgG species was observed in patients with sub-nephrotic-range proteinuria. We conclude that a loss of barrier size selectivity underlies proteinuria in diabetic glomerulopathy. In addition, either facilitated filtration of polyanions or preferential tubular reabsorption of polycations can be invoked to explain the final composition of urinary protein. Similar loss of size selectivity combined with enhanced fractional clearance of anionic IgG in a group of nondiabetic patients with nephrotic syndrome indicates that the foregoing abnormality of renal protein handling is not unique to diabetic glomerulopathy.
Structure, Function, and Immunogenicity of Human Insulinoma Cells
Authors: Thivolet Charles H , Demidem Aicha , Haftek Marek , Durand Annie , Bertrand Jean
Published: September 2013

Dissociated human insulinoma cells were plated onto plastic multiwell dishes. Cells were maintained for 1 mo on plastic with three passages. Cultures consisted of small colonies with some areas of stratification and few intercellular spaces. Ultrastructural studies indicated that cultured cells had epithelial features with desmosomes at cell-to-cell contacts and intermediate filaments in addition to secretory granules in the cytoplasm. Insulin and C-peptide were released in equimolar amounts in culture media. When challenged for 30 min with 16.7 mM glucose, 1 mM 3-isobutyl-1-methylxanthine, 4 mM tolbutamide, or 10−6 M glucagon, insulinoma cells responded by a 1.5-, 1.5-, 2-, or 3-fold increase, respectively, in insulin release above baseline levels. A 15-min challenge with 10−5 M isoproterenol increased insulin secretion by 1.85-fold. By indirect immunofiuorescence, an anti-insulin antibody reacted positively with cell cytoplasm, whereas anti-somatostatin and anti-glucagon antibodies did not. Insulinoma cell surface expressed class I MHC molecules but not class II molecules. Immediately after isolation, crude insulinoma cells were contaminated by 2% of DR+ cells from nonislet components that disappeared after several weeks in culture. The ability of insulinoma cells to stimulate allogenic T-lymphocyte proliferation was assessed by [3H]thymidine incorporation in mixed culture combinations. Crude insulinoma cells elicited a strong lymphoproliterative response with a stimulation index ranging between 3.5 and 7, whereas no stimulation was found after 1 mo in culture. It is postulated that absence of class II-positive cells in the stimulatory cell preparation conditioned this immune tolerance across the major histocompatibility barrier. These results obtained with human insulin-secreting cells underline the need of in vitro manipulation for the success of human islet allografts in insulin-dependent diabetes mellitus.
Downregulation of High-Density Lipoprotein Receptor in Human Fibroblasts by Insulin and IGF-I
Authors: Oppenheimer Mark J , Sundquist Karin , Bierman Edwin L
Published: September 2013

High-density lipoprotein (HDL3) particles bind to a cell surface receptor, thereby promoting the efflux of cholesterol from extrahepatic nonsteroidogenic cells. This receptor appears to be upregulated by increased cell cholesterol content and also may be responsive to the growth state of cells. Because insulin can be mitogenic, the effect of insulin on HDL-receptor function was tested. HDL-receptor activity of cholesterol-loaded fibroblasts was inhibited by insulin treatment. Insulin decreased HDL binding in a log-dose fashion (−25% at 67 nM insulin) in association with increases in [3H]thymidine incorporation into DNA. HDL-mediated cholesterol efflux from cholesterol-loaded cells was diminished by insulin treatment of cells in parallel with decreased HDL binding. Insulin induced reciprocal changes in HDL- and low-density lipoprotein (LDL)-receptor activity. In cells in which these receptors were upregulated by varying cell cholesterol content, insulin increased LDL binding (+ 88%) and decreased HDL binding (−24%). Insulinlike growth factor I (IGF-I, 100 ng/ml) also significantly decreased HDL binding and HDL-mediated cholesterol efflux to a comparable degree. Pooled human serum similarly induced a reduction in HDL binding to its receptor. These results are consistent with the hypothesis that growth factors in general, and insulin and IGF-I in particular, decrease HDL-receptor activity, possibly to promote retention of cholesterol needed for new membrane synthesis during cell proliferation. Such a mechanism could be partly responsible for accumulation of cholesteryl esters in arterial wall cells during atherogenesis in diabetes mellitus.
Compensatory Growth of Pancreatic β-Cells in Adult Rats After Short-Term Glucose Infusion
Authors: Bonner-Weir Susan , Deery Deanna , Leahy John L , Weir Gordon C
Published: September 2013

The extent to which adult pancreatic β-cells can respond in vivo to a sustained glucose stimulus by increasing their mass through either hyperplasia or hypertrophy has remained unanswered. Therefore, we studied the in vivo effect of short-term (96-h) hyperglycemia on the growth of β-cells by infusing adult rats with 35 or 50% glucose or 0.45% saline. After 96 h of glucose infusion, the β-cell mass, quantified by point-counting morphometrics of immunoperoxidase-stained paraffin sections, showed a 50% increase (9.57 ± 0.87 mg, n = 5, 50% glucose infused; 9.50 ± 1.23, n = 7, 35% glucose infused; 6.15 ± 0.55, n = 6, 0.45% saline infused). This growth was selective for β-cells; the non-β-cell mass was unchanged. The mitotic index, measured by accumulated mitotic frequency after a 4-h colchicine treatment, increased fivefold in glucose-infused animals compared to saline-infused animals. This enhanced replication of β-cells provides evidence for increase in cell number or hyperplasia. In addition, hypertrophy of the β-cells was also quantified. Mean cell volume, determined from the mean cell cross-sectional area measured planimetrically from low-magnification electron micrographs, increased to 150% of control values after 96 h of 50% glucose infusion. Seven days after the 96-h infusion, in reversal experiments, the β-cell mass had not returned to saline-infused levels. In addition, the non-β-cell mass of glucose-infused animals had increased. The mitotic index of the β-cells of glucose-infused rats was, however, significantly lower than that of the saline controls, but the mean cell volume of the β-cells remained elevated. Thus, with a short-term in vivo stimulus, adultβ-cells have a far greater capacity to respond with compensatory growth by hyperplasia and hypertrophy than has been appreciated before. Even 7 days after discontinuation of the stimulus, β-cell mass remains elevated.
Intravenous Glucose Tolerance and Pancreatic Islet β-Cell Function in Patients With Multiple Sclerosis During 2-yr Treatment With Cyclosporin
Authors: Robertson R Paul , Franklin Gary , Nelson Lorene
Published: September 2013

Cyclosporin is an immunosuppressive drug used with increasing frequency in patients with diabetes mellitus both as experimental primary therapy for insulin-dependent diabetes mellitus and as therapy accompanying pancreatic transplantation. However, reports have appeared contending that cyclosporin causes glucose intolerance and inhibits pancreactic islet β-cell function. Consequently, concern has been raised that the beneficial effects of immunosuppression may be offset by adverse metabolic effects of the drug. To address this issue, we examined intravenous glucose tolerance and pancreatic islet β-cell function in a group of nondiabetic multiple sclerosis patients before and during a 2-yr course of cyclosporin or placebo therapy. Patients were randomly assigned to one of the two drug groups and followed in a double-blind manner. Basal levels of glucose, insulin, and C-peptide as well as glucose disappearance rates and pancreatic islet β-cell function after stimulation with intravenous glucose and arginine were determined immediately before therapy and after 3 wk, 6 mo, 1 yr, and 2 yr of therapy. No abnormalities in these parameters were observed in the cyclosporin or the placebo-treated group. It appears that cyclosporin can be given in conventional doses for as long as 2 yr without encountering evidence for impaired glucose homeostasis. However, whether adverse effects will materialize over longer periods of drug use remains a question.
Experimental Atherosclerosis-Like Lesions Induced by Hyperinsulinism in Wistar Rats
Authors: Sato Yuzo , Shiraishi Sanshiro , Oshida Yoshiharu , Ishiguro Tetsuya , Sakamoto Nobuo
Published: September 2013

To elucidate the possible role of hyperinsulinism in the etiology of diabetic macroangiopathy, we studied the long-term effects of insulin injection on the arterial wall of the rat both biochemically and histologically. Fifty male Wistar rats were divided into two groups. One group was subjected to daily injection of insulin-zinc suspension (20 U/kg), and the other group was treated with saline. After 1 yr, all the animals were killed, and the lipid contents in the intimal media of their aortas were determined. Parts of the ascending aortic tissues were further examined by use of either light or electron microscopy. The triglyceride content of the insulin-treated rat aortas was significantly (P < .05) increased compared with that of the saline-treated rat aortas. As determined by light microscopy, the intimas of the aortas from the insulin-treated rats were significantly (P < .001) thickened, and the subendothelial tissues consisted of eosinophilic fiber bundles, amorphous ground substances, and irregularly arranged cells. These cells were identified by electron microscopy as having smooth muscle cell origin. All these findings suggest that atherosclerosis like lesions could be induced by long-term insulin injection in the aortas of the rat and that hyperinsulinism plays a certain role in the development of diabetic macroangiopathy.
Protease Activity in Pancreatic Islet Isolation by Enzymatic Digestion
Authors: McShane Philip , Sutton Robert , Gray Derek WR , Morris Peter J
Published: September 2013

Commercial Collagenase* prepared from Clostridium histolyticum is widely used in isolation of pancreatic islets. It is known that the enzyme is very impure and that there are substantial variations in effectiveness between batches. Our studies suggest that one of the impurities of importance in islet isolation is a protease that has not been very well characterized. Comparison of two batches of enzyme, one of which was known to give good yields of islets and the other poor yields, showed that they had very similar activity against collagen (measured by digestion of insoluble collagen followed by assay of soluble products with ninhydrin) but substantially different activities against azocasein as measured by optical density increase (measured by release of dye). Eighteen batches of Collagenase were examined for efficiency in islet isolation, and the yields obtained correlated with manufacturer's data of activity against casein. The data show that low caseinase activity is associated with performance in islet isolation (r = .5 after adjusting for collagenase activity). The effect of supplementing a batch of collagenase, known to be poor in isolating islets, with proteolytic enzymes was investigated. Trypsin and papain had apparently no effect, but dispase significantly increased yield. Dispase alone failed to digest pancreas. Size-exclusion high-performance liquid chromatography identified a peak associated with high protease activity and efficiency in islet isolation, having an Mr of ∼30,000, compared to 78,000 for collagenase. The protease, like collagenase, is inhibited by EDTA. Increased Ca2+ and Mg2+ (up to 10 mM) did not affect activity. Both the protease and collagenase are stable under normal use but are inactivated by heating at 56°C. Other properties, e.g., effect of temperature, pH, and dithiothreitol, were also investigated.
Automated Islet Isolation From Human Pancreas
Authors: Ricordi Camillo , Lacy Paul E , Scharp David W
Published: September 2013

An automated method for the isolation of human pancreatic islets is described. The procedure meets the following requirements: 1) minimal traumatic action on the islets, 2) continuous digestion in which the islets that are progressively liberated can be saved from further enzymatic action, 3) minimal human intervention in the digestion process, and 4) high yield and purity of the isolated islets. After purification of Ficoll gradients, an average of 2279 islets/g pancreas was obtained, with an average purity of 79% islets. The average volume and average insulin content of the final islet preparation were 348 mm3 and 93.4 U, respectively. The islets were morphologically intact with a normal degree of β-cell granulation, responded to glucose stimulation with a fivefold increase of insulin secretion over basal levels, and produced normoglycemia after transplantation into diabetic mice. The procedure is being used in the second phase of clinical trials of islet transplantation in patients with insulin-dependent diabetes mellitus.
Intraportally Transplanted Pancreatic Islets Revascularized From Hepatic Arterial System
Authors: Andersson Arne , Korsgren Olle , Jansson Leif
Published: September 2013

Isolated pancreatic islets were implanted intraportally into normoglycemic or alloxan-induced diabetic C57BL/6 mice. Each animal was grafted with 200–250 syngeneic islets. Four, 8, 14, or 56 days after the transplantation, 2 to 3 × 105 microspheres (10 μm diam) were injected into either the portal vein or the abdominal aorta of anesthetized animals. The presence of microspheres within the islet grafts was determined in stained sections of the livers. In general, the number of microspheres in the liver sections was >10 times higher in mice given microspheres via the portal route. When the microspheres were administered via the arterial system 2 wk after transplantation, ∼20–30% of the islet sections contained microspheres; only occasional spheres were found in the islets after intraportal administration. This was the case even when as many as 56 days had elapsed. Some but not all mice investigated 4 and 8 days after transplantation had islets that contained microspheres. The intraportally implanted islets in hyperglycemic animals had decreased revascularization compared with normoglycemic mice 2 wk after implantation. The results indicate that intraportally implanted syngeneic pancreatic islets become revascularized mainly from the hepatic artery and that this process starts as early as during the 1st wk postimplantation. Furthermore, hyperglycemia seems to influence this process.
Role of CD4+ T-Lymphocytes in Rejection by Mice of Fetal Pig Proislet Xenografts
Authors: Dennis Wilson J , Simeonovic Charmaine J , Ting John HL , Ceredig Rhodri
Published: September 2013

The role of T-lymphocyte subpopulations in the rejection of fetal pig proislet (islet precursor) xenografts in mice was examined with in vivo administration of monoclonal antibodies (MoAbs) to CD4+ (GK1.5) and CD8+ (49–11.1) T-lymphocyte subsets. The data indicate that the rejection process is T-lymphocyte dependent and mediated by CD4+ T-lymphocytes. The in vivo administration of MoAbs specific for graft-borne pig leukocytes failed to prevent xenog raft rejection in fully immunocompetent transplant recipients.