bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Analysis of Glucose Tolerance in Inbred Mouse Strains: Evidence for Polygenic Control
Authors: Kaku Kohei , Fiedorek Frederick T , Province Michael , Permutt M Alan
Published: September 2013

To determine genetic factors involved in diabetes susceptibility in inbred strains of mice, we initially evaluated differences in fed plasma glucose and insulin concentrations among six strains (AKR/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J). There was considerable variation in fed plasma glucose concentration, with C3H/HeJ mice the most glucose tolerant (174 ± 7 mg/dl) and C57BL/6J mice the least glucose tolerant (252 ± 7 mg/dl, P < .0001 vs. C3H/HeJ mice). Glycosylated hemoglobin of C57BL/6J mice (4.0 ± 0.06%) was also higher than that of C3H/HeJ mice (3.52 ± 0.06%, P < .0001). The fed plasma insulin concentration did not differ between these two strains. Glucose tolerance was further evaluated in overnight-fasted C3H/HeJ and C57BL/6J mice by an intraperitoneal glucose tolerance test (IPGTT). Although fasting plasma glucose did not differ, the most remarkable difference in plasma glucose during IPGTT between C57BL/6J and C3H/HeJ mice was noted at 30 min (489 ± 29 vs. 227 ± 20 mg/dl, P < .001). To determine the number of genes involved in the phenotypic difference in glucose tolerance, C57BL/6J males were crossed with C3H/HeJ females (F1, C3H/HeJ × C57BL/6J), and the F1 hybrid females were backcrossed with C57BL/6J males (backcrossed, F1 × C57BL/6J). Plasma glucose after 30 min on IPGTT was 219 ± 8 (n = 21), 456 ± 18 (n = 23), and 292 ± 13 (n = 23) mg/dl for C3H/HeJ, C57BL/6J, and F1 mice, respectively (P < .001 for all comparisons). The range of glucose levels on IPGTT of the backcrossed mice was 250–500 mg/dl. If a single gene were determining the difference in glucose tolerance in C57BL/6J mice, analysis of the plasma glucose level of individual backcrossed mice would show segregation into two classes, one like the F1 hybrid and the other like the inbred parent. This segregation was not observed; therefore, we conclude that modification of glucose tolerance in these two inbred strains is under polygenic control. Plasma insulin levels after 15 min on IPGTT for C57BL/6J mice were lower than those for C3H/HeJ mice (1.0 ± 0.09 vs. 1.3 ± 0.09 ng/ml, n = 6, P < .001). On the other hand, insulin levels after 30 min on IPGTT for C3H/HeJ (0.9 ± 0.08 ng/ml, n = 21), C57BL/6J (1.05 ± 0.1 ng/ml, n = 23), F1 (1.05 ± 0.1 ng/ml, n = 23), and backcrossed mice (1.0 ± 0.1 ng/ml, n = 23) were not different from each other. A positive correlation between plasma glucose and insulin concentrations was observed in C3H/HeJ (r = .42, P < .05) and F1 (r = .46, P < .05) mice, whereas a negative correlation was seen in C57BL/6J mice (r = −.45, P < .05). This suggests that glucose-stimulated insulin secretion may be significantly impaired in C57BL/6J mice, indicating one of the possible mechanisms for the phenotypic expression of glucose intolerance.
Abnormal Erythrocyte Charge in Diabetes Mellitus: Link with Microalbuminuria
Authors: Gambaro Giovanni , Baggio Bruno , Cicerello Elisa , Mastrosimone Stefania , Marzaro Giovanni , Borsatti Arturo , Crepaldi Gaetano
Published: September 2013

The anionic charge on the surface of the erythrocyte and the erythrocyte membrane content of sialic acid and acid glycosaminoglycans (GAGs) were evaluated in insulin-dependent diabetic patients who had albumin excretion rates < 300 mg/24 h. In these subjects a statistically significant reduction of erythrocyte anionic charge (RBCCh) and GAGs content in erythrocyte ghosts was shown. In view of the demonstration of a negative correlation between RBCCh and albuminuria after a lysine provocative test, these observations support the hypothesis that the onset of microalbuminuria in human diabetes is sustained by an alteration of glomerular charge and consequently of glomerular charge selectivity.
Two-Color Flow Cytometry Analysis of Activated T-Lymphocyte Subsets in Type I Diabetes Mellitus
Authors: Legendre Christoph M , Schiffrin Alicia , Weitzner Gabriel , Colle Eleanor , Guttmann Ronald D
Published: September 2013

We addressed the question of whether newly diagnosed type I (insulin-dependent) diabetes mellitus patients showed an increased number of DR (la+) T-lymphocytes compared with nondiabetic siblings and normal control subjects. Two-color flow cytometry measurements of peripheral-blood lymphocytes showed a slight but statistically significant increase in DR+ T-lymphocytes in diabetic subjects as well as the nondiabetic sibling control compared with the normal control subjects. This difference was not present in long-term—diabetic subjects. Thus, in addition to minor changes in this lymphocyte subset in peripheral blood, the sibling data demonstrate a lack of specificity for the disease; therefore, these measurements are probably of limited diagnostic usefulness.
Effect of Tolbutamide and Glyburide on cAMP-Dependent Protein Kinase Activity in Rat Liver Cytosol
Authors: Okuno Senji , Inaba Masaaki , Nishizawa Yoshiki , Inoue Akira , Morii Hirotoshi
Published: September 2013

The effect of sulfonylureas tolbutamide and glyburide on adenylate cyclase– and cAMP-dependent protein kinase (A-kinase) was examined in rat liver cytosol. Both tolbutamide and glyburide inhibited the A-kinase activity in a dose-dependent manner. Half-maximal inhibition was obtained at 10 mM with tolbutamide and at 0.2 mM with glyburide, indicating that glyburide was 50-fold as potent as tolbutamide. Neither tolbutamide nor glyburide affected [3H]cAMP binding to the protein kinase, but both inhibited the activity of catalytic units of the A-kinase. Lineweaver-Burk double-reciprocal plots revealed that the inhibitory effects of these drugs were noncompetitive with respect to the protein substrate histone, as well as to the phosphate-donor substrate ATP. Thus, tolbutamide and glyburide inhibited the A-kinase activity in rat liver cytosol, and it was suggested that, through the inhibition of A-kinase, the sulfonylureas would affect the carbohydrate metabolism in the liver. In fact, the relative potencies of these two drugs on A-kinase activity corresponded well with those of their reported antidiabetic effects.
Morphologic Evidence of Interactions Between Adult Ductal Epithelium of Pancreas and Fetal Foregut Mesenchyme
Authors: Dudek Ronald W , Lawrence Irvin E
Published: September 2013

First, tissue recombinants were used to determine the residual capability of adult endodermal pancreatic ductal epithelium to form islets. Second, adult epithelium was challenged by placing it between adult stroma and fetal mesenchyme to investigate the epithelial response (subsequently called challenge grafts). Trypsin was used to isolate adult mouse (heteroplastic) or rat (homoplastic) pancreatic ductal epithelium and fetal rat mesenchyme. All the adult epithelium was maximally stressed with alloxan to control for β-cell contamination. Fetal mesenchyme was layered with epithelium in vitro for at least 48 h. Subsequently, the recombinants were grafted into nude mice and allowed to develop for 4–6 wk. To assess development, grafts were removed and fixed for both light and electron microscopy. In general, the grafts became organized into vesicles, tubules, and buds, many of which were in the form of vascularized isletlike structures. Epithelium responded with mitosis and intercellular adhesion. The range of cytodifferentiation varied. Culture time, age, and adhesiveness of the fetal mesenchyme and the amount of adult epithelium used in making the recombinants influenced the degree of differentiation. Analysis of fine structure of isletlike structures revealed cells with small electron-dense granules. These observations provide evidence for activation of endodermal epithelium taken from an adult mammal.
Increased Dye Coupling in Pancreatic Islets From Rats in Late-Term Pregnancy
Authors: Sheridan Judson D , Anaya Patricia A , Parsons Jonathon A , Sorenson Robert L
Published: September 2013

Our previous studies have suggested that elevated lactogen, increased glucose-stimulated insulin secretion, and increased β-cell coupling are associated. To determine whether this association occurs under conditions of physiologically increased lactogen, we have studied the extent of dye coupling in rat islets during the later stage of pregnancy. These animals have high plasma lactogen levels in the form of placental lactogen, increased plasma insulin, and decreased plasma glucose. The fluorescent tracer, Lucifer yellow CH, was microinjected into central cells of islets from both pregnant and virgin rats, and the extent of transfer was quantitated by determining the projected area of dye spread. Two area measurements were made for each injection, one around the entire discernible fluorescent region (“outer”) and another around the distinct brighter region of cells surrounding the injected cell (“inner”). Pregnancy increased dye transfer, as determined by both measurements. The outer area of dye transfer was 9047 ± 775 μm2 for the islets from pregnant rats and 4699 ± 391 μm2 for the islets from virgin rats (P < .001). Similarly, pregnancy increased the inner area of dye transfer, 1447 ±161 μm2 for the islets f rom pregnant rats and 795 ± 80 μm2 for the islets from virgin rats (P < .001). These results support the hypothesis that elevated lactogen, increased glucose-stimulated insulin secretion, and increased β-cell dye coupling are associated under physiological conditions. The study indicates that enhanced β-cell coupling is part of the structural and functional adaptation that the islets undergo during a subject's pregnancy and demonstrates that the extent of β-cell coupling is regulated by a physiological condition.
Arachidonic Acid Metabolism and Insulin Secretion by Isolated Human Pancreatic Islets
Authors: Turk John , Hughes Jonathan H , Easom Richard A , Wolf Bryan A , Scharp David W , Lacy Paul E , McDaniel Michael L
Published: September 2013

Isolated human pancreatic islets converted [3H8]arachidonate to compounds with the high-performance liquid-chromatographic mobility of cyclooxygenase products, including prostaglandin E2(PGE2), PGF2α, and the lipoxygenase product 12-HETE. Human islet synthesis of PGE2, PGF2α, and 12-HETE from endogenous arachidonate was demonstrated with stable isotope dilution-gas chromatographic-negative ion-chemical ionization-mass spectrometric analysis. Pharmacologie inhibition of arachidonate metabolism by both lipoxygenase and cyclooxygenase pathways with BW 755C strongly suppressed glucose-induced insulin secretion from perifused human islets, and the selective cyclooxygenase inhibitor indomethacin enhanced insulin secretion. These findings are similar to those reported for islets isolated from rats and suggest that arachidonate metabolites may modulate glucose-induced insulin secretion in humans.
HLA and Genetics of IDDM: Holism vs. Reductionism?
Authors: Segall Miriam
Published: September 2013

Analysis of HLA-associated susceptibility to insulin-dependent diabetes mellitus (IDDM) has largely focused on identifying the susceptibility gene. Adherents of a countertrend have long suggested the importance of analysis of HLA haplotypes (combinations of alleles on 1 chromosome) rather than individual genes. Accumulating data suggest that the relationship between IDDM susceptibility and HLA is much more complex than a single susceptibility gene. Consideration of this question should include the possibilities that 1) more than one HLA gene is involved in determining susceptibility or resistance; 2) different alleles of the same gene may be associated with different pathogenetic mechanisms; and 3) different susceptibility-associated haplotypes, even if they share an allele at an IDDM-relevant locus, may behave differently in IDDM. A better understanding of the genetics, and perhaps the pathogenesis, of IDDM may be obtained by following up the clues offered by analysis of the association of HLA haplotypes (rather than individual alleles) with one another, with clinical features of IDDM, and with possible non-HLA-linked susceptibility factors.
Mechanisms of Nicotinamide and Thymidine Protection From Alloxan and Streptozocin Toxicity
Authors: LeDoux Susan P , Hall Carolyn R , Forbes Pam M , Patton Nancy J , Wilson Glenn L
Published: September 2013

A common mechanism has been proposed for the β-cell toxins alloxan (ALX) and streptozocin (STZ) involving the formation of single-strand breaks in DNA that lead to the overactivation of the enzyme poly(ADP-ribose) synthetase and the critical depletion of its substrate NAD. If the toxins act via this common mechanism, the poly(ADP-ribose) synthetase inhibitors nicotinamide and thymidine would be expected to affect the formation of DNA single-strand breaks in a similar fashion. To test the effects of these inhibitors, the formation of single-strand breaks in the DNA of insulin-secreting RINr cells was monitored by assessing changes in the supercoiling of nucleoids after exposure to STZ, ALX, or methylnitrosourea (MNU). With the inclusion of nicotinamide or thymidine and STZ or MNU, more single-strand breaks in RINr cell DNA were detected. These results would be expected if nicotinamide and thymidine acted through inhibition of poly(ADP-ribose) synthetase. However, when the inhibitors were used in combination with ALX, fewer single-strand breaks were present. This suggests a reduction in ALX-induced hydroxyl radicals available to interact with DNA. Because nicotinamide has been demonstrated to be a hydroxyl-radical scavenger, the ability of thymidine to scavenge hydroxyl radicals was investigated. Thymidine, like nicotinamide, was found to be a potent scavenger of hydroxyl radicals. Thus, the mechanisms by which nicotinamide and thymidine protect against the toxic effects of STZ or ALX appear different. These findings suggest that the actions of β-cell toxins are more complex than simply the overactivation of a single enzyme.
Restriction-Fragment–Length Polymorphism in Insulin-Receptor Gene and Insulin Resistance in NIDDM
Authors: McClain Donald A , Henry Robert R , Ullrich Axel , Olefsky Jerrold M
Published: September 2013

Restriction-enzyme analysis of genomic DNA from 52 White and Hispanic nondiabetic subjects and 51 subjects with non-insulin-dependent diabetes (NIDDM) was carried out with insulin-receptor cDNA probes. A polymorphic 5.8-kilobase SstI fragment was found in 12 (23.5%) of 51 NIDDM subjects but only in 4 (7.7%) of 52 nondiabetic control subjects. This association is significant by χ2-analysis (P < .05). Furthermore, the nondiabetic subjects with the polymorphism were found to have hyperinsulinemia and/or nondiagnostic glucose tolerance. The polymorphism is a genetic marker for a phenotype that is neither necessary nor, by itself, sufficient for NIDDM. Nevertheless, it may indicate that insulin resistance functionally related to an insulin-receptor gene polymorphism is the proximal cause of NIDDM in at least one subset of the population.
Diabetic Neuropathy in STZ-Induced Diabetic Rat and Effect of Allogeneic Islet Cell Transplantation: Morphometric Analysis
Authors: Sima Anders A F , Zhang Wei-Xian , Tze Wah J , Tai Joseph , Nathaniel Virgil
Published: September 2013

The effect of pancreatic islet cell allotransplantation on the development of diabetic neuropathy in streptozocin-induced diabetic ACI rats was examined morphometrically with light- and electron-microscopic procedures. Peripheral nerve function was evaluated by nerve conduction velocity and evoked muscle potential amplitude measurements. Diabetes was induced at 4 mo of age, and diabetic animals were transplanted by intracerebral and intraportal grafts 2 wk later. Diabetic animals with accepted grafts returned to euglycemia and showed a normal body-weight gain over the subsequent 14-mo observation period. Transplanted animals with accepted grafts and those in whom graft rejection was induced were compared with age-matched nontransplanted diabetic rats and nondiabetic control rats at 18 mo of age. Successful allotransplantation completely prevented axonal atrophy and the characteristic nodal and paranodal structural abnormalities in diabetic nerve, as well as the typical slowing of nerve conduction velocity. Our data suggest that islet cell allotransplantation is an effective therapeutic approach to the prevention of diabetic neuropathy.
Islet Cell Antigens: Extraction Studies and ELISA Analysis
Authors: Moncayo Roy , Kirsch Klaus , Pasquarello Tim , Pfeiffer Ernst Friedrich
Published: September 2013

Whole human and bovine pancreases were extracted in 20 mM Tris-HCI buffer without detergents and fractionated by high-speed centrifugation. The 80,000 × g supernatant was used to coat microtiter plates at a concentration of 5 μg protein/ml in phosphate-buffered saline. This solid-phase ELISA system was used for the detection of islet cell antigens defined by a series of monoclonal islet cell antibodies (HISL-1, -4, -5, -8, -14, and -19 and 4F2, 3G5, and A2B5). Both glycoprotein and glycolipid islet cell antigens in the total pancreatic extracts were detected by the monoclonal islet cell antibody in the ELISA system, indicating that epitope preservation had occurred during the extraction procedure. There was a good correlation between islet cell antigen quantitated by the ELISA system and the corresponding islet immunohistochemical reaction. Studies along these lines have the potential to facilitate the design of large-scale protocols for the purification of diabetes-related islet cell antigens to homogeneity.
Deficiency of Cardiac β-Adrenergic Receptor in Streptozocin-Induced Diabetic Rats
Authors: Nishio Yoshihiko , Kashiwagi Atsunori , Kida Yasuo , Kodama Mitsuaki , Abe Nanami , Saeki Yukikazu , Shigeta Yukio
Published: September 2013

The number of β-adrenergic receptors in cardiac myocytes isolated from rats made diabetic with streptozocin (STZ) for 10 wk was measured by use of a hydrophilic nonselective antagonist [3H]CGP 12177 and was found to decrease to 59% of the number in control rats (P < .05), without any change in affinity. Similarly, using [125I]iodocyanopindolol as a ligand, we found a decrease in the β-adrenergic-receptor number on cardiac plasma membrane isolated from the diabetic rats [29% decrease (P < .05) at 1 wk, 50% (P < .01) at 3 wk, and 49% (P < .01) at 10 wk compared with control rats]. However, the serum triiodothyronine level that had been known to modulate the β-adrenergic-receptor-adenylate cyclase system was decreased in the 1-wk-diabetic rats but not in the 10-wk-diabetic rats compared with each control group. Furthermore, there was no difference in urinary catecholamine excretion between diabetic and control groups. In the 10-wk-diabetic rats, the response of adenylate cyclase to isoproterenol was significantly defective (56% decrease compared with control rats; P < .05), although both the basal and the forskolin-stimulated maximum adenylate cyclase activities and a half-maximum concentration of isoproterenol for the stimulation of adenylate cyclase were similar in control and diabetic rats. On the other hand, both cholera toxin-dependent and islet-activating protein-dependent [32P]NAD incorporations into cardiac plasma membrane were markedly increased in the diabetic rats. The 2-wk insulin treatment improved not only the number of β-adrenergic receptors but also the response of adenylate cyclase to 10 μM isoproterenol. These results indicate that cardiac unresponsiveness to a β-adrenergic agonist in STZ-induced diabetic rats is specifically associated with a deficiency in the β-adrenergic-receptor concentration in cardiac plasma membrane.
Effect of Hyperinsulinemia on Urea Pool Size and Substrate Oxidation Rates
Authors: Tappy Luc , Owen Oliver E , Boden Guenther
Published: September 2013

Recently, indirect calorimetry has frequently been used together with hyperinsulinemic clamps. With few exceptions, however, no attention was paid in these studies to the possible effects of hyperinsulinemia on urea nitrogen (N) pool size and the consequences of such changes on the calculated rates of protein, lipid, and carbohydrate (CHO) oxidation. We have determined the effects of euglycemic-hyperinsulinemic clamps on urea N pool size, urinary N excretion, and rates of protein, lipid, and CHO oxidation (measured by indirect calorimetry) in six normal men. Insulin infusion (1 mU · kg−1 · min−1) increased peripheral venous insulin concentration from 7 ± 1.2 (mean ± SE) to 51 ±4 μU/ml. Glucose concentration was clamped at 84 ± 1.1 mg/dl. Between 0 (preclamp) and 360 min (end of clamp), blood urea N concentration decreased from 17.2 ± 1.1 to 11 ± 0.8 mg/dl (P < .001), and the urea N pool decreased from 604 ± 41 to 388 ± 30 mmol (P < .001). The urea N production rate decreased from 461 ± 91 (preclamp) to 91 ± 63 μmol/min during the last 4 h of the clamp (P < .05). Urinary N excretion remained unchanged (705 ±113 vs. 905 ±125 μmol/min, NS). Correction of urinary N excretion for insulin-induced reductions in the urea N pool resulted in the following changes in substrate oxidation rates (calculated for the last 4 h of the clamp). Protein oxidation rate decreased by 72 ± 8% (from 82.8 ± 10.9 to 25.7 ± 9 mg/min); lipid oxidation rate increased by 66 ± 16% (from 43.6 ± 11.9 to 61.7 ± 12.1 mg/min); and CHO oxidation rate increased by 12 ± 2% (from 167.8 ± 20.4 to 187.7 ± 21.6 mg/min). We conclude that euglycemic hyperinsulinemia decreases the urea N pool. If uncorrected, this will result in substantial overestimation of protein oxidation and underestimation of lipid and CHO oxidation rates.
Insulin Production and Glucose Metabolism in Isolated Pancreatic Islets of Rats With NIDDM
Authors: Portha Bernard , Giroix Marie-Hélène , Serradas Patricia , Welsh Nils , Hellerström Claes , Sener Abdullah , Malaisse Willy J
Published: September 2013

Rats with non-insulin-dependent diabetes mellitus (NIDDM) induced by neonatal injection of streptozocin are known to have a deficient insulin response to glucose. To evaluate to what extent this glucose insensitivity can be attributed to a pertubation of the islet glucose metabolism, we estimated the rates of glucose phosphorylation, glucose utilization, oxygen consumption, and glucose oxidation in islets isolated from normal and NIDDM rats and compared these values with rates of islet insulin biosynthesis and release in vitro. The data confirm that islets from rats with NIDDM display a deficient response to glucose of both insulin biosynthesis and release that is still present after an overnight culture of the islets at 5.5 mM glucose. Furthermore, they show that islets of these rats have 1) normal low- and high-Km glucose-phosphorylating activities and no major alteration of the glucose utilization rate, 2) decreased insulin release in response to glyceraldehyde, 3) decreased rates of basal respiration and glucose oxidation and a markedly reduced stimulation by glucose of both islet oxygen consumption and glucose oxidation, and 4) decreased glucose-stimulated net 45Ca uptake. We conclude that the relative unresponsiveness to glucose of islets from NIDDM rats is associated with, and perhaps due to, a deficient islet glucose metabolism. This defect is not due to gross alterations in the glycolytic pathway but probably reflects alteration in the islet mitochondria function.
Insulin-Mimetic Effects of Vanadate in Primary Cultures of Rat Hepatocytes
Authors: Jackson Timothy K , Salhanick Arthur I , Sparks Janet D , Sparks Charles E , Bolognino Mary , Amatruda John M
Published: September 2013

To evaluate possible mechanisms by which insulin inhibits hepatic apolipoprotein B (apoB) secretion, we incubated primary cultures of rat hepatocytes with sodium ort ho vanadate, a phosphotyrosine phosphatase inhibitor and insulin-mimetic agent. Vanadate (10 μM) and insulin (10 nM) inhibited the medium accumulation of apoB (secretion) by 21 and 37%, respectively, without increasing intracellular apoB. The effects of insulin and vanadate together were not additive. Both insulin and vanadate enhanced intracellular glycogen accumulation by 82 and 37%, respectively. Unlike insulin, vanadate, at a concentration that inhibited apoB secretion (10 μM), had no effect on intracellular lipogenesis, inhibited the secretion of newly synthesized hepatic proteins, and had a delayed onset and termination of action on inhibition of apoB secretion. At higher concentrations (40 and 80 μM), vanadate stimulated intracellular lipogenesis. In conclusion, our data indicate that vanadate mimics insulin action in hepatocytes with regard to the inhibition of medium accumulation of apoB. These data are consistent with the hypothesis that inhibition of apoB secretion may be secondary to an increase in phosphotyrosine content at its site of synthesis. The kinases responsible for this effect have not been identified. Several effects of vanadate, however, are different from those of insulin, suggesting a differential sensitivity to vanadate, a divergence of the signal transfer by insulin and vanadate at the insulin-receptor or postreceptor level, or both.
β-Endorphin–Induced Inhibition and Stimulation of Insulin Secretion in Normal Humans Is Glucose Dependent
Authors: Giugliano Dario , Cozzolino Domenico , Salvatore Teresa , Torella Roberto , D'Onofrio Felice
Published: September 2013

This study evaluated the effect of human β-endorphin on pancreatic hormone levels and their responses to nutrient challenges in normal subjects. Infusion of 0.5 mg/h β-endorphin caused a significant rise in plasma glucose concentrations preceded by a significant increase in peripheral glucagon levels. No changes occurred in the plasma concentrations of insulin and C-peptide. Acute insulin and C-peptide responses to intravenous pulses of different glucose amounts (0.33 g/kg and 5 g) and arginine (3 g) were significantly reduced by β-endorphin infusion (P < .01). This effect was associated with a significant reduction of the glucose disappearance rates, suggesting that the inhibition of insulin was of biological relevance. β-Endorphin also inhibited glucose suppression of glucagon levels and augmented the glucagon response to arginine. To verify whether the modification of prestimulus glucose level could be important in these hormonal responses to β-endorphin, basal plasma glucose concentrations were raised by a primed (0.5 g/kg) continuous (20 mg kg−1 · min−1) glucose infusion. After stabilization of plasma glucose levels (350 ± 34 mg/dl, f = 120 min), β-endorphin infusion caused an immediate and marked increase in plasma insulin level (peak response 61 ± 9 μU/ml, P < .01), which remained elevated even after the discontinuation of opioid infusion. Moreover, the acute insulin response to a glucose pulse (0.33 g/kg i.v.) given during β-endorphin infusion during hyperglycemia was significantly higher than the response obtained during euglycemia (171 ± 32 vs. 41 ± 7 μU/ml, P < .01). Therefore, during euglycemia, β-endorphin infused at low pharmacological dose in normal humans inhibits basal and nutrient-stimulated insulin secretion, raises plasma glucose and glucagon concentrations, and increases the glucagon response to arginine. Also, during hyperglycemia (glucose clamp), β-endorphin stimulates basal and glucose-induced insulin secretion. Hyperglycemia alters the reactivity of the β-cells to β-endorphin.
Rat Insulin-Receptor Kinase Activity Correlates With In Vivo Insulin Action
Authors: Bryer-Ash Michael
Published: September 2013

Tyrosine kinase activity of skeletal muscle-derived insulin receptors isolated from rats that had undergone euglycemic clamps at various insulin infusion rates was examined. Receptors were isolated under conditions designed to preserve their in vivo phosphorylation state, and their kinase activity toward histone was measured in the absence of in vitro exposure to insulin. Results showed that significant activation of the insulin-receptor kinase occurred after exposure in vivo to mean serum insulin concentrations as low as 34 ± 3.5 μU/ml and that maximal activation was achieved by insulin levels ≤2000 μU/ml. There was a highly significant correlation between receptor kinase activity and serum insulin concentration in the physiologic range (r = .92, P < .0001) and between kinase activity and glucose utilization rate (r = .74, P < .0001). These findings further support a role for the insulin-receptor kinase in insulin action in vivo, and this model provides a novel method for the study of the effect of factors known to influence insulin action on the insulin-receptor kinase under physiologic conditions.
Prevention of Diabetes in BB/Wor Rat by Single Transfusion of Spleen Cells: Parameters That Affect Degree of Protection
Authors: Burstein Dina , Mordes John P , Greiner Dale L , Stein Daniel , Nakamura Naoto , Handler Eugene S , Rossini Aldo A
Published: September 2013

Previous studies have shown that multiple transfusions of spleen cells from histocompatible nondiabetic donors prevent autoimmune diabetes mellitus in diabetes-prone (DP) BioBreeding/Worcester (BB/Wor) rats. In this study, a single transfusion of ≥50 × 106 cells from either diabetes-resistant (DR) BB/Wor or Wistar-Furth (WF) rats substantially reduced the incidence of diabetes when given to DP rats 27 or 46 days old but not 61 days old. Transfusion and protection were associated with the appearance of RT6+ donor lymph node cells in recipient rats. In vivo depletion of RT6+ T-lymphocytes in 150-day-old protected animals did not produce diabetes. DR BB/Wor and WF spleen cells were equally efficacious when given either intraperitoneally or intravenously. Mitogen-activated spleen cells were relatively less effective than untreated cells. We conclude that BB rat diabetes can be prevented by one transfusion of spleen cells from histocompatible DR and WF donors, and that the protective effect is dependent on recipient age and cell dose. The effect may be mediated by a population of RT6+ T-lymphocytes that, during a critical developmental period, regulate the expression of autoimmunity in these animals.
Insulin Secretion and cAMP Metabolism in HIT Cells: Reciprocal and Serial Passage-Dependent Relationships
Authors: Zhang Hui-Jian , Walseth Timothy F , Robertson R Paul
Published: September 2013

The HIT cell is a variably glucose-responsive clonal line of pancreatic islet β-cells. To ascertain whether insulin responsiveness to glucose, arginine, isoproterenol, forskolin, and K+ varied in a predictable fashion, full concentration-response curves with these agonists were examined with cells from a span of 25 passages. Basal and stimulated cAMP metabolism were also examined. The findings indicate that insulin responses to glucose diminish progressively with increasing passage number and that studies of glucose-induced insulin secretion should be limited to passages 81 and earlier. This defect in insulin secretion is a general rather than a glucose-specific phenomenon in that insulin responses to the other nonglucose secretagogues also diminished with increasing passage number. All changes in glucose-stimulated responses were limited to diminutions in maximal responses; no alterations in apparent half-maximal effective concentrations (EC50 s) were observed. In contrast to the continually diminishing insulin responsiveness observed, hormone inhibition by somatostatin of insulin secretion and basal cAMP metabolism remained intact throughout the passages examined. Interestingly, a reciprocal relationship between insulin responsiveness and cAMP responsiveness was observed. Dramatic cAMP responses to isoproterenol and forskolin were observed with the later passages. We conclude that loss of insulin responsivity in HIT cells is a passage-dependent process thatis serial rather than sporadic and global rather than glucose specific. Dramatic reciprocal changes in cAMP metabolism occur in the later passages.
Vasopressin in Rats With Genetic and Streptozocin-Induced Diabetes
Authors: Brooks David P , Nutting David F , Crofton Joan T , Share Leonard
Published: September 2013

Rats were administered streptozocin (STZ; 50 or 75 mg/kg i.v., tail vein) or vehicle. Approximately 2 wk later, venous and arterial catheters were implanted for subsequent (24 h later) vasopressin, electrolyte, and hemodynamic measurements. STZ-induced diabetic (STZ-D) rats demonstrated a dose-dependent increase in the plasma glucose concentration, plasma osmolality, and plasma vasopressin concentration. Mean arterial blood pressure (MABP) was unchanged, but heart rate was reduced. Diabetes-prone BB rats, maintained on or withdrawn from insulin treatment for 24–48 h, and diabetes-resistant rats were instrumented and studied as above. Spontaneous-diabetes–prone rats demonstrated increases in plasma glucose concentration and plasma osmolality similar to STZ-D rats but had significantly greater plasma vasopressin concentrations. The significant decrease in MABP observed in these animals probably contributed to the enhanced vasopressin response. We conclude that both osmotic and cardiovascular parameters play important roles in vasopressin secretion in diabetic rats.
Underestimation of Glucose Turnover Measured With [6-3H]- and [6,6-2 H2]- but not [6-14C]glucose During Hyperinsulinemia in Humans
Authors: McMahon M Molly , Schwenk W Frederick , Haymond Morey W , Rizza Robert A
Published: September 2013

Recent studies indicate that hydrogen-labeled glucose tracers underestimate glucose turnover in humans under conditions of high flux. The cause of this underestimation is unknown. To determine whether the error is time-, pool-, model-, or insulin-dependent, glucose turnover was measured simultaneously with [6-3H]-, [6,6-2H2]-, and [6-14C]glucose during a 7-h infusion of either insulin (1 mU · kg−1 · min−1) or saline. During the insulin infusion, steady-state glucose turnover measured with both [6-3H]glucose (8.0 ± 0.5 mg · kg−1 · min−1) and [6,6-2H2]glucose (7.6 ± 0.5 mg kg−1 min−1) was lower (P < .01) than either the glucose infusion rate required to maintain euglycemia (9.8 ± 0.6 mg · kg−1 · min−1) or glucose turnover determined with [6-14 C]glucose and corrected for Cori cycle activity (9.8 ± 0.7 mg · kg−1 · min1). Consequently “negative” glucose production rates (P <.01) were obtained with either [6-3 H]- or [6,6-2 H2]-but not [6-14 C]glucose. The difference between turnover estimated with [6-3 H]glucose and actual glucose disposal (or 14C glucose flux) did not decrease with time and was not dependent on duration of isotope infusion. During saline infusion, estimates of glucose turnover were similar regardless of the glucose tracer used. High-performance liquid chromatography of the radioactive glucose tracer and plasma revealed the presence of a tritiated nonglucose contaminant. Although the contaminant represented only 1.5% of the radioactivity in the [6-3H]glucose infusate, its clearance was 10-fold less (P <.001) than that of [6-3H]glucose. This resulted in accumulation in plasma, with the contaminant accounting for 16.6 ± 2.09 and 10.8 ± 0.9% of what customarily is assumed to be plasma glucose radioactivity during the insulin or saline infusion, respectively (P < .01). When corrected for the presence of the contaminant, glucose turnover determined with [6-3H]glucose during insulin infusion (9.5 ± 0.6 mg · kg−1 · min−1) no longer differed from either the glucose infusion rate or that determined with [6-14C]glucose. Therefore, the underestimation of glucose turnover during insulin infusion and negative glucose production rates observed with traditional methods to analyze plasma radioactivity and commercially available tracers is the result of an artifactual increase in [6-3H]glucose specific activity. The etiology of the underestimation of glucose turnover with [6,6-2H2]glucose remains to be determined.
Modulation of Prostaglandin Metabolism by K-MAP and Prevention of Toxic Effect of Cyclosporin on Pancreatic Islet Cells
Authors: Teraoka Satoshi , Kawai Tatsuo , Babazono Tetsuya , Fuchinoue Shohei , Honda Hiroshi , Nakajima Ichiro , Fujikawa Hiroyasu , Hayashi Taketoshi , Nakagawa Yoshihiko , Fujita Shogo , Tojinbara Tamotsu , Ota Kazuo
Published: September 2013

The injection of 25 mg/kg i.p. cyclosporin (CsA) for 3 wk caused marked functional and morphological deteriorations of pancreatic islet cells in Wistar rats that were prevented by the combined administration of p-aminobenzoic acid-N-D-mannoside sodium salt (K-MAP). In this article, the toxic effect of CsA on pancreatic islet cells and the preventive effect of K-MAP on CsA-associated islet cell toxicity were investigated. Prolonged hyperglycemia and depressed insulin secretion after the glucose challenge observed in CsA-treated rats could be prevented by the combined administration of 300 and 900 mg/kg K-MAP. Cytoplasmic vacuolizations and a decrease in the number of mitochondria, intact endoplasmic reticula, secretory granules, and insulin-positive cells, as revealed by peroxidase-antiperoxidase staining, could also be prevented by the administration of 900 mg/kg K-MAP. This preventive effect of K-MAP on CsA-associated islet cell toxicity may suggest the combined use of K-MAP with CsA in pancreas transplantation and treatment of insulin-dependent diabetes.
Factors Influencing Isolation of Islets of Langerhans
Authors: Burghen George A , Murrell Leonard R
Published: September 2013

More efficient methods of islet isolation must be developed for islet transplantation to become clinically routine. During collagenase dispersal of human pancreas, an amorphous, viscous, gellike material often develops and entraps large numbers of islets, thereby reducing the yield. When donor human pancreas is minced and treated with collagenase, the gel forms most abundantly if the digestion temperature is <35°C and if pH falls below 7.2 ± 0.2. Gel formation appears to be proportional to warm- or cold-ischemia time and may be related to tissue trauma during collection. Once gel has formed, trapped islets cannot be released by filtration, dilution, DNase, incubation temperature, or pH adjustment. These characteristics suggest that the material is gelatin derived from collagen released enzymatically from pancreatic stroma. We demonstrate that gelation is greatly reduced or eliminated when 1) the incubation medium includes glycerol—a common gelatin solvent—at 5% (vol/vol), 2) the minced tissue-to-total incubation volume ratio is ≥1:10, 3) free-islet exposure to pancreatic digestion products is minimized by frequent separation of islets, and 4) collagenase concentration is optimized by titration. Gelation is also minimized by maintaining 5) incubation temperature at 38 ± 1°C and 6) pH in the range 7.7–7.9. Variations in these physical and chemical conditions were analyzed by determining islet yields (stereoscopic microscope counts of serially diluted samples) and by insulin radioimmunoassay of acid alcohol extracts of isolated islets after separation through discontinuous Ficoll gradients. When isolation conditions are optimized as stated, we typically recover 3.3 ± 1.0 × 104 islets/g pancreas, corresponding to >106 islets per donor.
Viable Purified Islets of Langerhans From Collagenase-Perfused Human Pancreas
Authors: Warnock Garth L , Ellis Dwayne K , Cattral Mark , Untch Dixie , Kneteman Norman M , Rajotte Ray V
Published: September 2013

Human islets of Langerhans were isolated with the principles of collagenase perfusion via the pancreatic duct and gentle dissociation of tissue. The number of islets released was 161 × 103, distributed as 76 × 103large (>100-μm) and 85 × 103 small (<100-μm) islets. Recovery after Ficoll-gradient purification was 61% for the large islets and 42% for the small islets. The final islet volume was 240 μl, with purity of 70–90% (large islets) and.20–40% (small islets). Perifusion with glucose elicited a biphasic release of insulin, with the response rising sixfold from basal secretion. Implantation of pure islets under the kidney capsule of normal or streptozocin-induced diabetic nude mice resulted in human C-peptide secretion and partial or complete reversal of hyperglycemia, confirmed by histological recovery. The data show that these methods provide large quantities of viable purified human islets.
Tissue Culture of Human Fetal Pancreas; Effects of Nicotinamide on Insulin Production and Formation of Isletlike Cell Clusters
Authors: Sandler Stellan , Andersson Arne , Korsgren Olle , Tollemar Jan , Petersson Birger , Groth Carl-Gustav , Hellerström Claes
Published: September 2013

Human fetal pancreas (HFP) is a potential source of β-cells for transplantation to insulin-dependent diabetic patients. We have previously described a method for tissue culture of HFP that results in the in vitro development of isletlike cell clusters (ICCs) containing a minority of insulin-positive cells. Recently we found that nicotinamide, an inhibitor of poly(ADP-ribose) synthetase, induces an increased islet cell DNA replication both in vivo and in vitro. In this study, this culture technique was used to evaluate the effects of addition of 10 mM nicotinamide on HFP expiants cultured in RPMI-1640 medium plus 10% human serum. ICCs developed in 11 of 19 consecutive cultures with nicotinamide increased the yield of ICCs by 40%. Also, the insulin content of ICCs increased ∼50% with nicotinamide supplementation, although measurements of DNA indicated an unchanged number of cells in each ICC. Neither the rates of insulin release in response to 16.7 mM glucose plus 5 mM theophylline nor the (pro)insulin or total protein biosynthesis rates were affected by nicotinamide addition. The combined results of this study suggest that nicotinamide is useful for stimulating the formation of ICCs from HFP.
Insulin Sensitivity and Glucose Effectiveness in Long-Term Islet-Autotransplanted Dogs
Authors: Finegood Diane T , Warnock Garth L , Kneteman Norman M , Rajotte Ray V
Published: September 2013

Parameters determining glucose tolerance were quantified with Bergman's minimal-model method applied to frequently sampled intravenous glucose tolerance tests in five normal and five islet-autotransplanted mongrel dogs 8–10 mo posttransplantation. Despite normal fasting glucose and insulin levels, glucose tolerance was reduced in the islet-transplanted dogs (1.5 ± 0.4 vs. 4.2 ± 0.4%/min in normal controls, P < .002). The reduction in glucose tolerance was due to a reduced insulin response to glucose injection (4 ± 1 vs. 32 ± 5 μU/ml, P < .001) and reduced glucose effectiveness (1.7 ± 0.5 vs. 5.3 ± 0.6 × 102 min−1, P < .005) but not to a reduction in insulin sensitivity (8.4 ± 0.6 vs. 7.8 ± 0.7 × 104 min−1 · μU−1 · ml−1, P > .5). Our results suggest that reduced insulin secretory response as a result of islet transplantation may result in a defect in glucose's ability to promote its own disposal but not necessarily in a defect in insulin sensitivity.
Monitoring of Pancreas-Graft Perfusion by Radionuclide and Digital Subtraction Angiography
Authors: Rasmussen Knud , Burcharth Flemming , Thomsen Henrik S , Rygaard Henrik
Published: September 2013

The vascularity of a kidney transplant can be evaluated by intravenous radionuclide angiography. A normal functioning transplant should have an isotope histogram with a well-defined peak. Accordingly, a transplanted pancreas may be evaluated in the same way. By intravenous digital subtraction angiography and small amounts of contrast medium, arterial and venous structures can be visualized without catheterization of the arterial system. Five patients had combined kidney and pancreas transplantation. Intravenous angiography with 99mTc-pertechnetate was performed three times a week for the first 5 postoperative wk. Digital subtraction angiography with an intravenous bolus of 80 ml of a low-osmolar nonionic contrast medium (iopamidol) was performed late in the postoperative course or when severe impairment of pancreas-graft perfusion was discovered by radionuclide angiography. One patient had two episodes and three patients one episode of rejection of both kidney and pancreas. Impairment of the pancreas-graft perfusion always preceded or was associated with deterioration of the graft function. In all patients, digital subtraction angiography demonstrated the graft vessels in sufficient detail. No thrombotic complications were observed. We conclude that these two methods can be used for monitoring the kidney and pancreas-graft perfusion. The methods may be of great value, especially in the early postoperative period, when problems with organ function are frequent and early intervention is essential.
Magnetic Resonance Imaging and Computed Tomography in Long-Term-Functioning Duct-Occluded Pancreas Allotransplants
Authors: Schlumpf Rolf , Marincek Borut , Schulthess Gustav Von , Decurtins Marco , Largiadèr Felix
Published: September 2013

Duct-occluded segmental-pancreas transplants develop progressive fibrotic atrophy, even though endocrine function seems to be unaffected. To determine end-stage size of the graft and to evaluate magnetic resonance imaging (MRI) and computed tomography (CT), these imaging techniques were applied in eight patients with well-functioning intraperitoneal prolamine-injected segmental-pancreas transplants for 79, 48, 35, 20,19,19,18, and 10 mo. MRI was performed on a 1.5 Tesla system (Philips Gyroscan S15). T1- and T2-weighted images were acquired. CT (Siemens Somatom 2) was done before and after intravenous contrast agent. The graft was visualized in seven of eight patients with both techniques. Visualization with MRI (vs. CT) was considered excellent in 2 (vs. 1), good in 3 (vs. 6), and poor in 2 (vs. 0). The three grafts with function longer than 2 yr measured 3–4 cm in length; the remaining grafts measured 3–6 cm. Because of a marked decrease in size the transplants were no longer localized in Douglas' pouch but adjacent to or on top of the uterus or bladder, the position depending on the volume of these organs. The al log rafts exhibited an inhomogeneous structure with casual cystic degeneration visible with MRI due to a high signal intensity on T2-weighted images. This study suggests that shrinkage of the duct-occluded pancreatic segment due to exocrine atrophy may be terminated after ∼2 yr. It is concluded that thereafter an overshooting fibrosis causing late endocrine graft failure may not be anticipated.
Impact of Immunosuppression on Improvement of Results in Clinical Pancreas Transplantation Clinical Pancreas Transplantation
Authors: Melandri Marco , Dubernard Jean Michel , Sanseverino Roberto , Camozzi Luca , Lefrancois Nicole , Faure Jean Louis , Martin Xavier , Rocca Ennio La , Gelet Albert , Touraine Jean Louis
Published: September 2013

Since November 1975, 103 pancreas transplantations have been performed in 97 insulin-dependent diabetic patients. Pancreas and kidney were grafted simultaneously in 84 patients (plus 1 double retransplantation). Eighty-nine pancreas grafts were prepared by duct obstruction with neoprene, and 14 were pancreaticoduodenal grafts with enteric diversion in a Roux-en-Y loop. Five immunosuppressive protocols were subsequently used. With the latest protocols, patient and pancreas survival improved to 93 and 72% at 1 yr, respectively. The improvement in graft survival appeared to be particularly related to the reduction of the number of pancreas grafts lost in rejection. The patients treated with the last protocols, including cyclosporin A (CsA) and only low doses of steroids, showed a better glucose tolerance after provocative tests. Pancreas-graft function did not appear to be influenced by CsA treatment.