bluuebunny/crossref_metadata_2025 huggingface dataset:
Collagen Browning and Cross-Linking Are Increased in Chronic Experimental Hyperglycemia: Relevance to Diabetes and Aging
Authors: Monnier Vincent M , Sell David R , Abdul-Karim Fadi W , Emancipator Steven N
Published: September 2013

Diabetes and aging are associated with an increase in collagen-linked fluorescence and cross-linking that can be duplicated by incubating collagen with glucose. We have tested the hypothesis that browning and cross-linking can occur in vivo in rats by feeding them a diet containing 33% galactose. No significant increase in tail tendon browning or cross-linking, measured by tail tendon breaking time in urea, was observed after 3 mo of galactosemia. After 12 mo, however, twofold increases in tendon breaking time and collagen-linked chromophores absorbing>300 nm and fluorophores at 430 nm (excitation 355 nm, P < .001) analogous to those of diabetic and aging individuals were observed. The observed changes in collagen are attributed to the advanced Maillard (nonenzymatic glycosylation) reaction based on circumstantial evidence. With this premise, our data suggest that chronic galactosemia should be a powerful tool for investigating the role of the advanced Maillard reaction in complications of diabetes and aging.
Inhibition of Insulin Release In Vitro Mediated by Mononuclear Cells From Diabetic Patients Treated With Cyclosporin A or Placebo
Authors: Debray-Sachs Monique , Sai Pierre , Feutren Gilles , Lang François , Maugendre Didier , Boitard Christian , Hors Jacques , Bach Jean-François
Published: September 2013

Anti-β-cell–specific cell-mediated immunity was studied over a 12-mo period in 65 recently diagnosed diabetic patients randomly receiving either cyclosporin or placebo. Anti-β–cell cellular immunity was assessed by an in vitro test based on the inhibition of insulin release from cultured rat islet cells by patients' mononuclear cells. This β-cell–suppressive effect disappeared in cyclosporin A–treated patients within 1 mo and did not reappear during 12 mo of follow-up. Conversely, the suppressive effect persisted unchanged in placebo-treated patients during 12 mo of follow-up. These changes were predictive neither of cyclosporin A–induced remission nor of relapses. Results of the insulin-release inhibition test were not correlated to islet cell autoantibodies or HLA phenotype.
Ethanol and Its Novel Metabolites Inhibit Insulin Action on Adipocytes
Authors: Lomeo Flario , Khokher Mohammad A , Dandona Paresh
Published: September 2013

The effects of ethanol, acetaldehyde, and the two novel metabolites of ethanol 2,3-butanediol and 1,2-propanediol on basal and insulin-stimulated adipocyte lipogenesis and glucose oxidation in vitro were investigated. Whereas ethanol and acetaldehyde inhibited both processes at concentrations far greater than those found in alcoholic subjects, the two diols were extremely potent inhibitors of basal and insulin-stimulated adipocyte metabolism at concentrations far below those observed in alcoholic subjects. The incorporation of labeled glucose into the fatty acid moiety and glucose oxidation were inhibited at lower concentrations (0.25 μM) than those required to inhibit the incorporation of the labeled glucose into glycerol. The diols are therefore potent inhibitors of basal and insulin-stimulated adipocyte metabolism. This effect may be relevant to the pathogenesis of insulin resistance in alcoholic subjects.
Effect of Aldose Reductase Inhibition on Nerve Conduction Velocity and Resistance to Ischemic Conduction Block in Experimental Diabetes
Authors: Price David E , Airey C Mark , Alani Shakir M , Wales John K
Published: September 2013

The relationships between nerve polyol levels and both nerve conduction velocity (NCV) and resistance to ischemie conduction block (RICB) in streptozocin-induced diabetic rats were examined in two studies. In the first study, sciatic NCV and RICB of the tail nerve, assessed by measuring the time to disappearance of the nerve action potential after the tail was rendered ischemie, were measured in nondiabetic rats, untreated diabetic rats, and diabetic rats given Statil, an aldose reductase inhibitor (ARI). Sciatic NCV was lower in the untreated diabetic animals than in control animals (P < .05), and RICB of the tail nerve was greater (P < .001). Treatment with the ARI completely prevented the slowing of NCV but had no significant effect on the increase in RICB. In the second study, similar groups of rats were treated with either ARI, insulin, or myo-inositol. Sciatic NCV was lower in the untreated diabetic rats than in the nondiabetic rats (P < .001). In diabetic rats treated with the ARI and in those treated with insulin, NCV was greater than in the untreated diabetic rats (P < .05 and P < .001, respectively) and was not significantly different from the nondiabetic rats. NCV in the myo-inositol-treated rats was not significantly different from that in the untreated diabetic rats. RICB was assessed by measuring the decline in sciatic nerve action potential amplitude at minute intervals after death. The time to a 50% fall in amplitude (t50) in the untreated diabetic rats was increased by 93%, and the time to complete disappearance (t0) was increased by 100% compared with the nondiabetic control rats. The increase in t50and t0 was prevented by insulin but was unaffected by myo-inositol. After the onset of ischemia, sciatic nerve function in the diabetic rats given the ARI initially fell rapidly: t50 was 29% lower than that found in the untreated diabetic rats (P < .02). The rate of decline then slowed, and t0 was not significantly different from that found in the untreated diabetic rats. t50 strongly correlated with nerve sorbitol levels among the insulin-treated diabetic rats (r = .94) and the untreated diabetic rats (r = .68) but not with nerve or plasma glucose levels. Thus, in experimental diabetes, RICB may be related to polyol-pathway overactivity. To explain ARI's reduction of t50 but not t0 in diabetic rats, we hypothesize that ARI may have had an effect on the RICB of ischemia-sensitive large-amplitude myelinated nerve fibers but little effect on ischemia-resistant low-amplitude unmyelinated fibers.
Prevention of Spontaneous But Not of Adoptively Transferred Diabetes by Injection of Neonatal BB/Hooded Hybrid Rats With Splenocytes or Concanavalin A Blasts From Diabetes-Free Strains
Authors: Logothetopoulos John , Shumak Kenneth , Bailey Dennis
Published: September 2013

Spontaneous diabetes was fully prevented in 65 BB/hooded (BB/h) highly diabetes-prone hybrid rats that were given five intraperitoneal injections (25 to 30 × 106 cells/injection) of fresh splenocytes or concanavalin A (ConA)–activated cultured splenocytes (blasts) from the diabetes-free Wistar-Furth or Long-Evans strains during the first 2 postnatal wk. Rats remained under observation for up to the age of 180–200 days. Of 70 littermate controls that received no cell injections, 63 developed overt diabetes before the age of 180 days. One intraperitoneal injection (25 × 106 cells) of splenocytes or blasts given during the first 36 h after birth was not as effective as multiple injections in preventing overt diabetes. Mild insulitis was present in 4 of 59 “protected” rats; small, discrete mononuclear infiltrates in periductular connective tissue and/or between pancreatic acini were observed in 27. Nondiabetic BB/h rats that were protected with splenocytes or blasts from diabetes-free strains had the same degree of lymphopenia in peripheral blood and spleen as age-matched, insulin-treated diabetic BB/h rats, but the level of islet cell surface antibodies in their serum was significantly lower. The same neonatal injections that protected rats from the development of spontaneous diabetes were completely ineffective in preventing the adoptive transfer of diabetes later in life by the injection of blasts from acutely diabetic BB/h rats.
Measurement of Plasma Glucose, Free Fatty Acid, Lactate, and Insulin for 24 h in Patients With NIDDM
Authors: Reaven Gerald M , Hollenbeck Clarie , Jeng Chii-Yang , Wu Min Shung , Chen Yii-Der Ida
Published: September 2013

Fasting and postprandial plasma glucose, free fatty acid (FFA), lactate, and insulin concentrations were measured at hourly intervals for 24 h in 27 nonobese individuals—9 with normal glucose tolerance, 9 with mild non-insulin-dependent diabetes mellitus (NIDDM, fasting plasma glucose < 175 mg/dl), and 9 with severe NIDDM (fasting plasma glucose > 250 mg/dl). In addition, hepatic glucose production (HGP) was measured from midnight to 0800 in normal individuals and patients with severe NIDDM. Plasma glucose concentration was highest in patients with severe NIDDM, lowest in those with normal glucose tolerance, and intermediate in those with mild NIDDM (two-way ANOVA, P < .001). Variations in plasma FFA and lactate levels of the three groups were qualitatively similar, with lowest concentrations seen in normal individuals, intermediate levels in the group with mild NIDDM, and the highest concentration in those with severe NIDDM (two-way ANOVA, P < .001). Of particular interest was the observation that plasma FFA concentrations were dramatically elevated from midnight to 0800 in patients with severe NIDDM. The 24-h insulin response was significantly increased in patients with mild NIDDM, with comparable values seen in the other two groups. Values for HGP fell progressively throughout the night in normal individuals and patients with severe NIDDM, despite a concomitant decline in plasma glucose and insulin levels. Although the magnitude of the fall in HGP was greater in NIDDM, the absolute value was significantly (P < .001) greater than normal throughout the period of observation. These results demonstrate that there are differences in substrate level between individuals with normal glucose tolerance and patients with NIDDM and differing degrees of glucose intolerance, unrelated to ambient insulin level, and these changes persist over 24 h.
Renal Hemodynamics and Urinary Excretion of 6-Keto-Prostaglandin F1α and Thromboxane B2 in Newly Diagnosed Type I Diabetic Patients
Authors: Gambardella Sergio , Andreani Domenico , Cancelli Armando , Di Mario Umberto , Cardamone Italo , Stirati Giovanni , Cinotti Giulio Alberto , Pugliese Francesco
Published: September 2013

We have studied the functional importance of renal eicosanoids in renal hemodynamics of seven newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients by treatment with two structurally unrelated inhibitors of cyclooxygenase (i.e., piroxicam and sulindac). Glomerular filtration rate (GFR), renal plasma flow (RPF), daily urinary excretion of 6-keto-prostaglandin F1α (6-keto-PGF1α, the stable hydrolysis product of prostacyclin), and thromboxane B2 (TXB2, the stable hydrolysis product of thromboxane A2) were measured before, during, and after piroxicam (all patients) or sulindac (3 patients) treatment. Urinary excretion of 6-keto-PGF1α was significantly increased (P < .01) in diabetic patients compared with seven healthy subjects, whereas urinary excretion of TXB2 was unchanged. The baseline value of GFR was significantly (P < .01) higher in diabetic compared with normal volunteers, whereas baseline RPF was comparable in both groups. Piroxicam (20 mg/day) reduced urinary excretion of 6-keto-PGF1α and TXB2 by 65.7 ± 26 and 64.6 ± 33%, respectively. These biochemical changes were temporally associated with the ∼ 19% decrease in GFR (P < .01). A week after discontinuation of the drug, GFR and urinary excretion of 6-keto-PGF1α were still significantly (P < .05) reduced, whereas urinary excretion of TXB2 returned to control values. In contrast, urinary excretion of eicosanoids and renal function were not affected by sulindac (0.4 g/day) treatment. No functional changes were detected in healthy subjects despite a similar suppression of renal cyclooxygenase activity when they were treated with piroxicam.We conclude that altered GFR in newly diagnosed IDDM patients may depend, at least partially, on altered renal synthesis of vasodilator prostacyclin.
Cyclosporin A Suppresses Insulin Autoantibodies and Heterologous Insulin Antibodies in Type I Diabetic Children
Authors: Castaño Luis , Boîtard Christian , Bougnères Pierre-François
Published: September 2013

We report that cyclosporin A (CsA) suppresses the insulin autoantibodies that are present before insulin therapy in the sera of one-third of studied type I (insulin-dependent) diabetic children. CsA also reversibly blocks the production of antibodies after exogenous insulin injection, whereas high titers of heterologous insulin antibody are observed in all type I patients not receiving CsA.
Preferential Infiltration of Macrophages During Early Stages of Insulitis in Diabetes-Prone BB Rats
Authors: Lee Ki U , Kim Myung K , Amano Kazuhiko , Pak Chin Y , Jaworski Martine A , Mehta Jayant G , Yoon Ji-Won
Published: September 2013

The subpopulation of lymphoid cells at the different stages of insulitis in BB rats was determined by immunohistochemical techniques with various monoclonal antibodies, including the recently developed OX41, which distinguishes macrophages from T-lymphocytes, OX19 for pan–T-lymphocytes, W3/25 for both helper T-lymphocytes and macrophages, OX8 for cytotoxic T-lymphocytes and natural killer cells, and OX12 for B-lymphocytes. The major population of infiltrated cells found during the early stages of insulitis appeared to be macrophages. This preceded invasion by a mixed population of cells, including both T- and B-lymphocytes and/or natural killer cells. The preferential infiltration of macrophages during the early stages of insulitis strongly suggested that there might be an initial change in the target β-cells that precedes their immune destruction, although the amplification of immune response by activated T-lymphocytes and natural killer cells at a later stage seemed to be required for the clinical expression of the disease.
Pharmacokinetic Model of Circulating Covalent Aggregates of Insulin
Authors: Maislos Maximo , Bialer Meir , Mead Patricia M , Robbins David C
Published: September 2013

Covalent aggregates of insulin in blood of insulin-treated diabetic patients account for as much as 70% (mean 28 ± 3.6%) of serum insulin immunoreactivity. These aggregates may originate in therapeutic insulin, because similar substances account for 0.1–3% of these preparations. Larger amounts in blood imply that aggregates accumulate as a result of delayed clearance. To test and quantify this speculation, we calculated the plasma kinetics of this material in four normal volunteers who received large intravenous doses (30 mU · kg−1 · min−1) of beef-pork crystalline zinc insulin for 120 min. Insulin aggregate and monomer concentrations were measured in blood samples obtained at regular intervals throughout the infusion and during 4 h after discontinuation of insulin. Pharmacokinetic parameters were calculated from the data. Insulin aggregate and monomer serum t½ were 63.6 ± 6.9 and 34.3 ± 2.8 min, respectively, and clearances were 101 ± 10 and 232 ± 47 ml/min. Volume of distribution (Vβ) and volume at steady state (Vss) were 9.1 ± 1.8 and 8.2 ± 2.2 L for insulin aggregate and 11.6 ± 2.8 and 12.2 ± 3.6 L for insulin monomer, respectively. Mean residency time was 141 ± 14 and 114 ± 10 min for insulin aggregate and monomer, respectively [P < .01 for all parameters except Vβ (.014) and Vss (.012), aggregate vs. monomer]. Thus, in relation to insulin monomer, calculated pharmacokinetic parameters of the aggregate predict accumulation after insulin injection. Plasma t½ of the aggregate was almost double that of monomeric insulin; total-body clearance and the corresponding volumes of distribution were smaller.
Effect of Helper and/or Cytotoxic T-Lymphocyte Depletion on Low-Dose Streptozocin-Induced Diabetes in C57BL/6J Mice
Authors: Dayer-Métroz Marie-Dominique , Kimoto Makoto , Izui Shozo , Vassalli Pierre , Renold Albert E
Published: September 2013

The low-dose streptozocin (STZ) model of diabetes has been reported to involve direct STZ beta-cytotoxicity and/or immunologically mediated β-cell destruction. Because the T-lymphocyte dependency of such a model is controversial, we further assessed the role of T-lymphocytes by determining the occurrence and magnitude of hyperglycemia as well as the pancreatic insulin contents in both STZ-injected nude C57BL/6J male mice and STZ-injected euthymic C57BL/6J male mice selectively depleted in helper and/or cytotoxic T-lymphocytes with monoclonal antibodies (MoAbs). The effectiveness of MoAb treatment was assessed in lymph node cells by flow-microfluorometry analysis and in spleen cells by concanavalin A stimulation, allospecific cytotoxic T-lymphocyte activity, and T-lymphocyte lymphokine production. Sixteen days after the first STZ injection, hyperglycemia (plasma glucose > 200 mg/dl) occurred in significantly fewer helper T-lymphocyte-depleted mice (P < .005) or helper and cytotoxic T-lymphocyte–depleted mice (P < .001) than in non–MoAb-treated mice. However, a progressive increase in the number of mice with hyperglycemia ensued in all MoAb-treated groups, and 2 mo after STZ was administered, the prevalence of hyperglycemia, mean plasma glucose levels, and pancreatic insulin contents did not differ significantly from the values obtained in the non–MoAb-treated animals. Similarly, STZ-injected C57BL/6J male nude mice developed hyperglycemia that was associated with a marked decrease in pancreatic insulin contents on a time course comparable with that of STZ-injected euthymic C57BL/6J male mice depleted in helper or in helper and cytotoxic T-lymphocytes by MoAbs. Thus, selective helper T-lymphocyte depletion, either genetic or induced by MoAb treatment, although able to temporarily protect mice from the development of hyperglycemia, did not prevent the ultimate loss of the β-cell mass initiated by the direct toxic effect of STZ.
Demonstration of Altered Antithrombin III Activity Due to Nonenzymatic Glycosylation at Glucose Concentration Expected to be Encountered in Severely Diabetic Patients
Authors: Villanueva German B , Allen Nancy
Published: September 2013

The effect of nonenzymatic glycosylation on the kinetics and structure-function relationships of antithrombin III were investigated at normal physiologic concentrations of antithrombin III and glucose, which are 5.2 μM and 5 mM, respectively. The results were compared with antithrombin III incubated at the glucose concentration expected to be found in severely diabetic patients (15 mM). Antithrombin III incubated at 5 mM lost 33% of the heparin cofactor activity after 7 days, whereas antithrombin III incubated at 15 mM lost 50% for the same period. Under both conditions, half of the heparin cofactor activity was lost after 15 days. When D-[U-14C]glucose was used as tracer, ∼0.6 mol glucose/mol protein was incorporated after 10 days at both concentrations of glucose. A detailed evaluation of the kinetics of inhibition of thrombin by glycosylated antithrombin III revealed that the second-order rate constant is three times smaller than that of normal antithrombin III. On the basis of these data, it is concluded that glycosylated antithrombin III with 50% depressed heparin cofactor activity is three times weaker than normal antithrombin III as an inhibitor of thrombin. The implications of these observations with respect to the possible pathogenesis of thrombosis in diabetes are discussed.
Progression From Insulitis to β-Cell Destruction in NOD Mouse Requires L3T4+ T-Lymphocytes
Authors: Charlton Brett , Mandel Thomas E
Published: September 2013

The identity of the cells responsible for beta-cell destruction in type I (insulin-dependent) diabetes is still uncertain. L3T4+ T-lymphocytes have a role in the initiation of insulitis and in damaging transplanted allogeneic islets in nonobese diabetic (NOD) mice. The role of L3T4+ T-lymphocytes in destruction of beta-cells of the NOD mouse was studied in cyclophosphamide (CY)-induced diabetic NOD mice with a rat anti-L3T4 monoclonal antibody (MoAb). After administration of CY, most untreated animals became diabetic, whereas all antibody-treated animals remained normoglycemic. Insulitis was still present in MoAb-treated animals, but immunocytochemical staining showed rat antibody blocking the L3T4 antigen on T-lymphocytes. This study provides further evidence that L3T4+ T-lymphocytes are critical to the process of beta-cell destruction in NOD mice. The means by which L3T4+ cells exert their effect remains to be clarified.
Contributions of Absorbed Dietary Cholesterol and Cholesterol Synthesized in Small Intestine to Hypercholesterolemia in Diabetic Rats
Authors: Young Nancy L , Lopez Deborah , McNamara Donald
Published: September 2013

To determine how insulin deficiency leads to hypercholesterolemia, we examined cholesterol absorption, synthesis, and utilization in streptozocin-induced diabetic rats fed a grain-based diet ad libitum. Absorbed dietary cholesterol was determined from measurement of dietary cholesterol intake and previous data for cholesterol fractional absorption. Daily rates of cholesterol synthesis in the small intestine, liver, and periphery were calculated from recovery of labeled sterols after injection of 3H2O at six times during 24 h. Utilization of cholesterol for growth, fecal excretion, and bile acid synthesis were also determined. Absorbed dietary cholesterol in diabetic rats was double that in control rats. The contribution of absorbed cholesterol to total cholesterol production (sum of absorbed dietary cholesterol) and endogenous cholesterol synthesis) in control rats was 24% compared to 48% in diabetic rats. The increase in diabetic rats was due to overeating and, to a lesser extent, to increased fractional absorption. Overeating also induced intestinal hypertrophy and a twofold increase in cholesterol synthesis by the small intestine to 24% of whole-body production in diabetic rats. Consequently, the small intestine accounted for 72% of daily cholesterol input in diabetic rats compared to 37% in control rats, with diet accounting for two-thirds of the cholesterol input via the small intestine in both groups. In response to this increased input from the intestine, cholesterol synthesis in the periphery was 39% of whole-body production in control rats compared to 22% in diabetic rats, and synthesis in the liver was 26 and 6% of total cholesterol production in control and diabetic rats, respectively. The net effect of these changes was a decrease in whole-body synthesis equal to the increase in absorbed dietary cholesterol and no change in whole-body cholesterol production in diabetic rats. The need for cholesterol as substrate for bile acid synthesis was met largely by absorption from the diet in diabetic rats and by synthesis in the liver in control rats. The need for cholesterol in the periphery was entirely met by synthesis in situ in both groups. We conclude that 1) an increased fraction of the net cholesterol production is derived from intestinal sources and transported through the blood in diabetic rats, and 2) cholesterol absorbed from the diet contributes more to the hypercholesterolemia of diabetic rats than the increased cholesterol synthesis in the small intestine.
Treatment With Streptococcal Preparation (OK-432) Suppresses Anti-Islet Autoimmunity and Prevents Diabetes in BB Rats
Authors: Satoh Jo , Shintani Shigeki , Oya Keiichiro , Tanaka Shun-Ichi , Nobunaga Toshima , Toyota Takayoshi , Goto Yoshio
Published: September 2013

We have recently shown that a streptococcal preparation (OK-432) inhibits insulitis and prevents diabetes in nonobese diabetic (NOD) mice, an animal model of insulin-dependent diabetes mellitus (IDDM). We extended this study to another model of IDDM, namely BB rats. Male and female BB rats were injected weekly with 0.2 mg OK-432 i.p. starting from 5 to 6 wk and continuing through 20 or 30 wk of age. The cumulative incidence of IDDM over 20 wk in the OK-432-treated BB rats (4 of 54, 7.4%) was significantly (P < .01) lower than that found in the nontreated BB rats (13 of 47, 27.7%). We examined some of these rats as follows. All of the OK-432-treated BB rats tested showed normal glucose levels before and after oral glucose administrations, as did the nontreated and nondiabetic BB rats. Histological examination of pancreatic sections revealed that the OK-432-treated rats retained a greater number of intact islets without infiltration of the mononuclear cells than did the nontreated BB rats. A preliminary in vitro study further demonstrated that the cytotoxic activities of spleen cells against a rat insulinoma cell line, RIN, were suppressed in the OK-432-treated rat. However, the treatment of BB rats with OK-432 showed no suppressive effects in the spleen cell number, the responsiveness of spleen cells to concanavalin A, the populations of OX19+, W3/25+, and OX8+ peripheral blood lymphocytes, or in the titers of cell surface antibody against RIN. These results suggest that a nonimmunosuppressive immunomodulator such as OK-432 may be useful as an agent for immunotherapy of IDDM.
Sequence and Analysis of Promoter Region of Human Insulin-Receptor Gene
Authors: Mamula Paul W , Wong Kwok-Ying , Maddux Betty A , McDonald Alexander R , Goldfine Ira D
Published: September 2013

The promoter region of the human insulin-receptor (HINSR) gene was isolated from a human chromosome 19 bacteriophage library. With S1 nuclease mapping and primer-extension analysis, we identified multiple transcription-initiation sites. Dexamethasone, a known inducer of HINSR transcription, enhanced transcription of all major transcription-initiation sites. DNA sequence analysis indicated that the HINSR promoter has neither a TATA box nor a CAAT box. The HINSR promoter region contains six GGGCGG sequences that may be binding sites for the transcription factor Sp1. In addition, there were three TCCC sequences that were putative promoter regulatory regions. The HINSR gene promoter has structural similarity to the epidermal growth factor receptor gene promoter and has some features of the promoter of the meglutol (hydroxymethylglutaryl, HMG) CoA reductase gene and the early promoter of simian virus 40.
Genetic Control of Organ-Reactive Autoantibody Production in Mice by Obesity (ob) Diabetes (db) Genes
Authors: Yoon Ji-Won , Leiter Edward H , Coleman Douglas L , Kim Myung K , Pak Chin Y , McArthur Robert G , Roncari Daniel A K
Published: September 2013

Inbred strains of mice exhibited genetic and sex-dependent differences in spontaneous production of organ-reactive autoantibodies detected by indirect immunofluorescence. Antitestis autoreactivity was found primarily in sera from C57BL/6J (B6) mice, whereas antigastric autoreactivity was common to both CBA/J and 129/J strains. Autoantibodies against islet cell cytoplasmic antigens (ICAs) were uniquely expressed by C57BL/KsJ (BKs) males. Introduction of the diabetes (db) mutation into these various inbred-strain backgrounds induced expression of ICA, with stronger induction observed in males. The stress imposed by the db or obesity (ob) mutation induced ICA in BKs mice at a higher frequency than in B6 mice; this differential sensitivity was somehow related to a gene linked to the H-2 complex because BKs.B6 H-2b congenic mice resembled B6 mice. The db3J mutation increased the expression of these autoantibodies in 129/J mice, which, like B6, were H-2b and therefore presumably possessed the same H-2-linked inducibility allele as BKs. Cytotoxic autoantibodies against islet cell surface antigens were only observed in C3HeB/FeJ db/db males, and their presence was correlated with β-cell necrosis. It is concluded that db and/or ob genes appear to play an important role in the production of autoantibodies to islet cells, and sex-linked factor(s) may modify the phenotypic expression of the autoantibodies.
Adoptive Immunotherapy of Diabetes in Autologous Nonobese Diabetic Mice With Lymphoid Cells Ex Vivo Exposed to Cyclosporin Plus Interleukin 2
Authors: Formby Bent , Miller Nancy , Peterson Charles M
Published: September 2013

Between 12 and 26 wk of age, ∼80% of female nonobese diabetic (NOD) mice from the inbred Sansum colony develop lymphocytic insulitis and become overtly diabetic. The disease in this animal model is similar to human insulin-dependent diabetes mellitus (IDDM) in both genetics and autoimmune pathogenesis. Cyclosporin A (CsA) has been used for immunosuppression in IDDM of recent onset in humans but has several limiting side effects. Therefore, a different regimen for CsA immunosuppression was investigated. Autologous splenic lymphoid cells from 12-wk-old not-yet-diabetic female NOD mice were cultured for 72 h with CsA plus interleukin 2 (IL-2) before reinfusion into the animal from which they were isolated. After this treatment, only 2 (18%) of 11 mice became overtly diabetic during an observation period of 19 wk, while 18 (86%) of 21 age-matched control mice developed diabetes during the same observation period (P < .001). These data suggest an ex vivo preferential IL-2 activation of specific suppressor cells for the autoimmune process with CsA blockade of cytolytic/helper activities. Because the in vivo concentrations of CsA with this procedure would be negligible, these findings have implications for the potential nontoxic use of CsA in human protocols as well.
Characterization of Induction of Protooncogene c-myc and Cellular Growth in Human Vascular Smooth Muscle Cells by Insulin and IGF-I
Authors: Banskota Nirmal K , Taub Rebecca , Zellner Karen , Olsen Philip , King George L
Published: September 2013

Insulin and insulin-like growth factor I (IGF-I) are structurally related polypeptides that stimulate DNA synthesis and cellular proliferation, probably through a common pathway. Human arterial smooth muscle cells in culture demonstrated the presence of high-affinity receptors for both these hormones. Insulin and IGF-I both exhibited cross-reactivity to each other's receptors but with an affinity that is 100-fold less than for the homologous receptor. To examine more closely the receptor responsible for producing the growth effects, we used the polyclonal antibody against the insulin receptor, B2, and a monoclonal antibody to the IGF-I receptor, αIR3 . We studied the growth effects of insulin and IGF-I as measured by stimulation of c-myc, DNA synthesis, and cellular proliferation in the presence and absence of these antibodies. F(ab') fragments of the anti-insulin-receptor antibody at a concentration of 10 μg/ml were capable of displacing >90% of the bound insulin, thus establishing an effective insulin-receptor blockade. Under such blockade, insulin and IGF-I were both capable of doubling the amount of DNA synthesis and cell number in cultured human arterial smooth muscle cells. However, in the presence of a 1:2500 dilution of the monoclonal antibody αlR3 , which caused a 90% displacement of IGF-I bound to its receptor, both the insulin and IGF-I effects on stimulating DNA synthesis or cellular proliferation were inhibited by >90%. These findings demonstrate that the IGF-I receptor is the common pathway for the growth effects of both insulin and IGF-I. Induction of the protooncogene c-myc is recognized as an early response to many mitogenic stimuli. In human smooth muscle cells, insulin (1 × 10−7M) and IGF-I (1 × 10−9M) both cause a 5- to 10-fold increase in c-myc mRNA levels. Induction of c-myc could not be assessed under conditions of selective IGF-I-receptor blockade with the antibody αIR3, because, surprisingly, the antibody itself stimulated c-myc mRNA levels. This induction of c-myc with αIR3, which does not increase DNA synthesis or cellular proliferation, suggests that c-myc induction does not entirely correlate with the growth effects of these hormones.
Insulin-Receptor Gene and Its Expression in Patients With Insulin Resistance
Authors: Muller-Wieland Dirk , Taub Rebecca , Tewari Dinesh S , Kriaciunas Kristina M , Sethu S , Reddy K , Kahn C Ronald
Published: September 2013

We studied the structure of the insulin-receptor gene in normal individuals and in four unrelated patients with leprechaunism (Minn-1, Ark-1, Ark-2, Can-1) and four unrelated patients with the type A syndrome of insulin resistance, both disorders associated with genetic alterations in affinity, binding capacity, and kinase activity of the insulin receptor. Genomic cloning and Southern blot analysis indicate that the normal human insulin-receptor gene is ≥ 150 kilobases long and consists of a minimum of 17 exons, 6 in the genomic region of the α-subunit and 11 in the region of the β-subunit. Three of the patients, one with leprechaunism and two with type A syndrome, have decreases in insulin-receptor mRNA but on genomic blot analysis have no obvious abnormalities in the insulin-receptor gene. No distinctive pattern of restriction-fragment–length polymorphisms or evidence for major insertion or deletion mutations of the insulin-receptor gene was found in any of the patients. These data indicate that the insulin-receptor gene is > 35 times larger than coding regions and has a complex structure. Although leprechaunism and type A syndrome are most likely due to defects in the structure and expression of the insulin-receptor gene, they are likely to be associated with specific point mutations rather than major changes in gene structure.
Regulation of Carnitine Palmitoyltransferase Synthesis in Spontaneously Diabetic BB Wistar Rats
Authors: Brady Linda J , Brady Paul S
Published: September 2013

The long-term regulation of hepatic mitochondrial carnitine palmitoyltransferase (CPT) was studied in control, insulin-treated, and untreated spontaneously diabetic BB Wistar rats. The activity of CPT was elevated approximately twofold in the untreated diabetic rats. This corresponded to an approximately equivalent elevation in the immunoreactive CPT activity. mRNAcpT was assayed by reticulocyte lysate translation and by dot blot to a CPT oligonucleotide probe. The level of mRNACPT was approximately proportional to the observed CPT activity. A cDNA probe to CPT was developed, and transcriptional activity for CPT was assessed in isolated hepatic nuclei. Again, transcription of CPT mRNA was approximately proportional to the observed activity. We therefore conclude that at least part of the long-term regulation of hepatic CPT in spontaneously diabetic BB Wistar rats is the product of increased de novo synthesis of CPT protein brought about by regulation at the transcriptional level. Additional control of the amount of CPT may be via the regulation of RNA processing and turnover and enzyme insertion into the mitochondrial membrane.
Evidence for Multifactorial Origin of Diabetes-Induced Embryopathies
Authors: Sadler T W , Hunter E S , Wynn R E , Phillips L S
Published: September 2013

Serum factors characteristically altered in the diabetic state, e.g., glucose, ketone bodies (β-hydroxybutyrate), and somatomedin inhibitors, induce dysmorphogenesis with or without growth retardation in rodent embryos in whole-embryo culture. Furthermore, serum from diabetic animals, which contains combinations of altered factors, is teratogenic, thereby implying that the diabetic embryopathy is of a multifactorial origin. However, a detailed investigation with various combinations of factors at known concentrations to test this hypothesis has not been conducted. Therefore, we employed combinations of hyperglycemia (300 and 600 mg/dl; 16.6 and 33.3 mM), hyperketonemia (8 and 16 mM D-β-hydroxybutyrate), and an 800- to 1000-Mr somatomedin-inhibitor serum fraction (at an amount equal to that found in 0.05 and 0.1 ml of serum from a diabetic rat per deciliter culture medium), which represented doses with low teratogenic potential, to determine if interactions of the factors could occur that would increase the risk of malformations in mouse embryos exposed in whole-embryo culture during gastrulation and neurulation. The results demonstrate that glucose and D-β-hydroxybutyrate can act synergistically to produce growth retardation and additively to induce malformations. The addition of the somatomedin inhibitor exacerbates the induction of malformations produced by the ketone body and glucose. Thus, the origin of the diabetic embryopathy may be multifactorial, involving several altered maternal factors.
Pancreas-Transplant Outcome in Relation to Presence or Absence of End-Stage Renal Disease, Timing of Transplant, Surgical Technique, and Donor Source
Authors: Sutherland David ER , Moudry Kay C , Dunn David L , Goetz Fred C , Najarian John S
Published: September 2013

The differences in pancreas-transplant outcome according to recipient status, surgical approach, and donor source are illustrated by an analysis of results at one institution with experience in several categories. From July 1978 to January 1988, 210 pancreas transplants were performed, and 67 grafts are still functioning, the longest for 9.7 yr. Since October 1984, a uniform immunosuppressive protocol has been used, antilymphocyte globulin, cyclosporin, azathioprine, and prednisone for induction and the last three drugs for maintaining antirejection therapy. During this period, 110 pancreas transplants were performed, 62 in nonuremic non-kidney transplants, 28 in recipients of a previous kidney, and 20 simultaneous with a kidney; 64 with bladder and 43 with enteric drainage; and 25 from related and 85 from cadaver donors. The overall patient survival rate at 1 yr was 91%, and there were no significant differences between the various categories. Graft survival rates, however, differed between the various categories created by combinations of the above variables. With bladder drainage, 1-yr function rates were 58% (n = 30), 47% (n = 15), and 77% (n = 19) in recipients of a pancreas transplant alone, a pancreas after a kidney, or a simultaneous pancreas-kidney transplant; with enteric drainage, 1-yr function rates were 33% (n = 32) and 36% (n = 11) in the pancreas transplant alone and pancreas after kidney categories (enteric drainage was not done in double-transplant patients). The worst results were in recipients of enteric-drained cadaverdonor grafts because of the high propensity for rejection episodes of grafts from this donor source and an inability to diagnose rejection episodes early with this surgical approach. The best results were in recipients of bladder-drained cadaver-donor and enteric- or bladder-drained related-donor grafts. These approaches are currently exclusively applied; bladder drainage allows for early diagnosis and treatment of rejection based on urinary amylase monitoring, which is very important for transplants from cadaver donors, in which the incidence of rejection episodes is much higher than for transplants from related donors. An analysis of the results of pancreas transplantation in patients who, since October 1984, have been transplanted by the approaches in current use, shows overall patient and graft survival rates at 1 yr of 91 and 57%, respectively. One-year graft function rates within the various categories were 56% for 45 pancreas transplants alone, 45% for 22 pancreas transplants after a kidney, and 77% for 19 simultaneous pancreaskidney transplants. The results are best in uremic recipients of simultaneous pancreas-kidney transplants, but with quadruple immunosuppressive therapy, long-term function can also be achieved in more than half of recipients of pancreas transplants alone.
New Principle for Large-Scale Preparation of Purified Human Pancreas Islets
Authors: Winoto-Morbach Supandi , Ulrichs Karin , Leyhausen Gaby , MÜller-Ruchholtz Wolfgang
Published: September 2013

Because successful human islet transplantation requires large quantities of viable islets that must be separated from the highly immunogenic exocrine tissue and because handpicking is too time-consuming and laborious to be clinically relevant, a new approach for solving this problem has been established in rat models. It is based on the principle that magnetic microspheres (MMSs) coupled to lectins with binding specificity for the exocrine tissue portion are trapped in an electromagnetic field, thus providing effluent islets of a high degree of purity. In this study our aim was to adapt this principle to human islet preparations. In this context our prime interest was focused on a lectin suitable for human pancreatic tissue. Of 19 different lectins tested, only 1, Wisteria floribunda agglutinin (WFA), is suitable, as shown by immunofluorescence, MMS-lectin binding, and magnetic separation.
Effect of γ-Irradiation on Mouse Pancreatic Islet-Allograft Survival
Authors: Kanai Toshio , Porter Janis , Gotoh Mitsukazu , Monaco Anthony P , Maki Takashi
Published: September 2013

Elimination or inactivation of lymphoid tissue in the pancreatic islet preparation achieves prolongation of islet-allograft survival. In this study we examined the effect of γ-irradiation on mouse islet-allograft survival. In a B6AF1 isograft model, irradiation up to 2400 rad did not induce deterioration of islet function over 200 days, but greater doses caused cessation of graft function between 83 and 186 days. When DBA/2 crude islets were transplanted into B6AF1 recipients, all nonirradiated allografts were acutely rejected. Marked prolongation of allograft survival was achieved by islet irradiation with doses between 800 and 12,000 rad. With higher doses, significant numbers of allografts survived beyond the controls, but many lost function between 78 and 180 days, with none surviving >200 days. Irradiation with 16,000 rad caused acute radiation damage. Because most secondary islet allografts in recipient mice that lost primary islet-graft function between 84 and 195 days survived >100 days, late functional loss was probably due to the radiation injury. Combined use of recipient treatment with cyclosporin A and graft irradiation (2400 rad) achieved prolongation of DBA/2 islets in B6AF1 mice.
Segmental Duct-Obstructed Pancreas Grafts Versus Pancreaticoduodenal Grafts With Enteric Diversion
Authors: Martin Xavier , Dubernard Jean Michel , Sanseverino Roberto , Melandri Marco , Faure Jean Louis , Camozzi Luca , LeFrancois Nicole , Rocca Ennio , Gelet Alber
Published: September 2013

Between January 1985 and September 1987, we performed a prospective comparative study between segmental-pancreas transplantation with duct obstruction by neoprene (n = 17) and pancreaticoduodenal transplantation with enteric diversion to a Roux-en-Y intestinal loop (n = 14). All recipients had insulin-dependent diabetes. The immunosuppressive protocol consisted of low doses of the steroids cyclosporin A and azathioprine. Mean follow-up was 16.5 mo for the enteric-diversion group and 13.5 mo for duct-obstructed groups. Two-year patient and pancreas- and kidney-graft actuarial survival rates were 92.9, 75.5, and 74.2%, respectively, in the former group and 92.3, 58.4, and 63.7%, respectively, in the latter group (NS). Five whole-organ grafts were lost (3 vascular thromboses, 1 pancreatitis, 1 rejection), and four segmental grafts were lost (2 vascular thromboses, 1 bleeding, 1 patient's death with functional graft). More surgical complications occurred in the recipients of whole-organ grafts and were often related to the intestinal anastomosis. A satisfactory blood glucose control was observed at 3 mo and 1 yr in both groups. Provocative tests showed higher and prompter insulin secretion in patients with wholeorgan grafts. In patients with segmental grafts, the response was lower and delayed with a general tendency to impaired glucose tolerance. A marked hyperinsulinemia after meals was observed in whole-organ graft recipients. Slight nocturnal hyperinsulinemia was observed in both groups. At 1 yr, glycosylated hemoglobin was normal in both groups. The absence of a significant difference between the two groups, in terms of survival and graft function, and the lower surgical complication rate seen with segmental grafts have made us return to neo p re ne-injected segmental grafts.
Generation of Allospecific Cytolytic T-Lymphocytes Stimulated by Pure Pancreatic β-Cells in Absence of la+ Dendritic Cells
Authors: Stock Peter G , Meloche Mark , Ascher Nancy L , Chen Sally , Bach Fritz H , Sutherland David ER
Published: September 2013

A murine mixed islet-lymphocyte coculture system (MILC) was used to quantitate the immunogenicity of a pure population of pancreatic β-cells to more clearly define whether stimulator major histocompatibility complex (MHC) class ll-positive dendritic cells are a major component leading to islet immunogenicity. Pancreatic β-cells express MHC class I antigen but not class II antigen. These experiments compared the in vitro immunogenicity of fluorescence-activated cell sorted (FACS-IV) pure β-cells (MHC class I-positive cells only) relative to unpurified dispersed islet cells (MHC class I-positive cells and class II-positive cells). The results demonstrated the surprising finding that pure DBA/2J (H-2b) pancreatic β-cells stimulated a strong cytotoxic T-lymphocyte (CTL) response when exposed to C57BL/6 (H-2b) allosplenocytes in the MILC, similar to DBA/2J nonpurified dispersed islet cells. Furthermore, the stimulation of CTL by both purified β-cells and nonpurified dispersed islet cells was blocked by addition of MHC-specific anti-class I monoclonal antibody directed against stimulator MHC antigen. The data imply that the highly immunogenic MHC class II-positive passenger leukocytes present in the islets were not necessary for the generation of the immune response in the presence of MHC class l-positive β-cells. Although most of the pretreatment regimens attempting to decrease islet immunogenicity have been directed at eliminating the MHC class II-positive passenger leukocytes from the islets, this work suggests that modulation of MHC class I antigen may be an important approach.
Effects of Culture Conditions on Formation and Hormone Content of Fetal Porcine Isletlike Cell Clusters
Authors: Korsgren Olle , Sandler Stellan , Jansson Leif , Groth Carl-Gustav , Hellerström Claes , Andersson Arne
Published: September 2013

To establish methods for stimulation of the growth and differentiation of fetal endocrine pancreatic cells, a technique for the in vitro production of fetal porcine isletlike cell clusters (ICCs) was used. By varying the composition of the culture medium with different glucose concentrations and the addition to the culture medium of insulin, growth hormone (GH), amino acids, or nicotinamide, we estimated the formation of ICCs and their hormone content. High glucose content (28.0 mM) stimulated the formation of abundant ICCs that contained decreased amounts of insulin. In contrast, culture at a low (5.6-mM) glucose concentration increased the ICC insulin content but decreased the number of ICCs formed. Addition of seven times the normal amount of amino acids hampered both the formation of ICCs and their insulin content. Neither insulin nor GH supplementation of the medium influenced the ICC insulin content, but GH stimulated an abundant outgrowth of ICCs containing relatively high insulin concentrations. However, ICCs formed under these circumstances contained <10% of the insulin content of adult islets, and further work has to be carried out to identify factors responsible for further differentiation of the fetal porcine pancreas.