bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin-Receptor Autophosphorylation and Endogenous Substrate Phosphorylation in Human Adipocytes From Control, Obese, and NIDDM Subjects
Authors: Thies R Scott , Molina Joseph M , Ciaraldi Theodore P , Freidenberg Gary R , Olefsky Jerrold M
Published: September 2013

We identified a possible endogenous substrate (pp185) of the insulin-receptor kinase in human adipocytes by treating intact cells with insulin and immunoblotting the cellular extracts with polyclonal antiphosphotyrosine antibody. This 185,000-Mr protein was phosphorylated on tyrosine residues in response to insulin in both rat and human adipocytes. The time course of pp185 phosphorylation at 37°C was rapid and corresponded closely to insulin-receptor autophosphorylation but preceded insulin-stimulated glucose transport. Unlike many growth factor receptors, including the insulin receptor, pp185 was not adsorbed to wheat-germ agglutinin. We found that pp185 phosphorylation occurred at 12°C and that the phosphoprotein was associated with both cytoplasmic and membrane fractions at this temperature. Furthermore, pp185 phosphorylation was induced to the same extent as insulin by vanadate and hydrogen peroxide, compounds previously shown to mimic the biologic effects of insulin. In addition, dose-response analysis of insulin-stimulated glucose transport, receptor autophosphorylation, and pp185 phosphorylation resulted in ED50 values of 0.3, 12, and 12 ng/ml, respectively. These results demonstrate the magnitude of “spare” autophosphorylation and pp185 phosphorylation with respect to glucose transport stimulation in human adipocytes. To determine whether the insulin resistance characteristic of non-insulin-dependent diabetes mellitus (NIDDM) and obesity is associated with a defect in receptor autophosphorylation and/or endogenous substrate phosphorylation, we estimated the extent of β-subunit and pp185 phosphorylation in adipocytes from NIDDM, obese, and healthy subjects. Although the efficiency of coupling between receptor activation and pp185 phosphorylation was normal in obesity and NIDDM, the capacity for insulin-receptor autophosphorylation was ∼50% lower in NIDDM subjects compared with nondiabetic obese or lean subjects. Thus, the absolute level of pp185 phosphorylation in response to insulin stimulation would be lower in adipocytes from NIDDM subjects. We conclude that pp185 is directly phosphorylated by the insulin-receptor kinase in vivo and may serve as a mediating step to the biologic effects of insulin in human adipocytes. Furthermore, the extent of pp185 phosphorylation is compromised in adipocytes from NIDDM subjects and may contribute xto the insulin resistance associated with this disorder.
Handicaps to Host Defense: Effects of Hyperglycemia on C3 and Candida albicans
Authors: Hostetter Margaret K
Published: September 2013

The hyperglycemic patient remains persistently at risk for infectious complications. Whether ascribable to diabetes mellitus, to the administration of glucocorticoids, or to the infusion of hyperalimentation fluids, hyperglycemia may impair several mechanisms of humoral host defense, including such varied neutrophil functions as adhesion, chemotaxis, and phagocytosis. In addition, binding of glucose to the biochemically active site of the third component of complement C3 inhibits the attachment of this protein to the microbial surface and thereby impairs opsonization. Last, several pathogens frequently encountered in hyperglycemic patients possess unique mechanisms of virulence that flourish in the hyperglycemic environment. Most notable in this regard is the yeast Candida albicans, which expresses a glucose-inducible protein that is structurally and functionally homologous to a complement receptor on mammalian phagocytes. This protein promotes adhesion in the yeast and subverts phagocytosis by the host. Thus, hyperglycemia serves as a central mechanism in the predisposition of hyperglycemic patients to infection.
Ultraviolet Light Immunomodulation of Canine Islets for Prolongation of Allograft Survival
Authors: Kenyon Norma S , Strasser Sheryl , Alejandro Rodolfo
Published: September 2013

Ultraviolet (UV) light treatment of donor islets has been shown to be effective for the prolongation of islet allograft survival in rodent models. This study evaluated UV as an immunomodulator of canine islets. The effects of UV irradiation on islet secretory function in vitro revealed a trend of increasing basal insulin release with increasing doses of UV and a corresponding significant decrease in glucose-mediated insulin release (expressed as percentage of basal fractional insulin release) beginning at UV light exposures of 200–300 J/m2 (n = 3, P < 0.05). Proliferative responses to UV-irradiated allogeneic peripheral blood leukocytes and islets were significantly decreased by 53–112% (P < 0.05) in 27 of 29 mixed-lymphocyte cultures and by 35–74% (P < 0.05) in 4 of 5 mixed-lymphocyte islet culture experiments, respectively, beginning at 200–600 J/m2. Autotransplantation of nonirradiated (n = 8) and irradiated islets (600 J/m2 n = 6) resulted in a 1-mo graft survival rate of 75% for the control group and 50% for the irradiated group. Allotransplantation of irradiated islets (600 J/m2) into either nonimmunosuppressed recipients (1 donor to 1 recipient, n = 8) or recipients of subimmunosuppressive doses of cyclosporin (2 donors to 1 recipient, n = 4) resulted in 100% rejection by day 10. In contrast, when islets were cultured for 24 h postirradiation and transplanted into cyclosporin-treated pancreatectomized recipients (2 donors to 1 recipient), 3 of 7 grafts were prolonged beyond day 10 to days 16, 26, and >100. We conclude that direct UV irradiation of islets decreases canine islet immunogenicity and in combination with subtherapeutic dosages of cyclosporin can result in prolongation of islet allograft survival in this large-animal model.
In Vivo Relationship Between Insulin Clearance and Action in Healthy Subjects and IDDM Patients
Authors: Nijs Hubert G T , Radder Jasper K , Frölich Marijke , Krans H Michel J
Published: September 2013

The relationship between plasma clearance rate of insulin (PCR) and insulin-stimulated glucose disposal was investigated in 15 healthy subjects and 30 insulin-dependent diabetes mellitus (IDDM) patients with the sequential euglycemic (5 mMj clamp technique (insulin infusion rates of 0.5,1, 2, and 5 mU · kg−1 · min−1 in 2-h steps). In IDDM patients, insulin-stimulated glucose disposal was decreased at low insulinemia (steps 1–3), whereas at maximal insulinemia (step 4), insulin action was normal. In the healthy subjects, strong positive correlations were found for PCR versus steady-state glucose infusion rate (SSGIR): r = 0.71 (P < 0.005), 0.72 (P < 0.005), 0.72 (P < 0.005), and 0.78 (P < 0.001) for steps 1–4, respectively. In contrast, in the IDDM patients, no relationship was observed: r = 0.01, −0.03, 0.06, and 0.01 (NS) for steps 1–4, respectively. In univariate analyses of PCR, no differences were found between patient subgroups with values for percentage of tracer binding below or above 5% or insulin-antibody-binding capacities and equilibrium constants below or above the median. In multiple regression models, adjusting for insulin antibodies, preceding glycemic control (HbA1 or fructosamine), and duration of IDDM, correlations for PCR versusSSGIR remained nonsignificant. In conclusion, insulin action is correlated to insulin clearance in healthy subjects, suggesting a functional relationship from an in vivo perspective. No such relationship was present in patients with IDDM, even after adjusting for insulin antibodies, precedingglycemic control, and duration of IDDM.
Microvascular Reactivity to Norepinephrine at Different Arteriolar Levels and Durations of Streptozocin-Induced Diabetes
Authors: Morff Robert J
Published: September 2013

Microvascular disease is a hallmark of diabetes. Although the etiology of diabetic microangiopathy remains to be elucidated, numerous studies in experimental animals and human diabetic patients have suggested that a change in vascular reactivity to vasoactive agents is a component of the pathophysiology. Most previous studies have utilized indirect methods to study the microcirculation or have conducted studies of responses in large vessels. This study was designed to directly study in an intact microvascular preparation the effects of streptozocin-induced diabetes (STZ-D) on microvascular reactivity. The responses of arterioles in the cremaster muscle of pentobarbital sodium-anesthetized rats to topically applied norepinephrine were measured in STZ-D rats of 2, 4, 8,16, and 32 wk duration and in age-matched nondiabetic rats. Resting arteriolar diameters and mean arterial pressure were not affected by diabetes. In the STZ-D rats, larger (1A) arterioles were normally responsive after 2, 4, 8, and 16 wk of diabetes compared with nondiabetic rats. In contrast, the smaller 2A and 3A arterioles exhibited hypersensitivity to norepinephrine initially, but responses returned to normal sensitivity as the duration of diabetes progressed to the chronic stage. These results suggest that there are important functional changes in the responses of the microcirculation to norepinephrine that are associated with the development of diabetes and that these changes are anatomically specific and temporally dependent.
Operation of Randle's Cycle in Patients With NIDDM
Authors: Bevilacqua Stefano , Buzzigoli Giuseppe , Bonadonna Riccardo , Brandi Luigi S , Oleggini Marco , Boni Claudio , Geloni Mario , Ferrannini Eleuterio
Published: September 2013

It has been suggested that the insulin resistance of non-insulin-dependent diabetes mellitus (NIDDM) may be caused by substrate competition between glucose and free fatty acids (FFAs) (Randle's cycle). We measured substrate oxidation and energy metabolism in 10 nonobese untreated NIDDM patients with fasting glucose levels of 7–8 mM with indirect calorimetry in the basal state and during an isoglycemic-hyperinsulinemic (∼100 mU/L) clamp without (control) and with a concomitant infusion (∼0.35 mmol/min) of Intralipid, a triglyceride emulsion. In the control study, fasting rates of total glucose turnover ([3−3H]glucose) and glucose and lipid oxidation (9.4 ± 1.4, 7.3 ± 1.3, and 3.0 ± 0.4 μmol · kg−1 · min−1, respectively) were comparable with those of nondiabetic individuals. After insulin administration, lipid oxidation was normally suppressed (to 1.3 ± 0.3 μ · kg−1 · min−1 P < 0.01), as were the circulating levels of FFA, glycerol, and β-hydroxybutyrate, whereas glucose oxidation doubled (14.1 ± 1.8 μmol; · kg−1 · min−1 P <0.01). Because glycemia was clamped at 7.5 mM, endogenous glucose production (EGP) was completely suppressed, and total glucose disposal was stimulated (to 25.7 ± 5.2 μmol · kg−1 · min−1 P < 0.01 vs. baseline), but glucose clearance (3.6 ± 0.8 ml · kg−1 · min−1) was 30% reduced compared with normal. With concomitant lipid infusion, FFA, glycerol, and β-hydroxybutyrate all rose during the clamp; correspondingly, lipid oxidation was maintained at fasting rates (3.6 ± 0.2 μmol · kg−1 · min−1 P < 0.01 vs. control). As a consequence, the insulin-induced increase in glucose oxidation was abolished (7.9 ±1.3 μmol · kg−1 · min−1 P < 0.01 vs. control), and total glucose disposal was inhibited (21.8 ± 4.6 μmol · kg−1 · min−1 P < 0.05 vs. control) by an amount almost equal to the decrement in glucose oxidation. Lipid infusion did not detectably interfere with insulin-induced suppression of EGP. Energy expenditure failed to increase during the control insulin clamp but was significantly stimulated (∼10%, P < 0.01) by concomitant lipid administration (diet-induced thermogenesis). We conclude that in mildly hyperglycemic, nonobese NIDDM patients, excessive fatty substrate oxidation is unlikely to be responsible for the insulin resistance; increased lipid provision, however, enhances lipid oxidation and energy expenditure and inhibits glucose oxidation and total disposal. Thus, in this type of diabetes, Randle's cycle does not appear to be spontaneously overactive but can be induced acutely, with metabolic and energetic consequences similar to those observed in nondiabetic subjects.
Glucose Cycling in Islets From Healthy and Diabetic Rats
Authors: Khan Akhtar , Chandramouli Visvanathan , Östenson Claes-Göran , Löw Hans , Landau Bernard R , Efendić Suad
Published: September 2013

Pancreatic islets from healthy (control) and neonatally streptozocin-induced diabetic (STZ-D) rats, a model for non-insulin-dependent diabetes mellitus, were incubated with 3H2O and 5.5 or 16.7 m M glucose. At 5.5 mM glucose, no detectable [3H]glucose was formed. At 16.7 mM, 2.2 patom · islet−1 · h−1 of 3H was incorporated into glucose by the control islets and 5.4 · patom · islet−1 · h−1 by STZ-D islets. About 75% of the 3H was bound to carbon-2 of the glucose. Glucose utilization was 35.3 pmol · islet−1 · h−1 by the control and 19.0 pmol · islet−1 · h−1 by the STZ-D islets· Therefore, 4.5% of the glucose-6-phosphate formed by the control islets and 15.7% by the STZ-D islets was dephosphorylated. This presumably occurred in the β-cells of the islets catalyzed by glucose-6-phosphatase. An increased glucose cycling, i.e., glucose → glucose-6-phosphate → glucose, in islets of STZ-D rats may contribute to the decreased insulin secretion found in these animals.
Decreased Inner Cell Mass Proportion in Blastocysts From Diabetic Rats
Authors: Pampfer Serge , Hertogh René De , Vanderheyden Ivo , Michels Bénédicte , Vercheval Myriam
Published: September 2013

Late morulae and blastocysts were recovered from streptozocin-induced diabetic pregnant rats and individually examined for numbers of inner cell mass (ICM) cells and trophectoderm (TE) cells. Compared with embryos collected from control rats, exposure to maternal diabetes significantly decreased mean ICM cell number of blastocysts recovered on day 5 of gestation, but the TE population of these embryos remained unaffected. The mean ICM proportion was therefore significantly lower than that of control embryos. These differences were not observed between the two groups of morulae collected on day 5, suggesting that the distinctive susceptibility of the ICM was expressed after blastocyst formation. On day 6, a significant inhibitory effect of diabetes was observed on the growth of both ICM and TE cells, but because the reduction was more severe in the ICM than in the TE, the mean ICM proportion of these blastocysts was again significantly lower than in control embryos. A linear quadratic relationship was obtained between the numbers of ICM cells of individual blastocysts and their respective numbers of TE cells in each of the two experimental groups. However, the slope of the curve was slower in the diabetic group than the control group. The disturbed ICM cell growth in the blastocysts from diabetic rats was found to be associated with a significantly increased incidence of cell death predominantly located in the ICM. Because it is known that excessive reduction of the ICM is incompatible with normal embryogenesis after implantation, our results suggest that the differential sensitivity of ICM and TE cells in preimplantation blastocysts may contribute to the pattern of postimplantation defects described in diabetic pregnancies.
Atrial Structure and Plasma ANF Levels in Rats With Chronic Diabetes Mellitus
Authors: Todd Mary E , Hebden R Anthony , Gowen Brent , Tang Christine , McNeill John H
Published: September 2013

The effect of streptozocin-induced diabetes (STZ-D) on right atrial structure was investigated in male Wistar rats. STZ (55 mg/kg) or saline (1 ml/kg) was administered by intravenous injection 12 wk before the experimental studies. Tissue was sampled from four regions of the atrium, processed, and embedded in plastic. Quantitative stereological analysis indicated that in STZ-D rats, there was a significant diminution in size of the musculi pectinati (muscular ridges), which form a network making up the wall of the atrium. In addition, within the muscular ridges, there was a significant reduction in the relative proportion of cardiocytes within the cardiac tissue. The rest of the cardiac tissue consisted of interstitial regions, connective tissue, and blood vessels, which correspondingly increased. This suggests there was some form of cardiomyopathy. When atrial granularity was determined relative to cardiocyte volume density, a significant decrease (54%) was found in tissue from STZ-D rats. The blood pressure of conscious STZ-D rats was significantly lower than control rats, whereas right atrial pressure was not different. The level of resting plasma immunoreactive atrial natriuretic factor (ANF) in conscious STZ-D rats (98 ± 5 pg/ml) was significantly higher than in control rats (52 ± 7 pg/ml). The decreased atrial granularity could be related to the higher resting plasma ANF levels, suggesting a more rapid turnover or increased synthesis bypassing storage in the granular form.
Effects of Prolonged Pulsatile Hyperinsulinemia in Humans: Enhancement of Insulin Sensitivity
Authors: Ward Glenn M , Walters Jacqueline M , Aitken Patricia M , Best James D , Alford Frank P
Published: September 2013

Prolonged near-physiological pulsatile insulin infusion has a greater hypoglycemie effect than continuous insulin infusion. We have previously shown that continuous hyperinsulinemia induces insulin insensitivity. This study examines the mechanisms responsible for the greater hypoglycemie effect of pulsatile insulin administration, in particular, whether prolonged pulsatile hyperinsulinemia induces insulin insensitivity. Basally and 1 h after cessation of a 20-h pulsatile infusion of insulin (0.5 mU · kg−1 · min−1), eight nondiabetic human subjects were assessed for 1) glucose turnover with [3-3H]glucose, 2) insulin sensitivity by minimal-model analysis of intravenous glucose tolerance tests, and 3) monocyte insulin-receptor binding. The time-averaged plasma insulin levels were 30 ± 5 mU/L (mean ± SE) during the infusion, which was similar to the levels achieved in our previous continuous hyperinsulinemia study. However, the average rate of glucose infusion to maintain euglycemia was 55% greater than in the previous study. Hepatic glucose production was -5.2 ±1.4 μmol · kg −1 · min−1 during the infusion but returned to preinfusion levels 1 h after the infusion was stopped. Insulin sensitivity (Si) and glucose tolerance (rate of glucose disappearance, Kg) showed changes opposite in direction to our previous continuous hyperinsulinemia study (pre- vs. postinfusion Kg 1.5 ± 0.1 vs. 1.7 ± 0.2 min−1 × 102, NS; pre- vs. postinfusion S, 8.4 ± 2.3 vs. 11.8 ± 3.7 min−1 · mU1 · L × 104 P < 0.05). There was no change in glucose-mediated glucose disposal (2.0 ± 0.2 vs. 2.3 ± 0.4 min−1 × 102) or pancreatic β-cell responsiveness (1st phase, 1.8 ± 0.2 vs. 2.2 ± 0.4 μU · ml−1 · min−1 · mg−1 · dl; 2nd phase, 9.9 ± 1.0 vs. 16.6 ± 3.5 μU · ml−1 · min∼−2 · mg−1 · dl). Monocyte insulin-receptor binding showed a postinfusion decrease. Plasma nonesterified fatty acids were profoundly suppressed during the infusion (0.05 ± 0.01 mM) and remained significantly (P < 0.05) suppressed 1 h postinfusion (0.21 ± 0.06 mM) compared with preinfusion (0.54 ± 0.08 mM). We conclude that despite a reduction in monocyte insulin-receptor binding, prolonged moderate pulsatile hyperinsulinemia, in contrast to equivalent continuous hyperinsulinemia, induced enhancement of insulin sensitivity, causing a greater hypoglycemie effect and greater suppression of plasma nonesterified-fatty acid levels.
Application of HPLC in Disposition Study of A14-125I-Labeled Insulin in Mice
Authors: Sato Hitoshi , Tsuji Akira , Hirai Ken-Ichi , Kang Young Sook
Published: September 2013

To describe quantitatively the in vivo distribution and elimination of insulin, high-performance liquid chromatography (HPLC) separation was applied to the pharmacokinetic study of human insulin labeled with 125I at tyrosine A14 (A14-125I-insulin) as a tracer. Intact A14-125I-insulin levels were determined by HPLC and trichloroacetic acid (TCA) precipitation in plasma and various tissues after its intravenous bolus injection into mice. TCA precipitation consistently overestimated the intactness of A14-125I-insulin compared with HPLC, possibly due to the presence of both a TCA-precipitable intermediate degradation product of labeled insulin found in HPLC elution profiles and reported high-molecular-weight forms of labeled insulin in plasma. Thus, TCA precipitation gave a considerably lower total plasma clearance (Cltot) value than HPLC. The half-life of A14-125I-insulin was prolonged by a simultaneous injection of 8 U/kg unlabeled insulin, and labeled insulin behaved similarly to [14C]inulin (an extracellular fluid marker). The concentration time profiles of HPLC-separated labeled insulin in plasma were analyzed by a noncompartmental moment method, and both Cltot and steady-state apparent volume distribution (VDSS) of A14-125I-insulin were considerably decreased by unlabeled insulin coadministration. In particular, VDSS of labeled insulin decreased by 79%, similar to that of inulin (181 ml/kg), suggesting that the nonspecific binding of labeled insulin to tissues was so small that VD88 of labeled insulin was reduced to the extracellular fluid volume (∼20% of the body weight) when its receptor binding was blocked effectively by unlabeled insulin. This observation, together with the 63% reduction of CI,tot by unlabeled insulin coadministration, demonstrated that saturable, receptor-mediated processes of distribution and elimination are essentially involved in the pharmacokinetics of HPLC-separated A14-125I-insulin.
Prevention of Type I Diabetes in NOD Mice by Adjuvant Immunotherapy
Authors: Sadelain Michel W J , Qin Hui-Yu , Lauzon Jana , Singh Bhagirath
Published: September 2013

The nonobese diabetic (NOD) mouse is an excellent model of insulin-dependent (type I) human diabetes mellitus. We report that a single injection of complete Freund's adjuvant (CFA) given at an early age (5 wk) prevented the appearance of diabetes and greatly increased the life span of NOD mice without additional therapy. No treated mouse developed hyperglycemia by the age of 12 mo (n = 13), whereas all untreated mice died of diabetes before 8 mo of age (n = 38). All CFA-treated mice were alive and healthy at 12 mo of age. Some CFA-treated NOD mice that were monitored for long-term survival are still alive with no sign of disease at 18 mo of age (n = 5). Administration of CFA resulted in decreased in vitro splenic lymphocyte proliferative responses to alloantigen and mitogen. Cell-mixing experiments indicated that antigen-nonspecific inhibitory cells were elicited in the spleen and increased in the bone marrow. These regulatory cells were Thy-1− ; and nonadherent to nylon wool, as has been described for natural suppressor (NS) cells. These data lend support to a relationship between the boosting of endogenous NS activity and the establishment of tolerance to self in the context of autoimmunity. Our results suggest that early nonspecific immunotherapy of genetically predisposed individuals could prevent the development of autoimmune diabetes.
Persistence of Serum Antibodies to 64,000-Mr Islet Cell Protein After Onset of Type 1 Diabetes
Authors: Christie Michael R , Daneman Denis , Champagne Patricia , Delovitch Terry L
Published: September 2013

Antibodies to an islet protein of 64,000 Mr(64K antibodies) were measured in 15 diabetic children who were followed prospectively for up to 3 yr after onset of type 1 (insulin-dependent) diabetes. Of the 15 children, 12 were positive for 64K antibodies at diagnosis. Those patients who were negative for these antibodies at onset remained negative throughout the study. Modest increases in 64K antibodies were observed in 7 patients within 1 mo of diabetes onset, concomitant with an increase in C-peptide concentrations. All antibody-positive patients were still positive at the end of the study, with no significant decrease in antibody levels relative to those at diagnosis, whereas C-peptide concentrations decreased between 3 and 24 mo after onset. Islet cell antibodies, measured by immunochemical staining on sections of rat pancreas, were detected in 9 of 15 patients at onset, whereas only 3 of 11 patients were still positive after 3 yr. In an additional group of 11 patients with diabetes for 6–7 yr, when basal and stimulated C-peptide concentrations were undetectable, 4 patients were still positive for 64K antibodies. These results demonstrate that levels of 64K antibodies persist during the first 3 yr of diabetes, despite declining β-cell function and decreased immune responses to other islet antigens, but decrease during the next 3–4 yr as the remaining functional β-cells disappear.
Coexistence of Nerve Conduction Deficit With Increased Na+-K+-ATPase Activity in Galactose-Fed Mice: Implications for Polyol Pathway and Diabetic Neuropathy
Authors: Calcutt Nigel A , Tomlinson David R , Biswas Susmito
Published: September 2013

We measured motor nerve conduction velocity (MNCV), Na+-K+-ATPase activity, polyol-pathway metabolites, and myo-inositol in sciatic nerves from control mice, galactose-fed (20% wt/wt diet) mice, and galactose-fed mice given the aldose reductase inhibitor ponalrestat (300-mg/kg diet). Treatments were maintained for 4 wk. Galactose feeding was associated with a 21.5% reduction in MNCV (P < 0.001), which was almost completely prevented by ponalrestat. Galactose-fed mice showed an 81% increase in Na+-K+-ATPase (P < 0.01), an effect completely prevented by aldose reductase inhibition. Treatment of a separate galactose-fed group with sorbinil (300 mg/kg diet) also attenuated the MNCV deficit and prevented the increased Na+-K+-ATPase activity associated with galactosemia. Accumulation of galactitol in the nerves of galactose-fed mice was prevented by aldose reductase inhibition, but there were no alterations in myo-inositol levels in the sciatic nerves of any group. These data show that exaggerated flux through the polyol pathway can cause an MNCV deficit that is unrelated to either myo-inositol levels or Na+-K+-ATPase activity.
Biphasic Insulin Secretion After Intravenous but Not After Intraportal CCK-8 Infusion in Rats
Authors: Balkan B , Steffens A B , Strubbe J H , Bruggink J E
Published: September 2013

The effects of physiological doses of sulfated cholecystokinin-8 (CCK-8) on insulin secretion were investigated in unrestrained unanesthetized rats. The routes of administration were intravenous or intraportal infusion. Intravenous infusion (0.33–5.0 μg CCK-8 · kg−1 · 20 min−1) resulted in a biphasic response pattern consisting of a fast 1st-min rise in plasma insulin concentration and a slower second phase that lasted throughout the infusion. The first phase showed the same amplitude with all amounts of CCK-8 administered in this study, whereas the second phase exhibited dose dependency. Blood glucose levels were lowered during all infusions of CCK-8, although the second phase of insulin release was absent with the lowest dose. These results suggest a strong stimulatory effect of CCK-8 on the pancreatic β-cells, probably by changing the set point for glucose. The described effects of intravenous administration of CCK-8 cannot be produced when the infusion is given into the portal vein. Only very high concentrations of CCK-8 (15 μg · kg−1 · 20 min−1) produced a small increase in plasma insulin levels, indicating a strong CCK-8-eliminating mechanism in the liver. These results indicate that 1) CCK-8 evokes biphasic insulin release and a concomitant drop in glucose levels, and 2) CCK-8 acting on the β-cell in vivo is not of intestinal origin but is probably released by the pancreatic vagai branch.
Expression of Ly-6C by T Lymphocytes of NOD Mice After CD3-Complex Stimulation: Identification of Activated Cells During Insulitis of Prediabetic Mice
Authors: Herold Kevan C , Montag Anthony G , Meyer Shelley M , Wojcikowski Czeslaw , Fitch Frank W
Published: September 2013

Ly-6C is a differentiation antigen that distinguishes T-lymphocyte subsets. In concordance with previous results, splenocytes from NOD mice do not express the epitope recognized by anti-Ly-6C monoclonal antibodies (MoAbs), including MoAb HK1.4 in this study, and cannot be stimulated to proliferate in response to HK1.4. However, when splenocytes from NOD mice were stimulated in vitro with the anti-CD3 MoAb 145–2C11, T lymphocytes expressing Ly-6C were detected after 48 h of stimulation, with as many as 25% of lymphocytes expressing this antigen with prolonged passage in culture. Most of the cells expressing Ly-6C were Thy-1.2+, CD4+, and CD8 and proliferated after stimulation with HK1.4. To further understand the failure of NOD splenocytes to express Ly-6C, freshly isolated cells were stimulated with α/β-interferon (IFN-α/β) and IFN-γ. Although these lymphokines induced expression of Ly-6A and Ly-6C in splenocytes from C57BL/6J mice and Ly-6A in NOD cells, Ly-6C was not induced on NOD cells. Because Ly-6C expression on splenocytes was a marker of activation via the CD3 T-lymphocyte receptor complex, we also examined expression of Ly-6C on T lymphocytes within islets showing insulitis in vivo. Lymphocytes that were Ly-6C+ were identified within islets on histological sections of pancreas, whereas Ly-6C+ cells in the spleen from the same mouse could not be detected. Our findings imply functional abnormality in expression of Ly-6C in NOD mice. These results suggest that Ly-6C+ cells that are present in islets showing insulitis of the NOD mouse have been stimulated through the CD3 complex or associated molecules, and this marker will provide the means to identify and isolate antigen-specific cells from that site.
Intrinsic Cytotoxicity of Natural Killer Cells to Pancreatic Islets In Vitro
Authors: Akamura Naoto , Woda Bruce A , Tafuri Anna , Greiner Dale L , Reynolds Craig W , Ortaldo John , Chick William , Handler Eugene S , Mordes John P , Rossini Aldo A
Published: September 2013

BB rats develop spontaneous autoimmune insulin-dependent diabetes mellitus that is similar to human insulin-dependent diabetes. In this study, we used an in vitro islet cell cytotoxicity assay to study the possible role of natural killer (NK) cells and their soluble effector molecules in this disorder. First, the results demonstrated that in vivo treatment of acutely diabetic BB rats with anti-asialogangliosideM1 (an NK cell antiserum) but not with anti-T-lymphocyte antibodies reduces spleen cell cytotoxic activity to islets in vitro. Flow microfluorometry (FMF)-sorting experiments were then used to confirm that the splenic cytotoxic effector cell in acutely diabetic BB rats is a CD8+/CD5− NK cell. Further analysis demonstrated that both FMF-sorted NK cell populations from Wistar-Fu rt h rats and unfractionated spleen cells from athymic nu/nu rats with high intrinsic NK cell activity also exhibit high islet cell cytotoxic activity in vitro. Finally, we found that the kinetics and differential cytotoxic activity of NK cells toward islets in vitro could be mimicked by NK cell culture supernatants containing high levels of NK cytotoxic factor (NKCF). The islet cytotoxic activity of these culture supernatants was specifically inhibited by the addition of anti-NKCF monoclonal antibody. These results demonstrate that NK cells from diabetic and nondiabetic rats are cytotoxic to islet cells in vitro. They further suggest that this cytotoxic effect may be mediated in part through the production and release of soluble factors such as NKCF.
Serum Type IV Collagen Concentrations in Diabetic Patients With Microangiopathy as Determined by Enzyme Immunoassay With Monoclonal Antibodies
Authors: Matsumoto Eisaku , Matsumoto Gensaku , Ooshima Akira , Kikuoka Hiroyoshi , Bessho Hiroto , Miyamura Kei , Nanjo Kishio
Published: September 2013

In long-term diabetes mellitus, thickening of basement membrane in capillaries and small vessels is a characteristic lesion and plays an important role in the progression of diabetic microangiopathy. We have developed a sandwich enzyme immunoassay for human serum type IV collagen peptide with monoclonal antibodies. Previous studies suggested that collagen levels reflect the activity of fibrogenesis in basement membrane. Serum type IV collagen levels were measured in 137 non-insulin-dependent diabetic patients (aged 50–75 yr) with or without clinical signs of retinopathy, nephropathy, and/or neuropathy and 110 healthy subjects (aged 50–75 yr) without serological abnormality. Serum concentrations of type IV collagen were significantly higher (P < 0.01) in diabetic patients (mean ± SE 124.1 ± 4.1 ng/ml) than in healthy subjects (73.9 ± 2.2 ng/ml) and were increased with the prevalence or incidence of diabetic complications. In the patients with diabetic microangiopathy, serum type IV collagen levels became higher as clinical signs worsened. Especially in the patients with diabetic nephropathy, serum type IV collagen levels became higher with elevation of blood urea nitrogen, serum creatinine, and serum β2-microglobulin but not urinary excretion of β2- microglobulin and N-acetyl-β-glucosaminidase. These observations indicated that elevation of serum type IV collagen in diabetic nephropathy was related to glomerular filtration dysfunction rather than renal tubular dysfunction. However, the antigen, which can be detected by our assay system, did not exist in urine specimens of healthy subjects, and an intimate relationship was not observed between serum type IV collagen level and serum creatinine level. These results implied that the serum type IV collagen level reflects its increased synthetic activity in basement membrane rather than its reduced clearance rate. The concentration of serum type IV collagen peptide seems to be a useful, noninvasive, reproducible marker for assessment of the activity or progression of diabetic microangiopathy and for evaluation of therapeutic effects.
Effect of Continuous Versus Delayed Insulin Replacement on Sex Behavior and Neuroendocrine Function in Diabetic Male Rats
Authors: Steger Richard W , Kienast S Gail
Published: September 2013

The ability of insulin replacement to reverse the adverse effects of streptozocin-induced diabetes (STZD) on neuroendocrine and sexual function was tested in adult male rats. Rats were injected with STZ (50 mg/kg) or vehicle and then either started immediately on insulin (continuous) or allowed to remain untreated for 4 wk before insulin replacement was started (delayed). Replacement consisted of 5 IU/kg of insulin injected just before the lights were turned off and 2 IU/kg of insulin injected within 1 h of the lights being turned on. Copulatory behavior was tested 2, 4, 5, and 6 wk after induction of diabetes. Forty-five days after STZ administration, rats were killed for measurement of plasma hormone levels and hypothalamic catecholamine turnover and serotonin content. The STZ-D rats showed significant deficits in mount, intromission, and ejaculatory behaviors that were prevented by continuous insulin replacement. Delayed insulin replacement reversed the deficits in mount and intromission behaviors but not ejaculatory behavior. Plasma luteinizing hormone levels were unaffected by STZ or insulin treatment, but plasma testosterone and prolactin levels were both reduced in the diabetic animals. Continuous or delayed insulin replacement normalized both testosterone and prolactin levels. Median eminence, medial basal hypothalamus, anterior hypothalamus, and olfactory bulb rates of norepinephrine turnover were all reduced after STZ administration. Delayed insulin replacement restored norepinephrine turnover in all brain regions, whereas continuous insulin replacement enhanced norepinephrine turnover in the anterior hypothalamus and olfactory bulb but only partially blocked the effects of STZ in the median eminence and medial basal hypothalamus. STZ induced decreases in dopamine turnover in the medial basal hypothalamus and olfactory bulb, and these changes could not be prevented or reversed by insulin replacement. These results demonstrate that many but not all of the adverse effects of STZ-D on copulatory behavior and neuroendocrine function can be reversed by insulin replacement.
Elicitation of Sorbitol Accumulation in Cultured Human Proximal Tubule Cells by Elevated Glucose Concentrations
Authors: Bylander John E , Sens Donald A
Published: September 2013

The human proximal tubule (HPT) is the characteristic site within the kidney that mediates absorption of glucose. This study was designed to determine whether cultured HPT cells would respond to a hyperglycemic environment through activation of the polyol pathway. The results of this study clearly indicate that exposure of the HPT cells to an extracellular glucose concentration ≥11 mM results in substantial intracellular accumulation of sorbitol. This accumulation is inhibited by ∼70% by treatment with 100 μM sorbinil. When cells growing 24 h on 27.5 mM glucose were changed to medium containing 5.5 mM glucose, sorbitol concentration returned to the control level within 12 h. The activity of aldose reductase was increased by a factor of 1.6 by exposure to elevated glucose concentrations, and the relative reactivity of the enzyme with glucose as substrate was ∼0.1 compared with that of glyceraldehyde as substrate. Together, these results indicate that cultured cells derived from the HPT undergo activation of the polyol pathway when exposed to a hyperglycemic environment.
IGF-I–Stimulated Glucose Transport in Human Skeletal Muscle and IGF-I Resistance in Obesity and NIDDM
Authors: Dohm G Lynis , Elton Charles W , Raju Madhu S , Mooney Nitin D , DiMarchi Richard , Pories Walter J , Flickinger Edward G , Atkinson Samuel M , Caro José F
Published: September 2013

Based on the observation that insulinlike growth factor I (IGF-I) can stimulate glucose utilization in nondiabetic subjects and that the action of the IGF-I receptor is normal in the skeletal muscle of patients with noninsulin-dependent diabetes mellitus (NIDDM), it seems possible that IGF-I might provide an effective acute treatment for the hyperglycemia of NIDDM. Using our recently developed in vitro human muscle preparation, we investigated the hypothesis that IGF-I might be an effective alternative to insulin in stimulating glucose transport in diabetic muscle. Abdominal muscle samples from nonobese nondiabetic, obese nondiabetic, and obese NIDDM patients were obtained during elective abdominal surgery. Plasma levels of IGF-I in diabetic patients were lower than those in either of the nondiabetic groups. Binding studies with wheat-germ–agglutinin–chromatography–purified receptors demonstrated the presence of IGF-I receptors in human muscle, with IGF-I binding being ∼24% that of insulin. There was no change in IGF-I binding in muscle from obese or diabetic subjects, and the structural characteristics of the IGF-I receptor were not altered, as determined by electrophoretic mobility. IGF-I stimulated glucose transport approximately twofold in incubated muscle from control subjects, but there was no IGF-I stimulation of transport in muscle from obese subjects with or without NIDDM. These results confirm a previous report that human muscle contains receptors for IGF-I and demonstrate for the first time that IGF-I can stimulate glucose transport in human muscle. However, muscle from obese subjects with or without NIDDM is resistant to the action of IGF-I.
Equivalent In Vivo Biological Activity of Insulin Analogues and Human Insulin Despite Different In Vitro Potencies
Authors: Ribel Ulla , Hougaard Philip , Drejer Kirsten , Sørensen Anders R
Published: September 2013

In vivo biological potency of two human insulin analogues, AspB9,GluB27 insulin and AspB10 insulin with low and high affinity to the insulin receptor, respectively, was assessed by intravenous infusion of equimolar amounts in pigs, with the euglycemic clamp technique. Human insulin and the low- and high-affinity analogues showed equivalent glucose utilization rates in the steady state (mean ± SE 14.7 ± 1.4, 12.7 ± 1.5, and 12.2 ± 1.2 mg · kg−1 · min−1, respectively; n = 7). The corresponding plasma insulin levels, however, were markedly different (329 ± 25 and 856 ± 46 pM, P < 0.05; 197 ± 19 pM, P < 0.05). There was an inverse relationship between the insulin levels and the in vitro activities measured by binding to human hepatoma cells (HepG2; 100, 20, and 308%) or by incorporation of glucose into lipids in mouse free fat cells (100, 31, and 207%). The total amount of glucose infused during and after insulin infusion was equal for the three insulins, whereas glucose utilization as a function of time was somewhat different. By describing the individual plasma concentration courses with an open two-compartment model with elimination from the receptor compartment, the time courses for binding and elimination of the three insulins in the receptor compartment were estimated. The effect seems closely linked to the elimination of insulin from the receptors rather than to the amount of insulin bound to the receptors. In conclusion, the total effect of equimolar amounts of human insulin and the two insulin analogues on glucose utilization is equal regardless of the different receptor affinities of the insulins.
Demonstration of Lactogenic Receptors in Rat Endocrine Pancreases by Quantitative Autoradiography
Authors: Polak M , Scharfmann R , Ban E , Haour F , Postel-Vinay MC , Czernichow P
Published: September 2013

A direct effect of growth hormone and/or prolactin on the growth of the pancreatic β-cell has been proposed. This study assessed the presence of human growth hormone (hGH)-binding sites in male adult rat endocrine pancreas via quantitative autoradiography. The binding of 125I-labeled hGH was evaluated by receptor autoradiography on frozen-pancreas cryostat cut sections. The sections were incubated with 125I-hGH (10−10 M) for 75 min at room temperature, and nonspecific binding was determined in the presence of excess native hGH (5 × 10−7 M). The specificity of the binding was assessed in competition experiments with bovine GH and ovine prolactin. The autoradiograms were quantified with a computer-assisted imageprocessing system. The sections were then stained to visualize the endocrine islets. Nondiabetic control and streptozocin (STZ)-injected rats were used. Our results show that 1) there is specific binding of iodinated hGH in small areas of the pancreas, which appear as the Langerhans islets when the autoradiogram and the stained sections are superimposed; 2) the specificity of hGH binding in rat islets is lactogenic; 3) the density of the hGH-binding sites in the endocrine pancreas is estimated at 4.8 fmol/mg protein, with IC50 ranging from 0.98 to 2.50 nM; and 4) binding sites may be present on the β-cell, because specific binding disappears in STZ-injected rats. In conclusion, by use of a quantitative autoradiographic technique, we provide evidence for the presence of lactogenic receptors on rat β-cells.
Effects of Amino Acids on Glucose Disposal
Authors: Boden Guenther , Tappy Luc
Published: September 2013

Free fatty acids are known to inhibit carbohydrate disposal and oxidation. This action may play an important role in the pathophysiology of insulin resistance and non-insulin-dependent diabetes mellitus. To investigate whether amino acids (AAs) have similar actions, we determined the effects of an intravenously infused mixture of 15 AAs on carbohydrate disposal during euglycemichyperinsulinemic clamps associated with either basal or high glucagon concentrations in healthy male volunteers. Plasma glucose concentration was clamped at ∼4.7 mM (coefficient of variation 4.7%). Insulin infusion (7.18 pmol · kg−1 · min−1) raised serum insulin concentrations from 36–50 pM to between 300 and 600 pM. AA infusions (0.5 g · kg−1 h−1 · 4 h) raised plasma α-amino N2 concentrations about five- to six-fold. Infusion of AAs, somatostatin (somatotropin release inhibitory factor, SRIF), and high-glucagon replacement (3.0 ng · kg−1 · min−1) reduced the rate of exogenous glucose infusion needed to maintain euglycemia from 51.1 ± 7.2 μmol · kg−1 · min−1 (saline + SRIF + high glucagon) to 28.3 ±11.1 μmol · kg−1 · min−1 and stimulated endogenous glucose production (from 0 to ∼17 μmol · kg−1 · min−1). Thus, glucose disposal (exogenous infusion plus endogenous production of glucose) remained essentially unchanged. During infusion of AAs + SRIF + basal glucagon replacement (0.25 ng · kg−1 · min−1), endogenous glucose production remained completely suppressed, and the rates of exogenous glucose infusion did not change (compared with saline + SRIF + basal glucagon replacement). The data showed that 1) hyperaminoacidemia associated with hyperglucagonemia stimulated endogenous glucose production despite hyperinsulinemia, and 2) intravenous infusion of a mixture of 15 AAs had no inhibitory effect on insulin-stimulated total-body glucose disposal.
No Excess of DR*3/4 in Ashkenazi Jewish or Hispanic IDDM Patients
Authors: Rubinstein Pablo , Walker Mary , Mollen Norman , Carpenter Carol , Beckerman Svetlana , Suciu-Foca Nicole , McEvoy Robert , Ginsberg-Fellner Fredda
Published: September 2013

The gene frequencies, haplotype relative risks, and zygotic assortments of HLA-DR in three ethnically defined samples of insulin-dependent diabetes mellitus (IDDM) patients were determined in a prospective family study. Although DR3 and DR4 were positively associated with IDDM in the probands of 123 northern European, 94 Ashkenazi Jewish, and 49 New York Hispanic families, significant excess of DR*3/4 heterozygotes was observed only among the probands from families of northern European ancestry. There was also a significant decrease in the frequency of Bw62,DR4 haplotypes derived by northern European patients from their mothers compared with their fathers. This difference, together with data reported in the literature, suggests that the expressivity of the susceptible genotype(s) in IDDM patients may be modified by protective maternal effects associated with Bw62,DR4 and probably other DR4 haplotypes. Samples of IDDM patients from populations with high frequencies of these modifiers should have different DR-gene frequencies contributed by fathers andmothers, capable of accounting for the observed Hardy-Weinberg disequilibrium. We postulate that, because the mechanism of action of these modifiers is distinct from that of the susceptibility gene, the difference must be considered in devising strategies for elucidation of the mode of inheritance of the disease and for understanding the molecular nature of the susceptibility.
Evolution of Insulin Secretory Response to Glucose by Perifused Islets From Lean (FA/FA) Rats Chronically Infused With Glucose
Authors: Bedoya Francisco J , Jeanrenaud Bernard
Published: September 2013

Chronic infusion of nondiabetic rats with glucose for up to 7 days modified the insulin secretory response by subsequently perifused islets. Thus, on the 1st day of infusion with 40% glucose, the islets responded to 16.7 mM glucose with a 3.9-fold increase in insulin release during the first 10 min with no significant change during the second-phase insulin output compared with the control group. On the 2nd day, there was a 2-fold enhancement of insulin release during the initial 10 min of stimulation, the second phase being similar to control islets. On the 5th day of infusion, the pattern of insulin release was not significantly different from the control group. After 7 days of infusion, there was a 46% decrease in first-phase and a 33% decrease in second-phase insulin response to glucose. The response to 10 mM arginine plus 5 mM glucose was not modified by chronic glucose infusion. Priming experiments indicate that islets from rats infused for 7 days were not able to recover the normal pattern of secretion in vitro. Islets from 7-day glucose-infused rats contained 75% more protein and had a significantly higher insulin content than control islets, suggesting that insulin synthesis is not involved in the loss of the response observed. Glucose metabolism by the islets was modified by glucose infusion with a significant increase in glucose utilization and no changes in glucose oxidation, suggesting that alterations in mitochondrial oxidative events are involved in the phenomenon of desensitization.