bluuebunny/crossref_metadata_2025 huggingface dataset:
High-Molecular-Weight Aggregates of Therapeutic Insulin: In Vitro Measurements of Receptor Binding and Bioactivity
Authors: Robbins David C , Hirshman Michael , Wardzala Lawrence J , Horton Edward S
Published: September 2013

On the basis of a monomeric insulin standard, ∼28% of total circulating immunoreactive insulin in insulin-dependent diabetes mellitus (IDDM) is a covalent aggregate of insulin. This aggregate probably originates in therapeutic insulin preparations. In this study, the activity of these aggregates was compared with that of monomeric insulin with regard to behavior in the radioimmunoassay, binding to insulin receptors, and biologic activity in isolated rat adipose cells. Molar activity of the aggregate in the insulin radioimmunoassay was approximately twice (240%) that of monomeric insulin, whereas the log-logit slope produced by the aggregate was indistinguishable from that of monomeric insulin. Insulin-receptor binding was determined by displacement of 125I-labeled A14-insulin by insulin or insulin aggregate (10−10–10−5 M). The free-insulin and aggregate concentrations required for half-maximal displacement of 125I-insulin were 4.0 × 10−10 and 2.25 × 10−9 M, respectively. [1-14C]glucose incorporation into 14CO2, glyceride-glycerol, and fatty acids was measured over a wide range of insulin monomer and aggregate concentrations (0–8 nM). In the bioassay, the maximal rates of glucose metabolism were equal (normal responsiveness). However, the concentration of insulin aggregates producing half-maximal stimulation of glucose metabolism was threefold greater than that of insulin (140 vs. 46 pM, respectively), indicating decreased sensitivity of the adipose cells to the aggregates. This was associated with a sixfold decrease in the Kd for binding of aggregates to adipose cell insulin receptors compared with binding of monomeric insulin. Taken together, these findings may partially explain the apparent discrepancies between measured concentrations of circulating immunoreactive insulin and bioactive insulin in IDDM. These data are consistent with the hypothesis that the aggregates are largely composed of insulin dimers.
Structural β-cell Changes and Transient Hyperglycemia in Mice Treated With Compounds Inducing Inhibited Citric Acid Cycle Enzyme Activity
Authors: Boquist Lennart , Boquist Susanna , Ericsson Ingemar
Published: September 2013

An initial transient hyperglycemia was seen in mice injected with asparagine, fluoroacetate, hydroxylamine, or malonate plus methionine, whereas an initial triphasic blood glucose response and a transient “secondary” hyperglycemia were exhibited in those injected with hydroxylamine plus arsenite, and a delayed hypoglycemia was observed in those treated with fluoroacetate or arsenite. The glucose-induced insulin secretion was significantly decreased in isolated pancreatic islets incubated with hydroxylamine plus arsenite. Light and electron microscopy, pyroantimonate technique, and X-ray microanalysis disclosed mitochondrial damage, degeneration, and necrosis among the β-cells in the islets of mice injected with hydroxylamine plus arsenite. Glycogen depletion and microvesicular fatty change were seen in the liver of mice treated with fluoroacetate, arsenite, or hydroxylamine plus arsenite. These observations support the view that inhibition of the activity of citric acid cycle enzymes and associated reactions in the β-cells play a role in the induction of diabetic features.
Organ-Specific Autoimmunity and HLA-DR Antigens as Markers for β-Cell Destruction in Patients With Type II Diabetes
Authors: Groop Leif , Miettinen Aaro , Groop Per-Henrik , Meri Seppo , Koskimies Saija , Bottazzo Gian Franco
Published: September 2013

Islet cell antibodies (ICAs), thyrogastric antibodies, and HLA-DR antigens were determined in 204 patients with type II (non-insulin-dependent) diabetes controlled with diet and/or oral hypoglycemic agents (NIR) and in 108 age-matched patients who required insulin to control their hyperglycemia (IR). β-Cell function measured as C-peptide response to glucagon was evaluated in relation to the presence of ICAs and HLA-DR antigens. The IR patients differed from the NIR patients with respect to higher frequency of ICAs (P < .001), thyroid antibodies (P < .02), and the HLA antigen DR4 (P < .02). The highest frequency of ICAs and thyroid antibodies was observed in female insulin-treated subjects (51.2 and 46.4%). Patients who were heterozygous for HLA-DR3/DR4 showed significantly higher frequency of ICAs (P < .01) and complement-fixing ICAs (P < .001) than patients without the heterozygous form DR3/DR4. Neither the presence of ICA alone nor DR3/DR4 alone was associated with a significant impairment of β-cell function. However, when both ICA and DR3/DR4 were present in a diabetic individual, β-cell function was markedly impaired (P < .001), suggesting that both genetic and autoimmune factors are necessary to facilitate the process leading to β-cell destruction of the patients. Our findings suggest that type II diabetes is a heterogeneous disorder including at least two major subgroups, which can be further characterized by HLA-DR antigens and organ-specific antibodies
Descriptive and Mechanistic Considerations of Interleukin 1 and Insulin Secretion
Authors: McDaniel Michael L , Hughes Jonathan H , Wolf Bryan A , Easom Richard A , Turk John W
Published: September 2013

Insulin-dependent diabetes mellitus (IDDM) may be mediated in part by an autoimmune mechanism, as suggested by associated cytologie and serologie phenomena, e.g., insulitis, β-cell necrosis, and the presence of both islet cell and insulin antibodies. Immunological approaches to the prediction and intervention in the progression of β-cell destruction in this disease are under evaluation. A recent hypothesis is that cytokines, including interleukin 1 (IL-1), play causative roles in such autoimmune processes. Several studies have convincingly demonstrated that IL-1 is a potent modulator of β-cell function and can potentiate or inhibit glucose-induced insulin secretion, depending on the concentration and length of exposure to IL-1. IL-1 alone or in concert with other cytokines is cytotoxic to β-cells. The cellular mechanisms responsible for the potent effects of IL-1 on the β-cell are unknown and just beginning to emerge. Although speculative at this time, this perspective delineates cellular mechanisms that are likely to represent possible primary sites for the IL-1 action on β-cells. A mechanistic understanding of the effects of IL-1 on the β-cell may clarify its role in modulating insulin release in vivo or yield insight into the pathogenesis of IDDM.
Reduction of Insulin Clearance During Hyperglycemic Clamp: Dose-Response Study in Normal Humans
Authors: Tillil Hartmut , Shapiro E Timothy , Rubenstein Arthur H , Galloway John A , Polonsky Kenneth S
Published: September 2013

Insulin secretion and clearance were studied in eight normal subjects who underwent hyperglycemie clamp studies at plasma glucose levels of 120, 225, and 300 mg/dl on three occasions. Insulin secretion rates were calculated during a 1-h baseline period and during 3 h of glucose clamping from a two-compartmental analysis of peripheral C-peptide concentrations with individual kinetic parameters derived after intravenous bolus injections of biosynthetic human C-peptide. At the 300-mg/dl clamp level, the insulin secretion rate increased to a value 9.9 ± 0.7 times that of basal at the end of the clamp (mean ± SE), whereas over the same period, the peripheral insulin concentrations increased to a greater extent, reaching a value 15.4 ± 1.2 times that of basal (P = .002). This greater relative increase in the insulin concentration in comparison with the corresponding insulin secretion rate suggests a reduction in the clearance of endogenous insulin. A similar trend was seen at the 225-mg/dl clamp level, but the relative increase in the insulin concentration (9.9 ±1.5 times that of basal) was not significantly higher than the relative increase in the insulin secretion rate (8.1 ± 0.5 times that of basal, P = .17). At the 120-mg/dl clamp level, the relative increases in the insulin secretion rate (2.7 ± 0.2 times that of basal) and the insulin concentration (2.4 ± 0.2 times that of basal) were similar (P = .26), indicating no reduction in endogenous insulin clearance during moderate stimulation of insulin secretion. In conclusion, a reduction in endogenous insulin clearance occurs during greater stimulation of insulin secretion at higher glucose-clamp levels. These data suggest that endogenous insulin clearance is nonlinear and shows evidence of saturation at high physiologic insulin concentrations.
Activation of Smooth Muscle Cell Outgrowth From BB/Wor Rat Aortas
Authors: Larson David M , Haudenschild Christian C
Published: September 2013

An essential event in atherogenesis is the migration and proliferation of vascular smooth muscle cells (VSMCs), which contributes to the fibrocellular component of the atherosclerotic intimai thickening. We have been modeling this process by studying the outgrowth of VSMC from aortic expiants onto tissue-culture plastic substrate. Our hypothesis is that certain risk factors for atherosclerosis favor increases in subpopulations of cells with enhanced responsiveness to a variety of migratory and proliferative stimuli, in vivo and in vitro. As a known risk factor for atherosclerosis, diabetes might be reasonably postulated to induce such cell populations with altered susceptibility to additional noxious stimuli. We have tested this hypothesis with aortic expiants from rats of the spontaneous autoimmune diabetic BB/Wor group, including diabetes-resistant, diabetes-prone, and treated acutely (<2 wk after onset of hyperglycemie) and chronically (>2 wk) diabetic strains. As a group, the VSMCs from BB/Wor animals showed enhanced outgrowth in 0.1% serum (23–48%) compared with VSMCs from ordinary Wistar rats (<10%). Within the BB/Wor group, however, the rank order of enhanced outgrowth was diabetes-resistant > chronically diabetic > acutely diabetic subjects. When the outgrowth assay was performed in the presence of supplemental insulin (10 mU/ml), outgrowth was increased, especially for the diabetes-prone animals, giving a new rank order of diabetes-prone > acutely diabetic > diabetes-resistant > chronically diabetic subjects. These data suggest a genetic predilection in the entire BB/Wor rat group for increased VSMC responsiveness to migratory and proliferative stimuli, a sensitivity of these VSMCs to insulin, and the dissociation of this vascular cell effect from other manifestations of diabetes, e.g., hyperglycemia.
Insulin Autoantibodies and Insulin Assay
Authors: Armitage Mary , Wilkin Terence , Wood Peter , Casey Cecily , Loveless Ruth
Published: September 2013

Insulin antibodies are known to interfere with the radioimmunoassay of insulin. We tested intravenous glucose tolerance on 25 insulin autoantibody-positive (IAA+) patients and 25 IAA− controls, who were matched for sex, age, and body mass index, to establish if IAA could also interfere with insulin assay. Insulin content was measured in untreated serum, serum precipitated with polyethylene glycol (PEG, free insulin), and serum extracted with acid and precipitated with PEG (total insulin). The mean untreated first-phase insulin response (I1+3) for IAA+ patients was 172 ± 67.3 mU/L, significantly higher than the mean control value of 108 ± 47.5 (P < .001). After PEG precipitation, mean I1+3 in the patient group fell significantly to 105 ± 48.4 mU/L (P < .001), but the control value was unchanged (104 ± 45.5). The mean percentage fall after PEG precipitation was 36.9% (patients) and 2.9% (controls) (t = 8.3, P < .001). There was a strong correlation between the IAA titer and the interference in the insulin assay (r = .81). After total insulin extraction of IAA samples, there was a significant fall in mean I1+3 to 134 ± 55.4 mU/L (P < .001), but the control value was unchanged. IAA can significantly falsify insulin measurement, and care must be taken in the interpretation of insulin-release tests when IAA is present.
IDDM in BB Rats: Enhanced MHC Class I Heavy-Chain Gene Expression in Pancreatic Islets
Authors: Jeremy Ono Santa , Issa-Chergui Badia , Colle Eleanor , Guttmann Ronald D , Seemayer Thomas A , Fuks Abraham
Published: September 2013

Modulation in major histocompatibility complex (MHC) gene expression correlates with the inflammatory reactions that occur during graft rejection and autoimmune disease. We analyzed the expression of class I and II MHC genes in the pancreatic islets of prediabetic and newly diabetic BB rats by immunohistochemistry of tissue sections and Northern blotting of RNA extracted from isolated islets. We show that enhanced levels of MHC class I heavy-chain RNA are present in pancreatic islets before overt inflammation and the onset of insulin-dependent diabetes mellitus (IDDM) in the spontaneously diabetic BB rat. Immunohistochemical analysis revealed enhanced class I antigen expression throughout the pancreatic islets of newly diabetic animals but no induction of class II antigen on endocrine cells within the islet. Varying degrees of inflammatory infiltrate were observed in the sections exhibiting enhanced class I antigen expression or in nearby serial sections. Southern blot analysis revealed no restriction-fragment-length polymorphism or amplification of the endogenous class I heavy-chain genes compared with those of seroidentical disease-resistant Wistar-Furth rats. I-Aα and I-Eα hybridizing RNA appeared de novo before overt diabetes, although concomitantly with T-lymphocyte-receptor β-chain and interferon-γ gene hybridizing RNA and after MHC class I heavy-chain RNA enhancement was observed. These data indicate the possibility that enhanced class I heavy-chain gene expression plays a role in the progression of IDDM.
Effect of Coxsackievirus B4 Infection in Mice on Expression of 64,000-Mr Autoantigen and Glucose Sensitivity of Islets Before Development of Hyperglycemia
Authors: Gerling Ivan , Nejman Catherine , Chatterjee Nando K
Published: September 2013

Diabetogenic strains of Coxsackievirus B4 (CB4) produce a diabetes syndrome in susceptible mice that resembles insulin-dependent diabetes mellitus. To assess the possible role of autoimmunity, the expression of a 64,000-Mr islet antigen in SJL/J and CD1 mice infected with a diabetogenic strain of CB4 was monitored in early and late infection. Additionally, virus-induced abnormalities in glucose metabolism were correlated with several changes in purified islets to assess β-cell physiology. Over 80% of the mice exhibited subnormal blood glucose at 72 h postinfection (p.i.) and were hyperglycemie at 6 and 8 wk p.i. Islet yield in infected mice decreased by 29–47% at 72 h and 6 wk p.i. compared to noninfected mice. Insulin release stimulated by 16.7 mM glucose increased greater than twofold at 72 h p.i. but declined at 6 wk well below the level of noninfected mice. Likewise, residual islet insulin content after release also increased at 72 h and then declined. Total protein synthesis in the islets decreased by 30% at 72 h and by 60% at 6 wk p.i. Although the synthesis of five proteins of heterogeneous molecular weights, including tubulin, was severely depressed in the infected islets at 72 h p.i. compared with control islets or islets at 6 wk p.i., synthesis of the 64,000-Mr component and another protein of 36,000 Mr increased by two- to threefold. It is possible that CB4 infection may initiate or enhance an autoimmune reaction by increased expression of the 64,000-Mr antigen.
Biosynthetic Regulation of Endogenous Hamster Insulin and Exogenous Rat Insulin II in Transfected HIT Cells
Authors: Gold Gerald , Walker Michael D , Edwards Darrell L , Grodsky Gerold M
Published: September 2013

To investigate mechanisms underlying biosynthetic regulation of an insulin gene, the rat insulin II gene was introduced into hamster β-cells (HIT) by cotransfection with the neomycin phosphotransferaseselectable marker. The insulin gene fragment was 2.2 kilobases (kb) in length and contained all exons, introns, and ∼700 base pairs (bp) of 5'-flanking DNA and 300 bp of 3'-flanking DNA. The HIT cell was known to have endogenous hamster insulin production under regulation by glucose and dexamethasone. In a pool of stably transfected cells (HIT M62pR2), rat insulin II and hamster insulin were produced at comparable rates. Glucose (20 mM) stimulated cellular [3H]leucine labeling of both hamster insulin and rat insulin II by approximately twofold. Addition of 10−6 M dexamethasone to media containing 11.1 mM glucose inhibited biosynthesis of both hamster insulin and rat insulin II by >90%. Thus, with both positive and negative biosynthetic regulation, changes in the cellular labeling of exogenous rat insulin II were qualitatively and quantitatively similar to those of the endogenous hamster insulin. These data suggest that the 2.2-kb rat insulin II gene fragment contained sufficient information for both expression and apparently “normal” biosynthetic regulation of exogenous rat insulin II (when compared with endogenous hamster insulin) in response to glucose and dexamethasone.
Characterization of New Oral Antidiabetic Agent CS-045: Studies in KK and ob/ob Mice and Zucker Fatty Rats
Authors: Fujiwara Toshihiko , Yoshioka Shinji , Yoshioka Takao , Ushiyama Izumi , Horikoshi Hiroyoshi
Published: September 2013

CS-045 is a new oral antidiabetic agent that was effective in insulin-resistant diabetic animal models, including the KK mouse, the ob/ob mouse, and the Zucker fatty rat. CS-045 was not effective in the streptozocin-treated mouse, an insulin-deficient diabetic animal model. In fed KK mice, CS-045 lowered the plasma glucose levels in a dosedependent manner after a single oral administration, and the hypoglycemic effect lasted for at least 18 h. In normal rats, however, plasma glucose levels were not changed after administration of CS-045. CS-045 when given chronically (2 wk) to diabetic KK and ob/ob mice as a 0.2% food admixture dramatically improved hyperglycemia, hyperinsulinemia, and hypertriglyceridemia to near-normal values and decreased plasma lactate, free fatty acid, and ketone body levels without reducing food intake or body weight. In the obese Zucker fatty rat, oral administration of CS-045 had a similar effect in lowering plasma glucose, insulin, triglyceride, free fatty acid, lactate, and ketone body levels. The CS-045–treated Zucker fatty rats showed increased glucose tolerance and decreased insulin secretion in response to oral glucose. After 9 days of treatment, insulin binding to adipocyte plasma membranes from both CS-045-treated Zucker fatty rats and KK mice was increased. Furthermore, 2-deoxyglucose uptake in CS-045-treated adipocytes was increased and the insulin dose-response curve was shifted to the left. These findings suggest that CS-045 increases not only insulin sensitivity but also insulin responsiveness. Based on its pharmacological profile, CS-045 is a new orally effective antidiabetic agent that may reduce abnormalities of glucose and lipid metabolism in obese and non-insulin-dependent diabetes mellitus patients with insulin resistance.
Relationship of Hepatic Glucose Uptake to Intrahepatic Glucose Concentration in Fasted Rats After Glucose Load
Authors: Niewoehner Catherine B , Nuttall Frank Q
Published: September 2013

Glucose concentration gradients across the liver and hepatic blood flow were measured to characterize the relationship of hepatic glucose uptake to hepatic glucose concentration for 240 min after administration of a large oral glucose load to fasted rats. Extraction of glucose occurred only transiently, from 20 to 80 min after glucose administration. The liver changed from net glucose output to net glucose removal only when the intracellular hepatic glucose concentration exceeded 12.5 μmol/ml water. Even when arteriovenous glucose concentrations gradients were compatible with net direct hepatic uptake of glucose, the hepatic glucose concentration always exceeded the inflow glucose concentration. These data indicate that direct glucose uptake occurred against a concentration gradient when the liver is considered as a whole. The hepatic intracellular-to-extracellular glucose concentration gradient changed very little, suggesting that this is not being regulated by glucose, insulin, or other effectors. The mechanism by which the hepatic glucose concentration and net hepatic glucose uptake versus output are coordinated is unknown. The rate of glycogen synthesis was linear for 120 min after administration of the glucose load. This occurred in the presence of direct uptake of glucose early in the time course and later in the presence of net glucose output by the liver. Net direct uptake of glucose by the liver could account for, at most, 37–55% of the glycogen formed. Fractional extraction of both lactate and alanine decreased after glucose was given, but net hepatic uptake of these metabolites could account for 33–49 and 7–10%, respectively, of the glycogen formed, depending on plasma versus blood water flow. Although net glucose, lactate, and alanine uptake could account for netglycogen formation, uptake of these substrates alone could not account for net glucose as well as glycogen synthesis after glucose was given.
How, When, and Why to Predict IDDM
Authors: Maclaren Noel K
Published: September 2013

Insulin-dependent diabetes remains a serious disease replete with life-threatening complications. The onset of the disease is becoming increasingly predictable through the detection of its associated autoantibodies. The natural history of pathogenic events is attenuated in those with adult onset over that seen in infants and children. Immunosuppression therapies in newly diagnosed patients have been shown to induce remissions, whereas various intervention strategies in rodent models (BB rats and nonobese diabetic mice) can delay and even prevent the onset of diabetes. The stage is set for serious consideration of intervention trials to delay the clinical disease in humans.
Enhancement of Platelet Aggregation by Low-Density Lipoproteins From IDDM Patients
Authors: Watanabe Jun , Wohltmann Hulda J , Klein Richard L , Colwell John A , Lopes-Virella Maria F
Published: September 2013

Low-density lipoprotein (LDL) is known to enhance platelet sensitivity to some aggregating agents. In this study, we observed that LDL isolated from patients with insulin-dependent diabetes mellitus (IDDM) enhanced thrombin-induced platelet aggregation to a greater extent than LDL isolated from matched controls (P < .01). Thromboxane B2 production during aggregation was also significantly more enhanced by LDL isolated from IDDM than by control LDL (P < .01). There was no difference in the lipid composition (free and esterified cholesterol, total phospholipids, and triglycerides) of LDL isolated from diabetic and control subjects. In contrast, the extent of glycosylation of LDL isolated from diabetic patients was significantly greater than that observed in LDL from normal subjects (P < .01), and a positive correlation (r = .605, P < .01) between the degree of LDL glycosylation and the rate of platelet aggregation was observed. LDL glycosylated in vitro enhanced thrombin-, collagen-, and adenosine 5'-diphosphate-induced platelet aggregation to a greater extent than control LDL (P < .01). Although LDL glycosylated in vitro was taken up by platelets to a greater extent than control LDL (P < .05), the lipid composition (free chojesterol and phospholipid) of platelets was not significantly changed. We postulate that an increased degree of glycosylation of LDL may enhance its uptake by platelets and lead to increased platelet reactivity to aggregating agents, probably by altering the structure of the platelet membrane. The enhancement of platelet aggregation by LDL may contribute to the accelerated development of atherosclerosis in diabetes mellitus.
Modulation of Insulin Secretion From β-Cells by Phosphoinositide-Derived Second-Messenger Molecules
Authors: Zawalich Walter S
Published: September 2013

In isolated islets, the hydrolysis of membrane phosphoinositides (PI) participates in the transduction of both extracellular and intracellular signals into an effective insulin secretory response. A wide variety of potential second-messenger molecules are generated during the phospholipase C-mediated cleavage of these strategically situated membrane phospholipids. Several distinct but interrelated issues are addressed in this perspective. These include 1) methodological approaches utilized to assess PI turnover, 2) the synergistic relationship between PI-derived second messengers and cAMP, 3) the contribution of changing PI turnover rates to the biphasic pattern of insulin output induced by 20 mM glucose, and 4) the role played by PI turnover in the phenomenon of “memory” displayed by islets after prior stimulation with various agonists. The concept that events unique to PI turnover contribute to β-cell activation is well founded. Because of uncertainty regarding the exact nature of all PI-derived messengers, however, it is not yet possible to mold the available information into a comprehensive theory of β-cell activation. Future studies will have to address various important unresolved issues.
Increased Plasma IgA, sIgA, and C3- and IgA-Containing Immune Complexes With Renal Glomerular Deposits in Diabetic Rats
Authors: Miller Leon L , Izzo Mary Jane , Wemett Drusilla , Panner Bernard J , Schenk Eric A
Published: September 2013

Male Sprague-Dawley rats were fasted 18 h and given streptozocin (STZ; 60 mg/kg body wt i.p.). The resultant diabetes mellitus, not treated with insulin, was associated with persistent manifoldly increased plasma IgA levels, as measured by single-radial immunodiffusion after reduction with dithiothreitol and alkylation with iodoacetamide. Also observed were concurrent increases in plasma levels of secretory IgA (sIgA) and of C3- and IgA-containing immune complexes (C3-IgA-CIC). After 104 days without insulin treatment, six of the diabetic rats were given daily injections of 2 U of insulin for 11 days. Insulin treatment was associated with a precipitous decrease in plasma levels of IgA, sIgA, and C3-IgA-CIC. Cessation of insulin treatment resulted in restoration of greatly increased levels of all three IgA-containing species. Histoimmunofluorescence studies of kidneys from untreated rats with diabetes of 192–324 days revealed glomerular capillary wall and mesangial deposits reacting strongly with anti-IgA (α-chain-specific) antiserum. Kidneys from two of the diabetic rats (324 days) were tested with anti-rat C3 and anti-rat secretory component (SC) antisera, and they reacted positively. Control kidneys from normal rats examined simultaneously were negative. The concurrent changes in plasma levels of three IgA-containing species in the untreated STZ-induced diabetic rat and the demonstration of abnormal immunoreactive IgA-containing renal glomerular deposits make this experiment an attractive model for studying the possible role of disturbed IgA metabolism in the pathogenesis of diabetic nephropathy.
Incretin Effect Due to Increased Secretion and Decreased Clearance of Insulin in Normal Humans
Authors: Shuster Lynne T , Go Vay L W , Rizza Robert A , O'Brien Peter C , Service F John
Published: September 2013

To assess the contribution of changes in insulin secretion and clearance to the incretin effect (greater insulinemia after oral than after intravenous glucose), 10 healthy subjects were studied after oral glucose (1 g/kg body wt) and again when glucose was infused intravenously at rates to match arterialized plasma glucose concentrations after oral glucose. Although basal and integrated plasma glucose did not differ between oral and intravenous glucose, integrated responses of insulin (3.3 ± 0.5 vs. 1.8 ± 0.4 mU ml−1 · 240 min−1, P < .001), C-peptide (456.5 ± 58.5 vs. 327.9 ± 46.3 μg · ml−1 · 240 min−1, P = .002), gastric inhibitory polypeptide, (16.8 ± 3.5 vs. −2.8 ± 1.0 μg · ml−1 · 240 min−1, P < .001), and insulin secretion (6.6 ± 1.1 vs. 4.7 ± 0.7 U.240 min−1, P = .003) were greater with oral than intravenous glucose. However, insulin clearance, whether calculated as the molar ratio of integrated C-peptide to integrated insulin responses (6.9 ± 0.7 vs. 14.2 ± 3.8, P = .005) or from the formula insulin clearance equals insulin secretion divided by integrated insulin responses (1.1 ± 0.2 vs. 2.5 ± 0.7 L.min−1 · m−2, respectively, P = .002), was less for oral than for intravenous glucose. Therefore, the incretin effect is mediated both by increased secretion and decreased clearance of insulin.
Mechanisms of Nitrosourea-Induced β-Cell Damage: Activation of Poly (ADP-Ribose) Synthetase and Cellular Distribution
Authors: Wilson Glenn L , Hartig Phillip C , Patton Nancy J , LeDoux Susan P
Published: September 2013

It has been hypothesized that the critical step in streptozocin (STZ)-induced β-cell toxicity is the overactivation of the nuclear enzyme poly(ADP-ribose) synthetase resulting from DNA strand breaks. Overactivation of this enzyme leads to a lethal depletion of its substrate, NAD, in the β-cell. However, recently it has been shown that a lethal concentration of STZ and a nontoxic concentration of its nitrosoamide moiety methylnitrosourea (MNU) damage β-cell DNA to the same extent and cause comparable amounts of DNA strand breaks. This study was performed to determine whether STZ and MNU activate poly(ADP-ribose) synthetase to the same extent. Monolayer cultures of islet cells from neonatal rats were exposed to concentrations of MNU and STZ of 10−3 to 10−2 M. The results show that both chemicals caused comparable activation of the enzyme at all concentrations tested. These data demonstrate that activation of poly(ADP-ribose) synthetase alone is not the critical step in STZ-induced β-cell toxicity. Based on this finding, it appeared possible that STZ may be selectively sequestered into some critical site in the β-cell other than the nucleus. Therefore, studies were initiated with 14C-labeled STZ and MNU to determine whether STZ might be distributed in the β-cell differently than MNU. Total cellular DNA and protein from both RINr (clone 38) and islet cell monolayers were separated on hydroxylapatite columns after exposure to 14C-labeled chemicals. The amount of label incorporated into each fraction was determined by liquid scintillation spectrometry, and the ratio of label incorporated in protein to that in DNA was determined. The results showed that RINr cells, which are resistant to the toxic effects of STZ, have similar protein-to-DNA ratios for MNU (0.32 ± 0.04) and STZ (0.39 ± 0.16) at an equimolar concentration. However, in β-cells the ratio was approximately three times greater for STZ (0.60 ± 0.17) compared with that for MNU (0.21 ± 0.17). These data demonstrate that in β-cells, which are very sensitive to the toxic effects of STZ, many more of the reactive carbonium ions bind to protein. This finding indicates that in β-cells, STZ is sequestered differently than MNU.
Insulin Secretion and Hepatic Extraction in Humans by Minimal Modeling of C-Peptide and Insulin Kinetics
Authors: Cobelli Claudio , Pacini Giovanni
Published: September 2013

Methods for measuring insulin secretion and hepatic insulin extraction in vivo, e.g., hepatic vein catheterization, are invasive, and can be applied during steady state only. We introduce a noninvasive method for measuring in vivo insulin secretion and its extraction by the liver during an intravenous glucose tolerance test (IVGTT). This method is based on a minimal model of C-peptide secretion and kinetics that is used for interpreting plasma C-peptide concentration data during an IVGTT in normal humans. The model allows the reconstruction of the time course of insulin secretion and, used in conjunction with a minimal model of insulin delivery and kinetics (described in a previous study), provides a noninvasive measure of the time course of hepatic insulin extraction [H(t)]. The C-peptide model also provides a direct prehepatic measure of β-cell sensitivity to glucose, expressed by two parameters related to first (ϕIC)- and second (ϕIIC)-phase insulin secretion. In the 11 healthy volunteers we studied, these parameters were 61 ± 11 pM · min−1 · mg−1 · dl and 0.0154 ± 0.0034 pM · min−2 · mg−1 · dl, respectively. H(t) showed an initial decrement for ∼30–50 min (from a fasting value of 63 ± 8% to a nadir of 53 ± 9%) after the glucose stimulus, then a steady value of ∼62% was reestablished and maintained throughout the experiment. The validity of the C-peptide model was further assessed by comparing its estimate of the fractional plasma clearance rate (k01) with that obtained in experiments in which biosynthetic human C-peptide was administered. The k01 averaged 0.063 ± 0.007 min−1, virtually identical to 0.060 ± 0.002 min−1 found in other studies. Because of its noninvasiveness, this modeling-based method should prove useful in the clinical investigation of many pathophysiological states
Immunologic Aspects of Human Proinsulin Therapy
Authors: Fineberg S Edwin , Rathbun Mary J , Hufferd Suzan , Fineberg Naomi S , Spradlin C Thomas , Galloway John A , Frank Bruce H
Published: September 2013

We investigated the immunogenicity of human proinsulin (HPI) when used as the sole or principal insulin agonist in insulin-naive patients with insulin-dependent (type I) and non-insulin-dependent (type II) diabetes mellitus. Sixty-one patients (13 type I, 48 type II) were treated with rDNA human insulin (NPH HI with or without regular HI) and 53 were treated with HPI (8 type I, 45 type II). At 6 mo, virtually identical levels of HbA1c (5.2 vs. 5.3%, P = NS) were achieved. However, regular HI was added less often to the treatment regimen in HPI-treated patients (16 vs. 32 patients, P < .001). Overall, there was no significant increase in proinsulin-specific antibodies in either treatment group. However, 8 of 51 (1 transiently) patients in the HPI group developed low levels of binding of HPI (highest percentage bound was 5%). Two patients in the HI group developed very low levels of HPI binding (1.2 and 1.9%). Binding of HI (> 2.4%) was seen in both treatment groups; however, the prevalence of HI binding was less in the HPI group at 6 mo (39 of 60 in HI group vs. 20 of 51 in HPI group, P = .008). Concomitant treatment with regular HI did not affect the prevalence or level of binding of HPI or HI. We conclude that human proinsulin is a weak immunogen when used as the principal insulin agonist and may reduce both the formation of anti-HI antibodies and the need for concomitant therapy with regular HI.
Early Detection of Vascular Dysfunction in Type I Diabetes
Authors: Jorgensen Rachel G , Russo Libbie , Mattioli Leone , Moore Wayne V
Published: September 2013

Blood flow and transcutaneous oxygen tension was measured by venous occlusion plethysmography and a transcutaneous oxygen electrode before and after 5 min of arterial occlusion in the forearm of young adult subjects with type I (insulin-dependent) diabetes without overt evidence of angiopathy. In control subjects (n = 21), the forearm blood flow increased by ≥ 2.8-fold at 30 s after ischemia. Diabetic subjects with glycosylated hemoglobin (GHb) ≤ 9.5% (n = 15) exhibited a blood flow response that was not statistically different from normal control subjects. Diabetic subjects with GHb ≥ 12.5% (n = 23) did not exhibit an increase in the postischemic blood flow. When blood flow patterns for the first 14 diabetic subjects were examined regardless of GHb value, four patterns of response were noted: 1) normal pattern (n = 3), 2) normal postischemic rise in blood flow with a prolonged elevation (n = 3), 3) no postischemic rise (n = 4), and 4) variable baseline blood flow with a decrease in blood flow postischemia (n = 4). This approach indicated that a comparison of means obscured potentially meaningful abnormal patterns. Abnormalities in the response of the transcutaneous oxygen tension to ischemia were observed in both groups of diabetic patients, but the difference between diabetic patients in good and poor control was less obvious. We have defined an abnormal response of blood flow and transcutaneous oxygen tension to ischemia that may correlate to glycemic control and have identified several patterns of blood flow after ischemia that may be important in defining the etiology and natural history of diabetic angiopathy.
Increased Activity of Intestinal Acyl-CoA: Cholesterol Acyltransferase in Rats With Streptozocin-Induced Diabetes and Restoration by Insulin Supplementation
Authors: Jiao Sheng , Matsuzawa Yuji , Matsubara Kenji , Kihara Shinji , Nakamura Tadashi , Tokunaga Katsuto , Kubo Masaharu , Tarui Seiichiro
Published: September 2013

We examined the activities of intestinal acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol esterase, enzymes regulating cholesterol absorption, in rats with streptozocin-induced diabetes (STZ-D) to clarify the effect of diabetes on cholesterol absorption. Three weeks after the induction of diabetes, plasma cholesterol levels were slightly but significantly increased in diabetic rats compared with control animals, whereas a far more remarkable increase in plasma cholesterol was observed in diabetic rats when fed an atherogenic diet containing 1% cholesterol, 0.5% cholic acid, and 5% lard. Microsomal ACAT activity in intestinal mucosa was three times higher in diabetic than in control rats. However, no significant difference in the enzyme activity could be detected between diabetic animals fed control chow and those fed the atherogenic diet. Furthermore, insulin supplementation given to diabetic rats caused a reduction of enzyme activity to the levels found in control animals. In contrast, cholesterol esterase activity in rat intestinal mucosa was unaffected by either the induction of diabetes or the atherogenic diet feeding. In conclusion, we disclosed that apparent ACAT activity in intestinal mucosa is elevated in STZ-D rats. Therefore, we postulate that enhancement of CoA-dependent cholesterol esterification in the intestine might be one of the major factors responsible for hypercholesterolemia in diabetes.
Nonislet Pancreatic Autoantibodies in Sibship With Permanent Neonatal Insulin-Dependent Diabetes Mellitus
Authors: Ivarsson Sten-A , Marner Bente , Lernmark Åke , Nilsson Karl Olof
Published: September 2013

Neonatal insulin-dependent diabetes mellitus (IDDM) occurs rarely. A sibship of two HLA-Dw3/4-positive boys who developed IDDM within the 1st wk of life is described. Although the HLA-D region genotype would be consistent with IDDM associated with islet autoimmunity, islet cell antibodies were negative, but both boys exhibited the presence of a novel autoantibody that reacted specifically with a conspicuous, yet unidentified, determinant in the interstitial tissue among the acinar cells. The possible relationship between this acinar nonislet autoantibody and permanent neonatal diabetes remains to be established.
Molecular Mimicry Between Insulin and Retroviral Antigen p73: Development of Cross-Reactive Autoantibodies in Sera of NOD and C57BL/KsJ db/db Mice
Authors: Serreze David V , Leiter Edward H , Kuff Edward L , Jardieu Paula , Ishizaka Kimishige
Published: September 2013

Enzyme-linked immunosorbent assay (ELISA) was used to study temporal development of murine autoantibodies against insulin and both type C and intracisternal type A retroviral antigens. The nonobese diabetic (NOD) mouse, a model for autoimmune, insulin-dependent diabetes, was compared with a related, but diabetes-resistant, strain, nonobese normal (NON). Similarly, C57BL/KsJ db/db mice (insulin-resistant model of insulin-dependent diabetes and obesity) were compared with diabetes-resistant C57BL/6 db/db mice. NOD mice developed much higher autoantibody titers than did NON mice. Whereas type C autoantibodies in NOD developed to peak titer shortly after mice were weaned, autoantibodies against insulin and p73 (group-specific antigen of the intracisternal type A particle) did not develop until shortly before, or concomitant with, the development of hyperglycemia. Two NOD mice not developing hyperglycemia during the 40-wk study period were distinguished from the mice developing diabetes by a delayed onset of insulin (but not p73) autoantibodies. Our findings suggest that in NOD mice, the appearance of insulin and p73 autoantibodies signifies that extensive underlying necrosis of β-cells occurred. C57BL/KsJ db/db mice (with extensive β-cell necrosis and early hyperglycemia) developed much higher autoantibody titers to insulin and p73 than did the diabetes-resistant C57BL/6 db/db mice. However, the presence of autoantibodies in normoglycemic C57BL/KsJ +/db controls demonstrated that elevated autoantibody titers alone were insufficient to produce diabetes in this model. Absorption studies indicated that autoantibodies against p73 recognized a common epitope on insulin and IgE-binding factor. The potential significance of this molecular mimicry is discussed.
Familial NIDDM: Molecular-Genetic Analysis and Assessment of Insulin Action and Pancreatic β-Cell Function
Authors: Elbein Steven C , Ward W Kenneth , Beard James C , Permutt M Alan
Published: September 2013

Although the hereditary nature of non-insulin-dependent diabetes mellitus (NIDDM) is well recognized, the nature of the predisposing defect remains elusive. Individuals with a history of gestational diabetes had shown a reduced insulin-sensitivity index (S1) in the absence of fasting hyperglycemia. To determine whether this finding could result from an inherited defect of the insulin receptor, an NIDDM pedigree was ascertained through a former gestational-diabetic proband. The proband, her siblings, and her first cousins were clinically characterized for insulin sensitivity with the minimamodel–based S1 from a modified glucose tolerance test. Islet function was characterized by the incremental insulin response to 5 g i.v. arginine at baseline and at a plasma glucose level of 500–600 mg/dl. Genetic studies included linkage analyses for the insulin gene and the insulin-receptor gene with DNA polymorphisms (restriction-fragment–length polymorphisms, RFLPs) previously described. The pattern of inheritance in this large pedigree appeared to follow autosomal-dominant transmission. No defect in islet function was found, but as a group, third-generation family members had an S1 that was significantly lower than that of weight-matched control individuals, suggesting an inherited defect in insulin action. Genetic studies showed no sharing of insulin gene, insulin-receptor–gene alleles among the diabetic individuals, or insulin-receptor alleles among third-generation individuals with insulin insensitivity. The genetic analyses thus suggest that this pedigree has an inherited defect that is not linked to the insulin gene or the insulin-receptor gene. The diminished S1 may nonetheless suggest an inherited defect in insulin action.
Critical Mass of Purified Islets That Induce Normoglycemia After Implantation Into Dogs
Authors: Warnock Garth L , Rajotte Ray V
Published: September 2013

Twenty grafts of highly purified islets of Langerhans (mean ± SE vol 0.98 ± 0.3 ml, islet diam 122 ± 5 μm) were autoimplanted into the spleen or liver of totally pancreatectomized dogs. Portal venous pressure did not change significantly. Incremental doses of islets of 1000–3000 (n = 3), 3000–4000 (n = 6), 4000–5000 (n = 5), 5000–6000 (n = 3), and 6000–8000 (n = 3) per kilogram body weight resulted in corresponding fasting plasma glucose (PG) of 258 ± 18, 163 ± 13, 158 ± 17, 138 ± 15, and 108 ± 6 mg/dl. In 3 apancreatic control dogs, PG was 338 ± 9 mg/dl. One (normoglycemic) dog died of wound complications, and follow-up PG at 1 mo was 89 ± 5 mg/dl in 6 of 10 dogs that received 3000–5000 islets/kg and 91 ± 6 mg/dl in all 6 that received > 5000 islets/kg. K values 1 mo after surgery during glucose tolerance tests were 1.8 ± 0.3 for 6 spleen dogs and 1.6 ± 0.3 for 6 liver dogs. Six months after splenic implantation, PG was 75 ± 4 mg/dl and rose to > 350 mg/dl after splenectomy. These data define the critical number of purified dog islets of known size that is necessary to induce prolonged normoglycemia. Sufficient pure islets can be collected from 1 dog pancreas to correct diabetes after autoimplantation into the liver or spleen.
Selective Localization of Factor VIII Antigenicity to Islet Endothelial Cells and Expression of Class II Antigens by Normal Human Pancreatic Ductal Epithelium
Authors: Dib Sergio A , Vardi Pnina , Bonner-Weir Susan , Eisenbarth George S
Published: September 2013

We evaluated the expression of factor VIII and class II histocompatibility antigens on frozen sections of normal human and rat pancreases. The immunohistologic studies were performed with directly fluoresceinated anti-human factor VIII and monoclonal antibodies A2B5, 3G5 (anti-islet), L-243, I-2, OK1 (anti-human Ia-DR), Leu-10 (anti-human HLA-DQ), anti-human HLA-DP, and OX6 (anti-rat Ia). Islet endothelial cells of humans and Wistar, CD, and BB diabetes-prone rats could be distinguished from intra-acinar endothelial cells by markedly enhanced factor VIII immunoreactivity. Factor VIII–antibody staining of islet endothelial cells was specifically absorbed by prior incubation of anti-human factor VIII antibody with normal human plasma but not by incubation with factor VIII–deficient plasma. By double indirect immunofluorescence, normal human pancreatic ductal epithelium expressed Ia in five of six pancreases studied.
Characterization and Expression of Human HepG2/Erythrocyte Glucose-Transporter Gene
Authors: Fukumoto Hirofumi , Seino Susumu , Imura Hiroo , Seino Yutaka , Bell Graeme I
Published: September 2013

The human HepG2/erythrocyte glucose-transporter gene, including the promoter region, has been isolated and characterized. The gene, which is ∼35,000 base pairs, is interrupted by nine intervening sequences or introns. The sequence of the HepG2 glucose-transporter protein predicted from the gene sequence differs from that determined from the published cDNA sequence in having Leu rather than Phe at position 152. In addition, there are several other nucleotide differences between the gene and cDNA sequences in both the coding region and 3′-untranslated region that do not alter the amino acid sequence of the protein. The sequence of the promoter and the site of transcription initiation have also been determined. The promoter region includes a TATA motif and two binding sites for the transcription factor Spl as well as a sequence that is found in the promoter region of several phorbol ester–inducible genes.