bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Lipoprotein Lipase and Apolipoprotein E Secretion by Macrophages in Modulating Lipoprotein Uptake: Possible Role in Acceleration of Atherosclerosis in Diabetes
Authors: Kraemer Fredric B
Published: September 2013

Because the accumulation of lipid in macrophages is a characteristic feature of atherosclerosis, the mechanisms by which this lipid accumulation occurs have been intensively studied. This paper reviews the receptor- and non-receptor-mediated pathways that promote lipid accumulation in macrophages. Particular emphasis is placed on the contributions of two secretory products of macrophages, lipoprotein lipase and apolipoprotein E, to both receptor- and non-receptor-mediated uptake of triglyceride-rich lipoproteins by macrophages. The hormonal, lipid, and immunological factors that regulate the secretion of LpL and apoE by macrophages are discussed, as are how changes in the secretion of apoE and LpL that can modulate the uptake of triglyceride-rich lipoproteins by macrophages might influence the atherosclerotic process in people with diabetes.
Glucose and Insulin Chronically Regulate Insulin Action via Different Mechanisms in BC3H1 Myocytes: Effects on Glucose Transporter Gene Expression
Authors: Mayor Pilar , Maianu Lidia , Garvey W Timothy
Published: September 2013

We previously reported that, in primary cultured adipocytes, chronic exposure to glucose plus insulin impairs the insulin-responsive glucose transport system. In this study, we examined regulation of glucose transport in BC3H1 myocytes as a model for muscle and found important differences between BC3HI cells and adipocytes. In myocytes, chronic glucose exposure per se (25 mM) decreased basal glucose transport activity by 78% and insulin's acute ability to maximally stimulate transport by 68% (ED50 ∼2.5 mM; T½ ∼4 h). D-Mannose and 3-O-methylglucose diminished transport rates with ∼100 and 50% of the potency of D-glucose, respectively, wherea L-glucose, D-fructose, and D-galactose were inactive. Chronic glucose exposure also reduced cell surface insulin binding by 30% via an apparent decrease in receptor affinity, and this effect was associated with a comparable rightward shift in the insulin-glucose transport dose-response curve. In other studies, persistent stimulation with 15 nM insulin also decreased maximally stimulated glucose transport activity, which was independent and additive to the regulatory effect of glucose. Moreover, glucose and insulin-induced insulin resistance via different mechanisms. Glucose (25 mM) reduced the number of cellular glucose transporter proteins by 84% and levels of GLUT1 transporter mRNA by 50% (whether normalized to total RNA or CHO-B mRNA). In contrast, chronic insulin exposure led to a 2.1-fold increase in GLUT1 mRNA but did not alter cellular levels of transporter protein. Cotreatment with glucose prevented the insulin-induced rise in GLUT1 mRNA. BC3HI cells did not express GLUT4 mRNA thatencodes the major transporter isoform in skeletal muscle. In conclusion, in BC3H1 myocytes 1) glucose diminished insulin sensitivity by decreasing insulin receptor binding affinity and decreased basal and maximally insulin-stimulated glucose transport rates via cellular depletion of glucose transporters and suppression of GLUT1 mRNA; 2) chronic insulin exposure exerted an independent and additive effect to reduce maximal transport activity; however, insulin increased levels of GLUT1 mRNA and did not alter the cellular content of glucose transporters; and 3) although BC3H1 cells are commonly used as a model for skeletal muscle, studies examining glucose transport should be interpreted cautiously due to the absence of GLUT4 expression. Nevertheless, the data generally support the idea that, in non-insulindependent diabetes mellitus, hyperglycemia and hyperinsulinemia can induce or exacerbate insulin resistance in target tissues.
Effect of Free Fatty Acids on Insulin Receptor Binding and Tyrosine Kinase Activity in Hepatocytes Isolated From Lean and Obese Rats
Authors: Svedberg Jan , Björntorp Per , Smith Ulf , Lönnroth Peter
Published: September 2013

We demonstrated previously that high physiological concentrations of free fatty acids (FFA) rapidly decrease insulin binding, degradation, and action in isolated rat hepatocytes. In this study, hepatocytes from lean and obese Sprague-Dawley rats (Alab, Stockholm) were preincubated with or without 0.4 mM oleic acid, and the effect on insulin binding and tyrosine kinase activity was measured. In the absence of exogenous FFA, insulin binding was reduced in hepatocytes from obese compared with lean rats (mean ± SE reduction 44 ±7%, n = 8, P < 0.01). Furthermore, the inhibitory effect of oleic acid added to hepatocytes from lean rats (n = 8; 40 ± 9%, P < 0.01) was not seen in cells from obese rats. Treating obese rats with Etomoxir, a carnitine palmitoyl transferase I inhibitor, increased insulin binding to isolated hepatocytes by 41 ± 13% (n = 5, P < 0.05). There was no difference in total binding to partially purified insulin receptors from solubilized hepatocytes from lean and obese rats, whether cells were or were not preincubated with oleic acid. Tyrosine kinase activity of partially purified receptors from basal or insulin-stimulated cells was not affected by either obesity, treatment with Etomoxir, or preincubating the cells with oleic acid. Thus, both obesity and elevated ambient FFA levels are associated with impaired insulin cell surface binding to isolated hepatocytes, possibly through an effect of lipid oxidation on the internalization/recycling of the insulin-receptor complex without any perturbation of the receptor tyrosine kinase activity. The data suggest that the reduced insulin binding to hepatocytes from obese rats is due to elevated ambient FFA levels.
Cell-Specific Carbohydrate Metabolism Regulates S14 Gene Transcription
Authors: Goto Yoshinori , Mariash Cary N
Published: September 2013

Carbohydrate administration rapidly regulates hepatic mRNA-S14 content. Both sucrose and fructose but not glucose increase the transcription of hepatic mRNA-S14 in vivo. In primary hepatocyte cultures, mRNA-S14 transcription responds to either fructose or glucose. To test the hypothesis that the difference in hexose response is due to differences in cellular metabolism, we studied the regulation of this gene with a transient transfection assay system in Chinese hamster ovary (CHO) cells, hamster pancreatic β-cells (HIT), and primary hepatocytes. In HIT cells, glucose stimulation of the expression vector pS14CAT(5 kilobases [kb]) containing 4.9 kb of 5′-flanking DNA was threefold greater than fructose. Glucose also gave a fourfold greater response at 27.5 mM than at 2.2 mM. In CHO cells, pS14CAT(5 kb) showed a twofold greater response to fructose than to glucose. The differential response to the hexoses in the two cell lines is a result of cell-specific metabolism. Without glucose in the media, both CHO and HIT cells used pyruvate for energy. However, glucose addition to CHO cells enhances glycolysis and hexose shunt pathway activity while inhibiting pyruvate oxidation and S14 gene transcription. In contrast, addition of glucose to HIT cells leads to enhanced tricarboxylic acid cycle activity to oxidize pyruvate and an associated stimulation of S14 transcription. We confirmed these conclusions in primary hepatocyte cultures. Addition of 27.5 mM glucose led to a twofold increase in endogenous mRNA-S14 accumulation, a twofold increase in transfected pS14LUC (5 kb) activity, and a parallel twofold increase in pyruvate oxidation. Thus, the different transcriptional responses of the S14 gene to fructose and glucose are attributable to cell-specific metabolism. Our data show that the metabolic regulation of mRNA-S14 gene transcription correlates with the rate of cellular pyruvate oxidation.
Prevention of Autoimmune Diabetes With Nonactivating Anti-CD3 Monoclonal Antibody
Authors: Herold Kevan C , Bluestone Jeffery A , Montag Anthony G , Parih Ashu , Wiegner Aramy , Gress Ronald E , Hirsch Raphael
Published: September 2013

Autoreactive T cells mediate diabetes in animal models of insulin-dependent diabetes mellitus (IDDM) and are believed to cause the disease in humans. Therefore, immunotherapies directed against T cells are of particular interest for the treatment of IDDM. One candidate for such immunotherapy is anti-CD3 monoclonal antibodies (MoAbs), but clinical side effects are common with anti-CD3 treatment due to the ability of these MoAbs to activate T cells in vivo. However, F(a′)2 fragments of anti-CD3 are nonactivating and immunosuppressive. We evaluated the effects of whole anti-CD3 MoAb and F(ab′)2 fragments in the setting of experimental autoimmune diabetes. Treatment with whole MoAb or F(ab′)2 fragments significantly reduced the hyperglycemia induced with multiple low dosages of streptozocin (MDSDM; 232 ± 23 mg/dl, P < 0.01 and 235 ± 16 mg/dl, P < 0.01 vs. 325 ± 25 mg/dl, respectively) in male CD1 mice. Both whole MoAb and F(ab′)2 fragments suppressed the development of insulitis (P < 0.001). Treatment with whole MoAb resulted in marked weight loss (10.4 ± 1.5% of total body wt), and the mice appeared ill and listless, whereas, mice treated with F(ab′)2 fragments gained weight (4.9 ± 5.5% of total body wt) and appeared healthy. Treatment with whole MoAb caused activation of T cells in vivo as reflected by proliferation of freshly isolated spleen cells to recombinant interleukin-2. Depletion of T cells with whole MoAb was more pronounced than with F(ab′)2 fragments, and T-cell receptor (TCR) reexpression on remaining cells occurred with F(ab′)2 fragments within 48 h after F(ab′)2 treatment. Despite the expression of this surface molecule, signaling by the TCR complex was blocked because T cells stimulated with anti-CD3 MoAb failed to produce lymphokines. We conclude that treatment with either whole anti-CD3 MoAb or F(ab′)2 fragments suppresses the hyperglycemia and insulitis of MDSDM. The morbidity seen with whole MoAb does not occur with F(ab′)2 fragments and is correlated with the failure of the latter to activate T cells in vivo. Nonactivating forms of anti-CD3 MoAbs may be a useful form of immunosuppressive treatment for incipient IDDM because they do not cause morbidity associated with activation of T cells, which occurs with whole MoAb.
Mapping Diabetes-Susceptibility Genes: Lessons Learned From Search for DNA Marker for Maturity-Onset Diabetes of the Young
Authors: Cox Nancy J , Xiang Kun-San , Fajans Stefan S , Bell Graeme I
Published: September 2013

During our search for a marker for non-insulin-dependent diabetes mellitus (NIDDM) in a large multigenerational family with a form of NIDDM termed maturity-onset diabetes of the young (MODY), we learned a great deal that may serve to streamline the search for diabetes-susceptibility genes in other families. We describe here our experience and suggest strategies that may enhance the search for markers for other diabetes susceptibility genes with genetic linkage approaches.
Risk of Early-Onset Proliferative Retinopathy in IDDM Is Closely Related to Cardiovascular Autonomic Neuropathy
Authors: Krolewski Andrej S , Barzilay Joshua , Warram James H , Martin Blaise C , Pfeifer Michael , Rand Lawrence I
Published: September 2013

Determinants of proliferative diabetic retinopathy (PDR) that occur during the 2nd decade of insulin-dependent diabetes mellitus (IDDM) (early-onset PDR) were investigated in a nested case-control study. From an inception cohort of patients with juvenile-onset IDDM that now has 15–21 yr diabetes duration, the patients with PDR (cases, n = 74) were selected for study along with a random sample of the patients in the cohort without PDR (control subjects, n = 88). The risk of PDR was associated with poor glycemic control during the first 12 yr of diabetes. Relative to patients in the first quartile of the index of hyperglycemia, those in higher quartiles and nonattenders had a four- to fivefold risk of developing PDR. A striking relationship with cardiovascular autonomic neuropathy (CAN) was found. Relative to patients without CAN, patients with significant and mild CAN had odds ratios of 77.5 and 34.6, respectively. Patients with albumin excretion rates > 30 μgrams/min had moderately increased risk of PDR (ranging from 4-fold for microalbuminuria to 7-fold for proteinuria). In contrast, patients with impaired renal function had an extremely high risk of PDR. All 20 of these patients were cases, therefore the odds ratio was infinite. All three factors (poor glycemic control, CAN, and various stages of nephropathy) were associated with PDR in multiple logistic regression analysis. However, in models including glycemic control, the association between microalbuminuria or proteinuria and PDR was weakened. In conclusion, our findings are consistent with a hypothesis that the level of glycemia is a primary determinant of early-onset PDR. Autonomic neuropathy, almost universal amongcases, may be a strong risk factor for or a risk indicator of an etiologic process underlying the development of PDR. Conditions associated with advanced diabetic nephropathy, on the other hand, may accelerate the progression of nonproliferative retinopathy to PDR.
Glycerol Production and Utilization During the Early Phase of Human Obesity
Authors: Stunff C Le , Bougnères P F
Published: September 2013

To study the initial period of fat deposition in human obesity, we measured glycerol turnover in 12 children of 135–253% ideal body weight, who had continuously gained weight since the onset of obesity 2–9 yr previously. Hyperinsulinemia developed in these children depending on obesity duration (r = 0.74, P < 0.01). Whole-body glycerol production was twofold greater in the obese children (311 vs. 156 μmol·min−1 P < 0.01) and correlated with body fat (r = 0.67, P < 0.005). Normalization of glycerol flux to fat mass revealed that the rate of triglyceride hydrolysis was in fact lower in the adipose tissue of obese children (9.4 vs. 17.7 μmol·min−1/kg body fat) and correlated with plasma insulin (r = 0.64, P < 0.005). Euglycemic insulin clamps showed that the response of glycerol production to a unit increment in plasma insulin concentration was increased in obese children, suggesting increased insulin sensitivity of adipose tissue. As a direct consequence (r = 0.67, P < 0.025) of their elevated plasma glycerol concentration (65 ± 4 vs. 37 ± 2 μM, P < 0.05) obese children had an increased glycerol utilization by the whole body, as well as per unit of lean body mass (9.1 ± 1 vs. 6.5 ± 0.9 μmoles·min−1.kg lean body mass−1 P < 0.025).
Encapsulation of Insulin for Oral Administration Preserves Interaction of the Hormone with Its Receptor In Vitro
Authors: Roques Marina , Damgé Christiane , Michel Christine , Staedel Cathy , Cremel Gérard , Hubert Pierre
Published: September 2013

It has been shown that insulin associated with nanocapsules of isobutylcyanoacrylate retains biological activity after oral administration to diabetic rats from 6 to 21 days. Because part of this action is unexplained, we focused on the interaction of encapsulated insulin with the insulin receptor in vitro. We have shown that encapsulated insulin is able 1) to bind to insulin receptors both in rat liver plasma membranes and after solubilization from Chinese hamster ovary (CHO) cells transfected with the gene of human insulin receptor, 2) to accelerate 125I-labeled insulin dissociation from its receptor, and 3) to ensure transduction of a signal leading to stimulation of the β-subunit phosphorylation, with parameters similar to those of native insulin. In addition, encapsulated 125I-insulin was rapidly internalized in transfected CHO cells. Analysis of cell-associated radioactivity showed that encapsulated insulin remained largely intact (> 80%) after 3 h, whereas native insulin was mostly degraded. These data indicate that encapsulated insulin fulfills all the earliest events at the receptor level leading to biological actions and suggests that encapsulation protects insulin against insulin degradation inside the cells.
Anti-CD3 Immunotoxin Prevents Low-Dose STZ/Interferon-Induced Autoimmune Diabetes in Mouse
Authors: Vallera Daniel A , Carroll Stephen F , Brief Susan , Blazar Bruce R
Published: September 2013

Autoimmune diabetes was induced with an established model in which 3 daily injections of 95 mg/kg body wt/day streptozocin (STZ) and 2 × 104 U interferon-γ (IFN-γ) were administered to C57BL/6 mice. Diabetes onset was accompanied by precipitous increases in serum glucose levels and validated by immunoperoxidase studies showing diminished islets in pancreatic tissue sections. Administration of two to three doses of a monoclonal antibody (MoAb) or an immunotoxin (IT) directed against the CD3 ε-chain before STZ/IFN-γ treatment prevented increases in serum glucose and protected islets from damage. IT was made by crosslinking anti-CD3 to a low oligosaccharide-containing fraction of purified ricin toxin A chain (RTA; a catalytic inhibitor of protein synthesis) with a stabilized derivative of 2-iminothiolane. Protection was complete, long-lived, and selective because two different control ITs did not prevent diabetes onset. A second pan T-cell-reactive IT was synthesized by linking the MoAb anti-Ly1 to the same RTA toxin. Anti-Ly1 reacts with the murine homologue of human CD5. Anti-Ly1 RTA also protected against diabetes onset in a dose-dependent manner requiring higher doses and a longer schedule than anti-CD3 or anti-CD3 RTA. These studies demonstrate for the first time the importance of CD3+ and CD5+ cells in diabetes onset in the low-dose STZ/IFN-γ model and show that anti-CD3, anti-CD3 RTA, or anti-CD5 RTA may be useful in vivo for the treatment of diabetes or perhaps other T-cell-mediated autoimmune diseases. These data may have important therapeutic implications for early autoimmune diabetes in humans.
Focal Induction of IGF Binding Proteins in Proximal Tubules of Diabetic Rat Kidney
Authors: Bach Leon A , Cox Alison J , Mendelsohn Frederick A O , Herington Adrian C , Werther George A , Jerums George
Published: September 2013

Diabetes-associated kidney enlargement is associated with increased kidney insulinlike growth factor I (IGF-I) binding. IGF-I binds to the type I IGF receptor, which mediates most of its actions, and to specific binding proteins (IGFBPs), which modulate its actions. To explore the nature and extent of IGF-I binding in the kidney, in vitro autoradiography was used to map the distribution of IGF binding in control and diabetic rat kidney. Specificity studies were performed with increasing concentrations of unlabeled IGF-I, IGF-II, des(1–3)IGF-I (an IGF-I derivative that binds to receptors normally but with decreased affinity to binding proteins), and insulin. In control rats, diffuse binding was found throughout the kidney with increased density in the papilla. Binding specificity in the cortex and outer medulla was typical of the type I IGF receptor (IGF-I = des[1–3]IGF-I > IGF-II > > insulin). Binding in the outer medulla of diabetic kidney was typical of the type I IGF receptor. A marked focal increase in proximal tubular binding occurred in 13 of 22 postpubertal diabetic rats. Binding specificity of the proximal tubular binding was consistent with the predominance of an IGF binding protein (IGF-I = IGF-II > des[1–3]IGF-I with minimal displacement by insulin). Northern-blot analysis revealed increased IGFBP-1 and IGFBP-3 mRNA in cortical tissue from diabetic rats displaying increased proximal tubular binding but not from diabetic rats not displaying this phenomenon. As cell surface association of IGFBPs is linked to potentiation of IGF activity, a possible mechanism for potentiation of local IGF-I action may be provided.
Poly I:C Induces Development of Diabetes Mellitus in BB Rat
Authors: Sobel Douglas O , Newsome Joseph , Ewel Cynthia H , Bellanti Joseph A , Abbassi Val , Creswell Karen , Blair Owen
Published: September 2013

Polyinosinic polycytidilic acid (poly I:C), an inducer of α-interferon, accelerates the development of diabetes in diabetes-prone (DP) BioBreeding (BB) rats. This study investigates the effect of administering poly I:C to a diabetes-resistant (DR) strain of BB rats. We compared the incidence of diabetes, the degree of insulitis, the number of NK cells, helper-inducer cells, cytotoxic-suppressor cells, Ia+ T cells, RT6.1+ T cells, and NK cell bioactivity in DR rats treated with saline and with a 5 micrograms/g body wt (poly-5) dose and a 10 micrograms/g body wt (poly-10) dose of poly I:C. The incidence of diabetes was also compared with that of DP rats receiving poly-5. We found that both doses of poly I:C significantly induce the development of diabetes in the DR BB rat. However, treatment of DR rats with the higher dose induces a greater rate of development of diabetes and earlier onset of diabetes than the lower poly-5 dose. The rate of diabetes development and the mean age of onset were similar in poly-10–treated DR and poly-5–treated DP rats. A significant degree of insulitis occurred in all the poly I:C–treated DR rats, even those not developing diabetes. Peripheral blood NK cell number was greater in poly I:C than in saline-treated rats, after 2 wk of treatment and when killed. The percentage of OX19+ peripheral blood mononuclear cells expressing RT6.1 allotype or Ia antigen were similar in poly I:C– and saline-treated rats. of diabetes in poly-10–treated DR rats and poly-5–treated DP rats is consistent with a similar mechanism of poly I:C action in the DR and DP BB rats. Although the specific mechanism is not defined, NK cell numbers are elevated with poly I:C treatment. Alterations in RT6.1+ and la+ T cells do not appear to play a role.
NIDDM Associated With Mutation in Tyrosine Kinase Domain of Insulin Receptor Gene
Authors: Cocozza Sergio , Porcellini Antonio , Riccardi Gabriele , Monticelli Antonella , Condorelli Gianluigi , Ferrara Assiamira , Pianese Luigi , Miele Claudia , Capaldo Brunella , Beguinot Francesco , Varrone Stelio
Published: September 2013

A population of 103 patients with non-insulin-dependent diabetes mellitus (NIDDM) was screened for mutations in the tyrosine kinase domain of the insulin receptor gene. Patient genomic DNAs corresponding to exons 17–21 of the insulin receptor gene have been amplified by polymerase chain reaction and analyzed by denaturing gradient gel electrophoresis (DGGE). One patient was identified with an altered pattern of mobility of exon 20 in the DGGE assay. Direct sequence of amplified DNA showed a single nucleotide substitution in the codon 1152 (CGG – – > CAG), resulting in the replacement of Arg with Gln. Two bands appeared in the sequence of exon 20 of the insulin receptor (nucleotide position 3584), indicating that this patient was heterozygous for the mutation. Insulin binding to intact erythrocytes from the patient was in the normal range. Although autophosphorylation of the purified insulin receptor also seemed normal, its kinase activity toward the exogenous substrate poly Glu:Tyr (4:1) was undetectable. This mutation may impair insulin receptor kinase and contribute to insulin resistance in this patient.
Antibodies to Glutamic Acid Decarboxylase Discriminate Major Types of Diabetes Mellitus
Authors: Rowley Merrill J , Mackay Ian R , Chen Qiao-Yi , Knowles William J , Zimmet Paul Z
Published: September 2013

Insulin-dependent diabetes mellitus (IDDM) is marked by circulating antibodies to a 64,000-Mr islet cell antigen identified as glutamic acid decarboxylase (GAD). We describe a radioimmunoprecipitation assay with GAD isolated from pig brain. The sera tested were from 80 patients with IDDM including 26 with disease of recent onset and 54 with disease of longer duration (3–42 yr), 20 with non-insulin-dependent diabetes mellitus (NIDDM), and 55 nondiabetic subjects. Conventional assays for islet cell cytoplasmic antibodies were performed concurrently. The level of antibody in serum was expressed in units based on percentage reactivity of a standard reference serum. The frequency of antibody to GAD in IDDM was 69% in short-duration cases and 59% in long-duration cases. The latter was substantially higher than the frequency of islet cell cytoplasmic antibody. Antibodies to GAD were elevated (means ± 3 SD) in 5% NIDDM cases and in none of the nondiabetic subjects. A simple laboratory test with a defined autoantigen has substantial implications for population screening and early diagnosis of IDDM and for better understanding of its pathogenesis.
Correlates of Brain Edema in Uncontrolled IDDM
Authors: Durr Jacques A , Hoffman Willaim H , Sklar Allan H , El Gammal Taher , Steinhart Curt M
Published: September 2013

Blood glucose, plasma sodium, bicarbonate (HCO−3), vasopressin, and hematocrit were monitored before and during treatment in patients with uncontrolled insulin-dependent diabetes mellitus (IDDM). These parameters were correlated with simultaneous serial cranial computed tomography readings of brain edema. Six of seven patients had positive computed tomography readings for brain edema on admission. Initial brain edema correlated directly with blood glucose (r = 0.79, P = 0.033) and inversely with HCO−3 (r = −0.76, P = 0.047). At 6 h, brain edema still correlated with acidosis (HCO−3; r = −0.79, P = 0.033) but no longer with blood glucose. At that time, however, brain edema correlated with the rate of change in blood glucose (r = 0.915, P = 0.005). Results of interactive stepwise regression analysis suggest that the change in the calculated effective plasma osmolality plays a predominant role in the progression of brain edema during therapy (r = 0.995, P < 0.001). Thus, although hyperglycemia and acidosis probably predispose to diabetic brain edema, osmotic factors may be major predictors of its evolution. No relationships were detected between brain edema and initiation of insulin therapy, plasma vasopressin, or changes in hematocrit. The factors responsible for initial brain edema and its progression, statistically identified in this study, require reassessment of common theories that attribute brain edema exclusively to therapy.
Linkage Analysis of Insulin-Receptor Gene in Familial NIDDM
Authors: Elbein Steven C , Sorensen Lise K , Taylor Michelle
Published: September 2013

Although non-insulin-dependent diabetes mellitus (NIDDM) is clearly inherited, the mode of inheritance and genetic etiology remain unknown. Impaired insulin action is an important component of NIDDM, which may precede NIDDM onset, and appears to be inherited. Numerous defects of the insulin-receptor gene have been described in syndromes of extreme insulin resistance, and this gene is a strong candidate for genetic predisposition to NIDDM. To test this hypothesis, we examined 18 white pedigrees from Utah that had two or more siblings with NIDDM. For each pedigree, individuals not known to be affected were tested by standard oral glucose tolerance test, and diagnoses of NIDDM and impaired glucose tolerance were made by World Health Organization criteria. Each individual was typed for seven restriction-fragment–length polymorphism markers at the insulin-receptor locus, and marker phase was established by segregation. Linkage was examined with the LINKAGE program under six models, including autosomal dominant and autosomal recessive, with individuals with impaired glucose tolerance counted either as affected or of unknown status and with or without sporadic cases of diabetes. Under each model, linkage was significantly rejected. Neither inspection of individual pedigree log of odds scores nor formal tests of heterogeneity suggested a subgroup in which linkage of NIDDM and insulin-receptor gene was likely. In addition, sharing of insulin-receptor gene haplotypes among 108 affected sibling pairs drawn from the pedigrees did not deviate from that expected by chance alone. These data suggest that the insulin-receptor gene locus is unlikely to act as a major locus in the predisposition to NIDDM in most white pedigrees, although we cannot exclude a minor role for this locus under a highly polygenic model or a major role in rare pedigrees.
Simultaneous Insulinlike Growth Factor I and Insulin Resistance in Obese Zucker Rats
Authors: Jacob Ralph J , Sherwin Robert S , Greenawalt Kathleen , Shulman Gerald I
Published: September 2013

It has recently been shown that the ability of insulinlike growth factor I (IGF-I) to stimulate glucose uptake and to lower circulating amino acid levels is retained in insulin-resistant diabetic BB rats. To examine in vivo effects of IGF-I in obese Zucker rats (another model of insulin resistance) 6 obese and 6 lean rats received euglycemic IGF-I infusions (0.65 nmol · kg−1 · min−1). IGF-I-stimulated glucose uptake in obese rats was 50% lower than lean control rats (45.0 ± 2.8 vs. 92.2 ± 6.1 μmol · kg−1 · min−1, respectively), even though the rise in circulating IGF-I levels was greater in the obese group during IGF-I infusion. In addition, branched chain amino acid concentrations that declined by 45% in lean controls were not suppressed significantly in obese rats (392 ± 33 basal vs. 327 ± 29 microM at 90 min). Equivalent results were observed during euglycemic insulin clamps (12 pmol · kg−1 · min−1) in 7 obese and 11 lean rats. These studies demonstrate that obese Zucker rats are resistant to the effects of IGF-I and insulin on glucose and amino acid metabolism.
Insulin Can Rapidly Increase Cell Surface Insulin Binding Capacity in Rat Adipocytes: A Novel Mechanism Related to Insulin Sensitivity
Authors: Eriksson Jan , Lönnroth Peter , Smith Ulf
Published: September 2013

To elucidate the acute effect of insulin on its receptor, rat adipocytes were preincubated with insulin, washed with KCN to inhibit receptor cycling, and 125I-labeled insulin binding was measured. Preincubating cells from young insulin-sensitive rats with insulin increased cell surface binding up to approximately fourfold without changing apparent receptor affinity. This effect was rapid (t1/2 <5 min) and had a similar dose-response relationship as the effect on glucose transport. It was also energy dependent because preincubation with KCN completely abolished the effect of subsequent insulin exposure. The increased binding capacity was not recovered after cell solubilization or in partially purified receptors or isolated plasma membranes. Cells pretreated with insulin were less sensitive to the ability of trypsin to remove cell surface receptors, suggesting a conformational change of the receptors. This was also supported by the finding that the polyclonal binding in insulin-treated antibody B10 inhibited 125I-insulin binding in insulin-treated but not in control cells. Vanadate mimicked the effect of insulin to increase insulin binding, whereas concanavalin A, vasopressin, phorbol esters, or the adenosine analogue phenyl isopropyl adenosine was without effect. Insulin-resistant adipocytes from obese rats displayed no increase in cell surface binding after insulin treatment, despite normal tyrosine kinase activity in response to insulin. Thus, both insulin and vanadate elicit a rapid effect to markedly increase the number of cell surface insulin binding sites in intact rat adipocytes. This appears to occur independently of protein kinase C and the inhibitory GTP binding protein (Gi). Furthermore, the effect of insulin could not be demonstrated in insulin-resistant cells, suggesting that this mechanism may be of importance for the regulation of insulin sensitivity.
Treatment of Severely Diabetic Pancreatectomized Dogs Using a Diffusion-Based Hybrid Pancreas
Authors: Lanza Robert P , Borland Kermit M , Lodge Peter , Carretta Mauro , Sullivan Susan J , Muller Thomas E , Solomon Barry A , Maki Takashi , Monaco Anthony P , Chick William L
Published: September 2013

Long-term survival of dog islet allografts implanted in diabetic pancreatectomized dogs was achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes (nominal Mr exclusion of 50,000–80,000). Dog islets were isolated from the pancreases of outbred mongrel dogs by collagenase digestion. Chambers containing mean ± SE 316 ± 63K islet equivalents (mean islet volume, 558 ± 111 mm3, purity 90–95%) were peritoneally implanted into six totally pancreatectomized dogs. The dogs were monitored for glycemic control by fasting and postprandial blood glucose determinations, and responses to both intravenous glucose (intravenous glucose tolerance test 0.5 g/kg) and oral glucose (oral glucose tolerance test 1 g/kg). All of the dogs required appreciably lower dosages of exogenous insulin therapy for control of fasting blood glucose levels, with the mean daily insulin dose dropping from 38 ± 7 to 5 ± 1 U/day during the 1st wk. Three recipients required no insulin for >82, >68, and 51 days. Intravenous glucose tolerance test K values (decline in glucose levels, %%min) at 1 and 2 mo postimplantation were 2.7 ± 0.4 and 2.0 ± 0.5, respectively compared with 3.5 ± 0.5 before pancreatectomy. The glucose values during oral glucose tolerance tests at 2 wk, although returning to < 125 mg/dl (< 7.0 mM) by 2 h, exceeded the normal range, with peak values of 174 to 202 mg/dl (9.7 to 11.3 mM). These preliminary results are encouraging, and represent an important step in determining the feasibility of using this type of diffusion-based hybrid artificial pancreas as treatment for diabetes mellitus in humans.
Noninvasive Detection of Cardiac Sympathetic Nervous Dysfunction in Diabetic Patients Using [123I]Metaiodobenzylguanidine
Authors: Mäntysaari Matti , Kuikka Jyrki , Mustonen Juha , Tahvanainen Kari , Vanninen Esko , Länsimies Esko , Uusitupa Matti
Published: September 2013

The association between clinical autonomic dysfunction and myocardial MIBG accumulation was investigated. The study groups comprised 6 male diabetic patients with autonomic neuropathy (ANP+ group), 6 male diabetic patients without autonomic neuropathy (ANP- group), and 6 male nondiabetic control subjects. The mean age was comparable in all groups, and the subjects had no evidence of coronary heart disease. Reduced heart-rate variation in a deep-breathing test was used as a criterion for autonomic neuropathy. Immediately after injection, the peak net influx rate of MIBG to myocardium was significantly (P < 0.05) reduced in both diabetic groups. At 6 hr after MIBG injection, the MIBG uptake of the myocardium was significantly (P < 0.05) smaller in the ANP+ group than in the control group. In the ANP- group, the MIBG uptake of the myocardium was between that of the ANP+ group and that of the control group. Our data show that reduced myocardial MIBG accumulation is associated with autonomic dysfunction in diabetic patients, but it can occur to a lesser extent also in diabetic patients without apparent autonomic neuropathy. The measurement of the myocardial MIBG accumulation is a promising new method to detect cardiac sympathetic nervous dysfunction in diabetic patients.
A Rapid and Sensitive Radioimmunoassay for the Measurement of Proinsulin in Human Serum
Authors: Bowsher Ronald R , Wolny James D , Frank Bruce H
Published: September 2013

RIA methodology is used widely to measure proinsulin in human serum. However, some RIAs lack the sensitivity necessary to quantify proinsulin in unextracted serum and require long incubation periods. We developed an RIA with a sensitivity of 3.5 in <48 h. This was accomplished by using a nonequilibrium binding reaction at room temperature and PEG-assisted second antibody precipitation as the method for separating bound and free proinsulin. We obtained a specific antiproinsulin antibody by adsorbing the initial goat antiserum with human C-peptide–agarose. Proinsulin produced 50% displacement of tracer at 25.6 pM, whereas both human insulin and C-peptide failed to displace tracer at concentrations as high as 1 μM. We evaluated several cleaved derivatives of proinsulin for cross-reactivity with the antibody. B-chain–C-peptide cleaved derivatives (≤50% cross-reactivity) were more potent than A-chain–C-peptide cleaved derivatives (<5% cross-reactivity). However, all derivatives cleaved in the region from 56–60 failed to cross-react with the antiserum. These data indicate that a major antigenic determinant is present on the C-peptide region of proinsulin adjacent to the A-chain–C-peptide junction. After administration of an oral glycemic challenge, the mean fasting serum concentration of proinsulin in normal adults rose from 4.1 ± 0.28 to 23.6 ± 3.8 pM. We found a significant difference in the proinsulin concentrations in 6 adults before and after aglycemic challenge when two different antibodies were used in the RIA. Based on the antibodies different specificity for proinsulin, we concluded that B-chain-C-peptide junctional split forms of proinsulin comprise a significant portion of circulating proinsulin material after a glycemic challenge.
Brain D1 Dopamine Receptor in Alloxan-Induced Diabetes
Authors: Šalković Melita , Lacković Zdravko
Published: September 2013

Specific binding of [3H]SCH 23390 to dopamine D1 receptors in the striatum and olfactory tubercle in 14-day alloxan-induced diabetic rats was investigated. The Scatchard analysis revealed decreased D1 receptor density in the striatum (Bmax values were 548 ± 23 fmol/mg protein for the control and 466 ± 33 fmol/mg for the diabetic rats). No change was observed in the olfactory tubercle (Bmax; 299 ±27 fmol/mg for the control and 317 ± 32 fmo/mg for the diabetic rats). Thus, specific binding of [3H[SCH 23390 to striatal and olfactory tubercle membranes showed region-specific changes of brain dopamine D1 receptors in alloxan diabetic rats.
Elevated Plasma Concentrations of β-Cell Tropin (ACTH22–39) in Diet-Treated Type II Diabetic Patients
Authors: Levy Jonathan C , Morton John L , Davenport Michelle , Beloff-Chain Anne , Turner Robert C
Published: September 2013

β-Cell tropin, the pituitary peptide ACTH22–39, is a potent insulin secretagogue and stimulates lipogenesis in adipose tissue in rodents. Plasma β-cell tropin was measured fasting and after glucose infusion (5 mg · kg glucose ideal body weight−1 · min−1 for 90 min) in 10 mild diet-treated non-insulin-dependent (type II) diabetic subjects and 10 control subjects (body mass index) (BMI): 26.4 ± 3.2 and 24.1 ± 2.0 kg/m−2, NS, fasting plasma glucose 7.8 ± 2.7 mM and 4.7 ± 0.3 mM, respectively). The diabetic subjects had raised fasting plasma β-cell tropin compared with the normal subjects (geometric mean (1 SD range): 0.49 (0.25–0.96) nM and 0.17 (0.10–0.28) nM, respectively, P = 0.007). In response to the glucose infusion, plasma glucose rose higher in the diabetic subjects (mean ± 1 SD: 13.7 ± 3.1 and 9.6 ± 0.9 mM, P = 0.007) and plasma insulin was impaired in the diabetic compared with the nondiabetic subjects (geometric mean (1 SD range): 14 (8–26) and 34 (18–63), P <0.01). β-Cell tropin concentrations in the diabetic subjects rose to 1.31 (0.74–2.30) nM (P = 0.007), whereas β-cell tropin did not change in the normal subjects at 0.19 (0.11–0.91) nM. There was no overlap between glucose-stimulated plasma β-cell tropin in the two groups (P = 0.0002). Pituitary-adrenal function, as assessed by plasma cortisol, did not differ between the two groups when fasting and did not change after the glucose infusion. Though its physiological role is unclear, abnormal plasma β-cell tropin may be a feature of type II diabetes.
Markedly Reduced β-Cell Function Does Not Result in Insulin Resistance in Islet Autografted Dogs
Authors: Tobin Brian W , Lewis Jamie T , Lobin Bonnie L , Rajotte Raymond V , Finegood Diane T
Published: September 2013

Autotransplantation of islets of Langerhans has resulted in long-term normoglycemia in pancreatectomized dogs. This canine model is useful in evaluating both the progress of islet transplantation and the effect of a reduced islet mass upon the determinants of glucose tolerance: i.e., insulin secretion, insulin sensitivity, and glucose effectiveness. To determine the effect of a reduced islet mass on these factors, we measured the acuteinsulin response to arginine (AIRa) and glucose (AIRg), the slope of glycemic potentiation of AIRa (SP), insulin sensitivity (S1), and glucose effectiveness (SG) in control (CN), diabetic (DM), and pancreatectomized dogs rendered normoglycemic with transplanted autografts of islets of Langerhans (TX). Normal fasting plasma glucose (FPG) (TX 4.7 ± 0.2 mM; CN 4.9 ± 0.1 mM; P > 0.05) was maintained despite a markedly reduced insulin secretion in TX (AIRa 24%, AIRg 15%, and SP 11% of CN). All measures of insulin secretion were significantly correlated (SP vs. AIRg r = 0.80, P < 0.0001; AIRa vs. AIRg r = 0.92, P < 0.0001) across all animals, but none of the measures of secretion were significantly correlated with either the number of islets transplanted or time posttransplant (P > 0.10). Insulin sensitivity was normal in islet autografted dogs (TX: 136 ±12 min−1/(nmol/ml); CN: 101 ±11 min−1/(nmol/ml), P > 0.05) but SG was reduced (TX:±1.93 ± 0.28 × 100 min−1; CN: 3.53 ± 0.35 × 100 min−1 P < 0.05), as determined by the minimal-model method. In diabetic animals (FPG = 16.1 ±1.3 mM), insulin secretion was negligible by all measures (P > 0.05), and was associated with insulin resistance (S1 = 28 ± 8 min−1/(nmol/ml)) and reduced SG (1.72 ±0.11 × 100 min−1). These studies indicate that across a range of insulin secretion in dogs, thesecretagogues arginine and glucose provide similar estimates of β-cell function. This markedly reduced β-cell function does not result in insulin resistance when fasting normoglycemia is maintained, but is associated with a decrease in glucose action at basal insulin.
Tissue Kallikrein and Tonin Levels in Submandibular Glands of STZ-Induced Diabetic Rats and the Effects of Insulin
Authors: Chan Ka-Ming , Chao Julie , Proctor Gordon B , Garrett John R , Shori Deepak K , Anderson Leigh C
Published: September 2013

The levels of tissue kallikrein, tonin, and other kallikrein-like proteinases were determined in extracts of rat submandibular glands 3 mo after the induction of diabetes with STZ (65 mg/kg i.v.). Total kallikrein-like proteinase activity was assayed catalytically with the fluorogenic substrate DVLR-AFC. Tissue kallikrein was assayed by using the same substrate in the presence of SBTI. Activity of other kallikrein-like proteinases was defined as the difference between the total kallikrein-like activity and that of tissue kallikrein. Tonin was assayed by using the substrate ZVKKR-AFC in the presence of aprotinin. Results were compared with age-matched controls and with diabetic rats that had received daily insulin injections for the last week of the test period. The results showed that all activities were significantly reduced in diabetic glands compared with controls. Insulin treatment restored concentrations of tissue kallikrein activity, whereas the activities of tonin and other kallikrein-like proteinases were unchanged. RIA supported these findings. The results indicate that in rat submandibular glands, insulin affects the synthesis of kallikrein-like proteinases in different ways and, allowing for the slowness of the processes involved, insulin may exert a direct influence on the regulation of tissue kallikrein synthesis but only have indirect effects on the synthesis of tonin and the closely related kallikrein-like proteinases.
Characterization of the Extracellular Matrix—Containing Giant Perivascular Spaces in the NOD Mouse Thymus
Authors: Savino Wilson , Carnaud Claude , Luan Jian-Jian , Bach Jean-François , Dardenne Mireille
Published: September 2013

It is well established that the NOD mouse develops T-cell-dependent autoimmune type I diabetes that is abolished by neonatal Tx and enhanced by Tx at weaning. In a previous study, we demonstrated that the NOD thymus displays various abnormalities in the microenvironmental compartment, including abnormal distribution of epithelial cell subsets, precocious decline in thymic hormone production and formation of giant PVS. These latter structures present an internal ECM-containing network filled with T-cells and to a lesser extent B-cells. Herein we have investigated further the giant PVS and particularly the origin of the T-cells that colonize these structures. The thymic origin of intra-PVS T-cells was ascertained by distinct protocols. First, sublethal X-ray irradiation or HC treatment leading to cortical thymocyte depletion showed that intra-PVS lymphocytes were resistant, similar to medullary thymocytes. Second, adoptive transfer experiments that used newborn or adult irradiated Thy-1 congenic recipients demonstrated that intra-PVS accumulation of T-cells did not result from the reentry of peripheral mature T-cells into the thymus. Third, kinetic studies that used BrdUrd pulse chase revealed that labeled intra-PVS cells appear late, simultaneously with medullary thymocytes, and remain only transiently within the PVS. Thus, the kinetics of T-cell reconstitution of PVS was compatible with the progressive differentiation of T-cell precursors originating from the thymic cortex. In this respect, thegiant PVS of the NOD mouse thymus may represent a useful model to study the relationships between trafficking thymocytes and ECM proteins.
Muscle-Specific Expression of Human Insulin Receptor in Transgenic Mice
Authors: Benecke Heike , Flier Jeffrey S , Rosenthal Nadia , Siddle Kenneth , Klein Harald H , Moller David E
Published: September 2013

Variations in skeletal muscle insulin signaling are thought to have important effects on in vivo glucose homeostasis. To address the role of the insulin receptor in insulin action in muscle, we overexpressed human insulin receptors in the skeletal muscle of transgenic mice. A muscle-specific transgene (TMPE/HIR) was constructed by using promotor and enhancer elements derived from the rat MLC1/3 locus coupled to the intact protein-coding region of the human insulin-receptor cDNA. After testing the transgene for expression in cultured C2C12 myotubes, six founder mice transgenic for TMPE/HIR were generated. We determined that one line of mice had significant expression of human insulin-receptor mRNA in skeletal muscle. The analysisof several tissues from these mice by immunoprecipitation of labeled insulin receptors with a human-specific antireceptor antibody, revealed exclusive expression of human insulin receptors in skeletal muscle. Using both human-specific and non-species-specific anti-insulin receptor antibodies, we developed two immunoassays capable of quantitating the relative amounts of human and total insulin receptors in muscle. Compared with nontransgenic littermate controls, the total number ofinsulin receptors was increased by 30% in heterozygous transgenics and 68% in homozygotes. Human insulin-receptor protein contributed substantially to the total insulin-receptor pool present in transgenic muscle (42% for heterozygotes, 61% for homozygotes). Intraperitoneal glucose and insulin tolerance tests were performed with homozygous transgenic and nontransgenic littermate mice. Results with both approaches were significantly different for the two groups of mice, suggesting that the modest increase in insulin receptors in the muscle of transgenic mice causes a direct increase in insulin responsiveness. This study represents the first successful expression of human insulin receptors in transgenic mice. This model and others like it will provide valuable insights into the regulation of insulin-receptor expression and the role of insulin receptors in specific tissues or cell types in metabolism.