bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Suppression of Hepatic Glucose-6-Phosphatase with Carbohydrate Feeding
Authors: Newgard Christopher B , Foster Daniel W , McGarry J Denis
Published: September 2013

The mechanism by which exogenous glucose stimulates the incorporation of hepatic glucose-6-phosphate into glycogen in fasted rats has not been clearly delineated. We gave glucose intragastrically over a 3.5-h period during which liver glycogen was deposited at linear rates. Simultaneous primed continuous infusion of [2-3H] or [3-3H]glucose established that under these conditions absolute carbon flow through hepatic glucose-6-phosphatase was greatly suppressed.

After 1 h, hepatic [UDP-glucose] and [glucose-6-phosphate] had fallen by 50–60% and the former remained low throughout the experiment. By contrast, [glucose-6-phosphate] rebounded to its initial value by 2 h and remained at this level during the subsequent hour.

We interpret the data as follows. Exogenous glucose, in addition to acting as a precursor of liver glucose-6-phosphate, causes diversion of the latter away from free glucose formation and into glycogen synthesis. The fall in [UDP-glucose] is in accord with a glucose-induced activation of glycogen synthase, as proposed by Hers (Annu. Rev. Biochem. 1976; 45:167–89.). However, the fall-rise sequence of glucose-6-phosphate concentration constitutes the first direct evidence in vivo for simultaneous inhibition at the level of glucose-6-phosphatase.
The Human Glucagon Gene Is Located on Chromosome 2
Authors: Tricoli James V , Bell Graeme I , Shows Thomas B
Published: September 2013

DNA samples prepared from a panel of human-mouse cell hybrids with different numbers and combinations of human chromosomes were examined for the presence of the human preproglucagon gene by hybridization with a cloned segment of the human gene. The segregation of the human glucagon gene specific DNA fragment and human chromosome 2 in these cell hybrids indicated that the preproglucagon gene (designated GCG) is on chromosome 2 in humans.
The Effects of an Alpha-Glucoside Hydrolase Inhibitor on Glycemia and the Absorption of Sucrose in Man Determined Using a Tracer Method
Authors: Radziuk J , Kemmer F , Morishima T , Berchtold P , Vranic M
Published: September 2013

Acarbose, an alpha-glucosidase inhibitor, lowers the glycemie excursion following the ingestion of carbohydrates, in particular, sucrose. This was confirmed with increasing doses of acarbose (0, 50, and 100 mg) and the causes investigated. The absorption of the glucose moiety of sucrose was determined from plasma tracer concentrations when overnight-fasted normal subjects received a 100-g oral sucrose load labeled with sucrose ([1-14C]glucose) and a simultaneous intravenous infusion of [3-3H] glucose. As the dose of acarbose given with the sucrose load was increased from 0 to 100 mg, the percentage of the load appearing in the peripheral circulation decreased from 90% to 62%. Malabsorption was confirmed by the appearance of breath hydrogen. Simultaneously, absorption time increased from 243 to 411 min. Maximal glycemie excursions were therefore lowered from 64 to 31 mg/dl. The plasma concentrations of gastric inhibitory polypeptide and insulin decreased with the acarbose dose so that the fractional disappearance rate of glucose also decreased. However, the concentrations of glucagon-like immunoreactivity (GLI) rose, confirming the ileal appearance of malabsorbed sucrose.
Dermal Nerves in Human Diabetic Subjects
Authors: Johnson Peter C , Doll Susan C
Published: September 2013

The lack of a nerve tissue source that is easily, safely, and repeatedly available has been a major impediment to the study of human diabetic neuropathy. In this study dermal nerves from skin, obtained at biopsy and autopsy from the lower leg and at autopsy from the mid-abdomen, were subjected to quantitative electron microscopy to assess for diabetic perineurial cell basement membrane thickening, a change previously reported in sural nerve. A highly significant degree of perineurial cell basement membrane thickening was found in the diabetic subjects. Other structures in dermal nerves, such as axons, myelin, Schwann cells, and their organelles are also amenable to quantitative ultrastructural study. Skin biopsy is a way to obtain samples of peripheral nervous system tissue safely and repeatedly for the study of diabetic neuropathy.
Growth Retardation During Early Organogenesis in Embryos of Experimentally Diabetic Rats
Authors: Eriksson Ulf J , Lewis Nancy J , Freinkel Norbert
Published: September 2013

Embryos from rats rendered diabetic with streptozotocin for 1 wk or more before conception were examined on day 11.5 of gestation (i.e., at the 26–29-somite stage of normal rat embryonic development). The studies were designed to assess whether poorly regulated maternal diabetes is associated with demonstrable abnormalities even during this early phase of embryogenesis. We found that manifest retardations in growth and development were invariably present as judged by significant reductions in crown-rump length and somite number, respectively. Total protein and DNA content of the embryos were also reduced, although not symmetrically, so that protein/DNA ratios were increased. Gross dysmorphogenic lesions in neural tissue disproportional to the overall growth retardation at 11.5 days could not be demonstrated. The findings suggest that maternal diabetes can compromise intrauterine growth and development during the period preceding and coinciding with the establishment of circulation in the allantoic placenta. The possible multifactorial determinants remain to be elucidated. It also remains to be established whether the early embryotoxicity provides a setting conducive to the increased dysmorphogenesis that is traditionally recognized during the later stages of pregnancy complicated by diabetes.
Normal Insulin Sensitivity of the Islets of Langerhans in Obese Subjects with Resistance to Its Glucoregulatory Actions
Authors: Klimes Iwar , Nagulesparan Murugasu , Vasquez Barbara , Hidaka Hideki , Unger Roger H
Published: September 2013

The ability of varying levels of circulating insulin to suppress α- and Β-cell secretion was assessed by plasma glucagon and C-peptide measurement in 6 obese and 6 nonobese subjects maintained in a euglycemic state, with an insulin concentration elevated by 10, 20, or 100 μU/ml above basal levels by a primed-continuous infusion of insulin. The 10-μU/ml increase did not suppress C-peptide levels significantly in either group. However, incremental increases in plasma insulin of approximately 20 and 100 μU/ml above basal suppressed plasma C-peptide by 0.27 ± 0.14 and 0.53 ± 0.07 pmol/ml, respectively, in the obese subjects (14% and 31% of the basal values of 2.20 ± 0.18 and 2.19 ± 0.26 pmol/ml, respectively) and by 0.16 ± 0.06 and 0.17 ± 0.06 pmol/ml in the nonobese subjects (20% and 25% the basal values of 0.74 ± 0.11 and 0.78 ± 0.11 pmol/ml, respectively). Plasma glucagon levels were suppressed to a similar degree in each group in a dose-related manner during both the 20-μU/ ml and 100-μU/ml clamps. We were unable to identify an increment of insulin that suppressed C-peptide and/ or glucagon in one group but not in another. These data demonstrate inhibition of α- and Β-cell secretion by insulin within its physiologic range in both non-obese and obese man, and exclude insulin resistance of α- and β-cells in obese individuals. However, despite their much higher insulin levels before and during the insulin infusions, obese subjects had C-peptide levels that, at all times, were 3–3.5-fold higher than those of the nonobese subjects. This is consistent with a greater number and/or a higher secretory activity of β-cells in the obese subjects.
The Fate of Insulin in Rat Hepatocytes: Evidence for the Release of an Immunologically Active Fragment
Authors: Misbin Robert I , Almira Ernesto C
Published: September 2013

We have investigated the fate of 125I-insulin after binding by rat hepatocytes. Approximately 30% of the bound radioactivity dissociated from the cells as intact 125I-insulin; approximately 56% dissociated as 125and 125I-Tyr. The remaining radioactivity was recovered as peptides that we postulate are intermediate products of insulin metabolism. Experiments were performed in the presence of chloroquine (0.1 mM), an agent known to inhibit the intracellular processing of 125I-insulin. As expected, chloroquine increased the amount of radioactivity recovered as intact 125I-insulin (P < 0.005) and decreased the amount of 125I- and 125I-Tyr (P < 0.005). In addition, chloroquine decreased the amount of one of the insulin peptides (P < 0.005), but increased the amount of the other (P < 0.01). These data suggest the presence of two pathways of insulin metabolism in rat hepatocytes, one of which is inhibited by chloroquine. We have found a second pathway by which insulin is degraded due to the removal of several amino acids from the carboxyterminus of the B-chain. The resulting fragment bound poorly to insulin receptors on IM-9 cultured human lymphocytes, and probably has little if any biologic activity. However, this fragment bound well to anti-insulin antibody and constituted about 20% of the immunoreactive radioactivity that dissociated from the hepatocytes.
Pancreatic Alpha Cell Autoantibodies and Glucagon Response to Arginine
Authors: Winter William E , Maclaren Noel K , Riley William J , Unger Roger H , Neufeld Michel , Ozand Pinar T
Published: September 2013

The frequency and significance of cytoplasmic pancreatic alpha cell autoantibodies (ACA) were investigated in 2102 healthy controls, 879 patients with insulin-dependent diabetes mellitus (IDDM) who were negative for islet cell autoantibodies (ICA), and 1567 relatives of IDDM patients. ACA were found in approximately 1 in 200 people of all ages and were not significantly associated with IDDM, the IDDM-associated HLA phenotypes DR3 and DR4, or thyrogastric or adrenal autoantibodies. Of 11 ACA-positive patients studied by arginine stimulation tests, none had frank glucagon deficiency. Thus, ACA do not appear to be associated with defective alpha cell function or with IDDM.
Insulin Receptor Regulation and Desensitization in Rat Hepatoma Cells: Concomitant Changes in Receptor Number and in Binding Affinity
Authors: Crettaz Marco , Kahn C Ronald
Published: September 2013

We have studied the effects of chronic exposure to insulin on the binding and the biologic activity of the hormone using a well-differentiated cell line (Fao) derived from the Reuber H35 rat hepatoma. Prolonged incubation (24 h) with 10−6 M insulin produced a 20–25% decrease in binding of tracer concentrations (2 × 10−11 M) of125I-insulin, and a leftward shift of the curve for inhibition by unlabeled insulin. Scatchard analysis of the binding data revealed that a 75–80% decrease in the number of binding sites had occurred in the insulin-treated cells, but was accompanied by an increase in apparent receptor affinity. Kinetic studies suggested negative cooperativity in insulin binding and indicated that the change in affinity was accounted for by a decrease in the rate of dissociation. Both the decrease in receptor number and the increase in affinity were dependent on time, temperature, and the insulin concentration during the treatment period. Both effects were also blocked by cycloheximide, suggesting that they required new protein synthesis. Plasma membranes isolated from downregulated cells retained both the change in receptor number and affinity. Anti-receptor antibodies present in two human sera (B-2 and B-9) inhibited 125I-insulin binding in downregulated cells with equal or slightly greater sensitivity than in control cells.

The changes in insulin binding were accompanied by changes in insulin's biologic effects in these cells. Induction of tyrosine aminotransferase (TAT) activity by insulin, an effect that is observed in control cells within 4–6 h and is half-maximal with 5 × 10−10 M insulin, was totally abolished in downregulated cells. In addition, insulin increased two- to threefold the I-form of glycogen synthase in control cells, but failed to stimulate the enzyme in downregulated cells. These results indicate that prolonged exposure of Fao hepatoma cells to insulin produces a type of downregulation characterized by a decrease in insulin receptor number and a concomitant increase in receptor affinity. This is also accompanied by a total desensitization of the cells to insulin's effects on both tyrosine aminotransferase and glycogen synthase.
Gastric Inhibitory Polypeptide (GIP) and Insulin Release in the Obese Zucker Rat
Authors: Chan Catherine B , Pederson Raymond A , Buchan Alison M J , Tubesing Karen B , Brown John C
Published: September 2013

The involvement of the gut hormone GIP (gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide) in the hyperinsulinemia of the adult obese Zucker rat was investigated. Glucose, insulin, and GIP responses to oral glucose were compared in lean and obese rats. The sensitivity of the isolated, perfused pancreas to glucose and GIP was studied in basal and hyperglycemie conditions in lean and obese rats. Immunocytochemical studies of the gut and pancreas were also carried out.

The glucose and GIP responses to oral glucose were similar in lean and obese rats, but obese animals were hyperinsulinemic compared with lean controls under fasting conditions and after oral glucose. The isolated, perfused pancreas of obese Zucker rats had an elevated insulin response to 300 mg/dl glucose. GIP increased the insulin response to 300 mg/dl glucose threefold in both lean and obese rats. At basal glucose levels (80 mg/dl), GIP augmented insulin release in obese but not in lean rats. Immunocytochemical studies demonstrated the presence of enlarged islets in obese rats due to an increase in the B-cell mass. A-, D-, and PP-cells appeared normal. Obese and lean rats had similar numbers of GIP-containing cells in the gut. This study suggests that GIP may contribute to the fasting hyperinsulinemia characteristic of adult obese Zucker rats. GIP infusion to achieve levels equivalent to those seen in the basal state are capable of stimulating insulin release in the absence of hyperglycemia in the obese rat, which suggests an impairment of the regulatory mechanisms controlling the glucose-dependent insulinotropic action of GIP in these animals.
Effects of Alloxan-induced Diabetes on Corpus Luteum Function in the Pseudopregnant Rat
Authors: Garris David R , Whitehead Dan S , Morgan Carl R
Published: September 2013

The effects of alloxan-induced diabetes mellitus on rat ovarian structure and function were examined throughout pseudopregnancy (PSP). Animals received either saline (C) or alloxan (40 mg/kg) treatment on the day of proestrus (PA) preceding PSP or on day 1 (D-1A) of PSP (day 0 = ovulation). Serum samples were analyzed by radioimmunoassay for progesterone (P) and 17-β-estradiol (E) levels and compared with the corresponding changes in ovarian and uterine weights in C, PA, and D-1A rats. In addition, the effects of daily treatment with 6 IU ovine insulin (Al) on serum P levels were assessed in D-1A-treated rats and compared with controls. Alloxan treatment effectively elevated blood glucose levels (P ≤ 0.01) in PA and D-1 A groups as compared with controls or AI rats. Alloxan treatment reduced both ovarian and uterine weights of PA and D-1A groups as compared with C and Al rats. Serum P levels were significantly reduced in PA (P ≤ 0.01) and D-1 A (P ≤ 0.05–0.01) rats as compared with control rats throughout PSP. Daily insulin treatment reversed the suppressive effects of D-1 A treatment on serum P levels, but did not restore luteal function to control levels. Neither C nor D-1 A groups exhibited any marked differences in serum E levels throughout PSP. The results of these studies indicate that the administration of alloxan before the onset of PSP effectively inhibits luteal function, whereas D-1 A treatment induces early luteolysis as compared with controls. The associated declines in both uterine and ovarian weights reflect the impaired ovarian steroid production associated with diabetes. Since insulin partially restored luteal activity, it is suggested that diabetes-associated reproductive impairment is directly related to an insulin deficiency in the alloxan-treated rat.
Activation of Pyruvate Dehydrogenase Complex by Porcine and Biosynthetic Human Insulin in Cultured Human Fibroblasts
Authors: Buffington Cynthia K , Stentz Franke B , Kitabchi Abbas E
Published: September 2013

Cultured human fibroblasts represent an appropriate model for studying both insulin receptor interaction and hormone responsiveness. We have investigated the properties of the pyruvate dehydrogenase multienzyme complex (PDC) and have studied the effects of various concentrations of porcine and biosynthetic human insulin (BHI) on the activity of the enzyme. Under optimal conditions of the assay, both BHI and porcine insulin activated PDC in a dose-dependent fashion in which full activation of the enzyme was achieved with 10−8 M insulin. The half-maximal concentration for porcine and human insulin was similar, occurring at the level of 5 × 10−9 M for activation of the PDC of human fibroblasts.

We conclude that the PDC of cultured human fibroblasts is activated by both human and porcine insulin at a comparable physiologic concentration. Human fibroblasts may therefore serve as a useful model to study insulin action in isolated human tissue.
Phosphorylation of Insulin Receptors Solubilized from Rat Skeletal Muscle
Authors: Burant Charles F , Treutelaar Mary K , Landreth Gary E , Buse Maria G
Published: September 2013

A method has been developed to solubilize insulin receptors from skeletal muscles. Rat hindlimb muscles were rapidly frozen in liquid nitrogen, powdered, extracted with buffered Triton X-100, and partially purified by differential centrifugation followed by wheat germ agglutinin affinity chromatography. The solubilized receptors exhibit typical curvilinear Scatchard plots in insulin binding assays: rapid, Mn2+-dependent autophosphorylation of the β-subunit on exposure to insulin as well as insulin-stimulated kinase activity toward histone H2B. Furthermore, when intact soleus muscles were incubated in phosphate-depleted medium containing Na2H[32P]PO4, addition of insulin stimulated the in situ phosphorylation of the β-subunit of the insulin receptor. The ability to rapidly and efficiently isolate insulin receptors from skeletal muscle may permit investigation of factors that modulate insulin action in this tissue.
Comparison of an Immunohistochemical and Immunofluorescence Procedure to Detect Antibody to Pancreatic Islet Cells
Authors: Krell James , Rabin Bruce S
Published: September 2013

Antibody to the islet cells of the pancreas is detected in the serum of many patients with recent onset, insulin-dependent diabetes. Islet cell antibodies have been detected using the procedure of indirect immunofluorescence. We have adopted an immunohistochemical procedure using glucose oxidase to the histochemical identification of islet cell antibody. This procedure was found to be more sensitive than the immunofluorescence procedure, and, in addition, it was much easier to determine when a positive reaction was present. Thus, the glucose-oxidase immunohistochemical procedure for detecting antibodies to islet cells is preferable in comparison with indirect immunofluorescence.
Pre-Type I Diabetes: Linear Loss of Beta Cell Response to Intravenous Glucose
Authors: Srikanta S , Ganda O P , Gleason R E , Jackson R A , Soeldner J S , Eisenbarth G S
Published: September 2013

Twenty-one intravenous (i.v.) glucose tolerance tests were performed on nine subjects before the onset of overt type I diabetes mellitus. Islet cell antibodies (6 of 9 subjects) and elevated levels of la-positive T-lymphocytes (3 of 3 subjects studied) were detected during the prediabetic period. Elevations of fasting blood glucose and peak glucose during oral glucose tolerance tests were not observed until the year before onset of clinically overt diabetes. During the prediabetic period, there was a progressive loss of early-phase insulin release to i. v. glucose (rate of decline, 20–40 μU/ml insulin release/yr; correlation coefficient, 0.9).
Effect of Glycemic Control on Serum Insulin-like Growth Factors in Diabetes Mellitus
Authors: Merimee Thomas J , Gardner Donald F , Zapf J , Froesch E R
Published: September 2013

We investigated the effect of improving glycemie control on serum concentrations of insulin-like growth factors I and II (IGF-I and IGF-II). In 22 adults followed during an intensive home glucose monitoring program for 6 mo, no effect of improving control was seen on either IGF-I or IGF-II. Similar results were obtained in young diabetic children <10 yr of age and in diabetic adolescents with detectable puberty before entering the study. In older diabetic children without evidence of puberty before treatment (Tanner prepubertal stage 1), initial IGF-I concentrations were low, but increased during establishment of glycemie control. Puberty developed during therapy in this latter group.

Our data do not support a “global” effect of glycemie control on serum IGF-I in diabetic patients. Increases of IGF-I with better glycemie control appear most likely to occur when the metabolic consequences of diabetes have suppressed normal pubertal increases of IGF-I. IGF-II concentrations were unaffected by glycemie control in all subjects.
Possible Dissociation Between Insulin Binding and Insulin Action in Isolated Fetal Rat Hepatocytes
Authors: Sinha M K , Miller J D , Sperling M A , Suchy F J , Ganguli S
Published: September 2013

To directly examine the relationship between insulin receptors and insulin action in fetal tissue, we compared insulin receptor characteristics and insulin-mediated 14C-glucose incorporation into glycogen, as well as glycogen synthase activity, in freshly isolated hepatocytes from 21-day fetal (F) and adult (A) rats. Viability of hepatocytes was documented by trypan blue exclusion (> 90%), time-dependent 14C-leucine incorporation into protein, and dose-related incorporation of glucose into glycogen. Percent specific binding of 125I-insulin per unit protein was significantly higher in F than A liver plasma membranes (32.2 ± 0.3 versus 18 ± 2.4; P < 0.01) and Scatchard plots revealed twice the number of receptors in F. Similarly, receptor number per cell surface area was threefold higher in F than in A (150 versus 50 sites/μm2). At a fixed medium glucose concentration of 11.2 mM, insulin stimulated 14C-glucose incorporation into glycogen in a dose-related manner in A with an apparent Km of 1.0 ng/ml and Vmax at 5–10 ng/ml corresponding to 30–40% of total receptor occupancy; no effect was obtained in F with insulin up to 100 ng/ml. Net glucose incorporation into glycogen (nmol/106 cells/h) increased progressively with increasing medium glucose concentrations ranging from 1.4 to 27.8 mM; incorporation by F was significantly greater than by A at each glucose concentration. However, whereas insulin at 100 ng/ml significantly augmented net glucose incorporation at each glucose concentration in A, no effect of insulin was apparent in F. Finally, insulin at 10 and 100 ng/ml significantly increased the active (a form) and total (a + b form) glycogen synthase activity in A; the ratio of active to inactive forms (% I) also increased significantly in A. In contrast, there was no demonstrable augmentation by insulin of glycogen synthase activity in F hepatocytes. Thus, insulin-mediated incorporation of glucose into glycogen and insulin-stimulated glycogen synthase activity was not demonstrable in fetal rat hepatocytes despite the presence of an increased number of insulin receptors.
Establishment of Glucagon-producing Cells by Cell Hybridization
Authors: Takaki Ryosaburo , Ono Junko , Yokogawa Yasushi , Kumae Satoko , Nakamura Mitsuo , Koyama Hideki , Kawaoi Akira
Published: September 2013

Glucagon-producing cell lines were established by fusing pancreatic islet cells of adult hamster and 6-thioguanine-resistant hamster insulinoma cells. Under phase-contrast microscopy, the morphology of cultured hybrid cells was intermediate between those of the parental cells. The hybrid cells contained A-like granules, though few in number, and were stained with anti-glucagon antibody. The mode of chromosome number decreased to 78 or 79 by 3 mo after hybridization in comparison with the expected chromosome number of the heterokaryon of 104, and showed a minute decrease in 4 of 6 cell lines after 6 mo. The population doubling time ranged from 24 to 38 h, while that of parental insulinoma cells was 22.8 h. There was no correlation between the expression of cellular function and the stability of chromosome number or the length of population doubling time. The capacity of glucagon secretion was between that of the parental cells. The glucagon secreted into the medium, as assayed by the glucagon-specific antibody, was 0.6–2.5 ng/106 cells for 2 h, which was about 40% of total glucagon-like immunoreactivity secreted. Secretion of glucagon was not affected by high concentration of glucose, was markedly increased by theophylline, and was suppressed by exogenous insulin. All of the hybrid cells produced tumors on transplantation 6 mo after hybridization. The tumor-bearing hamsters exhibited high levels of plasma glucagon and blood glucose as well as a high level Of serum insulin.
Impaired Insulin Action in Rats with Non-insulin-dependent Diabetes
Authors: Levy Joseph , Gavin James R , Fausto Aurora , Gingerich Ronald L , Avioli Louis V
Published: September 2013

Studies were performed to test the hypothesis that impaired insulin action occurs as an acquired phenomenon in the streptozocin (STZ)-treated, non-insulin-dependent (NIDDM) diabetic rat model. A number of methods were used to evaluate impaired carbohydrate tolerance in these animals. Plasma glucose and insulin levels were measured at 6 and 14 wk of age, and insulin glucose tolerance tests were performed at 4 and 5 wk of age (before overt hyperglycemia ensues), and at 8 and 14 wk of age (after the animals manifest overt diabetes). The STZ-treated rats had higher plasma glucose levels than those of control animals (P < 0.001) at 6 and 14 wk of age, while their plasma insulin values were decreased to levels 73% of the controls (1.7 ng/ ml versus 2.3 ng/ml, P < 0.04).

Glucose disappearance rates after high (0.35 U/kg) and low (0.175 U/kg) insulin challenge were reduced in the experimental diabetic animals at all ages with both insulin doses. The data suggest that these animals have an early and progressive acquired impairment in insulin action, most compatible with a defect in cellular biologic response to insulin. The possibility that this abnormality is secondary to insulin deficiency is raised.
Application of a Rapid Enzyme-linked Immunosorbent Microassay (ELISA) to Study Human Anti-insulin Antibody
Authors: Nell Laura J , Virta Valerie J , Thomas James W
Published: September 2013

A rapid, reproducible enzyme-linked immunosorbent assay (ELISA) for antibody to insulin is characterized and used to study antibodies from insulin-treated diabetic subjects. No radioactivity is involved in the ELISA; rather, peroxidase-conjugated anti-human immunoglobulin is used to detect binding of antibodies to insulin-coated microplates. Color is produced by action of peroxidase on a substrate and an automated reader then measures binding spectrophotometrically. This ELISA was optimized to be at least as sensitive as measurement of antibody by direct 125I-insulin binding. Although detection of IgG directly binding to insulincoated plates in the ELISA does not reveal speciesspecific differences, avidity differences are shown in competitive inhibition with insulins from several species. Insulin-binding IgG was purified by affinity chromatography and used to construct a standard curve of the optical density (OD) in the ELISA relative to the concentration of antibody. This assay has been used to quantify and to characterize insulin antibody of sera from a large number of diabetic subjects, ranging from insulin resistant to those who had received only highly purified and human insulins. The assay is shown to be most useful to screen for insulin antibodies in resistant patients.
Whole Body De Novo Amino Acid Synthesis in Type I (Insulin-dependent) Diabetes Studied With Stable Isotope-labeled Leucine, Alanine, and Glycine
Authors: Robert Jean Jacques , Beaufrere Bernard , Koziet Joseph , Desjeux Jehan Francois , Bier Dennis M , Young Vernon R , Lestradet Henri
Published: September 2013

Dynamic aspects of whole body alanine and glycine metabolism have been explored in insulin-dependent (type I) diabetic subjects. Using a primed, continuous intravenous (i.v.) infusion of [2H3]alanine and [15N] glycine given simultaneously with [1-13C] leucine, whole body alanine and glycine fluxes and their rates of de novo synthesis were measured in 6 diabetic young men. Subjects were studied in the postabsorptive state, after blood glucose was clamped overnight at 15.2 ± 0.3 mM, and then, on the following night, at 5.9 ± 0.2 mM (insulin infusion rates of 0.24 ± 0.09 and 1.65 ± 0.20 U/h, respectively). In the normoglycemic state, leucine, alanine, and glycine fluxes averaged 88 ± 4, 378 ± 39, and 155 ± 8 μmol · kg−1 · h−1 respectively. Based on the leucine flux, alanine and glycine de novo synthesis rates were 264 ± 36 and 67 ± 8 ftmol · kg−1 · h−1. In the hyperglycemic state, leucine flux increased 23% (P < 0.01), alanine flux rose slightly (+ 5%) but significantly (P < 0.05), while alanine de novo synthesis and glycine flux remained unchanged and glycine de novo synthesis decreased by 33% (P < 0.001). These results show that small alterations in peripheral alanine inflow in the hyperglycemic state reflect increased proteolysis and suggest that increased circulating plasma glucose does not contribute to de novo alanine synthesis in the absence of adequate insulin effect and/or augmented glucose tissue uptake. These observations also reveal the importance of insulin in the maintenance of whole body leucine economy, since a lower rate of insulin administration was associated with an increased rate of leucine oxidation.
A Radioimmunoassay for Circulating Human Proinsulin
Authors: Cohen Robert M , Nakabayashi Tomio , Blix Petra M , Rue Paul A , Shoelson Steven E , Root Mary A , Frank Bruce H , Revers Robert R , Rubenstein Arthur H
Published: September 2013

A radioimmunoassay for human proinsulin (hPI) has been developed using biosynthetic hPI prepared by recombinant DNA technology as immunogen, standard, and tracer. The antiserum was raised in a guinea pig and then adsorbed against insulin and C-peptide conjugated to Sepharose to improve its specificity. After adsorption of the antiserum, the cross-reactivities to insulin and C-peptide were each <0.2%. Competition studies using in vitro enzymatically split forms of proinsulin demonstrated that the major antigenic determinant recognized was the junctional region between the B-chain of insulin and the C-peptide. The range of the assay extended from 10 to 150 fmol/tube, with a 50% displacement of 45–55 fmol/tube. This sensitivity proved suitable for measurements of serum hPI concentrations during infusion of biosynthetic hPI into normal subjects and type I diabetic subjects. Eightyfive of 89 serum samples from the normal subjects and each of 20 samples from diabetic subjects diluted in parallel with the hPI standard.

Since the direct assay sensitivity was not sufficient for measurement of endogenous hPI levels, a simple procedure for quantitative extraction of proinsulin-like material (PLM) from up to 40 ml of plasma on insulin antibody-Sepharose columns was developed. Log it-log slopes were calculated for dilutions of extracts of samples collected in the fasting state and 60 min after 75 g of oral glucose from eight healthy subjects. The slopes of 15 of the 16 samples did not differ significantly from the slope of the hPI standard. The fasting PLM concentration was 5.8 ± 3.0 fmol/ml (mean ± S.D, N = 8) and rose to 22.5 ± 6.7 fmol/ml (N = 6) 60 min after a 75-g oral glucose load. This assay will be useful in characterizing the metabolism and metabolic effects of biosynthetic human proinsulin and its intermediate forms and determining their therapeutic potential, and for measuring endogenous PLM in extracted plasma samples.
Insulin Mediator Stimulates Pyruvate Dehydrogenase of Intact Liver Mitochondria
Authors: Parker Janice C , Jarett Leonard
Published: September 2013

The effects of putative insulin mediators on the pyruvate dehydrogenase (PDH) activity of intact mitochondria isolated from rat liver were investigated. The mitochondria were judged intact on the basis of electron microscopic examination and demonstrated respiratory control. Only mitochondria having respiratory control ratios of >4, using succinate as a substrate, were used in these studies. Addition of physiologic concentrations of insulin to these mitochondria caused stimulation of PDH activity, attributed to generation of an insulin mediator from plasma membranes contaminating the mitochondrial preparation. Exogenous plasma membranes from rat adipocytes or liver caused further stimulation of PDH activity, which was proportional to the amount of plasma membranes added. Addition of insulin to the mixture of mitochondria and plasma membranes stimulated PDH still further. The stimulation was proportional to the insulin concentration, with maximal effects observed at 50 μU/ml insulin. Partially purified mediators from liver, muscle, H4-II-E hepatoma cells, and IM9 lymphocytes also stimulated PDH activity in intact mitochondria. Mediators prepared from insulin-treated liver, muscle, and cultured hepatoma cells stimulated PDH more than did mediators from the corresponding untreated source. Mediator from insulin-treated IM9 lymphocytes stimulated PDH less than did mediator from untreated IM9 Iymphocytes. These findings are consistent with the known effects of insulin on these tissues and with the reported effects of the various mediators on PDH activity in non-intact mitochondria. These observations support the proposal that these mediators are physiologically significant modulators of insulin's effects on PDH activity.
Redistribution of 125I-Insulin on the Surface of Rat Hepatocytes as a Function of Dissociation Time
Authors: Carpenter Jean-Louis , Fehlmann Max , Obberghen Emmanuel Van , Gorden Phillip , Orci Lelio
Published: September 2013

In the present experiments, we have correlated the distribution of 125I-insulin on the surface of rat hepatocytes with the dissociation of 125I-insulin from the cell. When 125I-insulin interacts with isolated rat hepatocytes at 15°C, an increasing proportion of the bound ligand becomes nondissociable under the influence of acid pH (6.0), trypsin (0.5 mg/ml), or an excess of unlabeled insulin (10−6 M). Under these conditions, only a small percentage of the labeled material is internalized as determined by quantitative electron microscope (EM) autoradiography. This progressive nondissociability of the ligand parallels its movement from microvilli to coated pits and its progressive concentration in these later surface specializations. These data suggest that receptors in different domains of the plasma membrane may have different dissociation rates for the ligand.
Insulin and Insulin Receptor Uptake into Rat Liver: Chloroquine Action on Receptor Recycling
Authors: Khan Masood N , Savoie Sylvine , Khan Rahat J , Bergeron John J M , Posner Barry I
Published: September 2013

In the present study, the effect of chloroquine on both insulin and receptor distribution was examined in vivo. Insulin injection (25 nmol/100 g body wt) caused a marked accumulation of both insulin and its receptor in purified hepatic Golgi fractions by 15 min postinjection. Percoll fractionation of parent Golgi fractions resolved two endocytic components of low (ρ = 1.040–1.050) and high (ρ = 1.053–1.064) density in which the relative distribution of insulin binding sites was unaltered by chloroquine. Chloroquine significantly accumulated in the high-density region of the Percoll gradient consistent with this being a low pH compartment. 125I-insulin accumulated first in the low-density (1 min) and subsequently in the high-density region (5–10 min) of Percollsubfractionated Golgi fractions. Chloroquine treatment caused marked accumulation of 125I-insulin in the highdensity compartment with substantial retention of radiolabel therein at 20 min postinjection. 125I-insulin extracted from the Percoll fractions was comparably intact in control and chloroquine-treated rats. These data suggest that the chloroquine-accumulating, high-density compartment of hepatic Golgi fractions is the site of dissociation of internalized insulin-receptor complexes before degradation of the ligand and receptor recycling.
Diet Can Prevent Diabetes in the BB Rat
Authors: Scott F W , Mongeau R , Kardish M , Hatina G , Trick K D , Wojcinski Z
Published: September 2013

When compared with laboratory chow, a defined, semipurified diet prevented diabetes, reduced the frequency of insulitis, increased thymus weight and total white blood cell count, and doubled thymus T-helper/T-suppressor cell ratios in diabetes-prone BB rats. These data show that the diabetic syndrome in BB rats may be prevented or delayed by changes in diet, which may occur through alteration of pathogenic defects in the immune system.
Insulin Stimulates DNA Synthesis in Cerebral Microvessel Endothelium and Smooth Muscle
Authors: Vinters Harry V , Berliner Judith A , Beck David W , Maxwell Karen , Bready James V , Cancilla Pasquale A
Published: September 2013

Experiments were performed to test the hypothesis that insulin stimulates DNA synthesis in cerebral microvessel endothelium and smooth muscle. Cultured endothelium and smooth muscle derived from isolated mouse cerebral microvessels were exposed to insulin in serum-free medium, and [3H]-thymidine incorporation in the cells was measured. Up to 40-fold stimulation of DNA synthesis in endothelium and fourfold stimulation in smooth muscle were observed. Stimulation became maximal in both cell types at an insulin concentration of approximately 104 ng/ml, although an effect was observed at much lower concentrations. Similar concentrations of insulin produced a less-dramatic (approximately twofold) increase in both endothelial and smooth muscle cell numbers. This effect of insulin, observed in microvessel endothelium and smooth muscle, but not in bovine aortic endothelium, emphasizes another way in which large- and small-vessel endothelia appear to differ.
Reduced Insulin Sensitivity in Nondiabetic, HLA-Identical Siblings of Insulin-dependent Diabetic Subjects
Authors: Raghu P , Johnston C , Beard J C , Bergman R , McCulloch D K , Palmer J P
Published: September 2013

Nondiabetic, HLA-identical siblings of insulin-dependent diabetic subjects have an increased risk of developing diabetes. Since insulin resistance is present in newly diagnosed diabetic subjects, we studied 12 HLA-identical siblings to determine whether they have impaired insulin sensitivity. Each sibling was carefully matched for age, sex, and body weight to a control from a nondiabetic family. The insulin sensitivity index (S1,) was determined after an intravenous glucose tolerance test (IVGTT) using the minimal model of insulin kinetics. The insulin sensitivity index was significantly lower in the HLA-identical siblings compared with their matched controls.
Platelet Function During Continuous Insulin Infusion Treatment in Insulin-dependent Diabetic Patients
Authors: Mayfield Ronald K , Halushka Perry V , Wohltmann Hulda J , Lopes-Virella Maria , Chambers Jerre K , Loadholt Claude B , Colwell John A
Published: September 2013

Patients with diabetes mellitus manifest increased in vitro platelet aggregation andincreased synthesis of the proaggregant and vasoconstrictor, thromboxane A2 (TXA2). We studied the effects of continuous insulin infusion treatment on platelet aggregation and arachidonic acid (AA)-stimulated platelet TXA2 synthesis (15 and 30 s post-AA, 1 mM) in 16 type I diabetic patients. Strict glycemic control was induced with the Biostator for 2 days and maintained for 12–14 days with continuous subcutaneous insulin infusion (CSII). The average premeal plasma glucose level (4/day) fell from 184 ± 15, before treatment, to 107 ± 6 mg/dl on the final day (P < 0.001). After control, platelet synthesis of TXA2, measured by radioimmunoassay of its stable metabolite, immunoreactive TXB2 (iTXB2), decreased in all patients (30 s: 276 ± 31 versus 199 ± 28ng iTXB2/ml/ 5 × 105 platelets; P < 0.05). The reductionin platelet iTXB2 synthesis (15 and 30 s) was greater in poorly controlled patients (HbA1c>12%; N = 8), and for all patients the decrease in iTXB2 (15 and 30 s) was correlated with the prestudy HbA1c level (15 s: r = 0.6; P < 0.01).

In contrast, platelet aggregation responses did not improve during intensive insulin treatment. The ED50 for AA (dose producing 50% maximum aggregation at 1 min) was unchanged after 2 wk of treatment and the ED50 for aggregation induced by ADP fell significantly inpatients with HbA1c 12% (2.8 ± 1.3 versus 1.2 ± 0.6 μM; P < 0.01). Other factors that were associated in this study with platelet aggregation responses were plasma lipoprotein levels and microvascular disease. In all patients before treatment, the ED50 for AA was inversely correlated with the LDL-cholesterol level(r = 0.57; P < 0.01). Although platelet aggregation did not improve during the period of intensive treatment, when the relations of microvascular disease and glycemic control to platelet aggregation were analyzed together in all patients, the ED50 for AA was greatest (P < 0.02) in thosepatients without microvascular disease and HbA1c <12%, indicating less platelet aggregability.

Thus, a brief period of CSII treatment reduced AA-stimulated platelet iTXB2 generation; however, the effects of this treatment on platelet aggregation appear to be complex and other extraplatelet factors also seem to influence the aggregation response.
The Effect of Insulin Treatment on Changes in Vascular Reactivity in Chronic, Experimental Diabetes
Authors: MacLeod K M
Published: September 2013

The influence of insulin treatment on the reactivity of aortae and mesenteric arteries from rats with chronic streptozocin (STZ)-induced diabetes was examined. Ninety days after theonset of diabetes, the responsiveness (developed tension [g]/cross-sectional area of tissue [mm2]) but not the sensitivity (pD2 value) of both aortae and mesenteric arteries from untreated rats to norepinephrine (NE) was significantly increased compared with age-matched, nondiabetic controls. However, responses of K+-depolarized preparations from untreated diabetic rats to increasing extracellular Ca2+ were unchanged. Treatment of diabetic animals with daily injections of insulin for 90 days, starting 3 days after STZ treatment, normalized blood glucose levels and body weights and completely prevented the increases in responsiveness of aortae and mesenteric arteries to NE. No significant differencesin systolic blood pressures, measured at weekly intervals, could be detected between nondiabetic,untreated diabetic, and insulin-treated diabetic rats. Insulin treatment of diabetic animals for 30 days, begun 90 days after the onset of diabetes, also normalized blood glucose levels and completely reversed the increases in the responsiveness of aortae and mesenteric arteries to NE. These results indicate that selective increases in the reactivity of aortae and mesenteric arteries to NE occur in diabetic rats before the development of hypertension. The ability of chronic insulin treatment to restore vascular responsiveness to NE to control levels suggests that the increased reactivity is a consequence of the diabetic state, and may predispose animals to the subsequentdevelopment of hypertension.
Fingerprint Analysis of Insulin and Proinsulins
Authors: Grau Ulrich
Published: September 2013

Proteolysis of insulin or (pre)proinsulin with S. aureus protease V8 in Tris buffer at neutral pH yields a characteristic pattern of peptide fragments that is resolved using high-performance liquid chromatography. Identification of the fragments of interest was achieved by comparison of insulins of different species, of modified insulins and of proinsulin and N-extended proinsulin, and by amino acid analysis.

The fingerprint method allows, for example, the simultaneous analysis of porcine and human insulin, the identification of a modified insulin generated in dosing devices, as well as the individual analysis of the two disulfide linkages between the A- and B-chain in refolded insulins.
The Effect of Met-Enkephalin and Naloxone on Somatostatin and Insulin Secretion from the Isolated, Perfused Rat Pancreas
Authors: Scalac-Guénot Dominique Di , McIntosh Christopher H S
Published: September 2013

The effect of met-enkephalin and the opiate antagonist, naloxone, on somatostatin and insulin secretion from the isolated, perfused rat pancreas has been studied during perfusion with 300 mg/dl glucose. In response to a gradient of met-enkephalin from 0 to 10−5 M, release of somatostatin was inhibited at low concentrations and stimulated at high concentrations. However, both low and high concentrations of metenkephalin stimulated insulin secretion. A gradient of met-enkephalin from 0 to 10−6 M caused only an inhibition of somatostatin release, whereas insulin release was stimulated.

The effects of met-enkephalin on somatostatin release were antagonized by naloxone (106 M). The metenkephalin-induced insulin secretion was partially, but not completely, blocked by naloxone. Naloxone (10−6 M) alone changed the endocrine secretions by decreasing somatostatin release and by stimulating insulin release. Met-enkephalin may, therefore, be a physiologic regulator of pancreatic endocrine secretion.
Insulin Gene Analysis in a Family with Maturity-onset Diabetes of the Young
Authors: Andreone Teresa , Fajans Stefan , Rotwein Peter , Skolnick Mark , Permutt M Alan
Published: September 2013

The insulin gene locus has been studied in a large kindred with maturity-onset diabetes of the young (MODY) characterized by hypoinsulinemia. DNA was isolated from peripheral leukocytes of 42 family members and 5 spouses. A highly polymorphic region in the 5'-flanking portion of the human insulin gene provided an opportunity for linkage analysis. The presence of three different length polymorphisms of +1600 base pairs (bp), −50 bp, and −150 bp different from the common size allele allowed haplotype assignment of insulin alleles. The hypothesis of linkage was tested by calculating the log of the ratio of the likelihood of the hypothesis of linkage to that of the hypothesis of nonlinkage (LOD score) at a given recombination distance between the insulin polymorphism and the diabetes locus. At a recombination frequency of 0.0, the LOD score was −14.50 and, therefore, the hypothesis of tight linkage can be strongly rejected.

This report is the third study of the relationship between the insulin locus and MODY; however, it is the first report in which a formal linkage analysis indicates with a high degree of probability no linkage between the insulin locus and hypoinsulinemia in a family. Because MODY is a heterogeneous disorder, it may be that different genotypes result in a composite phenotype. The lack of linkage between an insulin allele and MODY in a total of four families studied, however, suggests that the insulin locus is probably not a marker for the MODY phenotype. These results do not exclude the possibility that the insulin locus may be involved in the etiology of other forms of NIDDM.