bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin Synthesis Inhibitors Impair Hepatic Glucose Production in Response to Glucagon and Epinephrine Stimulation
Authors: Miller J D , Ganguli S , Sperling M A
Published: September 2013

To investigate whether inhibition of prostaglandin synthesis affects hormone-inducedglucose dynamics, we measured glucose turnover in response to glucagon alone (5 ng · kg−1 min−1) or combined with epinephrine (0.1 μg · kg−1 min−1) in conscious trained dogs (N = 6) on three separate occasions in each animal: (1) during a control saline infusion, (2) during infusion of indomethacin, and (3) during infusion of sodium sal icy late. Glucose production (Ra) and utilization (Rd) were determined by isotope dilution using the nonrecycling label 3-3H glucose. In controls, glucagon levels (IRG) rose from a basal of 44 ± 12 to 260 ± 40 pg/ml (mean ± SEM) during glucagon infusion; basal epinephrine levels (EPI) of 150 ± 20 pg/ml were unaffected by glucagon infusion but rose four- to fivefold during combined glucagon/epinephrine infusion. Plasma glucose rose transiently from 95 ± 1 to a peak of 136 ± 13 mg/dl after 20 min of glucagon; infusion of EPI resulted in a second glycemic response with a peak of 148 ± 9 mg/dl. Ra increased transiently from 2.9 ± 0.2 to a peak of 7.9 ± 1.4 mg ± 0.8 mg · kg−1 min−1 10 min after beginning EPI. With glucagon alone, Rd paralleled Ra but addition of EPI resulted in a relative fall in Rd. Insulin (IRI) rose from 9 ± 1 μU/ml to 29 ± 6 μU/ml with glucagon but IRI fell despite the second glycemic response during EPI. When either indomethacin or salicylate was infused, basal IRI, IRG, EPI, glucose, Ra and Rd were unaffected and were similar to controls. Although plasma levels of IRG and EPI during glucagon or glucagon plus epinephrine infusion were also similar to controls, the glycemic response was reduced (P < 0.05). This attenuation of glycemic response was due to a reduction of stimulated Ra (P < 0.05) and not to an increase in Rd. Changes in IRI paralleled the reduction in glycemic response. Thus, both indomethacin and salicylate blunt the glycemic response to glucagon and glucagon plus epinephrine by attenuating glucose production and not by enhancing glucose utilization or insulin secretion. These results with two prostaglandin synthesis inhibitors suggest that prostaglandins modulate the hepatic effects of glucagon and epinephrine.
Long-acting Insulin: Slow Release of Insulin from a Biodegradable Matrix Implanted in Diabetic Rats
Authors: Goosen Mattheus F A , Leung Yin F , O'Shea Geraldine M , Chou Sheng , Sun Anthony M
Published: September 2013

This report describes the development of a long-acting insulin accomplished by the slow release of hormone from an implantable, biodegradable matrix. Rats made diabetic with streptozotocin received a single subcutaneous implant of insulin-albumin microbeads that released biologically active insulin for periods up to 3 wk. The mean fasting blood glucose level for treated animals was 88 mg/dl as compared with 392 mg/dl for untreated diabetic controls. With a mean starting body weight of 187 g, treated animals gained weight reaching a mean weight of 228 g; in contrast, untreated animals lost weight to a mean of 175 g. When insulin-albumin microbeads were periodically implanted and removed, lower blood glucose levels were only associated with the presence of the implants. The microbead implants biodegraded in 4–8 wk, thus obviating the need for surgical removal. These results suggest that a long-acting insulin may be produced by the entrapment of insulin within a biodegradable matrix.
The Effect of Pancreatic Islet Transplantation and Insulin Therapy on Experimental Diabetic Autonomic Neuropathy
Authors: Schmidt Robert E , Plurad Santiago B , Olack Barbara J , Scharp David W
Published: September 2013

Rats with chronic streptozotocin (SZ) diabetes develop dilatation of the alimentary tract, loss of fecal consistency, and autonomic neuropathy involving unmyelinated axons of the extrinsic innervation of the small bowel. Diabetic autonomic neuropathy involving the ileal mesenteric nerves is characterized by modest to marked dilatation of axons by distinctive subcellular organelles identical to those described in experimental and clinical axonal dystrophies. Axonopathy is confined to the alimentary tract; examination of myelinated and unmyelinated axons of the sciatic (midthigh level) and distal somatic nerves of the tail of diabetic animals with prominent ileal axonopathy failed to demonstrate significant numbers of dystrophic axons.

The prevention or reversal of diabetic autonomic neuropathy by a variety of experimental manipulations clearly indicates that the lesions we have demonstrated in chronically SZ-induced diabetic animals were produced by diabetes and were not the result of a direct neurotoxic effect of the diabetogenic agent streptozotocin. Animals did not develop axonopathy after simultaneous administration of SZ and nicotinamide, a procedure which prevents pancreatic beta-cell necrosis and induction of diabetes while exposing the nervous system to a possible neurotoxic agent. Selected animals that were given SZ, became diabetic, and subsequently received daily insulin therapy or pancreatic islet transplantation also did not develop axonopathy. Transplantation of pancreatic islets 6 mo after induction of diabetes, a time at which mesenteric axonopathy was well developed, quickly reestablished normoglycemia, and within 3 mo resulted in nearly complete resolution of the neuropathy. Mild chronic diabetes maintained for 5–6 mo failed to produce significant levels of axonopathy. Diabetes-induced alteration in the gross appearance and weight of portions of the digestive tract and stool consistency were completely or partially prevented by institution of diabetic control.
Muscle Capillary Basement Membrane in Identical Twins Discordant for Insulin-dependent Diabetes
Authors: Barnett A H , Spiliopoulos A J , Pyke D A , Stubbs W A , Rowold E , Hoffmann P , Faller A , Kilo C , Miller J P , Williamson J R
Published: September 2013

Although hereditary factors clearly modulate susceptibility to develop diabetes, their role as determinants of vascular complications associated with diabetes remains unclear. These studies were undertaken to further assess the extent to which capillary basement membrane thickening (CBMT) is governed by metabolic derangements associated with relative or absolute insulin deficiency versus genetic determinants of vascular disease closely linked to but independent of those modulating susceptibility to develop relative or absolute insulin deficiency. Quadriceps muscle capillary basement membranes obtained by needle biopsy were examined in eight pairs of identical twins discordant for insulin-dependent diabetes (IOD) for 11–29 yr. Biopsy material from one of the diabetic twins was technically unsuitable for study.

The average CBM width of the IDD twins was found to be significantly thicker than that of their nondiabetic (ND) twin mates (t = 2.50, P < 0.025). Three IDD, but none of the ND twins, had basement membrane width values in excess of 95% upper tolerance intervals for age- and sex-matched controls with no family history of diabetes.

The absence of CBMT in all of the ND twins and in four of the IDD twins with diabetes of 15–24 yr duration argues against the existence, in this group of subjects, of hereditary determinants of diabetic vascular disease linked to those governing susceptibility to develop diabetes. In addition, the absence of CBMT in four subjects with IDD of 15–24 yr duration is consistent with evidence from other studies indicating that diabetic microangiopathy is not an inevitable consequence of the diabetic milieu. The finding that muscle CBMT and retinopathy were restricted to IDD twins attests to the importance of the diabetic milieu as a predisposing or permissive factor in the initiation and progression of diabetic vascular disease.
Lower Levels of Erythrocyte Membrane Fluidity in Diabetic Patients: A Spin Label Study
Authors: Kamada Tetsuro , Otsuji Shogo
Published: September 2013

The dynamic properties of intact erythrocyte membrane in diabetic patients were investigated by means of electron spin resonance using three stearic acid spin labels (SAL): 5-, 12-, and 16-SAL. Significantly lower levels of erythrocyte membrane fluidity were revealed with 16-SAL as a probe in diabetic patients when compared with normal controls. However, there were no significant differences in fluidity values using 5- or 12-SAL between the two groups. Therefore, it became obvious that the decrease in fluidity was located in deeper sites (hydrophobic region) of the erythrocyte double membrane in diabetic patients. It was strongly suggested that changes in the membrane cholesterol to phospholipid molar ratios are not a principal factor contributing to the fluidity change. A significant increase of sphingomyelin and decrease of phosphatidylethanolamine were found in the erythrocyte membrane of diabetic patients and an alteration in membrane phospholipidclasses and their acyl-chains could conceivably be related to the fluidity change.

There were no significant correlations betweenchange in membrane fluidity and most plasma lipids, plasma lecithin-cholesterol acyl-transferase activities erythrocyte glycosylated hemoglobin, erythrocyte adenosine triphosphate, fasting blood glucose or duration of the disease. Plasma cholesterol of high-density lipoprotein showed a significant negative correlation with the membrane fluidity values.

Some of the possible factors contributing to and the significance of the lower levels of erythrocyte membrane fluidity were discussed in conjunction with both metabolic and clinical aspects in diabetic patients.
Relationship Between Serum Glucose Level and the Metabolic Clearance Rate of Glucose in Non-insulin-dependent Diabetes Mellitus
Authors: Revers Robert R , Kolterman Orville G , Olefsky Jerrold M
Published: September 2013

The metabolic clearance rate of glucose (MCR) has been widely used to compare glucose disposal rates in subjects studied at different plasma glucose levels. This approach assumes that the glucose disposal rate varies linearly with the prevailing glucose concentration. In the present study the relationship between MCR and the serum glucose concentration was assessed in 10 patients with non-insulin-dependent diabetes mellitus (NIDDM). Glucose clamp studies were performed in each subject at the patient's fasting glucose concentration (mean = 280 ± 17 mg/dl) and at euglycemia (mean glucose level = 85 ±3 mg/dl) in the presence of a maximally effective steady-state serum insulin concentration (∼11,000 μU/ml). Six subjects were also studied at hyperglycemia and euglycemia at a steady-state insulin concentration of ∼400 μU/ml. As expected, glucose disposal rates were significantly greater in the presence of hyperglycemia as compared with euglycemia (415 ± 28 versus 193 ± 30 mg/M2/min at the higher insulin level and 377±35 versus 162 ± 26 mg/M2/min at the lower insulin level). The mean MCR for the 10 subjects studied at the higher insulin level was 2.27 ± 0.37 ml/kg/min at euglycemia and fell to 1.51±0.21 ml/kg/ at hyperglycemia. The mean MCR for the six subjects studied at the lower insulin level was 1.91 ± 0.31 ml/kg/min at euglyglycemia. Thus at hyperglycemia the mean MCR fell by 34% and 36% at the higher and lower insulin levels, respectively. When the individual data were analyzed, a highly significant positive relationship (r = −0.88, P < 0.001) was found between the magnitude of the MCR value at euglycemia and the degree of fall in MCR at hyperglycemia. Thus, the greater the insulin resistance (lower MCR at euglycemia), the smaller the reduction in MCR at hyperglycemia. These results indicate that (1) the metabolic clearance rate of glucose is not independent of changes in the serum glucose concentration, indicating that even in the presence of maximally effective insulin concentrations glucose uptake by peripheral tissues is a saturable process and (2) as type II diabetic patients become less insulin resistant, their glucose uptake mechanisms become more saturable.
Nonenzymatic Glycosylation Reduces the Susceptibility of Fibrin to Degradation by Plasmin
Authors: Brownlee Michael , Vlassara Helen , Cerami Anthony
Published: September 2013

The effect of nonenzymatic glycosylation on the susceptibility of fibrin to degradation by the specific fibrinolytic enzyme plasmin was evaluated using both a fibrin plate assay anda fluorogenic synthetic plasmin substrate assay. Data from both types of experiments demonstrate that nonenzymatic glycosylation reduces the susceptibility of fibrin to plasmin degradation. Acetylation and carbamylation have qualitatively similar effects, indicating that chemical modification of lysine amino groups is the underlying phenomenon responsible for the observed degradative defect produced by glucose. Experimental conditions that increased the rate of nonenzymatic protein glycosylation (higher monosaccharide concentration, glucose-6-phosphate) were associated with correspondingly greater degrees of resistance to degradation by plasmin. Such reduced degradation of nonenzymatically glycosylated proteins in vivo may contribute to the accumulation of fibrin and several other proteins observed in those tissues most frequently affected by the complications of diabetes.
Circulating Somatostatin Concentrations in Healthy and Non-insulin-dependent (Type II) Diabetic Subjects
Authors: Conlon J M , McCulloch A J , Alberti K G M M
Published: September 2013

A technically simple radioimmunoassay for somatosta-tin-like immunoreactivity (SLI) in human plasma, which uses polyethylene glycol 6000 to precipitate tracer-degrading enzymes and high molecular weight interfering components, is described and validated. Fasting concentrations of SLI in peripheral plasma were similar in healthy and non-insulin-dependent diabetic (NIDD) subjects (range 3–39 pg/ml) but showed fluctuations for individuals in both groups when sampled frequently over a 60-min period. The mean coefficient of variation of SLI concentration was, however, less (8.0%; range 3–13%) in the diabetic group than in age-matched healthy subjects (19.5%; range 11–44%) (P < 0.02). Thus the regulation of basal D cell activity or of the somatostatinergic nervous system may be altered in type II diabetic subjects. A hospital breakfast (550 kcal) was a weak stimulus for release of SLI in both healthy and diabetic subjects. The rise in plasma SLI was sustained over 120 min. An increment in plasma SLI over mean basal levels of 5.9 ± 0.8 pg/ml was observed in young (mean age 23 yr; N = 6) control subjects, 7.9 ± 2.0 pg/ml in older (mean age 55 yr; N = 7) control subjects and 5.9 ± 0.6 pg/ml (mean age 62 yr; N = 7) in diabetics. Oral glucose (75 g) produced only a transient and erratic rise in plasma SLI that was not significant at any time point for either healthy or NIDD subjects. It is concluded that further investigations are necessary beforehypotheses linking somatostatin deficiency with the hormonal and metabolic abnormalities of non-insulin-dependent diabetes can be accepted.
An Association Between Complement-Fixing Cytoplasmic Islet Cell Antibodies and Endogenous Insulin Secretion in Children with Insulin-dependent Diabetes Mellitus
Authors: Mustonen Aki , Knip Mikael , Åkerblom Hans K
Published: September 2013

Cytoplasmic islet cell antibodies and endogenous insulin secretion were studied in 184 children and adolescents having insulin-dependent diabetes mellitus (IDDM) in a cross-sectional study. The mean age of the subjects was 12.3 yr (range: 2.8–19.2 yr), and the mear duration of diabetes was 4.6 yr (range: 0.1–15.6 yr). Islet cell antibodies (ICA) were determined by both the indirect immunofluorescence (IF-ICA) and the complement-fixing (CF-ICA) methods. Forty-four patients (23.9%) were positive with respect to both IF- and CF-ICA, 54 patients (29.3%) had only IF-ICA, and 86 patients had no ICA. The patients having CF-ICA had a significantly higher endogenous insulin secretion in comparison with the patients who were only IF-ICA positive. The difference between the groups remained significant even when the age at onset of diabetes and the duration of the disease weretaken into account.

This finding, revealing an association between CF-ICA and endogenous insulin secretion, suggests that complement-fixing antibodies are seen only if the beta-cell mass is sufficiently preserved. The result contradicts the hypothesis, based on studies in vitro, that CF-ICA should be involved in the selective beta-cell damage in IDDM.
Rat Islet Mitochondrial Adenine Nucleotide Translocase and the Regulation of Insulin Secretion
Authors: Ewart R B L , Yousufzai S Y K , Bradford M W , Shrago E
Published: September 2013

Employing a preparation of rat islet mitochondria, phosphoenolpyruvate has been shown to interact with the mitochondrial adenine nucleotide translocase. Thus, phosphoenolpyruvate inhibited mitochondrial uptake of [14C]ADP and exchanged with intramitochon-drial [14C]ATP. A concentration-dependent inhibition of islet mitochondrial 45Ca2+ accumulation was seen when mitochondria were exposed to phosphoenolpyruvate with half-maximal inhibition observed at a phosphoenolpyruvate concentration of 0.2 mM.

In experiments employing whole islets, phosphoenolpyruvate content was shown to be significantly elevated at both 1 and 30 min after an increase in the medium glucose concentration from 2 to 20 mM. In these experiments, the estimated islet concentrations of phosphoenolpyruvate fell in the range of maximal sensitivity of the islet adenine nucleotide translocase to phosphoenolpyruvate-induced inhibition of Ca2+ accumulation. It is concluded that increased concentrations of islet phosphoenolpyruvate resulting from increased extracellular glucose concentration may act to trigger or promote glucose-stimulated insulin secretion by modifying the distribution of Ca2+ between the islet cy-tosolic and mitochondrial compartments in a transport reaction catalyzed by the adenine nucleotide translocase.
Mononuclear Leukocyte β2-Adrenergic Receptors and Adenylate Cyclase Sensitivity in Insulin-dependent Diabetes Mellitus
Authors: Sérusclat Pierre , Rosen Stephen G , Smith Edward B , Shah Suresh D , Clutter William E , Cryer Philip E
Published: September 2013

Patients with insulin-dependent diabetes mellitus (IDDM) have been found to have a heightened hyperglycemie response to epinephrine. To determine if patients with IDDM have increased sensitivity of cellular β2-adrenergic receptor-effector systems, we assessed β2-adrenergic receptors and adenylate cyclase sensitivities to isoproterenol in partially purified mononuclear leukocyte (MNL) plasma membranes from 10 patients with IDDM (without adrenergic neuropathy) and 10 matched nondiabetic controls. MNL β2-adrenergic receptor densities (Bmax = 48 ± 8 fmol [3H] DHA/mg protein in IDDM, 44 ± 3 fmol [3H] DHA/mg protein in controls) and binding affinities (apparent KD = 0.3 ± 0.07 nM in IDDM, 0.3 ± 0.04 nM in controls) did not differ. Further, MNL adenylate cyclase activities were not significantly different either at baseline (325 ± 86 pmol/mg protein/15 min in IDDM, 275 ± 49 pmol/mg protein/15 min in controls) or in response to isoproterenol (842 ± 229 pmol/mg protein/15 min in IDDM, 608 ± 86 pmol/mg protein/15 min in controls). Thus, the data do not support the presence of a generalized alteration of β-adrenergic receptors or adenylate cyclase sensitivity in IDDM. To the extent that MNL β-adrenergic receptors and adenylate cyclase activities reflect those of extravascular catecholamine target cells, these findings suggest that the heightened hyperglycemie response to epinephrine exhibited by patients with IDDM is not due to increased sensitivity of cellular β2-adrenergic receptor-effector systems and is best attributed to the altered hormonal milieu of the insulin-deficient state.
Ciglitazone, a New Hypoglycemic Agent: II. Effect on Glucose and Lipid Metabolisms and Insulin Binding in the Adipose Tissue of C57BL/6J-ob/ob and − + / ? Mice
Authors: Chang Albert Y , Wyse Beatrice M , Gilchrist Barbara J
Published: September 2013

The fat pads isolated from control and ciglitazone-treated C57BL/6J-ob/ob mice and their lean littermates (− + / ?) were incubated in vitro with glucose-1-14C/-5-3H in the presence of insulin. The formation of 14CO2 and 3H2O and the levels of free fatty acids and glycerol in the medium and the incorporation of 14C and 3H into esterified lipids and free fatty acids in the fat pads were measured. The basal rates of glucose-1-14C/-5-3H metabolism per unit weight were increased in the fat pads of ciglitazone-treated mice, more pronouncedly in the treated lean than in the treated obese. The insulin-dependent effects were enhanced in the treated ob/ob mice. To see the dose-response of insulin, a second experiment was carried out in which portions of the fat pads were incubated in vitro with glucose-1-14C in the presence of 0–40 ng/ml insulin and isolated adipo-cytes were used to estimate for 125l-insulin binding. The basal rates of 14CO2 and 14C-lipids formation per unit weight of fat pads were increased in both treated obese and treated lean groups but insulin-dependent elevation was seen only in the treated obese group. Ciglitazone significantly increased insulin binding capacity at the low-affinity sites in the adipocytes of obese mice but not in those of lean mice. The data showed that ciglitazone increased the basal rate of glucose metabolism, lipogenesis, insulin receptor numbers, and post-receptor responses in the C57BL/ 6J-ob/ob mice; it increased the basal rate of glucose metabolism and lipogenesis but not insulin sensitivity in the lean mice.
Impact of Insulin or Tolbutamide Treatment on 14C-Arachidonic Acid Conversion to Prostacyclin and/or Thromboxane in Lungs, Aortas, and Platelets of Streptozotocin-induced Diabetic Rats
Authors: Valentovic Monica A , Lubawy William C
Published: September 2013

Diabetes is associated with a dramatic increase in the risk of thrombotic and atherosclerotic disease. The underlying factors responsible for this predisposition as well as the mechanisms involved have yet to be elucidated. Two endogenous substances, prostacyclin (PGI2) and thromboxane (TXA2), have recently been shown to possess significant vascular and thrombotic activity and are known to be altered in atherosclerosis, thrombotic conditions, and diabetes. We determined the conversion of 14C-arachidonic acid (AA) to PGI2 and TXA2 by lungs, aortas, and platelets obtained from chemically induced diabetic rats. In addition, we investigated the ability of insulin or tolbutamide to reverse these changes. Streptozotocin (STZ)-injected rats developed blood glucose levels 2–4 times that seen in normoglycemic controls. Intact perfused lungs isolated from rats beginning 7 days after STZ treatment synthesized 22–30% less PGI2 from 14C-AA. The ratio of PGI2/ TXA2 was decreased in the diabetic rat lungs and was inversely proportional to plasma glucose levels at the time of death. Platelet TXA2 generation was increased 67% above control in diabetic rats while aortic PGI2 generation was decreased 28% below normoglycemic controls. Ten-day treatment with NPH insulin 20 U/kg s.c. in STZ-pretreated rats lowered plasma glucose toward normoglycemia more effectively than tolbutamide 200 mg/kg orally. Partial correction of the decreased pulmonary PGI2/TXA2 ratio seen in diabetic rats was produced by insulin and tolbutamide in proportion to their ability to lower blood glucose. At the doses employed, insulin caused aortic PGI2 and platelet TXA2 generation from 14C-AA to approach that seen in normoglycemic controls more effectively than tolbutamide. An inverse correlation with plasma glucose levels was observed for pulmonary PGI2/TXA2 ratios as well as aortic PGI2 generation, r = −0.72 and −0.74, respectively. Platelet TXA2 generation correlated posi-tively with plasma glucose, r = 0.69. This study indicates that metabolism of 14C-AA by pulmonary tissue, platelets, and aortas is altered by experimentally induced diabetes. Furthermore, these findings suggest that such alterations can be partially reversed by regulation Of the diabetic condition.
Transplantation of Fetal Rat Islets Into the Cerebral Ventricles of Alloxan-Diabetic Rats: Amelioration of Diabetes by Syngeneic but not Allogeneic Islets
Authors: McEvoy Robert C , Leung Pearl E
Published: September 2013

Syngeneic fetal rat islets were isolated and transplanted into alloxan-diabetic inbred male Lewis rats. The effect of transplantation of islets into the cerebral ventricles on the diabetic state of the recipients was compared with that of the conventional transplantation of islets homeotypically into the liver via the portal vein. Fourteen of fourteen rats surviving after stereo-taxic implantation of islets into the ventricles returned to normoglycemia; normoglycemia has been maintained for up to 34 wk. Glucose tolerance tests revealed an improved, although not completely normalized, pattern. Histologie examination of the brains of these recipients revealed clusters of intact islets in the ventricle. These data provided a physiologic basis for further investigation of the immunologically privileged nature of the intraventricular space as a site for implantation of allogeneic pancreatic islets. Islets from Wistar-Furth rats (major histocompatibility difference) or Fischer 344 rats (minor histocompatibility difference) were transplanted into the ventricles of alloxan-diabetic Lewis rats. There were only small and unsus-tained changes in body weight and blood and urine glucose of any of the rats receiving the allogeneic islets. Histologie examination of the ventricles of these rats 3 wk after transplantation revealed only glial scar tissue. These data suggest that the cerebral ventricles cannot serve as a privileged site for islet transplantation, at least using the type of islet preparation employed in these experiments.
Suppressive Effect of Antibodies to Immune Response Gene Products on the Development of Low-Dose Streptozotocin-induced Diabetes
Authors: Kiesel Ulrich , Kolb Hubert
Published: September 2013

Low-dose streptozotocin-induced diabetes in mice serves as a model of type Idiabetes. Suppression of the development of diabetes (hyperglycemia) in C3H/ He mice was achieved with in vivo administration of antibody reactive to Ir-gene products before streptozo-tocin treatment. A persistent effect was reached with two monoclonal antibodies directed against l-Ak gene products and, surprisingly, by an allo-antiserum to I-J determinants. These results suggest a role for I-A and I-J positive T-lymphocytes and/or macrophages in B-islet cell autoimmunity.
Identification of a Point Mutation in the Human Insulin Gene Giving Rise to a Structurally Abnormal Insulin (Insulin Chicago)
Authors: Kwok Simon C M , Steiner Donald F , Rubenstein Arthur H , Tager Howard S
Published: September 2013

Both insulin gene alleles of a diabetic patient with a mutant insulin were cloned in a lambda vector and their nucleotide sequences were determined. Nucleo-tide sequence analysis revealed, in one allele, a C (cytidylate) to G (guanylate) transversion in the codon for phenylalanine at position 25 of the insulin B-chain. This point mutation leads to the substitution of a leucine for phenylalanine accompanied by the loss of a restriction endonuclease Mboll recognition site and the creation of a new Rsal cleavage site at this position.
Fuel-induced Insulin Release In Vitro from Insulinomas Transplanted into the Rat Kidney
Authors: Hoenig Margarethe , Ferguson Duncan C , Matschinsky Franz M
Published: September 2013

We studied the release of insulin, glucagon, and somatostatin in response to glucose, glyceraldehyde (GA), and α-ketoisocaproate (KIC) from rat kidneys containing transplanted insulinomas. Kidneys were perfused about 11 wk after transplantation when the plasma glucose concentration of the fed animals had decreased from 180 ± 7 to 95.1 ± 9.9 mg/dl and plasma insulin concentrations had increased from 2.6 ± 0.5 to 14.2 ± 2.0 ng/ml. The insulin content of the tumorcontaining kidney ranged from 40 to 679 μg; the glucagon and somatostatin concentrations ranged from undetectable levels to 3.7 μg and 248 ng, respectively. The average response to 30 mM glucose and 10 mM GA was a four- to fivefold increase in insulin secretion, whereas 30 mM KIC caused a 16- to 28-fold increase. In vitro stimulation of the insulinoma with 30 mM glucose primed the β-cell response to a second stimulus following a short rest period. Cytochalasin B did not enhance this primed glucose response. Diazoxide inhibited glucose, GA, and KIC-stimulated insulin release. Glucose, GA, and KIC stimulated glucagon release in 2 of 17 insulinomas studied here.

Somatostatin release was not seen in any of the experiments. These findings show that this islet cell tumor transplanted under the kidney capsule releases insulin in response to physiologic and model fuel substances. Thus, this particular transplantable tumor offers an opportunity to study the biochemistry and biophysics that underlie fuel-stimulated insulin release.
Glycosylated Fetal Hemoglobin: Correlation with Hyperglycemia and Birth Weight in Infants of Diabetic Mothers
Authors: Feldman Felix , Rubin Harry , Nawabi Ismat , Abbondante Ann Marie , Posner Norman A , Stefanyshyn Nicholas I
Published: September 2013

Glycosylated fetal hemoglobin levels were measured in umbilical cord blood of normal infants and infants of diabetic mothers. The glycosylated fraction proved to be a stable compound; its level remained unchanged over a 19-day period. Exposure of fetal and adult hemoglobins to the same concentrations of glucose in vitro resulted in similar levels of glycosylated hemoglobins, suggesting that both types of hemoglobin are about equally reactive with glucose. Levels of glycosylated hemoglobin were significantly increased above normal in umbilical cord blood of infants of both Class A and Class B diabetic mothers. A significant relationship was found between macrosomia, reflected in birth weight ratios, and glycosylated hemoglobin from fetal erythrocytes in infants of diabetic mothers. While these data are consistent with the conclusion that glycosylated fetal hemoglobin levels are a function of fetal blood glucose concentrations in utero during the 2 mo before delivery, it is not known whether the glycosylated hemoglobin contributes to the abnormalities other than macrosomia found in infants of diabetic mothers.
Complement-Fixing Islet Cell Antibodies in the Spontaneously Diabetic BB Rat
Authors: Martin Diego R , Logothetopoulos John
Published: September 2013

Fourteen rats of the spontaneously diabetic BB line were bled from the retroorbital sinus approximately every 10 days. Sera taken from an early age up to 20 days after the onset of overt diabetes were assayed for complement-fixing antibodies against antigens of the surface of islet cells (CFA). Dispersed islet cells from normal Wistar rats prelabeled with 3H-leucine were used as targets. Target cells in suspension were incubated with heat-inactivated rat sera and then, after washing, exposed to guinea pig complement. Cytolytic “injury” was measured by the percentage of labeled cellular proteins released into the medium. Sera from sequential bleedings from eight normal Wistar rats and three rats from a nondiabetic BB subline were assayed to establish basal control cytolytic activity. The mean response ± SD obtained with all control sera was 7.7 ± 1.7%. A response exceeding the mean + 3 SD (12.8%) was considered significantly different from the basal value. Thirteen of the fourteen BB rats developed strongly positive sera. The cytolytic activity preceded the onset of overt diabetes. In several rats CFA appeared 4–8 wk preceding diabetes while in other rats CFA appeared 1–2 wk preceding the manifestation of the disease. These results indicate that CFA may contribute to the destruction of pancreatic islets directly or by attracting mononuclear cells.
Alloxan-induced Free Radical Production in Isolated Cells: Selective Effect on Islet Cells
Authors: Asayama Kohtaro , Nyfeler Fritz , English Denis , Pilkis Simon J , Burr Ian M
Published: September 2013

Chemiluminescence, as a direct measure of oxygen free radical production, induced in isolated cells, hepatocytes, and red cells by the action of alloxan has been measured. The assay system used luminol, 3 μM, for signal amplification. The buffer used was Krebs-Ringer bicarbonate with 16 mM Hepes, pH 7.4. This buffer did not react with alloxan in the absence of cells. Some chemiluminescence was noted from all cells in the absence of alloxan. In the presence of alloxan, reactions occurred within seconds and islet cells were significantly more reactive to alloxan than either red cells or hepatocytes as defined by alloxan dose-response curves with fixed cell numbers or fixed surface areas. These data indicate a cell specificity for an early action of alloxan perhaps mediated at the cell membrane.
Susceptibility to Insulin-dependent Diabetes Defined by Restriction Enzyme Polymorphism of HLA-D Region Genomic DNA
Authors: Owerbach David , Hägglöf Bruno , Lernmark Åke , Holmgren Gösta
Published: September 2013

DNA fragments complementary to cloned sequences encoding HLA-D region class II antigen α- and β-chains were determined by genomic blotting with DNA from HLA-typed members of 22 complete families, 12 of which had a proband with insulin-dependent diabetes mellitus (IDDM). Analysis of genotypes showed that the DNA sequences were linked to HLA-DR and permitted confirmation of recombinations in two families. Digestion with the restriction enzymes BamHI, EcoRI, and Pstl and hybridization with an HLA-D region β-chain cDNA probe confirmed a BamHI 3.7 kilobase (kb) fragment present at low frequency among diabetic individuals and a BamHI 3.2 kb fragment that was also decreased among the diabetic subjects compared with siblings (P < 0.05) as well as nonrelated control siblings (P < 0.02) and their parents (P < 0.01). BamHI 12.0 kb (P < 0.05) and 5.8 kb (P < 0.02, P < 0.02), EcoRI 20 kb (P < 0.05, P < 0.02), and Pstl 6.0 kb (P < 0.05) fragments were more frequent in diabetic individuals compared with nonrelated control siblings or their parents, respectively. Analysis of individual haplotypes revealed that HLA-DR4-containing chromosomes were heterogeneous among controls but that the diabetic individuals showed a similar pattern of restriction fragment length polymorphism. Genomic blotting of blood lymphocyte DNA with a cDNA clone encoding the chain of HLA-D region class II antigens permits detection of fragments that are strongly associated With IDDM.
Syngeneic Transplantation of Cryopreserved Fetal Mouse Proislets
Authors: Hegre Orion D , Simeonovic Charmaine J , Lafferty Kevin J
Published: September 2013

Proislets, derived from fetal mouse pancreata by collagenase digestion and subsequent organ culture, can be frozen to −196°C and stored in a viable condition before successful syngeneic transplantation.

Cryopreserved proislets are relatively undifferentiated morphologically, but continue to differentiate into mature islets after transplantation.
Breathing, Sleep, and Diabetic Autonomic Neuropathy
Authors: Catterall James R , Calverley Peter M A , Ewing David J , Shapiro Colin M , Clarke Basil F , Douglas Neil J
Published: September 2013

Sudden, unexplained deaths are relatively common in diabetic autonomie neuropathy. As disturbed autonomie function has been associated with sleep apnea, and sleep apnea with sudden death, we recorded breathing patterns, arterial oxygen saturation, and EEG during sleep in 8 male diabetic subjects with severe autonomie neuropathy and 8 age-matched, male diabetic subjects without autonomie neuropathy. None of the patients with autonomie neuropathy had more than 11 apneic episodes per night, and there were no significant differences between the two groups in the number of sleep apneas, the duration of individual apneic episodes, the total duration of irregular breathing during sleep, or the duration and quality of sleep. The arterial oxygen saturation when awake and the lowest arterial oxygen saturation during sleep were also similar in both groups.

Thus, diabetic patients with severe autonomie neuropathy have normal breathing patterns and oxygenation during sleep, and it is unlikely that sleep apnea causes these unexpected deaths.
A New Approach to the Detection of Autoantibodies Against Insulin Receptors that Inhibit the Internalization of Insulin into Human Cells
Authors: Yoshimasa Yasunao , Namba Yujiro , Hanaoka Masao , Kohno Michiaki , Okamoto Motozumi , Hattori Masakazu , Yamada Kazunori , Kuzuya Hideshi , Imura Hiroo
Published: September 2013

It has been shown that the conjugate of the fragment A of diphtheria toxin to insulin is cytotoxic to cultured cells bearing insulin receptors, apparently through the endocytosis of fragment A. We examined the effect of autoantibodies against insulin receptors on the cyto-toxicity of the conjugate. The conjugate was cytotoxic to a rat fibroblast cell line that was resistant to the intact toxin, and the cytotoxicity was inhibited by exogenous insulin, indicating that the fragment A underwent endocytosis through insulin receptors. Immunoglobulins from three patients with type B syndrome of insulin-resistant diabetes blocked the cytotoxicity of the conjugate to Chang's liver cells in a dose-dependent manner. When the cells were pretreated with the immunoglobulins, cytotoxicity of the conjugate was also blocked. These results suggest that autoantibodies against insulin receptors interfere with the binding of the conjugate to insulin receptors or with the endocytosis of fragment A after binding. This assay system seems useful for detecting autoantibodies against the determinants that are involved in the internalization of the ligand-receptor complex.
A Method for Isolation of Islets of Langerhans from the Human Pancreas
Authors: Gray D W R , McShane P , Grant A , Morris P J
Published: September 2013

A method has been developed for the isolation of islets of Langerhans from the human pancreas. The average number of islets isolated was 1011 islets per gram of pancreas (SD 475, range 752–2111), and the purity of the preparation as defined by histologie examination and specific staining for insulin varied from 10% to 40%. Islet structure was well preserved and the islets were shown to be viable by supravital staining, demonstration of insulin response to glucose, and by transplantation of isolated islets beneath the renal capsule of nude mice. The essential features of this technique for isolation of human islets include injection of a high concentration of collagenase (6 mg/ml) into the pancreatic duct under pressure, followed by a short incubation (23 min) at 39°C. The gland is then dispersed by a process of teasing and shaking, and the islets are separated by a two-stage process of filtration on a nylon mesh to remove the larger islets and centrifugation on a preformed Ficoll density gradient to separate the small islets.
Muscle Protein Turnover in the Perfused Hindquarters of Lean and Genetically Obese-Diabetic (db/db) Mice
Authors: Shargill Narinder S , Ohshima Kihachi , Bray George A , Chan Timothy M
Published: September 2013

The stability of the perfused mouse hindquarter was assessed for a period of 5 h, and the preparation shown to remain stable for metabolic studies over this time period. Muscle protein synthesis and degradation rates in lean and diabetic-obese (db/db) mice were measured using the in situ perfused hindquarter preparation. The rates of protein synthesis were 48% lower in the muscles of intact db/db mice than in the lean controls when expressed per gram TCA precipitable protein and 46% lower when expressed per gram dry weight. Adrenalectomy, which has been shown to restore the lean body mass of the db/db mice to normal, had the effect of returning protein synthesis rate in muscle of db/db mice to lean control values. Insulin at a dose of 1 mU/ml stimulated protein synthesis in lean mice only, showing that the process of protein synthesis in the db/db mice is also insensitive to insulin. Measurements of the rates of degradation of muscle protein showed no differences between lean and db/db mice. These findings suggest that the decreased lean body mass of db/db mice is the result of a defect in protein synthesis rather than due to altered degradation.
Simple and Reliable Conditions for Routine, Long-Term Culturing of Fetal Human Pancreatic Tissue Fragments
Authors: Farkas Gyula , Joó Ferenc
Published: September 2013

Fetal human pancreatic tissue fragments were isolated and cultured for 18 wk. Insulin production was almost continuous during this period. Multiplication of cells was observed at the second week, and these cells later aggregated as epithelioid cells and formed pseudoislets. The growth characteristics, insulin-like immunoreactivity, and endocrine properties of these cells were evidenced by light microscopy, immunocytochemistry, electron microscopic examination, and measurement of the total insulin content. These results indicate that long-term culture of fetal islets may be useful in clinical work and provides a possible method for increasing islet mass and reducing immunogenicity.
Closed-Loop Glycemic Control with a Wearable Artificial Endocrine Pancreas Variations in Daily Insulin Requirements to Glycemic Response
Authors: Shichiri Motoaki , Kawamori Ryuzo , Hakui Nobuyoshi , Yamasaki Yoshimitsu , Abe Hiroshi
Published: September 2013

We succeeded in miniaturizing a needle-type glucose monitoring system with characteristics suitable for application in a wearable, closed-loop control system. A wearable artificial endocrine pancreas (12 × 15 × 6 cm, 400 g) consisting of a sensor, a microcomputer system that calculates insulin and glucagon infusion rates, and two roller pumps was developed.

Continuous glucose monitoring by a glucose sensor inserted in the subcutaneous tissue of the forearm or abdomen of healthy and diabetic volunteers revealed that glucose concentrations in subcutaneous tissue were 10% lower than, but were highly correlated with, blood glucose concentrations in the range of 49–388 mg/dl.

Glycemic control was established in diabetic patients by intravenously infusing insulin in response to measured glucose concentrations on a moment-to-moment basis for a period of several days. By comparing the glycemic control obtained in each patient treated with multiple insulin injections or open-loop subcutaneous insulin infusion, the superiority of feedback control with the system was clearly demonstrated.

During continuous glycemic regulation, day-to-day variations of insulin requirements were recognized in both basal insulin infusion and postprandial insulin infusion rates in response to identical meals and exercise.

These data suggest the feasibility of long-term glycemic control in diabetic subjects with a wearable artificial endocrine pancreas, and indicate that to overcome changes in individual metabolic characteristics on a moment-to-moment basis, a closed-loop glycemic control system may be essential for ambulatory diabetic patients.
Effects on Glucose-induced Insulin Secretion of Lipoxygenase-derived Metabolites of Arachidonic Acid
Authors: Metz Stewart A , Murphy Robert C , Fujimoto Wilfred
Published: September 2013

Our previous data suggested that lipoxygenation of endogenously released arachidonic acid (AA) is a critical step in stimulus-secretion coupling in the pancreatic beta cell. In the current study using monolayer cultures of neonatal rat islet cells, exogenous arachidonic acid (AA) (5 μg/ml) potently stimulated insulin release in the presence of a substimulatory glucose concentration, and potentiated release induced by glucose. Since the latter stimulatory effect of AA is prevented by inhibitors of the lipoxygenase pathway, we examined the effects of various lipoxygenase pathway products on glucose-induced insulin secretion. The mediator was not one of the stable end-products of either limb of the lipoxygenase pathway: 12- or 5-hy-droxyeicosatetraenoic acid (HETE) (0.5–2000 ng/ml) did not alter insulin release, whereas 11-HETE, 15-HETE, leukotriene (LT)B4 and the δ6 trans isomers of LTB4, LTC4 and 11-trans LTC4 all inhibited insulin release. Furthermore, diethylcarbamazine, a selective leukotriene synthesis inhibitor, did not prevent AA- or glucose-induced insulin release, arguing against a role for LTs as the mediator of AA's stimulatory effect. However, the unstable intermediate 12-hydroperoxyei-cosatetraenoic acid (12-HPETE), and positional isomers of 12-HPETE, potentiated glucose-induced insulin secretion. In addition, insulin release induced by either AA or by glucose was amplified by diethylmaleate, an agent shown in other cell systems to bind reduced glutathione (GSH) and thereby to lead to intracellular accumulation of fatty acid hydroperoxides and epoxides (insulin release at glucose, 300 μmg/dl = 78 ± 2 μU/ml; glucose, 300 mg/dl + diethylmaleate, 1 m M = 131 ± 5, P < 0.001; glucose, 300 mg/dl + AA, 5 (μM/ml = 310 ± 9; P 0.001; glucose + diethylmaleate + AA = 537 ± 3; P < 0.001). In conjunction with our previously reported observation that several analogues of the unstable epoxide LTA4 (epoxyeicosatrienoic acids) increase insulin release, these data are compatible with the hypothesis that reactive, labile intermediates derived from the lipoxygenation of AA (and possibly other polyunsaturated fatty acids) could provide an “on” signal for glucose-induced insulin release. Glucose metabolism, by providing additional GSH, could convert this signal to an “off” response in a negative feedback loop.
Insulin Dose-Response Characteristics Among Individual Muscle and Adipose Tissues Measured in the Rat In Vivo with 3(H)2-Deoxyglucose
Authors: Hom Frederick G , Goodner Charles J
Published: September 2013

The dose-response characteristics of three skeletal muscles, three adipose tissue beds, and heart muscle to single i.v. injection of insulin were compared in vivo. Comparisons were made at 8 dose levels spanning the entire range for response by all tissues and for the integrated whole body response as reflected in the rate of disappearance of 3H-2-deoxyglucose from plasma. The insulin-sensitive tissues varied widely with respect to the magnitude of the maximal response and the sensitivity to insulin as judged by the effective dose 50% (ED 50). Among the muscles, a slow-twitch oxidative muscle, soleus, was more sensitive than the fast-twitch glycolytic muscle, extensor digitorum longus (EDL), while a mixed muscle, quadriceps femoris, displayed even lower sensitivity. Heart muscle sensitivity was comparable to EDL. Among the adipose sites, the rank order of sensitivity was subcutaneous > epididymal ≫ omental. The threshold for a detectable response to insulin was 0.013 U/kg rat.
Chemiluminescence as an Index of Drug-induced Free Radical Production in Pancreatic Islets
Authors: Asayama Kohtaro , English Denis , Slonim Alfred E , Burr Ian M
Published: September 2013

Chemiluminescence induced in isolated islets from rat pancreas by the diabetogenic drugs, alloxan and streptozotocin, has been measured. The assay system consisted of 3 μM of luminol, 10 islets, and 100 μM of alloxan or 500 μM of streptozotocin in 5 ml Krebs-Ringer bicarbonate buffer containing 16 mM of Hepes (pH 7.4). Alloxan-induced chemiluminescence appeared very rapidly and lasted more than 5 min. On the other hand, streptozotocin failed to produce chemiluminescence over a period of 60 min after addition. The presence of Superoxide dismutase (1000 U/ml) and/or catalase (100 U/ml) markedly suppressed alloxan-induced chemiluminescence. These results suggest that alloxan acts as an exogenous free radical generator in pancreatic islets, but that streptozotocin does not. The involvement of Superoxide anion and hydrogen peroxide in production of chemiluminescence by alloxan suggests that the hydroxyl radical may mediate this chemiluminescence.