bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Activity of Biosynthetic Human Proinsulin: Receptor Binding and Biologic Potency of Proinsulin and Insulin in Isolated Rat Adipocytes
Authors: Peavy Daniel E , Abram Jill D , Frank Bruce H , Duckworth William C
Published: September 2013

The receptor binding characteristics and biologic potency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with 125I(A14)-iodoinsulin for binding to adipocyte receptors at 15°C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 ± 0.8 × 10−7 M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 ± 0.3 × 10−9 M for insulin. These results were confirmed in direct competition studies using proinsulin and 125I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37°C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 ± 0.4 × 10−8 M, whereas the same response was observed at an insulin concentration of 5.2 ± 1 × 10−11 M. Proinsulin also demonstrated an antilipolytic potency that was <1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones. It is concluded that biosynthetic human proinsulin has approximately 1% the receptor binding affinity and biologic potency of insulin in the isolated rat adipocyte system.
Impaired Auditory Brainstem-evoked Responses in Insulin-dependent Diabetic Subjects
Authors: Fedele Domenico , Martini Alessandro , Cardone Claudio , Comacchio Francesco , Bellavere Federico , Molinari Giorgio , Negrin Paolo , Crepaldi Gaetano
Published: September 2013

Auditory brainstem-evoked responses (ABR) were recorded from the scalp of 30 normoacoustic insulin-dependent diabetic subjects, aged between 15 and 41 yr (29 ± 7 yr). Three different stimulus repetition rates (11, 37, and 87 cps) were used. The results were compared with those obtained from 20 age- and sex-matched, normoacoustic control subjects. In diabetic patients, metabolic control (mean daily plasma glucose, glycosylated hemoglobin) and the presence of retinopathy, nephropathy, and somatic neuropathy were also investigated. The latencies (ms) of ABR waves were significantly impaired in diabetic subjects as compared with normals. Peripheral transmission time (wave I) and central transmission time (waves I–V) were also significantly delayed in diabetic subjects. Moreover, by increasing stimulus repetition rates, a significant increase in waves I–V shift was observed in diabetic patients. ABR impairment was not related to glucose balance, to the duration of diabetes, or to the presence of the diabetic retinopathy, nephropathy, and somatic neuropathy.

In conclusion, diabetic neuropathy is characterized not only by somatic and autonomie nerve dysfunctions, but also by the early involvement of the central nervous system (CNS). ABR recording can represent a useful, noninvasive, simple procedure to detect both acoustic nerve and CNS damage.
Insulin and Multiplication-stimulating Activity Induce a Very Rapid Centrosomal Orientation Response to Wounding in Endothelial Cell Monolayers
Authors: Mascardo Renato N , Sherline Peter
Published: September 2013

Using serum-deprived monolayers of bovine aortic endothelial cells that were disrupted experimentally with a linear wound, we observed, by immunofluorescence microscopy, centrosomal perinuclear movement that is associated closely with cell orientation and locomotion. In the presence of the growth factors fetal bovine serum, insulin, or multiplication-stimulating activity (MSA), the centrosome rapidly (within 10 s) translocated and positioned itself between the nucleus and wound track in ≥70% of cells bordering the wound. Random centrosomal orientation was observed in control border cells (50%). This growth factor-stimulated centrosomal orientation response to wounding depended on growth factor concentration, high-energy phosphates, functional microtubules and microfilaments, and calcium-calmodulin interaction. In the absence of fetal bovine serum, insulin, or MSA, the border cells of the wounded endothelial cell monolayer exhibited a significant centrosomal orientation response 5–6 h after wounding.
Terbium, a Fluorescent Probe for Insulin Receptor Binding: Evidence for a Conformational Change in the Receptor Protein Due to Insulin Binding
Authors: Williams Paul F , Turtle John R
Published: September 2013

Terbium (Tb3+), an ion of the lanthanide series that has been used as a fluorescent probe for calcium (Ca2+) binding sites in proteins, binds to the proteins in both solubilized and purified human placental insulin receptor preparations. Tb3+ fluorescence was determined directly and the effect of insulin binding on Tb3+-enhanced fluorescence was studied without the need to separate bound and free ligands. Tb3+ behaved similarly to, but was more potent (50-fold) than, Ca2+ in increasing the insulin bound to its receptor.

When insulin bound to its receptor, the Tb3+ fluorescence of the receptor preparation decreased. When various insulin analogues were tested, the decrease in Tb3+ fluorescence was proportional to the biologic activity of the insulin analogues. In addition, Tb3+ could be displaced from insulin-sensitive sites by Ca2+, indicating that there were Ca2+ (and Tb3+) binding sites on or near the insulin receptor. These sites, when filled, were responsible for the increased insulin receptor affinity. The decrease in Tb3+ fluorescence after insulin binding may be indicative of a conformational change in the insulin receptor precipitated by the binding of insulin. This conformational change may be related to the release of Ca2+ by insulin binding, is associated with a decrease in insulin receptor affinity, and suggests that an allosteric mechanism involving both Ca2+ and insulin binding sites may be responsible for the observed changes in insulin receptor affinity.
In Vivo Chloroquine-induced Inhibition of Insulin Degradation in a Diabetic Patient with Severe Insulin Resistance
Authors: Blazar Bruce R , Whitley Chester B , Kitabchi Abbas E , Tsai Michael Y , Santiago Julio , White Neil , Stentz Franke B , Brown David M
Published: September 2013

We report on a 26-yr-old patient with an 11-yr history of insulin-dependent diabetes mellitus who exhibited insulin resistance with a requirement of up to 15,000 U of intravenous (i.v.) insulin/day. Attempts to diminish her insulin requirement by administration of sulfated insulin or Trasylol were unsuccessful, with the patient remaining resistant to subcutaneous (s.c.) and i.v. administration of pure pork insulin. Chloroquine phosphate therapy (500 mg twice a day) resulted in a decreased requirement for i.v. insulin (700 U/day as compared with the pretreatment requirement of 8400 U/ day). Accelerated insulin degradation in s.c. fat tissue of the patient before treatment with chloroquine was demonstrated. This activity was decreased by 64% during chloroquine therapy. Inhibition of insulin degrading activity (IDA) during chloroquine therapy was associated with reductions in the leukocyte lysosomal enzymes α-galactosidase and hexosaminidase-A but not hexosaminidase-B and β-glucuronidase. This study constitutes the first reported use of chloroquine for treatment of insulin resistance as a result of accelerated insulin degradation, and it provides evidence of the effectiveness of this agent in this rare condition.
Effect of Tolbutamide on Myocardial Metabolism and Mechanical Performance of the Diabetic Rat
Authors: Tan Boen Hie , Wilson Glenn L , Schaffer Stephen W
Published: September 2013

Exposure of the isolated, glucose-perfused rat heart to buffer containing 0.4 mM tolbutamide resulted in significant changes in both energy metabolism and myocardial contractility. In the nondiabetic, tolbutamide mediated only small increases in mechanical function at low atrial filling pressure, but this effect increased with increasing preload. By contrast, the stimulation of mechanical function resulting from exposure of the diabetic heart to tolbutamide was independent of preload. As a result, the tolbutamide-mediated, positive inotropic effect in the diabetic heart was greater at lower, but not higher, preload values than the effect in the nondiabetic. Moreover, the changes in energy metabolism initiated by tolbutamide were considerably larger in the diabetic. The most prominent effect was the mobilization of glycogen by tolbutamide in the diabetic, which was considerably greater than that observed in the nondiabetic. The drug also enhanced glucose utilization. The net effect of sulfonylurea exposure was to shift from preferential use of fatty acids as an energy source for contraction to use of glucose. Since the most prominent effect of the drug in the diabetic was the stimulation of glycogenolysis, it is concluded that tolbutamide can dramatically alter the metabolism of a tissue without acting through insulin.
Diabetes Induction by Subdiabetogenic Doses of Streptozotocin in BALB/cBOM Mice: Noninvolvement of Host B-Lymphocyte Functions
Authors: Blue Marie-Luise , Shin Seung-Il
Published: September 2013

Multiple subdiabetogenic doses of streptozotocin induce an insulin-dependent progressive hyperglycemia in genetically susceptible strains of mice. We have shown previously that T-cell-dependent autoimmune mechanisms play an obligatory role in this model of diabetogenesis by demonstrating that athymic nude mice and lethally irradiated euthymic mice are selectively resistant to diabetes induction and that the susceptibility can be reconstituted by grafting thymus in nude mice or by giving B-cell-depleted splenic lymphocytes to the irradiated mice.

In this report we investigate more directly the possible role of host B-cell functions in the induction of hyperglycemia. Mice were rendered selectively deficient in functional B lymphocytes by repeated injections of a polyclonal antiserum against mouse IgM, starting immediately after birth. These B-cell-suppressed mice had no detectable ability to produce antibodies against a test antigen but appeared to have normal levels of T cells. When treated with multiple low doses of streptozotocin, they developed progressive hyperglycemia in a manner indistinguishable from control mice with normal B-cell functions. These results suggest that host B cells, in contrast to host T cells, are not etiologically involved in the development of diabetes induced by multiple subdiabetogenic doses of streptozotocin.
Von Willebrand Factor (VIII R: Ag), Fibronectin, and Insulin-like Growth Factors I and II in Diabetic Retinopathy and Nephropathy
Authors: Lamberton R Preston , Goodman A David , Kassoff Aaron , Rubin Cherilyn L , Treble Donald H , Saba Thomas M , Merimee Thomas J , Dodds W Jean
Published: September 2013

We have measured plasma von Willebrand factor (VWF) as the factor Vlll-related antigen, plasma fibronectin, and two of the serum somatomedins, insulinlike growth factor I (IGF I) and IGF II, in 51 diabetic patients and 25 nondiabetic control subjects. VWF was significantly higher in the diabetic group than in the controls (173 ± 9% SEM versus 101 ± 9%, P < 0.001), as has been reported by others. However, within the diabetic group there was no significant difference in VWF between those patients without retinopathy, those with background or proliferative retinopathy, or those with macular edema. There was also no difference in VWF between the diabetic subjects with and those without proteinuria. These results rule against a previously advanced hypothesis that the increase in VWF in patients with diabetes is secondary to microangiopathy. No significant difference was observed in fibronectin, IGF I, or IGF II between the diabetic and control groups, between the diabetic group without retinopathy and the retinopathic subgroups, and between the diabetic subjects with and without proteinuria. In the diabetic patients, there was no correlation between diabetic control as assessed by glycosylated hemoglobin and glycosylated serum protein, and the plasma levels of VWF, fibronectin, IGF I, or IGF II. The results of this study strongly suggest that neither plasma VWF, fibronectin, IGF I, nor IGF II plays an important primary role in the pathogenesis of diabetic microvascular disease, although one or more of these factors might play a permissive role.
Anti-Beta-Cell Immunity in Insulinopenic Diabetic Dogs
Authors: Sai Pierre , Debray-Sachs Monique , Jondet Andreé , Gepts Willy , Assan Roger
Published: September 2013

Anti-islet immunity was studied in six spontaneously insulin-dependent diabetic (IDD) dogs, using mouse islets of Langerhans cells as targets, in vitro. Insulinopenia was demonstrated in all dogs by an i.v. glucose tolerance test. A significant lymphocytopenia was detected in the peripheral blood of this diabetic group. Pancreatic tissue from one of these animals was obtained shortly after death and the islets displayed a marked loss in beta cells without significant changes in the other types of islet cells. No insulitis was observed.

Circulating mononuclear cells from the diabetic dogs induced an increased basal insulin (IRI) release from islet cells and a suppressed stimulated IRI release. Damage to or depth of beta cells may account for these findings.

The stimulated IRI release was also suppressed when islets were incubated with the diabetic sera + complement, while the D-cell response to arginine was not altered, and the A-cell response was reduced but not abolished. A lysis of islet cells in the presence of IDD sera + complement was demonstrated by an increased release of 51Cr from labeled cells. These anomalies were observed neither when complement was heat-inactivated nor in the presence of control sera + complement.

Canine IDD may be a new animal model for the study of anti-islet cellular and humoral immunities.
Abnormal Islet and Adipocyte Function in Young B-cell-deficient Rats with Near-Normoglycemia
Authors: Trent D F , Fletcher D J , May J M , Bonner-Weir S , Weir G C
Published: September 2013

Sprague-Dawley male rats were injected at 2 days of age with streptozotocin (SZ). At 4 wk of age the fed plasma glucose concentration of the SZ group was 151 ± 6 mg/dl as compared with 133 ± 4 for the control group. The fed plasma insulin values were indistinguishable, however. In response to an intraperitoneal glucose challenge the SZ group had marked glucose intolerance and virtually no rise in plasma insulin. After a meal challenge the SZ group also had glucose intolerance, but plasma insulin responses were similar to those of the control. The pancreata of the 4-wk-old rats were perfused in vitro and the SZ group had essentially no response to glucose, but did respond to arginine. Adipocytes of the 4-wk-old SZ rats had impaired glucose conversion to CO2 similar to that seen in the more hyperglycemie 6-wk-old SZ rats. Castration carried out at about 3 wk of age did not influence the hyperglycemia seen at 6 wk of age and later. These data indicate that 4-wk-old SZ rats, while having near-normal plasma glucose levels and normal plasma insulin values, have clearly abnormal B-cell and adipocyte function. With increasing age and weight gain these SZ rats develop frank hyperglycemia.
Effect of EDTA on Insulin Binding and Insulin Action in Isolated Cardiocytes from Adult Rat: Evidence for a Functional Role of Low-Affinity Insulin Receptors
Authors: Eckel Jürgen , Reinauer Hans
Published: September 2013

Calcium-tolerant myocytes from the adult rat heart were used to study the effects of EDTA on insulin binding and insulin action. Treatment of cardiocytes with EDTA resulted in a 60% inhibition of insulin binding. This effect was partially reversible by subsequent addition of calcium or magnesium. Scatchard analysis of equilibrium binding data in the presence of calcium and magnesium showed a curvilinear plot with a high-affinity segment having a Kd of 5.7 × 10−10 mol/L. In the presence of EDTA a linear Scatchard plot was observed with a Kd of 5.6 × 10 −9 mol/L. The total number of insulin receptors remained unaltered under these conditions. In contrast to insulin binding, insulin internalizaron was not affected by EDTA treatment.

Insulin action was studied by measuring the effect of the hormone on the transport of 3-O-methylglucose. Half-maximal action occurred at an insulin concentration of 3 × 10−10 mol/L and 10−8 mol/L in control and EDTA-treated cells, respectively. Maximal transport stimulation, however, was not significantly different in both groups (130% and 106%, respectively).

In conclusion, low-affinity insulin receptors in cardiac myocytes mediate a biologic response comparable to that of high-affinity sites; moreover, they may be involved in the process of internalization in this tissue. The data suggest a functional role of low-affinity insulin receptors in cardiac muscle.
Islet Cell Cytoplasmic Antibodies in Macaca nigra
Authors: Howard Charles F , Fang Ta-Yun
Published: September 2013

Islet cell antibodies (ICA) have been measured in mature Macaca nigra. Of 30 nondiabetic monkeys, 26 (87%) were ICA-negative; of 43 monkeys with evidence of mild to severe hormonal or glycemie abnormalities, 39 (91%) were ICA-positive. Pancreatic islets were examined from biopsy and autopsy sections to assess cell deterioration and amyloid infiltration. No ICA were found in 13 of 18 (72%) monkeys with no evidence of amyloid, whereas 30 of 35 (86%) monkeys with islet amyloid and concurrent cell deterioration were ICA-positive. Association of ICA with metabolic and islet abnormalities was significant at P ≤ 0.001. ICA were specific for the islet cells in pancreatic sections; plasma preincubated with insulin, glucagon, or acetone extracts of tissues retained their ICA-positive reaction. The relationships of ICA in older monkeys to the islet lesion and to metabolic abnormalities could be relevant to similar situations in aging diabetic persons.
A Prospective Analysis of Islet-cell Cytotoxic Antibodies in Insulin-dependent Diabetic Children: Transient Effects of Plasmapheresis
Authors: Rabinovitch Alexander , MacKay Peter , Ludvigsson Johnny , Lernmark Åke
Published: September 2013

We determined the effects of plasmapheresis on cytotoxic antibodies to islet cells in 10 children (aged 11–16 yr) with newly diagnosed insulin-dependent diabetes mellitus (IDDM), as well as the plasma levels of antibodies over the next 30 mo and their relation to serum C-peptide concentrations. Complement-dependent, antibody-mediated cytotoxicity (C′AMC) in plasma was measured in a 51Cr release assay using monolayers of the rat islet cell line RINm5F. Cytotoxic antibodies decreased in most IDDM subjects treated by plasmapheresis four times within 2 wk of diagnosis; however, the decreases were small and lasted less than 2–3 days. Thus, both before and after plasmapheresis, 7 of the 10 IDDM children were C′AMC-positive (51Cr release > 2 SD above mean for 13 healthy children). After 18–30 mo, only 2 of the original 7 IDDM children with C′AMC-positive plasmas were still positive, and 1 of the original 3 IDDM children without significant cytotoxicity had become positive. Meal-stimulated serum C-peptide responses, measured from diagnosis to 18–30 mo later, did not correlate with C′AMC values. We conclude that (1) plasmapheresis has only transient effects on islet-cytotoxic antibody levels in children with IDDM; (2) these antibodies decrease in most, but not all, subjects over the first 18 mo after diagnosis; and (3) the level of cytotoxic antibodies in IDDM plasma at diagnosis has no predictive effect on residual B-cell function.
Interrelation of Platelet Vitamin E and Thromboxane Synthesis in Type I Diabetes Mellitus
Authors: Karpen C W , Cataland S , O'Dorisio T M , Panganamala R V
Published: September 2013

Platelet vitamin E content and thromboxane A2 (TxA2) synthesis have been investigated in type I diabetic subjects and age- and sex-matched controls. Platelets, but not plasma, from diabetic subjects contained significantly lower vitamin E levels and synthesized significantly greater amounts of TxA2 when challenged with collagen or thrombin than platelets from control subjects. Conversion of exogenously added arachidonic acid to TxA2 was unaltered between platelets from control and diabetic groups. Platelet vitamin E content from control and diabetic groups combined exhibited a significant negative linear correlation with collagen- and thrombin-induced TxA2 production. These data suggest that low platelet vitamin E levels could be a contributing factor to the increased thromboxane synthesis demonstrated by platelets from the above type I diabetic subjects.
Membrane Structural/Functional Properties of Adipocytes from Normal and Streptozotocin-diabetic Rats
Authors: Sauerheber Richard D , Kuhn Chris E , Hyslop Paul A
Published: September 2013

The uptake of 2-deoxy-D-[-14C]-glucose, in the presence and absence of insulin, was measured in adipocytes from normal and streptozotocin-diabetic animals, over a wide temperature range (25–45°C). Optimum temperatures for uptake in the presence of maximally stimulating insulin concentrations occurred near physiologic temperatures. Both high and low temperatures lead to progressive inhibition of insulin-sensitive uptake, whereas basal uptake was only marginally affected. It is possible that the apparent “resistance” to insulin action at high (febrile) temperatures may have pathologic significance.

The magnitude of 2-deoxy-D-glucose uptake at all temperatures in the diabetic adipocytes was much reduced, both in the presence and absence of insulin, on a per cell basis, compared with cells from age-matched control animals. However, the fold stimulation of uptake caused by insulin at 35°C is comparable in both normal and diabetic adipocytes (approximately 2–3-fold).

Photomicrographs of adipocytes were used to estimate the cell diameters of the average normal (50 μm) and diabetic (37 μm) cells. The diabetic adipocytes are not “resistant” to insulin action in terms of 2-deoxy-D-glucose uptake since the basal and insulin-stimulated uptake magnitudes per μm2 membrane surface area are identical in both normal and diabetic cells (within experimental error). It is possible that the decreased diabetic cell size reflects the reduced antilipolytic effects of chronic hypoinsulinemia rather than any inherent resistance to insulin action in the cell itself.

The basal and insulin-stimulated overall removal of glucose from the incubation medium by adipocytes, isolated from the normal and streptozotocin-treated rats, was also measured. Both the overall glucose uptake magnitude per μm2 membrane surface area and the fold increase in glucose uptake due to insulin were identical between cells obtained from the two groups of animals. Any intracellular metabolic resistance to the effects of exogenous insulin that may be present in cells from the diabetic rats does not appear to grossly interfere with the accelerated net utilization of glucose induced by exposure to the hormone in vitro.

Purified plasma membranes from normal and diabetic adipocytes were examined in spin label studies. No differences in the flexibility of either the 5-nitroxide stearate or the cholestane (cholesterol-like) spin labels were noted between the two animal groups at any temperature between 15 and 40°C Although the diabetic membrane may well exhibit differing structural/functional properties compared with controls, our data do not support the concept that gross alterations in the lipid structure of the membrane occur in this hyperglycemie condition.
Modulation by a Sulfonylurea of Insulin-dependent Glycogenesis, but Not of Insulin Binding, in Cultured Rat Hepatocytes: Evidence for a Postreceptor Mechanism of Action
Authors: Fleig Wolfgang E , Noether-Fleig Gaby , Fussgaenger Rolf , Ditschuneit Hans
Published: September 2013

To detect potential direct effects of the sulfonylurea glyburide on hepatic carbohydrate metabolism, we tested whether the drug was capable of modulating insulin binding and glycogenesis in primary cultured hepatocytes. After 24-h culture under serum- and hormone-free conditions, cells were incubated with or without 10−8 M insulin and/or glyburide (0.1–5.0 μg/ ml) for another 24 h. Then, specific 125I-insulin binding and basal and insulin-stimulated glycogen synthesis were determined. Acute addition of glyburide to previously untreated cells did not modulate any of these parameters. Incubation for 24 h with 2 μg/ml of glyburide did not affect the DNA and protein content of the dishes. Cellular glycogen content and basal glycogenesis also remained unchanged by glyburide in hepatocytes incubated in the absence of insulin, but glycogen content was increased and basal glycogen synthesis decreased in insulin-pretreated cells. In contrast, glyburide increased insulin-stimulated glycogenesis in a dose-dependent fashion in both insulin-pretreated and control cells by enhancing responsiveness, but not sensitivity, toward insulin. Pretreating hepatocytes with 10−8 M insulin caused a 40% reduction in specific insulin binding. Glyburide did not modulate insulin binding or degradation in control cells nor was insulin-induced regulation of insulin receptors affected. These results demonstrate a direct dose-dependent effect of a sulfonylurea on an insulin action toward hepatic carbohydrate metabolism, and suggest that this effect is mediated by a postreceptor mechanism.
Sequence of a cDNA Encoding Syrian Hamster Preproinsulin
Authors: Bell Graeme I , Sanchez-Pescador Ray
Published: September 2013

A complementary DNA (cDNA) library was prepared from messenger RNA (mRNA) isolated from Syrian hamster islets. Bacterial colonies containing hamster preproinsulin cDNA were identified by cross-hybridization with the human preproinsulin gene. The sequences of two of these established the complete sequence of Syrian hamster preproinsulin mRNA and predicted the sequence of the protein. Hamster preproinsulin is 110 amino acids and possesses 90.0% and 82.7% identity with the corresponding proteins of rats (either I or II) and human beings, respectively. Analysis of the hybridization of hamster preproinsulin cDNA to restriction endonuclease digests of hamster DNA suggests that there is only a single preproinsulin gene in this rodent, in contrast to rats and mice, which possess two nonallelic genes.
Calmodulin and Cyclic AMP: Possible Different Sites of Action of These Two Regulatory Agents in Exocytotic Hormone Release
Authors: Steinberg Jay P , Leitner J Wayne , Draznin Boris , Sussman Karl E
Published: September 2013

It may now be possible to identify certain intracellular events that impact specifically on secretion-granule fusion to the plasma membrane or on granule lysis. Secretion vesicles in isolated rat islets appear to translocate somatostatin (SRIF) receptors from the Golgi apparatus to the plasma membrane. We have proposed that secretion granule fusion to the plasma membrane can be determined by measuring recruitment of SRIF receptors to the surface membrane. Granule lysis can be assessed by measuring insulin release. To activate cyclic AMP (cAMP)-dependent pathways, we employed isobutylmethylxanthine (IBMX, 400 μM), glucagon (10 μM), and forskolin (20 μM), a diterpene activator of adenylate cyclase. These agents evoked rapid release of insulin (from 0.41 ± 0.02 to 1.88 ± 0.02; 0.41 ± 0.02 to 1.93 ± 0.08; and 0.41 ± 0.02 to 1.66 ± 0.03 μU/islet/ min, respectively, P < 0.001). There was no concomitant recruitment of SRIF receptors. Somatostatin (10 μg/ml), which inhibits cAMP-stimulated protein phosphorylation, suppresses insulin release evoked by IBMX, glucagon, or forskolin (inhibition: 80, 75, or 82%, respectively). In contrast, trifluoperazine (10 μM), an inhibitor of calmodulin, did not suppress insulin release induced through cAMP-dependent pathways. Trifluoperazine suppresses glucose-induced insulin release and the recruitment of SRIF receptors to the surface membrane, suggesting the possible role of calmodulin in promoting secretion-granule fusion with the plasma membrane.

These data suggest that granule fusion to the plasma membrane may be a calmodulin-directed function. In contrast, the major site of action of CAMP in polypeptide hormone secretion may be in promoting lysis of secretion granules and associated hormone discharge.
Effect of Ischemia and Temperature on Fetal Mouse Pancreas: Insulin Production In Vitro, and Function After Isotransplantation
Authors: Mandel T E , Koulmanda Maria
Published: September 2013

The effects of ischemia at varying temperatures on the survival of fetal islet endocrine cells was investigated by placing 17-day-old fetal mouse pancreata in organ culture after 2, 4, or 6 h at either 4°C, 22°C, or 37°C. Insulin secretion by the cells in vitro, the content of insulin in the cultured pancreata, and the ability of the cultured islets to reverse diabetes in syngeneic streptozotocin-diabetic mie were assessed. Fetal pancreas subjected to 2–6 h of ischemia at either 4°C or 22°C showed neither loss of insulin secretory capacity in vitro nor loss of ability to produce large functional grafts, and behaved identically to tissue not subjected to deliberate ischemia. In contrast, after 2 h of ischemia at 37°C, although some grafts functioned, their insulin content was reduced despite apparently normal prior insulin production in vitro, but 4 or 6 h at 37°C resulted in total loss of functional islet tissue. However, despite retention of functional capacity and the ability to produce large grafts with high insulin content after cold or room temperature ischemia, some loss of insulin storage capacity in vitro was noted by islets subjected to ischemie periods longer than 2 h even at 4°C. Thus, fetal pancreas can withstand prolonged periods of ischemia provided its temperature is reduced, and functional recovery can be demonstrated after transplantation.
Reduced Diabetogenic Effect of Streptozotocin in Rats Previously Adapted to a High-Protein, Carbohydrate-free Diet
Authors: Eizirik Décio L , Migliorini Renato H
Published: September 2013

The diabetogenic effects of streptozotocin (STZ) (40 or 60 mg /kg body wt, i.v.) were examined in rats that had been previously fed either a high-protein, carbohydrate-free (HP) diet (70% protein, 8% fat) or a control diet (66% carbohydrate, 16% protein, 8% fat), and maintained on the same diets after drug injection. Rats on the HP diet 15–21 days before STZ administration showed reduced mortality and decreased severity of diabetes as judged by rate of body-weight change, plasma glucose, urine volume and glycosuria, serum and pancreatic insulin, and morphology of islets of Langerhans. In rats on the control diet, values for serum and pancreatic insulin did not differ 2 and 14 days after 40 mg/kg of STZ. In contrast, in rats under the HP regimen, serum insulin levels doubled 14 days after STZ and pancreatic insulin content was almost 20 times higher than at day 2. Rats previously fed on the HP diet for 21 days, but transferred to the control diet 2 days after STZ injection, also showed reduced severity of diabetes, as indicated by rates of body-weight gain, urinary excretion of urea, and levels of serum and pancreatic insulin. However, in these animals the effects of the HP diet were not as marked as those observed in the rats that were maintained on the HP diet after STZ injection. These findings indicate that the rapid amelioration of diabetes in rats adapted to the HP diet results from the combination of two beneficial effects of the diet: (1) an initial partial protection against the diabetogenic activity of the drug at the time of exposure, and (2) a subsequent improvement of the induced diabetic state. The data suggest that adaptation of rats to a high-protein diet entails a decreased susceptibility of the beta cells to the cytotoxic action of STZ and an enhancement of the restorative capacity Of these cells.
Extended Allograft Survival of Islets Grafted into Intra-abdominally Placed Testis
Authors: Selawry Helena P , Whittington Karen
Published: September 2013

Isolated islets of ACI donor rats were cultured for 4 days at 37°C and then grafted into male diabetic Wistar-Lewis rats into three different organ sites without adjuvant immunosuppression. None of 6 recipients of the intraportal injection of 10 islets per gram of body weight became normoglycemic. Similarly, six rats that received islets injected under the renal capsule remained diabetic. None of six rats that received an intratesticular islet allograft became normoglycemic while these organs remained within the scrotum. By contrast, six rats that were transplanted with an identical number of islets into the testis, which were then surgically placed into the abdominal cavity, promptly became aglycosuric and have remained so for more than 50 days.
Increased Insulin Responsiveness In Vivo and In Vitro Consequent to Induced Hyperinsulinemia in the Rat
Authors: Trimble E R , Weir G C , Gjinovci A , Assimacopoulos-Jeannet F , Benzi R , Renold A E
Published: September 2013

The effect of constant, controlled hyperinsulinemia on in vivo and in vitro insulin responsiveness has been investigated in rats that have received insulin infusions through chronically implanted jugular vein catheters. Constant rates of insulin infusion during days 1–4 resulted in stable plasma insulin concentrations. The plasma glucoseinitially fell from 122 ± 3 to 53 ± 4 mg/dl. While the infusion rate was maintained constant,the plasma glucose continued to fall over the subsequent days so that on day 4 the plasma glucose, 40 ± 2 mg/dl, was significantly lower than that in the same animals on day 1 (P < 0.02). Subsequently, the rate of insulin infusion was decreased to maintain the plasma glucose level in the 35—40 mg/dl range. Plasma catecholamine levels were high in insulin-infused rats. On the eighth day an in vivo insulin tolerance test (0.5 U/kg) was performed. Insulin-infused rats responded with a hypoglycemia that was both more pronounced and longer sustained than in salineinfused controls.

Insulin responsiveness in vitro has been measured in isolated adipocytes. Adipocytes from epididymal fat pads were of similar size in the two groups of animals. Glucose uptake by adipocytes from insulin-infused rats was similar to that of controls under basal (zero insulin) conditions, but showed an increase in the maximum response to insulin. Glucose incorporation into total lipid and fatty acid was greater in adipocytes from insulin-infused rats than in controls under both basal (zero insulin) and insulin-stimulated conditions. Activities of the lipogenic enzymes acetyl CoA carboxylase and fatty acid synthetase were markedly increased in epididymal fat pads of insulin-infused rats.

Thus, insulin infusion for a period of 8 days did notresult in reduced insulin action in vivo; on the contrary, an apparent increase in the hypoglycemic effect of insulin was observed. In vitro, the maximum effect of insulin on glucose uptake and lipogenesis in the isolated adipocyte was markedly increased after in vivo insulin infusion.
Tolbutamide Stimulates Fructose-2, 6-Bisphosphate Formation in Perfused Rat Liver
Authors: Matsutani Akira , Kaku Kohei , Kaneko Toshio
Published: September 2013

Effect of tolbutamide on liver fructose-2,6-bisphosphate (F-2,6-P2) was examined in isolated perfused rat liver in situ with a flow-through method. Tolbutamide (1 mM) gradually increased liver F-2,6-P2 level from 7.4 ±1.6 to 21.2±1.6 pmol/mg wet wt for 20 min perfusion. The increase of liver F-2,6-P2 induced by tolbutamide was dose dependent and was significantly observed at 10 min perfusion. The maximum plateau level of F-2,6-P2 induced by 16.7 mM glucose was further increased with 1 mM tolbutamide. Glucagon (10−11 M) decreased the elevated level induced by 16.7 mM glucose,but this effect was completely inhibited with 2 mM tolbutamide. Cyclic AMP level of the liver throughout the perfusion with tolbutamide did not change. Carboxytolbutamide or gliclazide perfusion did not change significantly the liver F-2,6-P2 level; however, the results suggest that tolbutamide may increase the liver F-2,6-P2 level by affecting the phosphorylation state of fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase through cyclic AMP—dependent protein kinase, resulting in the stimulation of glycolysis and the inhibition of gluconeogenesis in the liver. Thus, the extrapancreatic action and the mechanism of action of different sulfonylureas may differ.
Thyroid Function and Prolactin Levels in Insulin-dependent Diabetic Children and Adolescents
Authors: Salardi Silvana , Fava Alessandra , Cassio Alessandra , Cicognani Alessandro , Tassoni Paola , Pirazzoli Piero , Frejaville Elisabetta , Balsamo Antonio , Cozzuti Enrico , Cacciari Emanuele
Published: September 2013

We evaluated serum thyroid hormones, TSH, and prolactin before and after induction of TRH and thyroid microsomal autoantibodies in 91 diabetic children and adolescents (mean age 11.11 ±4.13 yr), with illness ranging from a few days to 14.25 yr, and in 127 “short-normal” subjects (mean age 10.32 ± 3.18 yr). All were clinically euthyroid. The control pubertal subjects showed T4, rT3, TBG, and rT3/T3 ratio values that were significantly lower than those of prepubertal subjects. The PRL area was significantly higher in pubertal than in prepubertal females. In diabetic patients, differences between pubertal and prepubertal subjects were similar to those of controls regarding T4 levels and PRL area only. T3, T4, and fT3 appeared to be significantly lower than in controls, while the rT3/T3 ratio was higher. A negative correlation (r = −0.277, P = 0.009) between T3 and HbA1 levels was demonstrated. Furthermore, thyroid function was not different in subjects with or without retinal changes or in subjects with or without residual B-cell function. Microsomal autoantibodies were observed in 6.25% of the subjects examined, though none showed any clinical or humoral sign of impaired thyroid function. In conclusion, the lower T4 and rT3 values detected in pubertal controls suggest an increased efficacy of peripheral thyroid activity in this particular life span. Considering the fact that, in diabetic children, such a decrease in rT3 at puberty is not present and that the T3 value in diabetic children is persistently lower than in controls, it would seem that even diabetic children show a “low T3 syndrome”, as in adult diabetic subjects.
Decreased Synthesis of Interleukin-2 (IL-2) in Insulin-dependent Diabetes Mellitus
Authors: Zier Karen S , Leo Martha M , Spielman Richard S , Baker Lester
Published: September 2013

Synthesis of interleukin-2 (IL-2) by lymphocytes from 26 insulin-dependent diabetic subjects (IDDM) was compared with that by lymphocytes from 24 nondiabetic control subjects. The control group produced 1.001 ± 0.071 U/ml (mean ± SEM). The IDDM group, containing patients diagnosed between 5 days and 10 yr before testing, produced only 0.59 ± 0.050 U/ml (P < 0.002). IL-2 synthesis by 6 non-insulin-dependent diabetic subjects (NIDDM) was not decreased (mean ± SEM, 1.20 ± 0.04 U/ml). Moreover, decreased levels of IL-2 production was found with lymphocytes of patients in good control, as well as those in poor control. These data suggest that decreased IL-2 synthesis is specific for IDDM, not explainable solely as a consequence of poor metabolic control, and thus, might be involved in the pathogenesis of the disease.
Protein Phosphatase-1 and -2A Activities in Heart, Liver, and Skeletal Muscle Extracts from Control and Diabetic Rats
Authors: Foulkes J Gordon , Jefferson Leonard S
Published: September 2013

Protein phosphatase activities have recently been classified into two major groups of enzymes termed type 1 and type 2. In the present study, this classification scheme has been used to explore the types of protein phosphatase activities found in rat heart, liver, and skeletal muscle, and to determine the type of protein phosphatase activity affected by diabetes. Protein phosphatase activities have been measured under conditions designed to minimize the influence of effectors of these enzymes, and a thermostable protein phosphatase inhibitor, termed inhibitor-2, has been used as a probe to specifically inhibit type 1 protein phosphatase. The specific activity of protein phosphatase-1 in extracts of heart, liver, and skeletal muscle from control rats ranged between 0.34 and 0.44 U/mg protein. The specific activity of a type 2 enzyme, termed protein phosphatase-2A, was approximately the same as protein phosphatase-1 in the case of skeletal muscle extracts, but was about 50% higher than type 1 in extracts from liver and heart. The only significant effect of diabetes was on hepatic protein phosphatase-1 in which a 50% decrease in specific activity was noted. Therefore, the effect of diabetes appeared to be confined to protein phosphatase-1 and this effect was only seen in liver.
Naloxone Decreases Centrally Induced Hyperglycemia in Dogs: Evidence for an Opioid Role in Glucose Homeostasis
Authors: Ipp Eli , Garberoglio Carlos , Richter Harry , Moossa A Rahim , Rubenstein Arthur H
Published: September 2013

Intracerebroventricular (ICV) instillation of morphine and β-endorphin causes centrally induced hyperglycemia. Locally active, endogenous opioids in the central nervous system may, therefore, also be involved in the elevation of blood sugar. This possibility was tested by examining the glucoregulatory response to central glucoprivation induced by ICV administration of 2-deoxy-D-glucose (2DG) in dogs. Administration of 2DG resulted in a rise in plasma glucose and immunoreactive glucagon (IRG) of 108 ± 19 mg/dl and 70 ± 20 pg/ml, respectively. These changes were attenuated by the simultaneous central infusion of the opiate antagonist naloxone: plasma glucose levels increased by 77 ± 14 mg/dl and IRG by 43 3 pg/ml, both significantly different from the effect of 2DG alone (P < 0.05–0.01). These findings suggest that opiate receptors participate in the counterregulatory response to central glucoprivation. They also provide a mechanism by which endogenous opioid peptides may play a role in the central regulation of glucose homeostasis.
Abnormal Glucose Regulation of Insulin Secretion in Models of Reduced B-Cell Mass
Authors: Leahy J L , Bonner-Weir S , Weir G C
Published: September 2013

The influence of glucose on nonglucose-stimulated in sulin secretion was examined in two animal models with reduced B-cell mass: rats treated with streptozotocin as neonates (SZ) and rats with a partial pancreatectomy (Px). The effects of arginine and IBMX upon insulin secretion were studied at varying glucose con centrations using the in vitro, isolated, perfused pan creas.

In normal rats, as expected, 16.7 mM glucose caused a biphasic insulin release and arginine-stimulated in sulin secretion was markedly potentiated by an in crease in background glucose concentration. In the SZ model, 16.7 mM glucose caused a minimal insulin re sponse. However, arginine stimulated insulin release at 2.8 mM glucose as well as at higher glucose concen trations. A similar alteration in the glucose regulation of insulin secretion was shown with another secret agogue, IBMX, even though responses to this agent in the SZ rats exceeded those of the controls at both high- and low-background glucose concentrations. The less diabetic, Px model showed partial preservation of the glucose regulation of the B-cell response to arginine.

Thus, a reduction in B-cell mass leads not only to a toss of glucose-stimulated insulin secretion but also to impaired glucose regulation of nonglucose-stimulated insulin secretion.
Abnormal Products of the Human Insulin Gene
Authors: Tager Howard S
Published: September 2013

Heritable abnormalities in the insulin gene have often been considered in terms of their potential for contributing to diabetes. It is only within the last 5 yr, however, that evidence has demonstrated the existence of insulin gene mutations in man and the secretion of abnormal human insulins in affected individuals. HPLC analysis of insulin purified from serum by immunoaffinity chromatography and detection of insulin by radioimmunoassay have documented abnormal insulins in subjects from three separate families. HPLC analysis of these natural insulins and of semisynthetic insulin analogues have identified the accompanying amino acid substitutions in two of these cases: in one, leucine replaces phenylalanine B25; in the other, serine replaces phenylalanine B24. Both substitutions occur in an invariant tetrapeptide sequence within the active site of the hormone. Studies of the biologic activities of these analogues further suggest that replacements at position B25 result in the loss of an important side chain contact between the hormone and its receptor, whereas those at position B24 result in conformational changes in the insulin molecule as a whole. Two additional individuals have been identified to secrete abnormal intermediates of proinsulin conversion in which the C-peptide remains attached to the insulin A-chain and in which Arg65 has been replaced by a nonbasic amino acid. This result emphasizes the importance of dibasic amino acid pairs at prohormone conversion sites and provides clues about the evolution of hormone precursors. Thus, studies of the products of abnormal human insulin genes have provided insights into subjects as varied as insulin biosynthesis, structure-activity relationships in insulin recognition by receptors, and the physiology of insulin action.
Evidence for Separate Handling In Vivo of Different Regions of the Insulin Molecule Using A14- and B1-labeled Insulin Tracers
Authors: Cockram Clive S , Jones Richard H , Boroujerdi Massoud A , Sönksen Peter H
Published: September 2013

To compare the metabolic characteristics and degradation of insulin tracers labeled unselectively, selectively at the A14 position (A14-monoiodoinsulin), and selectively at the B1 position (B1-monoiodoinsulin), we have followed the time course of disappearance of intact (immunoprecipitable [IP] and trichloroacetic acid [TCA] precipitable) iodoinsulin after bolus injection into greyhounds. We have used noncompartmental analysis to determine metabolic clearance rate (MCR) and apparent distribution space (DS). We have also measured the appearance of non-IP- and non-TCA-precipitable fragments, and have developed a mathematical model using compartmental analysis to explain the observed differences.

B1-Monoiodoinsulin has a significantly higher MCR (16.3 ml/min/kg) than both A14-monoiodoinsulin (10.6 ml/min/kg) and unfractionated tracers (7.6 ml/min/kg) as determined by immunoprecipitation, and reaches the values observed for native insulin in greyhounds. MCR values obtained by TCA precipitation are approximately one-half of those obtained by IP for all 3 tracers. The concentration of non-IP fragments is significantly lower with B1-monoiodoinsulin than with the other tracers. Compartmental analysis suggests this to be due to greater intracellular retention of the B1 moiety during the experimental period.

We conclude that: (1) by the criterion of MCR, B1-monoiodoinsulin seems to behave more like native insulin than other preparations tested; (2) the reduced MCR of A14-monoiodoinsulin raises doubts about its validity as a tracer for insulin; (3) a high-molecular-weight product of insulin degradation, which includes both the B1 and the A14–A19 regions of the molecule, is released into the circulation; and (4) smaller fragments containing A14–A19 reappear in the circulation more rapidly than fragments containing B1. Thus, the use of specifically labeled insulin tracers allows investigation of the mechanisms of intracellular processing in Vivo.