Insulin immuno-neutralization in fed chickens: effects on liver and muscle transcriptome.

Chickens mimic an insulin-resistance state by exhibiting several peculiarities with regard to plasma glucose level and its control by insulin. To gain insight into the role of insulin in the control of chicken transcriptome, liver and leg muscle transcriptomes were compared in fed controls and "diabetic" chickens, at 5 h after insulin immuno-neutralization, using 20.7K-chicken oligo-microarrays. At a level of false discovery rate <0.01, 1,573 and 1,225 signals were significantly modified by insulin privation in liver and muscle, respectively. Microarray data agreed reasonably well with qRT-PCR and some protein level measurements. Differentially expressed mRNAs with human ID were classified using Biorag analysis and Ingenuity Pathway Analysis. Multiple metabolic pathways, structural proteins, transporters and proteins of intracellular trafficking, major signaling pathways, and elements of the transcriptional control machinery were largely represented in both tissues. At least 42 mRNAs have already been associated with diabetes, insulin resistance, obesity, energy expenditure, or identified as sensors of metabolism in mice or humans. The contribution of the pathways presently identified to chicken physiology (particularly those not yet related to insulin) needs to be evaluated in future studies. Other challenges include the characterization of "unknown" mRNAs and the identification of the steps or networks, which disturbed tissue transcriptome so extensively, quickly after the turning off of the insulin signal. In conclusion, pleiotropic effects of insulin in chickens are further evidenced; major pathways controlled by insulin in mammals have been conserved despite the presence of unique features of insulin signaling in chicken muscle.

Magnetic resonance imaging of transplanted mouse islets labeled with chitosan-coated superparamagnetic iron oxide nanoparticles.

Although only 10% of islet recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years after transplantation. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect Feridex-labeled islet grafts in rodents. In this study, we used a novel MRI contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles, to monitor mouse islet grafts. Male inbred C57BL/6 mice were used as donors and recipients of islet transplantation. The islet cytotoxicity was evaluated by fluorescein diacetate and propidium iodide staining for RAW cells incubated with CSPIO. After being incubated overnight with and without CSPIO (10 mg/mL), 300 islets were transplanted under the left kidney capsule of each mouse. After transplantation, 3.0-Tesla MRI of the recipients was performed biweekly until 19 weeks. At the end of study, the islet graft was removed for insulin and Prussian blue staining. The cell death rates in RAW cells did not increase with increasing CSPIO concentrations or incubation time. The grafts of CSPIO-labeled islets were visualized on MRI scans as distinct hypointense spots homogeneously located at the upper pole of left kidney. Their MRI signal was 30%-50% that of control islets and was maintained throughout the follow-up period. At 18 weeks, the histology of CSPIO-labeled islet graft revealed the insulin- and iron-stained areas to be almost identical. Our results indicate that isolated mouse islets labeled with CSPIO nanoparticles can be effectively and safely imaged by using MRI as long as 18 weeks after transplantation.

Increased insulin is not required for beta2-adrenoceptor-induced increases in mouse brain tryptophan.

The current study tested the hypothesis that beta(2)-adrenoceptor-mediated increases in brain tryptophan are caused by increased insulin secretion. Male mice were treated with streptozotocin (40 mg/kg) for 5 days to induce experimental diabetes. Control and diabetic mice were treated with the beta(2)-adrenoceptor agonist, clenbuterol (0.1 mg/kg), 1 h before selected brain regions were dissected for analysis by high performance liquid chromatography (HPLC) with electrochemical detection for tryptophan content, and plasma was collected for analysis of total and free tryptophan and glucose concentrations. Clenbuterol increased brain tryptophan and plasma glucose and decreased plasma total tryptophan but did not alter plasma free tryptophan. There were no significant differences in brain or plasma tryptophan between control and streptozotocin-treated mice. In a separate experiment, pretreatment of the mice with an insulin antibody did not prevent the clenbuterol-induced increases in brain tryptophan. These results suggest that beta(2)-adrenoceptor agonists increase brain tryptophan by a mechanism that does not involve changes in insulin.

Insulin-like peptides are not involved in maturation or functional recovery of neural circuits in the locust flight system.

We sought to manipulate maturation and functional recovery of locust flight circuitry by treating locusts with pharmacological doses of bovine anti-insulin and insulin. Anti-insulin treatment of maturing locusts caused reduced growth of the thoracic nervous system, lower body weight, and softer cuticles compared with control locusts. We were unable to block either maturation or recovery of flight circuitry with anti-insulin. We propose that insulin-related peptides are involved in growth and cuticular changes during adult maturation, but have no role in promoting neuronal sprouting during this period or as a result of injury.

Insulin depletion leads to adipose-specific cell death in obese but not lean mice.

Mutation of the obese gene produces obesity, hyperinsulinemia, and compensatory "overexpression" of the defective gene. As insulin activates obese gene expression, it seemed possible that hyperinsulinemia might be responsible for overexpression of the gene. To address this question we rapidly neutralized circulating insulin by injection of an insulin antibody. Unexpectedly, insulin depletion in obese (ob/ob or db/db) mice caused massive adipose RNA degradation confirmed by histological analysis to result from adipocyte cell death by a largely necrotic mechanism. This effect was not observed in lean littermates and was completely corrected by coadministration of insulin. Comparison of multiple tissues demonstrated that the effect was restricted to adipose tissue. Insulin depletion in obese mice by administration of streptozotocin also led to cell death, but this death was less extensive and appeared to be apoptotic in mechanism. Thus insulin may promote the survival side of the physiological balance between adipocyte survival and death.

The satiating potency of hepatic portal glucagon in rats is not affected by [corrected] insulin or insulin antibodies.

To characterize the interactive effects of acute prandial manipulations of insulin and glucagon on spontaneous feeding in adult male rats fed ad lib, glucagon (G) and insulin (1) or insulin antibodies (IAb) were confused into the hepatic portal vein during the first meal of the dark phase. Infusions (3-6 min, 33 microliters/min) were remotely controlled, and a computerized system recorded meal patterns. In Experiment 1, five separate factorial designs were used to test the effects of G (1.3 or 13 micrograms/meal) alone, I (1.3 or 2.7 mU/meal) alone, or both G + I. The peptides were infused either simultaneously or sequentially (G before I). The larger dose of G alone reduced meal size. I neither inhibited feeding nor increased the effects of either G dose. In one test, 13 micrograms/meal G did not block meal size when followed by 2.7 mU I, but this antagonism did not occur in a replication. In several tests, there was a trend for I to decrease the size of the spontaneous meal that followed the meal during which I was infused, but this was statistically significant only once. Intermeal intervals were not affected in any test. Experiment 2 tested coinfusions of 20 micrograms G and polyclonal IAb with an in vitro binding capacity of 40 mU rat insulin. G alone reduced meal size, IAb alone increased meal size, and G + IAb produced an additive effect. These data extend previous investigations of the satiating action of G and I in the rat and indicate 1. that exogenous I does not affect the satiating potency of G; 2. that exogenous G and endogenous I elicit an additive synergistic inhibition of spontaneous meal size; and 3. that G-induced I secretion does not mediate the satiating effect of G.

Ventromedial hypothalamic lesion-induced vagal hyperactivity stimulates rat pancreatic cell proliferation.

BACKGROUND & AIMS: Ventromedial hypothalamic (VMH) lesions cause an increase in DNA content in the rat pancreas. This study examined the role of cell proliferation in the mitotic response of the rat pancreas after VMH lesion formation. METHODS: Alterations in rat pancreatic DNA content, DNA synthesis, and labeling indices using antiproliferation cell nuclear antigen molecular antibody were measured 0,1,3, and 7 days after VMH lesion formation. Additionally, the effects of vagotomy, atropine, or anti-insulin antibody on VMH lesion-induced alterations in DNA synthesis were examined. Pancreatic samples were also treated with double immunostaining: first for PCNA and then for insulin, glucagon, and somatostatin. RESULTS: Pancreatic weight, DNA content, and DNA synthesis increased in animals receiving VMH lesions. Proliferation was primarily observed in islet B and acinar cells beginning 1 day after VMH lesion formation, reaching a maximum rate after 3 days. VMH lesion-induced stimulation of DNA synthesis was completely inhibited by vagotomy or atropine administration but not by anti-insulin antibody. CONCLUSIONS: Vagal hyperactivity produced by VMH lesions stimulated cell proliferation of rat pancreatic islet B and acinar cells primarily through a cholinergic receptor mechanism.

Immunological resistance to human biosynthetic insulin--effects of immunosuppression and plasmapheresis.

A 55-year-old gentleman, after being treated for a short time with a diet and with Chlorpropamide, was switched to purified porcine insulin due to ketonuria and ketoacidosis. After a year the patient developed immunological insulin resistance (mean daily insulin dose: 3.72 U/kg body weight; anti-insulin antibodies 78%). In order to lower anti-insulin antibodies human recombinant DNA insulin was introduced into further therapy. Contrary to expectations, the patient did not reduce whatsoever his anti-insulin antibodies and his daily insulin dose increased up to 5.63 U/kg body weight. Introduction of combined immunosuppressive therapy (prednisone plus azathioprine) together with plasmapheresis resulted in rapid lowering of daily insulin requirement and reduction in anti-insulin antibodies. Immunosuppressive therapy was continued with 10 mg of prednisone and a year later the patients insulin daily requirement was 0.66 U/kg BW while his antibodies were 18%. The possible causes of insulin resistance to human recombinant DNA insulin are discussed as well as the advantage of combined immunosuppressive therapy together with plasmapheresis that was used for rapid lowering of insulin daily requirement and anti-insulin antibodies titer.

Insulin antibody does not cause insulin resistance during glucose clamping in rats.

Although it has often been stated that insulin antibodies cause insulin resistance, this concept is still controversial. The effect of insulin antibody GP30, commonly used in insulin radioimmunoassay, on insulin action was investigated in Wistar rats in vivo by the euglycemic glucose clamp technique. As a preliminary experiment, the equilibrium time required for insulin antibody to bind with endogenous insulin was examined. One hundred microliters/kg insulin antibody took 60 min or more to attain equilibrium, but 10 microliters/kg insulin antibody almost immediately equilibrated with endogenous insulin. During a 60-min glucose clamp study, 2 mU/kg/min porcine insulin was infused with 100 microliters/kg insulin antibody. At steady state, during the last 20-min period, the mean glucose infusion rate was 2.10 +/- 0.85 mg/kg/min (n = 5, mean +/- SD), significantly lower than the 5.77 +/- 1.61 mg/kg/min of the control, indicating insulin resistance before equilibrium was reached. However, the glucose infusion rates during the clamp with 10 microliters/kg insulin antibody and 100 microliters/kg insulin antibody infused 75 min before the insulin were 6.10 +/- 1.44 and 7.12 +/- 1.19 mg/kg/min, respectively, no different from the control. In these instances, free insulin levels measured by radioimmunoassay using the polyethyleneglycol method were 43.8 +/- 20.4 and 15.4 +/- 6.1 microU/ml, respectively, lower than the control (77.0 +/- 16.1 microM/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin antibodies retard and insulin accelerates growth and differentiation in early embryos.

The physiologic function of insulin in early embryonic life is unknown. We have shown that insulin is present in unfertilized eggs and in chick embryos at 2-3 days of development, even before the emergence of the endocrine pancreas. To define insulin's role, we exposed 2-day-old chick embryos to anti-insulin antibodies and followed their development up to day 5. Antibody-treated embryos had a higher rate of growth retardation and death by days 3-5 of embryogenesis, compared with controls. Among the survivors, biochemical maturation was delayed at days 4 and 5; weight, protein, total creatine kinase activity, and creatine kinase-MB were decreased in antibody-treated embryos. By contrast, insulin (50 ng/embryo) administered to 2-day-old embryos yielded nearly symmetrical stimulatory results. These findings suggest that endogenous insulin plays a probable physiologic role regulating growth and differentiation in early embryos. In addition, the findings provide some clues to a possible function for insulin produced outside the organism's own beta cells.

Immunoprecipitation of 125I-insulin crosslinked receptor.

125I-insulin was covalently crosslinked to its receptor on human placental membrane fractions with disuccinimidyl suberate. The 125I-insulin crosslinked receptor was solubilized with Triton X-100 and used as a probe to determine autoantibodies to the insulin receptor in sera from patients with insulin resistance (type B syndrome). When the solubilized 125I-insulin: receptor complex was incubated with these sera and then with anti-human IgG serum, the complex was precipitated as a function of the amount of anti-receptor serum. Unlabelled insulin at concentrations less than or equal to 10 micrograms/ml did not affect immunoprecipitation of the complex, suggesting that a subpopulation of anti-receptor antibodies recognizes determinants outside the insulin binding region of the receptor molecule. 125I-insulin: receptor complex was precipitated also by the addition of anti-insulin antibodies. The effect of anti-insulin antibody was eliminated by the addition of excess unlabelled insulin. The immunoprecipitation assay using 125I-insulin crosslinked receptor was sensitive and convenient for detecting anti-receptor antibodies.

Somatostatin secretion from monolayer cultures of neonatal rat pancreas.

Monolayer cultures of neonatal rat pancreas have been characterized as an in vitro system for studying SRIF secretion. Marked 12- and 6-fold potentiation of SRIF release occurred with N-2-O-dibutyryl cAMP monosodium salt and theophylline, respectively. High glucose (300 mg/dl) stimulated SRIF release, whereas galactose was without effect. Exogenous insulin did not alter SRIF release, and the SRIF responses to theophylline and glucose were unaffected by the addition of antiinsulin serum to neutralize the insulin released by these agents. Arginine evoked a significant 2-fold increase in SRIF release. Exogenous glucagon produced slight but not significant stimulation of SRIF release. However, after exposure of the cultures to antiglucagon serum to diminish the concentration of glucagon in contact with the SRIF cells, exogenous glucagon produced a marked enhancement of SRIF secretion. These data suggest that glucose, arginine, glucagon, N-2-O-dibutyryl cAMP monosodium salt, and theophylline stimulate SRIF secretion, probably by direct effects on D cells or through mechanisms other than increased insulin secretion. Monolayer cultures of rat pancreas should provide a powerful in vitro system for studying pancreatic SRIF physiology.

Glucose metabolism in the newborn rat: the role of insulin.

The effect of the administration of anti-insulin serum to newborn rats, surgically delivered under ether anaesthesia at term, was examined with respect to liver glycogen concentration and plasma concentrations of glucose, lactate and free fatty acids. Newborn rats thus treated showed decreased liver glycogen concentrations and elevated plasma concentrations of glucose, lactate and free fatty acids compared to untreated control animals one hour later. These effects were dose-dependent with respect to the amount of anti-insulin serum administered. The simultaneous administration of glucagon with anti-insulin serum at birth was no more effective in mobilising glycogen stores than anti-insulin serum alone, although plasma glucose concentrations in these animals were higher and plasma lactate concentrations were lower. Either anti-insulin serum or glucagon abolished the postnatal hypoglycaemia observed in untreated neonatal rats. The rate of fall in plasma lactate concentrations after birth was stimulated in glucagon-treated rats but was retarded in rats treated with anti-insulin serum. Hormonal control over the initiation of glycogenolysis and gluconeogenesis in the newborn rat appears to be different, a fall in plasma insulin being the prime factor involved in triggering glycogen mobilization and a rise in plasma glucagon the prime event that initiates gluconeogenesis.