[Show abstract][Hide abstract] ABSTRACT: A fast-moving abnormal hemoglobin found in a diabetic patient was identified as β140 (H18) ALA→ASP. It comprised about 40 % of the total hemoglobin. The β-terminal glycation in the abnormal hemoglobin was estimated to be 3 times as much as that in Hb A in the same blood sample. The abnormal hemoglobin was slightly unstable. Oxygen affinity of the stripped hemoglobin was decreased, but that of red cells from the carrier was slightly higher than normal because of the reduced effect of 2,3-diphosphoglycerate.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the effect of sodium orthovanadate on the alterations of human erythrocytes insulin receptor autophosphorylation. Human erythrocytes were incubated with insulin in a cell system and then lysed. The autophosphorylated insulin receptors were measured with the aid of a two-site immunofluorometric assay and using a monoclonal anti-insulin receptor antibody to label the insulin receptors and a monoclonal anti-phosphotyrosine antibody to assess tyrosine phosphorylation. When the erythrocytes were treated with insulin and then reincubated in insulin-free medium, vanadate completely inhibited insulin receptor dephosphorylation, although it had no effect on in vitro receptor autophosphorylation. Thus insulin receptor tyrosine phosphatase activity is postulated to be [% (autophosphorylated insulin receptors with vanadate - autophosphorylated insulin receptors without vanadate)/total insulin receptors] under overall steady conditions in a cell system. Using this assay, the insulin receptor tyrosine phosphatase activities of 25 control and 32 diabetic subjects were studied. There was no significant difference in insulin receptor tyrosine phosphatase activity between control subjects and diabetic subjects (0.173 +/- 0.062 vs 0.209 - +/- 0.057 autophosphorylated insulin receptors units/insulin receptors units). The assay used in this study requires only 0.6 ml of whole blood, and so should be a useful tool for detecting patients who are insulin-resistant due to abnormal insulin receptor tyrosine phosphatase activity.
Diabetes Research and Clinical Practice 10/1998; 41(3):157-63. DOI:10.1016/S0168-8227(98)00076-X · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A method to detect the biological activity of serum insulin has been developed. This method, called a bioactive insulin assay, determines the ability of serum insulin to stimulate the autophosphorylation of insulin receptors in an intact cell system. For this, intact Chinese hamster ovary cells which overexpress the human insulin receptor are treated with serum and then lysed. Autophosphorylation of the insulin receptors is then measured by a two-site immunofluorometric assay using monoclonal anti-insulin receptor antibodies and europium-labeled anti-phosphotyrosine antibodies. The detection limit of this assay is 1 microU/ml of insulin. Dilution and recovery test inter- and intraassay coefficient variations are permissible. The amount of insulin determined by this assay correlated well with the amount of insulin detected by a traditional immunological assay for insulin (r = 0.94, P < 0.001). In the case of a mutant insulin, the insulin from a Wakayama subject, the biologically active insulin was found to constitute 9% of the immunologically reactive insulin. Since this assay specifically measures the amount of biologically active insulin present in serum, it should be particularly useful in monitoring active insulin in patients with various mutant insulins.
[Show abstract][Hide abstract] ABSTRACT: We have previously reported that corticotropin-releasing factor (CRF) is a potent stimulator of adrenocorticotropic hormone and cortisol secretion in the dog. Therefore, in the present study, we investigated the extrahypophysiotropic actions of CRF in this species. When CRF was injected into the third cerebral ventricle, it failed to inhibit food intake significantly at doses of 1.19, 3.57, and 11.9 nmol. This is in sharp contrast with the results in rodents. At the 3.57 and 11.9 nmol doses, CRF markedly stimulated the secretion of pancreatic polypeptide (PP), a hormone under vagal control, and at the highest dose CRF increased plasma glucose levels. These results suggest species differences in the feeding response to CRF and activation of the parasympathetic nervous system in the dog.
Journal of Endocrinology 03/1998; 156(2):359-64. DOI:10.1677/joe.0.1560359 · 3.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study, the effects of intracerebroventricular (ICV) NPY, [Leu31, Pro34]NPY and NPY13-36 have been evaluated with respect to anxiety in mice in the elevated plus maze. NPY had opposing effects on behavior, depending on the doses used. NPY decreased the normal preference for the closed arms of the maze at 0.7 nmol, indicating an anxiolytic effect; however, at 7 pmol NPY further increased the preference for the closed arm, indicating an anxiogenic effect. [Leu31, Pro34]NPY, a Y1-type receptor agonist, significantly reduced the preference for the closed arms at 70 pmol. NPY13-36, a Y2-type receptor agonist, significantly intensified the preference at 20 pmol. It has been demonstrated that NPY produces not only an anxiolytic effect via Y1-type receptors, but also an anxiogenic effect via Y2-type receptors. The time course of these NPY actions are quite different and the anxiogenic effect was observed only shortly after ICV NPY injection.
[Show abstract][Hide abstract] ABSTRACT: Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in behavior and its disorders. To understand the long-term modulation of neuronal functions by NPY, we raised transgenic mice created with a novel central nervous system (CNS) neuron-specific expression vector of human Thy- gene fragment linked to mouse NPY cDNA. In situ hybridization analysis demonstrated transgene-derived NPY expression in neurons (e.g., in the hippocampus, cerebral cortex, and the arcuate nucleus of the hypothalamus) in the transgenic mice. The modest increase of NPY protein in the brain was demonstrated by semiquantitative immunohistochemical analysis and by radioreceptor assay (115% in transgenic mice compared to control littermates). Double-staining experiments indicated colocalization of the transgene-derived NPY message and NPY protein in the same neurons, such as in the arcuate nucleus. The transgenic mice displayed behavioral signs of anxiety and hypertrophy of adrenal zona fasciculata cells, but no change in food intake was observed. The anxiety-like behavior of transgenic mice was reversed, at least in part, by administration of corticotropin-releasing factor (CRF) antagonists, alpha-helical CRF9-41, into the third cerebral ventricle. These results suggest that NPY has a role in anxiety and behavioral responses to stress partly via the CRF neuronal system. This genetic model may provide a unique opportunity to study human anxiety and emotional disorders.
Proceedings of the Association of American Physicians 01/1998; 110(3):171-82.
[Show abstract][Hide abstract] ABSTRACT: The secretion of pancreatic polypeptide (PP) is regulated by fluctuations in blood glucose concentrations and food intake, in which vagal-cholinergic mechanisms play an important role, especially for the cephalic phase of PP secretion. In this study, we examined whether central cholinergic mechanisms are also important for PP secretion by relaying information in the brain to the vagus nerve and the muscarinic cholinergic receptors in the pancreas. Atropine sulfate (20-200 micrograms) was administered into the lateral cerebral ventricle and its effects on the basal secretion of PP as well as the secretions stimulated by insulin-induced hypoglycemia (Actrapid MC, 0.25 U/kg) and a mixed meal (243 kcal) were studied in seven dogs. Intralateral cerebroventricular (ILV) atropine (100 and 200 micrograms) abolished the fluctuations in basal PP secretion without appearing in the plasma. Pretreatment with 20, 100, and 200 micrograms ILV atropine significantly decreased the PP response to insulin-induced hypoglycemia, with the integrated PP response to 58, 32, and 26% of that of controls respectively. Atropine (100 micrograms ILV) significantly reduced the postprandial PP secretion in both the cephalic and the gastrointestinal phases, whereas increased insulin and glucose levels were unaffected. Centrally administered atropine was able to suppress the basal secretion of PP as well as the secretions stimulated by hypoglycemia and food intake. These findings suggest that (1) the spontaneous release of PP is governed by an oscillating, central cholinergic tone, and (2) the stimulating PP secretion is, at least in part, regulated by the central cholinergic system.
Journal of Endocrinology 09/1997; 154(2):311-7. DOI:10.1677/joe.0.1540311 · 3.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a new ventriculoscope (Yamadori-type VII), a 2.1 mm calibrated fiberscope with a monopolar coagulator, that can be inserted from the cisterna cerebellomedullaris into the third cerebral ventricle of experimental animals, like dogs. With the improved performance of the neuroendoscope compared to its prototype, Yamadori III, it was possible to inspect clearly and to produce electrocoagulative lesions accurately on any part of the intraventricular structures with minimal injury to nearby vital brain areas.
[Show abstract][Hide abstract] ABSTRACT: We investigated the efficacy and the mechanism of action of EM523L, a nonpeptide motilin agonist (motilide), on the stimulation of gastric emptying and on the release of gut peptides after ingestion of a solid meat in normal controls (n = 8) and in diabetic patients (n = 8) with signs of neuropathy. A dose of 2 mg EM523L was administered IV over 15 min just after ingestion of a solid meal (200 kcal Gastric emptying was measured by a radionuclide technique. EM523L accelerated gastric emptying and markedly augmented postprandial pancreatic polypeptide (PP) response in both normal control and diabetic patients. This may suggest the mediation of the Vagal-cholinergic pathway to accelerate gastric emptying. The present study offers a promising therapeutic potential of the motilide in gastrointestinal motility disorders like those observed in diabetics mellitus.
[Show abstract][Hide abstract] ABSTRACT: The tissue sensitivity to insulin was evaluated using the hyperinsulinemic euglycemic clamp technique in a 44-year old male with Werner's syndrome associated with diabetes mellitus. In addition, the autophosphorylation of insulin receptors and the number of autophosphorylated insulin receptors were measured in his erythrocytes by a new enzyme-linked immunosorbent assay. He had an increased serum insulin level (30.5 microU/ml) in addition to hyperglycemia (226 mg/dl), suggesting that he had insulin-resistant diabetes mellitus. A clamp study revealed that his insulin-stimulated glucose disposal rate (2.80 mg/kg/min) was comparable to that in noninsulin-dependent diabetes mellitus (4.14 +/- 1.94 (SD) mg/kg/min, n = 23). The number of autophosphorylated insulin receptors per 300 microliters of erythrocytes was also identical to that in normal control subjects. In addition, the autophosphorylation of insulin receptors determined at six different insulin concentrations was within the normal range, even when it was expressed as per 300 microliters of erythrocytes as well as per unit receptor. These results demonstrate that insulin resistance in the patient with Werner's syndrome is caused by a defect that cannot be detected by means employed in this study, and suggest that the defect is present at the steps distal to the autophosphorylation of tyrosine kinase of insulin receptors.
[Show abstract][Hide abstract] ABSTRACT: A 60-kDa tyrosine-phosphorylated protein has been observed after insulin treatment of cells in immunoprecipitations of the GTPase-activating protein of Ras (called GAP) as well as the phosphatidylinositol 3-kinase. In the present studies, these two 60-kDa proteins have been shown to differ by limited proteolytic digestions as well as by immunoprecipitation with a monoclonal antibody. This monoclonal antibody was also utilized to show that the 60-kDa GAP-associated protein was rapidly phosphorylated in intact cells after insulin stimulation and to associate with GAP only after insulin treatment of the cells. In addition, the 60-kDa protein was found to be phosphorylated in vitro by the insulin receptor. Finally, the 60-kDa protein immunoprecipitated by this antibody was found not to react with a polyclonal antibody directed against a 62-kDa tyrosine-phosphorylated GAP-associated protein previously observed in src-transformed cells. These studies indicate that insulin stimulates the tyrosine phosphorylation of at least two distinct 60-kDa proteins, one that becomes associated with GAP and appears to be a direct substrate of the insulin receptor kinase and another that associates with the phosphatidylinositol 3-kinase.
Journal of Biological Chemistry 05/1994; 269(15):11498-502. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied the effects of intra-third cerebroventricular administration of neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) on the locomotor activity and the feeding and learning behavior of mice. NPY (0.3-10 micrograms), PYY (0.1-10 micrograms) and PP (3.0-10 micrograms) produced significant increases in locomotor activity. A significant decrease was then observed 15 min after administration of 10 micrograms of PYY. NPY, PYY and PP significantly increased food intake at 20 min and this effect continued for 2 to 4 hr at the high doses. The feeding response to PP family peptides were quite similar to that in locomotor activity with respect to dose-response, time course and peptide specificity. Learning behaviors were evaluated at three different stages of memory processing, acquisition, consolidation and retrieval, in a battery of step-down type passive avoidance tests. NPY and PYY had no effect on acquisition, but significantly improved consolidation at a dose of 0.03 and 0.3 microgram, respectively. NPY also improved retrieval at a dose of 0.03 microgram. The ranking order of potency in stimulating feeding and locomotor activity was PYY > NPY > PP, and in improving memory consolidation NPY > PYY > PP. These observations suggest that NPY and PYY influence different neural substrates in the brain involved in feeding and learning.
Journal of Pharmacology and Experimental Therapeutics 03/1994; 268(2):1010-4. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report a 27-year-old man with insulin-dependent diabetes mellitus and transient-pituitary-isolated gonadotropin deficiency. He had typical diabetic symptoms, and loss of libido of a 6-month duration. Although antibodies to islet cells or islet cell surface were not detected in his sera, daily urinary excretion of c-peptide immunoreactivity was extremely low, and antibodies to the pituitary AtT-20 cell were detected. The plasma responses of gonadotropin to a single and a repetitive luteinizing hormone-releasing hormone were extremely low, whereas testosterone concentrations in the serum and urine were low normal. After 6 months, the gonadotropin deficiency and loss of libido were not detected and antibodies to the AtT-20 cell was negative. We suspected that both insulin-dependent diabetes mellitus and transient-gonadotropin-deficiency might be an autoimmune mechanism.
Internal Medicine 02/1994; 33(1):27-30. DOI:10.2169/internalmedicine.33.27 · 0.90 Impact Factor