Convergence and divergence of the signaling pathways for insulin and phosphoinositolglycans

Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian tissues have been demonstrated in numerous in vitro studies to exert partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells. However, their ill-defined structures, heterogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism. Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale from the core glycan of a glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces cerevisiae has recently been shown to stimulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 microns) of the maximal insulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G. Müller et al., 1997, Endocrinology 138: 3459-3476). Consequently, we used this PIG-P for the present study in which we compare its intracellular signaling with that of insulin. The activation of glucose transport by both PIG-P and insulin in isolated rat adipocytes and diaphragms was found to require stimulation of phosphatidylinositol (PI) 3-kinase but to be independent of functional p70S6kinase and mitogen-activated protein kinase. The increase in glycerol-3-phosphate acyltransferase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase. This suggest that the signaling pathways for PIG-P and insulin to glucose transport and metabolism converage at the level of PI 3-kinase. A component of the PIG-P signaling pathway located up-stream of PI 3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell viability and the insulin-mimetic activity of sodium vanadate but completely blunted the insulin response. Incubation of the cells with either trypsin or NaCl alone was ineffective. The desensitized adipocytes were reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns) compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/salt extract was demonstrated to bind in a functional manner to the adipocyte plasma membrane of desensitized adipocytes via bipolar interactions. An excess of trypsin/salt extract inhibited PIG-P action in untreated adipocytes in a competitive fashion compatible with a receptor function for PIG-P of this protein. The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolated rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling by PIG-P. Furthermore, treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with other agents exerting partially insulin-mimetic activity, such as PI-specific phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine phosphorylation of the caveolar marker protein caveolin, which was apparently correlated with stimulation of lipogenesis. Strikingly, in adipocytes subjected to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride failed completely to provoke insulin-mimetic effects. A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins located in caveolae/detergent-insoluble complexes.     

Cytomorphometric markers for uterine cancer in intermediate cells

Precise microphotometric assessment of intermediate cells from patients with normal cervical cytology and from patients with dysplasia or carcinoma in situ shows the existence of small but consistent differences. Marker features for the presence of premalignant and malignant disease can be extracted from the cell images of "normal"-appearing intermediate cells. The marker features and their diagnostic classification potential are described.     

Effects of amphetamine and haloperidol on avoidance behavior and exploratory activity

The effect of graded doses of D-amphetamine and haloperidol were tested on retention of a one trial learning passive avoidance response, on extinction of pole-jumping active avoidance behavior and on open-field activity. Low doses of amphetamine (10 microgram/animal) increased passive avoidance latency when given s.c. 1 h prior to the retention test. Higher doses (20 and 1000 microgram/animal) caused a bimodal distribution of avoidance latencies. Haloperidol (0.03 or 1.0 microgram/animal) significantly attenuated passive avoidance behavior. Amphetamine caused a delay of extinction of pole-jumping avoidance behavior in a dose-dependent manner (10, 30 or 90 microgram per rat). Conversely, haloperidol induced a dose-dependent facilitation of extinction (0.03 or 0.1 microgram per rat). Open-field activity was not significantly affected by 30 microgram amphetamine or 0.03 microgram haloperidol; 90 microgram amphetamine significantly increased rearing activity and 0.1 microgram haloperidol decreased ambulation. The data show that passive and active avoidance behavior are sensitive measures to test the activity of psychomotor stimulant and neuroleptic drugs. Exploratory behavior allows more specific behavioral effects to be dissociated from locomotor influences.     

Vasopressin fragment, AVP-(4-8), improves long-term and short-term memory in the hole board search task

To investigate the neurotoxicity of acetaldehyde covalent adducts, immunohistochemical staining for acetaldehyde adducts using the antibody against acetaldehyde adducts, was performed in the cerebral cortex of ethanol-fed (withdrawal) mice. In the ethanol-fed mice, the degeneration in the cerebral cortex was found, while the protein epitope related to acetaldehyde was found in the cerebral cortex, liver and adrenal cortex. No histochemical and immunohistochemical changes in the tissues from the control mice were found. It is possible that acetaldehyde adducts may effect on the cerebral cortex as the neurotoxicity which cause psychosis such as delirium and hallucination after alcohol drinking.     

Conditioned fear-induced tachycardia in the rat: vagal involvement

The effects of conditioned fear on gross activity, heart rate, PQ interval, noradrenaline and adrenaline were studied in freely moving rats. Subcutaneous (s.c.) injections of atropine methyl nitrate (0.5 mg/kg) during rest resulted in a significant shortening of the PQ interval, indicating that the PQ interval can be used as a measure of vagal activity. Conditioned fear was induced by 10-min forced exposure to a cage in which the rat had previously experienced footshocks (5 x 0.5 mA x 3 s). In non-shocked controls, an increase in gross activity was found and a pronounced tachycardia, without changes in PQ interval. Conditioned fear rats showed immobility behaviour, associated with a less pronounced tachycardia and an increase in PQ interval. Noradrenaline was similarly increased in both groups, whereas adrenaline was increased in conditioned fear rats only. To further evaluate the role of the vagus, rats were exposed to conditioned fear after pre-treatment with atropine methyl nitrate (0.5 mg/kg, s.c.). Again, immobility was observed with a concomitant tachycardia, but without an increase in PQ interval. These results indicate that the autonomic nervous system is differentially involved in heart rate regulation in conditioned fear rats and in non-shocked controls: in non-shocked controls a predominant sympathetic nervous system activation results in an increase in heart rate, whereas in conditioned fear rats the tachycardiac response is attenuated by a simultaneous activation of sympathetic nervous system and parasympathetic nervous system.     

Neuropsychological performance and plasma cortisol, arginine vasopressin and oxytocin in patients with major depression

Understanding the nature of swallowing in persons without swallowing problems is a prerequisite to evaluating the nature and extent of dysphagia in persons with compromised swallowing. In order to determine how swallowing varies with age and with liquid bolus volume in women, we assessed 167 normal female swallowers videofluoroscopically and obtained multiple measures of swallowing function. The women in this study demonstrated a change in swallowing function with age, due primarily to an increase in pharyngeal transit and total duration of the motor response. The duration of closure and opening of valves in the upper aerodigestive tract also increased with age, and the duration of laryngeal elevation and hyoid movement peaked in the 60-79-year-old age groups. Bolus volume effects were quite consistent across most measures. As the bolus volume increased from 1 ml to 10 ml, transit times decreased and durations of valve closure and opening increased. The results of this study may be used to specify the relationship of swallowing function to age and liquid bolus volume in women, relationships that heretofore have been observed only in part and in smaller and more heterogeneous populations.     

Evidence of the cross-cultural stability of the factor structure of the SKT short test for assessing deficits of memory and attention

Many of the psychometric instruments employed in dementia research are adapted versions of tests developed in countries different from those in which they are applied. The validity of these instruments has been established in their countries of origin; however, there is little information available regarding their validity when transferred to other cultures. The SKT, a short cognitive performance test for the assessment of memory and attention deficits developed and validated in Germany, was administered in research centers in Chile, Greece, Russia, and England. SKT raw scores were factor analyzed with regard to a prespecified target structure, i.e., the factor solution found for a large German reference sample. The cross-cultural stability of the test was assessed using a statistical method that combined the perfect congruent weights approach and the bootstrapping technique. This procedure allowed for testing the similarity between factorial solutions obtained for the different centers. Results clearly indicate the factorial stability of the SKT in the countries participating in the study.     

Phosphoinositolglycan-peptides from yeast potently induce metabolic insulin actions in isolated rat adipocytes, cardiomyocytes, and diaphragms

Polar headgroups of free glycosyl-phosphatidylinositol (GPI) lipids or protein-bound GPI membrane anchors have been shown to exhibit insulin-mimetic activity in different cell types. However, elucidation of the molecular mode of action of these phospho-inositolglycan (PIG) molecules has been hampered by 1) lack of knowledge of their exact structure; 2) variable action profiles; and 3) rather modest effects. In the present study, these problems were circumvented by preparation of PIG-peptides (PIG-P) in sufficient quantity by sequential proteolytic (V8 protease) and lipolytic (phosphatidylinositol-specific phospholipase C) cleavage of the GPI-anchored plasma membrane protein, Gce1p, from the yeast Saccharomyces cerevisiae. The structure of the resulting PIG-P, NH2-Tyr-Cys-Asn-ethanolamine-PO4-6(Man1-2)Man1-2Man1-+ ++6Man1-4GlcNH(2)1-6myo-inositol-1,2-cyclicPO4, was revealed by amino acid analysis and Dionex exchange chromatography of fragments generated enzymatically or chemically from the neutral glycan core and is in accordance with the known consensus structures of yeast GPI anchors. PIG-P stimulated glucose transport and lipogenesis in normal, desensitized and receptor-depleted isolated rat adipocytes, increased glycerol-3-phosphate acyltransferase activity and translocation of the glucose transporter isoform 4, and inhibited isoproterenol-induced lipolysis and protein kinase A activation in adipocytes. Furthermore, PIG-P was found to stimulate glucose transport in isolated rat cardiomyocytes and glycogenesis and glycogen synthase in isolated rat diaphragms. The concentration-dependent effects of the PIG-P reached 70-90% of the maximal insulin activity with EC50-values of 0.5-5 microM. Chemical or enzymic cleavages within the glycan or peptide portion of the PIG-P led to decrease or loss of activity. The data demonstrate that PIG-P exhibits a potent insulin-mimetic activity which covers a broad spectrum of metabolic insulin actions on glucose transport and metabolism.