Regulation of insulin-stimulated GLUT4 translocation by Munc18c in 3T3L1 adipocytes

Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2- and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.     

Ultrastructural localization of GLUT 1 and GLUT 3 glucose transporters in rat brain

Owing to the complexity of the parathyroid hormone's metabolic interactions, clinical hypoparathyroidism is one of the most difficult of all endocrine disorders to treat. Therefore, causative treatment of this disorder by transplantation of parathyroid glands is highly desirable. We have recently documented the long-term in vivo function of iso- and allotransplanted rat parathyroid tissue without systemic immunosuppression in an animal model. In view of the potential clinical use of this method, human parathyroid tissue has been microencapsulated and transplanted over the highest immunological barrier. In a controlled, long-term animal study in the parathyroidectomized rat, the effect of microencapsulation on xenotransplanted human parathyroid tissue was evaluated over 30 weeks (native and microencapsulated parathyroid tissue = 40 rats respectively). Functionally, human parathyroid tissue was able to replace that of the rat. All animals that had received microencapsulated parathyroid tissue were normocalcemic for 16 weeks; 27/40 at the end of the study. In contrast, serum calcium concentrations dropped to post-parathyroidectomy levels within 4 weeks in those animals that had received native tissue only. Histologic evaluation of the explanted, functionally successful xenografts showed vital parathyroid tissue inside intact microcapsules surrounded by a small rim of fibroblasts. Avital fibrotic remnants were demonstrated in animals with non-encapsulated parathyroid tissue. Thus, we have established the feasibility of microencapsulation of human parathyroid tissue, preserving its viability over long periods in vivo even if xenotransplanted. In combination with an improved tissue culture method, transplantation of human parathyroid tissue and maintenance of its physiological function is reproducibly achieved without postoperative systemic immunosuppression over the highest transplantation barrier. This may be a crucial step towards the first clinical application of this method.     

Programmed cellular response to ionizing radiation damage

Typically, economic evaluation compares the costs and benefits of two or more interventions and seeks to identify the single superior option on the basis of relative cost-effectiveness. It is then anticipated that all patients will receive the more or most cost-effective option. This 'all or nothing' approach can be departed from when sub-groups of patient exist, defined on the basis of clinical or demographic characteristics which are considered to influence benefit, for whom an option is cost-effective whilst not being so for the population of patients as a whole. However, patients' preferences concerning the different process characteristics and outcomes of an intervention will also influence the benefit they derive from health care. This paper explores the concept of preference-based sub-group analysis in economic evaluation to assess the potential cost-effectiveness of using patients' preferences to determine treatment allocation. The clinical example used to explore these methods is the comparison of abdominal hysterectomy (AH) and transcervical resection of the endometrial (TCRE) for the treatment of menorrhagia.     

Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1-5 and SGLT1. The apparent Km of DHA transport via GLUT1 and GLUT3 was 1.1 +/- 0.2 and 1.7 +/- 0.3 mM, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2-4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, D-glucose, and 3-O-methylglucose among other hexoses while fructose and L-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-alpha-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2-8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.     

Endocrine response to surgical stress

The similar response is elicited by different surgical stress for the maintenance of homeostasis. The afferent neurogenic stimuli initiate the endocrine response. The production of inflammatory cytokines also stimulates the neuroendocrine system. Activation of the sympathoadrenal, hypothalmo-pituitary-adrenal axis, and renin-angiotensin-aldosterone-axis results in increased serum concentrations of stress hormones. The endocrine alterations provide the optimal hemodynamic, metabolic and immunologic response. Such response to surgical stress is highly complex. However, a better understanding of the mechanisms may lead to improved strategies of the treatment for critical surgical illness. This article focuses on the mechanisms of the neuroendocrine system activation by surgical injury and the role of the response for maintaining homeostasis.     

Dynactin phosphorylation is modulated in response to cellular effectors

In microgravity (microG) humans have marked changes in body fluids, with a combination of an overall fluid loss and a redistribution of fluids in the cranial direction. We investigated whether interstitial pulmonary edema develops as a result of a headward fluid shift or whether pulmonary tissue fluid volume is reduced as a result of the overall loss of body fluid. We measured pulmonary tissue volume (Vti), capillary blood flow, and diffusing capacity in four subjects before, during, and after 10 days of exposure to microG during spaceflight. Measurements were made by rebreathing a gas mixture containing small amounts of acetylene, carbon monoxide, and argon. Measurements made early in flight in two subjects showed no change in Vti despite large increases in stroke volume (40%) and diffusing capacity (13%) consistent with increased pulmonary capillary blood volume. Late in-flight measurements in four subjects showed a 25% reduction in Vti compared with preflight controls (P < 0.001). There was a concomittant reduction in stroke volume, to the extent that it was no longer significantly different from preflight control. Diffusing capacity remained elevated (11%; P < 0.05) late in flight. These findings suggest that, despite increased pulmonary perfusion and pulmonary capillary blood volume, interstitial pulmonary edema does not result from exposure to microG.     

Effect of mutations of murine lens alphaB crystallin on transfected neural cell viability and cellular translocation in response to stress

The relationship between exercise-induced lowering of plasma glutamine concentrations and proliferation of peripheral lymphocytes was investigated in male Wistar rats. The T-lymphocyte proliferative responses to the mitogen, concanavalin A, were determined by incorporation of radiolabelled thymidine into the DNA in vitro. The rats ran 2 h x day(-1), 6 days x week(-1) for 4 weeks. Analysis immediately after the final period of exercise showed T-lymphocyte proliferation to be significantly depressed, together with a marked decrease in plasma glutamine concentrations. There were also significant increases in serum corticosterone concentrations immediately after exercise. However, following 24-h recovery, this exercise-induced immunosuppression was not statistically significant when compared with the age-matched control group. In the second experiment, in order to clarify the importance of glutamine for immunological function in vivo, methionine sulfoximine, an effective inhibitor of glutamine synthetase was injected intraperitoneally (12.5 mg x kg body mass[-1]). Plasma glutamine concentrations were decreased 4 h after the injection, compared with the placebo control group, and this resulted in a significant decrease in the rate of T-lymphocyte proliferation. This treatment had no effects on serum corticosterone concentrations. These results would suggest that the chronic exercise-induced reduction in proliferation of peripheral T-lymphocytes is a transient reversible phenomenon, which returns to normal levels within 24 h of the final training period. It is also conceivable that this exercise-induced immunosuppression is associated with a decrease in circulating glutamine concentrations.     

Structural-functional chromosome organization in response to stress

Published data and the results of experiments conducted at the Laboratory of Genetics of Higher Nervous Activity, Pavlov Institute of Physiology, on the effect of stress on chromosome structure and function are reviewed briefly. Experiments were performed on inbred rats selected for excitation threshold and mutant Drosophila with altered metabolism of secondary mediators.     

Role of apoptotic response in cellular resistance to cytotoxic agents

To elucidate compositional changes of the pubic symphysis (PS) by aging, elements of pubic symphysis (PSs) removed from 26 cadavers were determined by inductively coupled plasma atomic-emission spectrometry. It was found that the relative contents (RCs) of calcium and phosphorus in women's PSs were about three- and five-fold amounts as compared with those in men's PSs, respectively. In contrast, the RCs of sulfur, magnesium, sodium, and iron in women's PSs were somewhat lower than those in men's PSs. The accumulations of calcium (Ca) and phosphorus (P) in women's PSs occurred mainly beyond the age of 70-yr-old, but did not occur in men's PSs.     

Glucose transporters in rat peripheral nerve: paranodal expression of GLUT1 and GLUT3

Peripheral nerve depends on glucose oxidation to energize the repolarization of excitable axonal membranes following impulse conduction, hence requiring high-energy demands by the axon at the node of Ranvier. To enter the axon at this site, glucose must be transported from the endoneurial space across Schwann cell plasma membranes and the axolemma. Such transport is likely to be mediated by facilitative glucose transporters. Although immunohistochemical studies of peripheral nerves have detected high levels of the transporter GLUT1 in endoneurial capillaries and perineurium, localization of glucose transporters to Schwann cells or peripheral axons in vivo has not been documented. In this study, we demonstrate that the GLUT1 transporter is expressed in the plasma membrane and cytoplasm of myelinating Schwann cells around the nodes of Ranvier and in the Schmidt-Lanterman incisures, making them potential sites of transcellular glucose transport. No GLUT1 was detected in axonal membranes. GLUT3 mRNA was expressed only at low levels, but GLUT3 polypeptide was barely detected by immunocytochemistry or immunoblotting in peripheral nerve from young adult rats. However, in 13-month-old rats, GLUT3 polypeptide was present in myelinated fibers, endoneurial capillaries, and perineurium. In myelinated fibers, GLUT3 appeared to be preferentially expressed in the paranodal regions of Schwann cells and nodal axons, but was also present in the internodal aspects of these structures. The results of the present study suggest that both Schwann cell GLUT1 and axonal and Schwann cell GLUT3 are involved in the transport of glucose into the metabolically active regions of peripheral axons.     

Drinking in response to cellular dehydration in the pigeon.

21 male White Carneau, White King, and Silver King pigeons received intraperitoneal injections of different amounts of hypertonic saline, and the amount of water that they drank was recorded. The volume of water drunk increased in proportion to the size of the hypertonic load, and on the average amounted to 97% of the volume of water needed to restore body fluids to isotonicity. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Aggresomes: a cellular response to misfolded proteins

Intracellular deposition of misfolded protein aggregates into ubiquitin-rich cytoplasmic inclusions is linked to the pathogenesis of many diseases. Why these aggregates form despite the existence of cellular machinery to recognize and degrade misfolded protein and how they are delivered to cytoplasmic inclusions are not known. We have investigated the intracellular fate of cystic fibrosis transmembrane conductance regulator (CFTR), an inefficiently folded integral membrane protein which is degraded by the cytoplasmic ubiquitin-proteasome pathway. Overexpression or inhibition of proteasome activity in transfected human embryonic kidney or Chinese hamster ovary cells led to the accumulation of stable, high molecular weight, detergent-insoluble, multiubiquitinated forms of CFTR. Using immunofluorescence and transmission electron microscopy with immunogold labeling, we demonstrate that undegraded CFTR molecules accumulate at a distinct pericentriolar structure which we have termed the aggresome. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes. Similarly, inhibition of proteasome function also prevented the degradation of unassembled presenilin-1 molecules leading to their aggregation and deposition in aggresomes. These data lead us to propose that aggresome formation is a general response of cells which occurs when the capacity of the proteasome is exceeded by the production of aggregation-prone misfolded proteins.     

The role of ceramide in the cellular response to cytotoxic agents

STUDY AIM: The aim of this study was to report the results of pharyngoesophageal reconstruction in extensive corrosive strictures and to describe an original conception concerning extensive chemical burns of the pharynx with involvement of the epiglottis, oro-hypopharyngeal junction and cricopharyngeal pinchcock. PATIENTS AND METHODS: A personal series of 253 esophageal reconstructions using the colon and ileum is reported. In 124 patients, the cervical anastomosis of the graft was performed at the level of the pharynx, and these cases with extensive pharyngeal lesions were the basis of this study. The anastomosis was performed with the hypopharynx in 27 patients, with the oropharynx in nine and a total reconstruction of the pharynx or "pharyngoplasty" was carried out in 69 patients. The pharyngoplasty was classified according to the approach, in anterior, posterior, lateral, superior (transmandibular) and inferior. In high strictures with epiglottic injury, epiglottectomy was necessary in order to prevent recurrence. A visceral pharyngoplasty was performed in 61 patients, using the colon in 42 and the ileum in 19, a skin reconstruction in six patients and a myocutaneous flap in two. RESULTS: The global postoperative mortality rate was 4.7%. Stenosis of the cervical anastomosis occurred in 4.9% of the whole series. With a follow-up from 6 months to 10 years, 70% of the patients resumed a normal oral diet, 21% had mild symptoms and 7% had poor results (patients with tracheostomy and gastrostomy). CONCLUSION: Extensive chemical burns of the pharynx are very severe and their treatment very difficult. For the author, total visceral pharyngeal reconstruction is considered to be the procedure of choice, using ileopharyngoplasty with realization of an ileal pouch. Good results were obtained in 70% of the patients with extensive corrosive strictures.     

Dissociation of GLUT4 translocation and insulin-stimulated glucose transport in transgenic mice overexpressing GLUT1 in skeletal muscle

Overexpression of the human GLUT1 glucose transporter protein in skeletal muscle of transgenic mice results in large increases in basal glucose transport and metabolism, but impaired stimulation of glucose transport by insulin, contractions, or hypoxia (Gulve, E. A., Ren, J.-M., Marshall, B. A., Gao, J., Hansen, P. A., Holloszy, J. O. , and Mueckler, M. (1994) J. Biol. Chem. 269, 18366-18370). This study examined the relationship between glucose transport and cell-surface glucose transporter content in isolated skeletal muscle from wild-type and GLUT1-overexpressing mice using 2-deoxyglucose, 3-O-methylglucose, and the 2-N-[4-(1-azi-2,2, 2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos-4-yloxy)-2-propyl amine exofacial photolabeling technique. Insulin (2 milliunits/ml) stimulated a 3-fold increase in 2-deoxyglucose uptake in extensor digitorum longus muscles of control mice (0.47 +/- 0.07 micromol/ml/20 min in basal muscle versus 1.44 micromol/ml/20 min in insulin-stimulated muscle; mean +/- S.E.). Insulin failed to increase 2-deoxyglucose uptake above basal rates in muscles overexpressing GLUT1 (4.00 +/- 0.40 micromol/ml/20 min in basal muscle versus 3.96 +/- 0.37 micromol/ml/20 min in insulin-stimulated muscle). A similar lack of insulin stimulation in muscles overexpressing GLUT1 was observed using 3-O-methylglucose. However, the magnitude of the insulin-stimulated increase in cell-surface GLUT4 photolabeling was nearly identical (approximately 3-fold) in wild-type and GLUT1-overexpressing muscles. This apparently normal insulin-stimulated translocation of GLUT4 in GLUT1-overexpressing muscle was confirmed by immunoelectron microscopy. Our findings suggest that GLUT4 activity at the plasma membrane can be dissociated from the plasma membrane content of GLUT4 molecules and thus suggest that the intrinsic activity of GLUT4 is subject to regulation.