Glucose-induced phosphorylation of the insulin receptor. Functional effects and characterization of phosphorylation sites.

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Abstract

Elevated glucose concentrations have been reported to inhibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts transfected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids (delta CT). In both cell lines, 25 mM glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuridine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor peptide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is independent of activation of the MAP kinase cascade. Proportionately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase activation and mitogenic signaling.

Authors

T S Pillay, S Xiao, J M Olefsky

Immunity to the G1 globular domain of the cartilage proteoglycan aggrecan can induce inflammatory erosive polyarthritis and spondylitis in BALB/c mice but immunity to G1 is inhibited by covalently bound keratan sulfate in vitro and in vivo.

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Abstract

Earlier work from this laboratory showed that the human proteoglycan aggrecan from fetal cartilages can induce a CD4+ T cell-dependent inflammatory polyarthritis in BALB/c mice when injected after removal of chondroitin sulfate chains. Adult keratan sulfate (KS)-rich aggrecan does not possess this property. We found that two CD4+ T cell hybridomas (TH5 and TH14) isolated from arthritic mice recognize bovine calf aggrecan and the purified G1 domain of this molecule, which also contains a portion of the interglobular domain to which KS is bound. These hybridoma responses to G1 are enhanced by partial removal of KS by the endoglycosidase keratanase or by cyanogen bromide cleavage of core protein. KS removal results in increased cellular uptake by antigen-present cells in vitro. After removal of KS by keratanase, G1 alone can induce a severe erosive polyarthritis and spondylitis in BALB/c mice identifying it as an arthritogenic domain of aggrecan. The presence of KS prevents induction of arthritis presumably as a result of an impaired immune response as observed in vitro. These observations not only identify the arthritogenic properties of G1 but they also point to the importance of glycosylation and proteolysis in determining the arthritogenicity of aggrecan and fragments thereof.

Authors

J Y Leroux, A Guerassimov, A Cartman, N Delaunay, C Webber, L C Rosenberg, S Banerjee, A R Poole

Congenital sucrase-isomaltase deficiency. Identification of a glutamine to proline substitution that leads to a transport block of sucrase-isomaltase in a pre-Golgi compartment.

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Abstract

Congenital sucrase-isomaltase deficiency is an example of a disease in which mutant phenotypes generate transport-incompetent molecules. Here, we analyze at the molecular level a phenotype of congenital sucrase-isomaltase deficiency in which sucrase-isomaltase (SI) is not transported to the brush border membrane but accumulates as a mannose-rich precursor in the endoplasmic reticulum (ER), ER-Golgi intermediate compartment, and the cis-Golgi, where it is finally degraded. A 6-kb clone containing the full-length cDNA encoding SI was isolated from the patient's intestinal tissue and from normal controls. Sequencing of the cDNA revealed a single mutation, A/C at nucleotide 3298 in the coding region of the sucrase subunit of the enzyme complex. The mutation leads to a substitution of the glutamine residue by a proline at amino acid 1098 (Q1098P). The Q1098P mutation lies in a region that is highly conserved between sucrase and isomaltase from different species and several other structurally and functionally related proteins. This is the first report that characterizes a point mutation in the SI gene that is responsible for the transport incompetence of SI and for its retention between the ER and the Golgi.

Authors

J Ouwendijk, C E Moolenaar, W J Peters, C P Hollenberg, L A Ginsel, J A Fransen, H Y Naim

The roles of insulin and glucagon in the regulation of hepatic glycogen synthesis and turnover in humans.

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Abstract

To determine the respective roles of insulin and glucagon for hepatic glycogen synthesis and turnover, hyperglycemic clamps were performed with somatostatin [0.1 micrograms/(kg.min)] in healthy young men under conditions of: (I) basal fasting) portal vein insulinemia-hypoglucagonemia, (II) basal portal vein insulinemia-basal glucagonemia, and (III) basal peripheral insulinemia-hypoglucagonemia. Synthetic rates, pathway (direct versus indirect) contributions, and percent turnover of hepatic glycogen were assessed by in vivo 13C nuclear magnetic resonance spectroscopy during [1-13C]glucose infusion followed by a natural abundance glucose chase in conjunction with acetaminophen to noninvasively sample the hepatic UDP-glucose pool. In the presence of hyperglycemia (10.4 +/- 0.1 mM) and basal portal vein insulinemia (192 +/- 6 pM), suppression of glucagon secretion (plasma glucagon, I:31 +/- 4, II: 63 +/- 8 pg/ml) doubled the hepatic accumulation of glycogen (Vsyn) compared with conditions of basal glucagonemia [I: 0.40 +/- 0.06, II: 0.19 +/- 0.03 mumol/(liter.min): P < 0.0025]. Glycogen turnover was markedly reduced (I: 19 +/- 7%, II: 69 +/- 12%; P < 0.005), so that net rate of glycogen synthesis increased approximately fivefold (P < 0.001) by inhibition of glucagon secretion. The relative contribution of gluconeogenesis (indirect pathway) to glycogen synthesis was lower during hypoglucagonemia (42 +/- 6%) than during basal glucagonemia (54 +/- 5%; P < 0.005). Under conditions of basal peripheral insulinemia (54 +/- 2 pM) and hypoglucagonemia (III) there was negligible hepatic glycogen synthesis and turnover. In conclusion, small changes in portal vein concentrations of insulin and glucagon independently affect hepatic glycogen synthesis and turnover. Inhibition of glucagon secretion under conditions of hyperglycemia and basal concentrations of insulin results in: (a) twofold increase in rate of hepatic glycogen synthesis, (b) reduction of glycogen turnover by approximately 73%, and (c) augmented percent contribution of the direct pathway to glycogen synthesis compared with conditions of basal glucagonemia.

Authors

M Roden, G Perseghin, K F Petersen, J H Hwang, G W Cline, K Gerow, D L Rothman, G I Shulman

Pulmonary epithelial cell expression of GM-CSF corrects the alveolar proteinosis in GM-CSF-deficient mice.

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Abstract

Mutation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene by homologous recombination caused alveolar proteinosis in mice. To further discern the role of GM-CSF in surfactant homeostasis, the synthesis of GM-CSF was directed to the respiratory epithelium of GM-CSF-hull mutant mice (GM-/-) with a chimeric gene expressing GM-CSF under the control of the promoter from the human surfactant protein-C (SP-C) gene. Transgenic mice bearing the SP-C-GM-CSF construct (SP-C-GM+) were bred to GM-/- mice resulting in complete correction of alveolar proteinosis in bitransgenic GM-/-, SP-C-GM+ mice. No effects of the transgene were found outside the lung. GM-CSF was increased in bronchoalveolar lavage fluid of the bitransgenic mice. Surfactant proteins-A and -B and phospholipid in bronchoalveolar lavage fluid were normalized in the GM-/-, SP-C-GM+ mice. SP-A, -B, and -C mRNAs were unaltered in lungs from GM-CSF-deficient and -replete mice. Expression of GM-CSF in respiratory epithelial cells of transgenic mice restores surfactant homeostasis in GM-/- mice. From these findings, we conclude that GM-CSF regulates the clearance or catabolism rather than synthesis of surfactant proteins and lipids.

Authors

J A Huffman, W M Hull, G Dranoff, R C Mulligan, J A Whitsett

Regulation of calcium channel expression in neonatal myocytes by catecholamines.

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Abstract

Expression of the dihydropyridine (DHP) receptor (alpha 1 subunit of L-type calcium channel) in heart is regulated by differentiation and innervation and is altered in congestive heart failure. We examined the transmembrane signaling pathways by which norepinephrine regulates DHP receptor expression in cultured neonatal rat ventricular myocytes. Using a 1.3-kb rat cardiac DHP receptor probe, and Northern analysis quantified by laser densitometry, we found that norepinephrine exposure produced a 2.2-fold increase in DHP receptor mRNA levels at 2 h followed by a decline to 50% of control at 4-48 h (P < 0.02). The alpha-adrenergic agonist phenylephrine and a phorbol ester produced a decline in mRNA levels (8-48 h). The beta-adrenergic agonist isoproterenol and 8-bromo-cAMP produced a transient increase in mRNA levels. After 24 h of exposure to isoproterenol, 3H-(+)PN200-110 binding sites increased from 410 +/- 8 to 539 +/- 39 fmol/mg (P < 0.05). The number of functional calcium channels, estimated by whole-cell voltage clamp experiments, was also increased after 24 h of exposure to isoproterenol. Peak current density (recordings performed in absence of isoproterenol) increased from -10.8 +/- 0.8 (n = 23) to -13.9 +/- 1.0 pA/pF (n = 27) (P < 0.01). Other characteristics of the calcium current (voltage for peak current, activation, and inactivation) were unchanged. Exposure for 48 h to phenylephrine produced a significant decline in peak current density (P < 0.01). We conclude that beta -adrenergic transmembrane signaling increases DHP receptor mRNA and number of functional calcium channels and that alpha - adrenergic transmembrane signaling produces a reciprocal effect. Regulation of cardiac calcium channel expression by adrenergic pathways may have physiological and pathophysiological importance.

Authors

T Maki, E J Gruver, A J Davidoff, N Izzo, D Toupin, W Colucci, A R Marks, J D Marsh

Transepithelial water permeability in microperfused distal airways. Evidence for channel-mediated water transport.

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Abstract

Water movement across the airway epithelium is important for regulation of the volume and composition of airspace fluid. A novel approach is reported here to measure osmotic and diffusional water permeability in intact airways. Small airways (100-200 microns diameter, 1-2 mm length) from guinea pig lung were microdissected and perfused in vitro using concentric glass holding and perfusion pipettes. For measurement of osmotic water permeability (Pf), the airway lumen was perfused wit PBS (300 mOsM) containing a membrane impermeable fluorophore, fluorescein sulfonate (FS), and the airway was bathed in solutions of specified osmolalities. Pf determination was based on the changes in FS fluorescence at the distal end of the airway resulting from transepithelial water transport. Pf was 4-5 x 10(-3) cm/s at 23 degrees C and independent of lumen flow rate (10-100 nl/min) and the magnitude and direction of the osmotic gradient (bath osmolality 50-600 mOsM). Temperature dependence measurements gave an activation energy of 4.4 kcal/mol (15-37 degrees C). Pf was not altered by 0.3 mM HgCl2 or 50 microM forskolin, but was increased to 31 x 10(-3) cm/s by 100 micrograms/ml amphotericin B, indicating that osmosis is not limited by unstirred layers. Diffusional water permeability (Pd) was measured by H2O/D2O (deuterium oxide) exchange using the H2O/D2O-sensitive fluorescent probe aminonapthelane trisulfonic acid in the lumen. Measured Pd was 3-6 x 10(-6) cm/s at 23 degrees C, indicating significant restriction to water diffusion by unstirred layers. Antibody localization of water channels showed strong expression of the mercurial-insensitive water channel (AQP-4) at the basolateral membrane of airway epithelial cells. These results provide functional evidence that water movement across the distal airway epithelium is mediated by water channels.

Authors

H G Folkesson, M A Matthay, A Frigeri, A S Verkman

Apoptotic depletion of CD4+ T cells in idiopathic CD4+ T lymphocytopenia.

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Abstract

Progressive loss of CD4+ T lymphocytes, accompanied by opportunistic infections characteristic of the acquired immune deficiency syndrome, ahs been reported in the absence of any known etiology. The pathogenesis of this syndrome, a subset of idiopathic CD4+ T lymphocytopenia (ICL), is uncertain. We report that CD4+ T cells from seven of eight ICL patients underwent accelerated programmed cell death, a process facilitated by T cell receptor cross-linking. Apoptosis was associated with enhanced expression of Fas and Fas ligand in unstimulated cell populations, and partially inhibited by soluble anti-Fas mAb. In addition, apoptosis was suppressed by aurintricarboxylic acid, an inhibitor of calcium-dependent endonucleases and proteases, in cells from four of seven patients, The in vivo significance of these findings was supported by three factors: the absence of accelerated apoptosis in persons with stable, physiologic CD4 lymphopenia without clinical immune deficiency; detection of serum antihistone H2B autoantibodies, one consequence of DNA fragmentation, in some patients; and its selectivity, with apoptosis limited to the CD4 population in some, and occurring among CD8+ T cells predominantly in those individuals with marked depletion of both CD4+ T lymphocytes linked to clinical immune suppression have evidence for accelerated T cell apoptosis in vitro that may be pathophysiologic and amenable to therapy with apoptosis inhibitors.

Authors

J Laurence, D Mitra, M Steiner, D H Lynch, F P Siegal, L Staiano-Coico

Oxidation of pristanic acid in fibroblasts and its application to the diagnosis of peroxisomal beta-oxidation defects.

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Abstract

Pristanic acid oxidation measurements proved a reliable tool for assessing complementation in fused heterokaryons from patients with peroxisomal biogenesis defects. We, therefore, used this method to determine the complementation groups of patients with isolated defects in peroxisomal beta-oxidation. The rate of oxidation of pristanic acid was reduced in affected cell lines from all of the families with inherited defects in peroxisomal beta-oxidation, thus excluding the possibility of a defective acyl CoA oxidase. Complementation analyses indicated that all of the patients belonged to the same complementation group, which corresponded to cell lines with bifunctional protein defects. Phytanic acid oxidation was reduced in fibroblasts from some, but not all, of the patients. Plasma samples were still available from six of the patients. The ratio of pristanic acid to phytanic acid was elevated in all of these samples, as were the levels of saturated very long chain fatty acids (VLCFA). However, the levels of bile acid intermediates, polyenoic VLCFA, and docosahexaenoic acid were abnormal in only some of the samples. Pristanic acid oxidation measurements were helpful in a prenatal assessment for one of the families where previous experience had shown that cellular VLCFA levels were not consistently elevated in affected individuals.

Authors

B C Paton, P C Sharp, D I Crane, A Poulos

Cryptococcal polysaccharides induce L-selectin shedding and tumor necrosis factor receptor loss from the surface of human neutrophils.

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Abstract

High titers of cryptococcal polysaccharides in the serum and spinal fluid and the lack of cellular infiltrates in the infected tissues are hallmarks of disseminated cryptococcosis. Cryptococcal polysaccharides given intravenously to mice inhibit the influx of leukocytes into sites injected with inflammatory mediators. The purpose of this investigation was to determine if cryptococcal polysaccharides, i.e., glucuronoxylomannan (GXM), galactoxylomannan, and mannoprotein, affect expression of molecules on the surface of neutrophils that are important in extravasation. GXM in the absence of serum was shown to induce human neurophils to shed L-selectin, a molecule needed in the first step of neutrophil movement into tissues. In the presence of serum, GXM caused a further shedding of L-selectin. Shedding of L-selectin was evident by reduced amounts of L-selectin on the neutrophils treated with GXM and by increased levels of soluble L-selectin in the GXM-treated neutrophil supernatants. GXM also stimulated neutrophils to have reduced expression of TNF receptor. In contrast, GXM-treated neutrophils showed increased levels of CD15 and CD11b, and unchanged CD16 expression. In the absence of serum, galactoxylomannan and mannoprotein did not affect L-selectin, TNF receptor, CD15, CD11b, or CD16 on neutrophils but did induce loss of L-selectin in the presence of serum. Our results indicate that cryptococcal polysaccharides, especially GXM, can cause shedding of L-selectin from the surface of neutrophils, and this may prevent neutrophils from attaching to the endothelial cell surfaces. Blockage of this early step in cell migration from the vessels into tissues may be responsible in part for reduced cellular infiltration into infected tissues of individuals with disseminated cryptococcosis.

Authors

Z M Dong, J W Murphy