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PubMed2003

Glutamine stimulates argininosuccinate synthetase gene expression through cytosolic O-glycosylation of Sp1 in Caco-2 cells.

Glutamine stimulates the expression of the argininosuccinate synthetase (ASS) gene at both the level of enzyme activity and mRNA in Caco-2 cells. Searching to identify the pathway involved, we observed that (i) the stimulating effect of glutamine was totally mimicked by glucosamine addition, and (ii) its effect but not that of glucosamine was totally blocked by 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of amidotransferases, suggesting that the metabolism of glutamine to glucosamine 6-phosphate was required. Moreover, run-on assays revealed that glucosamine was acting at a transcriptional level. Because three functional GC boxes were identified on the ASS gene promoter (Anderson, G. M., and Freytag, S. O. (1991) Mol. Cell Biol. 11, 1935-1943), the potential involvement of Sp1 family members was studied. Electrophoretic mobility shift assays using either the Sp1 consensus sequence or an appropriate fragment of the ASS promoter sequence as a probe demonstrated that both glutamine and glucosamine increased Sp1 DNA binding. Immunoprecipitation-Western blot experiments demonstrated that both compounds increased O-glycosylation of Sp1 leading to its translocation into nucleus. Again, the effect of glutamine on Sp1 was inhibited by the addition of DON but not of glucosamine. Taken together, the results clearly demonstrate that the metabolism of glutamine through the hexosamine pathway leads to the cytosolic O-glycosylation of Sp1, which, in turn, translocates into nucleus and stimulates the ASS gene transcription. Collectively, the results constitute the first demonstration of a functional relationship between a regulating signal (glutamine), a transcription factor (Sp1), and the transcription of the ASS gene.

Argininosuccinate Synthase↗
PubMed2004

Phosphorylation of mouse glutamine-fructose-6-phosphate amidotransferase 2 (GFAT2) by cAMP-dependent protein kinase increases the enzyme activity.

A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase (GFAT) was termed GFAT2 on the basis of this similarity. The mouse GFAT2 cDNA was cloned, and the protein was expressed with either an N-terminal glutathione S-transferase or His tag. The purified protein expressed in mammalian cells had GFAT activity. The Km values for the two substrates of reaction, fructose 6-phosphate and glutamine, were determined to be 0.8 mm for fructose 6-phosphate and 1.2 mm for glutamine, which are within the ranges determined for GFAT1. The protein sequence around the serine 202 of GFAT2 was conserved to the serine 205 of GFAT1, whereas the serine at 235 in GFAT1 was not present in GFAT2. Previously we showed that phosphorylation of serine 205 in GFAT1 by the catalytic subunit of cAMP-dependent protein kinase (PKA) inhibits its activity. Like GFAT1, GFAT2 was phosphorylated by PKA, but GFAT2 activity increased approximately 2.2-fold by this modification. When serine 202 of GFAT2 was mutated to an alanine, the enzyme not only became resistant to phosphorylation, but also the increase in activity in response to PKA also was blocked. These results indicated that the phosphorylation of serine 202 was necessary and sufficient for these alterations by PKA. GFAT2 was modestly inhibited (15%) by UDP-GlcNAc but not through detectable O-glycosylation. GFAT2 is, therefore, an isoenzyme of GFAT1, but its regulation by cAMP is the opposite, allowing differential regulation of the hexosamine pathway in specialized tissues.

Acetylglucosamine↗
PubMed2004

Neutrophils exhibit rapid agonist-induced increases in protein-associated O-GlcNAc.

A variety of cytoplasmic and nuclear proteins can be modified on serine and threonine residues by O-linked beta-N-acetylglucosamine (O-GlcNAc), although the effects of this modification on protein and cellular functions are not completely defined. The sugar donor for the O-GlcNAc transferase that catalyzes this post-translational modification is UDP-N-acetylglucosamine (UDP-GlcNAc), a product of the hexosamine biosynthesis pathway (HBP). Here, the dynamics of the O-GlcNAc modification are examined in the physiological context of agonist-induced signal transduction using neutrophils. Formylated Met-Leu-Phe (fMLF) is shown to stimulate a rapid and transient increase in protein O-GlcNAcylation in both immunoblot and immunofluorescence imaging assays using O-GlcNAc-specific antibodies. In high performance liquid chromatography analyses of HBP metabolic activity, short term exposure to an exogenous substrate of the HBP, glucosamine (GlcNH(2)), leads to increased GlcNH(2) 6-phosphate and then UDP-GlcNAc levels. The GlcNH(2) treatments also increase O-GlcNAcylation and augment the aforementioned fMLF-associated increase. In functional assays, GlcNH(2) pre-treatment selectively augments fMLF-induced chemotaxis but has little effect on respiratory burst activity. Furthermore, augmenting levels of O-GlcNAc in the absence of agonist is sufficient to stimulate chemotaxis. These data demonstrate that neutrophils possess a functionally significant O-GlcNAcylation pathway that is robustly induced by stimulation with agonist. We propose that O-GlcNAcylation plays an important role in rapid and dynamic neutrophil signal transduction, especially with respect to chemotaxis.

Acetylglucosamine↗
PubMed2005

Yeast glutamine-fructose-6-phosphate aminotransferase (Gfa1) requires methionine aminopeptidase activity for proper function.

Methionine aminopeptidase (MetAP) catalyzes the co-translational processing of initiator methionine from nascent proteins. A cellular requirement for MetAP activity is likely due to dysfunction of MetAP substrates that require methionine removal for proper protein function. Glutamine-fructose-6-phosphate aminotransferase (Gfa1) is an essential enzyme in yeast that catalyzes the first and rate-limiting step in hexosamine biosynthesis. The alpha-amino group of Gfa1 Cys-1 has been proposed to act as a nucleophile in the catalytic mechanism. We used two mutational strategies to evaluate whether removal of initiator methionine, catalyzed by MetAP, is required for Gfa1 function. Our results demonstrate that exposure of the alpha-amino group of Cys-1 is required for normal Gfa1 function as failure to do so results in decreased enzyme activity and slow growth. Further, either isoform of MetAP in yeast is sufficient for Gfa1 processing in vivo. These results are the first demonstration of an endogenous yeast protein that requires the exposure of the alpha-amino group by MetAP action for normal function. Additionally, Gfa1 will be a relevant target in therapeutic or physiological applications in which MetAP activity is inhibited.

Aminopeptidases↗
PubMed2005

Structure and kinetics of a monomeric glucosamine 6-phosphate deaminase: missing link of the NagB superfamily?

Glucosamine 6-phosphate is converted to fructose 6-phosphate and ammonia by the action of the enzyme glucosamine 6-phosphate deaminase, NagB. This reaction is the final step in the specific GlcNAc utilization pathway and thus decides the metabolic fate of GlcNAc. Sequence analyses suggest that the NagB "superfamily" consists of three main clusters: multimeric and allosterically regulated glucosamine-6-phosphate deaminases (exemplified by Escherichia coli NagB), phosphogluconolactonases, and monomeric hexosamine-6-phosphate deaminases. Here we present the three-dimensional structure and kinetics of the first member of this latter group, the glucosamine-6-phosphate deaminase, NagB, from Bacillus subtilis. The structures were determined in ligand-complexed forms at resolutions around 1.4 Angstroms. BsuNagB is monomeric in solution and as a consequence is active (k(cat) 28 s(-1), K(m(app)) 0.13 mM) without the need for allosteric activators. A decrease in activity at high substrate concentrations may reflect substrate inhibition (with K(i) of approximately 4 mM). The structure completes the NagB superfamily structural landscape and thus allows further interrogation of genomic data in terms of the regulation of NagB and the metabolic fate(s) of glucosamine 6-phosphate.

Aldose-Ketose Isomerases↗
PubMed2006

Separation and identification of major plant sphingolipid classes from leaves.

Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for approximately 64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.

Anions↗
PubMed2000

The pgdA gene encodes for a peptidoglycan N-acetylglucosamine deacetylase in Streptococcus pneumoniae.

Analytical work on the fractionation of the glycan strands of Streptococcus pneumoniae cell wall has led to the observation that an unusually high proportion of hexosamine units (over 80% of the glucosamine and 10% of the muramic acid residues) was not N-acetylated, explaining the resistance of the peptidoglycan to the hydrolytic action of lysozyme, a muramidase that cleaves in the glycan backbone. A gene, pgdA, was identified as encoding for the peptidoglycan N-acetylglucosamine deacetylase A with amino acid sequence similarity to fungal chitin deacetylases and rhizobial NodB chitooligosaccharide deacetylases. Pneumococci in which pgdA was inactivated by insertion duplication mutagenesis produced fully N-acetylated glycan and became hypersensitive to exogenous lysozyme in the stationary phase of growth. The pgdA gene may contribute to pneumococcal virulence by providing protection against host lysozyme, which is known to accumulate in high concentrations at infection sites.

Acetylation↗
PubMed2004

High-throughput functional genomics identifies genes that ameliorate toxicity due to oxidative stress in neuronal HT-22 cells: GFPT2 protects cells against peroxide.

We describe a novel genetic screen that is performed by transfecting every individual clone of an expression clone collection into a separate population of cells in a high-throughput mode. We combined high-throughput functional genomics with experimental validation to discover human genes that ameliorate cytotoxic responses of neuronal HT-22 cells upon exposure to oxidative stress. A collection of 5,000 human cDNAs in mammalian expression vectors were individually transfected into HT-22 cells, which were then exposed to H(2)O(2). Five genes were found that are known to be involved in pathways of detoxification of peroxide (catalase, glutathione peroxidase-1, peroxiredoxin-1, peroxiredoxin-5, and nuclear factor erythroid-derived 2-like 2). The presence of those genes in our "hit list" validates our screening platform. In addition, a set of candidate genes was found that has not been previously described as involved in detoxification of peroxide. One of these genes, which was consistently found to reduce H(2)O(2) -induced toxicity in HT-22, was GFPT2. This gene is expressed at significant levels in the central nervous system (CNS) and encodes glutamine-fructose-6-phosphate transaminase (GFPT) 2, a rate-limiting enzyme in hexosamine biosynthesis. GFPT has recently also been shown to ameliorate the toxicity of methylmercury in Saccharomyces cerevisiae. Methylmercury causes neuronal cell death in part by protein modification as well as enhancing the production of reactive oxygen species (ROS). The protective effect of GFPT2 against H(2)O(2) toxicity in neuronal HT-22 cells may be similar to its protection against methylmercury in yeast. Thus, GFPT appears to be conserved among yeast and men as a critical target of methylmercury and ROS-induced cytotoxicity.

Animals↗
PubMed1984

The simultaneous estimation of the amounts of protozoal, bacterial and dietary nitrogen entering the duodenum of steers.

Four steers were given straw and tapioca diets, twice daily, in a 4 X 4 Latin-square design. These diets, containing 4.2 g nitrogen/kg dry matter (DM), were further supplemented with either urea, decorticated groundnut meal (DCGM), untreated (UT) casein or formaldehyde-treated (FT) casein to give a total of 19.7 g N/kg DM and 10.5 MJ/kg DM daily. Concurrent samples of rumen bacteria and protozoa and abomasal digesta were collected for each period of the experiment and the concentrations of 2-aminoethyl phosphonic acid (AEPA), diaminopimelic acid (DAPA), total nitrogen (TN), total phosphorus (TP), amino acids and hexosamines were determined in the dried preparations. The nature of the dietary supplements had little effect on the concentrations of most of these constituents or on the total protozoal numbers. Abomasal digesta samples marked with polyethylene glycol (PEG) and chromic oxide for flow estimation were collected over 24 h, and the proportions of protozoal-N, bacterial-N and microbial-N estimated simultaneously using the markers AEPA, DAPA and RNA respectively. These digesta-N components were also estimated using an amino acid profiling (AAP) method which gave, in addition, estimates of the dietary and endogenous components. For the diets containing casein, the proportion of dietary casein was estimated directly using casein-P as a marker. Estimates of the respective mean proportions of microbial-N in abomasal digesta non-ammonia-N (NAN) for the diets containing urea, DCGM, UT casein or FT casein were: AEPA 0.56, 0.32, 0.27 and 0.16; DAPA 0.88, 0.70, 0.81 and 0.57; RNA 0.98, 0.85, 0.92 and 0.53. Giving FT casein significantly (P less than 0.001) increased the flow of casein-N at the abomasum and a significantly (P less than 0.001) greater proportion of casein-N was found in abomasal NAN (0.51 v. 0.09) where FT rather than UT casein was given. The AAP method gave results for the proportions of microbial- and dietary-N (where casein was given) which were, in general, slightly lower than those obtained using RNA and casein-P as markers. Agreement with estimates of bacterial protein (from DAPA) and of protozoal protein (from AEPA) was less satisfactory. Comparisons of the various estimates of the proportions of microbial-N in abomasal digesta suggested that the results obtained for protozoal-N by AEPA were overestimates. AEPA was found in mixed rumen bacteria which may have accounted in part for these overestimates. However, AEPA was not detected in any of the dietary ingredients.

Abomasum↗
PubMed1986

The effects of dietary acid stress on bone metabolism in young ovariectomized and intact rats.

1. Two studies were performed to determine the effects of acid stress and ovariectomy on bone metabolism in young rats. In Expt 1, eighteen female weanling Sprague-Dawley-descended rats were ovariectomized, placed in one of three dietary groups and given a diet containing (g/kg): 6 calcium and 3, 6 or 12 phosphorus for 10 weeks. In Expt 2, thirty-two female weanling Sprague-Dawley-descended rats were ovariectomized, and thirty-two were left intact. Eight rats from each group were given a diet containing (g/kg): 6 Ca and 3 P, 12 P, 3 P + 1 S or 3 P + 20 ammonium chloride for 10 weeks. 2. Feeding diets containing 12 g P/kg resulted in decreased serum Ca and increased urinary P with no changes in femur composition. Feeding high-sulphate and ammonium chloride diets resulted in increased urinary Ca and, when combined with ovariectomy, lower femur Ca and P with no changes in femur hydroxyproline or hexosamines. 3. The findings reflect the more rapid turnover of the amorphous calcium phosphate salts found in greater amounts in bones of younger animals than for more stable apatitic crystals that predominate in bones of older animals.

Acids↗
PubMed1975

An antigenic antimorphogenetic bone hydrophobic glycopeptide (AHG).

A hydrophobic glycopeptide, isolated from fresh marrow-free cortical bone of the rat, extinguished the bone morphogenetic response of insoluble bone matrix gelatin which otherwise invariably produces new bone from migrating mesenchymal cells after implantation in muscle of the rat. Incubation of the 10 to 100 mug of the glycopeptide per milligram of bone gelatin in phosphate or Tris-HCl buffer, pH = 7.4, for 24 hours recombines the hydrophobic polypeptide and gelatin. Incubated with doses of 50 to 100 mug, implants of gelatin in Sprague-Dawley recipients are rapidly resorbed by a lymphocyte-plasma cell-macrophage infiltrate without developing any new bone. With 10 to 20 mug, infiltrate appears and bone develops inversely in proportion to the dose. Bone develops at all dose levels only when bone matrix gelatin and glycopeptide were prepared from an inbred strain of Lewis rats and recipients were of the same strain, and thereby suggests that only allogeneic AHG, produces an antigenic antimorphogenetic resonse. The glycopeptide was isolated from bone together with lipids and other hydrophobic proteins or polypeptides by chloroform-methanol (1:1) extraction and further purified by chromatography on ion-exchange columns and molecular sieves. The molecular weight established by Sephadex G-100 filtration and by calibrated dialysis is about 5000 daltons. Analysis of the biologically active fraction shows 75% of proteinaceous material and close to 25% carbohydrate with trace amounts of hexosamines and sialic acid. Amino acid analysis shows high levels of alanine, valine and leucine in comparison to other chloroform-methanol extractable but immunogenically relatively-inactive fractions.

Amino Acids↗
PubMed1976

A single column packing for the gas-liquid chromatographic separation of various biologically important compounds.

The use of a single, commercially available column packing, TabsorbR, is described for the g.l.c. separation of a large number of different compounds. The resolution of the homologous members of the following series of compounds was achieved: (1) saturated fatty acids (C1-C18), (2) normal aliphatic saturated dicarboxylic acids (C2-C14), (3) normal aliphatic saturated alcohols (C1-C24), (4) normal aliphatic saturated amines (C1-C12), (5) the common amino acids except arginine, histidine and cysteine, (6) aliphatic hydrocarbons (C10-C20) and (7) monosaccharides. It should be noted that twenty-two monosaccharides including three hexosamines and two anhydrohexoses, could be resolved as alditol acetates in a single run. In addition, galacturonic, glucuronic and iduronic acids could be separated from one another as their 1,4-lactones. The resolution achieved in these series of compounds was found to be consistent and highly reproducible. It is of further interest that certain isomers of the higher fatty acids and hydrocarbons with one double bond could also be separated from the normal and saturated compounds, respectively. The applicability of "Tabsorb" for the g.l.c. separation, although noted above to be considerably broad, is by far not yet exhausted. These procedures which form the basis for the quantitative determinations of the various compounds studied as demonstrated by analysis of glycopeptides for neutral hexoses and proteins for the amino acids, can readily be adapted to preparative methods. From the biochemical point of view "Tabsorb" is an extremely versatile column packing in that it can be used for the identification of many of the common building blocks of natural products.

Alcohols↗
PubMedDate not supplied

Poly(vinylbenzyl chloride) microsphere synthesis and their chemical modifications.

Vinylbenzyl chloride (VBC) was dispersion polymerized to give monodisperse microspheres in the presence of poly(vinylpyrrolidone) (PVP) as a steric stabilizer. The effect of PVP concentration on the size and on microsphere stability during the polymerization process was investigated. Microsphere size was examined when co-stabilizer molecules were employed with PVP during the polymerization reaction. The built-in reactive chloromethyl groups of the microspheres were the sites of the nucleophilic reaction of two amino-group model molecules, glucosamine (G), a hexosamine implicated in processes of molecular recognition, and also bovine serum albumin (BSA). Elemental analyses and Fourier transform infrared spectroscopy (FTIR) spectra showed that poly(vinylpyrrolidone) was associated with the microsphere network. Elemental analyses, attenuated total reflection infrared spectroscopy (ATR-FTIR), and zeta potential measurements confirmed G and BSA links at the microsphere surface.

Capsules↗
PubMed2006

A preliminary study on antimetastatic activity of Thuja occidentalis L. in mice model.

The effect of Thuja occidentalis extract on the inhibition of lung metastasis induced by B16F-10 melanoma cells was studied in C57BL/6 mice. The extract was administered by three different modalities. A remarkable reduction in tumor-nodule formation was shown by simultaneous (74.4%) and prophylactic (71.5%) mode of administration. The effect was comparatively low in drug administration after tumor development (60.2%). Increased lung collagen hydroxyproline (21.13 microg/mg protein) in the metastasized lungs of control animals compared with normal animals (0.98 microg/mg protein) was significantly reduced in Thuja-treated animals. The elevated level of uronic acid (349.5 microg/100 mg tissue) and hexosamine contents in metastatic control animals was significantly reduced in the animals treated with alcoholic extract of Thuja. Similarly the elevated levels of serum sialic acid and serum gamma glutamyl transpeptidase activity in the untreated control animals was significantly reduced in the animals treated with the extract of Thuja. The lifespan of the Thuja treated animals also was seen to be significantly increased.

Animals↗
PubMed2000

Sucrose has no beneficial effects on wound healing in rats.

OBJECTIVE: To evaluate the effects of sucrose treatment on the formation of granulation tissue in a standard wound model. DESIGN: Animal study. SETTING: University hospital, Finland. ANIMALS: 32 male Sprague-Dawley rats divided into 4 groups. INTERVENTIONS: Implantation of viscose cellulose sponge subcutaneously, and daily injection of three concentrations of sucrose (0.01, 0.1 or 1 M) or vehicle for 7 days. MAIN OUTCOME MEASURES: The amount of granulation tissue measured by chemical analysis and histology. The amount and distribution of types I and III collagen assayed by immunofluorescence. RESULTS: None of the three concentrations altered the amounts of DNA, RNA, hydroxyproline, nitrogen, hexosamines, and uronic acids in granulation tissue. Neither improvement nor deterioration was seen in the growth of granulation tissue in histological specimens. The amount and distribution of types I and III collagen was similar in controls and sucrose-treated rats. Type III collagen was most abundant near newly-formed vessels. Neither sucrose nor fructose was found in wound fluid while the concentration of glucose was significantly lower in all test groups than in controls. CONCLUSIONS: Sucrose solution had neither beneficial nor deleterious effects on the amount of developing granulation tissue in an experimental wound model. The amount and distribution of types I and III collagens were also not altered by sucrose treatment.

Animals↗
PubMed2006

Hyperglycemia and glucosamine-induced mesangial cell cycle arrest and hypertrophy: Common or independent mechanisms?

The Hexosamine Pathway (HP) is one hypothesis proposed to explain glucose toxicity and the alterations observed during the course of diabetic microvascular complication development. Glucosamine is a precursor of UDP-N-Acetylglucosamine (UDP-GlcNAc), the main product of the HP that has often been used to mimic its activation. The transfer of a UDP-GlcNAc residue onto proteins (O-GlcNAc modification) represents the final step of the HP and is considered as a major mechanism by which this pathway exerts its signalling effects. While it is well accepted that the HP promotes extracellular matrix accumulation in the context of diabetic nephropathy, its involvement in the perturbations of cell cycle progression and hypertrophy of renal cells has been poorly investigated. Nevertheless, in a growing number of studies, the HP and O-GlcNAc modification are emerging as important regulators of cell cycle progression. This review will focus on the role of glucosamine and O-GlcNAc modification in cell cycle regulation in the context of diabetic nephropathy. Special emphasis will be given into the role of the HP as a potential mediator of the effects of high glucose on the perturbations of renal cell growth.

Animals↗
PubMed1987

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures.

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.

Animals↗
PubMed1995

Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells.

A phosphooligosaccharide has been proposed as a second messenger of insulin. It is believed to be structurally related to the carbohydrate moiety of phosphatidylinositol glycan anchors of many cell surface proteins. Herein we demonstrate that [32]phosphate in freshly isolated adipocytes and [3H]galactose in cultured hepatoma cells (H4IIE) labeled the same set of three different glycolipids. With all three, the radiolabel was made water soluble by phosphatidylinositol(glycan)-specific phospholipase C or D catalyzed hydrolysis. We isolated the three phospholipase C-released substances. One of them was susceptible to nitrous acid deamination, indicative of a hexosamine with a free amino group. This phosphooligosaccharide structure had an apparent molecular mass between tetra- and pentaglucose by gel filtration. By anion-exchange chromatography it was separated into two differently charged and interconvertible species. Adipocytes stimulated with insulin accumulated the nitrous acid sensitive phosphooligosaccharide: after stimulation the intracellular level of free phosphooligosaccharide increased threefold within 5 min, fell off during the next few minutes and then remained at a slightly elevated level. After insulin stimulation the intracellular concentration of free phosphooligosaccharide was > 1,000-fold higher than in the incubation medium. When prepared from rat livers on a preparative scale, the oligosaccharide was also found to exhibit insulinomimetic effects on protein phosphorylation of insulin target proteins in intact adipocytes. After subcellular fractionation of adipocytes the lipid-bound [32P]phosphooligosaccharide of the plasma membrane was found to be localized in plasma membrane domains apparently corresponding to caveolae. Lipid-bound [32P]phosphooligosaccharide was found also in the microsomal fraction.

Adipocytes↗