Separation of hexosamines, hexosaminitols and hexosamine-containing di- and trisaccharides on an amino acid analyser.
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Amino Acids↗
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Production of [3H]hexosamine-labeled proteoglycans by cultures of normal and diabetic skin fibroblasts: dilution of exogenous [3H]glucosamine by endogenous hexosamine from glucose and other sources.
Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with [3H]glucosamine and [35S]-sulfate for varying periods of time. Incorporation of 3H into macromolecules appearing in the medium was linear after approximately 45 min, and incorporation of 35S was linear after approximately 30 min. The amounts of 35S-proteoglycan formed by each of the cultures during 5-h incubations were compared and were found to be fairly similar for the six lines, varying from 0.08 to 0.14 nmol sulfate/microgram DNA. Isolated 3H,35S-glycosaminoglycans were then treated with chondroitin ABC lyase to characterize the location and degree of sulfation. Results indicated a considerable variation in completeness of chondroitin/dermatan sulfation and in proportions of 6-sulfation to 4-sulfation among the various lines. However these variations did not seem to be related to whether the cells were from normals or diabetics. 3H,35S-Labeled disaccharides were isolated and ratios of 3H to 35S determined in order to calculate the [3H]glucosamine dilution by endogenous glucosamine derived from glucose or other sources during the period of incubation. Dilutions varied widely from 160- to 635-fold among the different cell lines, but the variations did not seem to be related to whether the cells were from normals or diabetics.
Adult↗
Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (4) adenosine 3':5'-cyclic monophosphate independent stimulation by prostaglandin F2alpha.
The mechanism of the stimulatory effect of prostaglandin PG) F2alpha on the production of hexosamine-containing substances by cultured fibroblasts was studied with special reference to adenosine 3':5'-cyclic monophosphate (cAMP). At the stationary phase, the cells were exposed for 6 hrs to PGF2alpha, E1, cAMP or dibutyryl-cAMP in a wide range of concentrations. cAMP itself showed a slight stimulation on the production of hexosamine-containing substances, and the effect was enhanced by using the dibutyryl derivative. PGF2alpha had much a greater capacity than either the exogeneous cAMP or the dibutyryl-cAMP for enhancing the production of hexosamine-containing substances. To know whether cAMP is involved in the stimulatory effect of PGF2alpha, intracellular cAMP level was concomitantly measured in both PGF2alpha and PGE1 treated cultures. Although the cellular cAMP level in PGE1 treated cultures was much higher than that in the PGF2alpha treated cultures, the stimulatory effect on the production of hexosamine-containing substances in PGE1 treated cultures was always much smaller than that in the PGF2alpha treated cultures. Moreover, PGF2alpha had a significant stimulatory effect on the production of hexosamine-containing substances even at a low concentration as 100 pg/ml, which is small enough not to increase any cellular cAMP level. From these results, it was concluded that the stimulatory effect of PGF2alpha on the production of hexosamine-containing substances by cultured fibroblasts is not mediated by cAMP and is caused by a mechanism different from that caused by cAMP.
Animals↗
Gastric mucosal hexosamine content in various liver diseases.
Since gastric mucosal lesions are frequently encountered in patients with liver disease, we measured the levels of gastric mucosal hexosamine. In chronic hepatitis patients, hexosamine levels were reduced in both the antrum and corpus as compared with those in normal controls, while values in the advanced liver cirrhosis group (total bilirubin greater than 5 mg/dl) were lower than in the less advanced group. Although the presence or absence of esophageal varices had no influence on hexosamine, higher concentrations were found in patients with the red color sign (+) in comparison with those with negative red color sign (-). One month after endoscopic injection sclerotherapy of esophageal varices, hexosamine did not change, but decreases were seen in both the antrum and corpus at 3 months. We observed an increase in gastric mucosal blood flow after treatment with teprenone, a new antiulcerative agent, in normal controls. Gastric mucosal hexosamine increased significantly after teprenone treatment in both chronic hepatitis and liver cirrhosis groups. From these results, we conclude that hexosamine has a defensive action against gastric lesions in various liver diseases.
Anti-Ulcer Agents↗
Coordinated regulation of glutamine:fructose-6-phosphate amidotransferase activity by insulin, glucose, and glutamine. Role of hexosamine biosynthesis in enzyme regulation.
We reported previously that glutamine:F-6-P amidotransferase (GFAT) plays an integral role in the development of insulin resistance by directing the flow of incoming glucose into the hexosamine biosynthesis pathway. To determine whether the enzymatic activity of GFAT is altered during desensitization of the glucose transport system, we treated isolated rat adipocytes with various combinations of insulin, glucose, and glutamine. Treatment with insulin or glucose alone (or in combination) failed to reduce cytosolic GFAT activity after 4 h, whereas combined treatment with all three components elicited a progressive loss of GFAT activity that was rapid (t1/2 of 2 h) and extensive (70% loss). A pronounced loss of GFAT activity was also seen in cells exposed to glucosamine, an agent known to directly enter the hexosamine pathway (55% loss at 4 h, ED50 of 360 microM). Moreover, a close correlation was observed between the induction of desensitization and the loss of GFAT activity as a function of glucose, insulin, glutamine, and glucosamine concentrations. When total intracellular hexosamine products were measured, we found that hexosamine formation was unaltered by insulin or glucose (or a combination) but was elevated by greater than 4-fold in the presence of insulin, glucose, and glutamine (t1/2 of 22 min), a condition known to cause both desensitization and loss of GFAT activity. Additional studies indicated that the loss of GFAT activity under desensitizing conditions is not due to allosteric regulation since removal of potential allosteric factors from the cytosol of desensitized cells by G-25 column chromatography failed to restore enzyme activity. Overall, these studies indicate that 1) GFAT is an insulin-regulated enzyme; however, control of enzyme activity is not due to a direct action of insulin, but rather is mediated by insulin-induced enhancement of glucose uptake; 2) the routing of incoming glucose through the hexosamine pathway and the formation of hexosamine products appears to regulate GFAT activity; and 3) the progressive loss of GFAT activity over several hours is probably not due to allosteric regulation.
Adipose Tissue↗
Quantitative gas chromatographic measurement of glycosaminoglycan hexosamines in urine and plasma.
A method is described for the quantitative determination of urine and plasma glycosaminoglycans (GAGs) by gas chromatography of the acetylated amino sugars. GAGs were first recovered by precipitation from urine with alkyltrimethylammonium bromide and from plasma by mini-column chromatography after papain digestion. Urine samples (24) analysed for total hexosamines by gas chromatography and for uronic acid by colorimetry had a correlation coefficient of 0.85. The within-run coefficient of variation (C.V.) for nineteen samples from a pooled urine was 5.2% for total hexosamines and that for the ratio of galactosamine to total hexosamines was 3.7%. The corresponding C.V. values for twelve plasma samples from a common pool were 6.5 and 3.7%. The mean ratio of galactosamine to total hexosamine in ten pre-breakfast spot urines was 51.5%. The corresponding ratio in the plasma from twenty adolescent blood donors was 76.3% and the mean total hexosamine content of the GAGs was 47.36 mumol/l.
Adult↗
Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance.
Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate by the first and rate-limiting enzyme of the hexosamine pathway, glutamine:fructose-6-phosphate amidotransferase.
Animals↗
HPLC analysis of hexosamine phosphates in biological samples.
Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.
Animals↗
Effect of strain variation and growth phase of culture on dry weight and hexosamine content of cell wall layers of a marine pseudomonad.
Two variants of marine pseudomonad B-16 (ATCC 19855) differing in that one, variant 3, formed opaque colonies and the other, variant 7, formed translucent colonies were examined to determine if the variants differed in the amount and hexosamine content of their three outer cell wall layers. In both variants, the three outer layers of the cell wall, the loosely bound outer layer, the outer double-track layer, and the underlying (periplasmic space) layer contributed less to the dry weight of the cells when the cells were harvested in the stationary than in the logarithmic phase of growth. The hexosamine content of the layers of variant 3 increased dramatically as the cells went from the logarithmic to the stationary phase. The hexosamine content of the layers of variant 7 changed little by comparison. Thus cells of the variant which forms opaque colonies enrich the outer layers of their cell wall with hexosamine when grown to stationary phase.
Cell Wall↗
Insulin regulation of pyruvate kinase activity in isolated adipocytes. Crucial role of glucose and the hexosamine biosynthesis pathway in the expression of insulin action.
We recently identified glutamine:fructose-6-phosphate amidotransferase (GFAT) as an insulin-regulated enzyme in adipocytes. Moreover, we found that loss of GFAT activity is not due to a direct action of insulin but rather is mediated by enhanced glucose uptake and the subsequent routing of glucose through the hexosamine biosynthesis pathway. To assess whether other cytosolic enzymes are controlled through formation of hexosamine products, we treated adipocytes for 5 h with physiological concentrations of insulin (ED50 = 0.33 ng/ml), glucose (ED50 = 4.5 mM), and glutamine (ED50 = 4.4 mM) and then measured pyruvate kinase (PK) activity. Combined treatment resulted in a progressive (t 1/2 of 2.5 h) and marked (3-fold) increase in PK activity, whereas omission of one or more of these components failed to alter enzyme activity. Several lines of additional evidence implicated the hexosamine biosynthesis pathway in PK regulation; therefore, it appears that the M2 isoform of pyruvate kinase represents another enzyme regulated by insulin through stimulation of glucose uptake and formation of hexosamine products. Related studies revealed that enhancement of PK activity is dependent upon ongoing mRNA synthesis and de novo protein synthesis and is mediated by an increase in enzyme content. Considered together, these findings provide new insights into the cascade of metabolic events triggered by insulin and implicated a novel metabolic pathway in the pretranslational control of enzyme function.
Adipose Tissue↗
[Effect of hydrocortisone on the metabolic indices of hexosamine-containing biopolymers in the stomach].
The experimental findings showed that hydrocortisone effect on metabolism of hexosamine-containing biopolymers of the stomach was dose-dependent. When administered in a small dose (0.2 mg/kg body weight) it increased the activity of biosynthesis of hexosamines, which are necessary for the formation of hexosamine-containing biopolymers, and also increased the total content in the gastric wall of biopolymers, glycosaminoglycans and glycoproteins soluble in perchloric acid. Administration of a large dose of the hormone (50 mg/kg) produced opposite changes in the studied tissue. In patients with peptic ulcer of the stomach and duodenum, exacerbation of which occurred against the background of stress-induced hypercorticosteroidemia, there was observed a decrease of glycosaminoglycan content in the gastric juice and mucosa. A decrease of hexosamine content in glycoproteins was noted.
11-Hydroxycorticosteroids↗
Relaxant effects of hexosamines on isolated small human placental arteries.
Wharton's jelly contains large amounts of hyaluronic acid, and glucosamine is an important constituent of this macromolecule. In order to evaluate the placental vascular effects of this aminosugar, small chorionic and stem villous arteries were dissected from placental specimens obtained at normal term vaginal deliveries (n = 15). Ring preparations were mounted in organ baths, and isometric wall tensions were measured. Glucosamine and its epimer galactosamine (5 X 10(-4) to 10(-2) M) produced marked relaxation of contractions induced by PGF2 alpha (10(-5) M) in both chorionic and stem villous arteries. The effect was unchanged after pretreatment with atropin, propranolol and indomethacin. The relaxant effect of the neutral sugar mannose was less pronounced compared with that of the hexosamines. Total tissue concentrations of placental hexosamines have been reported within the range needed to produce placental vascular relaxation in the present study. However, the major part of these compounds is integrated in macromolecules, and the tissue level of free hexosamine is probably far below the total concentrations. Accordingly, the effects of hexosamines demonstrated in the present study might not be of physiological importance in the regulation of fetal placental medial smooth muscle tension.
Arteries↗
Mass spectrometry of hexosamine containing oligosaccharides as permethylated N-trifluoroacetyl derivatives.
Permethylated N-trifluoroacetyl hexosamine containing oligosaccharide alditols are favorable for analysis by mass spectrometry. Electron impact and fast atom bombardment mass spectra of these derivatives are characterized by strong primary and secondary fragments in which a positive charge is localized on the N-trifluoroacetyl hexosamine residue. From the mass spectra the monosaccharide sequence and the position(s) of substitution of a N-trifluoroacetyl hexosamine can be determined.
Carbohydrate Sequence↗
Identification of a novel hepraglycosylceramide with two fucose residues and a terminal hexosamine.
A polar fucose-containing glycosphingolipid fraction isolated from dog small intestine has been characterized by mass spectrometry of intact methylated, and methylated and reduced (LiAlH4) glycolipid. The native fraction, which was homogenous on thin-layer chromatography, was shown after methylation to be a mixture of two compounds. One was identified as a hexaglycoslyceramide with the following composition and sequence: fucose-hexose(fucose)-hexosamine-hexose-hexose-ceramide, with a terminal saccharide structure similar to blood group Leb determinants. The second compound was a novel heptaglycosyceramide with the sequence: hexosamine(fucose)-hexose-tfucose)-hexosamine-hexose-hexose-ceramide. This glycolipid was also detected in human small intestine and pancreas. The dog intestinal fraction had phytosphingosine as its major base and contained almost exclusively 2-hydroxy fatty acids (16 : 0--24 : 0). The fraction of human pancreas differed in having spingosine as its major base and normal fatty acids (16 : 0--24 :0) as major acids.
Animals↗
Stimulative effect of prostaglandins on production of hexosamine-containing substances by cultured fibroblasts (2) early effect of various prostaglandins at various doses.
Early effects of various prostaglandins on the production of hexosamine-containing substances by cultrued fibroblasts, which were derived from a rat carrageenin granuloma, were studied. At hte stationary phase, the cells were exposed for 6 h to one of the prostaglandin A1 (PGA1), A2, B1, B2, D2, F1 alpha, E1, E2 or arachidonic acid in various concentrations ranging from 0.01 to 10 microng/ml for all the stimuli and from 10 pg to 10 microng/ml for PGF2alpha. The activity of the cells in incorporating 3H-glucosamine into hexosamine-containing substances (acidic glycosaminoglycans and glycoproteins) during this period was compared with that of control cells. All the stimuli tested showed more or less stimulative effect on the synthesis of hexosamine-containing substances at their specific concentrations. PGF2alpha was found to be the most potent stimulant and its stimulative effect was found significant even at the low concentration of 100 pg/ml. PGD2, F1alpha and E2 were the next potent stimuli. Their optimum dose were aroung 1 microng/ml but they still had significant stimulation at the concentration of 0.01 microng/ml. Effect of PGE2 was rather mild. Stimulation by PGA1, A2, B1 and B2 or arachidonic acid was seen at high dose, and it seemed to be non-specific. The results suggested that these prostaglandins such as PGF2alpha, D2, F1alpha and E2 play some important role on regulating the production of intercellular ground substances.
Animals↗
Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (3) induction of hyaluronic acid synthetase by prostaglandin F2alpha.
The mechanism of the stimulatory effect of prostaglandin(PG)F2alpha on the production of hexosamine-containing substances by cultured fibroblasts was studied. Treatment of the cells with 1 microgram/ml of PGF2alpha resulted in a doubled net synthesis of acidic glycosaminoglycans during 20 hrs measured with uronic acid as index, and also resulted in 300 per cent increase of 3H-glucosamine incorporation into hexosamine-containing substances during the first 6 hrs. Fractionation of the PGF2alpha-stimulated hexosamine-containing substances with double isotope technique revealed that hyaluronic acid was the most stimulated component. Prior to the increase of hyaluronic acid, hyaluronic acid synthetase activity was found to be augmented by PGF2alpha as high as 4 times over the control. The augmentation of hyaluronic acid synthetase activity by PGF2alpha did not take place if actinomycin D was simultaneously present in the culture medium, suggesting that PGF2alpha induced the enzyme.
Animals↗
Simultaneous determination of neutral sugars and hexosamines in glycoproteins and acid mucopolysaccharides (glycosaminoglycans) by gas-liquid chromatography.
A reliable and reproducible method for the simultaneous determination of neutral sugars and hexosamines in glycoproteins and acid mucopolysaccharides has been described. It involves the following steps: the release of neutral sugars and hexosamines from biopolymers by resin-catalysed hydrolysis, the nitrous acid deamination of resin-bound hexosamines in this hydrolysate to aldoses, and the determination of these newly formed anhydroaldoses together with the pre-existing neutral sugars as aldononitrile acetates by gas chromatography. Applications of this method to analyses of glycoprotein from urine of schizophrenic patients, bovine thyroglobulin, human brain glycoprotein, chondroitin sulfates from bovine vitreous humor, and veal brain, and human umbilical cord hyaluronic acid are given.
Animals↗
Chitin biosynthesis during Blastocladiella zoospore germination: evidence that the hexosamine biosynthetic pathway is post-translationally activated during cell differentiation.
De novo construction of a chitinous cell wall accompanies Blastocladiella emersonii zoospore germination. At least an order of magnitude increase in total hexosamine occurs during germination. This increase is into polymer (chitin) and occurs on schedule in the presence of cycloheximide. Uridine-5'-diphospho-N-acetylglucosamine (UDPGlcNAc), both the end product of hexosamine biosynthesis and a substrate for chitin biosynthesis, is a potent inhibitor of the activity of the first pathway-specific enzyme of hexosamine biosynthesis in zoospore extracts. Certain uridine nucleotides, not perceptibly influencing the activity of the first enzyme per se, counteract the inhibitory effects of UDPGlcNAc. The concentration of UDPGlcNAc in the zoospore is sufficient to act as an inhibitor of the enzyme, but the amount of UDPGlcNAc is sufficient, by over an order of magnitude, to account for the chitin synthesized during germination. Both the production of UDPGlcNAc and its utilization for chitin synthesis appear to be post-translationally regulated in zoospores and during zoospore germination.
Blastocladiella↗