Tissue-specific effects of estrogen on glycerol channel aquaporin 7 expression in an ovariectomized mouse model of menopause.

OBJECTIVE: Elevated fat mass and redistribution of body fat are commonly observed in postmenopausal women. Aquaporin 7 (AQP7), a unique glycerol permeable integral membrane protein, has been associated with the onset of obesity. We hypothesized that estrogen supplementation could counteract this fat accumulation and redistribution through tissue-specific modulation of AQP7. METHODS: We measured fat depot weight, adipocyte size, and the expression of AQP7 and glycerol kinase (GK) in visceral and subcutaneous fat tissues of ovariectomized mice supplemented with or without 17ß-estradiol. RESULTS: Removal of the ovaries resulted in a significant decrease in AQP7 expression and an increase in GK expression in visceral adipocyte tissue; expression of AQP7 and GK in subcutaneous adipose tissue remained unaltered. Supplementation with estrogen significantly restored the visceral, but not subcutaneous, fat depot mass and adipocyte size to those of sham-operated mice. A marked increase in the expression of AQP7 and a reduction of GK were observed selectively in the visceral fat depots in estrogen-treated mice. CONCLUSIONS: Our results suggest that estrogen has tissue-specific effects on AQP7 expression, and modulation of AQP7 by estrogen alters the balance of adipocyte metabolism between adipose tissue depots.

MicroRNA-135b exerts oncogenic activity in glioblastoma via the inhibition of glycerol kinase 5 expression.

Glioblastoma is the most common primary malignancy of the adult central nervous system and is associated with a markedly poor prognosis. Elucidating the pathogenesis and molecular changes will assist in further understanding the pathogenesis and progression of the disease and offer novel targets for therapies. The present study demonstrated that the expression level of GK5 was lower in high-grade glioblastoma tissues compared with low-grade ones and it can promote proliferation in glioblastoma cells. The regulatory mechanism of GK5 in glioblastoma were also investigated. It was revealed that GK5 is a target of miR-135b in U87MG glioblastoma cells. Controry to GK5, the expression of miR-135b is upregulated in glioblastoma and its expression is positively associated with the grade of the disease. Finally, it was demonstrated that miR-135b promoted the proliferation of U87MG cells. Therefore, miR-135b may function as an oncogene by inhibiting GK5 in glioblastoma.

Human and murine glycerol kinase: influence of exon 18 alternative splicing on function.

Glycerol kinase (GK) is a key enzyme in glycerol metabolism with two alternatively spliced forms-one with an 87bp insertion corresponding to exon 18 (GK+EX18), and one lacking exon 18 (GK-EX18). We report the expression of GK+/-EX18 in various tissues and cell lines, as well as their enzymatic characteristics and subcellular localization. RT-PCR revealed differential expression in tissues and cell lines. Northern blot analysis revealed that both forms of the murine ortholog, Gyk, were highly expressed in murine heart and increased during embryonic development. K(m) values for glycerol for GK+/-EX18 were not significantly different, although GK-EX18 had a higher V(max) for glycerol. GK-EX18 had a lower K(m) and V(max) for ATP than GK+EX18. Immunofluorescence experiments showed that GK+EX18 co-localized to the mitochondria and the perinuclear region while GK-EX18 had a diffuse expression pattern. These data suggest specific and divergent roles for GK+EX18 and GK-EX18 in cellular metabolism and development.

sn-glycerol-1-phosphate-forming activities in Archaea: separation of archaeal phospholipid biosynthesis and glycerol catabolism by glycerophosphate enantiomers.

In Methanobacterium thermoautotrophicum, sn-glycerol-1-phosphate (G-1-P) dehydrogenase is responsible for the formation of the Archaea-specific backbone of phospholipids, G-1-P, from dihydroxyacetonephosphate (DHAP). The possible G-1-P-forming activities were surveyed in cell-free extracts of six species of Archaea. All the archaeal cell-free homogenates tested revealed the ability to form G-1-P from DHAP. In addition, activities of G-3-P-forming glycerol kinase and G-3-P dehydrogenase were also detected in four heterotrophic archaea, while glycerol kinase activity was not detected in two autotrophic methanogens. These results show that G-1-P is produced from DHAP by G-1-P dehydrogenase in a wide variety of archaea while exogenous glycerol is catabolized via G-3-P.

Quantification of the regulation of glycerol and maltose metabolism by IIAGlc of the phosphoenolpyruvate-dependent glucose phosphotransferase system in Salmonella typhimurium.

The amount of IIAGlc, one of the proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS), was modulated over a broad range with the help of inducible expression plasmids in Salmonella typhimurium. The in vivo effects of different levels of IIAGlc on glycerol and maltose metabolism were studied. The inhibition of glycerol uptake, by the addition of a PTS sugar, was sigmoidally related to the amount of IIAGlc. For complete inhibition of glycerol uptake, a minimal ratio of about 3.6 mol of IIAGlc to 1 mol of glycerol kinase (tetramer) was required. Varying the level of IIAGlc (from 0 to 1,000% of the wild-type level) did not affect the growth rate on glycerol, the rate of glycerol uptake, or the synthesis of glycerol kinase. In contrast, the growth rate on maltose, the rate of maltose uptake, and the synthesis of the maltose-binding protein increased two- to fivefold with increasing levels of IIAGlc. In the presence of cyclic AMP, the maximal levels were obtained at all IIAGlc concentrations. The synthesis of the MalK protein, the target of IIAGlc, was not affected by varying the levels of IIAGlc. The inhibition of maltose uptake was sigmoidally related to the amount of IIAGlc. For complete inhibition of maltose uptake by a PTS sugar, a ratio of about 18 mol of IIAGlc to 1 mol of MalK protein (taken as a dimer) was required.

[Changes in enzyme systems and lipogenesis metabolites in experimental tuberculosis]. / Izmenenie nekotorykh fermentnykh sistem i metabolitov lipogeneza pri éksperimental'nom tuberkuleze.

Lung tissue phospholipid metabolism has been studied under the conditions of experimental tuberculosis. These investigations were combined with the simultaneous studying of the qualitative and quantitative composition of individual phospholipids, of the changes in the content of glycerophosphate and dioxiacetonphosphate, as well as, of the abnormalities in the activity of glycerokinase and glycerophosphatedehydrogenase, which play an important role in the catalysis of the initial processes of phosphatidogenesis. Our data obtained have shown that formation of tuberculosis process is accompanied by the pronounced increase of glycerokinase and glycerophosphatedehydrogenase activities, which leads to the significant accumulation of the dioxiacetonphosphate and the free glycerol concentrations. It was established by us, that under the conditions of the pathology studied, the significant decrease of the quantity of phosphatidylcholines, phosphatidylethanolamines and phosphatidylserines took place, while the level of sphingomyelins was increased. The results of our investigations shed the light on the up-to-date understanding of the role of different fractions of phospholipids, as well as, of corresponding enzymatic systems in the pathogenesis of lung tuberculosis and in the formation of focus of necrosis in lung tissue.

Congenital adrenal hypoplasia and selective absence of pituitary luteinizing hormone: a new autosomal recessive syndrome.

Congenital hypoplasia of the adrenal glands (CHA) is a rare condition, particularly in the absence of a central nervous system (CNS) anomaly. Two major types of CHA have been described in the setting of an apparently normal CNS and pituitary: a cytomegalic type usually with X-linked recessive inheritance and a miniature adult type that, when hereditary, is an autosomal recessive trait. Glycerol kinase deficiency (GKD) is an X-linked recessive trait, and it may be associated with CHA and adrenal insufficiency, presumably because of deletion of adjacent X-linked loci. We report on three sibling infants, one male and two females, with normal CNS and lethal CHA of the miniature adult type, selective absence of pituitary LH; two of the infants also had glycerol kinase (GK) activity that was decreased but not in the GKD range. Restriction fragment length polymorphism (RFLP) analysis of X chromosome markers located at Xp21-p22 was carried out on the maternal grandfather, both parents, two of three affected infants, and a living normal brother. The results excluded the X-linked type of this disorder associated with GKD in this family. Autosomal recessive inheritance is most likely.

Peroxidase-coupled method for kinetic colorimetry of total creatine kinase activity in serum.

We describe a peroxidase-coupled method involving a colorimetric indicator reaction for determining the total activity of creatine kinase (EC 2.7.3.2) in serum. The kinetically favorable reverse reaction is exploited to generate adenosine 5'-triphosphate, which is used in the glycerol kinase-catalyzed phosphorylation of glycerol. The glycerol 3-phosphate so generated is oxidized in the presence of alpha-glycerophosphate oxidase to produce hydrogen peroxide, which is reduced in the presence of peroxidase with the simultaneous oxidation and coupling of 4-aminoantipyrene and 2-hydroxy-3,5-dichlorobenzenesulfonate to produce an intensely colored red chromogen. Results of the proposed method (y) correlate well with those of the Boehringer-Mannheim "CK-NAC UV" method as applied to the Hitachi 705 chemistry analyzer (y = 1.025 chi - 18.1, r = 0.9985, n = 100, range = 19-4531 U/L). The sensitivity of the method, based on molar absorptivities, is nearly fourfold that of procedures involving the reduction of NADP+.

Glycerol metabolism and osmoregulation in the salt-tolerant yeast Debaryomyces hansenii.

A glycerol-nonutilizing mutant of the salt-tolerant yeast Debaryomyces hansenii was isolated. When subjected to salt stress the mutant produced glycerol, and the internal level of glycerol increased linearly in proportion to increases of external salinity as in the wild-type strain. However, at increased salinity the mutant showed a more pronounced decrease of growth rate and growth yield and lost more glycerol to the surrounding medium than did the wild type. Uptake experiments showed glycerol to be accumulated against a strong concentration gradient, and both strains displayed similar kinetic parameters for the uptake of glycerol. An examination of enzyme activities of the glycerol metabolism revealed that the apparent Km of the sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5) was increased 330-fold for sn-glycerol 3-phosphate in the mutant. Based on the findings, a scheme for the pathways of glycerol metabolism is suggested.

Phospholipid synthesizing enzymes of dermatophytes. III. Glycerol kinase of dermatophytes.

Glycerol kinase, the key enzyme for glycerol use in phospholipid synthesis, was identified in cytosolic fractions of 2 dermatophytes, Microsporum gypseum and Epidermophyton floccosum. Ammonium sulfate was observed to activate and stabilize this enzyme in both dermatophytes. Two pH optima, 8.0 and 10.5, were observed for both dermatophyte enzymes. Glycerol kinase from M. gypseum was purified up to 33-fold with a 225% recovery by ammonium sulfate precipitation and gel filtration. The molecular weight of the enzyme was ca. 4.5 X 10(5). It had 2 pH optima of 8.0 and 10.5. The enzyme had Km values of 0.35 mM and 2.3 mM for glycerol and ATP. Reactivity of the enzyme for various nucleotides was ATP greater than TTP greater than GTP greater than ITP = CTP = UTP. Kinetic studies showed the enzyme to catalyze the reaction by the ping-pong mechanism. Fructose 1,6-bisphosphate and glucose-6-phosphate inhibited the enzyme competitively, whereas glucose was not inhibitory.

Lipid metabolism in fungi.

So far, reviews that have appeared on fungal lipids present data mainly on the lipid composition of these organisms and the influence of lipids on their physiology. These reviews provide little information about the enzymes of lipid metabolism in these organisms and it is assumed, by most workers, that lipid synthesis in all fungi takes place as in Saccharomyces cervesiae, the only fungus in which the complete pathways of phospholipid biosynthesis have been worked out. During the last few years, literature has accumulated on lipid metabolic enzymes of other fungi, as investigators became increasingly interested in this area of research. The present review, after an introduction, will be divided into different sections and each section will deal, comparatively, with various aspects of fungal lipid metabolism and physiology. This review will, therefore, bring out the differences or similarities of lipid metabolism in diverse fungal species.

Glycerol catabolic enzymes and their regulation in wild-type and mutant strains of Streptomyces coelicolor A3(2).

Assay procedure were developed for a soluble glycerol kinase (apparent Km (glycerol) 9 microM) and a probably membrane-associated, NAD-independent L-glycerol-3-phosphate dehydrogenase [apparent Km (L-glycerol 3-phosphate) 7 mM] present in Streptomyces coelicolor A3(2). Both enzymes were cold sensitive. They were co-ordinately induced (about 35-fold) by addition of glycerol to cultures growing on arabinose as sole carbon source. Induction was rifampicin sensitive. The dehydrogenase was absent from glycerol-sensitive mutants, and both kinase and dehydrogenase were absent from glycerol non-utilizing (but glycerol-resistant) mutants, demonstrating that the two enzymes are part of the major pathway of glycerol catabolism in S. coelicolor. Circumstantial evidence suggested that their inducer is glycerol 3-phosphate rather than glycerol. The enzymes were subject to co-ordinate repression by various carbon sources, of which glucose exerted the strongest effect (a fivefold repression). Previously described mutants resistant to 2-deoxyglucose, shown here to have very low glucose kinase activity, were defective in glucose repression of the glycerol enzymes.

Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187.

1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37 degrees C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately 50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 mug of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 mug of ionophore A23187/ml was about 10 muM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown.

Early development of adipose cell lipogenesis and glycerol utilization in Zucker obese rats.

A study of adipose cell metabolism was made at ages 5, 7, 10, and 14 wk of age in genetically obese Zucker rats. Adipose samples were surgically removed and used for in vitro adipose cell incubations and for characterization of enzyme patterns. Lipogenic capacity from glucose and enzymes normally associated with lipogenesis (malic enzyme, citrate cleavage enzyme and glucose-6-PO4 dehydrogenase) followed the same pattern of development. At 5 wk of age, the adipose cells of obese animals had a greater capacity for fat synthesis than the lean rats. At all other ages lipogenic activity and enzyme levels were either similar or less than the pair-fed lean littermates. Glycerol utilization by isolated fat cells was similar; however, adipose tissue glycerokinase was elevated in obese rats at 14 wk of age. It was concluded that there was no apparent change in specific lipogenic capacity of fat cells from the obese rat when compared to its lean littermate. It was also concluded that increased adipose glycerokinase activity in obese rats represented a secondary shift in metabolism.

Role of glycerol 3-phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken liver.

1. The metabolic role of hepatic NAD-linked glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was investigated vis-a-vis glyceride synthesis, glyceride degradation and the maintainence of the NAD redox state. 2. Five-week-old chickens were placed on five dietary regimes: a control group, a group on an increased-carbohydrate-lowered-fat diet, a group on a high-fat-lowered-carbohydrate diet, a starved group and a starved-refed group. In each group the specific activity (mumol/min per g wet wt. of tissue) of hepatic glycerol 3-phosphate dehydrogenase was compared with the activities of the beta-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, glycerol kinase (EC 2.7.1.30) and lactate dehydrogenase (EC 1.1.1.27). 3. During starvation, the activities of glycerol 3-phosphate dehydrogenase, glycerol kinase and lactate dehydrogenase rose significantly. After re-feeding these activities returned to near normal. All three activities rose slightly on the high-fat diet. Lactate dehydrogenase activity rose slightly, whereas those of the other two enzymes fell slightly on the increased-carbohydrate-lowered-fat diet. 4. The activity of the beta-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, a lipid-synthesizing enzyme, contrasted strikingly with the other three enzyme activities. Its activity was slightly elevated on the increased-carbohydrate diet and significantly diminished on the high-fat diet and during starvation. 5. The changes in activity of the chicken liver isoenzyme of glycerol 3-phosphate dehydrogenase in response to dietary stresses suggest that the enzyme has an important metabolic role other than or in addition to glyceride biosynthesis.