Increase in phosphoribosyl pyrophosphate induced by ATP and Pi depletion in hepatocytes

A series of compounds that induce depletion of ATP and Pi when added to isolated rat hepatocytes were found to cause a remarkable, although transient, elevation in the concentration of phosphoribosyl pyrophosphate (PRPP) in these cells. After the addition of 5 mM fructose, xylitol, tagatose, or D-xylulose, PRPP increased from a basal value of 6 +/- 1 nmol/g of cells to, respectively, 68 +/- 11, 42 +/- 11, 67 +/- 22, and 530 +/- 50 nmol/g of cells (means +/- SEM of 3-9 experiments). In each case, the increase in PRPP was preceded by a latency period of 5-10 min. PRPP reached maximal levels 15 min after the addition of fructose and 30 min after that of xylitol and D-xylulose, but continued to increase for as long as 60 min after the addition of tagatose. Most striking was that the increase in PRPP closely paralleled the restoration of intracellular Pi. Ribose 5-P increased about two- to fivefold after the addition of fructose, xylitol, and tagatose, and approximately 12-fold after D-xylulose. The mechanism by which ATP- and Pi-depleting compounds stimulate the activity of PRPP synthetase in isolated rat hepatocytes is discussed.     

The content of pentose-cycle intermediates in liver in starved, fed ad libitum and meal-fed rats.

Liver content of pentose-cycle intermediates and the activity of the three major cytoplasmic NADPH-producing enzymes and pentose-cycle enzymes were measured in three dietary states: 48 h-starved rats, rats fed on a standard diet ad libitum, and rats meal-fed with a low-fat high-carbohydrate diet. Measured tissue contents of pentose-cycle intermediates in starved liver were: 6-phosphogluconate, 4.7 +/- 0.5 nmol/g; ribulose 5-P, 3.7 +/- 0.5 nmol/g; xylulose 5-P, 4.3 +/- 0.4 nmol/g; sedoheptulose 7-P, 25.5 +/- 1.3 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 30.6 +/- 0.7 nmol/g. These values were in good agreement with values calculated from fructose 6-P and free glyceraldehyde 3-P, assuming the major transketolase, transaldolase, ribulose-5-P 3-epimerase and ribose-5-P isomerase reactions were all in near-equilibrium. Similar results were found in animals fed ad libitum. These relationships were not valid in animals fed on a low-fat high-carbohydrate diet, with tissue contents of metabolites in some cases being more than an order of magnitude higher than the calculated values. Measured tissue contents of pentose-cycle intermediates in these animals were: 6-phosphogluconate, 124.2 +/- 13.9 nmol/g; ribulose 5-P, 44.8 +/- 7.1 nmol/g; xylulose 5-P, 77.2 +/- 9.4 nmol/g; sedoheptulose 7-P, 129.9 +/- 10.1 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 157.0 +/- 11.3 nmol/g. In all animals, regardless of dietary state, tissue content of erythrose 4-P was less than 2 nmol/ml. Liver activities of glucose-6-P dehydrogenase and 6-phosphogluconate dehydrogenase were increased from 3.5 +/- 0.9 mumol/g and 7.3 +/- 0.5 mumol/min per g in starved animals to 13.2 +/- 1.1 and 10.5 +/- 0.7 mumol/min per g in low-fat high-carbohydrate-fed animals. Despite these changes, the activities of transaldolase (3.4 +/- 0.3 mumol/min per g), transketolase (7.8 +/- 0.2 mumol/min per g) and ribulose-5-P 3-epimerase (7.5 +/- 0.4 mumol/min per g) were not increased in meal-fed animals above those observed in starved animals (3.4 +/- 0.2, 7.1 +/- 0.3 and 8.6 +/- 0.4 mumol/min per g respectively). The increase in the activity of oxidative pentose-cycle enzymes in the absence of any change in the non-oxidative pentose cycle appeared to contribute to the observed disequilibrium in the pentose cycle in animals meal fed on a low-fat high-carbohydrate diet.     

Reaction mechanism of 21S dynein ATPase from sea urchin sperm. I. Kinetic properties in the steady state

21S Dynein ATPase [EC 3.6.1.3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [gamma-32P]ATP at various concentrations, 5 mM divalent cations, and 20 mM imidazole at pH 7.0 and 0 degrees C. The following results were obtained. 1. 21S Dynein had a latent ATPase activity of about 0.63 mumol Pi/(mg . min) in 1 mM ATP, 100 mM KCl, 4 mM MgSO4, 0.5 mM EDTA, and 30 mM Tris-HCl at pH 8.0 and 25 degrees C. Its exposure to 0.1% Triton X-100 for 5 min at 25 degrees C induced an increase in the ATPase activity to about 3.75 mumol Pi/(mg . min) and treatment at 40 degrees C for 5 min also induced a similar activation. 2. The double-reciprocal plot for the ATPase activity of dynein activated by the treatment at 40 degrees C consisted of two straight lines, while that of nonactivated 21S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21S dynein showed substrate inhibition at ATP concentrations above 0.1 mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the Vmax and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of Pi liberation. The apparent Pi-burst size was 1.0 mol/(10(6) g protein) and the true size was calculated to be 1.6 mol/1,250 K after correcting for the effect of Pi liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of Pi liberation of 1.4 mol/(10(6) g protein) in the presence of 54% (v/v) glycerol.     

The deposition of calcium pyrophosphate by NTP pyrophosphohydrolase of matrix vesicles from fetal bovine epiphyseal cartilage

About 5 mumol CaPPi/mg protein was deposited within 3 h in the presence of the reaction mixtures containing 1 mM ATP, 2 mM Ca2+, 1 mM Pi, and 17 micrograms of purified NTP pyrophosphohydrolase. At 1 mM ATP, 50% of the deposition was inhibited by 0.5-1 mM of various substrate and product analogues including AMP, ADP, and ethylene hydroxyl diphosphonate. The magnitude of inhibition on NTP pyrophosphohydrolase activity was in the order of AMP = CMP = ADP greater than adenosine greater than adenine greater than NAD = NADP. AMP, CMP, ADP, and adenosine are competitive inhibitors. The modes of inhibition by adenine, NAD, and NADP differ from the competitive inhibition. Ribose, 3'-AMP, 2'-AMP, and cAMP did not inhibit the enzyme activity.     

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (Pi), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC----gamma ATP (0.2 mM/s), Pi----gamma ATP (0.2 mM/s) and the conversion beta ATP----beta ADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 mumol/min per mg protein) and adenylate kinase (approx. 0.4 mumol/min per mg protein) suggest that the beta ATP----beta ADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from Pi and the rate of consumption of oxygen atoms (4 X 10(-3) mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.     

Fructose effect to suppress hepatic glycogen degradation

The effect of fructose on glycogen degradation was examined by measuring the flux of 14C from prelabeled glycogen in perfused rat livers. During 2-h refeeding of 24-h-fasted rats, newly synthesized hepatic glycogen was labeled by intraperitoneal injection of [U-14C] galactose (0.1 mg and 0.02 microCi/g of body weight). The livers of refed rats were then perfused in a nonrecirculating fashion for an initial 30 min with glucose alone (10 mM) for the following 60 min with glucose (10 mM) without (n = 5) or with fructose (1, 2, or 10 mM; n = 5 for each). When livers were exposed to fructose, release of label into the perfusate immediately declined and remained markedly suppressed through the end of perfusion (p less than 0.05). The suppression was dose-dependent; at steady state (50-70 min), label release was suppressed 45, 64, and 72% by 1, 2, and 10 mM fructose, respectively (p less than 0.0001). Suppression was not accompanied by significant changes in the activities of glycogen synthase or phosphorylase assessed in vitro. These results suggest the existence of allosteric inhibition of phosphorylase in the presence of fructose. Fructose 1-phosphate (Fru-1-P) accumulated in proportion to fructose (0.11 +/- 0.01 without fructose, 0.86 +/- 0.03, 1.81 +/- 0.18, and 8.23 +/- 0.60 mumol/g of liver with 1, 2, and 10 mM fructose, respectively; p less than 0.0001). Maximum inhibition of label release was 82%; the Fru-1-P concentration for half inhibition was 0.57 mumol/g of liver, well within the concentration of Fru-1-P attained during refeeding. We conclude that fructose enhances net glycogen accumulation in liver by suppressing glycogenolysis and that the suppression is presumably caused by allosteric inhibition of phosphorylase by Fru-1-P.     

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.     

Production of xylitol from D-xylulose by Mycobacterium smegmatis

Mycobacterium smegmatis transformed D-xylulose to xylitol in washed cell reactions under aerobic and anaerobic conditions. The yield of xylitol reached about 70% in anaerobic conditions (in N₂) by cells grown on media containing xylitol or D-mannitol. Cells immobilized with Ca-alginate had almost the same activity of xylitol production as washed cells.Xylitol was produced from D-xylose using commercial immobilized D-xylose isomerase from Bacillus coagulans and immobilized cells of M. smegmatis. From 10 g of D-xylose, 4 g of xylitol was produced and 5 g of D-xylose remained in the reaction mixture; no D-xylulose was detected.     

Novel enzymatic method for the production of xylitol from D-arabitol by Gluconobacter oxydans

Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.     

Thyroid hormone and dehydroepiandrosterone permit gluconeogenic hormone responses in hepatocytes

The importance of the sn-glycerol- 3-phosphate (G-3-P) electron transfer shuttle in hormonal regulation of gluconeogenesis was examined in hepatocytes from rats with decreased mitochondrial G-3-P dehydrogenase activity (thyroidectomized) or increased G-3-P dehydrogenase activity [triiodothyronine (T(3)) or dehydroepiandrosterone (DHEA) treated]. Rates of glucose formation from 10 mM lactate, 10 mM pyruvate, or 2.5 mM dihydroxyacetone were somewhat less in hypothyroid cells than in cells from normal rats but gluconeogenic responses to calcium addition and to norepinephrine (NE), glucagon (G), or vasopressin (VP) were similar to the responses observed in cells from normal rats. However, with 2. 5 mM glycerol or 2.5 mM sorbitol, substrates that must be oxidized in the cytosol before conversion to glucose, basal gluconeogenesis was not appreciably altered by hypothyroidism but responses to calcium and to the calcium-mobilizing hormones were abolished. Injecting thyroidectomized rats with T(3) 2 days before preparing the hepatocytes greatly enhanced gluconeogenesis from glyc erol and restored the response to Ca(2+) and gluconeogenic hormones. Feeding dehydroepiandrosterone for 6 days depressed gluconeogenesis from lactate or pyruvate but substantially increased glucose production from glycerol in euthyroid cells and restored responses to Ca(2+) in hypothyroid cells metabolizing glycerol. Euthyroid cells metabolizing glycerol or sorbitol use the G-3-P and malate/aspartate shuttles to oxidize excess NADH generated in the cytosol. The transaminase inhibitor aminooxyacetate (AOA) decreased gluconeogenesis from glycerol 40%, but had little effect on responses to Ca(2+) and NE. However, in hypothyroid cells, with minimal G-3-P dehydrogenase, AOA decreased gluconeogenesis from glycerol more than 90%. Thus, the basal rate of gluconeogenesis from glycerol in the euthyroid cells is only partly dependent on electron transport from cytosol to mitochondria via the malate/aspartate shuttle and almost completely dependent in the hypothyroid state, and the hormone enhancement of the rate in euthyroid cells involves primarily the G-3-P cycle. These data are consistent with Ca(2+) being mobilized by gluconeogenic hormones and G-3-P dehydrogenase being activated by Ca(2+) so as to permit it to transfer reducing equivalents from the cytosol to the mitochondria.     

Glucose phosphorylation is not rate limiting for accumulation of glycogen from glucose in perfused livers from fasted rats

Incorporation of Glc and Fru into glycogen was measured in perfused livers from 24-h fasted rats using [6-3H]Glc and [U-14C]Fru. For the initial 20 min, livers were perfused with low Glc (2 mM) to deplete hepatic glycogen and were perfused for the following 30 min with various combinations of Glc and Fru. With constant Fru (2 mM), increasing perfusate Glc increased the relative contribution of Glc carbons to glycogen (7.2 +/- 0.4, 34.9 +/- 2.8, and 59.1 +/- 2.7% at 2, 10, and 20 mM Glc, respectively; n = 5 for each). During perfusion with substrate levels seen during refeeding (10 mM Glc, 1.8 mumol/g/min gluconeogenic flux from 2 mM Fru), Fru provided 54.7 +/- 2.7% of the carbons for glycogen, while Glc provided only 34.9 +/- 2.8%, consistent with in vivo estimations. However, the estimated rate of Glc phosphorylation was at least 1.10 +/- 0.11 mumol/g/min, which exceeded by at least 4-fold the glycogen accumulation rate (0.28 +/- 0.04 mumol of glucose/g/min). The total rate of glucose 6-phosphate supply via Glc phosphorylation and gluconeogenesis (2.9 mumol/g/min) exceeded reported in vivo rates of glycogen accumulation during refeeding. Thus, in perfused livers of 24-h fasted rats there is an apparent redundancy in glucose 6-phosphate supply. These results suggest that the rate-limiting step for hepatic glycogen accumulation during refeeding is located between glucose 6-phosphate and glycogen, rather than at the step of Glc phosphorylation or in the gluconeogenic pathway.     

Construction and co-expression of plasmid encoding xylitol dehydrogenase and a cofactor regeneration enzyme for the production of xylitol from D-arabitol

The biotransformation of D-arabitol into xylitol was investigated with focus on the conversion of D-xylulose into xylitol. This critical conversion was accomplished using Escherichia coli to co-express a xylitol dehydrogenase gene from Gluconobacter oxydans and a cofactor regeneration enzyme gene which was a glucose dehydrogenase gene from Bacillus subtilis for system 1 and an alcohol dehydrogenase gene from G. oxydans for system 2. Both systems efficiently converted D-xylulose into xylitol without the addition of expensive NADH. Approximately 26.91 g/L xylitol was obtained from around 30 g/L D-xylulose within system 1 (E. coli Rosetta/Duet-xdh-gdh), with a 92% conversion yield, somewhat higher than that of system 2 (E. coli Rosetta/Duet-xdh-adh, 24.9 g/L, 85.2%). The xylitol yields for both systems were more than 3-fold higher compared to that of the G. oxydans NH-10 cells (7.32 g/L). The total turnover number (TTN), defined as the number of moles of xylitol formed per mole of NAD(+), was 32,100 for system 1 and 17,600 for system 2. Compared with that of G. oxydans NH-10, the TTN increased by 21-fold for system 1 and 11-fold for system 2, hence, the co-expression systems greatly enhanced the NADH supply for the conversion, benefiting the practical synthesis of xylitol.     

Modulation by glycerol of hepatic glycero-3-phosphate concentration, ketogenesis, and output of triglyceride and glucose in perfused livers from hyperthyroid and euthyroid rats

Decreased glycero-3-phosphate (glycero-3-P) concentration, decreased output of triglyceride and glucose, increased output of apolipoprotein A-I, and increased ketogenesis were observed with isolated perfused livers from triiodothyronine-treated rats in comparison to livers from euthyroid animals. Infusion of glycerol produced a concentration-dependent accumulation of glycero-3-P in perfused livers from hyperthyroid and euthyroid rats, which was considerably enhanced in the euthyroid group. The antiketogenic effect of glycerol in livers from triiodothyronine-treated rats was accompanied by increased output of glucose and triglyceride, while no change in the output of apolipoprotein A-I was observed. The reduction of ketogenesis (49%) in euthyroid livers by glycerol was not accompanied by increased triglyceride output, while with the largest amount of glycerol infused, decreased output of apolipoprotein A-I was seen. Output of triglycerides by livers from hyperthyroid rats correlated with hepatic concentration of glycero-3-P and was maximal at a glycero-3-P concentration (0.5 mumol/g), similar to that observed in livers from euthyroid rats in the absence of glycerol. Availability of glycero-3-P appears to be rate-limiting for synthesis and secretion of triglyceride by livers from hyperthyroid animals, whereas the glycero-3-P concentrations in euthyroid livers were sufficient to support maximal production of triglyceride limited only by the supply of free fatty acid.     

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.     

Early metabolic effects of platelet-derived growth factor and transforming growth factor-beta in rat liver in vivo

The short term metabolic effects of the in vivo administration of platelet-derived growth factor have been examined in the liver of the rat. Meal-fed male Wistar rats weighing between 150-180 g received an intraperitoneal injection of platelet-derived growth factor (17 units/100 g weight), transforming growth factor-beta (185 ng/100 g weight), or saline. At 5 min after injection, the livers were freeze-clamped. Samples of the tissue were subsequently assayed for metabolite content and enzyme activities. Platelet-derived growth factor injection caused an elevation in the liver content of pyruvate from 0.14 +/- 0.012 to 0.19 +/- 0.009 mumol/g wet weight liver (p less than or equal to 0.01) and an increase in the cytosolic phosphorylation potential [sigma ATP]/[sigma ADP][sigma Pi] from 6670 +/- 540 to 8970 +/- 750 (p less than or equal to 0.01). In addition an increase in the hepatic content of the hexose monophosphate pathway metabolites, 6-phosphogluconate (0.027 +/- 0.004 to 0.037 +/- 0.005 mumol/g wet weight) (p less than or equal to 0.05), ribulose 5-phosphate (0.013 +/- 0.001 to 0.017 +/- 0.001 mumol/g wet weight) (p less than or equal to 0.05) and combined sedoheptulose 7-phosphate and ribose 5-phosphate (0.052 +/- 0.007 to 0.062 +/- 0.004 mumol/g wet weight) (p less than or equal to 0.05) was observed. The elevation in the hexose monophosphate pathway metabolites resulted from a 1.3-fold elevation in the activity of glucose-6-phosphate dehydrogenase [EC 1.1.1.49] when measured in a crude homogenate. Kinetic analysis performed on partially purified glucose-6-phosphate dehydrogenase demonstrated no significant change in the Km of the enzyme for either NADP+ or glucose 6-phosphate, while a 2.4-fold increase in the Vmax was observed. In view of the rapidity of the change in total measured enzyme activity and increase in the Vmax of glucose-6-phosphate dehydrogenase, it is postulated that platelet-derived growth factor causes a covalent modification of the existing enzyme. Transforming growth factor-beta caused no change in the hepatic metabolite content in the treated animals when compared to saline treated controls.     

Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver. A 31phosphorus nuclear magnetic resonance study.

Fructose metabolism has been studied with 31P n.m.r. in perfused livers from rats starved for 48h. The time course of changes in liver ATP, Pi and sugar phosphate (fructose l-phosphate) concentrations, and intracellular pH were followed in each perfusion after infusion of fructose to give an initial concentration of either 5mM or 10mM. Rapid falls in the concentrations of ATP and Pi and intracellular pH occurred after infusion of fructose, reaching a minimum after 4-5 min, which was lower in the 10mM group than in the 5mM group. These changes were accompanied by a rapid rise in fructose 1-phosphate, reaching a plateau also after 4-5 min. At both concentrations of fructose, after the early falls, some recovery of ATP, Pi and intracellular pH occurred; this was complete for Pi and intracellular pH in the 5mM-fructose experiments (within 12-30 min). Complete restoration of ATP to the pre-fructose value was not achieved in either the 5mM of 10mM groups. Measurements of the uptake of lactate by the liver indicated that the fall in intracellular pH was caused primarily by production of protons accompanying the formation of lactate from fructose with possibly a transient contribution generated during the rise in fructose 1-phosphate.     

Microbial Production of Xylitol from Glucose

A microbiological method is described for the production of xylitol, which is used as a sugar substitute for diabetics. A sequential fermentation process yielded 9.0 g of xylitol from 77.5 g of glucose via D-arabitol and D-xylulose. Candida guilliermondii var. soya (ATCC 20216) consumed 5.1 g of D-xylulose and produced 2.8 g of xylitol per 100 ml. Pentitol production from D-xylulose by yeasts was divided into three types: I, yeast-produced xylitol; II, yeast-produced D-arabitol; and III, yeast-produced xylitol and D-arabitol. D-Xylulose, but not glucose, was dissimilated to xylitol by yeasts under aerobic conditions.     

Effects of glutathione depletion on gluconeogenesis in isolated hepatocytes

Glutathione-depleted hepatocytes, by incubation with diethylmaleate (DEM) or phorone (2,6-dimethyl-2,5-heptadiene-4-one), i.e., substrates of the GSH S-transferases (EC 2.5.1.18), showed rates of gluconeogenesis from various precursors significantly lower than controls; however the rate of glucose synthesis from fructose was similar to that of controls. Isolated hepatocytes from rats pretreated with those substrates 1 h before isolation to deplete hepatic glutathione (GSH) also showed a decrease of the rate of gluconeogenesis from lactate plus pyruvate. Incubation of hepatocytes with L-buthionine sulfoximine, a specific inhibitor of gamma-glutamyl-cysteine synthetase (EC 6.3.2.2), resulted in a decreased rate of gluconeogenesis from lactate plus pyruvate only when GSH values were lower than 1 mumol/g cells. Freeze-clamped livers from GSH-depleted rats showed a higher concentration of malate and glycerol 3-phosphate, indicating that GSH depletion probably affects phosphoenolpyruvate carboxykinase and glycerol-3-phosphate dehydrogenase activities. Several indicators of cell viability, such as lactate dehydrogenase leakage, malondialdehyde accumulation, ATP concentration, or urea synthesis from different precursors, were not affected by GSH depletion under the experimental conditions used here. Besides, the GSH/GSSG ratio remained unchanged in all cases.