Effects of insulin on perfused liver from streptozotocin-diabetic and untreated rats: 13C NMR assay of pyruvate kinase flux

The effects of insulin in vitro on perfused liver from streptozotocin-diabetic rats and their untreated littermates during gluconeogenesis from either [3-13C]alanine + ethanol or [2-13C]pyruvate + NH4Cl + ethanol were studied by 13C NMR. A 13C NMR determination of the rate of pyruvate kinase flux under steady-state conditions of active gluconeogenesis was developed; this assay includes a check on the reuse of recycled pyruvate. The preparations studied provided gradations of pyruvate kinase flux within the confines of the assay's requirement of active gluconeogenesis. By this determination, the rate of pyruvate kinase flux was 0.74 +/- 0.04 of the gluconeogenic rate in liver from 24-h-fasted controls; in liver from 12-h-fasted controls, relative pyruvate kinase flux increased to 1.0 +/- 0.2. In diabetic liver, this flux was undetectable by our NMR method. Insulin's hepatic influence in vitro was greatest in the streptozotocin model of type 1 diabetes: upon treatment of diabetic liver with 7 nM insulin in vitro, a partial reversal of many of the differences noted between diabetic and control liver was demonstrated by 13C NMR. A major effect of insulin in vitro upon diabetic liver was the induction of a large increase in the rate of pyruvate kinase flux, bringing relative and absolute fluxes up to the levels measured in 24-h-fasted controls. By way of comparison, the effects of ischemia on diabetic liver were studied by 13C NMR to test whether changes in allosteric effectors under these conditions could also increase pyruvate kinase flux. A large increase in this activity was demonstrated in ischemic diabetic liver.     

Simultaneous 13C and 31P NMR studies of perfused rat liver. Effects of insulin and glucagon and a 13C NMR assay of free Mg2+

Metabolism of [2-13C]pyruvate, [1,2-13C]ethanol, and NH4+ in the presence and absence of 7 nM insulin has been followed at 35 degrees C by alternate scan 13C and 31P NMR at 90.5 and 145.8 MHz, respectively, in isolated perfused liver from 16-h fasted rats. With this technique, 31P and 13C NMR spectra are recorded simultaneously so that both phosphate metabolites and 13C-labeled metabolites could be followed, noninvasively, in perfused liver to give a comprehensive view of the response to a variety of stimuli. 13C-labeled glycogen increased synchronously, at a rate of 17 mumol of glucose units/g of liver/h, with the synthesis of 13C-labeled glucose, which also proceeded at a rate of 17 mumol/g of liver/h; glycogenesis was essentially a gluconeogenic process under these conditions and was not affected by the presence of insulin. From the position of the 13C-labeled citrate peak observed in liver, the measurement of Kd for the citrate-Mg complex under our conditions, and the expression relating these quantities to the concentration of free Mg2+, the intracellular level of free Mg2+ is estimated to be 0.46 +/- 0.05 mM in perfused rat liver. After subsequent administration of glucagon, a rapid decrease in glycogen and citrate was seen by 13C NMR and a 44% increase in glycero-3-phosphocholine was seen by 31P NMR; increase in glycero-3-phosphocholine is consistent with stimulation of liver phospholipase activity by glucagon. The co-administration of two different 13C-labeled substrates introduced multiplet structure arising from spin-spin interaction between labeled adjacent carbons into the peaks of several key metabolites. 13C enrichments at specific carbons of citrate, glutamate, glutamine, beta-hydroxybutyrate, and glucose and the distribution of intensity within the multiplets of specific carbons were measured in spectra of perfusates and extracts of the freeze-clamped livers. Within the context of a first order model for fluxes into the Krebs cycle and into glucose, analytical expressions were written that describe the intensity distributions within the several multiplets. In this way, a set of simultaneous equations was generated and solved in general form; when the measured intensity ratios are substituted into these expressions, relative fluxes under the conditions of the experiment can be estimated. Because a redundancy of information is available, checks on self-consistency are built into the estimated fluxes.     

Use of 31P and 13C NMR to study enzyme mechanisms

A number of complex biochemical problems have been solved recently by application of new techniques in which 31P and 13C NMR spectroscopy is used. Oxygen isotope exchange phenomena were studied by these NMR methods and used to analyze individual mechanistic events in enzymatic reactions. The existence of intermediates in the reactions catalyzed by glutamine synthetase (EC 6.3.1.2) and carbamyl-phosphate synthetase (EC 2.7.2.9) has been established as well as the kinetic competence of these intermediates for each enzyme. The NMR theory and kinetic experiments required to conduct such studies are discussed.     

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.     

Binding of the natural substrates and products to KDO8P synthase: 31P and 13C solution NMR characterization

Proton decoupled 31P and 13C solution NMR experiments were applied to mixtures of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase, with each of its natural substrates, phosphoenolpyruvate and arabinose-5-phosphate (ASP), and product KDO8P to identify the formation of the enzyme-substrate and enzyme-product complexes. Effects arising from ligand interactions with the enzyme are reported via chemical shifts and line broadening with respect to those of the free ligands in solution, depending on the strength and dynamics of binding under thermodynamic equilibrium conditions. The characterization was done both at low and high field spectrometers, 200 and 500 MHz (1H frequencies), and in cases of 31P NMR measurements, it was demonstrated that only the low field spectrometer is capable of providing direct experimental evidence on the enzyme-ligand interactions. Since both the substrate A5P and the product KDO8P exhibit multiple anomeric forms in solution, evidence for the preference of recognition and binding of particular forms is sought.     

Application of 13C and 31P NMR to the study of hepatic metabolism

Alternate scan 13C and 31P NMR has been used to follow the metabolism of 13C-labeled substrates, in the presence and absence of insulin, in isolated perfused liver from fasted rats. Because both 31P and 13C NMR spectra are recorded almost simultaneously with this method, both phosphate metabolites and 13C-labeled metabolites are measured, noninvasively and repetitively, to give an immediate, broad survey of the hepatic response to a variety of stimuli. During the metabolism of [2-13C]pyruvate, [1,2-13C]ethanol, and NH4+, 13C-labeled glycogen increases synchronously with, and at the same rate as, the synthesis of 13C-labeled glucose; thus, glycogenesis was essentially a gluconeogenic process under our conditions and was unaltered by the presence of insulin. From the position of the 13C-labeled citrate peak observed in liver, the measurement of KD for the citrate-magnesium complex under our conditions, and the expression relating these quantities to the concentration of free Mg2+, the intracellular level of free Mg2+ is estimated to be 0.46 +/- 0.05 mM. Later administration of glucagon led to a rapid decrease in glycogen and citrate and a 44% increase in glycero-3-phosphocholine (GPC); increase in GPC is consistent with stimulation of liver phospholipase activity by glucagon. Simultaneous administration of two different 13C-labeled substrates, or one doubly labeled substrate, introduced multiplet structure arising from spin-spin interaction between labeled adjacent carbons into the peaks of several key metabolites. The 13C NMR intensity distributions within the several multiplets are used, within the context of a first-order model for fluxes into the Krebs cycle, to estimate relative fluxes under the conditions of the experiment.     

13C NMR studies of glycogen turnover in the perfused rat liver

To assess whether hepatic glycogen is actively turning over under conditions which promote net glycogen synthesis we perfused livers from 24-h fasted rats with 20 mM D-[1-13C]glucose, 10 mM L-[3-13C]alanine, 10 mM L-[3-13C]lactate, and 1 microM insulin for 90 min followed by a 75-min "chase" period with perfusate of the same composition containing either 13C-enriched or unlabeled substrates. The peak height of the C-1 resonance of the glucosyl subunits in glycogen was monitored, in real time, using 13C NMR techniques. During the initial 90 min the peak height of the C-1 resonance of glycogen increased at almost a constant rate reflecting a near linear increase in net glycogen synthesis, which persisted for a further 75 min if 13C-enriched substrates were present during the "chase" period. However, when the perfusate was switched to the unenriched substrates, the peak height of the C-1 resonance of glycogen declined in a nearly linear manner reflecting active glycogenolysis during a time of net glycogen synthesis. By comparing the slopes of the curve describing the time course of the net [1-13C] glucose incorporation into glycogen with the rate of net loss of 13C label from the C-1 resonance of glycogen during the "chase" period we estimated the relative rate of glycogen breakdown to be 60% of the net glycogen synthetic rate. Whether this same phenomenon occurs to such an appreciable extent in vivo remains to be determined.     

Chlorpromazine interaction with phosphatidylserines: a (13)C and (31)P solid-state NMR study

Niemann-Pick type C, or NPC for short, is an early childhood disease exhibiting progressive neurological degeneration, associated with hepatosplenomegaly in some cases. The disease, at the cellular level, is a result of improper trafficking of lipids such as cholesterol and glycosphingolipids (GSLs) to lysosome-like storage organelles (LSOs), which become engorged with these lipids. It is believed that the initial defect in trafficking, whether of cholesterol or a GSL, results in an eventual traffic jam in these LSOs. This leads to the retention of not only other lipids, but also of transmembrane proteins that transiently associate with the late endosomes (LE) in normal cells, on their way to other cellular destinations such as the trans-Golgi network (TGN). In this review, we discuss the biophysical properties of lipids and cholesterol that might determine their intracellular itineraries, and how these itineraries are altered in NPC cells, which have defects in the proteins NPC1 or NPC2. We also discuss some potential therapeutic directions being suggested by recent research.     

31P and 13C NMR studies of oxygen transfer during catalysis by 3-deoxy-D-manno-octulosonate cytidylyltransferase from Escherichia coli

[18O]3-Deoxy-D-manno-octulosonate (KDO), labeled at the anomeric oxygen, was prepared by exchange with [18O]H2O and used to follow the route of oxygen transfer during cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate (CMP-KDO) formation catalyzed by 3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase). The 31P-NMR signal of the phosphoryl group of CMP-KDO (-5.85 ppm), which appeared as a single resonance when CMP-KDO formation took place with unenriched KDO, appeared as two peaks when CMP-KDO formation took place in the presence of a mixture of [16O]-and [18O]KDO. These results demonstrate the retention of 18O during CMP-KDO formation. Confirmation that the labeled oxygen in CMP-KDO was retained in the "bridge" position between CMP and KDO came from 13C-NMR studies of CMP-KDO formed in the presence of 90% [2-13C, 18O] KDO. The prominent C-2 KDO resonance in CMP-KDO, which is normally a doublet at 101.4 ppm (Kohlbrenner, W.E., and Fesik, S.W. (1985) J. Biol. Chem. 260, 14695-14700), appeared as four peaks when a mixture of [2-13C,16O]- and [2-13C, 18O]KDO was used, confirming the direct bonding of 18O to the C-2 of KDO in CMP-KDO. These results are consistent with a nucleophilic displacement mechanism for CMP-KDO formation.     

Biochemical events occurring during the respiratory burst of macrophages: A 31P and 13C NMR study.

In a previous study, 31P NMR revealed that an intracellular acidification occurred during the respiratory burst of P388 D1 macrophages, but this NMR technique could not provide information about the localization of this event in cells. However, using a fluorescent pH-dependent probe, it was confirmed that this transient pH decrease does not occur in the cytosol but more probably occurs in relation to the function of endocytic vesicles. A 31P NMR study allowed us to evidence a transient increase in ADP phosphorylation at the beginning of the respiratory burst, possibly in connection with the initiation of the oxidase complex involved in superoxide anion production. A 13C NMR study of perchloric acid extracts from in vivo primed cells revealed an increase in glucose consumption due to Con A triggering. Sugar phosphates, which must be considered markers of the hexose monophosphate shunt involved in the respiratory burst, were also observed upon this activation process.     

31P NMR and 13C NMR studies of recombinant Saccharomyces cerevisiae with altered glucose phosphorylation activities

Manipulation of cellular metabolism to maximize the yield and rate of formation of desired products may be achieved through genetic modification. Batch fermentations utilizing glucose as a carbon source were performed for three recombinant strains of Saccharomyces cerevisiae in which the glucose phosphorylation step was altered by mutation and genetic engineering. The host strain (hxk1 hxk2 glk) is unable to grow on glucose or fructose; the three plasmids investigated expressed hexokinase PI, hexokinase PII, or glucokinase, respectively, enabling more rapid glucose and fructose phosphorylation in vivo than that provided by wild-type yeast.Intracellular metabolic state variables were determined by ³¹P NMR measurements of in vivo fermentations under nongrowth conditions for high cell density suspensions. Glucose consumption, ethanol and glycerol production, and polysaccharide formation were determined by ¹³C NMR measurements under the same experimental conditions as used in the ³¹P NMR measurements. The trends observed in ethanol yields for the strains under growth conditions were mimicked in the nongrowth NMR conditions.Only the strain with hexokinase PI had higher rates of glucose consumption and ethanol production in comparison to healthy diploid strains in the literature. The hexokinase PII strain drastically underutilized its glucose-phosphorylating capacity. A regulation difference in the use of magnesium-free ATP for this strain could be a possible explanation. Differences in ATP levels and cytoplasmic pH values among the strains were observed that could not have been foreseen. However, cytoplasmic pH values do not account for the differences observed among in vivo and in vitro glucose phosphorylation activities of the three recombinant strains.     

Importance of polyunsaturated acyl chains in chlorpromazine interaction with phosphatidylserines: a 13C and 31P solid-state NMR study

The polyunsaturated fatty acid docosahexaenoic acid (DHA, c22:6, n-3) is found at a level of about 50% in the phospholipids of neuronal tissue membranes and appears to be crucial to human health. Dipalmitoyl phosphatidylcholine (DPPC, 16:0/16:0 PC), 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) and the DHA containing 1-stearaoyl-2-docosahexenoyl phosphatidylserine (SDPS) were used to make DPPC (60%)/POPS (29%)/SDPS (11%) bilayers with and without 10 mol% chlorpromazine (CPZ), a cationic, amphiphilic phenothiazine. The T1 relaxation measurements make it clear that the saturated acyl chains carbons (palmitic, stearic and most of the oleic chain) and the choline head group are not affected by CPZ addition. The observed increased signal intensity and T1-values of DHA indicate reduced mobility of C4 and C5 due to CPZ binding. 31P NMR spectra confirm that CPZ binding to the phosphatidylserines in the bilayer enhances phospholipid head group mobility.     

Study of phospholipid structure by 1H, 13C, and 31P dipolar couplings from two-dimensional NMR.

Various motionally averaged 31P-1H, 13C-1H, 1H-1H, and 31P-13C dipolar couplings were measured for natural-abundance and unoriented phosphocholine in the L alpha phase. The couplings were obtained and assigned by a variety of advanced and partly novel two-dimensional solid-state NMR experiments. Whereas 31P-1H and 31P-13C dipolar couplings provide long-range structural constraints, geminal 1H-1H couplings and the signs of 13C-1H couplings are important new elements in a segmental order-tensor analysis of the lipid headgroup and glycerol backbone. The implications of these measured dipolar couplings for the conformational exchange of the lipid headgroup and the bending of the headgroup from the glycerol backbone are discussed. These dipolar couplings are also analyzed semiquantitatively in terms of the segmental order tensor.     

31P NMR saturation-transfer and 13C NMR kinetic studies of glycolytic regulation during anaerobic and aerobic glycolysis

31P NMR saturation-transfer techniques have been employed in glucose-grown derepressed yeast to determine unidirectional fluxes in the upper part of the Embden-Meyerhof-Parnas pathway. The experiments were performed during anaerobic and aerobic glycolysis by saturating the ATP gamma resonances and monitoring changes in the phosphomonoester signals from glucose 6-phosphate and fructose 1,6-bis-phosphate. These experiments were supplemented with 13C NMR measurements of glucose utilization rates and 13C NMR label distribution studies. Combined with data obtained previously from radioisotope measurements, these 31P and 13C NMR kinetic studies allowed estimation of the net glycolytic flow in addition to relative flows through phosphofructokinase (PFK) and Fru-1,6-P2ase during anaerobic and aerobic glycolysis. The 31P NMR saturation-transfer results are consistent with previous results obtained from measurements of metabolite levels, radioisotope data, and 13C NMR studies [den Hollander, J.A., Ugurbil, K., Brown, T.R., Bednar, M., Redfield, C., & Shulman, R.G. (1986a) Biochemistry 25, 203-211], providing additional support for in vivo measurement of the flows during glycolysis.     

Detection of antineoplastic agent induced cardiotoxicity by 31P NMR of perfused rat hearts

Development of dose dependent chronic irreversible cardiotoxicity is a key problem encountered in chemotherapy with adriamycin. Here it has been demonstrated that infusion of this agent produced distinct and largely irreversible changes in levels of phosphate metabolites and substantial acidosis that are detected by 31P NMR of the Langendorf perfused heart. Administration of the antioxidant, butylated hydroxytoluene minimizes these spectral changes but does not substantially diminish the antineoplastic activity of adriamycin. Bisantrene (CL 216,942), a noncardiotoxic anthracene with antineoplastic activity, produces only minor perturbations of the 31P spectrum of the perfused rat heart. These studies demonstrate the potential utility of employing 31P NMR to monitor acute or chronic cardiotoxicity in the perfused rat heart and for developing noninvasive in vivo NMR techniques for monitoring cardiotoxicity in experimental animals and humans.     

13C n.m.r. studies of gluconeogenesis in rat liver suspensions and perfused mouse livers

Our early 31P n.m.r. studies of compartmentation in suspensions of rat liver cells have been extended by following fructose-1-phosphate peaks, known to be in the cytosol, which gave the same pH as the Pi peak previously assigned to the cytosol. Gluconeogenesis have been followed from [13C]glycerol labelled at C1,3 or at C2 and from labelled [3-13C]alanine. With the glycerol substrate it was possible to follow the label into alpha-glycerophosphate and to determine its distribution in the glucose formed. To a first approximation (i.e. 90%) the glucose level could be followed from its original glycerol position, e.g. [1,3-13C]glycerol to strongly labelled positions 1, 3, 4 and 6 of glucose. Slightly more than 10% of the label was scrambled (i.e. 10% movement of C2 to C1 and ca. 10% of C1 was lost, the remainder being unchanged). These are consistent with a flux through the pentose shunt, dominated by the transketolase pathway. With [3-13C]alanine, about 14 resonances are assigned to different carbons of the intermediates beta-hydroxybutyrate, acetoacetate, lactate, pyruvate, glutamate, glutamine, asparate, as well as C2-alanine, while another 7 resonances are observed from the different anomeric carbons of glucose. The effects of thyroid hormone treatment of the rats upon numerous in vivo rates are clearly observed and will be illustrated.     

13C, 15N, and 31P NMR studies on 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans

The interaction between the apoprotein of 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans and the prosthetic group FAD has been investigated by 13C, 15N, and 31P NMR techniques. The FAD prosthetic group was selectively enriched in 13C and 15N isotopes by adding isotopically labeled riboflavin derivatives to the growth medium of riboflavin-requiring mutant cells. In the oxidized state the chemical shift of the C(7) and C(8) atoms indicates that the xylene moiety of the isoalloxazine ring is embedded in a hydrophobic environment. The polarization of the isoalloxazine ring as a whole is, however, much more comparable to that of free flavin in a polar and protic environment than to free flavin in an apolar environment. The polarization of the ring system can be ascribed to strong hydrogen bonds between the apoprotein and the two carbonyl groups. The binding of the competitive inhibitor, 6-hydroxy-D-nicotine, influences the resonances of the C(4a) and the N(5) atoms strongly. It is suggested that these shifts are due to a strong hydrogen-bonding interaction between the N(5) atom and the inhibitor. On reduction all resonances, except those of the C(10a) and the N(1) atoms, shift upfield, indicating the increased electron density in the ring system. In the dithionite-reduced enzyme, the ring system is bent at the N(5) position. Due to the bending of the N(5) atom and the sp2 hybridized N(10) atom, electron density from the N(10) atom is reallocated at the C(4) carbonyl group. In contrast, in the substrate-reduced enzyme the N(5) atom is almost completely sp2 hybridized, yielding a rather planar isoalloxazine ring.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111

Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR. All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone. The remaining fraction was shown to be phosphatidylethanolamine. The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents. The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions. The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups. Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions. In summary, the heterogeneity of E. coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.