31P nuclear magnetic resonance evidence for the regulation of intracellular pH by Ehrlich ascites tumor cells.

The phenomenon of intracellular pH (pHin) regulation in cultured Ehrlich ascites cells was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. Measurements were made with a Bruker WH 360 wide bore NMR spectrometer at a 31P frequency of 145.78 MHz. Samples at a density of 10(8) cells ml-1 were suspended in a final volume of 2 ml of growth medium in 10 mm diameter NMR tubes. Intracellular pH was calculated from the chemical shifts of either intracellular inorganic phosphate (Piin) or intracellular 2-deoxyglucose-6-phosphate (2dG6Pin). The sugar phosphate was used as a pH probe to supplement the Piin measurements, which could not always be observed. When available, the pHin calculated from the Piin peak was identical within experimental error to the pHin calculated from the 2dG6Pin peak. Intracellular pH was measured to be more alkaline than the medium at an external pH (pHex) below 7.1. Typical values were pHin = 7.00 for pHex = 6.50. These measurements were constant for times up to 165 min using well-energized, respiring cells. This pH gradient was seen to collapse immediately upon onset of anaerobic shock. Above a pHex of 7.2 there was no significant difference between pHin and pHex. These results unequivocally demonstrate the steady state nature of the pH regulation and its dependence upon energization.

Proton magnetic resonance of transfer RNA.

The temperature dependence of the areas under the proton magnetic resonance spectra of unfractionated yeast transfer RNA in 1.0 molar NaCl is a consequence of salt-induced aggregation and does not constitute a monitor of the melting of secondary molecular structure. Such melting can be observed by following the widths of the resonances in the various regions of the spectra. Peaks attributable to dihydrouracil and the methyl groups of the methylated bases are detected in the spectra of unfractionated transfer RNA and alanine transfer RNA.

In vivo phosphorus-31 nuclear magnetic resonance reveals lowered ATP during heat shock of Tetrahymena.

Cells synthesize a characteristic set of proteins--heat shock proteins--in response to a rapid temperature jump or certain other stress treatments. The technique of phosphorus-31 nuclear magnetic resonance spectroscopy was used to examine in vivo the effects of temperature jump on two species of Tetrahymena that initiate the heat shock response at different temperatures. An immediate 50 percent decrease in cellular adenosine triphosphate was observed when either species was jumped to a temperature that strongly induces synthesis of heat shock proteins. This new adenosine triphosphate concentration was maintained at the heat shock temperature.

Quantitation of hepatic glycogenolysis and gluconeogenesis in fasting humans with 13C NMR.

The rate of net hepatic glycogenolysis was assessed in humans by serially measuring hepatic glycogen concentration at 3- to 12-hour intervals during a 68-hour fast with 13C nuclear magnetic resonance spectroscopy. The net rate of gluconeogenesis was calculated by subtracting the rate of net hepatic glycogenolysis from the rate of glucose production in the whole body measured with tritiated glucose. Gluconeogenesis accounted for 64 +/- 5% (mean +/- standard error of the mean) of total glucose production during the first 22 hours of fasting. In the subsequent 14-hour and 18-hour periods of the fast, gluconeogenesis accounted for 82 +/- 5% and 96 +/- 1% of total glucose production, respectively. These data show that gluconeogenesis accounts for a substantial fraction of total glucose production even during the first 22 hours of a fast in humans.

Cellular applications of 31P and 13C nuclear magnetic resonance.

High-resolution nuclear magnetic resonance (NMR) studies of cells and purified mitochondria are discussed to show the kind of information that can be obtained in vivo. In suspensions of Escherichia coli both phosphorus-31 and carbon-13 NMR studies of glycolysis and bioenergetics are presented. In rat liver cells the pathways of gluconeogenesis from carbon-13-labeled glycerol are followed by carbon-13 NMR. In the intact liver cells cytosolic and mitochondrial pH's were separately measured by phosphorus-31 NMR. In purified mitochondria the internal and external concentrations of inorganic phosphate, adenosine diphosphate, and adenosine triphosphate were determined by phosphorus-31 NMR while the pH difference across the membrane was measured simultaneously.

In vivo carbon-13 nuclear magnetic resonance studies of mammals.

Natural abundance carbon-13 nuclear magnetic resonances (NMR) from human arm and rat tissues have been observed in vivo. These signals arise primarily from triglycerides in fatty tissue. Carbon-13 NMR was also used to follow, in a living rat, the conversion of C-1-labeled glucose, which was introduced into the stomach, to C-1-labeled liver glycogen. The carbon-13 sensitivity and resolution obtained shows that natural abundance carbon-13 NMR will be valuable in the study of disorders in fat metabolism, and that experiments with substrates labeled with carbon-13 can be used to study carbohydrate metabolism in vivo.

NMR studies of human brain function.

Recent advances in magnetic resonance imaging and spectroscopy make it possible to measure localized changes in human brain activity and metabolism in single subjects during sensory stimulation and cognition. Differences between stimulated and unstimulated subjects can be visualized to a resolution of mm3 in less than 1s, a significant improvement over the more established method, positron emission tomography. Magnetic resonance spectroscopy of the human brain, measuring fluxes in several cm3, has followed changes in metabolic rates during visual stimulation.

31P nuclear magnetic resonance measurements of muscle glucose-6-phosphate. Evidence for reduced insulin-dependent muscle glucose transport or phosphorylation activity in non-insulin-dependent diabetes mellitus.

To assess the rate-limiting step in muscle glycogen synthesis in non-insulin-dependent diabetes mellitus (NIDDM), the concentration of glucose-6-phosphate (G6P) was measured by 31P nuclear magnetic resonance (NMR) during a hyperglycemic-hyperinsulinemic clamp. Six subjects with NIDDM and six age weight-matched controls were studied at similar steady-state plasma concentrations of insulin (approximately 450 pmol/liter) and glucose (11 mmol/liter). The concentration of G6P in the gastrocnemius muscle was measured by 31P NMR. Whole-body oxidative and nonoxidative glucose metabolism was determined by the insulin-glucose clamp technique in conjunction with indirect calorimetry. Nonoxidative glucose metabolism which under these conditions is a measure of muscle glycogen synthesis (1990. N. Engl. J. Med. 322:223-228), was 31 +/- 7 mumol/(kg body wt-min) in the normal subjects and 13 +/- 3 mumol/(kg body wt-min) in the NIDDM subjects (P less than 0.05). The concentration of G6P was higher (0.24 +/- 0.02 mmol/kg muscle) in the normal subjects than in the NIDDM subjects (0.17 +/- 0.02, P less than 0.01). Increasing insulin concentrations to insulin 8,500 pmol/liter in four NIDDM subjects restored the glucose uptake rate and G6P concentrations to normal levels. In conclusion, the lower concentration of G6P in the diabetic subjects despite a decreased rate of nonoxidative glucose metabolism is consistent with a defect in muscle glucose transport or phosphorylation reducing the rate of muscle glycogen synthesis.

Increased rate of gluconeogenesis in type II diabetes mellitus. A 13C nuclear magnetic resonance study.

UNLABELLED: To quantitate hepatic glycogenolysis, liver glycogen concentration was measured with 13C nuclear magnetic resonance spectroscopy in seven type II diabetic and five control subjects during 23 h of fasting. Net hepatic glycogenolysis was calculated by multiplying the rate of glycogen breakdown by the liver volume, determined from magnetic resonance images. Gluconeogenesis was calculated by subtracting the rate of hepatic glycogenolysis from the whole body glucose production rate, measured using [6-3H]glucose. Liver glycogen concentration 4 h after a meal was lower in the diabetics than in the controls; 131 +/- 20 versus 282 +/- 60 mmol/liter liver (P < 0.05). Net hepatic glycogenolysis was decreased in the diabetics, 1.3 +/- 0.2 as compared to 2.8 +/- 0.7 mumol/(kg body wt x min) in the controls (P < 0.05). Whole body glucose production was increased in the diabetics as compared to the controls, 11.1 +/- 0.6 versus 8.9 +/- 0.5 mumol/(kg body wt x min) (P < 0.05). Gluconeogenesis was consequently increased in the diabetics, 9.8 +/- 0.7 as compared to 6.1 +/- 0.5 mumol/(kg body wt x min) in the controls (P < 0.01), and accounted for 88 +/- 2% of total glucose production as compared with 70 +/- 6% in the controls (P < 0.05). IN CONCLUSION: increased gluconeogenesis is responsible for the increased whole body glucose production in type II diabetes mellitus after an overnight fast.

Simultaneous synthesis and degradation of rat liver glycogen. An in vivo nuclear magnetic resonance spectroscopic study.

Using 13C nuclear magnetic resonance spectroscopic methods we examined in vivo the synthesis of liver glycogen during the infusion of D-[1-13C]glucose and the turnover of labeled glycogen during subsequent infusion of D-[1-13C]glucose. In fasted rats the processes of glycogen synthesis and degradation were observed to occur simultaneously with the rate of synthesis much greater than degradation leading to net glycogen synthesis. In fed rats, incorporation of infused D-[1-13C]glucose occurred briskly; however, over 2 h there was no net glycogen accumulated. Degradation of labeled glycogen was greater in the fed versus the fasted rats (P less than 0.001), and the lack of net glycogen synthesis in fed rats was due to degradation and synthesis occurring at similar rates throughout the infusion period. There was no indication that suppression of phosphorylase a or subsequent activation of glycogen synthase was involved in modulation of the flux of tracer into liver glycogen. We conclude that in both fed and fasted rats, glycogen synthase and phosphorylase are active simultaneously and the levels of liver glycogen reached during refeeding are determined by the balance between ongoing synthetic and degradative processes.

Metabolic and functional consequences of inhibiting adenosine deaminase during renal ischemia in rats.

The concentrations of renal ATP have been measured by 31P-nuclear magnetic resonance (NMR) before, during, and after bilateral renal artery occlusion. Using in vivo NMR, the initial postischemic recovery of ATP increased with the magnitude of the residual nucleotide pool at the end of ischemia. ATP levels after 120 min of reflow correlated with functional recovery at 24 h. In the present study the effect of blocking the degradation of ATP during ischemia upon the postischemic restoration of ATP was investigated. Inhibition of adenosine deaminase by 80% with the tight-binding inhibitor 2'-deoxycoformycin led to a 20% increase in the residual adenine nucleotide pool. This increased the ATP initial recovery after 45 min of ischemia from 52% (in controls) to 62% (in the treated animals), as compared to the basal levels. The inhibition also caused an accelerated postischemic restoration of cellular ATP so that at 120 min it was 83% in treated rats vs. 63% in untreated animals. There was a corresponding improvement in the functional recovery from the insult (increase of 33% in inulin clearance 24 h after the injury). Inhibition of adenosine deaminase during ischemia results in a injury similar to that seen after a shorter period of insult.

Mechanism of liver glycogen repletion in vivo by nuclear magnetic resonance spectroscopy.

In order to quantitate the pathways by which liver glycogen is repleted, we administered [1-13C]glucose by gavage into awake 24-h fasted rats and examined the labeling pattern of 13C in hepatic glycogen. Two doses of [1-13C]glucose, 1 and 6 mg/g body wt, were given to examine whether differences in the plasma glucose concentration altered the metabolic pathways via which liver glycogen was replenished. After 1 and 3 h (high-dose group) and after 1 and 2 h (low-dose group), the animals were anesthetized and the liver was quickly freeze-clamped. Liver glycogen was extracted and the purified glycogen hydrolyzed to glucose with amyloglucosidase. The distribution of the 13C-label was subsequently determined by 13C-nuclear magnetic resonance spectroscopy. The percent 13C enrichment of the glucosyl units in glycogen was: 15.1 +/- 0.8%(C-1), 1.5 +/- 0.1%(C-2), 1.2 +/- 0.1%(C-3), 1.1 +/- 0.1%(C-4), 1.6 +/- 0.1%(C-5), and 2.2 +/- 0.1%(C-6) for the high-dose study (n = 4, at 3 h); 16.5 +/- 0.5%(C-1), 2.0 +/- 0.1%(C-2), 1.3 +/- 0.1%(C-3), 1.1 +/- 0.1%(C-4), 2.2 +/- 0.1%(C-5), and 2.4 +/- 0.1%(C-6) in the low-dose study (n = 4, at 2 h). The average 13C-enrichment of C-1 glucose in the portal vein was found to be 43 +/- 1 and 40 +/- 2% in the high- and low-dose groups, respectively. Therefore, the amount of glycogen that was synthesized from the direct pathway (i.e., glucose----glucose-6-phosphate----glucose-1-phosphate----UDP-glucose---- glycogen) was calculated to be 31 and 36% in the high- and low-dose groups, respectively. The 13C-enrichments of portal vein lactate and alanine were 14 and 14%, respectively, in the high-dose group and 11 and 8%, respectively, in the low-dose group. From these enrichments, the minimum contribution of these gluconeogenic precursors to glycogen repletion can be calculated to be 7 and 20% in the high- and low-dose groups, respectively. The maximum contribution of glucose recycling at the triose isomerase step to glycogen synthesis (i.e., glucose----triose-phosphates----glycogen) was estimated to be 3 and 1% in the high- and low-dose groups, respectively. In conclusion, our results demonstrate that (a) only one-third of liver glycogen repletion occurs via the direct conversion of glucose to glycogen, and that (b) only a very small amount of glycogen synthesis can be accounted for by the conversion of glucose to triose phosphates and back to glycogen; this suggests that futile cycling between fructose-6-phosphate and fructose-1,6-diphosphate under these conditions is minimal. Our results also show that (c) alanine and lactate account for a minimum of between 7 and 20% of the glycogen synthesized, and that (d) the three pathways through which the labeled flux is measured account for a total of only 50% of the total glycogen synthesized. These results suggest that either there is a sizeable amount of glycogen synthesis via pathway(s) that were not examined in the present experiment or that there is a much greater dilution of labeled alanine/lactate in the oxaloacetate pool than previously appreciated, or some combination of these two explanations.

On the measurement of pH in Escherichia coli by 31P nuclear magnetic resonance.

The 31P high resolution NMR spectra of concentrated suspensions of Escherichia coli cells have been measured at 145.8 MHz. The position of the orthophosphate resonance is used as a measure of internal and external pH. In accord with Paddan, Zilberstein and Rottenberg ((1976) Eur. J. Biochem. 63, 533--541) it is shown that when properly energized the internal pH is 7.5 +/- 0.1. By synchronizing the NMR data acquisition with 3-s bursts of O2 it is possible to measure the internal pH with a time resolution of about 1 s. It is shown that at 20 degrees C the pH remains constant for times longer than 15 s after the oxygen is discontinued and it decays in several minutes.

Nuclear magnetic resonance studies of muscle and applications to exercise and diabetes.

Natural-abundance 13C nuclear magnetic resonance (NMR) spectroscopy is a noninvasive technique that enables in vivo assessments of muscle and/or liver glycogen concentrations. When directly compared with the traditional needle biopsy technique, NMR was found to be more precise. Over the last several years, we have developed and used 13C-NMR to obtain information about human glycogen metabolism both under conditions of altered blood glucose and/or insulin and with exercise. Because NMR is noninvasive, we have been able to obtain more data points over a specified time course, thereby dramatically improving the time resolution. This improved time resolution has enabled us to document subtleties of the resynthesis of muscle glycogen after severe exercise that have not been observed previously. An added advantage of NMR is that we are able to obtain information simultaneously about other nuclei, such as 31P. With interleaved 13C- and 31P-NMR techniques, we have been able to follow simultaneous changes in muscle glucose-6-phosphate and muscle glycogen. In this article, we review some of the work that has been reported by our laboratory and discuss the relevance of our findings for the management of diabetes.

Dynamic magnetic resonance imaging of the rat brain during forepaw stimulation.

A magnetic resonance (MR) imaging brain mapping method was used to localize an activated volume of brain tissue in chloralose-anesthetized rats during electrical stimulation of the forepaw. Physiologically-induced changes are characterized by alterations of the magnetic properties of blood as determined by the oxygenation state of hemoglobin. Stimulation of the left forepaw led to an increase in MR signal intensity of the contralateral frontal and parietal cortices, which corresponded to forelimb motor and somatosensory areas. The activation was contiguous in coronal planes between +5 and +2 mm anterior to the bregma, and its volume was calculated to be 20-30 mm3. Each activated region was revealed using a paired t-test statistical analysis method and the activated volume was calculated from regions exposed by thresholding at p < 0.005. Physiologically-induced fractional signal changes, delta S/S, in the motor and somatosensory areas were 0.06 +/- 0.04 and 0.17 +/- 0.06, respectively.

NMR determination of the TCA cycle rate and alpha-ketoglutarate/glutamate exchange rate in rat brain.

A mathematical model of cerebral glucose metabolism was developed to analyze the isotopic labeling of carbon atoms C4 and C3 of glutamate following an intravenous infusion of [1-13C]glucose. The model consists of a series of coupled metabolic pools representing glucose, glycolytic intermediates, tricarboxylic acid (TCA) cycle intermediates, glutamate, aspartate, and glutamine. Based on the rate of 13C isotopic labeling of glutamate C4 measured in a previous study, the TCA cycle rate in rat brain was determined to be 1.58 +/- 0.41 mumol min-1 g-1 (mean +/- SD, n = 5). Analysis of the difference between the rates of isotopic enrichment of glutamate C4 and C3 permitted the rate of exchange between alpha-ketoglutarate (alpha-KG) and glutamate to be assessed in vivo. In rat brain, the exchange rate between alpha-KG and glutamate is between 89 +/- 35 and 126 +/- 22 times faster than the TCA cycle rate (mean +/- SD, n = 4). The sensitivity of the calculated value of the TCA cycle rate to other metabolic fluxes and to concentrations of glycolytic and TCA cycle intermediates was tested and found to be small.

NMR determination of intracerebral glucose concentration and transport kinetics in rat brain.

The concentration of intracerebral glucose as a function of plasma glucose concentration was measured in rats by 13C NMR spectroscopy. Measurements were made in 20-60 min periods during the infusion of [1-13C]D-glucose, when intracerebral and plasma glucose levels were at steady state. Intracerebral glucose was found to vary from 0.7 to 19 mumol g-1 wet weight as the steady-state plasma glucose concentration was varied from 3 to 62 mM. A symmetric Michaelis-Menten model was fit to the brain and plasma glucose data with and without an unsaturable component, yielding the transport parameters Km, Vmax, and Kd. If it is assumed that all transport is saturable (Kd = 0), then Km = 13.9 +/- 2.7 mM and Vmax/Vgly = 5.8 +/- 0.8, where Vgly is the rate of brain glucose consumption. If an unsaturable component of transport is included, the transport parameters are Km = 9.2 +/- 4.7 mM, Vmax/Vgly = 5.3 +/- 1.5, and Kd/Vgly = 0.0088 +/- 0.0075 ml mumol-1. It was not possible to distinguish between the cases of Kd = 0 and Kd greater than 0, because the goodness of fit was similar for both. However, the results in both cases indicate that the unidirectional rate of glucose influx exceeds the glycolytic rate in the basal state by 2.4-fold and as a result should not be rate limiting for normal glucose utilization.

1H NMR studies of glucose transport in the human brain.

The difference between 1H nuclear magnetic resonance (NMR) spectra obtained from the human brain during euglycemia and during hyperglycemia is depicted as well-resolved glucose peaks. The time course of these brain glucose changes during a rapid increase in plasma glucose was measured in four healthy subjects, aged 18-22 years, in five studies. Results demonstrated a significant lag in the rise of glucose with respect to plasma glucose. The fit of the integrated symmetric Michaelis-Menten model to the time course of relative glucose signals yielded an estimated plasma glucose concentration for half maximal transport, Kt, of 4.8 +/- 2.4 mM (mean +/- SD), a maximal transport rate, Tmax, of 0.80 +/- 0.45 micromol g-1 min-1, and a cerebral metabolic glucose consumption rate (CMR)glc of 0.32 +/- 0.16 micromol g-1 min-1. Assuming cerebral glucose concentration to be 1.0 micromol/g at euglycemia as measured by 13CMR, the fit of the same model to the time course of brain glucose concentrations resulted in Kt = 3.9 +/- 0.82 mM, Tmax = 1.16 +/- 0.29 micromol g-1 min-1, and CMRglc = 0.35 +/- 0.10 micromol g-1 min-1. In both cases, the resulting time course equaled that predicted from the determination of the steady-state glucose concentration by 13C NMR spectroscopy within the experimental scatter. The agreement between the two methods of determining transport kinetics suggests that glucose is distributed throughout the entire aqueous phase of the human brain, implying substantial intracellular concentration.

The flux from glucose to glutamate in the rat brain in vivo as determined by 1H-observed, 13C-edited NMR spectroscopy.

The rate of incorporation of carbon from [1-13C]glucose into the [4-CH2] and [3-CH2] of cerebral glutamate was measured in the rat brain in vivo by 1H-observed, 13C-edited (POCE) nuclear magnetic resonance (NMR) spectroscopy. Spectra were acquired every 98 s during a 60-min infusion of [1-13C]glucose. Complete time courses were obtained from six animals. The measured intensity of the unresolved [4-13CH2] resonances of glutamate and glutamine increased exponentially during the infusion and attained a steady state in approximately 20 min with a first-order rate constant of 0.130 +/- 0.010 min-1 (t1/2 = 5.3 +/- 0.5 min). The appearance of the [3-13CH2] resonance in the POCE difference spectrum lagged behind that of the [4-13CH2] resonance and had not reached steady state at the end of the 60-min infusion (t1/2 = 26.6 +/- 4.1 min). The increase observed in 13C-labeled glutamate represented isotopic enrichment and was not due to a change in the total glutamate concentration. The glucose infusion did not affect the levels of high-energy phosphates or intracellular pH as determined by 31P NMR spectroscopy. Since glucose carbon is incorporated into glutamate by rapid exchange with the tricarboxylic acid (TCA) cycle intermediate alpha-ketoglutarate, the rate of glutamate labeling provided an estimate of TCA cycle flux. We have determined the flux of carbon through the TCA cycle to be approximately 1.4 mumols g-1 min-1. These experiments demonstrate the feasibility of measuring metabolic fluxes in vivo using 13C-labeled glucose and the technique of 1H-observed, 13C-decoupled NMR spectroscopy.

Oxidative glucose metabolism in rat brain during single forepaw stimulation: a spatially localized 1H[13C] nuclear magnetic resonance study.

In the alpha-chloralose-anesthetized rat during single forepaw stimulation, a spatially localized 1H[13C] nuclear magnetic resonance spectroscopic method was used to measure the rate of cerebral [C4]-glutamate isotopic turnover from infused [1,6-(13)C]glucose. The glutamate turnover data were analyzed using a mathematical model of cerebral glucose metabolism to evaluate the tricarboxylic acid (TCA) cycle flux (V(TCA)). During stimulation the value of V(TCA) in the sensorimotor region increased from 0.47 +/- 0.06 (at rest) to 1.44 +/- 0.41 micromol x g(-1) x min(-1) (P < 0.01) in the contralateral hemispheric compartment (24 mm3) and to 0.65 +/- 0.10 micromol x g(-1) x min(-1) (P < 0.03) in the ipsilateral side. Each V(TCA) value was converted to the cerebral metabolic rates of glucose oxidation (oxidative-CMR(glc)) and oxygen consumption (CMR(O2)). These rates were corrected for partial-volume based on activation maps obtained by blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI). The percent increase and the absolute value of oxidative-CMR(glc) in the activated regions are similar to values reported previously for total-CMR(glc) using the same activation paradigm. This indicates that the large majority of energy required for brain activation, in going from the resting to an activated state, is supplied by glucose oxidation. The level of activity during stimulation is relevant to awake animals because the oxidative-CMR(glc) (1.05 +/- 0.28 micromol x g(-1) x min(-1); current study) is in the range of total-CMR(glc) previously reported for awake rats undergoing physiologic activation (0.7-1.4 micromol x g(-1) x min(-1)). It is concluded that oxidative glycolysis is the main source of energy for increased brain activity and a positive BOLD fMRI signal-change occurs in conjunction with a large increase in CMR(O2).