Direct assessment of liver glycogen storage by 13C nuclear magnetic resonance spectroscopy and regulation of glucose homeostasis after a mixed meal in normal subjects.

Despite extensive recent studies, understanding of the normal postprandial processes underlying immediate storage of substrate and maintenance of glucose homeostasis in humans after a mixed meal has been incomplete. The present study applied 13C nuclear magnetic resonance spectroscopy to measure sequential changes in hepatic glycogen concentration, a novel tracer approach to measure postprandial suppression of hepatic glucose output, and acetaminophen to trace the pathways of hepatic glycogen synthesis to elucidate the homeostatic adaptation to the fed state in healthy human subjects. After the liquid mixed meal, liver glycogen concentration rose from 207 +/- 22 to 316 +/- 19 mmol/liter at an average rate of 0.34 mmol/liter per min and peaked at 318 +/- 31 min, falling rapidly thereafter (0.26 mmol/liter per min). The mean increment at peak represented net glycogen synthesis of 28.3 +/- 3.7 g (approximately 19% of meal carbohydrate content). The contribution of the direct pathway to overall glycogen synthesis was 46 +/- 5 and 68 +/- 8% between 2 and 4 and 4 and 6 h, respectively. Hepatic glucose output was completely suppressed within 30 min of the meal. It increased steadily from 60 to 255 min from 0.31 +/- 32 to 0.49 +/- 18 mg/kg per min then rapidly returned towards basal levels (1.90 +/- 0.04 mg/kg per min). This pattern of change mirrored precisely the plasma glucagon/insulin ratio. These data provide for the first time a comprehensive picture of normal carbohydrate metabolism in humans after ingestion of a mixed meal.     

13C-nuclear magnetic resonance spectroscopy studies of hepatic glucose metabolism in normal subjects and subjects with insulin-dependent diabetes mellitus.

To determine the effect of insulin-dependent diabetes mellitus (IDDM) on rates and pathways of hepatic glycogen synthesis, as well as flux through hepatic pyruvate dehydrogenase, we used 13C-nuclear magnetic resonance spectroscopy to monitor the peak intensity of the C1 resonance of the glucosyl units of hepatic glycogen, in combination with acetaminophen to sample the hepatic UDP-glucose pool and phenylacetate to sample the hepatic glutamine pool, during a hyperglycemic-hyperinsulinemic clamp using [1-13C]-glucose. Five subjects with poorly controlled IDDM and six age-weight-matched control subjects were clamped at a mean plasma glucose concentration of approximately 9 mM and mean plasma insulin concentrations approximately 400 pM for 5 h. Rates of hepatic glycogen synthesis were similar in both groups (approximately 0.43 +/- 0.09 mumol/ml liver min). However, flux through the indirect pathway of glycogen synthesis (3 carbon units-->-->glycogen) was increased by approximately 50% (P < 0.05), whereas the relative contribution of pyruvate oxidation to TCA cycle flux was decreased by approximately 30% (P < 0.05) in the IDDM subjects compared to the control subjects. These studies demonstrate that patients with poorly controlled insulin-dependent diabetes mellitus have augmented hepatic gluconeogenesis and relative decreased rates of hepatic pyruvate oxidation. These abnormalities are not immediately reversed by normalizing intraportal concentrations of glucose, insulin, and glucagon and may contribute to postprandial hyperglycemia.     

Impaired hepatic glycogen synthesis in glucokinase-deficient (MODY-2) subjects.

All glucokinase gene mutations identified to date have been localized to exons that are common to the pancreatic and hepatic isoforms of the enzyme. While impaired insulin secretion has been observed in glucokinase-deficient subjects the consequences of this mutation on hepatic glucose metabolism remain unknown. To examine this question hepatic glycogen concentration was measured in seven glucokinase-deficient subjects with normal glycosylated hemoglobin and 12 control subjects using 13C nuclear magnetic spectroscopy during a day in which three isocaloric mixed meals were ingested. The relative fluxes of the direct and indirect pathways of hepatic glycogen synthesis were also assessed using [1-13C]glucose in combination with acetaminophen to noninvasively sample the hepatic UDP-glucose pool. Average fasting hepatic glycogen content was similar in glucokinase-deficient and control subjects (279+/-20 vs 284+/-14 mM; mean+/-SEM), and increased in both groups after the meals with a continuous pattern throughout the day. However, the net increment in hepatic glycogen content after each meal was 30-60% lower in glucokinase-deficient than in the control subjects (breakfast, 46% lower, P < 0.02; lunch, 62% lower, P = 0.002; dinner; 30% lower, P = 0.04). The net increment over basal values 4 h after dinner was 105 +/-18 mM in glucokinase-deficient and 148+/-11 mM in control subjects (P = 0.04). In the 4 h after breakfast, flux through the gluconeogenic pathway relative to the direct pathway of hepatic glycogen synthesis was higher in glucokinase-deficient than in control subjects (50+/-2% vs 34+/-5%; P = 0.038). In conclusion glucokinase-deficient subjects have decreased net accumulation of hepatic glycogen and relatively augmented hepatic gluconeogenesis after meals. These results suggest that in addition to the altered beta cell function, abnormalities in liver glycogen metabolism play an important role in the pathogenesis of hyperglycemia in patients with glucokinase-deficient maturity onset diabetes of young.     

Monitoring beta-lactamase activity in vivo by 13C nuclear magnetic resonance spectroscopy

A 13C-labeled cephalothin, 7 beta-(2-thienylacetamido)-3-[acetoxy-13C1]methyl-3-cephem-4- carboxylate (compound 1), has been prepared and used to monitor beta-lactamase activities by 13C nuclear magnetic resonance spectroscopy. Time-elapsed spectral analysis of the reaction of the labeled cephalothin with the TEM-2 beta-lactamase purified from Escherichia coli revealed the progressive loss of the cephalothin acetyl resonance at 176.8 ppm and accumulation of an acetate signal at 184.3 ppm. Spectral results identical to those observed in the in vitro experiment were obtained when compound 1 was incubated with cell suspensions of E. coli JSR-O (pBR322), which contains the plasmid-encoded TEM-2 beta-lactamase, and Enterobacter cloacae strains that contain a class I chromosomal beta-lactamase. Pseudo-first-order rate constants for the lactamase-catalyzed formation of acetate from cephalothin in vivo were obtained by integration of the 13C-acetyl resonances of compound 1 during timed incubations with cell preparations. These results constitute the first demonstration of the ability to monitor beta-lactamase activity in viable cells by nuclear magnetic resonance spectroscopy.     

Fatty acid distribution in systems modeling the normal and diabetic human circulation. A 13C nuclear magnetic resonance study.

A nonperturbing 13C nuclear magnetic resonance (NMR) method was used to monitor the equilibrium distribution of carboxyl 13C-enriched fatty acids (FA) between distinct binding sites on human serum albumin, native human lipoproteins, and/or phospholipid model membranes, under conditions that mimic the normal and diabetic human circulation. Two variables pertinent to the diabetic circulation were examined: FA/albumin mole ratio (as elevated in insulin deficiency and/or nephrosis) and pH (as decreased in acidosis). 13C NMR spectra for samples containing carboxyl 13C-enriched palmitate, human serum albumin, and phospholipid vesicles or native lipoproteins (all samples at pH 7.4, 37 degrees C) exhibited up to six carboxyl NMR resonances corresponding to FA bound to distinct binding sites on albumin and nonalbumin components. When the sample FA/albumin mole ratio was 1, three FA carboxyl resonances were observed (182.2, 181.8, and 181.6 ppm; designated peaks beta, gamma, and beta', respectively). These resonances corresponded to FA bound to three distinct high-affinity binding sites on human serum albumin. When the sample mole ratio value exceeded 1, additional carboxyl resonances corresponding to FA bound to phospholipid vesicles (179.0 ppm, peak phi), lipoproteins (180.7 ppm, peak sigma), and lower affinity sites on albumin (183.8 ppm, peak alpha; 181.9 ppm, peak gamma'), were observed. The intensity of peaks phi and sigma increased with increasing mole ratio or decreasing pH. Using Lorentzian lineshape analysis, the relative mole quantities of FA bound to albumin and nonalbumin binding sites were determined. Plots of the fraction of FA associated with nonalbumin components as a function of FA/albumin mole ratio were linear and extrapolated to the abscissa at a mole ratio value of 1. This pattern of FA distribution was observed regardless of the type of nonalbumin acceptor used (phospholipid vesicles, human high- or low-density lipoproteins) or the type of FA used (palmitate, oleate, or stearate), and provided evidence for negative cooperativity for human serum albumin upon binding of 1 mol of FA per mole albumin. These in vitro NMR results suggest that the threshold FA/albumin mole ratio value for alterations in FA distributions in the human circulation may be 1, rather than 3, as previously held. The pathophysiological implications of these findings are discussed.     

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.     

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

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.     

Quantitative analysis of glycogen repletion by nuclear magnetic resonance spectroscopy in the conscious rat

In order to directly determine the amount of label exchange that occurs in the tricarboxylic cycle from labeled alanine and lactate after the ingestion of a glucose load [1-13C]glucose was administered by continuous intraduodenal infusion to awake catheterized rats to achieve steady state jugular venous glycemia (160 mg/dl) for 180 min. Liver was freeze-clamped at 90 and 180 min, and perchloric acid extracts of the liver were subjected to 13C and 1H nuclear magnetic resonance analysis. Dilution in the oxaloacetate pool was determined by comparing the intrahepatic 13C enrichments of C2, C3 positions of glutamate with the C2, C3 positions of alanine and lactate. In addition steady state flux equations were derived for calculation of relative fluxes through pyruvate dehydrogenase/TCA cycle flux and pyruvate kinase flux/total pyruvate utilization. After glucose ingestion in a 24-h fasted rat direct conversion of glucose was responsible for 34% of glycogen. The intrahepatic dilution factor for labeled pyruvate in the oxaloacetate pool was 2.4. Using this factor, alanine and lactate contributed approximately 55% to glycogen formation. Pyruvate dehydrogenase flux ranged between 24 and 35% of total acetyl-coenzyme A (CoA) production and pyruvate kinase flux relative to total pyruvate utilization was approximately 40%.     

Primaquine: metabolism by microorganisms and 13C nuclear magnetic resonance assignments.

Primaquine (I), a 6-methoxy-8-aminoquinoline derivatives used for the treatment of malaria, has been subjected to metabolic studies using microorganisms. A total of 77 microorganisms (fungi and Streptomyces spp.) were evaluated for their ability to metabolize primaquine. Of these, 23 were found to convert primaquine to one or more metabolites (thin-layer chromatography analysis). Preparative scale fermentation of primaquine with four different microorganisms resulted in the isolation of two metabolites, identified as 8-(4-acetamido-1-methylbutylamino)-6-methoxyquinoline (II) and 8-(3-carboxyl-1-methylpropylamino)-6-methoxyquinoline (III). The structures of the metabolites were proposed based primarily on a comparison of the 13C nuclear magnetic resonance spectra of II and the methyl ester of III (IV) with that of primaquine. The structures of both metabolites II and III were confirmed by direct comparison with authentic samples.     

Human cerebral osmolytes during chronic hyponatremia. A proton magnetic resonance spectroscopy study.

The pathogenesis of morbidity associated with hyponatremia is postulated to be determined by the state of intracellular cerebral osmolytes. Previously inaccessible, these metabolites can now be quantitated by proton magnetic resonance spectroscopy. An in vivo quantitative assay of osmolytes was performed in 12 chronic hyponatremic patients (mean serum sodium 120 meq/liter) and 10 normal controls. Short echo time proton magnetic resonance spectroscopy of occipital gray and parietal white matter locations revealed dramatic reduction in the concentrations of several metabolites. In gray matter, myo-inositol was most profoundly reduced at 49% of control value. Choline containing compounds were reduced 36%, creatine/phosphocreatine 19%, and N-acetylaspartate 11% from controls. Similar changes were found in white matter. Recovery of osmolyte concentrations was demonstrated in four patients studied 8-14 wk later. These results are consistent with a reversible osmolyte reduction under hypoosmolar stress in the intact human brain and offer novel suggestions for treatment and monitoring of this common clinical event.     

A study of patients with alcoholic liver disease by 31P nuclear magnetic resonance spectroscopy

1. Patients with a history of alcohol abuse were studied by 31P n.m.r. spectroscopy of the liver in vivo, and the results were related to the pattern of disease assessed by standard biochemical and histological techniques. 2. The ratios of metabolites measured from the 31P n.m.r. spectra were abnormal in patients with alcoholic hepatitis but not in those with fatty change or cirrhosis in the absence of hepatitis. In particular, the levels of phosphomonoesters were raised, with respect either to Pi, or to adenosine 5'-triphosphate. The level of phosphomonoesters showed a significant positive correlation with the severity of alcoholic hepatitis, assessed by histology. 3. The ratio of Pi to adenosine 5'-triphosphate was used as a measure of the energy status of the hepatocytes, and was unchanged between patients and controls.     

Cutaneous blood flow during a hypoglycaemic clamp in insulin-dependent diabetic patients and healthy subjects.

1. A marked cutaneous vasodilatation has been shown to occur in healthy subjects, but not in insulin-dependent diabetic patients, in response to hypoglycaemia induced by a rapid intravenous bolus injection of insulin. 2. In the present study cutaneous blood flow in response to a gradual decline in blood glucose concentration was investigated in eight young adult diabetic patients and in eight age- and sex-matched control subjects. After a hyperinsulinaemic euglycaemic clamp for 40 min, hypoglycaemia was induced (plasma glucose concentration 2 mmol/l) by a standardized stepwise reduction in the intravenous glucose infusion. 3. Blood flow was measured by using a laser Doppler sensor and a cutaneous O2 electrode placed on the medial aspect of the forearm, and a laser Doppler sensor placed on the forehead. 4. No significant change in cutaneous blood flow occurred during euglycaemic hyperinsulinaemia. 5. In control subjects a marked increase in blood flow during hypoglycaemia was observed in the forearm by both methods. No corresponding change was observed in the forehead. 6. In the diabetic patients the increase in cutaneous blood flow was absent in both the forearm and forehead. 7. It is concluded that hypoglycaemia, but not hyperinsulinaemia, is associated with a regional cutaneous vasodilatation in healthy control subjects. This cutaneous vasodilatation is absent in diabetic patients.     

Use of proton nuclear magnetic resonance spectroscopy in detection and study of organic acidurias

We used high-resolution proton nuclear magnetic resonance spectroscopy to detect, identify, and study the major normal and abnormal organic acid metabolites in urine from patients with propionic acidemia, methylmalonic aciduria, branched-chain ketoaciduria, isovaleric acidemia, and glutaric aciduria type I. Characteristic and diagnostic spectra were obtained at 400 MHz for each disorder in all the patients studied and neutral and basic compounds, including amino acids and acylcarnitines, were also detected. The technique is rapid (10 min) and requires small samples (0.5 mL) and no preliminary extraction or derivative preparation. We believe that it is particularly suitable for the rapid and acute diagnosis of inborn errors of metabolism, especially the organic acidurias, and for acute pediatric clinical care, when rapid monitoring of major metabolic alterations is required in a time scale suitable to influence directly and immediately the therapy of the patients concerned.     

A review of 23Na nuclear magnetic resonance spectroscopy for the in vitro study of cellular sodium metabolism

Changes in intracellular sodium have been associated with a number of different diseases. Consequently, various methods have been used to quantify the level of intracellular sodium concentrations. Traditional methods like flame photometry and ion-selective electrodes are destructive or invasive, thereby potentially altering the intracellular sodium levels. There has been an increasing interest in evaluating the method of 23Na nuclear magnetic resonance in recent years, since this method allows for non-invasive continuous monitoring of intracellular sodium in cell suspensions and tissues. A phenomenological approach to basic theory, review of methodology, applications to the in vitro study of cellular sodium metabolism, and difficulties of interpretation of this analytical modality is presented.     

Synthesis of muscle glycogen during recovery after prolonged severe exercise in diabetic and non-diabetic subjects.

Glycogen synthesis rate in skeletal muscle studied in six juvenile diabetic and six non-diabetic males ingesting a carbohydrate rich diet during 12 h of resting recovery after exhaustive bicycle exercise. The diabetic subjects took their regular insulin. Blood samples and muscle biopsies were obtained at rest prior to exercise, immediately after cessation of exercise and after 2,4,6.9 and 12 h of recovery. A marked decrease in muscle glycogn content was observed in response to exercise in both groups of subjects. Mean glycogen utilization rate was the same in the two groups. Glycogen synthesis rate during the first 4 h or recovery was 6.4 +/- 0.6 mmol glucosyl units/kg w.w./h in the diabetic subjects and 7.2 +/- 0.7 mmol glycosyl units/kg w.w./h in the non-diabetic subjects. During the next 8 h glycogen synthesis rate was approximately 1/3 of that being 2.0 +/- 0.3 and 2.4 +/- 0.5 mmol glucosyl units/kg w.w./h in the two groups respectively. Glycogen synthetase I-activity increased markedly in response to exercise in both groups of subjects. However, no differences were observed between the groups. No significant differences in muscle glucose 6-phosphate concentrations were observed between the two groups. Plasma glucose levels were significantly higher in the diabetic than in the non-diabetic subjects. It is concluded that glycogen synthesis during recovery following prolonged severe exercise can proceed at the same rate in diabetic subjects taking their regular insulin as in non-diabetic subjects.     

Skeletal muscle metabolism in heart failure: a 31P nuclear magnetic resonance spectroscopy study of leg muscle

1. The gastrocnemius muscle of seven patients with mild to moderate chronic heart failure and of five healthy control subjects was studied using 31P nuclear magnetic resonance spectroscopy. Spectra were collected at rest and during an incremental, symptom-limited, exercise protocol. Blood flow was measured in the same study during brief interruptions to exercise. 2. The phosphocreatine/(phosphocreatine plus inorganic phosphate) ratio was lower in patients with heart failure than in control subjects at an exercise rate of 1.5 W, although intracellular pH and blood flow were similar. 3. The cytosolic free adenosine 5'-diphosphate concentration was markedly increased in patients with heart failure exercising at 1.5 W compared with control subjects exercising at the same workload. 4. Although the maximum workload achieved by patients with heart failure was less than half of that reached by control subjects, the pH and the phosphocreatine/(phosphocreatine plus inorganic phosphate) ratio were lower in patients with heart failure at maximal load. Blood flow was less at maximal exercise in patients with heart failure than in control subjects in keeping with the reduced work load. 5. The phosphocreatine depletion induced in the gastrocnemius muscle by exercise was more severe than previously described in the forearm of patients with heart failure. 6. Metabolic abnormalities in skeletal muscle may contribute to exercise intolerance in heart failure, particularly during submaximal exercise.