Which prior knowledge? Quantification of in vivo brain 13C MR spectra following 13C glucose infusion using AMARES.

The recent developments in high magnetic field 13C magnetic resonance spectroscopy with improved localization and shimming techniques have led to important gains in sensitivity and spectral resolution of 13C in vivo spectra in the rodent brain, enabling the separation of several 13C isotopomers of glutamate and glutamine. In this context, the assumptions used in spectral quantification might have a significant impact on the determination of the 13C concentrations and the related metabolic fluxes. In this study, the time domain spectral quantification algorithm AMARES (advanced method for accurate, robust and efficient spectral fitting) was applied to 13 C magnetic resonance spectroscopy spectra acquired in the rat brain at 9.4 T, following infusion of [1,6-(13)C2 ] glucose. Using both Monte Carlo simulations and in vivo data, the goal of this work was: (1) to validate the quantification of in vivo 13C isotopomers using AMARES; (2) to assess the impact of the prior knowledge on the quantification of in vivo 13C isotopomers using AMARES; (3) to compare AMARES and LCModel (linear combination of model spectra) for the quantification of in vivo 13C spectra. AMARES led to accurate and reliable 13C spectral quantification similar to those obtained using LCModel, when the frequency shifts, J-coupling constants and phase patterns of the different 13C isotopomers were included as prior knowledge in the analysis.

Effects of infused amino acids on glucose production and utilization in healthy human subjects.

Amino acids have been reported to increase endogenous glucose production in normal human subjects during hyperinsulinemia: however, controversy exists as to whether insulin-mediated glucose disposal is inhibited under these conditions. The effect of an amino acid infusion on glucose oxidation rate has so far not been determined. Substrate oxidation rates, endogenous glucose production, and [13C]glucose synthesis from [13C]bicarbonate were measured in six normal human subjects during sequential infusions of exogenous glucose and exogenous glucose with (n = 5) or without (n = 5) exogenous amino acids. Amino acids increased endogenous glucose production by 84% and [13C]glucose synthesis by 235%. Glucose oxidation estimated from indirect calorimetry decreased slightly after amino acids, but glucose oxidation estimated from [13C]glucose-13CO2 data was increased by 14%. It is concluded that gluconeogenesis is the major pathway of amino acid degradation. During amino acid administration, indirect calorimetry underestimates the true rate of glucose oxidation, whereas glucose oxidation calculated from the 13C enrichment of expired CO2 during [U-13C]glucose infusion does not. A slight stimulation of glucose oxidation during amino acid infusion, concomitant with an increased plasma insulin concentration, indicates that amino acids do not inhibit glucose oxidation.

Effects of infused fructose on endogenous glucose production, gluconeogenesis, and glycogen metabolism.

To determine the mechanisms that prevent an increase in gluconeogenesis from increasing hepatic glucose output, six healthy women were infused with [1-13C]fructose (22 mumol.kg-1.min-1), somatostatin, insulin, and glucagon. In control experiment, non-13C-enriched fructose was infused at the same rate without somatostatin, and [U-13C]glucose was infused to measure specifically plasma glucose oxidation. Endogenous glucose production (EGP, [6,6-2H]glucose), net carbohydrate oxidation (CHOox, indirect calorimetry), and fructose oxidation (13CO2) were measured. EGP rate did not increase after fructose infusion with (13.1 +/- 1.2 vs. 12.9 +/- 0.3 mumol.kg-1.min-1) and without (10.3 +/- 0.5 vs. 9.7 +/- 0.5 mumol.kg-1.min-1) somatostatin, despite the fact that gluconeogenesis increased. Nonoxidative fructose disposal, corresponding mainly to glycogen synthesis, was threefold net glycogen deposition, the latter calculated as fructose infusion minus CHOox (14.8 +/- 1.1 and 4.3 +/- 2.0 mumol.kg-1.min-1). It is concluded that 1) the mechanism by which EGP remains constant when gluconeogenesis from fructose increases is independent of changes in insulin and 2) simultaneous breakdown and synthesis of glycogen occurred during fructose infusion.

Effect of major hepatectomy on glucose and lactate metabolism.

BACKGROUND: The liver plays an important role in glucose and lactate metabolism. Major hepatectomy may therefore be suspected to cause alterations of glucose and lactate homeostasis. METHODS: Thirteen subjects were studied: six patients after major hepatectomy and seven healthy subjects who had fasted overnight. Glucose turnover was measured with 6,6(2)H glucose. Lactate metabolism was assessed using two complementary approaches: 13C-glucose synthesis and 13CO2 production from an exogenous 13C-labeled lactate load infused over 15 minutes were measured, then the plasma lactate concentrations observed over 185 minutes after lactate load were fitted using a biexponential model to calculate lactate clearance, endogenous production, and half-lives. RESULTS: Three to five liver segments were excised. Compared to healthy controls, the following results were observed in the patients: 1) normal endogenous glucose production; 2) unchanged 13C-lactate oxidation and transformation into glucose; 3) similar basal plasma lactate concentration, lactate clearance, and lactate endogenous production; 4) decreased plasma lactate half-life 1 and increased half-life 2. CONCLUSIONS: Glucose and lactate metabolism are well maintained in patients after major hepatectomy, demonstrating a large liver functional reserve. Reduction in the size of normal liver parenchyma does not lead to hyperlactatemia. The use of a pharmacokinetic model, however, allows the detection of subtle alterations of lactate metabolism.

Kinetics of dexamethasone-induced alterations of glucose metabolism in healthy humans.

Six healthy human subjects were studied during three 75-g oral, [13C]glucose tolerance tests to assess the kinetics of dexamethasone-induced impairment of glucose tolerance. On one occasion, they received dexamethasone (4 x 0.5 mg/day) during the previous 2 days. On another occasion, they received a single dose (0. 5 mg) of dexamethasone 150 min before ingestion of the glucose load. On the third occasion, they received a placebo. Postload plasma glucose was significantly increased after both 2 days dexamethasone and single dose dexamethasone compared with control (P < 0.05). This corresponded to a 20-23% decrease in the metabolic clearance rate of glucose, whereas total glucose turnover ([6,6-2H]glucose), total (indirect calorimetry) and exogenous glucose oxidation (13CO2 production), and suppression of endogenous glucose production were unaffected by dexamethasone. Plasma insulin concentrations were increased after 2 days of dexamethasone but not after a single dose of dexamethasone. In a second set of experiments, the effect of a single dose of dexamethasone on insulin sensitivity was assessed in six healthy humans during a 2-h euglycemic hyperinsulinemic clamp. Dexamethasone did not significantly alter insulin sensitivity. It is concluded that acute administration of dexamethasone impairs oral glucose tolerance without significantly decreasing insulin sensitivity.

Effects of infused amino acids and lipids on glucose metabolism in healthy lean humans.

The effects of infusion of a triglyceride emulsion (which induces peripheral insulin resistance) and amino acids (which stimulate gluconeogenesis) on glucose metabolism were investigated in healthy lean humans during exogenous infusion of glucose. One group of subjects (n = 5) was infused for 7.5 h with 11.1 mumol/kg/min glucose; during the last 4 h, amino acids were also infused at a rate of 3.33 mg/kg/min. A second group of subjects (n = 5) was infused with glucose+lipids (Lipovenös, 10% 10 ml/min) for 7.5 h and amino acids were added during the last 4 h. Infusion of lipids suppressed the increase in glucose oxidation observed during infusion of glucose alone (delta glucose oxidation: -2.1 +/- 1.1 vs. + 4.5 +/- 1.4 mumol/kg/min; P < 0.05) and during infusion of glucose+amino acids (delta glucose oxidation: + 1.6 +/- 1.4 vs. + 10.6 +/- 1.2 mumol/kg/min; P < 0.05). Gluconeogenesis (determined from 13C glucose synthesis during infusion of 13C bicarbonate) increased from 1.1 +/- 0.2 mumol/kg/min during infusion of glucose and 1.6 +/- 0.3 during infusion of glucose+lipids to 3.2 +/- 0.4 and 3.1 +/- 0.4, respectively, when amino acid infusion was superimposed (P < 0.05 in both instances). Plasma glucose concentrations were identical during infusion of glucose alone or glucose+amino acids, with or without lipids. Insulin concentrations were significantly increased by lipids both during infusion of glucose alone and of glucose+amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of adrenergic blockade on hepatic glucose production during ethanol administration.

Acute ethanol administration stimulates sympathetic nervous system activity. The present study was designed to determine whether this sympathetic activation affects glycogenolysis and total hepatic glucose production (HGP) during ethanol-induced inhibition of gluconeogenesis. Nineteen volunteers participated in four protocols. Two protocols aimed to study--using combined infusion of [6,6-2H2]glucose and [U-13C]glucose, VCO2 and 13CO2 measurements--the effects of ethanol infusion alone (n = 10) or with propranolol (n = 6) or phentolamine infusion (n = 4) on HGP, glucose disposal (Rd), glucose oxidation [13C]Glcox and non-oxidative glucose disposal (NOGD = Rd - [13C]Glcox). The fourth protocol assessed the effects of saline infusion alone on HGP. Using ethanol, HGP decreased by 23%, Rd by 20% and glycaemia by 9% (all P &lt; 0.001); heart rate increased by 10%, whereas blood pressure remained unchanged. The effects were not observed with saline, except a slight (10%) decrease in HGP (P &lt; 0.01 vs. ethanol). Ethanol did not affect [13C]Glcox but decreased NOGD by 73% (P &lt; 0.001). Propranolol or phentolamine did not alter any of the effects of ethanol on glucose metabolism, but decreased mean arterial pressure. Propranolol prevented the ethanol-induced increase in heart rate. In conclusion, ethanol decreased blood glucose by decreasing HGP, presumably by inhibiting gluconeogenesis. Sympathetic activation prevented the decrease in blood pressure produced by ethanol but did not stimulate glycogenolysis.

Metabolic fate of fructose ingested with and without glucose in a mixed meal.

Ingestion of pure fructose stimulates de novo lipogenesis and gluconeogenesis. This may however not be relevant to typical nutritional situations, where fructose is invariably ingested with glucose. We therefore assessed the metabolic fate of fructose incorporated in a mixed meal without or with glucose in eight healthy volunteers. Each participant was studied over six hours after the ingestion of liquid meals containing either 13C-labelled fructose, unlabeled glucose, lipids and protein (Fr + G) or 13C-labelled fructose, lipids and protein, but without glucose (Fr), or protein and lipids alone (ProLip). After Fr + G, plasma 13C-glucose production accounted for 19.0% ± 1.5% and 13CO2 production for 32.2% ± 1.3% of 13C-fructose carbons. After Fr, 13C-glucose production (26.5% ± 1.4%) and 13CO2 production (36.6% ± 1.9%) were higher (p < 0.05) than with Fr + G. 13C-lactate concentration and very low density lipoprotein VLDL 13C-palmitate concentrations increased to the same extent with Fr + G and Fr, while chylomicron 13C-palmitate tended to increase more with Fr + G. These data indicate that gluconeogenesis, lactic acid production and both intestinal and hepatic de novo lipogenesis contributed to the disposal of fructose carbons ingested together with a mixed meal. Co-ingestion of glucose decreased fructose oxidation and gluconeogenesis and tended to increase 13C-pamitate concentration in gut-derived chylomicrons, but not in hepatic-borne VLDL-triacylglycerol (TG). This trial was approved by clinicaltrial. gov. Identifier is NCT01792089.

Effects of hypothermia on brain glucose metabolism in acute liver failure: a H/C-nuclear magnetic resonance study.

Mild hypothermia has a protective effect on brain edema and encephalopathy in both experimental and human acute liver failure. The goals of the present study were to examine the effects of mild hypothermia (35°C) on brain metabolic pathways using combined 1H and 13C-Nuclear Magnetic Resonance (NMR) spectroscopy, a technique which allows the study not only of metabolite concentrations but also their de novo synthesis via cell-specific pathways in the brain. :1H and 13C NMR spectroscopy using [1-13C] glucose was performed on extracts of frontal cortex obtained from groups of rats with acute liver failure induced by hepatic devascularization whose body temperature was maintained either at 37°C (normothermic) or 35°C (hypothermic), and appropriate sham-operated controls. At coma stages of encephalopathy in the normothermic acute liver failure animals, glutamine concentrations in frontal cortex increased 3.5-fold compared to sham-operated controls (P < 0.001). Comparable increases of brain glutamine were observed in hypothermic animals despite the absence of severe encephalopathy (coma). Brain glutamate and aspartate concentrations were respectively decreased to 60.9% ± 7.7% and 42.2% ± 5.9% (P < 0.01) in normothermic animals with acute liver failure compared to control and were restored to normal values by mild hypothermia. Concentrations of lactate and alanine in frontal cortex were increased to 169.2% ± 15.6% and 267.3% ± 34.0% (P < 0.01) respectively in normothermic rats compared to controls. Furthermore, de novo synthesis of lactate and alanine increased to 446.5% ± 48.7% and 707.9% ± 65.7% (P < 0.001), of control respectively, resulting in increased fractional 13C-enrichments in these cytosolic metabolites. Again, these changes of lactate and alanine concentrations were prevented by mild hypothermia. Mild hypothermia (35°C) prevents the encephalopathy and brain edema resulting from hepatic devascularization, selectively normalizes lactate and alanine synthesis from glucose, and prevents the impairment of oxidative metabolism associated with this model of ALF, but has no significant effect on brain glutamine. These findings suggest that a deficit in brain glucose metabolism rather than glutamine accumulation is the major cause of the cerebral complications of acute liver failure.

Carbohydrate and Amino Acid Metabolism in the Eucalyptus globulus-Pisolithus tinctorius Ectomycorrhiza during Glucose Utilization1

The metabolism of [1-13C]glucose in Pisolithus tinctorius cv Coker & Couch, in uninoculated seedlings of Eucalyptus globulus bicostata ex Maiden cv Kirkp., and in the E. globulus-P. tinctorius ectomycorrhiza was studied using nuclear magnetic resonance spectroscopy. In roots of uninoculated seedlings, the 13C label was mainly incorporated into sucrose and glutamine. The ratio (13C3 + 13C2)/13C4 of glutamine was approximately 1.0 during the time-course experiment, indicating equivalent contributions of phosphoenolpyruvate carboxylase and pyruvate dehydrogenase to the production of α-ketoglutarate used for synthesis of this amino acid. In free-living P. tinctorius, most of the 13C label was incorporated into mannitol, trehalose, glutamine, and alanine, whereas arabitol, erythritol, and glutamate were weakly labeled. Amino acid biosynthesis was an important sink of assimilated 13C (43%), and anaplerotic CO2 fixation contributed 42% of the C flux entering the Krebs cycle. In ectomycorrhizae, sucrose accumulation was decreased in the colonized roots compared with uninoculated control plants, whereas 13C incorporation into arabitol and erythritol was nearly 4-fold higher in the symbiotic mycelium than in the free-living fungus. It appears that fungal utilization of glucose in the symbiotic state is altered and oriented toward the synthesis of short-chain polyols.

In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth

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A non-invasive assessment of hepatic glycogen kinetics and post-absorptive gluconeogenesis in man.

A novel approach to the study of hepatic glycogen kinetics and fractional gluconeogenesis in vivo is described. Ten healthy female subjects were fed an iso-caloric diet containing 55% carbohydrate energy with a 13C abundance of 1.083 atom percent for a 3-day baseline period; then, a diet of similar composition, but providing carbohydrate with a 13C abundance of 1.093 atom percent was started and continued for 5 days. Resting respiratory gas exchanges, urinary nitrogen excretion, breath 13CO2 and plasma 13C glucose were measured every morning in the fasting state. The enrichment in 13C of hepatic glycogen was calculated from these measured data. 13C glycogen enrichment increased after switching to a 13C enriched carbohydrate diet, and was identical to the 13C enrichment of dietary carbohydrates after 3 days. The time required to renew 50% of hepatic glycogen, as determined from the kinetics of 13C glycogen enrichment, was 18.9 +/- 3.6 h. Fractional gluconeogenesis, as determined from the difference between the enrichments of glucose oxidized originating from hepatic glycogen and plasma glucose 13C was 50.8 +/- 5.3%. This non-invasive method will allow the study of hepatic glycogen metabolism in insulin-resistant patients.

Effects of lactate infusion on hepatic gluconeogenesis and glycogenolysis.

Endogenous glucose production rate (EGPR) remains constant when lactate is infused in healthy humans. A decrease of glycogenolysis or of gluconeogenesis from endogenous precursors or a stimulation of glycogen synthesis, may all be involved; This autoregulation does not depend on changes in glucoregulatory hormones. It may be speculated that alterations in basal sympathetic tone may be involved. To gain insights into the mechanisms responsible for autoregulation of EGPR, glycogenolysis and gluconeogenesis were measured, with a novel method (based on the prelabelling of endogenous glycogen with 13C glucose, and determination of hepatic 13C glycogen enrichment from breath 13CO2 and respiratory gas exchanges) in healthy humans infused with lactate or saline. These measurements were performed with or without beta-adrenergic receptor blockade (propranolol). Infusion of lactate increased energy expenditure, but did not increase EGPR; the relative contributions of gluconeogenesis and glycogenolysis to EGPR were also unaltered. This indicates that autoregulation is attained, at least in part, by inhibition of gluconeogenesis from endogenous precursors. beta-adrenergic receptor blockade alone (with propranolol) did not alter EGPR, glycogenolysis or gluconeogenesis. During infusion of lactate, propranolol decreased the thermic effect of lactate but EGPR remained constant. This indicates that alterations of beta-adrenergic activity is not required for autoregulation of EGPR.

Role of Na+-K+-ATPase in insulin-induced lactate release by skeletal muscle.

Hyperinsulinemia increases lactate release by various organs and tissues. Whereas it has been shown that aerobic glycolysis is linked to Na+-K+-ATPase activity, we hypothesized that stimulation by insulin of skeletal muscle Na+-K+-ATPase is responsible for increased muscle lactate production. To test this hypothesis, we assessed muscle lactate release in healthy volunteers from the [13C]lactate concentration in the effluent dialysates of microdialysis probes inserted into the tibialis anterior muscles on both sides and infused with solutions containing 5 mmol/l [U-13C]glucose. On one side, the microdialysis probe was intermittently infused with the same solution additioned with 2.10(-5) M ouabain. In the basal state, [13C]lactate concentration in the dialysate was not affected by ouabain. During a euglycemic-hyperinsulinemic clamp, [13C]lactate concentration increased by 135% in the dialysate without ouabain, and this stimulation was nearly entirely reversed by ouabain (56% inhibition compared with values in the dialysate collected from the contralateral probe). These data indicate that insulin stimulates muscle lactate release by activating Na+-K+-ATPase in healthy humans.