Regional glucose metabolism and glutamatergic neurotransmission in rat brain in vivo

Multivolume (1)H-[(13)C] NMR spectroscopy in combination with i.v. [1,6-(13)C(2)]glucose infusion was used to detect regional glucose metabolism and glutamatergic neurotransmission in the halothane-anesthetized rat brain at 7 T. The regional information was decomposed into pure cerebral gray matter, white matter, and subcortical structures by means of tissue segmentation based on quantitative T(1) relaxation mapping. The (13)C turnover curves of [4-(13)C]glutamate, [4-(13)C]glutamine, and [3-(13)C]glutamate + glutamine were fitted with a two-compartment neuronal-astroglial metabolic model. The neuronal tricarboxylic acid cycle fluxes in cerebral gray matter, white matter, and subcortex were 0.79 +/- 0.15, 0.20 +/- 0.11, and 0.42 +/- 0.09 micromol/min per g, respectively. The glutamate-glutamine neurotransmitter cycle fluxes in cerebral gray matter, white matter, and subcortex were 0.31 +/- 0.07, 0.02 +/- 0.04, and 0.18 +/- 0.12 micromol/min per g, respectively. The exchange rate between the mitochondrial and cytosolic metabolite pools was fast relative to the neuronal tricarboxylic acid cycle flux for all cerebral tissue types.     

The contribution of GABA to glutamate/glutamine cycling and energy metabolism in the rat cortex in vivo

Previous studies have shown that the glutamate/glutamine (Glu/Gln) neurotransmitter cycle and neuronal glucose oxidation are proportional (1:1), with increasing neuronal activity above isoelectricity. GABA, a product of Glu metabolism, is synthesized from astroglial Gln and contributes to total Glu/Gln neurotransmitter cycling, although the fraction contributed by GABA is unknown. In the present study, we used (13)C NMR spectroscopy together with i.v. infusions of [1,6-(13)C(2)]glucose and [2-(13)C]acetate to separately determine rates of Glu/Gln and GABA/Gln cycling and their respective tricarboxylic acid cycles in the rat cortex under conditions of halothane anesthesia and pentobarbital-induced isoelectricity. Under 1% halothane anesthesia, GABA/Gln cycle flux comprised 23% of total (Glu plus GABA) neurotransmitter cycling and 18% of total neuronal tricarboxylic acid cycle flux. In isoelectric cortex, glucose oxidation was reduced >3-fold in glutamatergic and GABAergic neurons, and neurotransmitter cycling was below detection. Hence, in both cell types, the primary energetic costs are associated with neurotransmission, which increase together as cortical activity is increased. The contribution of GABAergic neurons and inhibition to cortical energy metabolism has broad implications for the interpretation of functional imaging signals.     

Increased tricarboxylic acid cycle flux in rat brain during forepaw stimulation detected with 1H[13C]NMR.

NMR spectroscopy was used to test recent proposals that the additional energy required for brain activation is provided through nonoxidative glycolysis. Using localized NMR spectroscopic methods, the rate of C4-glutamate isotopic turnover from infused [1-(13)C]glucose was measured in the somatosensory cortex of rat brain both at rest and during forepaw stimulation. Analysis of the glutamate turnover data using a mathematical model of cerebral glucose metabolism showed that the tricarboxylic acid cycle flux [(V(TCA)] increased from 0.49 +/- 0.03 at rest to 1.48 +/- 0.82 micromol/g/min during stimulation (P < 0.01). The minimum fraction of C4-glutamate derived from C1-glucose was approximately 75%, and this fraction was found in both the resting and stimulated rats. Hence, the percentage increase in oxidative cerebral metabolic rate of glucose use (CMRglc) equals the percentage increases in V(TCA) and cerebral metabolic rate of oxygen consumption (CMRO2). Comparison with previous work for the same rat model, which measured total CMRglc [Ueki, M., Linn, F. & Hossman, K. A. (1988) J. Cereb. Blood Flow Metab. 8, 486-4941, indicates that oxidative CMRglc supplies the majority of energy during sustained brain activation.     

Comparative 13C and 31P NMR assessment of altered metabolism during graded reductions in coronary flow in intact rat hearts.

13C NMR spectroscopy may offer a unique ability to characterize the metabolic response to graded reduction in coronary flow since it allows repeated, nondestructive identification of products of intermediary metabolism in the same heart. The sensitivity of 13C parameters of glucose metabolism was compared with changes in levels of phosphocreatine, ATP, and pH as determined by 31P NMR in the intact, beating rat heart model during graded reductions in coronary flow. Experiments were performed during 60 min of perfusion with [1-13C]glucose (5 mM) at normal flow (15 ml/min) and at the reduced flow rates of 5 and 2 ml/min. During flow at 5 ml/min, isovolumic developed pressure fell to 51 +/- 4% of control. Although phosphocreatine, ATP, and pH were not changed, [3-13C]lactate was increased (1.46 +/- 0.12 mumol/g of wet weight vs. 0.63 +/- 0.08 during normal flow). In addition, the time to 50% maximum enrichment of [2-13C]glutamate was prolonged (17 +/- 1 min vs. 9 +/- 1 min during normal flow), indicating that glucose-supported flux through the tricarboxylic acid (TCA) cycle was decreased. The relative anaplerotic contribution to citrate synthase-supported TCA flux was increased from 6% to 35%. These 13C metabolic changes could not be reproduced by reduced [1-13C]glucose delivery in the absence of ischemia, although similar reduced TCA flux indices were reproduced in additional hearts when workload was reduced by low calcium (0.7 mM) perfusion. Therefore, the information provided by 13C NMR spectroscopy can be a more sensitive indicator of flow-induced alterations in cardiac metabolism than that provided by the much more commonly used 31P NMR technique.     

1H-[13C] NMR measurements of [4-13C]glutamate turnover in human brain.

A limitation of previous methods for studying human brain glucose metabolism, such as positron emission tomography, is that metabolic steps beyond glucose uptake cannot be studied. Nuclear magnetic resonance (NMR) has the advantage of allowing the nondestructive measurement of 13C distribution in specific carbon positions of metabolites. In this study 1H-[13C] NMR spectroscopy in conjunction with volume localization was used to measure the rate of incorporation of 13C isotope from infused enriched [1-13C]glucose to human brain [4-13C]glutamate. In three studies C4 glutamate turnover time constants of 25, 20, and 17 min were measured in a 21-cm3 volume centered in the region of the visual cortex. Based on an analysis of spectrometer sensitivity the spatial resolution of the method can be improved to < 4 cm3. In conjunction with metabolic modeling and other NMR measurements this method can provide a measure of regional rates of the brain tricarboxylic acid cycle and other metabolic pathways.     

13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS)

Cellular metabolism of glucose is required for stimulation of insulin secretion from pancreatic beta cells, but the precise metabolic coupling factors involved in this process are not known. In an effort to better understand mechanisms of fuel-mediated insulin secretion, we have adapted 13C NMR and isotopomer methods to measure influx of metabolic fuels into the tricarboxylic acid (TCA) cycle in insulinoma cells. Mitochondrial metabolism of [U-13C3]pyruvate, derived from [U-13C6]glucose, was compared in four clonal rat insulinoma cell 1-derived cell lines with varying degrees of glucose responsiveness. A 13C isotopomer analysis of glutamate isolated from these cells showed that the fraction of acetyl-CoA derived from [U-13C6]glucose was the same in all four cell lines (44 +/- 5%, 70 +/- 3%, and 84 +/- 4% with 3, 6, or 12 mM glucose, respectively). The 13C NMR spectra also demonstrated the existence of two compartmental pools of pyruvate, one that exchanges with TCA cycle intermediates and a second pool derived from [U-13C6]glucose that feeds acetyl-CoA into the TCA cycle. The 13C NMR spectra were consistent with a metabolic model where the two pyruvate pools do not randomly mix. Flux between the mitochondrial intermediates and the first pool of pyruvate (pyruvate cycling) varied in proportion to glucose responsiveness in the four cell lines. Furthermore, stimulation of pyruvate cycling with dimethylmalate or its inhibition with phenylacetic acid led to proportional changes in insulin secretion. These findings indicate that exchange of pyruvate with TCA cycle intermediates, rather than oxidation of pyruvate via acetyl-CoA, correlates with glucose-stimulated insulin secretion.     

Fatty acid regulation of glucose metabolism in the intact beating rat heart assessed by carbon-13 NMR spectroscopy: the critical role of pyruvate dehydrogenase

Although the myocardium is capable of utilizing both glucose and fatty acid substrates, glucose metabolism is inhibited in the presence of fatty acid during normal perfusion conditions. Fatty acid regulation of glucose utilization in intact beating rat hearts was studied with 13C-enriched substrates and 13C and 31P NMR spectroscopy at 8.5 T. During [1-13C]glucose and insulin perfusion, the 13C appeared in alanine, lactate and the glutamate isotopomers, indicating glycolytic flux through pyruvate and glucose-supported tricarboxylic acid (TCA) cycle oxidation, respectively. Following the addition of hexanoic acid, 1 mM, [1-13C]glucose metabolism proceeded through the hexokinase and phosphofructokinase reactions, as evidenced by continued production of [3-13C]alanine and [3-13C]lactate, but was completely inhibited at the pyruvate dehydrogenase (PDH) reaction as evidenced by a lack of appearance of the 13C label in the glutamate isotopomers. This inhibition of PDH was associated with increased PCr/ATP levels and was readily reversed by removal of hexanoic acid. Addition of dichloroacetate, 5 mM, which increases the active form of PDH, to fatty acid and glucose containing perfusate reinstituted carbon flux through the PDH reaction, indicating that the mechanism of fatty acid cessation of PDH flux is by reversible inactivation of the PDH enzyme complex. Thus the point of inhibition and mechanism of action of fatty acid modulation of glucose metabolism can be continuously and non-destructively studied in the intact beating heart with 13C and 31P NMR and is primarily attributable, in this model, to reversible PDH enzyme inactivation.     

Glutamine and glucose metabolism in human peripheral lymphocytes

The metabolism of glutamine and glucose in resting and concanavalin A-stimulated human peripheral lymphocytes was investigated. Glutamine was metabolized at a high rate by resting and mitogen-stimulated human peripheral lymphocytes and the major end-products of glutamine metabolism were glutamate, aspartate, CO2, and ammonia: the carbon from glutamine may contribute about 21% to respiration. Concanavalin A enhanced the formation of all end-products except glutamate, indicating that more glutamine was metabolized beyond the stage of glutamate in the mitogen-stimulated cells. Mitogenic stimulation caused an increase in the rates of glucose utilization, lactate production and 14CO2 from variously labeled [14C] glucose. Concanavalin A caused an increase in the oxidation of pyruvate as indicated by the enhanced release of 14CO2 from [2-14C]-, [3,4-14C]-, and [6-14C]-glucose. When both glucose and glutamine were presented to the cells, the rates of utilization of both substrates increased and the increased rates of glucose and glutamine utilization could be accounted for mainly by increased rates of lactate and glutamate production, respectively.     

Metabolic adaptation to feeding and fasting during lactation in humans.

The aim of these studies was to determine the metabolic adaptation to fasting and feeding during lactation. Normal lactating (L) and nonlactating (NL) women (n = 6 each) were studied using infusions of [U-13C]glucose and [2-13C]glycerol during: 1) a 24-h fast, and 2) ingestion of Sustacal (protocol 1). In addition, 8 L and 6 NL women were studied during infusion of [6,6-2H2]glucose and ingestion of a glucose meal containing [1-13C]glucose (protocol 2). Protocol 1: Glucose production rate (GPR) during fasting was 33% higher in the L women (12.5 +/- 1.0 vs. 9.4 +/- 0.5 micromol x kg(-1) x min(-1); P < 0.03). Fractional gluconeogenesis (GNG), GNG rate, glucose, lactate, beta- hydroxybutyrate, FFA, insulin, and C-peptide were similar in both groups during feeding and fasting, but glycogenolysis was 50% higher in fasting L women. Protocol 2: Although GPR was slightly increased in the L group (L, 1.8 +/- 0.2 micromol x kg(-1) x min(-1); NL, 1.2 +/- 0.2 micromol x kg(-1) x min(-1); P < 0.04), no other differences were observed in splanchnic and systemic metabolism of ingested glucose between L and NL women. Insulin concentrations were lower in L women compared with controls (L, 15 +/- 3 microU/ml; NL, 28 +/- 6 microU/ml; P = 0.05). In conclusion, the increased glucose demands of lactation are met by increased GPR as a result of increased glycogenolysis but not GNG or by increased use of FFA. During feeding, lactating women handle oral carbohydrates normally but have increased insulin sensitivity.     

Lower arterial glucose concentrations in lambs with aortopulmonary shunts after an 18-hour fast.

Spontaneously occurring hypoglycemia has been described in children with severe acute congestive heart failure. Hypoglycemia may be the result of an increase in glucose utilization in tissues, a decrease in glucose production, or a decrease in the dietary intake of nutrients. To determine whether hypoglycemia may also occur in congenital heart disease with volume overloading, we investigated glucose metabolism during and after an 18-hour fast in nine lambs with an aortopulmonary left-to-right shunt and nine control lambs. Plasma levels of hormones involved in the endocrine control of glucose metabolism were determined. The glucose production rate (rate of appearance [Ra]) was studied using [U-13C]glucose. Gluconeogenesis through the Cori cycle was estimated by measuring glucose 13C recycling. The arterial glucose concentration (3,409 +/- 104 v 4,338 +/- 172 micromol/L, P < .001) and Ra of glucose (16.97 +/- 0.89 v 25.49 +/- 4.28 micromol x min(-1) x kg(-1), P < .05) were lower in shunt versus control lambs. There were no differences in hormone levels between control and shunt lambs. Fractional glucose 13C recycling via the Cori cycle (6.9% +/- 2.8% v 7.1% +/- 2.5%) and gluconeogenesis from pyruvate and lactate (1.24 +/- 0.58 v 1.95 +/- 0.67 micromol x min(-1) x kg(-1)) were similar in both groups of lambs. The sum of glycogenolysis and gluconeogenesis from precursors other than pyruvate and lactate was lower in shunt versus control lambs (15.73 +/- 1.07 v 23.54 +/- 4.27 micromol x min(-1) x kg(-1), P < .05). In conclusion, after an 18-hour fast, the arterial glucose concentration is lower in lambs with aortopulmonary shunts. This lower glucose concentration is associated with a decreased glucose production rate. In shunt lambs, glycogenolysis is decreased, while there is no difference in gluconeogenesis or hormonal control.     

In vivo carbon-13 nuclear magnetic resonance studies of heart metabolism.

Guinea pig heart metabolism was studied in vivo by 13C NMR at 20.18 MHz. High-quality proton-decoupled 13C NMR spectra with excellent signal-to-noise ratios and resolution could be obtained in 6 min. Natural-abundance spectra showed resonances that could be assigned to fatty acids, but glycogen was not seen. During intravenous infusion of D-[1-13C]glucose and insulin, the time course of myocardial glycogen synthesis was followed serially for up to 4 hr. Anoxia resulted in degradation of the labeled glycogen within 6 min and appearance of 13C label in lactic acid. Infusion of sodium [2-13C]acetate resulted in incorporation of label into the C-4, C-2, and C-3 positions of glutamate and glutamine, reflecting "scrambling" of the label expected from tricarboxylic acid cycle activity. Examination of the 31P NMR spectrum of the guinea pig heart in vivo demonstrated no change in the high-energy phosphates during the time periods of the 13C NMR experiments. Our studies indicate that 13C NMR is a unique non-destructive tool for the study of heart metabolism in vivo.     

Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia

Alterations in energy substrate utilization during reperfusion of ischemic hearts can influence the functional recovery of the myocardium. Energy substrate preference by the reperfused myocardium, however, has received limited attention. Therefore, we measured oxidation rates of glucose and palmitate during reperfusion of ischemic hearts. Isolated working rat hearts were perfused with 1.2 mM palmitate and 11 mM [14C]glucose, 1.2 mM [14C]palmitate and 11 mM glucose, or 11 mM [14C]glucose alone, at an 11.5 mm Hg preload and 80 mm Hg afterload. Hearts were subjected to 60-minute aerobic perfusion or 25-minute global ischemia followed by 60-minute aerobic reperfusion. Steady-state oxidative rates of glucose or palmitate were determined by measuring 14CO2 production. In hearts perfused with glucose alone, oxidative rates during reperfusion were not significantly different than nonischemic hearts (1,008 +/- 335 vs. 1,372 +/- 117 nmol [14C]glucose oxidized/min/g dry wt, respectively). In the presence of palmitate, glucose oxidation was markedly reduced in reperfused and nonischemic hearts (81 +/- 11 and 101 +/- 15 nmol [14C]glucose oxidized/min/g dry wt, respectively). Palmitate oxidation rates were not significantly different in reperfused compared with nonischemic hearts (369 +/- 55 and 455 +/- 50 nmol [14C]palmitate oxidized/min/g dry wt, respectively). [14C]Palmitate was incorporated into myocardial triglycerides to a greater extent in reperfused ischemic hearts than in nonischemic hearts (26.0 and 13.8 mumol/g dry wt, respectively). Under the perfusion conditions used, palmitate provided over 90% of the ATP produced from exogenous substrates. Addition of the carnitine palmitoyltransferase I inhibitor, ethyl 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (Etomoxir, 10(-6) M), during reperfusion stimulated glucose oxidation and improved mechanical recovery of ischemic hearts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of decreased glutamine supply on protein and amino acid metabolism in hepatic tissue: a study using isolated perfused rat liver

Glutamine deficiency, a common finding in severe illness, has a negative influence on immune status, protein metabolism, and disease outcome. In several studies, a close relationship between glutamine, branched-chain amino acid (BCAA), and protein metabolism was demonstrated. The aim of the present study was to investigate the effect of glutamine deficiency on amino acid and protein metabolism in hepatic tissue using a model of isolated perfused rat liver (IPRL). Parameters of protein metabolism and amino acid metabolism were measured using both recirculation and single pass technique with L-[1-(14)C]leucine and [1-(14)C]ketoisocaproate (KIC) as a tracer. Glutamine concentration in perfusion solution was 0.5 mmol/L in control and 0 mmol/L in the glutamine-deficient group. The net release of glutamine (about 11 micromol/g/h) and higher net uptake of most of the amino acids was observed in the glutamine-deficient group. There was an insignificant effect of lack of glutamine on hepatic protein synthesis, proteolysis, and the release of urea. However, significantly lower release of proteins by the liver perfused with glutamine-deficient solution was observed. The lack of glutamine in perfusion solution caused a significant decrease in leucine oxidation (6.66 +/- 1.04 v 13.67 +/- 2.38, micromol/g dry liver/h, P <.05) and an increase in KIC oxidation (163.7 +/- 16.5 v 92.0 +/- 12.9 micromL/g dry liver/h, P <.05). We conclude that decreased delivery of glutamine to hepatic tissue activates glutamine synthesis, decreases resynthesis of essential BCAA from branched-chain keto acids (BCKA), increases catabolism of BCKA, and has an insignificant effect on protein turnover in hepatic tissue.     

Determination of pathways of glycogen synthesis and the dilution of the three-carbon pool with [U-13C]glucose.

Rats were infused with glucose at 30 mg/min, containing 18% enriched [U-13C]glucose and [1-14C]- and [3-3H]glucose. The mass isotopomer patterns of 13C-labeled blood glucose and liver glycogen were determined by gas chromatography/mass spectroscopy. The contribution of the direct pathway to glycogen was calculated from the three tracers, and the values by all three were nearly identical, about 50%. The 14C specific activity in carbon 6 of glycogen glucose was about 6% that of carbon 1. The [3H]glucose/[1-14C]glucose ratio in glycogen was 80-90% that in blood glucose. The enrichment of 13C and the specific activity of 14C in glycogen formed by the indirect path were 20-25% of glycogen formed directly from glucose. The dilution is of two kinds: (i) an exchange of labeled carbon with unlabeled carbon in the tricarboxylic acid cycle and (ii) dilution by unlabeled nonglucose carbon. Methods to calculate the two types of dilution are presented. In control rats the dilution factor by exchange in the tricarboxylic acid cycle is 1.4, and the dilution by unlabeled carbon is 2.5- to 3.0-fold, with the overall dilution about 4-fold. In rats preinjected with glucagon, the dilution through the tricarboxylic acid cycle was unaffected but that by nonglucose carbon was decreased.     

The quantification of gluconeogenesis in healthy men by (2)H2O and [2-(13)C]glycerol yields different results: rates of gluconeogenesis in healthy men measured with (2)H2O are higher than those measured with [2-(13)C]glycerol

The quantification of gluconeogenesis (GNG) by (2)H2O and [2-(13)C]glycerol and the mass isotopomer dilution analysis of glucose does not involve assumptions regarding the enrichment of the oxaloacetate precursor pool. To compare these two methods we measured GNG in six healthy postabsorptive males under identical, strictly standardized, eucaloric conditions, once after oral administration of (2)H2O and once during a primed continuous infusion of [2-(13)C]glycerol. Endogenous glucose production (EGP) was measured by infusion of [6,6-(2)H(2)]glucose. EGP was not different after (2)H2O administration or during [2-(13)C]glycerol infusion (12.2 +/- 0.7 vs. 11.7 +/- 0.3 micromol/kg.min). However, GNG measured after (2)H2O administration was significantly higher than that during [2-(13)C]glycerol infusion (7.4 +/- 0.7 vs. 4.9 +/- 0.6 micromol/kg.min; P = 0.03), representing approximately 60% and 41% of EGP, respectively. The (2)H2O study was repeated during primed continuous infusion of unlabeled glycerol, showing that infusion of glycerol at the rate used in the [2-(13)C]glycerol method does not affect the measurement of GNG with (2)H2O, viz. 7.4 +/- 0.7 without glycerol vs. 7.6 +/- 0.9 micromol/kg.min with glycerol, representing approximately 60% vs. 62% of EGP. In conclusion, GNG measured by (2)H(2)O yields higher results than those measured by [2-(13)C]glycerol. This discrepancy is not merely caused by infusion of glycerol per se. Rather, the discrepancy between both methods probably relates to conceptual problems in underlying assumptions in one or both methods.     

Enhanced glutamine and glucose metabolism in cultured rat splenocytes stimulated by phorbol myristate acetate plus ionomycin.

Metabolism of glutamine and glucose was studied in normal rat splenocytes cultured for 48 hours in the presence and absence of a mixture of the mitogens, phorbol myristate acetate (PMA) + ionomycin (Iono). 3H-Thymidine uptake by splenocytes was stimulated more than 500-fold by PMA + Iono. After culture, cells were incubated for 2 hours in the presence of either 2 mmol/L [U-14C]glutamine +/- 5 mmol/L glucose or 5 mmol/L [U-14C]glucose +/- 2 mmol/L glutamine in Krebs-Ringer HEPES buffer. Glutamine was metabolized mainly to ammonia, glutamate, aspartate, and CO2, and these products were all increased (P less than .01) by twofold to 2.5-fold in stimulated cells. Glucose was metabolized mainly to lactate and, to a lesser extent, to pyruvate and CO2. Lactate production from glucose was increased (P less than .01) by 2.4-fold in stimulated cells, without changes in pyruvate or CO2 production. In unstimulated, cultured splenocytes, glutamine was not quantitatively as important as glucose in the provision of adenosine triphosphate (ATP), as calculated on the basis of measured metabolites. However, in stimulated cells, glutamine became a much more important energy substrate, providing similar amounts of ATP to those from glucose. The oxidation of glutamine via the Krebs cycle was the major pathway for glutamine-derived ATP production, while lactate production from glucose accounted for the major part of glucose-derived ATP in PMA+Iono-stimulated splenocytes. Thus, we suggest glutamine plays a dual metabolic role in these cells, as both an important fuel and an essential source of carbon and nitrogen precursors for biosynthetic processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of lipolysis during acute GH exposure increases insulin sensitivity in previously untreated GH-deficient adults

OBJECTIVE: Previous studies evaluating the lipolytic effect of GH have in general been performed in subjects on chronic GH therapy. In this study we assessed the lipolytic effect of GH in previously untreated patients and examined whether the negative effect of enhanced lipolysis on glucose metabolism could be counteracted by acute antilipolysis achieved with acipimox. METHODS: Ten GH-deficient (GHD) adults participated in four experiments each, during which they received in a double-blind manner: placebo (A); GH (0.88+/-0.13 mg) (B); GH+acipimox 250 mg b.i.d. (C); and acipimox b.i.d. (no GH) (D), where GH was given the night before a 2 h euglycemic, hyperinsulinemic clamp combined with infusion of [3-(3)H]glucose and indirect calorimetry. RESULTS: GH increased basal free fatty acid (FFA) levels by 74% (P=0.0051) and insulin levels by 93% (P=0.0051). This resulted in a non-significant decrease in insulin-stimulated glucose uptakes (16.61+/-8.03 vs 12.74+/-5.50 micromol/kg per min (s.d.), P=0.07 for A vs B). The rates of insulin-stimulated glucose uptake correlated negatively with the FFA concentrations (r=-0.638, P<0.0001). However, acipimox caused a significant improvement in insulin-stimulated glucose uptake in the GH-treated patients (17.35+/-5.65 vs 12.74+/-5.50 micromol/kg per min, P=0.012 for C vs B). The acipimox-induced enhancement of insulin-stimulated glucose uptake was mainly due to an enhanced rate of glucose oxidation (8.32+/-3.00 vs 5.88+/-2.39 micromol/kg per min, P=0.07 for C vs B). The enhanced rates of glucose oxidation induced by acipimox correlated negatively with the rate of lipid oxidation in GH-treated subjects both in basal (r=-0.867, P=0.0093) and during insulin-stimulated (r=-0.927, P=0.0054) conditions. GH did not significantly impair non-oxidative glucose metabolism (6.86+/-5.22 vs 8.67+/-6.65 micromol/kg per min, P=NS for B vs A). The fasting rate of endogenous glucose production was unaffected by GH and acipimox administration (10.99+/-1.98 vs 11.73+/-2.38 micromol/kg per min, P=NS for B vs A and 11.55+/-2.7 vs 10.99+/-1.98 micromol/kg per min, P=NS for C vs B). On the other hand, acipimox alone improved glucose uptake in the untreated GHD patients (24.14+/-8.74 vs 16.61+/-8.03 micromol/kg per min, P=0.0077 for D vs A) and this was again due to enhanced fasting (7.90+/-2.68 vs 5.16+/-2.28 micromol/kg per min, P=0.01 for D vs A) and insulin-stimulated (9.78+/-3.68 vs 7.95+/-2.64 micromol/kg per min, P=0.07 for D vs A) glucose oxidation. CONCLUSION: The study of acute administration of GH to previously untreated GHD patients provides compelling evidence that (i) GH-induced insulin resistance is mainly due to induction of lipolysis by GH; and (ii) inhibition of lipolysis can prevent the deterioration of insulin sensitivity. The question remains whether GH replacement therapy should, at least at the beginning of therapy, be combined with means to prevent an excessive stimulation of lipolysis by GH.     

Important role of the hepatic vagus nerve in glucose uptake and production by the liver

We examined the role of the hepatic vagus nerve in hepatic and peripheral glucose metabolism. To assess endogenous glucose production (EGP), hepatic uptake of first-pass glucose infused intraportally (HGU), and the metabolic clearance rate of glucose (MCR), rats were subjected to hepatic vagotomy (HV, n = 7) or sham operation (SH, n = 8), after 10 days, they were then subjected to a euglycemic-hyperinsulinemic clamp together with a portal glucose load in the 24-hour fasting state. Metabolic parameters were determined by the dual-tracer method using stable isotopes. During the experiment, [6,6-2H2]glucose was continuously infused into the peripheral vein. To maintain euglycemia (4.5 mmol/L), insulin (54 pmol x kg(-1) x min(-1)) and glucose were infused peripherally after the 90-minute tracer equilibration and 30-minute basal periods, and glucose containing 5% enriched [U-13C]glucose was infused intraportally (50 micromol x kg(-1) x min(-1)) for 120 minutes (clamp period). EGP was significantly higher in HV rats versus SH rats during the basal period (64.3 +/- 7.6 v 43.6 +/- 5.3 micromol x kg(-1) x min(-1), P < .005)) and was comparable to EGP in SH rats during the clamp period (9.3 +/- 21.5 v 1.1 +/- 11.7 micromol x kg(-1) x min(-1)). HGU was reduced in HV rats compared with SH rats during portal glucose infusion (5.9 +/- 2.4 v 10.1 +/- 3.2 micromol x kg(-1) x min(-1)). The MCR in HV rats was significantly higher than in SH rats in the basal period (11.0 +/- 2.0 v 7.9 +/- 0.8 mL x kg(-1) x min(-1), P < .01)) and was comparable to the MCR in SH rats during the clamp period (41.9 +/- 10.0 and 36.6 +/- 5.7 mL x kg(-1) x min(-1)). We conclude that innervation of the hepatic vagus nerve is important for the regulation of hepatic glucose production in the postabsorptive state and HGU in the postprandial state.     

Paradoxical changes of muscle glutamine release during hyperinsulinemia euglycemia and hypoglycemia in humans: further evidence for the glucose-glutamine cycle

Insulin suppresses and counterregulatory hormones increase proteolysis. Therefore, if proteolysis were a major factor determining amino acid fluxes in plasma, one would expect release of glutamine into plasma to be suppressed by insulin under euglycemic conditions and to be stimulated under hypoglycemic conditions. However, release of glutamine into plasma remains unaltered or increases during euglycemic hyperinsulinemia and decreases during insulin-induced hypoglycemia. To investigate the mechanisms for these paradoxical observations and the role of skeletal muscle, we infused overnight fasted volunteers with [U-(14)C] glutamine and measured release of glutamine into plasma, its removal from plasma, and forearm glutamine net balance, fractional extraction, uptake and release during 4-hour euglycemic ( approximately 5.0 mmol/L, n = 7) and hypoglycemic ( approximately 3.1 mmol/L, n = 8) hyperinsulinemic ( approximately 230 pmol/L) clamp experiments. During the euglycemic clamps, plasma glutamine uptake and release (both P <.05) and forearm muscle glutamine fractional extraction (P <.05), uptake (P <.02) and release (P <.01) all increased, whereas forearm glutamine net balance remained unchanged. The increase in muscle glutamine release (from 1.85 +/- 0.26 to 2.18 +/- 0.30 micromol. kg(-1). min(-1)) accounted for approximately 60% of the increase in total glutamine release into plasma (from 5.54 +/- 0.47 to 6.10 +/- 0.64 micromol. kg(-1). min(-1)) and correlated positively with the increase in muscle glucose uptake (r = 0.80, P <.03). During the hypoglycemic clamps, plasma glutamine uptake and release and forearm glutamine release remained unaltered, but forearm glutamine fractional extraction and uptake decreased approximately 25% (both P <.01) so that forearm glutamine net release increased from 0.37 +/- 0.06 to 0.61 +/- 0.09 micromol. kg(-1). min(-1) (P <.03). We conclude that skeletal muscle is largely responsible for the increased release of glutamine into plasma during euglycemic hyperinsulinemia in humans, and that this may be due to increased conversion of glucose to glutamine as part of the glucose-glutamine cycle; during hypoglycemic hyperinsulinemia decreased glutamine uptake by skeletal muscle may be important for providing substrate for increased glutamine gluconeogenesis.     

Metabolic insights into the hepatoprotective role of N-acetylcysteine in mouse liver

The hepatoprotective mechanisms of N-acetylcysteine (NAC) in non-acetaminophen-induced liver injury have not been studied in detail. We investigated the possibility that NAC could affect key pathways of hepatocellular metabolism with or without changes in glutathione (GSH) synthesis. Hepatocellular metabolites and high-energy phosphates were quantified from mouse liver extracts by 1H- and 31P-NMR (nuclear magnetic resonance) spectroscopy. 13C-NMR-isotopomer analysis was used to measure [U-13C]glucose metabolism through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC). NAC (150-1,200 mg/kg) increased liver concentrations of GSH from 8.60 +/- 0.48 to a maximum of 12.95 +/- 1.03 micromol/g ww, whereas hypotaurine (HTau) concentrations increased from 0.05 +/- 0.02 to 9.95 +/- 1.12 micromol/g ww. The limited capacity of NAC to increase GSH synthesis was attributed to impaired glucose metabolism through PC. However, 300 mg/kg NAC significantly increased the fractional 13C-enrichment in Glu (from 2.08% +/- 0.26% to 4.00% +/- 0.44%) synthesized through PDH, a key enzyme for mitochondrial energy metabolism. This effect could be uncoupled from GSH synthesis and was associated with the prevention of liver injury induced by tert-butylhydroperoxide and 3-nitropropionic acid. In conclusion, NAC (1) has a limited capacity to elevate GSH synthesis; (2) increases HTau formation linearly; and (3) improves mitochondrial tricarboxylic acid (TCA) cycle metabolism by stimulation of carbon flux through PDH. This latter effect is independent of the capacity of NAC to replete GSH stores. These metabolic actions, among other yet unknown effects, are critical for NAC's therapeutic value and should be taken into account when deciding on a wider use of NAC.