Upregulation of Krebs cycle and anaerobic glycolysis activity early after onset of liver ischemia.

The liver is a highly vascularized organ receiving a dual input of oxygenated blood from the hepatic artery and portal vein. The impact of decreased blood flow on glucose metabolism and how hepatocytes could adapt to this restrictive environment are still unclear. Using the left portal vein ligation (LPVL) rat model, we found that cellular injury was delayed after the onset of liver ischemia. We hypothesized that a metabolic adaptation by hepatocytes to maintain energy homeostasis could account for this lag phase. Liver glucose metabolism was characterized by 13C- and 1H-NMR spectroscopy and analysis of high-energy metabolites. ALT levels and caspase 3 activity in LPVL animals remained normal during the first 12 h following surgery (P<0.05). Ischemia rapidly led to decreased intrahepatic tissue oxygen tension and blood flow (P<0.05) and increased expression of Hypoxia-inducible factor 1-alpha. Intrahepatic glucose uptake, ATP/ADP ratio and energy charge level remained stable for up to 12 h after ligation. Entry of glucose in the Krebs cycle was impaired with lowered incorporation of 13C from [U-13C]glucose into glutamate and succinate from 0.25 to 12 h after LPVL. However, total hepatic succinate and glutamate increased 6 and 12 h after ischemia (P<0.05). Glycolysis was initially reduced (P<0.05) but reached maximum 13C-lactate (P<0.001) and 13C-alanine (P<0.01) enrichments 12 h after LPVL. In conclusion, early liver homeostasis stems from an inherent ability of ischemic hepatocytes to metabolically adapt through increased Krebs cycle and glycolysis activity to preserve bioenergetics and cell viability. This metabolic plasticity of hepatocytes could be harnessed to develop novel metabolic strategies to prevent ischemic liver damage.

Increased brain lactate is central to the development of brain edema in rats with chronic liver disease.

BACKGROUND & AIMS: The pathogenesis of brain edema in patients with chronic liver disease (CLD) and minimal hepatic encephalopathy (HE) remains undefined. This study evaluated the role of brain lactate, glutamine and organic osmolytes, including myo-inositol and taurine, in the development of brain edema in a rat model of cirrhosis. METHODS: Six-week bile-duct ligated (BDL) rats were injected with (13)C-glucose and de novo synthesis of lactate, and glutamine in the brain was quantified using (13)C nuclear magnetic resonance spectroscopy (NMR). Total brain lactate, glutamine, and osmolytes were measured using (1)H NMR or high performance liquid chromatography. To further define the interplay between lactate, glutamine and brain edema, BDL rats were treated with AST-120 (engineered activated carbon microspheres) and dichloroacetate (DCA: lactate synthesis inhibitor). RESULTS: Significant increases in de novo synthesis of lactate (1.6-fold, p<0.001) and glutamine (2.2-fold, p<0.01) were demonstrated in the brains of BDL rats vs. SHAM-operated controls. Moreover, a decrease in cerebral myo-inositol (p<0.001), with no change in taurine, was found in the presence of brain edema in BDL rats vs. controls. BDL rats treated with either AST-120 or DCA showed attenuation in brain edema and brain lactate. These two treatments did not lead to similar reductions in brain glutamine. CONCLUSIONS: Increased brain lactate, and not glutamine, is a primary player in the pathogenesis of brain edema in CLD. In addition, alterations in the osmoregulatory response may also be contributing factors. Our results suggest that inhibiting lactate synthesis is a new potential target for the treatment of HE.

Hepatocellular apoptosis in mice is associated with early upregulation of mitochondrial glucose metabolism.

Hepatocyte death due to apoptosis is a hallmark of almost every liver disease. Manipulation of cell death regulatory steps during the apoptotic process is therefore an obvious goal of biomedical research. To clarify whether metabolic changes occur prior to the characteristic apoptotic events, we used ex vivo multinuclear NMR-spectroscopy to study metabolic pathways of [U-(13)C]glucose in mouse liver during Fas-induced apoptosis. We addressed whether these changes could be associated with protection against apoptosis afforded by Epidermal Growth Factor (EGF). Our results show that serum alanine and aspartate aminotransferase levels, caspase-3 activity, BID cleavage and changes in cellular energy stores were not observed before 3 h following anti-Fas injection. However, as early as 45 min after anti-Fas treatment, we observed upregulation of carbon entry (i.e. flux) from glucose into the Krebs-cycle via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) (up to 139% and 123% of controls, respectively, P < 0.001). This was associated with increased glutathione synthesis. EGF treatment significantly attenuated Fas-induced apoptosis, liver injury and the late decrease in energy stores, as well as the early fluxes through PDH and PC which were comparable to untreated controls. Using ex vivo multinuclear NMR-spectroscopic analysis, we have shown that Fas receptor activation in mouse liver time-dependently affects specific metabolic pathways of glucose. These early upregulations in glucose metabolic pathways occur prior to any visible signs of apoptosis and may have the potential to contribute to the initiation of apoptosis by maintaining mitochondrial energy production and cellular glutathione stores.

Organic osmolytes in hyponatremia and ammonia toxicity.

Hyperammonemia (HA) is a major and commonly observed feature of hepatic encephalopathy. Furthermore, hyponatremia is an important pathogenetic factor in patients with hepatic encephalopathy. Both conditions have some features in common, such as the release of organic osmolytes, which might be an adaptive mechanism against cell swelling. However, the consequence of a possible relationship between osmoregulatory response in hyperammonemia and hyponatremia is not completely understood. This review gives a short introduction into the pathogenesis of hepatic encephalopathy and hyponatremia. For a comparison of both pathological events, some basics on cellular osmo- and volume regulation are explained, in particular as the mechanisms involved in the adaption of the cell to volume changes can be different under both pathological conditions. The role of brain glutamine and organic osmolytes in hyponatremia and hyperammonemia and their combination are discussed based on findings in experimental animal models, and finally on data obtained from primary astrocytes in culture. The observations that the decrease of brain organic osmolytes in astrocytes not adequately compensate for an increased intracellular osmolarity caused by glutamine are consistent with results obtained after chronic hyponatremia in rats, in which the release of osmolytes does not protect from ammonia-induced brain edema. Furthermore, a decrease in intracellular osmolarity is attributed both to the release and a reduced de novo synthesis of amino acids.

Novel mechanisms of protection against acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine.

UNLABELLED: Acetaminophen (APAP) overdose is a major cause of acute liver failure. The glutathione (GSH) precursor N-acetylcysteine (NAC) is used to treat patients with APAP overdose for up to 48 hours. Although it is well established that early treatment with NAC can improve the scavenging of the reactive metabolite N-acetyl-p-benzoquinone imine, protective mechanisms at later times remain unclear. To address this issue, fasted C3Heb/FeJ mice were treated with 300 mg/kg APAP and then received intravenously 0.65 mmol/kg GSH or NAC at 1.5 hours after APAP. The animals were sacrificed at 6 hours. APAP alone caused severe liver injury with peroxynitrite formation and DNA fragmentation, all of which was attenuated by both treatments. However, GSH (-82%) was more effective than NAC (-46%) in preventing liver injury. Using nuclear magnetic resonance spectroscopy to measure tissue adenosine triphosphate (ATP) levels and the substrate flux through the mitochondrial Krebs cycle, it was observed that the reduced liver injury correlated with accelerated recovery of mitochondrial GSH content, maintenance of ATP levels, and an increased substrate supply for the mitochondrial Krebs cycle compared with APAP alone. NAC treatment was less effective in recovering ATP and mitochondrial GSH levels and showed reduced substrate flux through the Krebs cycle compared with GSH. However, increasing the dose of NAC improved the protective effect similar to GSH, suggesting that the amino acids not used for GSH synthesis were used as mitochondrial energy substrates. CONCLUSION: Delayed treatment with GSH and NAC protect against APAP overdose by dual mechanisms-that is, by enhancing hepatic and mitochondrial GSH levels (scavenging of reactive oxygen and peroxynitrite)-and by supporting the mitochondrial energy metabolism.

L-ornithine and phenylacetate synergistically produce sustained reduction in ammonia and brain water in cirrhotic rats.

UNLABELLED: Treatment of hyperammonemia and hepatic encephalopathy in cirrhosis is an unmet clinical need. The aims of this study were to determine whether L-ornithine and phenylacetate/phenylbutyrate (administered as the pro-drug phenylbutyrate) (OP) combined are synergistic and produce sustained reduction in ammonia by L-ornithine acting as a substrate for glutamine synthesis, thereby detoxifying ammonia, and the phenylacetate excreting the ornithine-derived glutamine as phenylacetylglutamine in the urine. Sprague-Dawley rats were studied 4 weeks after bile duct ligation (BDL) or sham operation. Study 1: Three hours before termination, an internal carotid sampling catheter was inserted, and intraperitoneal saline (placebo), OP, phenylbutyrate, or L-ornithine were administered after randomization. BDL was associated with significantly higher arterial ammonia and brain water and lower brain myoinositol (P < 0.01, respectively), compared with sham-operated controls, which was significantly improved in the OP-treated animals; arterial ammonia (P < 0.001), brain water (P < 0.05), brain myoinositol (P < 0.001), and urinary phenylacetylglutamine (P < 0.01). Individually, L-ornithine or phenylbutyrate were similar to the BDL group. In study 2, BDL rats were randomized to saline or OP administered intraperitoneally for 6 hours or 3, 5, or 10 days and were sacrificed between 4.5 and 5 weeks. The results showed that the administration of OP was associated with sustained reduction in arterial ammonia (P < 0.01) and brain water (P < 0.01) and markedly increased arterial glutamine (P < 0.01) and urinary excretion of phenylacetylglutamine (P < 0.01) in each of the OP treated groups. CONCLUSION: The results of this study provide proof of the concept that L-ornithine and phenylbutyrate/phenylacetate act synergistically to produce sustained improvement in arterial ammonia, its brain metabolism, and brain water in cirrhotic rats.

Sustained hydrogen peroxide stress decreases lactate production by cultured astrocytes.

Oxidative stress and disrupted energy metabolism are common to many pathological conditions of the brain. Because astrocytes play an important role in the glucose metabolism of the brain, we have investigated whether sustained oxidative stress affects astroglial glucose metabolism with cultured primary rat astrocytes as a model system. Cultured astrocytes were exposed to a sustained concentration of approximately 50 muM H(2)O(2) in the presence of [U-(13)C]glucose, and cellular and extracellular contents of lactate and glucose were analysed by enzymatic assays and NMR spectroscopy. Exposure of the cells to sustained H(2)O(2) stress for up to 120 min significantly lowered the rate of lactate accumulation in the media to 61% +/- 14% of that in cultures incubated without peroxide. In addition, the ratio of lactate release to glucose consumption was lowered in peroxide-treated astrocytes to 77% +/- 13% of that in control cells, and the specific activity of glyceraldehyde-3-phosphate dehydrogenase had declined to about 10% of control cells within 90 min. In addition, the (13)C enrichment of intracellular and extracellular [(13)C]lactate was about 30% and 95%, respectively, and was not affected by the presence of peroxide, demonstrating that two metabolic pools of lactate are present in cultured astrocytes. The decreased rate of lactate production by astrocytes that have been exposed to peroxide stress is a new example of an alteration by oxidative stress of an important metabolic pathway in astrocytes. Such alterations could contribute to the pathological conditions that have been connected with oxidative stress and disrupted energy metabolism in the brain.

Altered fatty acid metabolism and composition in cultured astrocytes under hyperammonemic conditions.

In vitro 1H- and 13C-NMR spectroscopy was used to investigate the effect of ammonia on fatty acid synthesis and composition in cultured astrocytes. Cells were incubated 3 and 24 h with 5 mM ammonia in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. An increase of de novo synthesized fatty acids and the glycerol subunit of lipids was observed after 3 h treatment with ammonia (35% and 40% over control, respectively), the initial time point examined. Both parameters further increased significantly to 85% and 60% over control after 24 h ammonia treatment. Three hours incubation with ammonia increased the synthesis of diacylglycerides, while formation of triacylglycerides was decreased (40% over and 15% under control, respectively). The degradation of fatty acids was not affected by ammonia treatment. Furthermore, ammonia caused alterations in the composition of fatty acids, e.g. increased mono- and decreased polyunsaturated fatty acids (85% over and 15% under control concentrations, respectively). The decrease of polyunsaturated fatty acids was even more pronounced in isolated astrocytic mitochondria (39% lower than controls). Our results suggest ammonia-induced abnormalities in astrocytic membranes, which may be related to astrocytic mitochondrial dysfunction in hyperammonemic states. Most of the observed effects of ammonia on fatty acid synthesis and composition were ameliorated when glutamine synthetase was inhibited by methionine sulfoximine, supporting a pathological role of glutamine in ammonia toxicity. This study further emphasizes the importance of investigating the relative contribution of exogenous ammonia, effects of glutamine and of glutamine-derived ammonia on astrocytes and astrocytic mitochondria.

Impaired oxidation of branched-chain amino acids in the medial thalamus of thiamine-deficient rats.

Thiamine, in its diphosphate form, is a required cofactor for enzymes of glucose metabolism and branched-chain alpha-ketoacid dehydrogenase (BCKDH). Although metabolic impairments in glucose metabolism have been found to occur in selectively vulnerable brain regions of the thiamine-deficient (TD) brain, the effects of thiamine deficiency on BCKDH have not been studied. BCKDH activity was assayed radiochemically in brain extracts of vulnerable (medial thalamus; MT) versus non-vulnerable (frontal cortex; FC) brain regions of rats made TD by administration of the central thiamine antagonist, pyrithiamine. A significant regional variation in BCKDH within the TD rat brain was noted, with a higher capacity for branched-chain amino acid oxidation in FC compared to MT: BCKDH activity was significantly reduced in MT of TD rats, resulting in selective accumulation of BCAAs in this brain region. Leucine concentrations were elevated over fivefold in the MT of symptomatic TD rats, compared with pair-fed control (PFC) rats. Impaired branched-chain ketoacid metabolism in rats may contribute to the neuronal dysfunction and ultimate thalamic neuronal cell death observed in thiamine deficiency.

Organochlorines inhibit acetaminophen glucuronidation by redirecting UDP-glucuronic acid towards the D-glucuronate pathway.

Industry-derived organochlorines are persistent environmental pollutants that are a continuing health concern. The effects of these compounds on drug metabolism are not well understood. In the current study we present evidence that the inhibition of acetaminophen (APAP) glucuronidation by minute concentrations of organochlorines correlates well with their ability to stimulate the d-glucuronate pathway leading to ascorbate synthesis. A set of 6 arylated organochlorines, including 5 PCB (polychlorinated biphenyl) congeners, were assessed for their effects on APAP glucuronidation in isolated hepatocytes from male Sprague-Dawley rats. The capacity of each organochlorine to inhibit APAP glucuronidation was found to be directly proportional to its capacity to stimulate ascorbate synthesis. PCB153, PCB28 and bis-(4-chlorophenyl sulfone) (BCPS) in increasing order were the most effective organochlorines for inhibiting APAP glucuronidation and stimulating the d-glucuronate pathway. None of the 3 inhibitors of APAP glucuronidation were able to alter the expression of UGT1A6, UGT1A7 and UGT1A8 (the major isoforms responsible for APAP glucuronidation in the rat), however, their efficacy at inhibiting APAP glucuronidation was proportional to their capacity to deplete UDP-glucuronic acid (UDPGA). BCPS-mediated inhibition of APAP glucuronidation in isolated hepatocytes had non-competitive characteristics and was insensitive to the inactivation of cytochrome P450. The effective organochlorines were also able to selectively stimulate the hydrolysis of UDPGA to UDP and glucuronate in isolated microsomes, but could not inhibit APAP glucuronidation in microsomes when UDPGA was in excess. We conclude that organochlorines are able to inhibit APAP glucuronidation in hepatocytes by depleting UDPGA via redirecting UDPGA towards the d-glucuronate pathway. Because the inhibition is non-competitive, low concentrations of these compounds could have long term inhibitory effects on the glucuronidating capacity of hepatocytes.

Region-selective alterations of glucose oxidation and amino acid synthesis in the thiamine-deficient rat brain: a re-evaluation using 1H/13C nuclear magnetic resonance spectroscopy.

Thiamine deficiency provides an effective model of selective neuronal cell death. (1)H and (13)C-NMR was used to investigate the effects of thiamine deficiency on the synthesis of amino acids derived from [1-(13)C]glucose in vulnerable (medial thalamus; MT) compared to non-vulnerable (frontal cortex; FC) brain regions. Following 11 days of thiamine deficiency, a time-point associated with the absence of significant neuronal cell death, regional concentrations of glutamate, glutamine and GABA remained unaffected in FC and MT; however, decreased levels of aspartate in MT at this time-point were a predictor of regional vulnerability. De novo synthesis of glutamate and GABA were unaffected at 11 days of thiamine deficiency, while synthesis of [2-(13)C]aspartate was significantly impaired. Glucose loading, which has been shown to exacerbate symptoms in patients with thiamine deficiency, resulted in further decreases of TCA cycle flux and reduced de novo synthesis of glutamate, aspartate and GABA in thiamine-deficient (TD) rats. Isotopomer analysis revealed that impaired TCA cycle flux and decreased aspartate synthesis due to thiamine deficiency occurred principally in neurons. Glucose loading deteriorated TD-related decreases in TCA cycle flux, and concomitantly reduced synthesis of aspartate and glutamate in MT.

Mice deficient in Neu4 sialidase exhibit abnormal ganglioside catabolism and lysosomal storage.

Mammalian sialidase Neu4, ubiquitously expressed in human tissues, is located in the lysosomal and mitochondrial lumen and has broad substrate specificity against sialylated glycoconjugates. To investigate whether Neu4 is involved in ganglioside catabolism, we transfected beta-hexosaminidase-deficient neuroglia cells from a Tay-Sachs patient with a Neu4-expressing plasmid and demonstrated the correction of storage due to the clearance of accumulated GM2 ganglioside. To further clarify the biological role of Neu4, we have generated a stable loss-of-function phenotype in cultured HeLa cells and in mice with targeted disruption of the Neu4 gene. The silenced HeLa cells showed reduced activity against gangliosides and had large heterogeneous lysosomes containing lamellar structures. Neu4(-/-) mice were viable, fertile and lacked gross morphological abnormalities, but showed a marked vacuolization and lysosomal storage in lung and spleen cells. Lysosomal storage bodies were also present in cultured macrophages preloaded with gangliosides. Thin-layer chromatography showed increased relative level of GD1a ganglioside and a markedly decreased level of GM1 ganglioside in brain of Neu4(-/-) mice suggesting that Neu4 may be important for desialylation of brain gangliosides and consistent with the in situ hybridization data. Increased levels of cholesterol, ceramide and polyunsaturated fatty acids were also detected in the lungs and spleen of Neu4(-/-) mice by high-resolution NMR spectroscopy. Together, our data suggest that Neu4 is a functional component of the ganglioside-metabolizing system, contributing to the postnatal development of the brain and other vital organs.

Glucose loading precipitates focal lactic acidosis in the vulnerable medial thalamus of thiamine-deficient rats.

Glucose loading in thiamine-deficient patients is known to precipitate Wernicke's Encephalopathy; however, the mechanisms responsible have not been fully elucidated. Lactate accumulation occurs in brains of thiamine-deficient rats. In order to determine whether glucose loading in thiamine-deficient rats causes selective lactic acidosis in vulnerable brain structures, cerebral pH was measured autoradiographically using 14-labeled 5,5-dimethyloxazolidine-2, 4-dione ([(14)C]DMO) in the medial thalamus, a vulnerable brain region, versus cerebral cortex, a brain region that is spared in thiamine deficiency. Following administration of a glucose load, regional lactate levels and de novo lactate synthesis measured by (1)H-(13)C-NMR spectroscopy, increased significantly to 21.86 +/- 3.04 mumol/g (wet weight) in the medial thalamus (p < 0.001) and pH in this brain region was decreased significantly from 7.08 +/- 0.04 to 6.87 +/- 0.05 (p < 0.001). No such changes were observed in cerebral cortex following a glucose load. These results demonstrate that the increased production and accumulation of brain lactate result in acidosis following glucose loading in thiamine deficiency. Alterations of brain pH could contribute to the pathogenesis of thalamic neuronal damage and consequent cerebral dysfunction in Wernicke's Encephalopathy.

Alteration in kidney glucose and amino acids are implicated in renal pathology in MRL/lpr mice.

This study has employed high-resolution NMR spectroscopy of kidney extracts to study alterations in the concentrations of amino acids and glucose in systemic lupus erythematosus (SLE). We used the well-established mouse model of SLE, MRL/lpr, and their congenic controls, MRL/+. There was a substantial increase in the tissue concentration of branched-chain amino acids (133%), aromatic amino acids (134%) and glutathione (122%) in the lupus mice, compared to the controls. Since increased glucose can lead to fibrosis, we used [1-(13)C] glucose as a tracer to study its transport into the kidney. Significant increases in the levels of [1-(13)C] glucose (200% of controls) were observed in the MRL/lpr mice 15 min after its injection. 13C NMR spectra demonstrated that the 13C-label from [1-(13)C] glucose was not incorporated into glycolytic and Krebs cycle related metabolites within 15 min. Furthermore, we found that the expression of the profibrotic cytokine, TGFbeta and the regulatory transcription factor Smad3 are significantly enhanced in MRL/lpr mice compared to the MRL/+ controls. The mRNA and protein expression of extracellular matrix proteins, fibronectin, laminin, and collagen IV were upregulated in the MRL/lpr mice compared to the controls. All these changes were significantly reduced by the complement (C) inhibitor, Crry. Our results suggest that C activation causes increased glucose concentration in the kidney, which can lead to the observed hyperglycemia. This may be one of the important factors that cause increased extracellular matrix (ECM) deposition through the TGFbeta signaling in lupus mice and thereby lead to glomerulosclerosis that translates into increased kidney disease.

The anaplerotic flux and ammonia detoxification in hepatic encephalopathy.

Metabolic alterations in the brain underly many of the mechanisms leading to acute and chronic Hepatic Encephalopathy (HE). Controversy exists about the role of glutamine accumulation as a causal factor in HE. Glutamine formation contributes to detoxify ammonia, whereby anaplerotic mechanisms in the astrocytes have to be sufficient to replenish Krebs cycle intermediates. The application of ex vivo high-resolution nuclear magnetic resonance (NMR) spectroscopy permits direct measurements of metabolites and different metabolic pathways. Ex vivo (13)C-NMR studies in experimental animal models of acute and chronic HE have provided new insights. In an experimental rat model of ALF, (13)C isotopomer analysis of glucose metabolism showed that alterations of glucose flux through astrocytic pyruvate carboxylase might be linked to the pathogenesis of ALF as a limited anaplerotic flux in the brain, but not in the muscle, correlates with the development of brain edema. Moreover, (13)C-NMR data from a rat model of mild HE demonstrated relative differences in the pathway of glucose through pyruvate carboxylase in thalamus compared to frontal cortex, which might explain the vulnerability of this brain region compared to thalamus. These findings further support that glutamine accumulation might be not the primary cause of neurological symptoms in HE, and show that anaplerotic mechanisms could be essential for ammonia detoxification in HE.

Nmr spectroscopic analysis of regional brain energy metabolism in manganese neurotoxicity.

A central question in manganese neurotoxicity concerns the focal neuronal damage in the globus pallidus. In the present study, we investigated specific pathways of [1-(13)C]glucose as well as of [2-(13)C]acetate in this brain region and the frontal cortex following 4-day manganese treatment by high-resolution NMR spectroscopy. Following administration of 50 mg/kg/day manganese, glutamine concentration in the globus pallidus was decreased to 67% of control values but increased in frontal cortex by 56%. Manganese treatment also caused pronounced changes in glutamine-glutamate-GABA interconversion in which region-selective differences were observed in the isotopomer pattern of GABA compared with that of glutamine when including the astrocyte-specific substrate [2-(13)C]acetate. In particular, decreased (13)C-labeled glutamine, synthesized from [1-(13)C]glucose, paralleled accumulation of (13)C-labeled GABA in globus pallidus but not in frontal cortex. On the other hand, increased synthesis of glutamine from [2-(13)C]acetate showed that GABA accumulation was not due to increased synthesis from astrocytic glutamine. Furthermore, treatment with manganese resulted in a selective decrease in N-acetyl-aspartate in the globus pallidus. These data illustrate the potential importance of alterations in neuronal metabolic function. In particular, neuronal metabolic derangements and regional differences in the ability of astrocytes to fulfill their contribution to the glutamine-glutamate-GABA cycle during the early phase of manganese neurotoxicity may be crucial in determining the severity of cellular injury.

Nuclear magnetic resonance studies of energy metabolism and glutamine shunt in hepatic encephalopathy and hyperammonemia.

Hepatic encephalopathy (HE) in both acute and chronic liver failure is more likely a reversible functional disease rather than an irreversible pathological lesion of brain cells. Metabolic alterations underlie many of the mechanisms leading to HE. This paper summarizes in vivo and ex vivo (1)H-, (13)C-, and (15)N-nuclear magnetic resonance (NMR) spectroscopy data on patients and experimental models of HE. In vivo NMR spectroscopy provides a unique opportunity to study metabolic changes noninvasively in the brain in vivo, and to quantify various metabolites in localized brain areas, and ex vivo NMR permits the high-resolution measurement of metabolites and the identification of different metabolic pathways. In vivo and ex vivo (1)H-NMR investigations consistently reveal severalfold increases in brain glutamine and concomitant decreases in myo-inositol, an important osmolyte in astrocytes. An osmotic disturbance in these cells has long been suggested to be responsible for astrocyte swelling and brain edema. However, ex vivo (13)C-NMR studies have challenged the convention that glutamine accumulation is the major cause of brain edema in acute HE. They rather indicate a limited anaplerotic flux and capacity of astrocytes to detoxify ammonia by glutamine synthesis and emphasize distortions of energy and neurotransmitter metabolism. However, recent (15)N-NMR investigations have demonstrated that glutamine fluxes between neurons and astrocytes are affected by ammonia. Further NMR studies may provide novel insights into the relationship between brain edema and/or astrocyte pathology and changes in inter- and intracellular glutamine homeostasis, which may secondarily alter brain energy metabolism.

Endotoxemia produces coma and brain swelling in bile duct ligated rats.

UNLABELLED: This study explores the hypothesis that the inflammatory response induced by administration of lipopolysaccharide (LPS) exacerbates brain edema in cirrhotic rats; and if so whether this is associated with altered brain metabolism of ammonia or anatomical disturbance of the blood-brain barrier. Adult Sprague-Dawley rats 4 weeks after bile duct ligation (BDL)/Sham-operation, or naïve rats fed a hyperammonemic diet (HD), were injected with LPS (0.5 mg/kg, intraperitoneally) or saline, and killed 3 hours later. LPS administration increased brain water in HD, BDL, and sham-operated groups significantly (P < 0.05), but this was associated with progression to pre-coma stages only in BDL rats. LPS induced cytotoxic brain swelling and maintained anatomical integrity of the blood-brain barrier. Plasma/brain ammonia levels were higher in HD and BDL rats than in sham-operated controls and did not change with LPS administration. Brain glutamine/myoinositol ratio was increased in the HD group but reduced in the BDL animals. There was a background pro-inflammatory cytokine response in the brains of cirrhotic rats, and plasma/brain tumor necrosis factor alpha (TNF-alpha) and IL-6 significantly increased in LPS-treated animals. Plasma nitrite/nitrate levels increased significantly in LPS groups compared with non-LPS controls; however, frontal cortex nitrotyrosine levels only increased in the BDL + LPS rats (P < 0.005 versus BDL controls). CONCLUSION: Injection of LPS into cirrhotic rats induces pre-coma and exacerbates cytotoxic edema because of the synergistic effect of hyperammonemia and the induced inflammatory response. Although the exact mechanism of how hyperammonemia and LPS facilitate cytotoxic edema and pre-coma in cirrhosis is not clear, our data support an important role for the nitrosation of brain proteins.

Direct molecular and spectroscopic evidence for increased ammonia removal capacity of skeletal muscle in acute liver failure.

BACKGROUND/AIMS: It has been proposed that, in acute liver failure, skeletal muscle adapts to become the principle organ responsible for removal of blood-borne ammonia by increasing glutamine synthesis, a reaction that is catalyzed by the cytosolic ATP-dependent enzyme glutamine synthetase. To address this issue, glutamine synthetase expression and activities were measured in skeletal muscle of rats with acute liver failure resulting from hepatic devascularization. METHODS: Glutamine synthetase protein and gene expression were investigated using immunoblotting and semi-quantitative RT-PCR analysis. Glutamine synthetase activity and glutamine de novo synthesis were measured using, respectively, a standard enzymatic assay and [13C]-nuclear magnetic resonance spectroscopy. RESULTS: Glutamine synthetase protein (but not gene) expression and enzyme activities were significantly up-regulated leading to increased de novo synthesis of glutamine and increased skeletal muscle capacity for ammonia removal in acute liver failure. In contrast to skeletal muscle, expression and activities of glutamine synthetase in the brain were significantly decreased. CONCLUSIONS: These findings demonstrate that skeletal muscle adapts, through a rapid induction of glutamine synthetase, to increase its capacity for removal of blood-borne ammonia in acute liver failure. Maintenance of muscle mass together with the development of agents with the capacity to stimulate muscle glutamine synthetase could provide effective ammonia-lowering strategies in this disorder.

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.