Itzhak Nissim

mTOR Regulates Lysosomal ATP-Sensitive Two-Pore Na+ Channels to Adapt to Metabolic State

Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here, we identify an endolysosomal ATP-sensitive Na(+) channel (lysoNa(ATP)). The channel is a complex formed by two-pore channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosomes membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNa(ATP) have much reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cells metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.

Fructose-1,6-bisphosphatase opposes renal carcinoma progression

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is characterized by elevated glycogen levels and fat deposition. These consistent metabolic alterations are associated with normoxic stabilization of hypoxia-inducible factors (HIFs) secondary to von Hippel–Lindau (VHL) mutations that occur in over 90% of ccRCC tumours. However, kidney-specific VHL deletion in mice fails to elicit ccRCC-specific metabolic phenotypes and tumour formation, suggesting that additional mechanisms are essential. Recent large-scale sequencing analyses revealed the loss of several chromatin remodelling enzymes in a subset of ccRCC (these included polybromo-1, SET domain containing 2 and BRCA1-associated protein-1, among others), indicating that epigenetic perturbations are probably important contributors to the natural history of this disease. Here we used an integrative approach comprising pan-metabolomic profiling and metabolic gene set analysis and determined that the gluconeogenic enzyme fructose-1,6-bisphosphatase 1 (FBP1) is uniformly depleted in over six hundred ccRCC tumours examined. Notably, the human FBP1 locus resides on chromosome 9q22, the loss of which is associated with poor prognosis for ccRCC patients. Our data further indicate that FBP1 inhibits ccRCC progression through two distinct mechanisms. First, FBP1 antagonizes glycolytic flux in renal tubular epithelial cells, the presumptive ccRCC cell of origin, thereby inhibiting a potential Warburg effect. Second, in pVHL (the protein encoded by the VHL gene)-deficient ccRCC cells, FBP1 restrains cell proliferation, glycolysis and the pentose phosphate pathway in a catalytic-activity-independent manner, by inhibiting nuclear HIF function via direct interaction with the HIF inhibitory domain. This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing FBP1 from previously identified tumour suppressors that are not consistently mutated in all tumours.

Glutathione turnover in cultured astrocytes: Studies with [15N]glutamate

Abstract: The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20–40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady‐state incubation medium with 0.05 mM L‐cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.

Endogenous synthesis of galactose in normal men and patients with hereditary galactosaemia

Despite restricted ingestion of lactose, patients with galactose-1-phosphate uridyltransferase deficiency have raised concentrations of galactose metabolites in blood and urine. Endogenous production of galactose may underlie this phenomenon. Using isotopically labelled galactose in a continuous intravenous infusion, we employed the steady-state flux method to calculate endogenous galactose production rate in three normal men and three patients with classic galactosaemia. We found that galactosaemic patients and normal subjects synthesise gram quantities of galactose per day. The rate of synthesis ranged from 0.53-1.05 mg/kg per h. Endogenous production of galactose may be an important factor in the pathogenesis of the complications of the brain and ovary, and could explain the persistent elevation of galactose metabolites in patients despite dietary restriction of galactose.

Newer aspects of glutamine/glutamate metabolism: the role of acute pH changes

This review focuses on the role of acute pH changes in the regulation of Gln/Glu metabolism in the kidney, liver, and brain. Alterations of proton concentration ([H+]) profoundly affect flux through phosphate-dependent glutaminase (PDG) or glutamate dehydrogenase (GDH), the primary enzymes responsible for mitochondrial metabolism of glutamine and glutamate, respectively. In the kidney, acute acidosis stimulates Gln uptake and its metabolism via the PDG pathway. The Glu formed from Gln can be removed via 1) oxidative deamination through the GDH reaction, 2) transamination reactions, and 3) transport of Glu from intracellular to extracellular compartment, thereby diminishing the intramitochondrial pool of glutamate sufficiently to stimulate flux through the PDG pathway. Converse changes may occur with increased pH. In the liver, acidosis diminishes the rate of Gln and Glu metabolism via the PDG and GDH pathways, but stimulates glutamine synthesis (i.e., glutamine recycling). Alkalosis has little effect. Hepatic Gln metabolism via the PDG pathway has a central role in ureagenesis via 1) supplementation of nitrogen for the synthesis of carbamyl phosphate, and 2) providing glutamate for N-acetylglutamate synthesis. In the brain, Gln/Glu metabolism links ammonia detoxification and energy metabolism via 1) detoxification of ammonia and excess glutamate by glutamine synthesis in astrocytes, 2) formation and export of glutamine to neurons where it is metabolized to glutamate and GABA, and 3) production of α-ketoglutarate and lactate from Glu and their transport to neurons. Changes in intracellular pH associated with changes in cellular [K+] may have a key role in the regulation of these processes of glial-neuronal metabolism of Gln/Glu metabolism.This review focuses on the role of acute pH changes in the regulation of Gln/Glu metabolism in the kidney, liver, and brain. Alterations of proton concentration ([H(+)]) profoundly affect flux through phosphate-dependent glutaminase (PDG) or glutamate dehydrogenase (GDH), the primary enzymes responsible for mitochondrial metabolism of glutamine and glutamate, respectively. In the kidney, acute acidosis stimulates Gln uptake and its metabolism via the PDG pathway. The Glu formed from Gln can be removed via 1) oxidative deamination through the GDH reaction, 2) transamination reactions, and 3) transport of Glu from intracellular to extracellular compartment, thereby diminishing the intramitochondrial pool of glutamate sufficiently to stimulate flux through the PDG pathway. Converse changes may occur with increased pH. In the liver, acidosis diminishes the rate of Gln and Glu metabolism via the PDG and GDH pathways, but stimulates glutamine synthesis (i.e., glutamine recycling). Alkalosis has little effect. Hepatic Gln metabolism via the PDG pathway has a central role in ureagenesis via 1) supplementation of nitrogen for the synthesis of carbamyl phosphate, and 2) providing glutamate for N-acetylglutamate synthesis. In the brain, Gln/Glu metabolism links ammonia detoxification and energy metabolism via 1) detoxification of ammonia and excess glutamate by glutamine synthesis in astrocytes, 2) formation and export of glutamine to neurons where it is metabolized to glutamate and GABA, and 3) production of alpha-ketoglutarate and lactate from Glu and their transport to neurons. Changes in intracellular pH associated with changes in cellular [K(+)] may have a key role in the regulation of these processes of glial-neuronal metabolism of Gln/Glu metabolism.

Astrocyte leucine metabolism: significance of branched-chain amino acid transamination.

Abstract: We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a Vmax of 53.6 ± 3.2 nmol/mg of protein/min and a Km of 449.2 ± 94.9 µM. Virtually all leucine transport was found to be Na+ independent. Astrocytic accumulation of leucine was much greater (3×) in the presence of α‐aminooxyacetic acid (5 mM), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to α‐ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent Km for leucine conversion to extracellular KIC was a medium [leucine] of 58 µM with a Vmax of ∼2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine + α‐ketoglutarate ? KIC + glutamate) in astrocytes, but flux from leucine → glutamate is more active than that from glutamate → leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the “buffering” of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.

Mechanism of Hyperinsulinism in Short-chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency Involves Activation of Glutamate Dehydrogenase

The mechanism of insulin dysregulation in children with hyperinsulinism associated with inactivating mutations of short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) was examined in mice with a knock-out of the hadh gene (hadh−/−). The hadh−/− mice had reduced levels of plasma glucose and elevated plasma insulin levels, similar to children with SCHAD deficiency. hadh−/− mice were hypersensitive to oral amino acid with decrease of glucose level and elevation of insulin. Hypersensitivity to oral amino acid in hadh−/− mice can be explained by abnormal insulin responses to a physiological mixture of amino acids and increased sensitivity to leucine stimulation in isolated perifused islets. Measurement of cytosolic calcium showed normal basal levels and abnormal responses to amino acids in hadh−/− islets. Leucine, glutamine, and alanine are responsible for amino acid hypersensitivity in islets. hadh−/− islets have lower intracellular glutamate and aspartate levels, and this decrease can be prevented by high glucose. hadh−/− islets also have increased [U-14C]glutamine oxidation. In contrast, hadh−/− mice have similar glucose tolerance and insulin sensitivity compared with controls. Perifused hadh−/− islets showed no differences from controls in response to glucose-stimulated insulin secretion, even with addition of either a medium-chain fatty acid (octanoate) or a long-chain fatty acid (palmitate). Pull-down experiments with SCHAD, anti-SCHAD, or anti-GDH antibodies showed protein-protein interactions between SCHAD and GDH. GDH enzyme kinetics of hadh−/− islets showed an increase in GDH affinity for its substrate, α-ketoglutarate. These studies indicate that SCHAD deficiency causes hyperinsulinism by activation of GDH via loss of inhibitory regulation of GDH by SCHAD.

Astrocyte Metabolism of [15N]Glutamine: Implications for the Glutamine-Glutamate Cycle

Abstract: The metabolism of glutamine was studied in cultured astrocytes by incubating these cells with [2‐15N]‐glutamine and using gas chromatography‐mass spectrometry to quantitate the transfer of 15N to other amino acids. We found that astrocytes simultaneously synthesize and consume [2‐15N]glutamine, with the respective synthetic and utilization rates being approximately equal (ca. 13.0 nmol min‐1 mg protein‐1). Considerable 15N was transferred to alanine and a significant amount to the essential amino acids leucine, tyrosine, and phenylalanine, the latter process denoting active reamination of cognate ketoacids. A net export of alanine into the medium was noted. Astrocyte glutamine utilization appeared to be mediated via both the phosphate‐activated glutaminase (PAG) pathway and the glutamine aminotransferase pathway, the activity of which was about half that of PAG. The glutamine concentration in the incubation medium determined whether net synthesis or utilization of this amino acid occurred. When glutamine was omitted from the medium, net synthesis occurred. When it was present at a high (5 mM) level, net consumption was observed. At a physiologic (0.5 mM) concentration, neither net synthesis nor consumption was noted, although the 15N data indicated that glutamine was actively metabolized. An implication of this work is that astrocytes clearly are capable of both synthesizing and utilizing glutamine, and current concepts of a glutamate‐glutamine cycle functioning stoichiometrically between astrocytes and neurons may be an oversimplification.

Ketogenic diet, amino acid metabolism, and seizure control.

The ketogenic diet has been utilized for many years as an adjunctive therapy in the management of epilepsy, especially in those children for whom antiepileptic drugs have not permitted complete relief. The biochemical basis of the dietary effect is unclear. One possibility is that the diet leads to alterations in the metabolism of brain amino acids, most importantly glutamic acid, the major excitatory neurotransmitter. In this review, we explore the theme. We present evidence that ketosis can lead to the following: 1) a diminution in the rate of glutamate transamination to aspartate that occurs because of reduced availability of oxaloacetate, the ketoacid precursor to aspartate; 2) enhanced conversion of glutamate to GABA; and 3) increased uptake of neutral amino acids into the brain. Transport of these compounds involves an uptake system that exchanges the neutral amino acid for glutamine. The result is increased release from the brain of glutamate, particularly glutamate that had been resident in the synaptic space, in the form of glutamine. These putative adaptations of amino acid metabolism occur as the system evolves from a glucose‐based fuel economy to one that utilizes ketone bodies as metabolic substrates. We consider mechanisms by which such changes might lead to the antiepileptic effect.

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu‐cine furnishes α‐NH2 groups for glutamate synthesis via branched‐chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography‐mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2‐15N]glutamine/[15N]leucine suggested that at least one‐fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 16N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [16N]‐isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched‐chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α‐ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ∼50% in only 5 min. Extracellular concentrations of α‐ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched‐chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was ∼17 times greater than the rate of [1‐14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a‐ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.