Deborah A. Berkich

Neuroglial metabolism in the awake rat brain: CO2 fixation increases with brain activity

Glial cells are thought to supply energy for neurotransmission by increasing nonoxidative glycolysis; however, oxidative metabolism in glia may also contribute to increased brain activity. To study glial contribution to cerebral energy metabolism in the unanesthetized state, we measured neuronal and glial metabolic fluxes in the awake rat brain by using a double isotopic-labeling technique and a two-compartment mathematical model of neurotransmitter metabolism. Rats (n = 23) were infused simultaneously with 14C-bicarbonate and [1-13C]glucose for up to 1 hr. The 14C and 13C labeling of glutamate, glutamine, and aspartate was measured at five time points in tissue extracts using scintillation counting and 13C nuclear magnetic resonance of the chromatographically separated amino acids. The isotopic 13C enrichment of glutamate and glutamine was different, suggesting significant rates of glial metabolism compared with the glutamate-glutamine cycle. Modeling the 13C-labeling time courses alone and with 14C confirmed significant glial TCA cycle activity (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(V_{\mathrm{PDH}}^{(\mathrm{g})},{\sim}0.5\) \end{document} μmol · gm-1 · min-1) relative to the glutamate-glutamine cycle (VNT) (∼0.5-0.6 μmol · gm-1 · min-1). The glial TCA cycle rate was ∼30% of total TCA cycle activity. A high pyruvate carboxylase rate (VPC, ∼0.14-0.18 μmol · gm-1 · min-1) contributed to the glial TCA cycle flux. This anaplerotic rate in the awake rat brain was severalfold higher than under deep pentobarbital anesthesia, measured previously in our laboratory using the same 13C-labeling technique. We postulate that the high rate of anaplerosis in awake brain is linked to brain activity by maintaining glial glutamine concentrations during increased neurotransmission.

Nitrogen shuttling between neurons and glial cells during glutamate synthesis

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of α‐ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the α‐amino group to glutamate. Measurements of synthesis of 14C‐glutamate and of 14C‐glutamine from H14CO3 have shown that 14C‐amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mm. The specific radioactivity of 14C‐glutamine indicated that ∼30% of glutamine was derived from 14CO2 fixation. Using gabapentin, an inhibitor of the cytosolic branched‐chain aminotransferase, synthesis of 14C‐glutamate and 14C‐glutamine from H14CO3− was inhibited by 31%. These results suggest that transamination of α‐ketoglutarate to glutamate in Müller cells is slow, the supply of branched‐chain amino acids may limit flux, and that branched‐chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H14CO3−. 14C‐metabolites in brain extracts were measured to determine net incorporation of 14CO2 and specific radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14CO2 fixation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for ∼60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.

Substrate–Enzyme Competition Attenuates Upregulated Anaplerotic Flux Through Malic Enzyme in Hypertrophied Rat Heart and Restores Triacylglyceride Content. Attenuating Upregulated Anaplerosis in Hypertrophy

Recent work identifies the recruitment of alternate routes for carbohydrate oxidation, other than pyruvate dehydrogenase (PDH), in hypertrophied heart. Increased carboxylation of pyruvate via cytosolic malic enzyme (ME), producing malate, enables “anaplerotic” influx of carbon into the citric acid cycle. In addition to inefficient NADH production from pyruvate fueling this anaplerosis, ME also consumes NADPH necessary for lipogenesis. Thus, we tested the balance between PDH and ME fluxes in hypertrophied hearts and examined whether low triacylglyceride (TAG) was linked to ME-catalyzed anaplerosis. Sham-operated (SHAM) and aortic banded rat hearts (HYP) were perfused with buffer containing either 13C-palmitate plus glucose or 13C glucose plus palmitate for 30 minutes. Hearts remained untreated or received dichloroacetate (DCA) to activate PDH and increase substrate competition with ME. HYP showed a 13% to 26% reduction in rate pressure product (RPP) and impaired dP/dt versus SHAM (P<0.05). DCA did not affect RPP but normalized dP/dt in HYP. HYP had elevated ME expression with a 90% elevation in anaplerosis over SHAM. Increasing competition from PDH reduced anaplerosis in HYP+DCA by 18%. Correspondingly, malate was 2.2-fold greater in HYP than SHAM but was lowered with PDH activation: HYP=1419±220 nmol/g dry weight; HYP+DCA=343±56 nmol/g dry weight. TAG content in HYP (9.7±0.7 &mgr;mol/g dry weight) was lower than SHAM (13.5±1.0 &mgr;mol/g dry weight). Interestingly, reduced anaplerosis in HYP+DCA corresponded with normalized TAG (14.9±0.6 &mgr;mol/g dry weight) and improved contractility. Thus, we have determined partial reversibility of increased anaplerosis in HYP. The findings suggest anaplerosis through NADPH-dependent, cytosolic ME limits TAG formation in hypertrophied hearts.

Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes.

Abstract: CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C‐labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C]glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate →α‐ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C‐labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of α‐[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of α‐ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of α‐ketoglutarate to glutamate may frequently be the rate‐limiting step in this process of glutamate synthesis.

Mitochondrial transport proteins of the brain.

In this study, cellular distribution and activity of glutamate and γ‐aminobutyric acid (GABA) transport as well as oxoglutarate transport across brain mitochondrial membranes were investigated. A goal was to establish cell‐type‐specific expression of key transporters and enzymes involved in neurotransmitter metabolism in order to estimate neurotransmitter and metabolite traffic between neurons and astrocytes. Two methods were used to isolate brain mitochondria. One method excludes synaptosomes and the organelles may therefore be enriched in astrocytic mitochondria. The other method isolates mitochondria derived from all regions of the brain. Immunological and enzymatic methods were used to measure enzymes and carriers in the different preparations, in addition to studying transport kinetics. Immunohistochemistry was also employed using brain slices to confirm cell type specificity of enzymes and carriers. The data suggest that the aspartate/glutamate carriers (AGC) are expressed predominantly in neurons, not astrocytes, and that one of two glutamate/hydroxyl carriers is expressed predominantly in astrocytes. The GABA carrier and the oxoglutarate carrier appear to be equally distributed in astrocytes and neurons. As expected, pyruvate carboxylase and branched‐chain aminotransferase were predominantly astrocytic. Insofar as the aspartate/glutamate exchange carriers are required for the malate/aspartate shuttle and for reoxidation of cytosolic NADH, the data suggest a compartmentation of glucose metabolism in which astrocytes catalyze glycolytic conversion of glucose to lactate, whereas neurons are capable of oxidizing both lactate and glucose to CO2 + H2O.

Energy sources for glutamate neurotransmission in the retina: absence of the aspartate/glutamate carrier produces reliance on glycolysis in glia

The mitochondrial transporter, the aspartate/glutamate carrier (AGC), is a necessary component of the malate/aspartate cycle, which promotes the transfer into mitochondria of reducing equivalents generated in the cytosol during glycolysis. Without transfer of cytosolic reducing equivalents into mitochondria, neither glucose nor lactate can be completely oxidized. In the present study, immunohistochemistry was used to demonstrate the absence of AGC from retinal glia (Müller cells), but its presence in neurons and photoreceptor cells. To determine the influence of the absence of AGC on sources of ATP for glutamate neurotransmission, neurotransmission was estimated in both light‐ and dark‐adapted retinas by measuring flux through the glutamate/glutamine cycle and the effect of light on ATP‐generating reactions. Neurotransmission was 80% faster in the dark as expected, because photoreceptors become depolarized in the dark and this depolarization induces release of excitatory glutamate neurotransmitter. Oxidation of [U‐14C]glucose, [1‐14C]lactate, and [1‐14C]pyruvate in light‐ and dark‐adapted excised retinas was estimated by collecting 14CO2. Neither glucose nor lactate oxidation that require participation of the malate/aspartate shuttle increased in the dark, but pyruvate oxidation that does not require the malate/aspartate shuttle increased to 36% in the dark. Aerobic glycolysis was estimated by measuring the rate of lactate appearance. Glycolysis was 37% faster in the dark. It appears that in the retina, ATP consumed during glutamatergic neurotransmission is replenished by ATP generated glycolytically within the retinal Müller cells and that oxidation of glucose within the Müller cells does not occur or occurs only slowly.

A1 adenosine receptor antagonism improves glucose tolerance in Zucker rats

The A1 adenosine receptor (A1ar) antagonist 1,3-dipropyl-8-( p-acrylic)phenylxanthine (BW-1433) was administered to lean and obese Zucker rats to probe the influence of endogenously activated A1ars on whole body energy metabolism. The drug induced a transient increase in lipolysis as indicated by a rise in serum glycerol in obese rats. The disappearance of the response by day 7 of chronic studies was accompanied by an increase in A1ar numbers. Glucose tolerance tests were administered to rats treated with BW-1433. Peak serum insulin levels and areas under glucose curves (AUGs) were 34 and 41% lower in treated obese animals than in controls, respectively, and 19 and 39% lower in lean animals. With chronic administration (6 wk), AUGs decreased 47 and 33% in obese and lean animals, respectively. There was no effect of BW-1433 in either lean or obese rats on weight gain or percent body fat. Thus the major sustained influence of whole body A1ar antagonism in both lean and obese animals was an increase in whole body glucose tolerance at lower levels of insulin.The A1 adenosine receptor (A1ar) antagonist 1,3-dipropyl-8-(p-acrylic)-phenylxanthine (BW-1433) was administered to lean and obese Zucker rats to probe the influence of endogenously activated A1ars on whole body energy metabolism. The drug induced a transient increase in lipolysis as indicated by a rise in serum glycerol in obese rats. The disappearance of the response by day 7 of chronic studies was accompanied by an increase in A1ar numbers. Glucose tolerance tests were administered to rats treated with BW-1433. Peak serum insulin levels and areas under glucose curves (AUGs) were 34 and 41% lower in treated obese animals than in controls, respectively, and 19 and 39% lower in lean animals. With chronic administration (6 wk), AUGs decreased 47 and 33% in obese and lean animals, respectively. There was no effect of BW-1433 in either lean or obese rats on weight gain or percent body fat. Thus the major sustained influence of whole body A1ar antagonism in both lean and obese animals was an increase in whole body glucose tolerance at lower levels of insulin.

Whole-brain glutamate metabolism evaluated by steady-state kinetics using a double-isotope procedure: effects of gabapentin

Cerebral rates of anaplerosis are known to be significant, yet the rates measured in vivo have been debated. In order to track glutamate metabolism in brain glutamatergic neurons and brain glia, for the first time unrestrained awake rats were continuously infused with a combination of H14CO3– and [1−13C]glucose in over 50 infusions ranging from 5 to 60 min. In whole‐brain extracts from these animals, the appearance of 14C in brain glutamate and glutamine and appearance of 13C in the C‐4 position of glutamate and glutamine were measured as a function of time. The rate of total neuronal glutamate turnover, the anaplerotic rate of synthesis of glutamine and glutamate from H14CO3–, flux through the glutamate/glutamine cycle, and a minimum estimate of whole‐brain anaplerosis was obtained. The rate of synthesis of 14C‐glutamate from H14CO3– was 1.29 ± 0.11 nmoles/min/mg protein, whereas the rate of synthesis of 14C‐glutamine was 1.48 ± 0.10 nmoles/min/mg protein compared to total glutamate turnover of 9.39 ± 0.73 nmoles/min/mg protein. From the turnover rate of glutamine, an upper limit for flux through the glutamate/glutamine cycle was estimated at 4.6 nmoles/min/mg protein. Synthesis of glutamine from H14CO3– was substantial, amounting to 32% of the glutamate/glutamine cycle. These rates were not significantly affected by a single injection of 100 mg/kg of the antiepileptic drug gabapentin. In contrast, acute administration of gabapentin significantly lowered incorporation of H14CO3– into glutamate and glutamine in excised rat retinas, suggesting metabolic effects of gabapentin may require chronic treatment and/or are restricted to brain areas enriched in target enzymes such as the cytosolic branched chain aminotransferase. We conclude that the brain has a high anaplerotic activity and that the combination of two tracers with different precursors affords unique insights into the compartmentation of cerebral metabolism.

Evaluation of brain mitochondrial glutamate and alpha-ketoglutarate transport under physiologic conditions

Some models of brain energy metabolism used to interpret in vivo 13C nuclear magnetic resonance spectroscopic data assume that intramitochondrial α‐ketoglutarate is in rapid isotopic equilibrium with total brain glutamate, most of which is cytosolic. If so, the kinetics of changes in 13C‐glutamate can be used to predict citric acid cycle flux. For this to be a valid assumption, the brain mitochondrial transporters of glutamate and α‐ketoglutarate must operate under physiologic conditions at rates much faster than that of the citric acid cycle. To test the assumption, we incubated brain mitochondria under physiologic conditions, metabolizing both pyruvate and glutamate and measured rates of glutamate, aspartate, and α‐ketoglutarate transport. Under the conditions employed (66% of maximal O2 consumption), the rate of synthesis of intramitochondrial α‐ketoglutarate was 142 nmol/min·mg and the combined initial rate of α‐ketoglutarate plus glutamate efflux from the mitochondria was 95 nmol/min·mg. It thus seems that much of the α‐ketoglutarate synthesized within the mitochondria proceeds around the citric acid cycle without equilibrating with cytosolic glutamate. Unless the two pools are in such rapid exchange that they maintain the same percent 13C enrichment at all points, 13C enrichment of glutamate alone cannot be used to determine tricarboxylic acid cycle flux. The α‐ketoglutarate pool is far smaller than the glutamate pool and will therefore approach steady state faster than will glutamate at the metabolite transport rates measured.

Effects of adenosine receptor antagonism on protein tyrosine phosphatase in rat skeletal muscle.

Earlier studies have shown that whole body adenosine receptor antagonism increases skeletal muscle insulin sensitivity in insulin-resistant Zucker rats. To find which steps in the insulin signaling pathway are influenced by adenosine receptors, muscle from lean and obese Zucker rats, treated for 1 week with the adenosine receptor antagonist, 1,3-dipropyl-8-(4-acrylate)-phenylxanthine (BWA1433), were analyzed. All rats were first anesthetized and injected intravenously (i.v.) with 1 IU of insulin. About 3 min later the gastrocnemius was freeze clamped. Insulin receptors were partially purified on wheat germ agglutinin (WGA) columns and insulin receptor kinase activity measured in control and BWA1433-treated lean and obese Zucker rats. Protein tyrosine phosphatase (PTPase) activity was also analyzed in subcellular fractions, including the cytosolic fraction, a high-speed particulate fraction and the insulin receptor fraction eluted from WGA columns. Administration of BWA1433 increased insulin receptor kinase activity in obese but not lean Zucker rats. PTPase activities were higher in the untreated obese rat muscle particulate fractions than in the lean rat particulate fractions. The BWA1433 administration lowered the PTPase activity of the obese rats but not the lean rats. Although the PTPase activity in WGA eluate fractions containing crude insulin receptors were similar in lean and obese animals, BWA1433 administration was found to lower the PTPase activities in the fractions obtained from obese but not from the lean rats. PTPases may be upregulated in muscles from obese rats due to activated adenosine receptors. Adenosine receptor blockade, by reducing PTPase activity, may thereby increase insulin signaling.