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 twocompartment mathematical model of neurotransmitter metabolism. Rats (n ϭ 23) were infused simultaneously with ). The glial TCA cycle rate was ϳ30% of total TCA cycle activity. A high pyruvate carboxylase rate (V PC , ϳ0.14 -0.18 mol ⅐ gm Ϫ1 ⅐ min Ϫ1) 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 13 C-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.

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 a-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the a-amino group to glutamate. Measurements of synthesis of 14 C-glutamate and of 14 C-glutamine from H 14 CO 3 have shown that 14 C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The speci®c radioactivity of 14 C-glutamine indicated that ,30% of glutamine was derived from 14 CO 2 ®xation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of 14 C-glutamate and 14 C-glutamine from H 14 CO 3 2 was inhibited by 31%. These results suggest that transamination of a-ketoglutarate to glutamate in Mu È ller cells is slow, the supply of branchedchain amino acids may limit¯ux, 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 H 14 CO 3 2 . 14 C-metabolites in brain extracts were measured to determine net incorporation of 14 CO 2 and speci®c radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14 CO 2 ®xation. 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.

Abstract-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 13 C-palmitate plus glucose or 13 C 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 (PϽ0.05). 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 HYPϩDCA by 18%. Correspondingly, malate was 2.2-fold greater in HYP than SHAM but was lowered with PDH activation: HYPϭ1419Ϯ220 nmol/g dry weight; HYPϩDCAϭ343Ϯ56 nmol/g dry weight. TAG content in HYP (9.7Ϯ0.7 mol/g dry weight) was lower than SHAM (13.5Ϯ1.0 mol/g dry weight). Interestingly, reduced anaplerosis in HYPϩDCA corresponded with normalized TAG (14.9Ϯ0.6 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.