LE JOURNAL CANADIEN DES SCIENCES NEUROLOGIQUES

Quebec Cooperative Study on Friedreich's Ataxia Effect of , and Insulin on Cerebral Amino Neurotransmitters

ROGER F. BUTTERWORTH, FRANCE LANDREVILLE, EDITH HAMEL, ANDREA MERKEL, FRANCOIS GIGUERE, ANDRE BARBEAU

SUMMARY: Treatment of rats with INTRODUCTION modified might be expected to show asparagine or glutamine caused substantial associated abnormalities of glucose increases in glutamine concentrations in There is increasing evidence that the . This appears to be the cerebellum and medulla oblongata. Insulin dicarboxylic amino glutamate case; it is now well established that treatment caused a diminution of gluta­ and aspartate may function as excita­ abnormalities of glucose metabolism mate and GA BA in these regions of brain. tory neurotransmitters in the mamma­ are frequently present in inherited Since it is now well-established that lian central nervous system. One glutamine is a very efficient precursor of ataxia (Shapcott et al., 1976; Thoren, region of brain in which there is 1962; Barbeau et al., 1976). There are the neurotransmitter pool of glutamate in convincing evidence for a neurotrans­ mammalian brain, treatment with aspara­ reports (Blass et al., 1970; Lonsdale et gine or glutamine could be of therapeutic mitter role for these amino acids is al., 1969; Wick et al., 1977) in which (replacement) value in the treatment of cerebellum where, using biochemical hereditary ataxia was found to be neurological disorders such as Friedreich's and electrophysiological indices, the associated with an inherited defect of ataxia, in which cerebral glutamate con­ presence of glutamatergic and aspar- pyruvate decarboxylase, the first en­ centrations have been found to be dimin­ tagergic neurons has been postulated zyme of the pyruvate dehydrogenase ished. in the parallel fibers originating from complex. Another report suggested the granule cells (Roffler-Tarlow and (Rodriguez-Budelli and Kark, 1978) Sidman, 1978) and in climbing fibers that Friedreich's ataxia may be the RfiSUMfi: L'injection d'asparagine ou de originating from the inferior olive result of an inherited defect of glutamine, chez le rat, cause une augmen­ (Guidotti et al., 1975; Butterworth et lipoamide dehydrogenase, the third tation substantiate de la concentration de al., 1978). enzyme present in both the pyruvate la glutamine au niveau du cervelet et de la Recent studies by the Quebec and a-ketoglutarate dehydrogenase medulla oblongata. Une injection d'insu- Cooperative Study of Friedreich's line cause une diminution du glutamate et complexes. This finding, however, was du GABA dans ces mimes regions du Ataxia have shown (Huxtable et al., not confirmed by others (Filla et al., cerveau. ttant donne qu'il est maintenant 1979) that glutamate, aspartate and 1978; Melancon et al., 1978; Stumpfet bien etabli que la glutamine est un the metabolically related inhibitory al., 1979). precurseur tres efficace du pool neuro- GABA are present in The search continues for the pri­ transmetteur de glutamate, dans le cerveau diminished concentrations in cere­ mary defect in most of the inherited des mammiferes, le traitement a I'aspara- bellar hemispheres and vermis of ataxias. Meanwhile, there is a consi­ gine ou la glutamine pourrait etre une patients dying with Friedreich's ataxia. approche therapeutique (remplacement) derable body of evidence, both of a Diminished aspartate concentration direct and indirect nature, to suggest pour le traitement de desordres neurolo- had previously been reported in the that the problems of coordination of giques comme I'Ataxie de Friedreich, dans cerebella of patients diagnosed as laquelle, les concentrations de glutamate movement (ataxia) may be related to having a dominantly inherited cere­ cerebral sont diminuees. the changes in concentration of the bellar ataxia (Perry et al., 1977). amino acid neurotransmitters gluta­ Furthermore, cerebellar glutamate con­ centrations were reportedly reduced in mate and aspartate found in this class genetically ataxic mice (Weaver- of degenerative diseases of the central Staggerer) (Roffler-Tarlov and Sid­ nervous system. man, 1978; McBride et al., 1976) and in Surprisingly little work has been experimental ataxia produced by 3- done to attempt to modify the con­ acetyl pyridine-induced degeneration centration of these amino acids in of cerebellar climbing fibers in the rat specific regions of the brain. The (Guidotti et al., 1975). present study was undertaken to investigate the possibility that the The amino acids glutamate and precursor amino acids glutamine and From the Clinical Research Institute of Montreal. aspartate are synthesized in nerve asparagine, both known to cross the Reprint requests for the complete supplement on terminals from glucose (Shank and blood-brain barrier by the neutral Friedreich's Ataxia (phase three) to: Dr. Andre Aprison, 1979) so that genetically Barbeau, Clinical Research Institute of Montreal, 110 amino acid carrier system (Oldendorf Pine Avenue West, Montreal, Quebec, Canada, H2W inherited disorders in which these and Szabo, 1976) may cause increases IR7. amino acids are found to be selectively in glutamate and aspartate respec-

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EFFECT OF VARIOUS TREATMENTS ON AMINO ACID EFFECT OF VARIOUS TREATMENTS ON AMINO ACID CONCENTRATIONS IN CEREBELLUM CONCENTRATIONS IN MEDULLA OBLONGATA

£ too Q.

GABA GLUTAMINE ASPARTATE GLUTAMATE GABA GLUTAMINE ASPARTATE GLUTAMATE Figure I Figure 2 Legend for Figures 1 & 2 Appropriate vehicle-treated control group (see text for details). zz Treatment with asparagine (1 g. per kg., per o.s.) 2 h. prior to sacrifice. 22 Treatment with glutamine (2 g. per kg., i.p.) 1 h. prior to sacrifice. Treatment with insulin (2 i.u. per kg., i.p.) 2 h. prior to sacrifice (24 h. fasted rats) II Treatment with insulin (5 i.u. per kg., i.p.) 2 h. prior to sacrifice (24 h. fasted rats) ** p<0.01, * p<0.05, N.S. no significantly different from control group.

tively. In addition, the effect of insulin Tissues were stored in liquid nitrogen RESULTS on cerebral amino acid distribution until the time of assay. Regions were L-aspargine in a dose of 1 g. per kg. was included as it had previously been separately homogenised in 10 vol. had no effect on glutamate, aspartate reported that treatment with the HC104 (0.48M) and the amino acids or GABA concentrations in either hormone led to increases in aspartate GABA, glutamate, aspartate and cerebellum or medulla oblongata in brain (Tews et al., 1965). Cerebel­ glutamine assayed by the dansyl (Figures 1 and 2) in rats sacrificed 2 h. lum and medulla oblongata were the microtechnique previously described following administration. regions of brain chosen for study since (Butterworth et al., 1978). Radio­ There was, however, a small but sig­ it is these regions that are most labeled 14C-glutamate, aspartate and nificant increase in the concentration frequently implicated in the inherited glutamine, as well as 3H-dansyl of glutamine in cerebellum following ataxias. chloride were purchased from New administration of asparagine. No England Nuclear, l4C-GABA from changes were found in glutamine in MATERIALS AND METHODS Amersham Searle, insulin (NPH) from medulla oblongata. Adult male Sprague-Dawley rats Connaught Labs, and L-asparagine L-Glutamine in a dose of 2 g. per kg. weighing 200 - 250 g. were injected and L-glutamine from Sigma. Micro- produced the changes in amino acids with the solutions of amino acid or polyamide tic plates were purchased shown in Figures 1 and 2. As can insulin in the doses and via the route from Schleicher and Schuell. All clearly be seen, 1 h. following adminis­ shown below: solvents were reagent grade and tration of glutamine, the most signifi­ Groups of 8 rats were used through­ double-distilled deionised water was cant effect (p<0.001) was a 2 - 3 fold out. Control animals for each treat­ used throughout. increase in glutamine in both cerebel- ment received equivalent volumes of the appropriate vehicle. Rats were NUTRITIONAL sacrificed 1 h. after administration of TREATMENT STATE DOSE ROUTE VEHICLE glutamine and 2 h. after asparagine or Asparagine* Fed 1 g. per kg. p.o. 0.4% methyl insulin; brains were rapidly removed cellulose in and, within 30 sec. cerebellum and saline medulla oblongata dissected on ice. Glutamine Fed 2 g. per kg. i.p. saline Insulin Fasted 2 i.u. per kg. i.p. saline •Amino acids refered to in this study are L-isomers in Insulin Fasted 5 i.u. per kg. i.p. saline all cases.

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lum and medulla oblongata. At first The most significant change (p < 0.01) formed via aspartate into oxaloacetate sight it might be argued that the was a diminished glutamate. then converted into glutamate either increase in cerebral glutamine may be by transamination or via the tricar­ caused by contamination with blood DISCUSSION boxylic acid (TCA) cycle (Figure 3) glutamine in the brain. Wurtman and The fact that none of the treatments with subsequent formation of gluta­ co-workers (Liebschutz et al., 1977) investigated caused significant in­ mine. Secondly these transformations recently showed that blood glutamine creases in the concentrations of the could have taken place in the periphery was 2.2 jumoles/ml 1 h. following dicarboxylic amino acids glutamate and the glutamine formed uptaken by administration of glutamine 2 g. per and aspartate supports the notion that brain. Both asparagine and glutamine kg. i.p.; i.e.: conditions identical to these substances, known to possess are transported into brain with ap­ those used in the present experiment. neurotoxic properties, are effectively proximately equal affinity for the Assuming that each gram of rat brain prevented from excessive accumula­ neutral amino acid carrier system contains 20 /ul of blood (Liebschutz et tion in brain either by a) active (Oldendorf and Szabo, 1976). transport out of brain or b) conversion al., 1977), one can calculate that 0.05 The highly significant increases in into (and perhaps storage as) gluta­ jumoles per g. of brain glutamate is due glutamine in both cerebellum and to blood contamination. This repre­ mine and N-acetyl aspartate. medulla oblongata following adminis­ sents less than 1% so that the changes Figure 2 shows that insulin causes tration of the amino acid confirm shown in Figures 1 and 2 are clearly significant decreases in the amino previous reports on its accumulation not due to blood glutamine. In acids glutamate and GABA, confirm­ in whole brain (Liebschutz et al., 1977; addition to the changes in cerebral ing previous reports (Tews et al., Schwerin et al., 1950). The observed glutamine following administration of 1965). Increased aspartate reported decreases in concentrations of the the amino acid there were substantial with convulsive doses of insulin was other amino acids at first sight suggests decreases in the other amino acids in not apparent at the dose used in this that glutamine may be inhibiting brain: glutamate and GABA levels study. It has been suggested that uptake of the dicarboxylic amino acids were significantly decreased (p > 0.01) glutamate may be consumed as an into brain, but uptake of in medulla oblongata and aspartate alternative (endogenous) energy source has been shown to be unaffected by was significantly decreased in both in insulin hypoglycemia. glutamine; in fact the two amino acids brain regions. Asparagine had no effect on aspar­ are transported into brain by different Insulin administered to 24 h. fasted tate concentrations in either of the two carrier mechanisms (Oldendorf and rats in doses of 2 or 5 i.u. per kg., i.p. regions of brain. The only effect of Szabo, 1976). The fact that both produced changes in cerebral amino asparagine administration was a signi­ glutamate and its cerebral metabolite acids in medulla oblongata only. The ficant increase in cerebellar glutamine. GABA are reduced suggests that changes (decreased glutamate, aspar­ There are two possible mechanisms to glutamate uptake, unlike that of tate and GABA) were apparent only explain this finding: firstly asparagine aspartate, may be inhibited by glu­ following the highest dose of insulin. could be taken up by brain, trans­ tamine. Clearly more work is neces­ sary to adequately resolve these questions. In particular, studies are needed to determine whether there are regional differences in the uptake of METABOLIC RELATIONSHIPS BETWEEN GLUCOSE AND AMINO ACID glutamine as has recently been demon­ NEUROTRANSMITTERS IN BRAIN strated for /3-hydroxybutyrate (Haw­ GLUCOSE kins and Biebuyck, 1979). Glutamine I is thought to be of major importance as a precursor of the neurotransmitter j (GLYCOLYSIS) pool of glutamate in brain, being superior to glucose in this regard PYRUVAT-im E (Hamberger et al., 1979). The present \ study clearly shows that glutamine ASPARAGINE ACETYL CoA (and asparagine) can effectively cause / accumulation of glutamine at least in ^-•-ASPARTATE; OXALOACETATE ~\. CITRATE some regions of brain. It is not incon­ MALATE ceivable, therefore that glutamine and f (TCA CYCLE) asparagine may find a therapeutic FUMARATE \ application in certain neurological ISOCITRATE)cin diseases found to associated with \ diminished cerebral glutamate concen­ ••SUCCINATE J trations. Friedreich's ataxia is an V aKETOaUTARATE • : GLUTAMATE = • GLUTAMINE SUCCINYL CoA example of one such disease. Further studies to evaluate these possibilities (GA8A SHUNT) GABA are in progress.

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