1. Introduction
Many amino acids and/or their derivatives are neurotransmitters. Hence, metabolic perturbations in the brain, affecting the levels of amino acids, often have neurological consequences, and vice versa. However, systemic consequences of changed levels of specific amino acids or related enzymes are not easily predictable. For instance, administration of arginine or nitric oxide synthase inhibitors at cerebral infarction may cause opposite physiological outcomes [
1]. The glutamate-induced excitotoxicity could be either aggravated [
2] or alleviated [
1] by nitric oxide signaling. Obviously, one should take into account that generation of nitric oxide involves an intercept between metabolism of lysine and arginine [
3], which, in their turn, are tightly linked to other amino acids through multiple intercepts in the amino acid metabolism. In particular, the transporters for the amino acid influx are usually common for a group of amino acids which thus compete for their intracellular transport.
This work is dedicated to elaboration of systemic markers of the changed metabolism of amino acids, resulting from the brain response to the physiological or pathological challenges. To achieve this goal, we consider the two important features of the amino acid metabolism. First, mitochondrial tricarboxylic acid (TCA) cycle actively participates both in the amino acid degradation and de novo biosynthesis of the amino acid precursors, such as 2-oxoglutarate and oxaloacetate. Under maximal energy demands, the flux through the cycle is limited by the highly regulated multienzyme complex of 2-oxoglutarate dehydrogenase (OGDHC) [
4,
5,
6], which strongly impacts on the amino acid metabolism in the brain and cerebellar neuronal cells in culture [
7,
8,
9,
10]. Based on the tight interconnection between the 2-oxo and amino acids, which may contribute to the common neurological symptoms upon the impaired degradation of 2-oxo acids [
4,
6], we consider dependence of the brain amino acid levels on the OGDHC activity as a systemic marker of mitochondrial metabolism. Second, specific (patho) physiological settings may strongly contribute to different systemic outcomes of the same treatment, because organization of metabolism under these settings may vary. Indeed, the tissue-specific expression of enzymes in a pathway is an important factor in predicting the metabolic changes in health and disease [
11], and the expression pattern may vary even in the same tissue in different (patho) physiological states. Indeed, inhibition of 2-oxoglutarate-dehydrogenase, through which glutamate is degraded in the TCA cycle, may increase or decrease the glutamate levels in the rat brain cortex, dependent on pregnancy, which in turn defines the level of OGDHC activity [
7,
8,
12]. Levels of another amino acid of signaling importance, homoarginine, are also affected in pregnancy [
13]. Thus, pregnant rats provide a good model of physiological differences in organization of metabolic networks, important for central nervous system functions. On the other hand, influence of specific inhibition of the brain OGDHC on biochemical, physiological, and behavioral parameters of experimental animals strongly depend on the pathological conditions, such as acute hypoxia or ethanol intoxication [
12,
14]. Because hypoxia is the most common pathogenic factor known to perturb the high-impact signaling by glutamate, (homo) arginine/nitric oxide, and their interaction, we use our well-established model of acute hypobaric hypoxia to study the changes in the brain amino acid metabolism under pathological conditions.
Because of the significant regional heterogeneity of the brain metabolism and signaling, we focus our investigation on the easily isolated brain structure, cerebellum, which is also well-characterized regarding its physiological functions. In particular, cerebellum is involved in compensatory responses of brain to impaired movement control [
15], which also occurs in rats exposed to acute hypobaric hypoxia. The movement disorders in Parkinson disease affecting cerebellum [
16,
17] have been associated with perturbations in cerebellar pool of amino acids and their signaling [
18]. These biochemical changes in cerebellum may be further translated into behavioral changes because of high interconnectivity in the brain [
16,
19].
In our analysis of systemic response in the healthy and hypoxia-affected brain, we take into account that correlated changes of certain metabolites may provide more information on biosystems than single markers [
20,
21,
22,
23]. As a result, the present study demonstrates that the metabolic interdependence of the brain amino acids and OGDHC provides systemic markers of different physiological and pathological states, which complement the information based on analysis of traditional metabolic markers, such as average levels of metabolites or enzymatic activities. Even when single indicators do not significantly change because of homeostatic mechanisms employed by biosystems, the patterns of metabolic correlations reliably estimate systemic changes, helping to suggest the affected pathways.
4. Discussion
In this work, cerebellar metabolism of amino acids and its dependence on the TCA-cycle-limiting OGDHC are shown to be affected by physiological settings (pregnancy) and metabolic stress (acute hypobaric hypoxia). Pregnancy increases cerebellar levels of glutamate and tryptophan (
Table 1) and decreases overall interdependence of the studied components of amino acid metabolism, compared to their interdependence in non-pregnant rats (
Table 4).
Hormonal changes during pregnancy are known to coordinate a broad range of physiological adaptations, from the supply of nutrients and oxygen for the fetus growth in utero to specific patterns of parental behavior [
32,
33]. Changed metabolism of amino acids in the brain of the pregnant females may be involved in these adaptations, as many of amino acids are neurotransmitters or their precursors. The increases in cerebellar glutamate and tryptophan in pregnant rats, observed in this work, are consistent with independent studies on the pregnancy-imposed changes in glutamatergic and serotonergic signaling. In fact, pregnancy is known to change expression of glutamate receptors to address specific physiological challenges faced by pregnant females [
34,
35]. Our data on the pregnancy-increased cerebellar content of tryptophan are in line with the activation of serotonergic signaling observed during pregnancy, because tryptophan is the serotonin precursor [
36]. The interaction between the levels of serotonin and estrogens also underlies sex-dimorphic prevalence of the serotonin-linked diseases, including migraine, depression, eating disorders and pregnancy-associated pathologies [
36,
37].
Our previous studies pointed to certain relationship between the amino acid levels in the blood plasma and brain [
23], which may be used for translation of findings on the brain samples in animal models to human studies. For instance, increased content of tryptophan in cerebellum of pregnant rats, shown in our study (
Table 1) corresponds to the findings in humans, which indicate that in maternal plasma, tryptophan catabolites and related compounds change in pregnancy [
38,
39]. This study suggests the neurotransmitters-dependent adaptation of maternal body to the fetus growth. As considered above, such adaptation is obvious also from our findings (
Table 1). Biomarker significance of the amino acid levels in human plasma and urine has also been explored in other studies [
40,
41,
42]. Their findings point to the potential diagnostic significance of the changes in multivariate metabolic profiles, including amino acids, for prediction of gestational diabetes [
42]. Besides, the pregnancy-dependent changes in the content of amino acids and their metabolites in plasma and urine point to variation of these parameters, dependent on the increased amino acid demands for the growing fetus [
40,
41].
The pregnancy-induced changes in cerebellar pool of amino acids (
Table 1) are associated with different responses of cerebellar amino acid metabolism to hypoxia in the pregnant and non-pregnant rats (
Table 2,
Table 3 and
Table 4). Strong negative correlations between the amino acid levels and OGDHC activity, which are a hallmark of cerebellar metabolism after hypoxia of non-pregnant rats, are not induced by hypoxia in the pregnant rats (
Table 3 and
Table 4). Simultaneously, hypoxia significantly increases average levels of cerebellar amino acids in the non-pregnant rats only (
Table 2). Because the observed biochemical changes in the cerebellar metabolism of amino acids are associated with a higher resistance to hypoxia in the non-pregnant vs. pregnant rats (
Figure 1), the metabolic rearrangement in the cerebellum of non-pregnant rats is of adaptive significance. In particular, increased flux of the amino acids degraded through OGDHC (Glu, Gln, Arg, His, Pro) and of the branched-chain amino acids Val and Ile may generate succinyl-CoA for the substrate level phosphorylation in mitochondria. Generation of ATP at the expense of succinyl-CoA may help overcoming energy deficits upon hypoxia which impairs oxidative phosphorylation. Increased degradation of amino acids in the TCA cycle whose flux is limited by OGDHC, is in good accordance with the negative correlations between the OGDHC activity and the levels of cerebellar amino acids after hypoxia, in contrast to normal metabolism (
Table 3). It is worth noting in this regard that hypoxic tolerance is associated with the mTOR-dependent autophagy [
43]. Increased autophagy may generate the higher amino acid levels after hypoxia (
Table 2) to use them for the substrate-level ADP phosphorylation in the hypoxic brain of non-pregnant rats. Autophagy is also coupled to pro-survival function of mitochondrial fission under energy stress [
44,
45,
46,
47]. However, the homeostatic and pro-survival functions of mTOR, autophagy and mitochondrial fission are highly conditional, with overactivation of these processes also mediating the brain damage by hypoxia [
48,
49,
50].
Different levels of activation of mTOR, autophagy and mitochondrial fission may be required for their pro-survival and death-inducing outcomes in different physiological states. In this regard, no increase in the brain amino acid levels by hypoxic exposure of pregnant rats (
Table 2) was due to a higher damaging potential of the autophagy stimulation in this physiological state. No adaptation to hypoxia by increased degradation of amino acids through OGDHC in pregnant rats is also evident from the hypoxia-induced decrease in the interdependences between the levels of OGDHC activity and/or amino acids, whereas in the non-pregnant rats hypoxia increases these interdependences (
Table 4). The different action of hypoxia in the two physiological states is obvious from comparison of the hypoxia-induced changes in the summarized and average correlation coefficients or in the number of significant correlations in the pregnant and non-pregnant rats (
Table 4). In view of the increased sensitivity of pregnant vs. non-pregnant rats to hypoxia (
Figure 1), the stability of the biochemical parameters in the cerebellum of the pregnant rats exposed to hypoxia (
Table 2) along with the absence of negative correlations between the OGDHC activity and levels of amino acids (
Table 3 and
Table 4) manifest limitations of the hypoxic adaptation through increased degradation of amino acids in the pregnant vs. non-pregnant rats. These findings indicate that the pregnancy-imposed changes in the cerebellar amino acid pool (
Table 2) and metabolism (
Table 3 and
Table 4) are associated with decreased stress adaptability, in line with other studies showing decreased perception of stress in pregnant females [
51,
52,
53].
It is worth noting that the response of the metabolic network of pregnant rats to hypoxia, undetectable from the average levels of amino acids (
Table 2), is evident from the correlation analysis (
Table 4). Hence, average levels of cerebellar amino acids are not as sensitive indicators of metabolic changes, as the cumulative parameters characterizing the interdependence of components of the corresponding metabolic network.
It has been noted previously that correlating metabolites may not only be of diagnostic significance [
23], but also help deciphering the yet unknown or poorly characterized synthetic and regulatory pathways [
20]. In this regard, the pregnancy-induced changes in metabolic correlations of cerebellar lysine are of interest, because gestational diabetes is associated with plasma levels of lysine and tyrosine [
54], both of them correlating to each other much stronger in cerebellum of the pregnant than non-pregnant rats (
Table 3). Besides, lysine catabolism is related to biosynthesis of homoarginine which is elevated in normal pregnancy [
13], with some studies linking its elevation to pregnancy disorders, including preeclampsia [
55]. Physiological manifestations of pre-eclampsia and eclampsia, associated with nearly one-tenth of all maternal deaths [
56], involve changed cerebral hemodynamics and hypertensive encephalopathy [
57,
58,
59], potentially linked to impaired signaling by homoarginine, known as a predictor of cardiovascular risk and mortality [
3]. Alternatively, homoarginine is synthesized from arginine and glycine. Because these amino acids are highly correlated independent of pregnancy and/or hypoxia (
Table 3), metabolism of cerebellar lysine appears to be a more likely contributor to specific adaptations imposed by pregnancy. This is further supported by the fact that lysine is known as an antagonist of a serotonin receptor [
60], with serotonergic signaling increased in pregnancy [
36,
37] and depressed upon increased synthesis of homoarginine from lysine [
61].