Biosynthesis and Degradation of Free D-Amino Acids and Their Physiological Roles in the Periphery and Endocrine Glands

Abstract

It was long believed that D-amino acids were either unnatural isomers or laboratory artifacts, and that the important functions of amino acids were exerted only by L-amino acids. However, recent investigations have revealed a variety of D-amino acids in mammals that play important roles in physiological functions, including free D-serine and D-aspartate that are crucial in the central nervous system. The functions of several D-amino acids in the periphery and endocrine glands are also receiving increasing attention. Here, we present an overview of recent advances in elucidating the physiological roles of D-amino acids, especially in the periphery and endocrine glands.

1. INTRODUCTION

Apart from glycine (Gly), all proteinogenic amino acids contain asymmetric α-carbon atoms and can exist as L-form and D-form stereoisomers. In the past, D-amino acids were considered either unnatural isomers or laboratory artifacts, despite their discovery in bacteria and insects. Consequently, it was believed that D-amino acids did not play a significant role in the physiology of most organisms. However, recent advances in analytical methods for separating chiral amino acids revealed that living organisms contain several D-amino acids, either free or as components of proteins. In particular, free D-serine (D-Ser) and D-aspartate (D-Asp) are present in a wide variety of mammalian tissues and cells at relatively high concentrations^1–11) and have been studied most extensively.

D-Ser is mainly found in the mammalian forebrain and is present at high concentrations throughout life, but it is also present in the cerebellum during the early postnatal period. In mammals, D-Ser is synthesized by Ser racemase (EC 5.1.1.16), an enzyme that produces D-Ser from L-Ser in a pyridoxal 5′-phosphate-dependent manner.^12–14) Accumulating evidence demonstrates that D-Ser plays an essential role in regulating brain function by acting as an endogenous ligand for the N-methyl-D-Asp (NMDA) receptor, a member of the L-glutamate (L-Glu) receptor family, or the δ2 Glu receptor.^15–17) Indeed, perturbation of D-Ser levels in the nervous system has been implicated in the pathophysiology of various neuropsychiatric disorders including schizophrenia,^18–20) Alzheimer’s disease,^12,21) and amyotrophic lateral sclerosis.^22,23)

D-Asp is present at high levels in multiple mammalian tissues and cells, particularly those of the central nervous, neuroendocrine, and endocrine systems. Several lines of evidence suggest that D-Asp plays crucial roles in regulating developmental processes, hormone secretion, and steroidogenesis.^24–26) In addition, D-Asp stimulates the NMDA receptor by acting as an endogenous ligand.^27–30) Recent studies suggest that D-Asp acts as a signaling molecule in the nervous and neuroendocrine systems, in part by binding to the NMDA receptor, and plays essential roles in regulating brain functions.^25,26,31) This was further supported by recent reports demonstrating that D-Asp levels in the prefrontal cortex and/or striatum of post-mortem brains of schizophrenic patients are significantly lower than those of individuals not suffering from a psychiatric illness.^32,33)

In addition to D-Ser and D-Asp, several other D-amino acids, such as D-alanine (D-Ala), D-glutamate (D-Glu), D-leucine (D-Leu), D-proline (D-Pro), D-arginine (D-Arg), D-methionine (D-Met), D-valine (D-Val), and D-tryptophane (D-Trp), are also present in various tissues and cells in mammals, albeit at relatively low levels.^3,8,34–44) However, their physiological roles in vivo remain to be fully clarified.

As described above, D-Ser and D-Asp function as important bioactive substances in the central nervous system. Additionally, investigations on the functions of several D-amino acids in the periphery and endocrine glands have gained attention in recent years. In the present article, we first summarize our understanding of the biosynthesis and degradation of free D-amino acids, and then review recent advances in studies on the physiological roles of D-amino acids in the periphery and endocrine glands.

2. BIOSYNTHESIS AND DEGRADATION OF D-AMINO ACIDS IN MAMMALS

2.1. Biosynthesis and Degradation of D-Ser in Mammals

As described above, in mammals, D-Ser is synthesized by Ser racemase, an enzyme, that catalyzes interconversion between D-Ser and L-Ser in a pyridoxal 5′-phosphate-dependent manner (Fig. 1A). Since the discovery of free D-Ser in mammals, much attention has been paid to the origins and synthetic pathways of this D-amino acid. In 1999, Wolosker et al. first identified mammalian Ser racemase and purified it from rat brain.¹⁴⁾ Ser racemase cDNAs have since been cloned from various organisms including mammals such as human,⁴⁵⁾ rat,⁴⁶⁾ and mouse,⁴⁷⁾ and recombinant forms of human and mouse enzymes have been purified and functionally characterized.^48–59) Human, rat, and mouse Ser racemases are 36–37 kDa and comprise 340, 333, and 339 amino acids, respectively. Ser racemase protein and mRNA are present in several mammalian tissues, particularly brain, liver, and kidney.^{46,47,60–62)} In the mammalian brain, Ser racemase is predominantly detected in forebrain regions, such as the cerebral cortex and hippocampus,^47,61,63) and it is localized in neurons as well as astrocytes and oligodendrocytes.^{47,61,63–68)}

Fig. 1. Reactions Catalyzed by D-Amino Acid Metabolic Enzymes

(A) Reaction catalyzed by amino acid racemase. Amino acid racemase catalyzes interconversion between D-amino acid and L-amino acid using either a pyridoxal 5′-phosphate-dependent or a pyridoxal 5′-phosphate independent mechanism. (B) Reactions catalyzed by D-amino acid oxidase (DAO) or D-Asp oxidase (DDO). DAO and DDO catalyze the dehydrogenation of D-amino acid to generate the corresponding imino acid, coupled with the reduction of FAD. FAD reoxidizes spontaneously in the presence of oxygen, producing hydrogen peroxide, while the imino acid is nonenzymatically hydrolyzed to the 2-oxo acid and ammonia. (C) Reaction catalyzed by D-Glu cyclase (DGLUCY). DGLUCY reversibly converts D-Glu to 5-oxo-D-Pro and H₂O.

Recently, four independent groups established Ser racemase knockout (KO) mice.^63,69–71) In these mice, D-Ser levels in the brain are reduced to 10–20% of those in wild-type control mice, indicating that D-Ser in the brain is predominantly synthesized by Ser racemase. None of these KO mice appear to have any obvious defects, but neurotransmission and behavior mediated by the NMDA receptor are altered in these mice, as expected. Specifically, Ser racemase KO mice exhibit a schizophrenia-like phenotype and have impaired spatial memory.^69,70) In addition, these KO mice exhibit reduced prepulse inhibition, decreased sociability, and elevated anxiety compared with wild-type mice. Furthermore, in cultured microglial cells, expression of the Ser racemase gene is induced following treatment with amyloid-β peptide, coupled with enhanced D-Ser release from cells.²¹⁾ Importantly, injection of NMDA and amyloid-β peptide into the forebrain results in less brain damage in Ser racemase KO mice than wild-type mice,¹²⁾ and cerebral ischemia also causes less brain damage in the KO mice.⁷²⁾ These injections and ischemia result in overstimulation of the NMDA receptor, which evokes excessive firing of neurons. Thus, in the KO mice, the resultant decrease in D-Ser level largely alleviates NMDA receptor-mediated neurotoxicity. More detailed information on Ser racemase and Ser racemase KO mice can be found in various review articles.^73–80)

In mammals, D-Ser is degraded by D-amino acid oxidase (DAO; also abbreviated as DAAO; EC 1.4.3.3). DAO is an FAD-containing flavoprotein that catalyzes the oxidative deamination of D-amino acids to generate 2-oxo acids, hydrogen peroxide, and ammonia (Fig. 1B). DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids, including D-Ser and D-Ala. However, DAO does not act on L-amino acids or acidic D-amino acids. This enzyme was first discovered in pig kidney by Krebs in 1935.⁸¹⁾ DAO cDNAs have since been cloned from various organisms, including mammals such as human,⁸²⁾ rat,⁸³⁾ and mouse,⁸⁴⁾ and recombinant forms of these enzymes have been purified and functionally characterized.^85–87) Human, rat, and mouse DAOs are 39 kDa and comprise 347, 346, and 345 amino acids, respectively. In mammals, DAO is mainly present in kidney, liver, and brain,^66,88–100) but it is absent in mouse liver.¹⁰¹⁾ Further studies revealed that DAO is localized to the epithelial cells of proximal renal tubules in the kidney and to hepatocytes in the liver.^{88,92,97–100)} In the brain, DAO is predominantly detected in the glial cells of the cerebellum, pons, medulla oblongata, and spinal cord, as well as choroid plexus epithelial cells.^66,90,91,96) Mammalian DAO is believed to regulate levels of several neutral and basic D-amino acids of internal and external origin, including D-Ser and D-Ala, in various organs, although its physiological roles in vivo remain to be fully clarified. For instance, DAO expressed in choroid plexus epithelial cells may regulate D-amino acid concentrations in cerebrospinal fluid. In this context, it was reported that expression levels of DAO are higher in choroid plexus epithelial cells of schizophrenia patients than controls.⁹⁶⁾

A mutant mouse strain that lacks DAO activity due to a missense mutation (ddY/DAO⁻) has been established.¹⁰²⁾ The ddY/DAO⁻ mice grow and behave normally, and their life span is not different from that of wild-type (ddY/DAO⁺) mice. The ddY/DAO⁻ mice also produce the same number of offspring as ddY/DAO⁺ mice. Significant differences were not observed in the gross or fine structures of the kidney and brain between ddY/DAO⁻ and ddY/DAO⁺ mice. Moreover, no significant differences were observed in the expression of the NR1 subunit of the NMDA receptor, Asc-1 transporter (a D-Ser transporter), GlyT1 transporter (a Gly transporter), and Ser racemase.¹⁰³⁾ In addition, uptake of D-Ser in synaptosomes prepared from the cerebellum was not different between ddY/DAO⁻ and ddY/DAO⁺ mice. Taken together, these results suggest that there is no significant compensatory effect for the lack of DAO. However, a wide variety of D-amino acids are present in various tissues and physiological fluids of ddY/DAO⁻ mice.^{38,104–107)} For instance, a large amount of D-Ser was detected in the cerebrum, cerebellum, kidney, liver, serum, and urine of ddY/DAO⁻ mice; a large amount of D-Ala was detected in the cerebrum, hippocampus, hypothalamus, pituitary gland, pineal gland, cerebellum, medulla oblongata, serum, and urine; a large amount of D-Leu was detected in the cerebrum, hippocampus, hypothalamus, pineal gland, cerebellum, medulla oblongata, and serum; and a large amount of D-Pro was detected in the cerebrum, hippocampus, hypothalamus, pituitary gland, pineal gland, cerebellum, medulla oblongata, and serum. By contrast, except for D-Ser in the cerebrum, these D-amino acids are present at relatively low levels in ddY/DAO⁺ mice. These observations indicate that several D-amino acids are constantly degraded by DAO in ddY/DAO⁺ mice, consistent with the idea that DAO degrades several neutral and basic D-amino acids of internal and external origin.

Meanwhile, responses to nociceptive stimuli were different between ddY/DAO⁻ and ddY/DAO⁺ mice, suggesting enhanced function of the NMDA receptor in ddY/DAO⁻ mice.^108,109) Indeed, the excitatory post-synaptic currents mediated by the NMDA receptor were augmented in ddY/DAO⁻ mice compared with ddY/DAO⁺ mice.¹⁰⁸⁾ Long-term synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), is considered a fundamental process in learning and memory, and the NMDA receptor is believed to play a pivotal role in this process. Importantly, LTP in the CA1 area of the hippocampus was augmented in ddY/DAO⁻ mice compared with ddY/DAO⁺ mice, and ddY/DAO⁻ mice displayed better performance in Morris water maze tests.¹¹⁰⁾ In addition, ddY/DAO⁻ mice displayed elevated anxiety-like behavior, believed to be associated with altered NMDA receptor function.¹¹¹⁾ Thus, ddY/DAO⁻ mice have contributed to research on the physiological roles of DAO and the etiology of neuronal diseases related to the NMDA receptor, including schizophrenia. More detailed information on DAO and ddY/DAO⁻ mice can be found in various review articles.^{78,112–120)}

2.2. Biosynthesis and Degradation of D-Asp in Mammals

Since the discovery of free D-Asp in mammals,²⁾ much attention has been paid to the origins and synthetic pathways of this D-amino acid, but its complete biosynthetic pathway in mammals remains to be elucidated. Biosynthesis of D-Asp was first demonstrated in cultured rat pheochromocytoma PC-12 cells.¹²¹⁾ PC-12 cells contain D-Asp, and levels (both intracellular and in the cell culture medium) increase with culture duration, even if it is not added to the culture medium. This observation clearly indicates that D-Asp is indeed synthesized in mammalian cells. D-Asp synthesis has also been observed in MPT1 cells (a subclone of PC-12 cells),¹²²⁾ rat pituitary tumor GH₃ cells,¹²³⁾ and human cervical adenocarcinoma HeLa cells,¹²⁴⁾ whereas the biosynthesis of D-Asp is not observed in mouse fibroblast Swiss 3T3 cells, human neuroblastoma NB-1 cells, or human embryonic kidney 293 cells.^121,125) Furthermore, in primary cultures of rat embryonic neurons, high levels of endogenous D-Asp were detected, and [¹⁴C]-D-Asp biosynthesis was demonstrated using [¹⁴C]-L-Asp as the precursor molecule.¹²⁶⁾ Treatment of these neuronal cultures with amino-oxyacetic acid markedly inhibited the accumulation of [¹⁴C]-D-Asp. Since amino-oxyacetic acid is a potent inhibitor of pyridoxal 5′-phosphate-dependent enzymes, this result suggests that one such enzyme is involved in the conversion of L-Asp to D-Asp. A likely candidate for this enzyme is an Asp racemase (EC 5.1.1.13) that catalyzes interconversion between D-Asp and L-Asp (Fig. 1A), and a pyridoxal 5′-phosphate-dependent Asp racemase has indeed been cloned and characterized from the bivalve Scapharca broughtonii.^127–130) A second Asp racemase has also been cloned from the sea slug Aplysia californica.¹³¹⁾ Recently, a putative Asp racemase was identified in mouse by Kim et al., and the recombinant form of the corresponding protein exhibited pyridoxal 5′-phosphate-dependent Asp racemase activity.¹³²⁾ Glutamic-oxaloacetic transaminase 1-like 1 (Got1l1) encodes this protein, and the mouse GOT1L1 is a 46 kDa protein comprising 404 amino acids. Notably, the deduced amino acid sequence of the mouse GOT1L1 is more homologous to that of mammalian L-Asp aminotransferase (EC 2.6.1.1) than to mammalian Ser racemases and both S. broughtonii and A. californica Asp racemases, whereas S. broughtonii and A. californica Asp racemases share relatively high amino acid sequence identity with mammalian Ser racemases. Therefore, it appears that mouse GOT1L1, a putative Asp racemase, and S. broughtonii and A. californica Asp racemases have evolved convergently from different ancestral proteins to acquire a similar catalytic mechanism.

In mice, GOT1L1 protein is expressed abundantly in the brain, heart, and testis, followed by the adrenal gland, and is expressed at negligible levels in the liver, lung, kidney, and spleen.¹³²⁾ Immunohistochemical analysis revealed coincident localization of GOT1L1 and D-Asp in the mouse brain, pituitary gland, hippocampus, pineal gland, adrenal medulla, and testis. Interestingly, depletion of GOT1L1 in the adult mouse hippocampus by retrovirus-mediated expression of GOT1L1-targeting short hairpin RNA elicited profound defects in dendritic development and neuron survival.¹³²⁾ Thus, GOT1L1 appears to play an essential role in neuronal development, consistent with the previously proposed role of D-Asp in neurogenesis and brain development.^126,133)

In both rat and human genomes, genes annotated as homologous to the mouse Got1l1 are designated Got1l1 and GOT1L1, respectively, and the proteins encoded by these genes are presumed to be responsible for the biosynthesis of D-Asp. However, the lengths of the predicted amino acid sequences of mouse, rat, and human GOT1L1s (404, 315, and 421 amino acids, respectively) are not conserved. In our study,¹²⁴⁾ we investigated whether rat and human GOT1L1s are involved in D-Asp synthesis. First, the correlation between cellular D-Asp content and the expression level of GOT1L1 mRNA was examined in several rat and human cell lines. Expression levels of mRNA encoding D-Asp oxidase (DDO; also abbreviated as DASPO; EC 1.4.3.1), the sole degradative enzyme that acts on free D-Asp in mammals, were also measured in these cell lines. Second, the effect of GOT1L1 gene knockdown on D-Asp biosynthesis was examined, but the results failed to show any correlation between the expression levels of GOT1L1 and D-Asp content in either rat or human cell lines, even if the expression level of DDO is considered.¹²⁴⁾ Furthermore, during culture of GH₃ cells that are known to synthesize D-Asp as described above,¹²³⁾ knockdown of the GOT1L1 gene did not reduce total D-Asp content (D-Asp in cells plus D-Asp in the culture media), confirming that GOT1L1 contributes little, if at all, to the biosynthesis of D-Asp in rat and human cells.¹²⁴⁾

In another report,¹³⁴⁾ a GOT1L1 KO mouse was generated, and amino acid levels in wild-type and KO mice were compared. No significant differences were observed in amino acid content in the hippocampus or testis. Furthermore, the recombinant form of mouse GOT1L1 was produced, but it was difficult to obtain large amounts of protein in soluble form.¹³⁴⁾ However, the recombinant protein failed to produce D-Asp from L-Asp, but it exhibited L-Asp aminotransferase activity. Thus, the overall picture of D-Asp biosynthesis in mammals remains to be elucidated.

It is noteworthy that A. californica Asp racemase shows racemase activity towards both Asp and Ser,¹³¹⁾ while S. broughtonii Asp racemase showed no racemase activity towards Ser.^127,129) Therefore, Ser racemase in mammals may catalyze the conversion of L-Asp to D-Asp; indeed, recombinant forms of mouse and human Ser racemases exhibit detectable Asp racemase activity.^53,54,59) Moreover, overexpression of rat Ser racemase in PC-12 cells resulted in increased intracellular D-Asp content compared with control cells, demonstrating that Ser racemase functions as an Asp racemase.⁵³⁾ In another study,⁵³⁾ Ser racemase KO PC-12 cells were constructed using a CRISPR/Cas9 genome editing system, and the contribution of endogenous Ser racemase to D-Asp biosynthesis was investigated. The Ser racemase KO cells exhibited decreased intracellular D-Ser levels but production of D-Asp was unaffected. By contrast, Ser racemase KO mice showed significantly decreased D-Asp levels in their frontal cortex, hippocampus, and striatum, where Ser racemase is normally highly expressed, while D-Asp levels in the cerebellum and testis remained unchanged.^53,135) Taken together, these results suggest that Ser racemase indeed acts as a D-Asp biosynthetic enzyme in some tissues, and additional enzyme(s) for D-Asp synthesis are likely present in mammals.

In mammals, D-Asp is degraded by DDO, as described above. In 1949, Still et al. first reported the activity of DDO in rabbit kidney extracts.¹³⁶⁾ Like DAO, DDO is an FAD-containing flavoprotein that catalyzes the oxidative deamination of D-amino acids to generate 2-oxo acids, hydrogen peroxide, and ammonia (Fig. 1B). However, DDO is highly specific for acidic D-amino acids, and degrades not only D-Asp but also D-Glu in vitro; DDO does not act on L-amino acids or neutral and basic D-amino acids. DDO is present in a wide variety of eukaryotes including humans, and DDO cDNAs have been cloned from various organisms including human,^137,138) rat,¹³⁹⁾ and mouse.¹⁴⁰⁾ Recombinant forms of these enzymes have been purified and functionally characterized.^{86,137–139,141–145)} Human, rat, and mouse DDOs are 38 kDa proteins comprising 341 amino acids. In mammals, DDO activity is highest in the kidney, followed by the liver and brain, and is low in other peripheral tissues.^146,147) Further studies revealed that DDO is localized to the epithelial cells of proximal renal tubules in the kidney and to hepatocytes in the liver.¹⁴⁸⁾ DDO levels in the kidney and liver are relatively low at birth and rapidly increase thereafter.^146,149,150) This is in contrast to D-Asp levels in these tissues, which decrease as development proceeds.^2,149) This is also true in the brain, in which the D-Asp content in the cerebrum rapidly decreases as development proceeds,⁷⁾ while the DDO content rapidly increases.¹⁴⁶⁾ Enzymatic histochemical analysis of rat brain tissues revealed that DDO is present in several regions, including the choroid plexus, ependyma, hippocampus, dentate gyrus, olfactory bulb, pituitary gland, granule cell layer, and white matter of the cerebellum.¹⁵¹⁾ Notably, the DDO activity is predominantly detected in nerve cells, such as hippocampal pyramidal neurons and olfactory epithelial neurons, tissues in which D-Asp immunoreactivity is not observed. Thus, the location and activity levels of DDO correlate inversely with the presence of D-Asp, suggesting that, in mammals, DDO degrades endogenous D-Asp as a physiological substrate, thereby regulating cellular levels of this D-amino acid.

Recently, two independent groups established DDO KO mice displaying significantly elevated levels of D-Asp in several tissues.^152,153) Although the DDO KO mice did not show any change in life span, several phenotypes were observed. Recent findings obtained from studies using DDO KO mice collectively suggest that D-Asp acts as a signaling molecule in the nervous system by binding to the NMDA receptor, and plays an essential role in regulating brain functions.^{27,154–158)} In neurological assessments such as the Morris water maze and contextual fear conditioning, altered behaviors are observed in DDO KO mice.¹⁵⁵⁾ D-Asp levels in the hippocampus of DDO KO mice are markedly higher than in wild-type mice.¹⁵⁴⁾ Furthermore, in DDO KO mice, significant suppression is observed in synaptic depotentiation following LTP induction of the hippocampus, a process in which the NMDA receptor is believed to play a pivotal role. Similarly, high-frequency stimulation of corticostriatal fibers induces LTD in corticostriatal slices of wild-type mice but not of DDO KO mice.¹⁵⁵⁾ In the corticostriatal system, enhanced NMDA receptor signaling is presumed to block the induction of LTD, and D-Asp is known to stimulate NMDA receptor activity by binding to the L-Glu-binding site.^28–30) Thus, it is likely that NMDA receptor-mediated neurotransmission is enhanced by elevated concentrations of D-Asp in DDO KO mice.

On the other hand, DDO KO mice exhibit decreased immobility duration in the Porsolt forced-swim test, a model of depression, suggesting that genetic ablation of DDO has a specific antidepressant effect.¹⁵⁸⁾ In addition, deficits in prepulse inhibition induced by amphetamine, a potent dopamine releaser, or by MK-801, a noncompetitive antagonist of the NMDA receptor, are attenuated in DDO KO mice.¹⁵⁵⁾ These results suggest that increased levels of D-Asp in the brain protect against sensorimotor gating deficits, a defect that is observed in schizophrenic patients. More detailed information on DDO and DDO KO mice can be found in various review articles.^{25,31,78,159–165)}

2.3. Biosynthesis and Degradation of D-Ala in Mammals

In mammals, D-Ala has been found in a wide variety of peripheral tissues and physiological fluids.^{3,38–44,166)} However, a D-Ala synthesis enzyme has not been identified in mammals. A likely candidate is Ala racemase (EC 5.1.1.1) that catalyzes interconversion between D-Ala and L-Ala (Fig. 1A), and pyridoxal 5′-phosphate-dependent Ala racemases from several eukaryotes including the yeast Schizosaccharomyces pombe and the Kuruma prawn Marsupenaeus japonicus have been cloned and characterized.^167,168) However, there is no known gene in mammalian genomes that is orthologous to the Ala racemase gene, and it remains to be determined whether D-Ala is synthesized in mammals.

In mammals, D-Ala is degraded by DAO, as described above. In fact, D-Ala levels in a wide variety of tissues and physiological fluids are markedly higher in ddY/DAO⁻ mice than in ddY/DAO⁺ mice.^{38,104–107)}

2.4. Biosynthesis and Degradation of D-Glu in Mammals

The biosynthetic pathway of D-Glu in mammals remains unclear. Glu racemase (EC 5.1.1.3), which catalyzes the interconversion of D-Glu and L-Glu (Fig. 1A), is likely involved. Indeed, we recently identified the cDNA encoding L-Ser dehydratase-like as the first mammalian clone with Glu racemase activity.¹²⁵⁾ This rat L-Ser dehydratase-like enzyme has been deposited in mammalian databases as a protein of unknown function, and its amino acid sequence shares approx. 60% identity with that of L-Ser dehydratase (EC 4.3.1.17; formerly 4.3.1.13). The rat L-Ser dehydratase-like enzyme is a 35 kDa protein comprising 329 amino acids. Rat L-Ser dehydratase-like is expressed in Escherichia coli, and the enzymatic properties of the recombinant were characterized. The results revealed a multifunctional enzyme with pyridoxal 5′-phosphate-dependent Glu racemase activity in addition to L-Ser/L-threonine (L-Thr) dehydratase activity, hence it was abbreviated as STDHgr (L-Ser/L-Thr dehydratase [Glu racemase]).¹²⁵⁾ Further experiments using cultured mammalian cells confirmed that D-Glu was synthesized and L-Ser and L-Thr were decomposed. It was also found that STDHgr contributes to the homeostasis of several other amino acids.¹²⁵⁾

As described above, D-Glu is degraded by DDO in vitro. However, the amounts of D-Glu are almost the same in DDO KO and wild-type mice,³⁶⁾ implying that D-Glu is not degraded by DDO in vivo. This result is in marked contrast to the observation that DDO KO mice display elevated concentrations of D-Asp in various tissues and physiological fluids.³⁶⁾ Meanwhile, D-Glu cyclase (DGLUCY; EC 4.2.1.48) is another enzyme that acts on D-Glu and degrades this D-amino acid; DGLUCY reversibly converts D-Glu to 5-oxo-D-Pro and H₂O (Fig. 1C). However, DGLUCY does not act on L-Glu, 5-oxo-L-Pro, D-Ser, D-Asp, or D-Ala. Recently, DGLUCY cDNA was cloned from mice,³⁴⁾ and recombinant mouse DGLUCY protein has been purified and functionally characterized.^34,169,170) Mouse DGLUCY is a 66 kDa protein comprising 617 amino acids. In mouse, DGLUCY expression is highest in the heart, followed by the kidney and liver, and expression is low in other tissues.³⁴⁾ DGLUCY KO mice have been established,³⁴⁾ and importantly, a significant increase in D-Glu was observed in DGLUCY KO mouse hearts compared with wild-type mouse hearts, indicating that D-Glu is degraded by DGLUCY in vivo. The idea that DGLUCY plays a physiologically relevant role as an enzyme that degrades D-Glu is supported by a recent report showing that DGLUCY is a metalloenzyme that preferentially acts on D-Glu rather than 5-oxo-D-Pro.¹⁶⁹⁾

3. EXPRESSION AND LOCALIZATION OF D-AMINO ACIDS IN THE PERIPHERY AND ENDOCRINE GLANDS

3.1. Expression and Localization of D-Ser in the Periphery and Endocrine Glands

D-Ser has been identified in a wide variety of peripheral tissues and physiological fluids including kidney, liver, spleen, thymus, lung, heart, muscle, oral cavity, gastric juice, saliva, serum, plasma, urine, and endocrine glands, such as the pineal gland, pituitary gland, hypothalamus, adrenal gland, testis, epididymis, and pancreas.^{3,7,38,39,41–43,166,171)} D-Ser persists at relatively stable concentrations throughout life in all tissues examined, with the exceptions of lung and heart. In rat lung and heart, D-Ser content decreases to trace levels as development proceeds.⁷⁾ Although the presence and localization of D-Ser in the nervous system have been investigated in detail, little is known about changes in D-Ser levels during development or its localization in the periphery and endocrine glands. However, expression and localization of Ser racemase in kidney, cartilage, bone, corpus cavernosum, lower esophageal sphincter, and skin have been investigated in detail, as described below. In cells expressing Ser racemase in these tissues, D-Ser may be biosynthesized and play a critical role in their distinct physiology.

3.2. Expression and Localization of D-Asp in the Periphery and Endocrine Glands

D-Asp occurs in a wide variety of peripheral tissues and physiological fluids including kidney, liver, spleen, thymus, lung, heart, muscle, oral cavity, gastric juice, saliva, blood, serum, plasma, urine, and endocrine glands, such as the pineal gland, pituitary gland, hypothalamus, thyroid gland, adrenal gland, testis, epididymis, ovary, placenta, and pancreas.^{2,3,5,7,8,35,42–44,153,166,172–182)} In addition, D-Asp has also been found in the kidney, liver, heart, spleen, adrenal gland, testis, ovary, and pancreas of chickens, pigeons, mallard ducks, frogs, lizards, and newts.^183–192)

3.3. Expression and Localization of D-Ala in the Periphery and Endocrine Glands

D-Ala is present in various peripheral tissues and physiological fluids including kidney, liver, spleen, thymus, lung, heart, bone marrow, muscle, oral cavity, gastric juice, saliva, serum, plasma, urine, and endocrine glands such as the pineal gland, pituitary gland, hypothalamus, thyroid gland, adrenal gland, testis, and pancreas.^{3,38–44,166)} In rat tissues, the highest D-Ala content was observed in the anterior pituitary gland, and the second highest in the pancreas.⁴⁰⁾

3.4. Expression and Localization of D-Glu in the Periphery and Endocrine Glands

In addition to D-Ser, D-Asp, and D-Ala, D-Glu has also been detected in numerous peripheral tissues and physiological fluids including kidney, liver, spleen, thymus, lung, heart, oral cavity, gastric juice, saliva, urine, and endocrine glands such as the pineal gland, pituitary gland, hypothalamus, adrenal gland, testis, and pancreas.^{8,34,35,42,43)} Establishing the biological significance of this D-amino acid in the periphery and endocrine glands requires further investigation.

3.5. Expression and Localization of Other D-Amino Acids in the Periphery and Endocrine Glands

In addition to D-Ser, D-Asp, D-Ala, and D-Glu, D-Leu, D-Pro, D-Arg, D-Met, D-Val, and D-Trp have also been found in kidney, heart, oral cavity, gastric juice, saliva, plasma, pineal gland, and testis in mammals, including humans.^{3,34,41–43,166)} Determining the biological significance of these D-amino acids in the periphery and endocrine glands requires more research.

4. PHYSIOLOGICAL ROLES OF D-SER AND D-GLU IN PERIPHERAL TISSUES

4.1. D-Ser in the Kidney

In the rat kidney, expression of Ser racemase is high in the pelvis and medulla, but relatively low in the cortex.¹⁹³⁾ Immunohistochemical analysis of rat kidney using a specific anti-Ser racemase antibody revealed expression predominantly in the muscle layer of the renal pelvis, and to a lesser extent the uroepithelial layer. Intrapelvic administration of D-Ser causes an increase in substance P release and afferent renal nerve activity in rats but has no effect on systemic arterial pressure.¹⁹³⁾ Furthermore, increasing intrapelvic pressure elevates afferent renal nerve activity, and therefore diuretic and natriuretic responses. These changes in renal function are significantly attenuated by prior administration of MK-801. In the rat kidney, the NR1 and NR2C subunits, but not the NR2A, NR2B, or NR2D subunits, are expressed, primarily in the pelvic nerve bundles and basolateral proximal tubules.^193–195) Thus, it is proposed that D-Ser plays a crucial role in hemodynamic regulation in kidney, including via regulation of renorenal reflex control of body fluid through the NMDA receptor¹⁹³⁾ (Table 1).

Table 1. Tissue Distribution, Quantities, and Physiological Functions of Free D-Amino Acids in the Periphery and Endocrine Glands

Amino acids	Tissues	Quantitiesa)	Proposed functions	References
D-Ser	Rat kidney	18 ± 1 nmol/g wet mass at 7 weeks old b) (3.0)	Modulation of hemodynamic regulation, including renorenal reflex control of body fluid, through NMDA receptor signaling	7,193)
	Rat and mouse cartilage	ND	Suppression of chondrogenic differentiation through binding to NMDA receptors composed of NR1 and NR3A subunits, and involving runt-related transcription factor-2 transcriptional activity	203,204)
	Rat and mouse bone	ND	Suppression of osteoclastogenesis through a mechanism involving competitive inhibition of extracellular L-Ser uptake via ATB0, + and/or ASCT2	206)
	Rat corpus cavernosum	ND	Modulation of non-adrenergic, non-cholinergic neurogenic relaxation through NMDA receptor signaling	211)
	Rat lower esophageal sphincter	ND	Modulation of non-adrenergic, non-cholinergic neurogenic contraction through NMDA receptor signaling	214)
	Mouse skin	ND	Modulation of the differentiation and/or barrier function of epidermal keratinocytes	216)
D-Asp	Rat pineal gland	2026 ± 148 nmol/g wet mass at 6 weeks oldb) (23.9)	Suppression of the synthesis and secretion of melatonin in pinealocytes	35,217–219)
	Rat anterior pituitary gland	137 ± 32 nmol/g wet mass at 6 weeks oldc) (4.69)	Enhancement of the synthesis and secretion of prolactin in mammotrophs	123,177,220,221)
	Mouse pituitary gland, intermediate lobe	ND	Suppression of α-melanocyte-stimulating hormone levels by regulation of the biosynthesis of proopiomelanocortin in melanotrophs	153)
	Rat hypothalamus	15.0 ± 1.9 nmol/g wet mass at 6 weeks oldb) (0.32)	Modulation of the production of oxytocin and/or vasopressin in magnocellular neurons	35,225–227)
	Rat adrenal gland	157 ± 57 nmol/g wet mass at 1 week oldb) (14.6)	Modulation of the development and maturation of steroidogenesis in the adrenal cortex	7,228)
		608 ± 70 nmol/g wet mass at 3 weeks oldb) (45.9)
		301 ± 35 nmol/g wet mass at 7 weeks oldb) (44.0)
		28 ± 2 nmol/g wet mass at 14 weeks oldb) (6.0)
	Rat testis	130 ± 14 nmol/g wet mass at 6 weeks oldb) (17.9)	Enhancement of testosterone synthesis in Leydig cells by stimulation of steroidogenic acute regulatory protein gene expression	35,232,233)
	Human pre-ovulatory follicular fluid	19.1 ± 1.9 nmol/mL at 22–34 years old c)	Quality control of oocytes	236)
		10.9 ± 1.2 nmol/mL at 35–40 years oldc)
D-Ala	Rat anterior pituitary gland	86.4 ± 9.9 nmol/g wet mass at 9 weeks oldb) (2.5)	Modulation of the production of adrenocorticotropic hormone in corticotrophs	40,238)
	Rat pancreas	29.2 ± 5.0 nmol/g wet mass at 9 weeks oldb) (1.3)	Modulation of the production of insulin in β cells of the Islets of Langerhans	40,239–241)

a) Proportions of D-amino acids of the total quantity [D-form/(D-form + L-form) × 100%] are shown in parentheses. b) Data are means ± standard error. c) Data are means ± standard deviation. ND, not determined.

As described above, in the kidney, DAO is localized to the epithelial cells of proximal renal tubules. Accordingly, a large proportion of D-amino acids reabsorbed after filtration at the glomerulus is degraded by DAO present at the tubules. Thus, levels of D-amino acids in the blood and urine are closely correlated with renal function, and D-amino acids have been recognized as novel biomarkers of nephropathy. For example, levels of D-Ser and L-Ser in the serum of patients with severe renal failure are significantly increased and decreased, respectively, compared with those of healthy volunteers, resulting in a marked increase in the ratio of D-Ser/L-Ser in the serum of patients with severe renal failure compared with that of healthy volunteers¹⁹⁶⁾ (Figs. 2A–C). Moreover, in a study using renal ischemia-reperfusion injury mice as an experimental model of acute kidney injury, ischemia-reperfusion injury raised the concentration of D-Ser in serum 20 h after reperfusion and lowered the concentration of D-Ser in urine 4 h after reperfusion.¹⁹⁶⁾ On the other hand, ischemia–reperfusion injury caused a rapid decrease in the concentration of L-Ser in serum 4 h after reperfusion and increased the concentration of L-Ser in urine 8 h after reperfusion. As a result, the ratio of D-Ser/L-Ser in serum and urine was significantly increased and decreased, respectively, 4 h after reperfusion, corresponding to the acute phase of renal failure (Fig. 2D). By contrast, the concentrations of creatinine, kidney injury molecule-1, and neutrophil gelatinase-associated lipocalin in urine, all considered biomarkers of nephropathy, were not altered significantly 4 h after reperfusion, but were changed significantly 8 h after reperfusion¹⁹⁶⁾ (Figs. 2E–G). These results suggest that the ratio of D-Ser/L-Ser is a novel biomarker of ischemic acute kidney injury. In addition, D-Ser is a useful biomarker for diagnosis of chronic kidney disease. A longitudinal cohort study on 108 chronic kidney disease patients demonstrated that the ratio of D-Ser/L-Ser in plasma was significantly associated with kidney function (estimated glomerular filtration ratio).¹⁹⁷⁾ Taken together, these findings strongly suggest that the ratio of D-Ser/L-Ser is a novel and useful biomarker of nephropathy, enabling more sensitive detection of acute kidney injury and chronic kidney disease than other promising biomarkers. Indeed, recent studies indicate that the ratio of D-Ser/L-Ser serves as a vital biomarker of nephropathy.^198–202)

Fig. 2. Involvement of Ser Content in Renal Function

(A–C) Serum levels of Ser enantiomers in healthy donors and patients with severe renal failure. D-Ser (A) and L-Ser (B) in serum of healthy volunteers (n = 4) and patients with renal failure (n = 4) were measured. The ratio of D-Ser/L-Ser (n = 4) is also shown (C). * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t-test). Figures are modified from Ref. 196). For details, see main text and Ref. 196). (D–G) Effects of renal ischemia-reperfusion injury on several biomarkers in mouse urine. Figures D–G show the ratios of D-Ser/L-Ser (D), concentrations of creatinine (E), kidney injury molecule-1 (KIM-1) (F), and neutrophil gelatinase-associated lipocalin (NGAL) (G) in the urine of sham-operated control mice and mice after renal ischemia-reperfusion injury. Results are means ± standard error of the mean (n = 5–7). ** p < 0.01, *** p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparison test). NS, not significant. Figures are modified from Ref. 196). For details, see main text and Ref. 196).

4.2. D-Ser in Cartilage and Bone

Expression of Ser racemase is observed in cartilage, such as that of the neonatal rat tibia, cultured rat costal chondrocytes, and cultured mouse embryonic metatarsals (isolated before vascularization).^203,204)In situ hybridization analysis of cultured mouse embryonic metatarsals revealed that Ser racemase is highly expressed in both hypertrophic and calcified chondrocytes, but not in proliferating or pre-hypertrophic chondrocytes. Interestingly, the NMDA receptor NR3A subunit, in addition to the NR1 and NR2D subunits, is expressed in cultured rat costal chondrocytes, while the NR2A, NR2B, NR2C, and NR3B subunits of the receptor are not expressed in these cells.^203,204) In contrast to an NMDA receptor composed of NR1 and NR2 subunits, an NMDA receptor composed of NR1 and NR3 subunits functions as an excitatory Gly receptor that is unaffected by L-Glu or NMDA, but inhibited by D-Ser.²⁰⁵⁾ Indeed, sustained exposure of cultured rat costal chondrocytes to D-Ser suppresses several chondrocytic maturation processes, including upregulation of osteopontin, a chondrocytic maturation marker protein.²⁰⁴⁾ Analysis of promoter activity in cultured cells forced to express the NR1 and NR3A subunits suggested that the inhibitory effect of D-Ser on the Gly-stimulated activation of the NMDA receptor (composed of NR1 and NR3A subunits) upregulates the transcriptional activity of runt-related transcription factor-2, resulting in an increase in the expression of osteopontin. It has therefore been proposed that D-Ser negatively regulates cellular differentiation by acting as an endogenous antagonist at the Gly-binding site of NMDA receptors composed of NR1 and NR3A subunits in chondrocytes, and is involved in runt-related transcription factor-2 transcriptional activity²⁰⁴⁾ (Table 1).

It has also been posited that D-Ser has a suppressive effect on chondrogenic differentiation through inhibition of the transcriptional activity of sry-type HMG box 9, which is essential for the differentiation of mesenchymal progenitor cells into chondrocytes.²⁰³⁾ Furthermore, in addition to the possible physiological role of D-Ser in cartilage, a potential role in bone was also suggested by the same research group.²⁰⁶⁾ Briefly, it was proposed that D-Ser plays an essential role in osteoclastogenesis through a mechanism involving inhibition of extracellular L-Ser uptake via ATB^{0, +} and/or ASCT2, both of which can transport extracellular D-Ser, as well as L-Ser, into cells in a competitive manner,^207–210) following synthesis of D-Ser and its subsequent release from adjacent osteoblasts (Table 1).

4.3. D-Ser in the Corpus Cavernosum and Lower Esophageal Sphincter

Expression of Ser racemase, but not DAO, is observed in rat corpus cavernosum.²¹¹⁾ Immunogold electron microscopy was used to demonstrate the cellular localization of Ser racemase in the cavernosal nerve membrane. An organ bath study using pre-contracted rat corpora cavernosa strips demonstrated that administration of D-Ser, but not L-Ser, causes an increase in the electrical field-induced non-adrenergic, non-cholinergic neurogenic relaxation of the tissue.²¹¹⁾ The NMDA receptor is expressed in mammalian corpus cavernosum,^212,213) and consistently, the ability of D-Ser to cause relaxation of the corpus cavernosum is inhibited by several antagonists of the NMDA receptor or the nitric oxide synthase inhibitor N^G-L-nitro-arginine methyl ester. Based on this, it was proposed that D-Ser plays a critical role in modulating neurogenic relaxation of the corpus cavernosum through activation of the NMDA receptor (Table 1), and these findings may open new therapeutic avenues for the treatment of impotence.²¹¹⁾

In addition to the role of D-Ser in modulating neurogenic relaxation of the corpus cavernosum, the possible involvement of D-Ser in non-adrenergic, non-cholinergic neurogenic contraction of the lower esophageal sphincter has also been reported.²¹⁴⁾ Expression of Ser racemase, but not DAO, is observed in the rat lower esophageal sphincter, and immunofluorescence analysis of this tissue revealed that Ser racemase is present in the submucosa. However, an organ bath study using pre-contracted rat lower esophageal sphincter strips demonstrated that, in contrast to the aforementioned relaxing effect of D-Ser on rat corpus cavernosum, administration of D-Ser has no significant effect on electrical field-induced non-adrenergic, non-cholinergic relaxation of the tissue.²¹⁴⁾ By contrast, administration of D-Ser or L-Ser to isolated rat lower esophageal sphincter strips causes an increase in electrical field-induced non-adrenergic, non-cholinergic neurogenic contraction of this tissue.

Because the NMDA receptor is expressed in the mammalian lower esophageal sphincter²¹⁵⁾ and the aforementioned effect of D-Ser on its contraction is inhibited by MK-801,²¹⁴⁾ the NMDA receptor may mediate the effect of D-Ser on the contraction of this tissue (Table 1). However, addition of L-Asp-β-hydroxamate, a Ser racemase inhibitor, to lower esophageal sphincter strips has no significant effect on the electrical field-induced contraction of the tissue, while the aforementioned effect of L-Ser on the contraction is inhibited by this compound.²¹⁴⁾ Further studies are needed to clarify whether endogenous D-Ser plays an important role as a co-agonist of the NMDA receptor in modulation of the neurogenic contraction of the lower esophageal sphincter.

4.4. D-Ser in Skin

Expression of Ser racemase has also been observed in the epidermis of mouse skin.²¹⁶⁾ Immunohistochemical analysis of mouse skin using a specific anti-Ser racemase antibody suggested that Ser racemase is mainly present in the cornified layer, and to a lesser extent in the granular layer, of the epidermis. Indeed, the concentration of D-Ser in confluent cultured keratinocytes (which represent the terminal differentiation state of keratinocytes) derived from the skin of wild-type mice was significantly higher than that in cells derived from the skin of Ser racemase KO mice, whereas intracellular L-Ser levels did not differ between wild-type and Ser racemase KO mice.²¹⁶⁾ Furthermore, expression levels of involucrin and transglutaminase 3, both of which are involved in the differentiation and/or barrier function of epidermal keratinocytes, in the epidermis of Ser racemase KO mice were significantly higher than those in the epidermis of wild-type mice. Moreover, a greater degree of transepidermal water loss and slower barrier recovery following tape stripping were also observed in the skin of Ser racemase KO mice compared with that of wild-type mice. The possible involvement of D-Ser synthesized by Ser racemase in the cornified layer in the differentiation of epidermal keratinocytes and formation of the skin barrier (Table 1) warrants further investigation.

4.5. D-Glu in the Heart

It was recently reported that DGLUCY expression in the heart was lower in a mouse model of heart failure.³⁴⁾ Consistent with the fact that DGLUCY degrades D-Glu to 5-oxo-D-Pro and H₂O, a significantly higher concentration of D-Glu was observed in DGLUCY KO mouse hearts (187.3 ± 11.5 nmol/g tissue, mean ± standard deviation) than in wild-type mouse hearts (31.0 ± 1.3 nmol/g tissue, mean ± standard deviation) at 13 weeks of age. Thus, D-Glu and/or 5-oxo-D-Pro are presumably correlated with cardiac function, and interest is growing in their unique metabolic pathways (in this context, we first identified DGLUCY as the degradative enzyme of D-Glu in mammals).³⁴⁾ As described above, we recently identified the cDNA encoding STDHgr as the first mammalian clone with Glu racemase activity.¹²⁵⁾ Experiments using cultured mammalian cells confirmed that STDHgr indeed acts as a D-Glu biosynthetic enzyme in vivo. Taken together, these findings suggest that D-Glu and/or 5-oxo-D-Pro play essential role(s) in cardiac function through their metabolism by DGLUCY and STDHgr, and are likely involved in the onset and/or exacerbation of heart failure. Future identification and characterization of D-Glu transporters and the specific pathways of D-Glu and 5-oxo-D-Pro release will contribute to our understanding of the physiological and pathological functions of these D-amino acids. Moreover, from a pathophysiological and pharmacological standpoint, detailed analysis of D-Glu and 5-oxo-D-Pro in biological samples is also required to understand the correlation between these D-amino acids and cardiac function, including the risk of onset of heart failure, as well as the biological significance of these D-amino acids in mammals.

5. PHYSIOLOGICAL ROLES OF D-ASP AND D-ALA IN ENDOCRINE GLANDS

5.1. D-Asp in the Pineal Gland

Substantial amounts of D-Asp are present in the pineal gland of mammals including mice, rats, sheep, and pigs^{3,35,172,174,182)} (Table 1). In particular, the D-Asp content of the adult rat pineal gland is very high,^3,174) but there are regional differences in D-Asp immunoreactivity. Specifically, immunohistochemical analysis of the rat pineal gland using a specific anti-D-Asp antibody revealed intense immunoreactivity in the distal (caudal) region of the gland, but minimal staining in the proximal (rostral) region.²¹⁷⁾ In addition, D-Asp is localized to the cytoplasm of pinealocytes, which constitute approx. 80% of the cells in this gland. Because pinealocytes in the distal region of the gland are involved in the synthesis and secretion of melatonin, the presence of D-Asp in this location suggests that it regulates one or both processes. Indeed, addition of D-Asp to primary cultured rat pinealocytes suppresses the norepinephrine-induced secretion of melatonin in a dose-dependent manner.^218,219) Moreover, cultured pinealocytes can efficiently take up D-Asp from the culture medium, and D-Asp is released from cells following stimulation with norepinephrine. It thus appears that both melatonin secretion and D-Asp release from pinealocytes are enhanced by stimulation with norepinephrine, after which melatonin secretion is suppressed by the action of the released D-Asp on cells (Table 1). This negative feedback mechanism may regulate norepinephrine-induced melatonin secretion in the pineal gland.

5.2. D-Asp in the Pituitary Gland and Hypothalamus

Substantial amounts of D-Asp have also been detected in the pituitary gland of mice, rats, sheep, and pigs^{5,7,35,44,172,173,177,181,182)} (Table 1). In the pituitary gland of adult rats, D-Asp immunoreactivity was evident in the anterior and posterior lobes, but not in the intermediate lobe,^151,177) and D-Asp immunoreactivity was observed predominantly in prolactin-producing mammotrophs or a closely-related cell type in the anterior. Expression of D-Asp in this cell type suggests that D-Asp plays a regulatory role in the synthesis and secretion of prolactin in the anterior lobe of the gland. This conclusion is supported by the observation that addition of D-Asp to a prolactin-producing clonal strain of rat pituitary tumor cells (GH₃ cells) enhances thyrotropin-releasing hormone-induced secretion of prolactin in a dose-dependent manner.¹²³⁾ Secretion of prolactin from isolated pituitary glands or dispersed anterior pituitary cells in adult rats is also enhanced by incubation with D-Asp.^220,221)

L-Glu transporters are presumed to be involved in D-Asp uptake because the affinity of these transporters for D-Asp is similar to their affinities for L-Glu and L-Asp; however, their affinity for D-Glu is so low that it is essentially not transported at all.^222–224) Because prolactin-producing cells do not express any known L-Glu transporters, it is likely that those in the anterior lobe of the gland synthesize their own D-Asp, rather than importing it after it is synthesized elsewhere. Indeed, intraperitoneally administered D-Asp is not incorporated into prolactin-producing cells in the rat pituitary gland, but is taken up by endothelial cells.¹⁷⁸⁾ Furthermore, alterations in the expression levels of L-Glu transporters in the gland do not correlate with development-related changes in D-Asp levels. Taking these pieces of evidence together, it appears that D-Asp is synthesized in the anterior lobe of the pituitary gland, where it acts on prolactin-producing cells as an autocrine or paracrine regulator to enhance prolactin secretion by these cells (Table 1). The possible involvement of D-Asp in the secretion of growth hormone and luteinizing hormone in the hypothalamo-hypophysial system has also been proposed.^172,173,181)

In the posterior lobe of the rat pituitary gland, intense D-Asp immunoreactivity has also been observed in the nerve processes and termini of magnocellular neurons.^151,225) The cell bodies of magnocellular neurons are present in the supraoptic and paraventricular nuclei of the hypothalamus, where D-Asp immunoreactivity is also observed. In magnocellular neurons, D-Asp appears to modulate the production of oxytocin and/or vasopressin. Indeed, D-Asp stimulates the release of oxytocin from rat hypothalamic explants.²²⁵⁾ Moreover, intraperitoneal injection of D-Asp leads to greater expression of oxytocin and vasopressin in supraoptic and paraventricular nuclei of the rat hypothalamus.²²⁶⁾ Within the magnocellular neurons of the rat supraoptic nucleus, D-Asp immunoreactivity is observed in the nucleoli,²²⁷⁾ where it is predominantly associated with heterochromatin, but not with other subcellular structures of the nucleus and soma. These observations have led to the proposal that D-Asp directly interacts with DNA and/or acts on nuclear protein(s) involved in regulating gene transcription, and thereby contributes to the regulation of gene expression in the hypothalamo-hypophyseal system²²⁷⁾ (Table 1).

In contrast to the anterior and posterior lobes, D-Asp immunoreactivity is not observed in the intermediate lobes of mouse, rat, and pig pituitary glands.^{151,153,177,182)} This may be due to high expression of DDO in this region. Indeed, in DDO KO mice, intense D-Asp immunoreactivity is observed in the intermediate lobe, as well as in the anterior and posterior lobes.¹⁵³⁾ The intermediate lobe of the gland is almost exclusively composed of melanotrophs, which produce proopiomelanocortin, the sole source of pituitary α-melanocyte-stimulating hormone, a member of the melanocortin peptide family. In DDO KO mice, expression levels of proopiomelanocortin and α-melanocyte-stimulating hormone in the intermediate lobe of the gland are substantially lower than in wild-type controls.¹⁵³⁾ Furthermore, phenotypic changes that can be ascribed to the effects of α-melanocyte-stimulating hormone, such as greater body mass, sexual deficits, and lower self-grooming, are observed in DDO KO mice. It thus appears that D-Asp suppresses α-melanocyte-stimulating hormone expression by regulating the biosynthesis of proopiomelanocortin in the intermediate lobe of the pituitary gland, and thereby participates in the generation of several phenotypes that can be ascribed to the actions of α-melanocyte-stimulating hormone (Table 1).

5.3. D-Asp in the Adrenal Gland

D-Asp is present in the adrenal glands of chickens, rats, sheep, and pigs, and changes in D-Asp levels during development and its localization in rat tissues have been investigated in detail. In the rat adrenal gland, a transient increase in D-Asp has been observed: D-Asp levels at 1 week of age are relatively low, but increased markedly at 3 weeks of age, before rapidly declining⁷⁾ (Table 1). Immunohistochemical analysis using a specific anti-D-Asp antibody revealed that, in the rat adrenal cortex, D-Asp is present in different cell types during distinct periods of development.²²⁸⁾ At 3 weeks of age, D-Asp immunoreactivity is prominent in the cytoplasm of cells in the two inner zones (zona fasciculata and zona reticularis) but negligible in the outermost zone (zona glomerulosa) of the cortex. By contrast, at 8 weeks of age, intense D-Asp immunoreactivity is observed in the zona glomerulosa, whereas a lower level is observed in the zona fasciculata and zona reticularis. Thus, the pattern of adrenocortical D-Asp distribution appears to reverse as development proceeds. The zona fasciculata and zona reticularis secrete glucocorticoids, while the zona glomerulosa secretes mineralocorticoids. Moreover, the maturation periods of these regions differ. Thus, the aforementioned temporal and site-specific patterns of D-Asp concentration suggest that it is involved in the development and maturation of steroidogenesis in the adrenal cortex (Table 1).

5.4. D-Asp in the Testis

Substantial amounts of D-Asp have been found in the testis of mammals including mice, rats, and pigs^{5,7,35,44,166,174–176,229,230)} (Table 1). In the adult rat testis, intense D-Asp immunoreactivity was observed in the cytoplasm of germ cells within the seminiferous tubules, particularly in elongated spermatids, the most mature germ cells.¹⁶⁶⁾ Intraperitoneally administered D-Asp is not incorporated into the seminiferous tubules, but instead accumulates in the interstitial spaces of the testis; therefore, it appears that germ cells in the testicular seminiferous tubules synthesize their own D-Asp, rather than importing it after it has been synthesized elsewhere. Furthermore, D-Asp levels in the testicular venous plasma are higher than those in peripheral plasma, suggesting that D-Asp synthesized in the testicular seminiferous tubules is then secreted.²³¹⁾ In addition, secreted D-Asp appears to act on interstitial Leydig cells to modulate testosterone synthesis. Indeed, addition of D-Asp to primary cultured rat testicular Leydig cells enhances human chorionic gonadotropin-induced synthesis of testosterone in a dose-dependent manner.²³²⁾ Moreover, cultured Leydig cells are able to efficiently take up D-Asp from the culture medium through L-Glu transporters, and the amount of D-Asp in cells correlates with the increase in testosterone production. When cultured Leydig cells are treated with L-cysteine sulfinic acid, an inhibitor that prevents uptake of amino acids by the L-Glu transporter, both uptake of D-Asp and D-Asp-mediated enhancement of testosterone production are concomitantly suppressed in a dose-dependent manner.²³²⁾ Therefore, it appears that D-Asp enters Leydig cells through L-Glu transporters, and then enhances testosterone synthesis (Table 1). Enhancement of testicular testosterone synthesis by D-Asp has also been demonstrated in both pigs and humans.^181,230)

In Leydig cells, testosterone synthesis is initiated by the transport of intracellular cholesterol to the inner mitochondrial membrane, where it is converted to pregnenolone by intramitochondrial CYP-mediated side-chain cleavage. Subsequently, pregnenolone is converted to testosterone via the steroidogenic pathway in the endoplasmic reticulum. Cholesterol delivery to the inner mitochondrial membrane is the rate-limiting step in testosterone synthesis, and the key regulatory protein that facilitates this process is steroidogenic acute regulatory protein. Interestingly, addition of D-Asp to cultured Leydig cells enhances the steady state mRNA and protein levels of steroidogenic acute regulatory protein, thereby increasing testosterone synthesis.²³³⁾ Thus, it appears that D-Asp enhances testosterone synthesis in Leydig cells by promoting steroidogenic acute regulatory protein gene expression, resulting in greater testosterone release and effects on germ cells within the seminiferous tubules. Through this feedback mechanism, D-Asp in germ cells may regulate the production and secretion of testosterone (Table 1), which subsequently promotes the differentiation and maturation of germ cells in the testis. The involvement of D-Asp in testosterone synthesis in the testis has also been demonstrated by studies on seasonal-breeding vertebrates including mallard ducks, frogs, and lizards.^{183,184,188–190,192)}

5.5. D-Asp in the Ovary

Substantial amounts of D-Asp are present in mouse, rat, and sheep ovaries,^5,153,172) and D-Asp has also been detected in frog and lizard ovaries.^{191,192,234,235)} In female lizards, exogenous D-Asp induces a significant decrease in testosterone levels in the ovary and plasma, while it enhances follicular production of estradiol by upregulating local aromatase activity.²³⁴⁾ Similar findings were reported following studies on female frogs.^192,235) Therefore, it has been proposed that D-Asp is involved in the maturation of oocytes in these species.^191,192) In this context, in female human patients undergoing in vitro fertilization, the D-Asp content of pre-ovulatory follicular fluid is lower in older than in younger patients (Table 1). This decrease in D-Asp content appears to reflect a reduction in oocyte quality and fertilization competence. Thus, D-Asp may be involved in the quality control of oocytes in mammals (Table 1). Overall, these findings provide some insight into the physiological and pathological roles of D-Asp and their relationships with the pathophysiology of infertility; however, establishing the details of these relationships requires further investigation.

5.6. D-Asp in the Pancreas

The pancreas is an exocrine organ that produces digestive zymogens and enzymes, but also contains endocrine tissue. A number of hormones are secreted from the endocrine Islets of Langerhans, which comprise approx. 1% of cells in this tissue. D-Asp has been detected in the pancreases of rats, chickens, and pigeons,^5,35,187) and in the adult rat pancreas D-Asp immunoreactivity is predominantly observed in the Islets.²³⁷⁾ Among the cell types that constitute the Islets, D-Asp is found in the highest concentrations in glucagon-secreting α cells and a subpopulation of pancreatic polypeptide-secreting PP cells. Thus, it is likely that D-Asp plays a regulatory role in the synthesis and/or secretion of glucagon and pancreatic polypeptide in the pancreas. However, the role of D-Asp in the endocrine function of the pancreas requires further investigation.

5.7. D-Ala in the Pituitary Gland and Pancreas

Immunohistochemical analysis of the adult rat pituitary gland using a specific anti-D-Ala antibody revealed that D-Ala immunopositive cells are present principally in the anterior lobe, whereas no significant staining was observed in the intermediate and posterior lobes.²³⁸⁾ Among the cell types that constitute the anterior pituitary gland, D-Ala immunoreactivity was observed predominantly in adrenocorticotropic hormone-producing corticotrophs, while in the adult rat pancreas, D-Ala immunoreactivity was observed principally in the insulin-secreting β cells of the Islets.²³⁹⁾ Because both adrenocorticotropic hormone and insulin regulate blood glucose, expression of D-Ala in these cell types suggests that D-Ala levels may be associated with blood glucose concentration in mammals (Table 1). Consistently, circadian variation in D-Ala levels is inversely correlated with that of plasma insulin levels in rats.²⁴⁰⁾ Moreover, stimulation of isolated rat Islets with glucose induces the release of D-Ala from the tissue.²⁴¹⁾

6. CONCLUDING REMARKS

As described above, D-Ser plays a critical role in regulating brain function by acting as an endogenous ligand for the NMDA receptor or the δ2 Glu receptor. In addition, D-Asp acts as a signaling molecule in the nervous system, in part by binding to the NMDA receptor, and plays essential roles in regulating brain functions. Thus, D-Ser and D-Asp have been shown to function as important bioactive substances in the central nervous system. Additionally, the functions of D-Ser, D-Asp, D-Ala, and D-Glu in the periphery and endocrine glands have gained attention in recent years. In the present article, we review recent advances in studies on the physiological roles of these D-amino acids in the periphery and endocrine glands, focusing on D-Ser in the kidney, cartilage and bone, corpus cavernosum and lower esophageal sphincter, and skin; D-Asp in the pineal gland, pituitary gland and hypothalamus, adrenal gland, testis, ovary, and pancreas; D-Ala in the pituitary gland and pancreas; and D-Glu in the heart. Progress in analytical methods for the enantiomeric determination of amino acids and the expression of metabolic enzymes of D-amino acids has enabled the effective and sensitive analysis of these D-amino acids and their metabolic enzymes in biological samples. In addition, the establishment of Ser racemase KO mice, ddY/DAO⁻ mice, DDO KO mice, and DGLUCY KO mice is of great importance. These mouse models have enabled the gradual discovery of several physiological functions of D-Ser, D-Asp, D-Ala, and D-Glu in mammals. Consequently, it has been proposed that these D-amino acids play crucial roles as important bioactive substances in the physiology of the periphery and endocrine glands, as well as the central nervous system. However, studies on D-amino acids in the periphery and endocrine glands lag far behind those in the central nervous system. Further studies are required to clarify the physiological and pathological roles of D-amino acids in the periphery and endocrine glands.

Acknowledgments

This work was supported by JSPS KAKENHI (Grant Number: JP21K06085) to MK.

Conflict of Interest

The authors declare no conflict of interest.

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