Amino Acid Metabolism and Autophagy in Skeletal Development and Homeostasis

Abstract

Bone is an active organ that is continuously remodeled throughout life via formation and resorption; therefore, a fine-tuned bone (re)modeling is crucial for bone homeostasis and is closely connected with energy metabolism. Amino acids are essential for various cellular functions as well as an energy source, and their synthesis and catabolism (e.g., metabolism of carbohydrates and fatty acids) are regulated through numerous enzymatic cascades. In addition, the intracellular levels of amino acids are maintained by autophagy, a cellular recycling system for proteins and organelles; under nutrient deprivation conditions, autophagy is strongly induced to compensate for cellular demands and to restore the amino acid pool. Metabolites derived from amino acids are known to be precursors of bioactive molecules such as second messengers and neurotransmitters, which control various cellular processes, including cell proliferation, differentiation, and homeostasis. Thus, amino acid metabolism and autophagy are tightly and reciprocally regulated in our bodies. This review discusses the current knowledge and potential links between bone diseases and deficiencies in amino acid metabolism and autophagy.

1. Introduction

Continuous bone formation and resorption are critical processes for the maintenance of healthy bones throughout life and are closely intertwined with energy metabolism, which comprises a series of metabolic pathways that generate energy in the form of adenosine triphosphate (ATP) from nutrients such as carbohydrates, fats, and proteins. Both the anabolic and catabolic metabolic pathways are catalyzed by numerous enzymes that require co-factors and ATP itself for their own activation [1]. In addition to enzymatic activities, proteins, which are combinations of > 20 amino acids, serve as functional molecules (e.g., cellular components, receptors, cytoskeleton, and growth factors) in the cell, extracellular matrix, and circulation systems. The amino acids for their production are supplied through the degradation of dietary and/or cellular proteins, as well as synthesis via metabolic pathways such as glycogenesis and the tricarboxylic acid (TCA) cycle (a.k.a. citric acid cycle); the TCA cycle is dependent on the carbohydrate and fatty acid metabolic pathways, which are important for bone homeostasis [2,3]. Amino acids also act as precursors of bioactive molecules such as neurotransmitters, second messengers, and cytokines. Therefore, dysregulation of amino acid metabolism may result in various pathologies, including those affecting bone tissue and the skeleton [4–6].

Amino acid levels can also be sustained through autophagy, a cellular system for the degradation and recycling of intracellular proteins and organelles [7]. The ULK1 complex (a.k.a. ATG13-ULK1/2-FIP200-ATG101 complex) acts as a pre-initiation complex in the autophagy pathway, which is activated through AMP-dependent protein kinase (AMPK) and inactivated through the core protein of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Under amino acid/nutrient starvation conditions, AMPK dephosphorylates ULK1 and activates the ULK1 complex. Beclin-1 (BECN1), a homolog of yeast Atg6 (autophagy-related 6) that is involved in autophagy, endocytosis, and apoptosis [8,9], interacts with Barkor/ATG14, PI3K/VPS34, VPS15, and AMBRA at the endoplasmic reticulum (ER) membrane, and forms the class III phosphoinositide 3 kinase (PI3K) class III complex I (the PI3KC3-C1 complex), which initiates formation of the isolation membrane (a.k.a. phagophore). An active ULK1 complex is known to phosphorylate BECN1 and PI3K/VPS34 in PI3KC3-C1. Conversely, interaction with B-cell lymphoma 2 (BCL2) inhibits the formation of the PI3KC3-C1 complex, while nutrient starvation induces dissociation of the Beclin-1–BCL2 complex, initiating autophagy [8,9]. At the ER, phosphorylated PI3K/VPS34 catalyzes the conversion of phosphatidylinositol (PI) to phosphatidylinositol-3-phosphate [PI(3)P] (the structure called omegasome). The accumulation of PI(3)P then promotes nucleation of the omegasome from the ER.

There are two ubiquitin-like conjugation pathways, ATG12–5 and ATG8, crucial for the autophagy regulation in autophagosome formation. ATG7 acts as an E1-like enzyme to activate both ATG12–5 and ATG8 pathways during initiation and elongation of the autophagosome membrane. ATG3 and ATG10 then act as E2-like enzymes in the ATG8 and ATG12–5 pathways, respectively. The ATG12–5 complex conjugates with ATG16L (the ATG12–5:ATG16L complex) and acts as an E3-like ligase to catalyze phosphatidylethanolamine (PE) to ATG8, transforming the inactivated cytosolic form (type I) into the activated membrane-bounded form (type II). There are three homologs of yeast Atg8 in mammals: LC3 (microtube-associated protein 1 light chain 3), GABARAP (γ-aminobutyric acid receptor-associated protein), and GATE16 (a.k.a. GABARAPL2; Golgi-associated ATPase enhancer of 16 KDa). Among them, LC3 is the best characterized in autophagy. The ATG12–5:ATG16L complex binds to the outer membrane only; by contrast, LC3-II binds to both outer and inner membranes of the isolation membrane in order to promote the elongation of the autophagic membrane (capturing unnecessary proteins/organelles) for autophagosome formation. These autophagosomes fuse with lysosomes (called autolysosomes) and degrade/recycle unnecessary proteins/organelles for supplying amino acid, lipids, and ATP [10–12].

The steady-state level of autophagy is involved in clearance and turnover of both organelles and proteins; however, in case of nutrient starvation, autophagy can be greatly induced to generate amino acids from proteins in order to meet the cellular needs [7,13]. Amino acid metabolism, its metabolites, and intracellular amino acid levels, are all involved in the regulation of autophagy, and vice versa, under physiological and pathological conditions [14–18]. In addition, recent studies show that autophagy is involved in antioxidant protection [19,20]. Under oxidative stress, reactive oxygen species (ROS) oxidize the cysteine residues in SQSTM1/p62 and ATG4 and promotes degradation of ubiquitinated proteins and lipidation of ATG8, respectively; thus, oxidative stress induces autophagic activity [21–23]. The KEAP1–NRF2 system plays a role in cellular defense against ROS, nitric oxide, and electrophilic stresses [20,24]. KEAP1 (Kelch-like ECH associated protein 1), an adaptor protein of Cullin-3 E3-like ligase, degrades NRF2 (erythroid 2-related factor 2), a transcription factor regulating the expression of anti-stress genes [25,26]. Under stress conditions, KEAP1 is inactivated, which allows NRF2 to translocate into the nuclei. Interestingly, SQSTM1/p62 can bind to KEAP1 by competing with NRF2, resulting in the stabilization and consequent translocation of NRF2 to the nuclei [24,26,27]. Thus, autophagy and KEAP1–NRF2 system are closely associated each other. On the other hand, hypoxia conditions activate both hypoxia-inducible factor 1-alpha (HIF-1α)-dependent anti-oxidative activity and ATG5-dependent mitophagy, the selective degradation of mitochondria by autophagy [28]. In this review, we discuss how amino acid metabolic aberrations, including those due to deficiencies in the autophagic machinery, lead to bone disease.

2. Role of Autophagy in Bone Development and Homeostasis

A growing number of studies suggest that autophagy is associated with bone and cartilage development and homeostasis [29,30]. For instance, mice with an osteoblast-specific deletion of the gene coding for autophagy-related 5 (Col1a1-Cre;Atg5^F/F mice), a protein crucial for the formation of autophagosomes, exhibit reduced bone formation and mineralization [31]. Moreover, mice deficient for Atg7 in osteoblasts (Osx1-Cre;Atg7^F/F mice) exhibit low bone mass and spontaneous fractures through the suppression of bone remodeling due to disrupted osteocyte survival and maturation [32]. Similarly, mice with a deletion of Atg7 in osteocytes (Dmp1-Cre;Atg7^F/F mice) exhibit low bone mass and reduced bone remodeling due to increased oxidative stress [33]. In cartilage, deletion of the Atg7 genes results in shorter bone length and growth retardation; for example, Prx1-Cre;Atg7^F/F and ^TamCol2a1-Cre;Atg7^F/F mice show accumulation of type II procollagen in the ER of chondrocytes, resulting in ER stress mediated by fibroblast growth factor 18 [34,35]. Chondrocytes in either Col2-Cre;Atg7^F/F, Col2-Cre;Atg5^F/F, or ^TamCol2a1-Cre;Atg7^F/F mutant mice show increased apoptotic cell death in the growth plate [35,36]. By contrast, Col11a2-Cre;Atg7^F/Fmice exhibit severe growth retardation, due to failure in the transition of mesenchymal cells to chondrocytes, with accumulation of glycogen granules, but not elevated ER stress, in chondrocytes of the growth plate [37].

In cathepsin K-expressing osteoclasts, mice with loss of the beclin1 gene (CstK-Cre;Becn1^F/F mice), which codes for a critical factor in the regulation of autophagy and cell death, show impaired osteoclast function and bone resorption as well as dysregulated chondrogenesis [38]. In addition, knockdown of Becn1 in mouse bone marrow-derived macrophages suppresses osteoclastogenesis [39]. Osteoclasts from mice with a monocyte-specific deletion of Atg5 (Lyz2-Cre;Atg5^F/F mice) also show impaired bone resorption, caused by reduced lysosome secretion from the ruffled border of osteoclasts, which results in increased trabecular bone volume in long bones [40]. Moreover, mutations in SQSTM1/p62, a known substrate of selective autophagy, cause Paget’s disease of bone (PDB), which is characterized by accelerated bone remodeling leading to dysregulated bone formation and weaker bones [41–43]. Sqstmt1 null mice (a.k.a. p62^−/− mice) and mice with a point mutation that substitutes proline with leucine at codon 394 of Sqstm1 (p.P394L), equivalent to the p.P392L SQSTM1 mutation in humans [44], also show accelerated osteoclastogenesis, which recapitulates PDB in humans [45,46].

mTOR, a core protein of the mTOR complex 1 (mTORC1) and complex 2 (mTORC2), acts as a serine/threonine kinase. mTORC1, which is activated by nutrients or growth factors, inhibits autophagy [10,47], and the amino acid–mTORC1–autophagy axis is closely associated with amino acid/protein intake into the cells [14,48]. Recent studies have demonstrated that mTOR plays key roles in amino acid-induced signaling via the Rag guanosine triphosphatases (GTPases) (a.k.a. Rags) [49–53]. Under amino acid starvation conditions, SQSTM1/p62, which is separated from the lysosomal membrane, binds to LC3-II at the isolation membrane, leading to degradation of SQSTM1/p62by the autophagosome. On the other hand, under amino acid-rich conditions, mTORC1 binds to SQSTM1/p62 on the lysosomal membrane and induces PI(3)P degradation. Active mTORC1 phosphorylates ULK1/2 and ATG13, leading to inactivation of the ULK1 complex that suppresses autophagic pathway.

Supplementation of L-glutamine, which is uptaken through the SLC1A5 transporter, promotes mTORC1 pathway and inhibits autophagic activation. On the other hand, inhibition of SLC1A5 suppresses mTORC1 activation and activates autophagy. L-type amino acid exchanger SLC7A5 requires mTORC1 signaling activation through exchanging intracellular L-glutamine to extracellular essential amino acid [54]. Thus, increase of intracellular L-glutamine level promotes exchanging L-glutamine to other amino acids, leading to the activation of mTORC1 and inhibition of the autophagic activity.

Leucine, a branched-chain amino acid, is catabolized by methylcrotonoyl-CoA carboxylase 1 (MCCC1) to acetyl-CoA. Inhibition of MCCC1 suppresses mTORC1 activity and activates autophagy through reduction of acetyl-CoA levels via acetylation of RAPTOR by an acetyltransferase EP300 [54].

An increasing number of studies show that mTOR plays crucial roles in osteoblasts and chondrocytes [55–58]. During endochondral ossification, mTORC1 signaling is detectable in pre-hypertrophic/hypertrophic chondrocytes, pre-osteoblasts, and osteoblasts in the primary spongiosa [59]. Mice with a conditional deletion of Mtor in skeletal mesenchymal cells (Prx1-Cre;Mtor^F/F) show diminished mTORC1 activity and die at birth with short limbs and exencephaly due to ossification defects in the long bones, calvaria, and sternum [59]. Mice with a pre-osteoblast specific deletion of Mtor (Osx1-Cre;Mtor^F/F) show defects in osteoblast differentiation through suppression of RUNX2 expression. Finally, mice deficient for Mtor in Osterix-positive osteoblasts exhibit low trabecular bone mass with ossification defects that resemble cleidocranial dysplasia in humans [60]. Regulatory-associated protein of mTOR (RAPTOR), a component of mTORC1, binds to mTOR [61], and mice with deficient Raptor in skeletal mesenchymal cells (Prx1-Cre;Raptor^F/F) exhibit bone phenotypes similar to Prx1-Cre;Mtor^F/F mice [59]. In addition, Osx1-Cre;Raptor^F/F mice exhibit a bone phenotype similar to Osx1-Cre;Mtor^F/F mice, with suppressed differentiation in pre-osteoblasts and chondrocytes in the growth plate [56,60,62]. Interestingly, in Raptor mutant osteoblasts the mTORC1 pathway is suppressed without affecting autophagic activity, while the mTORC2 pathway is activated [56]. The suppression of mTORC1 in osteoblasts also affects B-lymphopoiesis by reduced expression of Cxcl12 and Il7, which are key cytokines for B-lymphocyte differentiation [63]. Mice with a deletion of Raptor in osteocytes (Dmp1-Cre;Raptor^F/F) show normal bone development but slightly inhibited bone resorption, resulting in an increase of trabecular bone mass [64]. In addition, mice with a mesenchymal cell-specific deletion of Rictor (Prx1-Cre;Rictor^F/F) exhibit short and thinner long bones due to a delay in chondrocyte differentiation in the growth plate, as well as calvarial defects due to suppression of osteoblastic differentiation [65]. Thus, mTOR signaling is indispensable for chondrocyte and osteoblast differentiation.

Mice with an osteoclast-specific deletion of Raptor (Lyz2-Cre;Raptor^F/F) exhibit osteopenia due to accelerated osteoclastogenesis through the suppression of mTORC1 signaling [66,67]. By contrast, Ctsk-Cre;Raptor^F/F mice present increased bone mass due to suppression of osteoclastogenesis [68]. Therefore, the osteoclast phenotype in these mice is controversial for unknown reasons, and Raptor may have different functions at each specific stage of osteoclast differentiation. On the other hand, mice with a conditional deletion of mTORC1 inhibitor Tsc in osteoclasts (Lyz2-Cre;Tsc1^F/F mice and Ctsk-Cre;Tsc1^F/F mice) show increased bone mass and reduced osteoclast activity [66,69]; bone marrow mesenchymal cells isolated from Tsc1 mutant mice show suppression of osteoclastogenesis [67]. On the other hand, mice with a deletion of Tsc2, which forms a heterodimer with TSC1 to regulate the mTORC1 pathway, in osteoblasts (OC-Cre;Tsc2^F/F) exhibit an increase of cortical and trabecular bone thickness and bone mass. Because TSC2 regulates the insulin signaling pathway via AKT (a.k.a. protein kinase B), a loss of Tsc2 in osteoblasts lead to insulin resistance, resulting in the suppression of osteoblast proliferation and differentiation and disorganized bone formation. The normalization of mTORC1 signaling by haploinsufficiency of Mtor in Tsc mutant mice (OC-Cre;Tsc2^F/F;Mtor^F/+ mice) can partially rescue the bone phenotype [58]. These genetic mouse models for autophagy and their bone phenotypes are summarized in Table 1.

Table 1.

Skeletal defects associated with deficiencies in autophagy

Gene	Targeted cell type	Genotype	Phenotype	References
Atg5	Osteoblast	Col1a1-Cre;Atg5 F/F	Reduced bone formation and mineralization	[16]
Atg7	Osteoblast	Osx1-Cre;Atg7 F/F	Low bone mass and spontaneous fractures Suppressed bone remodeling Disrupted osteocyte survival and maturation	[17]
Slc7a5	Osteoblasts	Osx-Cre;Slc7a5 F/F	No bone phenotype	[35]
Atg7	Osteocytes	Dmp1-Cre;Atg7 F/F	Low bone mass and reduced bone remodeling Increased oxidative stress	[18]
Atg7	Skeletal mesenchymal cells	Prx1-Cre;Atg7 F/F	Accumulated type II procollagen in chondrocytes ER stress	[19, 20]
Atg7	Chondrocytes	Tam Col2a1-Cre;Atg7 F/F	Accumulated type II procollagen in chondrocytes ER stress	[19, 20]
		Col2a1-Cre;Atg7 F/F	Increased apoptotic cell death in the growth plate	[19, 20]
		Col2a1-Cre;Atg5 F/F	Increased apoptotic cell death in the growth plate	[20, 21]
		Col2a1-Cre;Atg7 F/F	Increased apoptotic cell death in the growth plate	[20, 21]
		Col11a2-Cre;Atg7 F/F	Severe growth retardation	[20, 21]
Becn	Osteoclasts	CstK-Cre;Becn1 F/F	Impaired osteoclast function and bone resorption Dysregulated chondrogenesis	[23]
Atg5	Osteoclasts	Lyz2-Cre;Atg5 F/F	Impaired bone resorption Reduced lysosome secretion from osteoclasts Increased trabecular bone volume of long bones	[25]
Slc7a5	Osteoclasts	Tnfrsf11a-Cre;Slc7a5 F/F	Low bone mass and increased bone resorption Accelerated osteoclastogenesis Suppression of mTORC1 pathway	[35]
Slc7a5	Osteoclasts	Lyz2-Cre;Slc7a5 F/F	Low bone mass and increased bone resorption Accelerated osteoclastogenesis Suppression of mTORC1 pathway	[35]
Raptor	Osteoclasts	Lyz2-Cre;Raptor F/F	Osteopenia Accelerated osteoclastogenesis Suppressed mTORC1 signaling	[37]
Raptor	Osteoclasts	Ctsk-Cre;Raptor F/F	Increased bone mass Suppressed osteoclastogenesis	[38]
Tsc1	Osteoclasts	Lyz2-Cre;Tsc1 F/F	Increased bone mass and reduced osteoclast activity	[37]
Tsc1	Osteoclasts	Ctsk-Cre;Tsc1 F/F	Increased bone mess and reduced osteoclast activity	[39]
P62	all cell types	P62 −/−	Accelerated osteoclastogenesis	[30]
P62	all cell types	P62 P394L/P394L	Accelerated osteoclastogenesis	[31]

3. Role of Amino Acids in Bone Development and Homeostasis

There are 20 proteogenic amino acids and numerous non-proteogenic amino acids in humans. The specific combination of amino acid determines the characteristics and functions of proteins in eukaryotes and microorganisms. Nine of the amino acids (phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine) are categorized as essential amino acids and cannot be synthesized in the human body. For those amino acids that can be produced by cells, their precursors are intermediates or metabolites generated during glycolysis and the TCA cycle. As shown in Figure 1, phenylalanine, tyrosine, and tryptophan arise from chorismate, which derives from phosphoenolpyruvate, a glycolysis metabolite. Isoleucine, valine, leucine, and alanine are converted from pyruvate, whereas methionine, threonine, lysine, and asparagine are synthesized from oxaloacetate. Glutamine, proline, and arginine arise from α-ketoglutarate, which is an intermediate of the TCA cycle. Glucose 6-phosphate is converted to ribose-5-phosphate, and then eventually to histidine, whereas cysteine, glycine, and serine arise from 3-phosphoglycerate, an intermediate of glycolysis. Beyond the abovementioned metabolic pathways, metabolites generated during degradation of amino acids give rise to precursors for other biosynthesis pathways or bioactive molecules. From the perspective of the degradation pathway, amino acids are also classified, based on their catabolites, as glycogenic or ketogenic: 14 glycogenic amino acids are catabolized to either pyruvate or TCA cycle metabolites, two ketogenic amino acids are catabolized to either acetyl-CoA or acetoacetyl-CoA, and five amino acids are both glycogenic and ketogenic (Figure 1). The metabolic reactions can be converted bidirectionally. For example, around 40% of glutamate in bone marrow stromal cells are derived from glutamine [70]. Human bone diseases associated with a failure in amino acid metabolism are summarized in Table 2 (see sections below for details). Mice with deficient amino acid metabolism and their bone phenotypes are summarized in Table 3.

Figure 1.

Amino acid metabolic pathway. This figure was drawn based on ARGININE BIOSYNTHESIS (mmu00220), ALANINE, ASPARATE AND GLUTAMATE METABOLISM (mmu00250), GLYCINE, SERINE AND THREONINE METABOLISM (mmu00260), CYSTEINE AND METHIONINE METABOLISM (mmu00270), VALINE, LEUCINE AND ISOLEUCINE DEGRADATION (mmu00280), LYSINE DEGRADATION (mmu00310), ARGININE BIOSYNTHESIS (mmu00220), ARGININE AND PROLINE METABOLISM (mmu00330), HISTIDINE METABOLISM (mmu00340), TYROSINE METABOLISM (mmu0350), PHENYLALANINE METABOLISM (mmu00360), and TRYPTOPHAN METABOLISM (mmu00380) pathway maps obtained from the KEGG website.

Table 2.

Human bone diseases associated with a failure in amino acid metabolism

Amino acid name	Genes	Disease name	Bone defects	References
Alanine	GPT	Nonalcoholic fatty liver disease	Lower bone mineral density	[40]
Proline	PYCR2		Microcephaly, facial dysmorphism, and developmental delay	[42]
Asparagine	ASNS		Epilepsy, developmental delay, and progressive microcephaly	[46]
Phenylalanine	PAH	Phenylketonuria	Osteopenia	[85–87]
Tyrosine		Dopamine-related diseases (e.g., Parkinson’s disease, Schizophrenia)	Osteoporosis	[93–95]

Table 3.

Mouse bone phenotypes associated with amino acid metabolic defects

Amino acid	Targeted genes	Bone defects	References
Alanine	Gpt −/−	Increased bone mineral contents	IMPC
Proline	Pycr2 −/−	Low bone mineral density	IMPC
Cysteine	Cod1 −/−	Short snout, facial asymmetry, irregular calvaria shape, and kyphosis	[48]
Histidine	Hdc −/−	Increased bone mineral contents and increased cortical bone thickness Accelerated bone formation and suppressed osteoclastogenesis	[71]
Methionine	Cbs −/−	Pointed snout, osteoporosis, delayed endochondral ossification, and small body	[76–79]
Phenylalanine	Pah enu2/enu2	Low mineral density, low trabecular volume, imbalance of bone homeostasis Suppressed osteogenic differentiation	[88]
Tyrosine	Slc6a3 −/−	Reduced bone mass and strength	[99]
Tyrosine	Slc6a4 −/−	Reduced bone mass and strength	[100]
Tryptophan	Ido −/−	Osteopenia	[112]

3.1. Alanine (L-alanine)

The (inter)conversion between pyruvate and alanine is catalyzed by glutamate dehydrogenase and alanine aminotransferase 1 (ALT1, a.k.a. glutamic-pyruvic transaminase), which are encoded by the GPT gene. ALT1 acts on the reversible transamination between alanine/α-ketoglutarate and pyruvate/glutamate. Gpt^−/− female mice, but not male mice, exhibit increased bone mineral content [reported by the International Mouse Phenotyping Consortium (IMPC), accessible at https://www.mousephenotype.org]. In addition, patients with nonalcoholic fatty liver disease and increased serum levels of ALT1 show lower bone mineral density compared with healthy individuals or patients with normal ALT1 levels [71].

In addition, alanine-glyoxylate aminotransferase 2 (AGXT2) catalyzes the conversion between alanine/glyoxylate and pyruvate/glycine. Mice with a deficiency for the Agxt2 gene (Agxt2^−/− mice) exhibit increased vasodilation in skeletal and cardiac muscles [72].

3.2. Arginine (L-arginine) and Proline

In the TCA cycle, alpha-ketoglutarate is converted into glutamic acid by glutamate dehydrogenase. Glutamic acid is further converted to either glutamine by glutamine synthase, to proline by pyrroline 5-carboxylate reductase 1 and 2 (PYCR1 and 2), or to citrulline (via ornithine), and then arginine, in the urea cycle, with formation of urea from toxic catabolite ammonia in the liver. Thus, arginine, ornithine, and citrulline are all substrates of the urea cycle.

Proline is catabolized into ornithine by proline dehydrogenase 1 (PRODH) and ornithine aminotransferase (OAT), or into glutamic acid by delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDH, a.k.a. ALDH4A); ornithine is further converted into glutamic acid by OAT.

Arginine enhances osteogenesis in human mesenchymal stem cells through upregulation of expression of osteogenic transcription factors: RUNX2, DLX5, and OSX (Osterix) [73]. Patients with autosomal recessive mutations in the PYCR2 gene display microcephaly, seizures, facial dysmorphism, developmental delay, and cerebral atrophy [74], and Pycr2^−/− mice exhibit low bone mineral density and decreased grip strength (reported by the IMPC). The catabolism of arginine involves its conversion to glutamic acid by OAT or cycling through the urea cycle. Mutations in OAT cause gyrate atrophy, characterized by progressive retinal atrophy and cataracts, which may cause blindness [75,76].

3.3. Asparagine and Aspartic Acid and Lysine

Oxaloacetate, an intermediate in the TCA cycle, is converted into aspartic acid by transaminase, then to asparagine, a substrate of the TCA cycle, by asparagine synthetase (ASNS). Alternatively, aspartic acid can be converted into methionine by methionine synthase via homocysteine, with co-factor cobalamin (vitamin B12), into threonine by threonine synthase, and into lysine by diaminopimelate decarboxylase. Patients with ASNS mutations display epilepsy, developmental delay, progressive microcephaly, and reduced brain volume [77]. Alpha aminoadipic semialdehyde synthase (AASS) converts L-lysine to saccharopine in the mitochondria. The final metabolite of lysine is acetyl-CoA, which enters the TCA cycle. A known mutation in AASS causes hyperlysinemia, which typically results in no health issues [78]. Patients with lysinuric protein intolerance (LPI), an autosomal recessive disorder of cationic amino acid (arginine, lysine, and ornithine) transport caused by mutations in the SLC7A7 (solute carrier family 7 member 7) gene present with low bone mass, delayed bone development and osteoporosis, muscle weakness, pulmonary alveolar proteinosis, and kidney dysfunction [79–81]. Slc7a7 null mice exhibit bone mineralization defects and delayed development in the kidneys, lungs and liver, as seen in LPI patients [82].

3.4. Cysteine

Cysteine is catabolized to taurine and pyruvate, which are in turn catalyzed by cysteine dioxygenase (CDO), aspartate aminotransferase, cysteine sulfonic acid decarboxylase (CSAD), 3-mercaptopyruvate sulfurtransferase (MPST), and hypotaurine dehydrogenase.Cdo1^−/− mice exhibit short snout, facial asymmetry, irregular calvaria shape, kyphosis, and hyperlaxity of the paws with low levels of taurine and excessive cysteine sulfate in adults [83]. Mpst^−/− mice show anxiety-like behavior without any morphological abnormality in the brain [84], but no bone-related defects. Studies with animal models also suggest that taurine supplementation can improve bone formation [85–89]. Moreover, exogenous taurine accelerates osteogenic differentiation in human mesenchymal stem cells [90]. Taken together, taurine and taurine synthesis seem to be related to bone development and homeostasis. N-acetyl-L-cysteine (NAC) is an amino acid derivative that plays a role in the cellular antioxidant capabilities [91]. NAC, which is incorporated into cells through cysteine transporter SLC3A1 or the NAC carrier AE1 (anion exchange protein 1), is deacetylated into L-cysteine, which is transformed into glutathione, an antioxidative molecule inhibiting intracellular ROS activity, by a glutamate cysteine ligase and glutathione synthetase in the cytosol [92,93]. Pre-treatment with NAC renders the cells resistant to oxidative stress in cell viability, cell proliferation, and osteogenic differentiation in rat femur bone marrow-derived osteoblast-like cells [94]. Moreover, in vivo transplantation of the pre-treated bone marrow-derived osteoblast-like cells with NAC to bone defects in rat long bone accelerates bone regeneration through suppression of ROS [94,95]. On the other hand, a treatment with NAC in bone marrow-derived monocytes suppresses osteoclastogenesis [96].

The members of the Forkhead box class O (FoxO) family of transcription factors play roles in various cellular functions (e.g. cell proliferation, differentiation, apoptosis, resistance for oxidative stress) in a variety of cells and tissues. FoxO1 promotes osteogenic differentiation of mesenchymal stem cells through RUNX expression. Therefore, mice with a deficiency for FoxO1 in Osteocalcin (a.k.a. BGLAP)-expressing osteoblasts (Bglap-Cre;FoxO1^F/F) exhibit low bone mass due to differentiation defects and increased apoptosis in osteoblasts through increased intracellular ROS [98]. As expected, treatment with NAC normalizes intracellular ROS levels and osteogenic differentiation in Bglap-Cre;FoxO1^F/F mice [98]. By contrast, overexpression of Foxo1 suppresses pre-osteoblast proliferation, suggesting that FoxO1 acts bi-directionally in osteogenesis [97].

3.5. Glutamine and L-Glutamic acid (Glutamate)

In the TCA cycle, alpha-ketoglutarate is converted into glutamic acid by glutamate dehydrogenase [(GLUD1 and GLUD2 (only in humans)]. Glutamic acid is further converted into glutamine by glutamine synthase.

Glutamic acid is known a neurotransmitter in the central nervous system and a precursor of gamma-aminobutyric acid (GABA); its conversion to GABA is catalyzed by glutamate decarboxylase 1 (GAD1; a.k.a. GAD67) with co-factor vitamin B6.Glutamic acid receptors are expressed in osteoblasts and osteoclasts [99–102], and intracellular glutamic acid metabolism plays crucial roles in various aspects of metabolic homeostasis in bone [103]. For instance, the inhibition of glutamate transporter as well as the blockage of the receptor increases the number of bone resorption pits, while glutamic acid has no effect on survival or activity of mature osteoclasts [103]. In addition, glutamic acid can significantly increase osteoblast differentiation and mineralization, while inhibition of the transport shows no effect on osteoblasts [103]. Blockage of the receptors inhibits osteoblast maturation and the mineralization activity, while stimulation of the receptors accelerates bone mineralization in vitro and in vivo [101]. These evidences suggest that glutamic acid may be more important in osteoclasts rather than in osteoblasts. Moreover, patients with autosomal recessive GAD1 mutations exhibit neuronal developmental defects such as early-onset epilepsy and intellectual disability, cleft palate, and scoliosis (a sideways curvature or twist of the spine) [104]. While scoliosis can be caused by conditions such as cerebral palsy and muscular dystrophy, a failure of spine formation and osteoporosis with fractures in the spine can cause scoliosis. The cause of most scoliosis remains unclear. Gad1 null mice exhibit cleft palate and immature lung and die at birth likely due to a respiratory failure [105,106].

Glutamine metabolism also plays roles in several cellular processes in bone cells. For instance, WNT7B-mediated β-catenin independent WNT signaling activates osteoblast function through activation of mTORC1 signaling [107]. The accelerated bone formation in mice with constitutively activated Wnt7b in osteoblasts (Osx-Cre;R26R-Wnt7b) is restored by deletion of Raptor (Osx-Cre;R26R-Wnt7b-Raptor^F/F) [107]. The glutamine-dependent anaplerosis is catalyzed by glutaminase (GLS; kidney type) and glutaminase 2 (GLS2; liver type) in the mitochondria, converting glutamine into glutamate. Interestingly, WNT3A-mediated β-catenin-independent, but mTORC1-dependent, signaling accelerates osteoblast differentiation through increased GLS-dependent glutamine catabolism in cultured osteoblast progenitors [108]. Mice with a deficiency for Gls in skeletal mesenchymal cells (Prx1-Cre;Gls^F/F) exhibit a decrease in the osteoblast number and bone formation rate without affecting osteoclast number and bone resorption, resulting in reduction of bone mass in long bones [70]. In addition, deletion of Gls in skeletal stem cells (Lepr-Cre;Gls^F/F) show reduced bone mass similar to Prx1-Cre;Gls^F/F mice; however, mice with an osteoblast-specific deletion of Gls (Bglap-Cre;Gls^F/F) show normal bone formation and resorption [70]. These results suggest that GLS is required for the fate determination of skeletal stem cells to osteoblasts, but not for cellular functions in osteoblasts. Osteoclastogenesis of bone marrow macrophages is accelerated by glutamine in a dose-dependent manner [109]. Mice with chondrocyte-specific deletion of Gls (Col2a1-Cre;Gls^F/F) exhibit a growth arrest in the growth plate after birth [110]. These chondrocyte’s phenotypes are restored by supplementation of α-ketoglutarate, which is a metabolite in the TCA cycle [110]. On the other hand, the enzymatic activity of glutamine synthetase (GS) is suppressed by canonical WNT/β-catenin signaling induced by either WNT3A or lithium chloride (LiCl) in MG-63 cells, a human osteoblast like-osteosarcoma cell line, and rat bone marrow mesenchymal stem cells [111].

In addition, glutamine influences bone healing and regeneration. An intravenous injection of glutamine/alanine accelerates formation of fibrocartilaginous callus and hyalin cartilage in fractured bone in rats [112]. Aging of mesenchymal stem cells (MSCs) leads to decrease in the osteogenic differentiation capacity through suppression of estrogen-related receptor alpha (ERRα) and glutaminases (GLS), resulting in age-associated osteoporosis. In the mitochondria, glutamine is catabolized to glutamic acid by GLS in the anaplerotic pathway. While it has been known that ERRα, an orphan nuclear receptor, regulates target genes, a recent study suggests that ERRα directly regulates Gls expression via mTROC1 pathway [113]. Interestingly, overexpression of Erra and Gls in aged MSCs can restore decreased osteogenic differentiation and glutamine consumption, indicating that glutamine anaplerosis in the mitochondria is crucial for osteogenic differentiation in MSCs [113].

3.6. Glycine

The conversion reactions between glycine and serine are reversibly catalyzed by serine hydroxymethyltransferase 1 and 2 (SHMT1 and SHMT2), with co-factor vitamin B6. In the liver, glycine is degraded to CO₂ and NH₃ with co-conversion of tetrahydrofolate to 5,10-) methylenetetrahydrofolate by the glycine cleavage system. While Shmt1 or Shmt2 null mice have been generated, detailed bone analyses have not been performed yet.

3.7. Histidine

Histidine is converted to histamine, a biological amine that regulates immune response and neurotransmission, by histidine decarboxylase (HDC), with co-factor vitamin B6. Hdc^−/− mice exhibit abnormal neurological responses, a suppressed immune system, increased bone mineral contents, and increased cortical bone thickness due to accelerated bone formation and suppressed osteoclastogenesis [114]. The upregulated expression of HDC in monocytes from patients with osteoporosis is negatively correlated with bone mineral density [115], suggesting that histamine may activate osteoclasts for bone resorption. T-cell ubiquitin ligand-2 (TULA-2), a histidine phosphatase that is expressed in pre- and mature-osteoclasts, suppresses osteoclast differentiation [116]. Mice with a double knockout of the Tula and Tula2 genes exhibit low bone mass due to an increase of the osteoclast number and activity [116].

3.8. Isoleucine, Leucine, and Valine

The degradation of isoleucine, leucine, and valine is catabolized by branched-chain amino acid transaminase 1 and 2 [BCAT1 (cytosolic) and BCAT2 (mitochondrial)] and branched-chain α-keto acid dehydrogenase [BCKDH; homodimer of alpha and beta (BCKDHA and BCKDHB)]. Leucine and isoleucine are eventually converted to acetyl-CoA, while isoleucine and valine are converted to succinyl-CoA; both acetyl-CoA and succinyl-CoA are transported into the TCA cycle. Mutations in either BCAT1 or BCAT2 cause hypervalinemia and hyperleucin-isoleucinemia. Although Bcat1^−/− mice have been generated, their phenotype still needs to be analyzed. However, it is known that mice with a point mutation in Bcat2 or with a deficiency of Bcat2 (Bcat2^−/−mice) exhibit decreased body weight [117,118]. In humans, mutations in either BCKDHA or BCKDHB cause maple syrup urine disease, which is characterized by neurodegeneration but no bone defect [119].Bckdha^−/− and Bckdhb^−/−null mice have been generated, but no phenotypic data have been reported. A recent study shows that MC3T3-E1 cells, an osteoblast cell line, generate adenosine triphosphate (ATP) through glycolysis and dependent upon branched chain amino acids [120]. Exogenous supplementation of leucine suppresses cell proliferation in MC3T3-E1 pre-osteoblast cells through cell senescence and DNA damage [121].

3.9. Methionine

Methionine is catabolized to S-adenosylmethionine (SAM) by methionine adenosyltransferase, and then to homocysteine; SAM acts as a substrate for methylation of multiple macromolecules such as DNA, RNA, and proteins. Homocysteine is then converted into cysteine, together with serine and with vitamin B6 as co-factor, by cystathionine beta-synthase (CBS) and cystathionin-γ-lyase (CTH, a.k.a. CSE) through reverse-transulfurylation. Cbs null mice exhibit pointed snout, osteoporosis, delayed endochondral ossification, hepatic steatosis, microphthalmia, small body, cardiovascular defects, and hyperhomocysteinemia/homocystinuria, which are also seen in patients with classical homocystinuria [122–125]. Cth null mice exhibit myofiber atrophy when cysteine is not supplemented from diets [126], and autosomal recessive CTH mutations cause primary cystathioninuria in humans [127]. Methionine-restricted diets increase longevity and lifespan by improving metabolism with reduced body size in mice and rats. Mice fed with methionine-restricted diets show weaker bones, lower bone density, and thinner trabecular bones compared to a normal diets group due to suppression of osteoblast differentiation, by suppressing Runx2, Opg and Dmp1, without affecting the number of osteoblasts and osteoclasts [128,129]. Interestingly, several microRNAs (small non-coding RNAs containing ~22 nucleotides), which alter Runx2 expression, are elevated in osteoblasts from mice fed with methionine-restricted diets [129].

Homocysteine can also be transformed back to methionine by methionine synthase (MTR; 5-methyltetrahydrofolate-homocysteine methyltransferase), with vitamin B12 as co-factor. In this conversion, 5’-methyltetrahydrofolate is transformed into tetrahydrofolate, a component of folic acid metabolism. Single-nucleotide polymorphisms (SNPs) in the MTR gene are associated with non-syndromic cleft lip with/without cleft palate [130].

3.10. Phenylalanine and Tyrosine

L-phenylalanine is converted into L-tyrosine by phenylalanine hydroxylase (PAH), or into phenethylamine, a neuromodulator, by DOPA decarboxylase (DDC). L-tyrosine is then converted by DDC into p-tyramine, also a neuromodulator, by tyrosine hydroxylase (TH) into L-DOPA, or by tyrosine aminotransferase (TAT) and fumarylacetoacetate hydrolase (FAH) into fumarate. L-DOPA can be further converted into dopamine by DDC. In the end, dopamine can be transformed into several catecholamines that act as neurotransmitters in the central and peripheral nervous systems.

Mice with a point mutation in Pah (phenylalanine hydroxylase) exhibit phenylketonuria, microcephaly, and impaired learning and motor coordination that recapitulates phenylketonuria in humans [131]. Osteopenia, risk of fracture, and low bone mineral density have also been reported in some patients with phenylketonuria [132–134]. Pah mutant (ENU-induced point mutation) mice show low mineral density, low trabecular volume, an imbalance in bone homeostasis through upregulation of parathyroid hormone PTH, and suppression of osteogenic differentiation [135].

DDC is a multifunctional enzyme that acts with co-factor vitamin B6, converting L-DOPA to dopamine, L-phenylalanine to phenethylamine, L-tyrosine to tyramine, L-histidine to histamine, L-tryptophan to tryptamine, and 5-hydroxytryptophan (5-HTP) to serotonin. All of its metabolites act as neuromodulators or neurotransmitters.

The majority of mice deficient for tyrosine hydroxylase (Th^−/− mice) die at the embryonic stage, while some that survive exhibit impaired locomotor activity and growth retardation [136,137]. Mice with a knock-in mutation in the human gene (Th^R233H/R233H), which results in its deficiency, survive but exhibit hypotension, hypokinesia, and impaired motor coordination, similar to what is observed in human patients [138].

Dopamine-related diseases (e.g., Parkinson’s disease, schizophrenia) pose a risk of osteoporosis [139–141], and previous studies show that dopamine can induce osteogenic differentiation via dopamine receptors [142–144]. Moreover, mice with a deficiency of either dopamine transporter (Slc6a3^−/− mice) or serotonin transporter (Slc6a4^−/−mice) show reduced bone mass and strength [145,146]. The skeletal phenotype has not yet been studied in Ddc or Th mutant mice.

Mutations in TAT cause tyrosinemia type II (a.k.a. Richner-Hanhart syndrome), which is characterized by photophobia, neurological dysfunction, and hyperkeratosis [147,148]. Tat^−/− mice have been generated and still need to be analyzed. Finally, mutations in FAH cause tyrosinemia type I, with symptoms ranging from neonatal death to survival beyond 20 years old. Milder cases show hepatocellular carcinoma, rickets, and renal failure [149,150], which are also seen in Fah mutant mice [151,152].

3.11. Serine

Metabolite 3-phosphoglycerate is converted into 3-phosphoserine by 3-phospho-hydroxypyruvate-glutamate transaminase (PSAT), with co-conversion of glutamate to α-ketoglutarate, and then to L-serine by phosphoserine-phosphatase (PSPH). L-serine is further converted into glycine by serine hydroxymethyltransferase 1 and 2 (SHMT1 and SHMT2) with co-factor vitamin B6, or into cysteine by cystathionine beta-synthase (CBS) and cystathionin-γ-lyase (CTH), also with co-factor vitamin B6, which is called as transsulfuration pathway. During the conversion of serine to glycine, tetrahydrofolate is transformed to 5,10-methylenetetrahydrofolate as part of the folic acid metabolism. Degradation of serine is a reverse reaction to glycine or conversion to pyruvate by serine dehydratase (SDS, a.k.a. SDH), with co-factor vitamin B6.

3.12. Threonine

Threonine is converted into pyruvate by threonine dehydrogenase (TDH) or acetyl-CoA and glycine by thiolysis. In humans, TDH is inactive. Instead, threonine is converted into α-ketobutylate by serine dehydratase (SDS), with co-factor vitamin B6. Both Tdh and Sds mutant mice have been generated but not analyzed yet (reported by the IMPC).

3.13. Tryptophan

Tryptophan is a precursor of bioactive molecules, particularly those that act in the brain: 1) serotonin (5-HT), which derives from 5-HTP; 2) melatonin, a derivative of 5-HT; and 3) niacin, which results from quinolinic acid (QA), kynurenic acid (KYNA), and picolinic acid [153–155]. Tryptophan and its metabolites have been suggested to be associated with osteoporosis [156,157]. Mice with suppressed degradation of tryptophan (Ido^−/− mice) exhibit osteopenia with an imbalance in the number of osteoblasts and osteoclasts [158]. Exogenous picolinic acid induces osteoblastogenesis in human mesenchymal stem cells [158]; however, excessive kynurenine induces osteoclastogenesis and bone loss in vivo [159].

Serotonin is produced at either the central nervous system or peripheral organs independently. Brain-derived neurotransmitter serotonin accelerates bone formation through inhibition of bone resorption; however, gastrointestinal-derived hormonal serotonin inhibits bone formation through suppression of osteoblast proliferation [160,161]. A human cohort study showed that long-term daily niacin intake is negatively associated with hip bone mineral density and risk of hip fracture [162]. The intestinal enterobacteria catabolize tryptophan to indole, 3-indolepropionic acid (IPA), or indole-3-carboxaldehyde (I3A), and these molecules are transfused into or taken up by the intestinal epithelium to enter the bloodstream and then transfer to the brain and liver.

3.14. Amino acid transporter and receptors

Amino acid-mediated signaling is induced by transporters and receptors. In bones, expression of amino acid transporters and receptors varies by cell type and developmental stage. For instance, glutamine transporters GLT-1 (a.k.a. EAAT2) and EAAT4 are expressed in differentiated mouse osteoclasts. By contrast, the cysteine-glutamate antiporter SLC7A11 is expressed in pre-osteoclasts although it disappears in mature osteoclasts [163]. Interestingly, exogenous supplementation of glutamate in RAW264.7 cells, an osteoclast cell line, suppresses osteoclastogenesis [163]. Inhibition of SLC7A11 by sulfasalazine enhances osteogenic differentiation in human bone marrow MSCs and suppresses bone loss in ovariectomized mice [164]. In mouse osteoblasts, glutamate transporters (SLC1A1 and SLC1A3) and glutamate receptor (GLUR2/−3/−4) are expressed [165]. Inhibition of glutamate transporters accelerates osteoblast differentiation with increased extracellular glutamate level. Importantly, a treatment with either glutamine or a glutamate receptor agonist accelerates osteoblast differentiation [165]. The glutamate-aspartate transporter GLAST1 (a.k.a. EAAT1) is expressed in osteocytes and osteoblasts in an extracellular glutamate’s dose-dependent manner [166,167]. However, Glast null mice show normal bone formation/resorption and tooth formation [168]. Mice with an osteoclast-specific deletion of the L-type amino acid transporter 1 gene (Tnfrsf11a-Cre;Slc7a5^F/F and Lyz2-Cre;Slc7a5^F/F mice) exhibit low bone mass and increased bone resorption due to accelerated osteoclastogenesis through suppression of the mTORC1 pathway [169]. By contrast, mice with osteoblast-specific deletion of Slc7a5 (Osx-Cre;Slc7a5^F/F mice) show normal bone formation and resorption [169].

N-methyl-D-aspartate (NMDA) receptor, a glutamate receptor, is expressed in both neuronal and non-neuronal tissues including bones and cartilages. NMDA-mediated glutamatergic signaling plays crucial roles in the differentiation of chondroblasts, osteoblasts, and osteoclasts [170,171]. Eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4), a member of EIF2α (eukaryotic initiation factor 2 alpha), responds to amino acid deprivation and acts as an amino acid sensor. Under ER stress, phosphorylated EIF2α activates ATF4 (activating transcription factor 4) that regulates target gene’s expression [172]. Elf2ak4 null (Elf2ak4^−/−) mice exhibit low bone mass, osteoblast proliferation defects, and reduction of bone turnover without any effects on osteogenic differentiation [173]. In addition, gene expression of amino acid transporters (such as Slc1a1, Slc1a5, Slc7a5, and Slc38a2) is suppressed in bone marrow MSCs from Elf2ak4^−/−mice, resulting in reduced cell proliferation [173]. ELF2α phosphorylated by either amino acid deprivation, oxidative stress, or ER stresses upregulates expression of ATF4 [174], a CREB family transcription factor that plays roles in osteoblast differentiation, bone matrix synthesis, and osteoclast differentiation. The EIF2α–ATF4 signaling pathway is also induced by parathyroid hormone (PTH) or bone morphogenetic protein 2 (BMP2), and stimulates osteogenic differentiation and proliferation in osteoblasts [175,176]. In addition, a variety of signaling pathways are involved in the regulation of ATF4 through Ras, which binds small molecules GTP and GDP interchangeably and can hydrolyze GTP to GDP. For instance, mice with a conditional deficiency for Neurofibromin 1 (NF1), a GTPase-activating protein known as a negative regulator of Ras signaling,in osteoblasts (Col1a(2.3kb)-Cre;Nf1^F/F mice; hereafter Nf1 cKO mice) exhibit increases of bone volume, bone formation, and bone resorption through upregulation of ATF4 phosphorylation and Osteocalcin expression. As a line of this evidence, mice with overexpression ofAtf4 under the type I collagen promotor driver control [Col1a1(2.3kb)-Atf4] display a bone phenotype similar to Nf1 cKO mice [177]. By contrast, Atf4^−/−mice display decreased bone volume, bone formation, and bone resorption [177]. Consistent with these evidences, low-protein diets in Nf1 cKO mice and a high-protein diet in Atf4^−/− mice can rescue the bone phenotypes [177].

4. Conclusion

An accumulating number of studies indicate that amino acid metabolism is crucial for bone development and homeostasis. Recent genetic studies highlight the link between bone diseases and metabolic disorders affected by abnormal amino acid metabolism. The molecular mechanisms and interactions between bone and non-bone cells in these networks remain to be determined. In this review, we focused on recent findings related to amino acid metabolism in bone homeostasis and diseases. As suggested by several studies, nutritional and pharmacological approaches targeting amino acid metabolism and related pathways may be suitable novel targets for the prevention and treatment of bone diseases.

Highlights.

• Recent genetic studies highlight the link between bone diseases and metabolic disorders affected by abnormal amino acid metabolism.

• Autophagy plays crucial roles in bone development and homeostasis

• Amino acid metabolism is crucial for bone development and homeostasis.

• As suggested by several studies, nutritional and pharmacological approaches targeting amino acid metabolism and related pathways may be suitable novel targets for the prevention and treatment of bone diseases.