Amino Acid Transport and Metabolism Regulate Early Embryo Development: Species Differences, Clinical Significance, and Evolutionary Implications

In this review we discuss the beneficial effects of amino acid transport and metabolism on pre- and peri-implantation embryo development, and we consider how disturbances in these processes lead to undesirable health outcomes in adults. Proline, glutamine, glycine, and methionine transport each foster cleavage-stage development, whereas leucine uptake by blastocysts via transport system B0,+ promotes the development of trophoblast motility and the penetration of the uterine epithelium in mammalian species exhibiting invasive implantation. (Amino acid transport systems and transporters, such as B0,+, are often oddly named. The reader is urged to focus on the transporters’ functions, not their names.) B0,+ also accumulates leucine and other amino acids in oocytes of species with noninvasive implantation, thus helping them to produce proteins to support later development. This difference in the timing of the expression of system B0,+ is termed heterochrony—a process employed in evolution. Disturbances in leucine uptake via system B0,+ in blastocysts appear to alter the subsequent development of embryos, fetuses, and placentae, with undesirable consequences for offspring. These consequences may include greater adiposity, cardiovascular dysfunction, hypertension, neural abnormalities, and altered bone growth in adults. Similarly, alterations in amino acid transport and metabolism in pluripotent cells in the blastocyst inner cell mass likely lead to epigenetic DNA and histone modifications that produce unwanted transgenerational health outcomes. Such outcomes might be avoided if we learn more about the mechanisms of these effects.

The leucine-NH4+ uptake regulator Any1 limits growth as part of a general amino acid control response to loss of La protein by fission yeast

The sla1+ gene of Schizosachharoymces pombe encodes La protein which promotes proper processing of precursor-tRNAs. Deletion of sla1 (sla1Δ) leads to disrupted tRNA processing and sensitivity to target of rapamycin (TOR) inhibition. Consistent with this, media containing NH4+ inhibits leucine uptake and growth of sla1Δ cells. Here, transcriptome analysis reveals that genes upregulated in sla1Δ cells exhibit highly significant overalp with general amino acid control (GAAC) genes in relevant transcriptomes from other studies. Growth in NH4+ media leads to additional induced genes that are part of a core environmental stress response (CESR). The sla1Δ GAAC response adds to evidence linking tRNA homeostasis and broad signaling in S. pombe. We provide evidence that deletion of the Rrp6 subunit of the nuclear exosome selectively dampens a subset of GAAC genes in sla1Δ cells suggesting that nuclear surveillance-mediated signaling occurs in S. pombe. To study the NH4+-effects, we isolated sla1Δ spontaneous revertants (SSR) of the slow growth phenotype and found that GAAC gene expression and rapamycin hypersensitivity were also reversed. Genome sequencing identified a F32V substitution in Any1, a known negative regulator of NH4+-sensitive leucine uptake linked to TOR. We show that 3H-leucine uptake by SSR-any1-F32V cells in NH4+-media is more robust than by sla1Δ cells. Moreover, F32V may alter any1+ function in sla1Δ vs. sla1+ cells in a distinctive way. Thus deletion of La, a tRNA processing factor leads to a GAAC response involving reprogramming of amino acid metabolism, and isolation of the any1-F32V rescuing mutant provides an additional specific link.

Acetylation of L-leucine switches its carrier from the L-amino acid transporter (LAT) to organic anion transporters (OAT)

ABSTRACTN-acetyl-DL-leucine is an analogue of the alpha amino acid leucine with a chiral stereocenter. The active L-enantiomer of the racemate is currently under development for rare neurological disorders. Here we present evidence that a selective recognition of N-acetyl-L-leucine versus L-leucine by different uptake transporters significantly contributes to the therapeutic effects of N-acetyl-L-leucine. A previous study of the pharmacokinetics of racemic N-acetyl-DL-leucine and N-acetyl-L-leucine revealed D-L enantiomer competition and saturation kinetics, best explained by carrier-mediated uptake. The strategy we used was to first analyze the physicochemical properties associated with good oral bioavailable drugs and how these are alerted by N-acetylation by comparing N-acetyl-L-leucine with L-leucine. Using in silico computational chemistry we found that N-acetylation has a profound impact on certain physicochemical properties that can rationalize why N-acetyl-L-leucine is drug-like compared to L-leucine. Our calculations show that at physiological pH, L-leucine is a zwitterion, whereas N-acetyl-L-leucine is present as mainly an anion. Specifically, N-acetylation removes a charge from the nitrogen at physiological pH and N-acetyl-L-leucine is an anion that is then a substrate for the organic anion transporters. We examined N-acetyl-L-leucine uptake in human embryonic kidney cells overexpression candidate organic anion transporters (OAT) and pharmacological inhibitors. We found that N-acetyl-L-leucine is a translocated substrate for OAT1 and OAT3 with low affinity (Km ~10 mM). In contrast, L-leucine is known to be transported by the L-type Amino Acid Transporter (LAT) with high affinity (Km ~0.2 mM) and low capacity. The clinical consequence is that L-leucine uptake becomes saturated at 50-fold lower concentration than N-acetyl-L-leucine. These results demonstrate a mechanism of action that explains why N-acetyl-L-leucine is effective as a drug and L-leucine itself is not.

Membrane localization of paralogous leucine permeases Bap2 and Bap3 is regulated by Bul1

Timeliness in expression and degradation of the nutrient permeases is crucial for any organism. In Saccharomyces cerevisiae while transcriptional regulation of permeases has been studied in great detail, post translational events such as trafficking and turnover are poorly understood. We found loss of a leucine permease BAP2, but not other permeases lead to severe growth retardation in presence of glucose or galactose but not in medium containing glycerol and lactate. Leucine prototrophy suppressed the growth retardation, showing BAP2 and LEU2 are synthetically lethal. We discovered that loss of BUL1, an arrestin involved in trafficking of diverse permeases suppressed this lethality. The suppression was dependent on another leucine permease, BAP3. Further experiments revealed that in bul1Δ cells, both BAP2 and BAP3 accumulated in plasma membrane and their turnover is reduced. Based on our results and what is known, we propose that BUL1 regulates TORC1 activity by controlling the leucine uptake.

Erythroid Cells Adapt to L-Leucine Scarcity By Reducing Hemoglobin Production Via the mTORC1/4E-BP Pathway

In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. Here, we find that maturing erythroid cells increase L-leucine uptake via transcriptional up-regulation of the L-leucine transporter, LAT3. Inadequate L-leucine uptake by L-leucine starvation or LAT3 inhibition triggers a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells via the mTORC1/4E-BP pathway. CRISPR-mediated deletion of 4E-BPs in murine erythroid cells renders them resistant to mTORC1 and LAT3 inhibition, markedly restoring hemoglobinization. Our complementary results demonstrate that globins are direct translational mTORC1 targets during normal development. This pathway is distinct from the previously reported translational regulatory mechanisms mediated by the heme-regulated inhibitor (HRI) kinase or by severe amino acid deprivation via the general control nonderepressible 2 (GCN2) kinase. We propose that, in red cells, mTORC1 serves as a homeostatic sensor coupling hemoglobin production to sufficient L-leucine uptake. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.