Provision Of Essential Amino Acids Research Articles

Enhanced gut microbiome supplementation of essential amino acids in Diploptera punctata fed low-protein plant-based diet.

Building on our previous work, we investigate how dietary shifts affect gut microbial essential amino acid (EAA) provisioning in the lactating cockroach Diploptera punctata. To that end, we fed cockroaches three distinct diets: a plant-only Gari diet composed of starchy and granulated root tuber Yucca (Manihot esculenta), a dog food diet (DF), and a cellulose-amended dog food (CADF) diet. We anticipated that the high carbohydrate, low protein Gari would highlight increased microbial EAA supplementation to the host. By day 28, we observed distinct profiles of 14 bacterial families in the insect gut microbiomes of the three dietary groups. CADF-fed insects predominantly harbored cellulolytic and nitrogen-fixing bacteria families Streptococcaceae and Xanthomonadaceae. In contrast, Gari-fed insects were enriched in anaerobic lignocellulolytic bacteria families Paludibacteraceae andDysgonomonadaceae, while DF-fed insects had a prevalence of proteolyticanaerobes Williamwhitmaniaceae and sulfate-reducing bacteria Desulfovibrionaceae. Furthermore, we confirmed significantly higher EAA supplementation in Gari-fed insects than in non-Gari-fed insects based on δ13C-EAA offsets between insect and their diets. The δ13C-EAA offsets between DF and CADF were nearly indistinguishable, highlighting the relevance of using the plant-based Gari in this experiment to unequivocally demonstrate this function in this insect. These results were underscored by lower standard metabolic rate (SMR) relative to the DF insect in Gari-fed (intermediate SMR and dietary quality) and CADF (least SMR and dietary quality) insects. The influence of diet on EAA provisioning and SMR responses in insects underscores the need for further exploration into the role of gut microbial functions in modulating metabolic responses.

Microbial gene expression during hibernation in arctic ground squirrels: greater differences across gut sections than in response to pre-hibernation dietary protein content.

Obligate seasonal hibernators fast for 5-9months depending on species yet resist muscle atrophy and emerge with little lean mass loss. The role of the gut microbiome in host nitrogen metabolism during hibernation is therefore of considerable interest, and recent studies support a role for urea nitrogen salvage (UNS) in host-protein conservation. We were interested in the effect of pre-hibernation diet on UNS and the microbial provision of essential amino acids (EAAs) during hibernation; therefore, we conducted a study whereby we fed arctic ground squirrels (Urocitellus parryii) pre-hibernation diets containing 9% vs. 18% protein and compared the expression of gut bacterial urease and amino acid (AA) metabolism genes in 4 gut sections (cecum mucosa, cecum lumen, small intestine [SI] mucosa, and SI lumen) during hibernation. We found that pre-hibernation dietary protein content did not affect expression of complete bacterial AA pathway genes during hibernation; however, several individual genes within EAA pathways were differentially expressed in squirrels fed 18% pre-hibernation dietary protein. Expression of genes associated with AA pathways was highest in the SI and lowest in the cecum mucosa. Additionally, the SI was the dominant expression site of AA and urease genes and was distinct from other sections in its overall microbial functional and taxonomic composition. Urease expression in the gut microbiome of hibernating squirrels significantly differed by gut section, but not by pre-hibernation dietary protein content. We identified two individual genes that are part of the urea cycle and involved in arginine biosynthesis, which were significantly more highly expressed in the cecum lumen and SI mucosa of squirrels fed a pre-hibernation diet containing 18% protein. Six bacterial genera were responsible for 99% of urease gene expression: Cupriavidus, Burkholderia, Laribacter, Bradhyrizobium, Helicobacter, and Yersinia. Although we did not find a strong effect of pre-hibernation dietary protein content on urease or AA metabolism gene expression during hibernation, our data do suggest the potential for pre-hibernation diet to modulate gut microbiota function during hibernation, and further investigations are warranted.

Role of the gut microbiome in mediating standard metabolic rate after dietary shifts in the viviparous cockroach, Diploptera punctata.

Diet may be a significant determinant of insect gut microbiome composition. However, the extent to which dietary shifts shape both the composition and relevant functions of insect gut microbiomes, and ultimately impact host energy balance (i.e. metabolic phenotype), is not well understood. We investigated the impacts of diet switching on Diploptera punctata females maintained on a dog food (DF) diet relative to those fed a comparatively sub-optimal cellulose-amended dog food (CADF) diet for 4weeks. After this period, dietary shift resulted in a significantly higher average mass-specific standard metabolic rate (SMR) in CADF-fed females compared with DF-fed females. We also uncovered significant 13C-enrichment in DF-fed insect samples relative to CADF-fed insect samples and lowered bacterial essential amino acid (EAA) provisioning in CADF-fed samples. Differences in SMR and EAA provisioning were not accompanied by significant differences in overall microbiome composition between the two groups. However, cellulolytic and nitrogen-fixing bacterial families dominant in wild omnivorous cockroaches and wood-feeding termites were significantly enriched in CADF-fed females than in DF-fed females, at the end of the study. We propose that these changes in microbiome composition after dietary shifts are associated with changes in EAA provisioning and possibly SMR. Further studies are needed to comprehensively understand the relative importance of gut microbial functions among the complexity of factors known to underscore SMR responses in insects under varying dietary conditions.

Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in Trypanosoma brucei.

Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T.brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.

Can (13)C stable isotope analysis uncover essential amino acid provisioning by termite-associated gut microbes?

Gut-associated microbes of insects are postulated to provide a variety of nutritional functions including provisioning essential amino acids (EAAs). Demonstrations of EAA provisioning in insect-gut microbial systems, nonetheless, are scant. In this study, we investigated whether the eastern subterranean termite Reticulitermes flavipes sourced EAAs from its gut-associated microbiota. δ13CEAA data from termite carcass, termite gut filtrate and dietary (wood) samples were determined following 13C stable isotope analysis. Termite carcass samples (−27.0 ± 0.4‰, mean ± s.e.) were significantly different from termite gut filtrate samples (−27.53 ± 0.5‰), but not the wood diet (−26.0 ± 0.5‰) (F(2,64) = 6, P < 0.0052). δ13CEAA-offsets between termite samples and diet suggested possible non-dietary EAA input. Predictive modeling identified gut-associated bacteria and fungi, respectively as potential major and minor sources of EAAs in both termite carcass and gut filtrate samples, based on δ13CEAA data of four and three EAAs from representative bacteria, fungi and plant data. The wood diet, however, was classified as fungal rather than plant in origin by the model. This is attributed to fungal infestation of the wood diet in the termite colony. This lowers the confidence with which gut microbes (bacteria and fungi) can be attributed with being the source of EAA input to the termite host. Despite this limitation, this study provides tentative data in support of hypothesized EAA provisioning by gut microbes, and also a baseline/framework upon which further work can be carried out to definitively verify this function.

Perspective: Protein: What Kind, How Much, When?

An adequate intake of dietary proteins is fundamental for muscle trophism by ensuring the provision of essential amino acids and by stimulating protein synthesis via the mammalian target of rapamycin pathway. Older adults are at high risk for inadequate protein ingestion. Moreover, the current recommended dietary allowance for protein (0.8 g/kg/d) may not be sufficient for preserving muscle mass in older individuals.1

Clinical use of amino acids as dietary supplement: pros and cons

Nitrogen supply is pivotal for the maintenance of life. Amino acids can be utilized to synthesize both glucose and lipids. The opposite, i.e., production of amino acids from either one of them, is not possible in the absence of other amino acids as donors of nitrogen. The quality of amino acid content in protein has been re-evaluated recently, and the relevance of essential amino acids has been repeatedly underlined. Essential amino acid requirements in different mammals are not identical, and ratios among them should be taken into account when projecting an efficient formulation. Recent research has demonstrated that genes respond to different qualities and quantities of nutritional supply, and increased provision of essential amino acids increases lifespan in animal experiments through mitochondriogenesis and maintenance of elevated rates of synthesis of anti-oxidant molecules. Moreover, genetic expression of key controllers of synthesis, like mTOR, may be particularly important for understanding skeletal muscle maintenance. Losses of muscle mass and impaired immune function are related to reduced protein supply, and there is increasing evidence that regular essential amino acid intake as part of an oral diet is effective in reversing muscle catabolism, promoting muscle anabolism, and restoring immunological function. Therefore, the use of amino acids as supplements to diet would be expanding in the near future. Is this safe? Few data are available on amino acid toxicity, and only one essential amino acid may be considered to have clinically relevant toxicity: methionine, because it is transformed into a toxic intermediate, homocysteine, when cysteine synthesis is required by metabolic needs. Matching of stoichiometric ratios between methionine and cysteine may solve the problem of supplying sufficient amounts of sulfur to the body. Arginine and glutamine are two non-essential amino acids than can become “conditionally essential” because of elevated needs during pathological conditions, and metabolism may not be able to maintain their concentrations at sufficient levels to match metabolic requirements. Chronic exogenous arginine supplementation has not proven to exert positive clinical effects in different trials, and sequential articulation of the knowledge of introduction of arginine-driven transcriptional, translational, and epigenetic adaptations may give us a key for interpreting those puzzling results.

Supplementing l-leucine to a low-protein diet increases tissue protein synthesis in weanling pigs

Recent work with young pigs shows that reducing dietary protein intake can improve gut function after weaning but results in inadequate provision of essential amino acids for muscle growth. Because acute administration of L-leucine stimulates protein synthesis in piglet muscle, the present study tested the hypothesis that supplementing L-leucine to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in multiple organs of post-weaning pigs. Eighteen 21-day pigs (Duroc×Landrace×Yorkshire) were fed low-protein diets (16.9% crude protein) supplemented with 0, 0.27 or 0.55% L-leucine (total leucine contents in the diets being 1.34, 1.61 or 1.88%, respectively). At 35 days of age, protein synthesis was determined using the [2H] phenylalanine flooding-dose technique. Additionally, total and phosphorylated levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eIF4E-binding protein-1 (4E-BP1) were measured in longissimus muscle and liver. Compared with the control group, dietary supplementation with 0.55% L-leucine for 2 weeks increased (P<0.05): (1) the phosphorylated levels of S6K1 and 4E-BP1; (2) protein synthesis in skeletal muscle, liver, the heart, kidney, pancreas, spleen, and stomach; and (3) daily weight gain by 61%. Dietary supplementation with 0.27% L-leucine enhanced (P<0.05) protein synthesis in proximal small intestine, kidney and pancreas. These novel findings provide a molecular basis for designing effective nutritional means to increase the efficiency of nutrient utilization for protein accretion in neonates.

The role of intracellular symbiotic bacteria in the amino acid nutrition of embryos from the black bean aphid,Aphis fabae

Aphids are well-known for their symbiotic relationship with intracellular bacteria of the genus Buchnera (γ-Proteobacteria). The symbiosis has a nutritional basis in that the bacteria supplement the aphid diet of phloem sap through the provision of essential amino acids. To date, few studies have considered the spatial complexity of the association, particularly the delineation of the symbiosis into embryo and maternal compartments. Here, we generate aposymbiotic (bacteria-free) embryos of the black bean aphid, Aphis fabae (Scopoli) (Hemiptera: Aphididae), as our experimental model and demonstrate that embryos reared in culture media require an external supply of essential amino acids. Analysis of individual amino acid deletions from the culture medium indicate that the key individual amino acids for embryo growth are phenylalanine and valine derived from the maternal tissues, and tryptophan derived from Buchnera. These results are discussed in relation to our current limited understanding of nutrient supply to aphid embryos.

Impaired translation initiation activation and reduced protein synthesis in weaned piglets fed a low-protein diet

Weanling mammals (including infants) often experience intestinal dysfunction when fed a high-protein diet. Recent work with the piglet (an animal model for studying human infant nutrition) shows that reducing protein intake can improve gut function during weaning but compromises the provision of essential amino acids (EAA) for muscle growth. The present study was conducted with weaned pigs to test the hypothesis that supplementing deficient EAA (Lys, Met, Thr, Trp, Leu, Ile and Val) to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in tissues. Pigs were weaned at 21 days of age and fed diets containing 20.7, 16.7 or 12.7% crude protein (CP), with the low-CP diets supplemented with EAA to achieve the levels in the high-CP diet. On Day 14 of the trial, tissue protein synthesis was determined using the phenylalanine flooding dose method. Reducing dietary CP levels decreased protein synthesis in pancreas, liver, kidney and longissimus muscle. A low-CP diet reduced the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) in skeletal muscle and liver while increasing the formation of an inactive eIF4E·4E-BP1 complex in muscle. Dietary protein deficiency also decreased the phosphorylation of mammalian target of rapamycin (mTOR) and the formation of an active eIF4E·eIF4G complex in liver. These results demonstrate for the first time that chronic feeding of a low-CP diet suppresses protein synthesis in animals partly by inhibiting mTOR signaling. Additionally, our findings indicate that supplementing deficient EAA to low-protein diets is not highly effective in restoring protein synthesis or whole-body growth in piglets. We suggest that conditionally essential amino acids (e.g., glutamine and arginine) may be required to maintain the activation of translation initiation factors and optimal protein synthesis in neonates.

On the functional significance of symbiotic microorganisms in the Homoptera: a comparative study of Acyrthosiphon pisum and Nilaparvata lugens

Summary All phloem-feeding Homoptera possess symbiotic microorganisms. Although the phylogenetic position and anatomical location of the micro- organisms differ, the underlying theme of the symbiosis is the same; the microorganisms improve the nutritional quality of the diet through the provision of essential amino acids. The symbiosis has been well documented in aphids, but little information is available from other homopteran groups. The impact of the loss of bacterial symbionts in the pea aphid Acyrthosiphon pisum Harris and eukaryotic yeast-like symbionts in the Asian rice brown planthopper Nilaparvata lugens Stal was examined in parallel. The weight and relative growth rate of aphids and planthoppers was significantly reduced by symbiont loss, and characteristic features of aposymbiotic pea aphids, so-called ‘metabolic signatures’, were, for the first time, observed in aposymbiotic N. lugens. For example, the amount of protein per unit fresh weight was reduced by 26 and 10%, and the free amino acid levels increased 1.8- and 1.4-fold, in aposymbiotic A. pisum and N. lugens, respectively. In addition, the concentration of the amino acid glutamine was elevated in the tissues of aposymbiotic insects. The data are discussed in the context of our current understanding of the nutritional role of the symbiosis and the mechanisms of nitrogen metabolism in the two insect species. It is concluded that the metabolic adjustments of the insects to symbiont loss are broadly equivalent.

On the functional significance of symbiotic microorganisms in the Homoptera: a comparative study of Acyrthosiphon pisum and Nilaparvata lugens

SummaryAll phloem‐feeding Homoptera possess symbiotic microorganisms. Although the phylogenetic position and anatomical location of the micro‐ organisms differ, the underlying theme of the symbiosis is the same; the microorganisms improve the nutritional quality of the diet through the provision of essential amino acids. The symbiosis has been well documented in aphids, but little information is available from other homopteran groups. The impact of the loss of bacterial symbionts in the pea aphid Acyrthosiphon pisum Harris and eukaryotic yeast‐like symbionts in the Asian rice brown planthopper Nilaparvata lugens Stål was examined in parallel. The weight and relative growth rate of aphids and planthoppers was significantly reduced by symbiont loss, and characteristic features of aposymbiotic pea aphids, so‐called ‘metabolic signatures’, were, for the first time, observed in aposymbiotic N. lugens. For example, the amount of protein per unit fresh weight was reduced by 26 and 10%, and the free amino acid levels increased 1.8‐ and 1.4‐fold, in aposymbiotic A. pisum and N. lugens, respectively. In addition, the concentration of the amino acid glutamine was elevated in the tissues of aposymbiotic insects. The data are discussed in the context of our current understanding of the nutritional role of the symbiosis and the mechanisms of nitrogen metabolism in the two insect species. It is concluded that the metabolic adjustments of the insects to symbiont loss are broadly equivalent.

Injection of essential amino acids substitutes for bacterial supply in aposymbiotic pea aphids (Acyrthosiphon pisum)

AbstractThe symbiotic bacteria Buchnera contribute to the nutrition of pea aphids, Acyrthosiphon pisum, through the provision of essential amino acids which are lacking in the diet. However, chemically defined diets, containing nutritionally adequate amounts of essential amino acids, fail to rescue aposymbiotic aphids, in which the bacteria have been disrupted with antibiotics. In this study the injection of a mixture of essential amino acids into the haemocoel of aposymbiotic aphids was shown to alleviate, at least partially, the impact of symbiont loss. Specifically, the total amino acid content in the tissues of aposymbiotic aphids was reduced by approximately 40% to levels comparable with symbiotic insects, and there was a 1.7‐fold increase in the number of embryos, suggesting that the availability of essential amino acids promotes aphid protein synthesis by rejuvenating the free amino acid pool of aposymbiotic aphids. In addition, a similar effect on the total amino acid content was observed when phenylalanine alone, but not glutamine, lysine or tryptophan, was injected into the haemocoel of aposymbiotic aphids, and there was also a significant increase in the number of embryos following injection of phenylalanine or tryptophan alone. The impact of amino acid injection on the embryo complement of aposymbiotic aphids was limited to an increase in the number of embryos, with no increase in basal embryo size. It is proposed that older embryos may rely on their own complement of symbiotic bacteria for essential amino acid provisioning. Taken together, the data highlight the importance of bacterial provisioning of essential amino acids, particularly the aromatic amino acids, in the intact symbiosis.

Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera.

Most aphids possess intracellular bacteria of the genus Buchnera. The bacteria are transmitted vertically via the aphid ovary, and the association is obligate for both partners: Bacteria-free aphids grow poorly and produce few or no offspring, and Buchnera are both unknown apart from aphids and apparently unculturable. The symbiosis has a nutritional basis. Specifically, bacterial provisioning of essential amino acids has been demonstrated. Nitrogen recycling, however, is not quantitatively important to the nutrition of aphid species studied, and there is strong evidence against bacterial involvement in the lipid and sterol nutrition of aphids. Buchnera have been implicated in various non-nutritional functions. Of these, just one has strong experimental support: promotion of aphid transmission of circulative viruses. It is argued that strong parallels may exist between the nutritional interactions (including the underlying mechanisms) in the aphid-Buchnera association and other insect symbioses with intracellular microorganisms.