Increase In Amino Acid Levels Research Articles

Serum and CSF metabolomics analysis shows Mediterranean Ketogenic Diet mitigates risk factors of Alzheimer’s disease

Alzheimer’s disease (AD) is influenced by a variety of modifiable risk factors, including a person’s dietary habits. While the ketogenic diet (KD) holds promise in reducing metabolic risks and potentially affecting AD progression, only a few studies have explored KD’s metabolic impact, especially on blood and cerebrospinal fluid (CSF). Our study involved participants at risk for AD, either cognitively normal or with mild cognitive impairment. The participants consumed both a modified Mediterranean Ketogenic Diet (MMKD) and the American Heart Association diet (AHAD) for 6 weeks each, separated by a 6-week washout period. We employed nuclear magnetic resonance (NMR)-based metabolomics to profile serum and CSF and metagenomics profiling on fecal samples. While the AHAD induced no notable metabolic changes, MMKD led to significant alterations in both serum and CSF. These changes included improved modifiable risk factors, like increased HDL-C and reduced BMI, reversed serum metabolic disturbances linked to AD such as a microbiome-mediated increase in valine levels, and a reduction in systemic inflammation. Additionally, the MMKD was linked to increased amino acid levels in the CSF, a breakdown of branched-chain amino acids (BCAAs), and decreased valine levels. Importantly, we observed a strong correlation between metabolic changes in the CSF and serum, suggesting a systemic regulation of metabolism. Our findings highlight that MMKD can improve AD-related risk factors, reverse some metabolic disturbances associated with AD, and align metabolic changes across the blood-CSF barrier.

Exogenous Spermidine and Amino-Ethoxyvinylglycine Improve Nutritional Quality via Increasing Amino Acids in Rice Grains.

Polyamines and ethylene are key regulators of the growth and development, quality formation, and stress response of cereal crops such as rice. However, it remains unclear whether the application of these regulators could improve the nutritional quality via increasing amino acids in rice grains. This study examined the role of exogenous polyamines and ethylene in regulating amino acid levels in the milled rice of earlier-flowered superior grain (SG) and later-flowered inferior grain (IG). Two rice varieties were field grown, and either 1 mmol L-1 spermidine (Spd) or 50 μmol L-1 amino-ethoxyvinylglycine (AVG) was applied to panicles at the early grain-filling stage. The control check (CK) was applied with deionized water. The results showed that the Spd or AVG applications significantly increased polyamine (spermine (Spm) and Spd) contents and decreased ethylene levels in both SG and IG and significantly increased amino acid levels in the milled rice of SG and IG relative to the CK. Collectively, the application of Spd or AVG can increase amino acid-based nutritional quality and grain yield via increasing polyamine (Spm and Spd) contents and reducing ethylene levels in both SG and IG of rice.

Influence of lactic-acid bacteria feed supplementation on free amino acid levels in serum and feces of rainbow trout (Oncorhynchus mykiss, Walbaum 1792)

IntroductionThe influence of intestinal microbiota, particularly lactic-acid bacteria, on digestion and nutrient metabolic activities has been extensively studied. In this research, we investigated the effects of supplementing the feed of rainbow trout (O. mykiss) with P. acidilactici or L. lactis probiotics. A Control group without probiotic supplementation was also included. The study aimed to evaluate growth, feed conversion indices, free amino acid levels in serum and feces, and the relative gene expression of amino acid solute carrier transporters (SLC).MethodsRainbow trout weighing 73.9±10 g were fed with a basal commercial diet supplemented with mono-strains of P. acidilactici or L. lactis at a dosage of 5 x 108 CFU per gram of feed for 60 days. The trout were reared in 100-liter tanks with independent closed recirculation systems, with a water replacement rate of 80% of the total volume daily and a controlled temperature of 15 ± 0.3 °C. The proximate composition of the basal diet consisted of crude protein (49.8%), fat (32.1%), fiber (1.31%), ash (13%), and moisture (8.3%) on a dry matter basis. The diet was provided at a rate of 2% of bodyweight per day.ResultsNo significant differences (p > 0.05) were observed in growth and feed conversion indices between the experimental groups. However, significant differences in free amino acid levels in feces and serum samples (p ≤ 0.05) were observed. The P. acidilactici group exhibited significantly higher levels of glutamate, lysine, proline, and tyrosine in feces samples, and higher levels of arginine, iso/leucine, phenylalanine, proline, serine, threonine, and valine in serum samples compared to the Control group. Additionally, the supplemented probiotic groups showed significant downregulation (p ≤ 0.05) of theslc6a19 and slc7a9 genes, which encode transporters for neutral and cationic amino acids.DiscussionThe autochthonous rainbow trout strain, P. acidilactici, demonstrated more pronounced effects on amino acid levels in feces and serum compared to the non-autochthonous L. lactis strain. However, the increased amino acid levels, particularly in the serum, resulting from P. acidilactici supplementation, did not lead to improved trout growth or feed conversion. Nonetheless, lactic-acid bacteria supplementation induced measurable metabolic effects, as evidenced by elevated levels of specific amino acids in the serum and feces.Future research should focus on evaluating these probiotic effects using specialized diets and considering the observed metabolic effects in this study.

Adjusted Thr: Lys Ratio Improved the Performance and Efficiency of Japanese Quail

Two hundred and forty Japanese quail aged 125 days were randomly allocated to five treatment groups based on laying (%) and quail’s weight (90.71 ± 1.8% egg/day × 100 and 178.05 ± 9.38 g, respectively), each of which included six replicates of eight quails. The diets were formulated based on corn, soybean meal, and industrial amino acids. An optimal diet achieves 100% of amino acids required by the quail requirements, except for threonine. Five treatments were made: 20% less amino acid; 10% less amino acid; optimal diet; 10% more amino acid; and 20% more amino acids than those in the optimal diet. The increase in amino acid levels in a fixed Lys: amino acid ratio led to histological alterations in the liver and uterine epithelium, reduction in blood lipid peroxidation, lower hepatic HSP70 gene expression, and the performance of laying Japanese quail. The optimal diet based on the NRC with an adjusted Thr: Lys 78 ratio (Lys 1.0%) improved the performance and efficiency of Japanese quail from 125 to 230 days of age.

Effect of Salinity and Sodicity on Vegetable Production and Remedial Measures: A Review

The word salinity comes from the Latin word salinium which means "salt cellar" and it means "position or quality of being". Sodicity indicates the amount of sodium converted to calcium and magnesium in soil. High sodicity suppresses plant growth due to sodium toxicity and nutrient imbalance in plants, as well as low availability of mineral nutrients in the soil. Salt stress is the cause of the slow growth and growth of plants and leads to changes in yield and quality in a variety of crops. Plants provide a complex response to salt and changes in the morphology, physiology and metabolism of plants are observed. The effect of salt on various vegetable plants namely beetroot, cabbage, capsicum, kabuli chana, coriander, fenugreek, lettuce, onion, tomato, potato was reviewed. Salinity was adversely damaged as a result of salinity: Seed formation, survival percent, phonological attribute, growth and yield, its components, dry and fresh weight were affected. Photosynthesis and respiratory rates of plants were reduced. Salinity reduced total carbohydrate, fatty acid, and protein content but notably increased amino acid levels. The growth of asparagus and tomatoes was more concentrated in sodic soils than saline soils, indicating that asparagus and tomatoes are sensitive to sodicity. Beans also die in sodic soils, indicating that the beans are very sensitive to sodicity. The negative effects of alkaline water on the addition of gypsum and FYM have shown a significant increase in plant growth and yield limits.

Bacterial Endophytes as a Promising Approach to Enhance the Growth and Accumulation of Bioactive Metabolites of Three Species of Chenopodium Sprouts.

Sprouts are regarded as an untapped source of bioactive components that display various biological properties. Endophytic bacterium inoculation can enhance plant chemical composition and improve its nutritional quality. Herein, six endophytes (Endo 1 to Endo 6) were isolated from Chenopodium plants and morphologically and biochemically identified. Then, the most active isolate Endo 2 (strain JSA11) was employed to enhance the growth and nutritive value of the sprouts of three Chenopodium species, i.e., C. ambrosoides, C. ficifolium, and C. botrys. Endo 2 (strain JSA11) induced photosynthesis and the mineral uptake, which can explain the high biomass accumulation. Endo 2 (strain JSA11) improved the nutritive values of the treated sprouts through bioactive metabolite (antioxidants, vitamins, unsaturated fatty acid, and essential amino acids) accumulation. These increases were correlated with increased amino acid levels and phenolic metabolism. Consequently, the antioxidant activity of the Endo 2 (strain JSA11)-treated Chenopodium sprouts was enhanced. Moreover, Endo 2 (strain JSA11) increased the antibacterial activity against several pathogenic bacteria and the anti-inflammatory activities as evidenced by the reduced activity of cyclooxygenase and lipoxygenase. Overall, the Endo 2 (strain JSA11) treatment is a successful technique to enhance the bioactive contents and biological properties of Chenopodium sprouts.

A Time-Course Comparison of Skeletal Muscle Metabolomic Alterations in Walker-256 Tumour-Bearing Rats at Different Stages of Life.

Cancer cachexia is a severe wasting condition that needs further study to find ways to minimise the effects of damage and poor prognosis. Skeletal muscle is the most impacted tissue in cancer cachexia; thus, elucidation of its metabolic alterations could provide a direct clue for biomarker research and be applied to detect this syndrome earlier. In addition, concerning the significant changes in the host metabolism across life, this study aimed to compare the metabolic muscle changes in cachectic tumour-bearing hosts at different ages. We performed 1H-NMR metabolomics in the gastrocnemius muscle in weanling and young adult Walker-256 tumour-bearing rats at different stages of tumour evolution (initial, intermediate, and advanced). Among the 49 metabolites identified, 24 were significantly affected throughout tumour evolution and 21 were significantly affected regarding animal age. The altered metabolites were mainly related to increased amino acid levels and changed energetic metabolism in the skeletal muscle, suggesting an expressive catabolic process and diverted energy production, especially in advanced tumour stages in both groups. Moreover, these changes were more severe in weanling hosts throughout tumour evolution, suggesting the distinct impact of cancer cachexia regarding the host’s age, highlighting the need to adopting the right animal age when studying cancer cachexia.

Enteropeptidase inhibition improves obesity by modulating gut microbiota composition and enterobacterial metabolites in diet-induced obese mice

Enteropeptidase is a transmembrane serine protease localized in the lumen of the duodenum that acts as a key enzyme for protein digestion. SCO-792 is an orally available enteropeptidase inhibitor that has been reported to have therapeutic effects on obesity and diabetes in mice. However, the mechanism underlying the therapeutic effect of SCO-792 has not yet been fully elucidated. In this study, we evaluated the role of gut microbiota on SCO-792-induced body weight (BW) reduction in high-fat diet-induced obese (DIO) mice.Chronic administration of SCO-792 substantially decreased BW and food intake in DIO mice. While the pair-fed study uncovered food intake-independent mechanisms of BW reduction by SCO-792. Interestingly, antibiotics-induced microbiota elimination in the gut canceled SCO-792-induced BW reduction by nearly half without affecting the anorectic effect, indicating the involvement of gut microbiota in the anti-obesity mechanism that is independent of food intake reduction. Microbiome analysis revealed that SCO-792 altered the gut microbiota composition in DIO mice. Notably, it was found that the abundance of Firmicutes decreased while that of Verrucomicrobia increased at the phylum level. Increased abundance of Akkermansia muciniphila, a bacterium known to be useful for host metabolism, was observed in SCO-792-treated mice. Fecal metabolome analysis revealed increased amino acid levels, indicating gut enteropeptidase inhibition. In addition, SCO-792 was found to increase the level of short-chain fatty acids, including propionate, and bile acids in the feces, which all help maintain gut health and improve metabolism. Furthermore, it was found that SCO-792 induced the elevation of colonic immunoglobulin A (IgA) concentration, which may maintain the microbiota condition, in DIO mice.In conclusion, this study demonstrates the contribution of microbiota to SCO-792-induced BW reduction. Enteropeptidase-mediated regulation of microbiota, enterobacterial metabolites, and IgA in the gut may coordinately drive the therapeutic effects of SCO-792 in obesity.

Amino Acids Regulate Cisplatin Insensitivity in Neuroblastoma.

Simple SummaryNeuroblastomas mostly show poor response to the Cisplatin therapy. Amino acids serve as building blocks for proteins, which are acquired either through diet or protein breakdown. Our study reveals high amino acid pools and dependence of Cisplatin-tolerant neuroblastomas cells on amino acids for their survival, especially, in drug treated conditions. Our study also demonstrates that response of neuroblastomas to Cisplatin can be improved by decreasing cellular amino acid levels either by reducing amino acid supplements or by applying autophagy inhibitor, Hydroxychloroquine. Thus, our findings establish that neuroblastomas can be sensitized to Cisplatin by targeting amino acid metabolism.Neuroblastoma are pediatric, extracranial malignancies showing alarming survival prognosis outcomes due to their resilience to current aggressive treatment regimens, including chemotherapies with cisplatin (CDDP) provided in the first line of therapy regimens. Metabolic deregulation supports tumor cell survival in drug-treated conditions. However, metabolic pathways underlying cisplatin-resistance are least studied in neuroblastoma. Our metabolomics analysis revealed that cisplatin-insensitive cells alter their metabolism; especially, the metabolism of amino acids was upregulated in cisplatin-insensitive cells compared to the cisplatin-sensitive neuroblastoma cell line. A significant increase in amino acid levels in cisplatin-insensitive cells led us to hypothesize that the mechanisms upregulating intracellular amino acid pools facilitate insensitivity in neuroblastoma. We hereby report that amino acid depletion reduces cell survival and cisplatin-insensitivity in neuroblastoma cells. Since cells regulate their amino acids levels through processes, such as autophagy, we evaluated the effects of hydroxychloroquine (HCQ), a terminal autophagy inhibitor, on the survival and amino acid metabolism of cisplatin-insensitive neuroblastoma cells. Our results demonstrate that combining HCQ with CDDP abrogated the amino acid metabolism in cisplatin-insensitive cells and sensitized neuroblastoma cells to sub-lethal doses of cisplatin. Our results suggest that targeting of amino acid replenishing mechanisms could be considered as a potential approach in developing combination therapies for treating neuroblastomas.

UJI PENINGKATAN KADAR ASAM AMINO PADA TAPE KETAN HITAM DENGAN PENAMBAHAN SARI BUAH NANAS DENGAN METODE SPEKTROFOTOMETRI SINAR TAMPAK

Amino acid levels in sticky rice tape are still relatively low, while amino acids themselves are food substances for body growth. Pineapple contains the enzyme bromelain. This enzyme is one of the protease enzymes that can hydrolyze protein, therefore it can increase amino acid levels. This study aims to determine the increase in amino acid levels in black sticky rice tape with the addition of pineapple juice and to determine the effect of adding pineapple juice to amino acid levels in black sticky rice tape. In the process of making black sticky rice tape, 25 ml 50 ml of pineapple juice was added per 500 grams each, while one treatment did not add pineapple juice as a control. Black glutinous rice that has been fermented for 2-3 days was tested for increased levels of amino acids by using visible light spectrophotometry. The sample of black glutinous tape without the addition of pineapple juice obtained an average content of 4.73%, on the sample of black glutinous tape with the addition of 25 ml of pineapple juice the average content obtained was 6.50%, and the sample of black sticky rice tape with the addition of 50 ml pineapple juice the average level obtained was 7.76% and gave the effect of increasing amino acid levels on black sticky rice tape.

Anti-inflammatory, analgesic and acute toxicity effects of fermented soybean

BackgroundTempeh is a widely known fermented soybean that contains elevated level of bioactive contents. Our previous study has shown that anaerobic fermented Nutrient Enriched Soybean Tempeh (NESTE) with increase amino acid and antioxidant levels possessed better hepatoprotective effect than raw soybean.MethodsIn this study, the anti-inflammatory effect of the NESTE aqueous extract and raw soybean aqueous extract (SBE) were evaluated by quantifying the inhibition of IL-1β, TNF-α and nitric oxide (NO) secretion in LPS treated RAW 264.7 cell in vitro. On the other hand, in vivo oral acute toxicity effect of the extract was tested on mice at the dose of 5000 mg/kg body weight. In vivo oral analgesic effect of both aqueous extracts at 200 and 1000 mg/kg body weight was evaluated by the hot plate test.ResultsIn the in vitro anti-inflammatory study, 5 mg/mL NESTE was able to inhibit 25.50 ± 2.20%, 35.88 ± 3.20% and 28.50 ± 3.50% of NO, IL-1β and TNF-α production in LPS treated RAW 264.7 cells without inducing cytotoxic effect on the cells. However, this effect was lower than 4 μg/mL of curcumin, which inhibited NO, IL-1β and TNF-α production by 89.50 ± 5.00%, 78.80 ± 6.20% and 87.30 ± 4.00%, respectively. In addition, 1.5 to 2.5-fold increase of latency period up to 120 min for mice in the hot plate test was achieved by 1000 mg/kg NESTE. The analgesic effect of NESTE was better than 400 mg/kg of acetyl salicylic acid, which only increased ~ 1.7-fold of latency period up to 90 min. Moreover, NESTE did not show acute toxicity (no LD50) up to 5000 mg/kg body weight.ConclusionNESTE is a nutritious food ingredient with potential anti-inflammatory and analgesic effects.

The Klebsiella pneumoniae citrate synthase gene, gltA, influences site specific fitness during infection.

Klebsiella pneumoniae (Kp), one of the most common causes of healthcare-associated infections, increases patient morbidity, mortality, and hospitalization costs. Kp must acquire nutrients from the host for successful infection; however, the host is able to prevent bacterial nutrient acquisition through multiple systems. This includes the innate immune protein lipocalin 2 (Lcn2), which prevents Kp iron acquisition. To identify novel Lcn2-dependent Kp factors that mediate evasion of nutritional immunity during lung infection, we undertook an InSeq study using a pool of >20,000 transposon mutants administered to Lcn2+/+ and Lcn2-/- mice. Comparing transposon mutant frequencies between mouse genotypes, we identified the Kp citrate synthase, GltA, as potentially interacting with Lcn2, and this novel finding was independently validated. Interestingly, in vitro studies suggest that this interaction is not direct. Given that GltA is involved in oxidative metabolism, we screened the ability of this mutant to use a variety of carbon and nitrogen sources. The results indicated that the gltA mutant has a distinct amino acid auxotrophy rendering it reliant upon glutamate family amino acids for growth. Deletion of Lcn2 from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, corresponding to increased fitness of the gltA mutant in vivo and ex vivo. Accordingly, addition of glutamate family amino acids to Lcn2+/+ bronchioloalveolar lavage fluid rescued growth of the gltA mutant. Using a variety of mouse models of infection, we show that GltA is an organ-specific fitness factor required for complete fitness in the spleen, liver, and gut, but dispensable in the bloodstream. Similar to bronchioloalveolar lavage fluid, addition of glutamate family amino acids to Lcn2+/+ organ lysates was sufficient to rescue the loss of gltA. Together, this study describes a critical role for GltA in Kp infection and provides unique insight into how metabolic flexibility impacts bacterial fitness during infection.

Molecular breath analysis supports altered amino acid metabolism in idiopathic pulmonary fibrosis.

Diagnosis of idiopathic pulmonary fibrosis (IPF) is complex and its pathogenesis is poorly understood. Recent findings indicate elevated levels of proline and other amino acids in lung tissue of IPF patients which may also be of diagnostic value. Following these findings, we hypothesized that such altered metabolic profiles would be mirrored in exhaled breath and could therefore be captured non-invasively in real time. We aimed to validate these results using real-time exhaled breath analysis by secondary electrospray ionization-mass spectrometry, which can provide a non-invasive, painless and fast insight into the metabolism. Breath analysis was performed in a matched 1:1 case-control study involving 21 patients with IPF and 21 control subjects. We found significantly (P < 0.05) elevated levels of proline, 4-hydroxyproline, alanine, valine, leucine/isoleucine and allysine in breath of IPF patients, whereas pyroglutamic acid and phenylalanine did not show significant differences. This coincides with the amino acid's abundance in pulmonary tissue indicating that our observations reflect progressing fibrosis. In addition, amino acid levels correlated across subjects, further supporting a common underlying pathway. We were able to obtain a cross-validated area under the curve of 0.86, suggesting that these increased amino acid levels in exhaled breath have the potential to be used as biomarkers for IPF. We could validate previous findings of elevated lung tissue amino acid levels in IPF and show that online breath analysis might be a practical tool for a rapid screening for IPF.

The bioaccumulation of waterborne zinc in tissues of silver catfish (Rhamdia quelen) and its effect on biochemical parameters.

Silver catfish (Rhamdia quelen) is a fish species with neotropical distribution, and is a potential model organism to study polluted environment. The aim of this study is to analyze the response of silver catfish to environmental concentrations of waterborne zinc (Zn) over 96h. Significant metal accumulation was seen in gill, intestine and liver tissues. No significant accumulation was seen in muscle tissue. Lipid peroxidation increased in the brain, and decreased in the muscle and liver at all levels of exposure. Zinc exposure led to decreased protein carbonyl levels in the brain and increased levels in the liver. The activity of catalase in the liver was reduced for all exposed groups. Glutathione S-transferase activity decreased in the brain at the highest level of exposure and in the liver at all Zn concentrations tested. Non-protein thiols increased in the muscle and in the gills after exposure. Ascorbic acid levels increased in the brain and in the gills. Exposure to Zn also altered the metabolic parameters, causing decreased lactate and ammonia levels in the muscle, and decreased glycogen in the liver. Zinc exposure increased ammonia and amino acid levels in the liver, and increase glycogen and amino acid levels in muscle tissue. Our results demonstrate that exposure to environmentally relevant concentrations of Zn led to accumulation of metals in the tissues of silver catfish, with significant changes in biochemical parameters.

Functional properties of fermented milk produced with plant proteins

In this study, storage-related changes in the physico-chemical and sensory properties of non-fat fermented milk drinks that contain soy protein isolate (SPI), pea protein isolate (PPI), wheat gluten (WG) and rice protein (RP) were investigated. The addition of plant protein additives to milk beverage affected the pH, titratable acidity %, syneresis, viscosity, dry matter, ash, color (L, b values), protein and amino acid contents (p < 0.01). During storage, the viscosity values showed an increase and protein additives increased amino acid levels in fermented milks beverage. The highest essential amino acid content was found in drinks containing SPI, with the most prevalent amino acids being lysine, leucine, isoleucine, methionine and threonine. The taste of fermented milks with PPI was preferred. In general, plant protein additives improved the sensory attributes. (p < 0.01). In conclusion, the use of plant protein additives in non-fat fermented milk drink formulations was shown to improve the physico-chemical and sensory properties and to increase the nutritional value of the formulations and therefore could be an alternative strategy for the development of new healthy dairy products.

Diet density during the first week of life: Effects on energy and nitrogen balance characteristics of broiler chickens

This study aimed to determine effects of diet density on growth performance, energy balance, and nitrogen (N) balance characteristics of broiler chickens during the first wk of life. Effects of diet density were studied using a dose-response design consisting of 5 dietary fat levels (3.5, 7.0, 10.5, 14.0, and 17.5%). The relative difference in dietary energy level was used to increase amino acid levels, mineral levels, and the premix inclusion level at the same ratio. Chickens were housed in open-circuit climate respiration chambers from d 0 to 7 after hatch. Body weight was measured on d 0 and 7, whereas feed intake was determined daily. For calculation of energy balances, O2 and CO2 exchange were measured continuously and all excreta from d 0 to 7 was collected and analyzed at d 7. Average daily gain (ADG) and average daily feed intake (ADFI) decreased linearly (P = 0.047 and P < 0.001, respectively), whereas gain to feed ratio increased (P < 0.001) with increasing diet density. Gross energy (GE) intake and metabolizable energy (ME) intake were not affected by diet density, but the ratio between ME and GE intake decreased linearly with increasing diet density (P = 0.006). Fat, N, and GE efficiencies (expressed as gain per unit of nutrient intake), heat production, and respiratory exchange ratio (CO2 to O2 ratio) decreased linearly (P < 0.001) as diet density increased. Energy retention, N intake, and N retention were not affected by diet density. We conclude that a higher diet density in the first wk of life of broiler chickens did not affect protein and fat retention, whereas the ME to GE ratio decreased linearly with increased diet density. This suggests that diet density appears to affect digestibility rather than utilization of nutrients.

ρ⁰ Cells Feature De-Ubiquitination of SLC Transporters and Increased Levels and Fluxes of Amino Acids.

Solute carrier (SLC) transporters are a diverse group of membrane transporter proteins that regulate the cellular flux and distribution of endogenous and xenobiotic compounds. Post-translational modifications (PTMs), such as ubiquitination, have recently emerged as one of the major regulatory mechanisms in protein function and localization. Previously, we showed that SLC amino acid transporters were on average 6-fold de-ubiquitinated and increased amino acid levels were detected in ρ0 cells (lacking mitochondrial DNA, mtDNA) compared to parental cells. Here, we elucidated the altered functionality of SLC transporters and their dynamic ubiquitination status by measuring the uptake of several isotopically labeled amino acids in both human osteosarcoma 143B.TK- and ρ0 cells. Our pulse chase analysis indicated that de-ubiquitinated amino acid transporters in ρ0 cells were accompanied by an increased transport rate, which leads to higher levels of amino acids in the cell. Finding SLC transport enhancers is an aim of the pharmaceutical industry in order to compensate for loss of function mutations in these genes. Thus, the ubiquitination status of SLC transporters could be an indicator for their functionality, but evidence for a direct connection between de-ubiquitination and transporter activity has to be further elucidated.

Multicompartment analysis of protein-restricted phenylketonuric mice reveals amino acid imbalances in brain.

The mainstay of therapy for phenylketonuria (PKU) remains dietary protein restriction. Developmental and neurocognitive outcomes for patients, however, remain suboptimal. We tested the hypothesis that mice with PKU receiving protein-restricted diets would reveal disruptions of brain amino acids that shed light on these neurocognitive deficits. Phenylalanine hydroxylase-deficient (PKU) mice and parallel controls (both wild-type and heterozygous) were fed custom diets containing 18, 6, and 4% protein for 3weeks, after which tissues (brain, liver, sera) were collected for amino acid analysis profiling. Phenylalanine (phe) was increased in all tissues (p < 0.0001) of PKU mice and improved with protein restriction. In sera, decreased tyrosine (p < 0.01) was corrected (defined as not significantly different from the level in control mice receiving 18% chow) with protein restriction, whereas protein restriction significantly increased many other amino acids. A similar trend for increased amino acid levels with protein restriction was also observed in liver. In brain, the effects of protein restriction on large neutral amino acids (LNAAs) were variable, with some deficit correction (threonine, methionine, glutamine) and no correction of tyrosine under any dietary paradigm. Protein restriction (4% diet) in PKU mice significantly decreased lysine, arginine, taurine, glutamate, asparagine, and serine which had been comparable to control mice under 18% protein intake. Depletion of taurine, glutamate, and serine in the brain of PKU mice with dietary protein restriction may provide new insight into neurocognitive deficits of PKU.

An Impaired Respiratory Electron Chain Triggers Down-regulation of the Energy Metabolism and De-ubiquitination of Solute Carrier Amino Acid Transporters

Hundreds of genes have been associated with respiratory chain disease (RCD), the most common inborn error of metabolism so far. Elimination of the respiratory electron chain by depleting the entire mitochondrial DNA (mtDNA, ρ(0) cells) has therefore one of the most severe impacts on the energy metabolism in eukaryotic cells. In this study, proteomic data sets including the post-translational modifications (PTMs) phosphorylation and ubiquitination were integrated with metabolomic data sets and selected enzyme activities in the osteosarcoma cell line 143B.TK(-) A shotgun based SILAC LC-MS proteomics and a targeted metabolomics approach was applied to elucidate the consequences of the ρ(0) state. Pathway and protein-protein interaction (PPI) network analyses revealed a nonuniform down-regulation of the respiratory electron chain, the tricarboxylic acid (TCA) cycle, and the pyruvate metabolism in ρ(0) cells. Metabolites of the TCA cycle were dysregulated, such as a reduction of citric acid and cis-aconitic acid (six and 2.5-fold), and an increase of lactic acid, oxalacetic acid (both twofold), and succinic acid (fivefold) in ρ(0) cells. Signaling pathways such as GPCR, EGFR, G12/13 alpha, and Rho GTPases were up-regulated in ρ(0) cells, which could be indicative for the mitochondrial retrograde response, a pathway of communication from mitochondria to the nucleus. This was supported by our phosphoproteome data, which revealed two main processes, GTPase-related signal transduction and cytoskeleton organization. Furthermore, a general de-ubiquitination in ρ(0) cells was observed, for example, 80S ribosomal proteins were in average threefold and SLC amino acid transporters fivefold de-ubiquitinated. The latter might cause the observed significant increase of amino acid levels in ρ(0) cells. We conclude that an elimination of the respiratory electron chain, e.g. mtDNA depletion, not only leads to an uneven down-regulation of mitochondrial energy pathways, but also triggers the retrograde response.

Muscle-specific loss of Bmal1 leads to disrupted tissue glucose metabolism and systemic glucose homeostasis.

BackgroundDiabetes is the seventh leading cause of death in the USA, and disruption of circadian rhythms is gaining recognition as a contributing factor to disease prevalence. This disease is characterized by hyperglycemia and glucose intolerance and symptoms caused by failure to produce and/or respond to insulin. The skeletal muscle is a key insulin-sensitive metabolic tissue, taking up ~80 % of postprandial glucose. To address the role of the skeletal muscle molecular clock to insulin sensitivity and glucose tolerance, we generated an inducible skeletal muscle-specific Bmal1−/− mouse (iMSBmal1−/−).ResultsProgressive changes in body composition (decreases in percent fat) were seen in the iMSBmal1−/− mice from 3 to 12 weeks post-treatment as well as glucose intolerance and non-fasting hyperglycemia. Ex vivo analysis of glucose uptake revealed that the extensor digitorum longus (EDL) muscles did not respond to either insulin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) stimulation. RT-PCR and Western blot analyses demonstrated a significant decrease in mRNA expression and protein content of the muscle glucose transporter (Glut4). We also found that both mRNA expression and activity of two key rate-limiting enzymes of glycolysis, hexokinase 2 (Hk2) and phosphofructokinase 1 (Pfk1), were significantly reduced in the iMSBmal1−/− muscle. Lastly, results from metabolomics analyses provided evidence of decreased glycolytic flux and uncovered decreases in some tricarboxylic acid (TCA) intermediates with increases in amino acid levels in the iMSBmal1−/− muscle. These findings suggest that the muscle is relying predominantly on fat as a fuel with increased protein breakdown to support the TCA cycle.ConclusionsThese data support a fundamental role for Bmal1, the endogenous circadian clock, in glucose metabolism in the skeletal muscle. Our findings have implicated altered molecular clock dictating significant changes in altered substrate metabolism in the absence of feeding or activity changes. The changes in body composition in our model also highlight the important role that changes in skeletal muscle carbohydrate, and fat metabolism can play in systemic metabolism.