A Theileria annulata parasite with a single mutation, methionine 128 to isoleucine (M128I), in cytochrome B is resistant to buparvaquone.

Tropical theileriosis is a fatal leukemic-like disease of cattle caused by the tick-transmitted protozoan parasite Theileria annulata. The economics of cattle meat and milk production is severely affected by theileriosis in endemic areas. The hydroxynaphtoquinone buparvaquone (BPQ) is the only available drug currently used to treat clinical theileriosis, whilst BPQ resistance is emerging and spreading in endemic areas. Here, we chronically exposed T. annulata-transformed macrophages in vitro to BPQ and monitored the emergence of drug-resistant parasites. Surviving parasites revealed a significant increase in BPQ IC50 compared to the wild type parasites. Drug resistant parasites from two independent cloned lines had an identical single mutation, M128I, in the gene coding for T. annulata cytochrome B (Tacytb). This in vitro generated mutation has not been reported in resistant field isolates previously, but is reminiscent of the methionine to isoleucine mutation in atovaquone-resistant Plasmodium and Babesia. The M128I mutation did not appear to exert any deleterious effect on parasite fitness (proliferation and differentiation to merozoites). To gain insight into whether drug-resistance could have resulted from altered drug binding to TaCytB we generated in silico a 3D-model of wild type TaCytB and docked BPQ to the predicted 3D-structure. Potential binding sites cluster in four areas of the protein structure including the Q01 site. The bound drug in the Q01 site is expected to pack against an alpha helix, which included M128, suggesting that the change in amino acid in this position may alter drug-binding. The in vitro generated BPQ resistant T. annulata is a useful tool to determine the contribution of the various predicted docking sites to BPQ resistance and will also allow testing novel drugs against theileriosis for their potential to overcome BPQ resistance.

Valine improves mitochondrial function and protects against oxidative stress.

Among the branched-chain amino acids, leucine and isoleucine have been well studied for their roles in improving mitochondrial function and reducing oxidative stress. However, role of valine in mitochondrial function regulation and oxidative stress management remains elusive. This study investigated valine effect on mitochondrial function and oxidative stress in vitro. Valine increased expression of genes involved in mitochondrial biogenesis and dynamics. It upregulates mitochondrial function at complexes I, II, and IV levels of electron transport chain. Flow cytometry studies revealed, valine reduced oxidative stress by significantly lowering mitochondrial reactive oxygen species and protein expression of 4-hydroxynonenal. Functional role of valine against oxidative stress was analyzed by XFe96 Analyzer. Valine sustained oxidative phosphorylation and improved ATP generation rates during oxidative stress. In conclusion, our findings shed more light on the critical function of valine in protecting mitochondrial function thereby preventing mitochondrial/cellular damage induced by oxidative stress.

Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice.

Low-protein diets promote health and longevity in diverse species. Restriction of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine recapitulates many of these benefits in young C57BL/6J mice. Restriction of dietary isoleucine (IleR) is sufficient to promote metabolic health and is required for many benefits of a low-protein diet in C57BL/6J males. Here, we test the hypothesis that IleR will promote healthy aging in genetically heterogeneous adult UM-HET3 mice. We find that IleR improves metabolic health in young and old HET3 mice, promoting leanness and glycemic control in both sexes, and reprograms hepatic metabolism in a sex-specific manner. IleR reduces frailty and extends the lifespan of male and female mice, but to a greater degree in males. Our results demonstrate that IleR increases healthspan and longevity in genetically diverse mice and suggests that IleR, or pharmaceuticals that mimic this effect, may have potential as a geroprotective intervention.

The Potential Effects of Isoleucine Restricted Diet on Cognitive Impairment in High-Fat-Induced Obese Mice via Gut Microbiota-Brain Axis.

SCOPE: Obesity induced by high-fat diet (HFD) can cause lipid metabolism disorders and cognitive impairment. Isoleucine restriction can effectively alleviate lipid metabolism disorders caused by HFD but the underlying mechanisms on cognition are unknown. METHODS AND RESULTS: Thirty 3-month-old C57BL/6J mice are divided equally into the following groups: the control group, HFD group, and HFD Low Ile group (67% reduction in isoleucine in high fat feeds). Feeding for 11 weeks with behavioral testing, which shows that isoleucine restriction attenuates HFD-induced cognitive dysfunction. As observed by staining, isoleucine restriction inhibits HFD-induced neuronal damage and microglia activation. Furthermore, isoleucine restriction significantly increases the relative abundance of gut microbiota, decreases the proportion of Proteobacteria, and reduces the levels of lipopolysaccharide (LPS) in serum and brain. Isoleucine restriction reduces protein expression of TLR4/MyD88/NF-κB signaling pathway and inhibits upregulation of proinflammatory cytokine genes and protein expression in mice brain. In addition, isoleucine restriction significantly improves insulin resistance in the brain as well as synaptic plasticity impairment. CONCLUSION: Isoleucine restriction may be a potential intervention to reduce HFD-induced cognitive impairment by altering gut microbiota, reducing neuroinflammation, insulin resistance, and improving synaptic plasticity in mice brain.

Transcranial direct-current stimulation confers neuroprotection by regulating isoleucine-dependent signalling after rat cerebral ischemia-reperfusion injury.

Isoleucine is a branched chain amino acid. The role of isoleucine in cerebral ischemia-reperfusion injury remains unclear. Here, we show that the concentration of isoleucine is decreased in cerebrospinal fluid in a rat model of cerebral ischemia-reperfusion injury, the rat middle cerebral artery occlusion (MCAO). To our surprise, the level of intraneuronal isoleucine is increased in an in vitro model of cerebral ischemia injury, the oxygen-glucose deprivation (OGD). We found that the increased activity of LAT1, an L-type amino acid transporter 1, leads to the elevation of intraneuronal isoleucine after OGD insult. Reducing the level of intraneuronal isoleucine promotes cell survival after cerebral ischemia-reperfusion injury, but supplementing isoleucine aggravates the neuronal damage. To understand how isoleucine promotes ischemia-induced neuronal death, we reveal that isoleucine acts upstream to reduce the expression of CBFB (core binding factor ß, a transcript factor involved in cell development and growth) and that the phosphatase PTEN acts downstream of CBFB to mediate isoleucine-induced neuronal damage after OGD insult. Interestingly, we demonstrate that direct-current stimulation reduces the level of intraneuronal isoleucine in cortical cultures subjected to OGD and that transcranial direct-current stimulation (tDCS) decreases the cerebral infarct volume of MCAO rat through reducing LAT1-depencent increase of intraneuronal isoleucine. Together, these results lead us to conclude that LAT1 over activation-dependent isoleucine-CBFB-PTEN signal transduction pathway may mediate ischemic neuronal injury and that tDCS exerts its neuroprotective effect by suppressing LAT1 over activation-dependent signalling after cerebral ischemia-reperfusion injury.

Methionine-isoleucine dichotomy at a key position in scorpion toxins inhibiting voltage-gated potassium channels.

Previous studies have identified some key amino acid residues in scorpion toxins blocking potassium channels. In particular, the most numerous toxins belonging to the α-KTx family and affecting voltage-gated potassium channels (KV) present a conserved K-C-X-N motif in the C-terminal half of their sequence. Here, we show that the X position of this motif is almost always occupied by either methionine or isoleucine. We compare the activity of three pairs of peptides that differ just by this residue on a panel of KV1 channels and find that toxins bearing methionine affect preferentially KV1.1 and 1.6 isoforms. The refined K-C-M/I-N motif stands out as the principal structural element of α-KTx conferring high affinity and selectivity to KV channels.

Glutamine, proline, and isoleucine support maturation and fertilisation of bovine oocytes.

Successful in-vitro production of bovine embryos relies on meiotic maturation of oocytes in vitro (IVM) before they can be fertilised. High levels of IVM are currently achieved using a complex medium that contains all 20 common amino acids, namely TCM199, but can also be achieved using a simple inorganic salt solution containing non-essential amino acids, proline, and glutamine. Further simplification of the amino acid content of medium used for IVM could lead to a more defined medium that provides reproducible IVM. The aim of this study was, therefore, to determine the minimal amino acid requirements for bovine oocyte nuclear maturation, as measured by progression to metaphase II (MII) of meiosis. Supplementation of a simple medium composed of inorganic salts (M1 medium) with multiple amino-acid combinations showed that M1 containing glutamine, proline, and isoleucine resulted in nuclear maturation comparable to that of TCM199 (57.4 ± 3.4% vs 67% ± 1.7%, respectively) but was reduced when cystine (Cys2) to that seen with M1 alone (38.0 ± 2.2%). Viability of oocytes matured in this simplified medium was equal to those matured in TCM199 since the same proportion of zygotes with 2 pronuclei were observed following fertilisation in medium containing no amino acids (33.9 ± 6.5% vs 33.3 ± 3.6%, respectively). Addition of glutamine, proline and isoleucine to fertilisation medium also increased the proportion of zygotes but did not increase blastocyst development rates. Thus, a defined medium containing only glutamine, proline and isoleucine is sufficient for oocyte maturation and successful fertilisation.

Bioefficacy of isolated compound l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester from entomopathogenic actinobacteria Actinokineospora fastidiosa against agricultural insect pests, human vector mosquitoes, and antioxidant activities.

Spodoptera litura and Helicoverpa armigera are polyphagous pests of agricultural crops in the Asian tropics since these pests have been responsible for massive crop and carry economic losses and low commodity production. At the same time, mosquitoes are vectors for numerous dreadful diseases, which is the most important group of insect for their public health concern. Using synthetic insecticides to control the pests can lead to contamination of land surface and groundwater and impact beneficial soil organisms and nontarget species. Applications of bioactive compounds are received considerable attention across the world as alternatives to synthetic insecticides. In the current study, actinobacterial secondary metabolite was isolated from Actinokineospora fastidiosa for the first time. The effect of actinobacterial metabolite (l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester) was assessed on agricultural pest S. litura and H. armigera, mosquito vectors larvae Ae. aegypti, An. stephensi, and Cx. quinquefasciatus. The bioactive fraction was characterized through UV, FTIR, and NMR analysis. GC-MS analyses reveal the existence of a bioactive compound with a respective retention time of 19.740 responsible for larvicidal activity. The bioefficacy of the l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester showed high antifeedant activity on S. litura (80.80%) and H. armigera (84.49%); and larvicidal activity on S. litura (82.77%) and H. armigera (88.00%) at 25 µg/mL concentration, respectively. The effective LC50 values were 8.07 µg/mL (F = 2.487, r2 = 0.988, P ≤ 0.05) on S. litura and 7.53 µg/mL (F = 123.25, r2 = 0.951, P ≤ 0.05) on H. armigera. The mosquito larvicidal effect of isolated compounds l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester treated against Ae. aegypti, An. stephensi, and Cx. quinquefasciatus the obtained percentage mortality was 96.66, 83.24, 64.52, 50.00, and 40.00% against Ae. aegypti; 100.00, 86.22, 73.81, 65.37, and 56.24% against An. stephensi; 100.00, 90.00, 76.24, 68.75, and 56.23% against Cx. quinquefasciatus. The mosquito larvae of Ae. aegypti obtained LC50 value was 13.25 µg/mL, F = 28.50, r2 = 0.90; on An. stephensi was 10.19 µg/mL, F = 15.55, r2 = 0.83, and Cx. quinquefasciatus was 9.68 µg/mL, F = 20.00, r2 = 0.87. Furthermore, l-isoleucine-, N-allyloxycarbonyl-, and dodecyl ester-treated larvae produced significant pupicidal activity on S. litura (62.71%) and H. armigera (66.50%) at 25 µg/mL, along with increased larval and pupal duration as compared to control group. Treated larvae revealed obliteration in the midgut epithelial cells and destruction of microvilli was noticed as compared to the control. The isolated compounds l-isoleucine, N-allyloxycarbonyl-, and dodecyl ester did not produce any significant mortality on zebrafish embryos in all tested concentrations on biosafety observation. The potential microbial isolated molecule may fit well in IPM programs. Since the risk to human health, the environment, etc. is unknown.

Understanding the interactive effects of dietary leucine with isoleucine and valine in the modern commercial broiler.

A study was conducted to understand the relationship among dietary branched-chain amino acids (BCAA) on the performance of Ross 344 × 708 male broilers. A total of 2,592 d-old male chicks were randomly placed into 144-floor pens according to a 23 full factorial central composite design (CCD) with 20 treatments (14 treatments and 6 center points). Each treatment consisted of varying digestible Ile:Lys (52 to 75), Val:Lys (64 to 87), and Leu:Lys (110 to 185) ratios. Birds and feed were weighed at 20 and 34 d of age to determine body weight gain (BWG), feed intake, and feed conversion ratio (FCR). At 35 d of age, feather amino acid composition and carcass characteristics were evaluated. Data were analyzed as CCD using the surface response option of JMP v. 15. Body weight gain (1,332 g; P < 0.001; R2 = 0.93) and FCR (1.54; P = 0.002; R2 = 0.88) were optimized at the lowest Leu:Lys ratio (110) with moderate Val:Lys (78 to 79) and Ile:Lys (65 to 66) ratios. Poorer BWG and FCR were observed as Leu:Lys ratio increased while increasing Val:Lys and Ile:Lys ratios alleviated the poor performance. Carcass (71.5%; P = 0.031; R2 = 0.76) and breast yield (26.7%; P < 0.001; R2 = 0.96) were maximized at the highest Leu:Lys ratio. This effect was complemented by increasing Ile:Lys ratio beyond 68. Lower Ile:Lys and Val:Lys ratios were required to maximize carcass and breast yield at the lowest Leu:Lys ratio. However, this strategy yielded less meat than providing a high Leu:Lys ratio diet. Dietary BCAA had little effect on altering the composition of feather protein and amino acid (P > 0.10). These results suggest that optimum BCAA ratios to Lys may vary depending on response criteria and demonstrate the importance of maintaining proper Val and Ile ratios centered on dietary Leu. Live performance can be optimized in diets with low Leu:Lys ratios; however, meat yield can be enhanced by increasing dietary Leu:Lys along with Ile:Lys ratios.

Oudemansiella raphanipies Polysaccharides Improve Lipid Metabolism Disorders in Murine High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease.

Oudemansiella raphanipies, also called "Edible Queen," is a mushroom that possesses antioxidant, anti-inflammatory, anti-bacterial, anti-tumor and immunity-enhancing properties. The present study aimed to assess the effect of O. raphanipies-derived polysaccharide (ORPS) on the progression of nonalcoholic fatty liver disease (NAFLD) in mice. We studied the structure of ORPS-1 by high-performance gel permeation chromatography (HPGPC), ion chromatography-mass spectrometry (GC-MS), and Fourier transform-infrared spectroscopy (FT-IR). ORPS-1 mainly comprised galactose, fucose, glucose, mannose, and xylose, following an 18:6:6:4:1 molar ratio. In addition, the therapeutic effect as well as a potential mechanism of ORPS-1 in the treatment of high-fat diet (HFD)-induced NAFLD were investigated. The results showed that ORPS-1 improved liver function, ameliorated liver steatosis, and reduced lipid droplet accumulation in HFD mice. A metabolomics approach with GC-MS was utilized to evaluate liver improvement by ORPS-1 treatment. Principal component analysis showed that liver metabolic profiling was significantly altered by HFD feeding or treatment with an intermediate dose of ORPS-1 in mice compared with that of control mice. By investigating the metabolic pathways with identified biomarkers, various pathways such as steroid biosynthesis, valine, leucine, and isoleucine biosynthesis, glycerol phospholipid metabolism, glyceride metabolism, and arginine and proline metabolism in HFD mice were observed to be significantly influenced by ORPS-1 treatment. The results indicate ORPS-1 metabolic effects on liver tissues, provide methods for assessing the molecular impact of ORPS-1 on NAFLD, and suggest the potential mechanism underlying its health benefits.

Phenotype microarray analysis reveals the biotransformation of Fusarium oxysporum f.sp. lycopersici influenced by Bacillus subtilis PBE-8 metabolites.

Herein, Bacillus subtilis PBE-8's biocontrol efficacy was evaluated through physiological and metabolic approaches against Fusarium oxysporum f.sp. lycopersici (FOL). The study elaborates on PBE-8's cell-free filtrate (CFF) antifungal activity through mycelial growth inhibition, metabolite profiling, and substrates utilization patterns. Additionally, under different CFF concentrations, reduction in spore count (94%-55%), biomass (50%), and cytoplasmic bulbous protrusions in mycelia were also observed. Furthermore, the effect of bacterial CFF on FOL metabolism was confirmed through GC-MS. CFF suppresses the concentration of aliphatic amino acids like L-valine, L-leucine, L-Isoleucine, glycine, and fatty acids such as linoleic acid and α- linolenic acid during the co-culturing conditions, which are essential for pathogenicity and resistance against host's systemic acquired resistance. The phenotype microarray assay revealed that CFF-treated FOL shows phenotype loss in 507 (56.58%) out of 896 substrates. Among 507, twenty-seven substrates showed significant phenotype loss, among which four substrates such as L-glutamic acid, L-glutamine, ammonia, and L-arginine are common in different crucial metabolic pathways of FOL, like alanine, aspartate, and glutamate metabolism, arginine and proline, carbon metabolism, arginine biosynthesis, nitrogen metabolism, amino-acyl tRNA synthesis, and biosynthesis of amino acids. The results suggest that PBE-8 CFF has certain antifungal metabolites that hinder the fungal metabolic pathways.

Toxicity effects of chlorantraniliprole in zebrafish (Danio rerio) involving in liver function and metabolic phenotype.

Chlorantraniliprole (CAP), a representative bisamide insecticide, is widely used in rice fields around the world, posing potential toxicity risks to aquatic organisms. In this study, we examined the effects of exposure to CAP on growth and metabolic phenotype of zebrafish (Danio rerio) and oxidative stress and apoptosis in the liver of zebrafish (Danio rerio). First, we identified that CAP had a low bioaccumulation in zebrafish. Subsequently, growth phenotype analysis revealed that CAP could significantly increase liver weight and liver index in zebrafish. In addition, we found that CAP exposure could cause significant changes in indicators of oxidative stress, resulting in a significant increase in the content of malondialdehyde (MDA), causing oxidative stress in the liver of zebrafish. Meanwhile, the expression levels of apoptosis-related genes were also significantly changed and apoptosis was promoted in the liver of zebrafish with CAP exposure. Importantly, the results of metabolomics analysis shown that CAP exposure could significantly disrupt the metabolic phenotype of zebrafish, interfering with multiple metabolic pathways, mainly including valine, leucine and isoleucine biosynthesis and degradation, alanine, aspartate and glutamate metabolism and d-glutamine and D-glutamate metabolism. Last but not least, correlation analysis identified strong links between changes in liver function involving oxidative stress and apoptosis and changes in metabolic phenotype of zebrafish following CAP exposure. In brief, these results indicate that potential environmental risks of CAP to aquatic organisms should receive more attention.

Transcriptome Analysis Reveals the Algicidal Mechanism of Brevibacillus laterosporus against Microcystis aeruginosa through Multiple Metabolic Pathways.

It is widely accepted that eutrophication has played an important role in the formation of harmful cyanobacterial blooms in recent decades, which impacts water quality and ecological environment and causes huge economic losses. Algicidal bacteria have a promising application prospect in controlling cyanobacterial blooms in aquaculture water. Here, the process of the algicidal bacterium Brevibacillus laterosporus strain Bl-zj acting on Microcystis aeruginosa was explored using transcriptome analysis to elucidate the algicidal mechanism. The results of the co-culture of bacterium and alga showed a strong alga-lysing effect of B. laterosporus against M. aeruginosa with an extreme morphology deformation of the algal cells. A total of 2744 differentially expressed genes of B. laterosporus were identified, which were mainly involved in the metabolism of amino acid, carbohydrate, and lipid. In the co-cultured group, the expression of genes mainly enriched in valine, leucine and isoleucine degradation, and fatty acid degradation were significantly increased. However, the expression of the genes related to ribosome were mainly inhibited. Transcriptome analysis showed that B. laterosporus obtained ATP and energy by the degradation of valine, leucine, isoleucine, and fatty acids, and destroyed algal cells by efflux pump transporters, secretion of hydrolytic enzymes, antibiotics, proteases, and other secondary metabolites, resulting in algal death and achieving the algicidal effect.

Intervention with isoleucine or valine corrects hyperinsulinemia and reduces intrahepatic diacylglycerols, liver steatosis, and inflammation in Ldlr-/-.Leiden mice with manifest obesity-associated NASH.

Non-alcoholic steatohepatitis (NASH) is associated with a disturbed metabolism in liver, insulin resistance, and excessive accumulation of ectopic fat. Branched-chain amino acids (BCAAs) may beneficially modulate hepatic lipids, however, it remains unclear whether individual BCAAs can attenuate already established NASH and associated oxidative-inflammatory stress. After a 26 weeks run-in on fast food diet (FFD), obese Ldlr-/-.Leiden mice were treated for another 12 weeks with either valine or isoleucine (3% of FFD) and then compared to FFD controls. Valine and isoleucine did not affect obesity, dyslipidemia, gut permeability, or fecal fatty acid excretion, but significantly reduced hyperinsulinemia. Valine and isoleucine reduced ALT, CK18-M30, and liver steatosis with a particularly pronounced suppression of the microvesicular component (-61% by valine and -71% by isoleucine). Both BCAAs decreased intrahepatic diacylglycerols and 4-hydroxynonenal immunoreactivity, a marker for oxidative stress-induced lipid peroxidation. Functional genomics analysis demonstrated that valine and isoleucine affected BCAA metabolism genes, deactivated master regulators of anabolic pathways related to steatosis (e.g., SREBPF1), and activated master regulators of mitochondrial biogenesis (e.g., PPARGC1A) and lipid catabolism (e.g., ACOX1, AMPK). This correction of critical metabolic pathways on gene expression level was accompanied by a significant decrease in histological liver inflammation, and suppression of FFD-stimulated cytokine and chemokine proteins KC/CXCL1, MCP-1/CCL2, and MIP-2/CXCL2 and their pathways. In conclusion, dietary intervention with either valine or isoleucine corrected liver diacylglycerols, gene expression of multiple metabolic processes, and reduced NASH histology with profound hepatoprotective effects on oxidative stress and inflammatory proteins.

The effect of standardized ileal digestible isoleucine:lysine in diets containing 20% dried distillers grains with solubles on finishing pig performance and carcass characteristics.

In order to determine the standardized ileal digestible (SID) Ile:Lys requirement in finishing diets containing 20% DDGS, a 56-d study was conducted utilizing 2,268 pigs (DNA 600 x Topigs Norsvin 70, initially 82.3 kg). A total of six dietary treatments were fed, including a standard corn-soybean meal (SBM) diet and five diets containing 20% DDGS with SID Ile:Lys ratios of 55%, 60%, 65%, 70%, and 75%. Dietary treatments were assigned to pens, incompletely balancing for previous treatment, with each treatment being replicated 14 times. Pair-wise comparisons were used to evaluate the impact of dietary treatment on performance and carcass traits, whereas single degree of freedom orthogonal polynomials were used to evaluate dose response of SID Ile:Lys in 20% DDGS diets. Increasing the SID Ile:Lys ratio in diets containing 20% DDGS did not impact pig growth performance criteria in a quadratic or linear fashion (P ≥ 0.18). However, increasing the SID Ile:Lys ratio in 20% DDGS diets resulted in decreased back fat (BF; Quadratic, P = 0.01), increased loin depth (Quadratic, P = 0.03), and tended to increase percent lean (Quadratic, P = 0.07) with optimal carcass parameters occurring when 65% SID Ile:Lys was supplied in 20% DDGS diets. Pigs fed the corn-SBM diet had a similar final body weight (BW; P = 0.26) and cumulative average daily gain (ADG; P = 0.12) compared to pigs fed a 20% DDGS diet containing 70% SID Ile:Lys ratio and 3% greater cumulative average daily feed intake (ADFI) compared to pigs receiving diets with SID Ile:Lys ratios of 65% and 75% (P ≤ 0.01). In conclusion, these results suggest that when feeding 20% DDGS in late finishing swine diets, a SID Ile:Lys ratio of 70% should be utilized when attempting to achieve similar overall growth performance relative to a corn-SBM diet.

The use of corn by-products, such as dried distillers grains with solubles (DDGS), from the ethanol industry in swine rations is a common practice when economical. However, the inclusion of these by-products can potentially impact the nutrient requirements of pigs. It is important to understand how one should adjust diets to meet these changes in nutrient requirements and to minimize the reduction in growth performance. Previous research has indicated that the excess of a specific amino acid, leucine, can negatively impact the performance of pigs through the increased breakdown of other amino acids, more specifically valine and isoleucine. This increased breakdown of valine and isoleucine can result in inadequate amounts being available for protein deposition and, therefore, reduce growth performance. In the present study, late finishing pigs fed a diet containing 20% DDGS required a ratio of 70% between the amino acids isoleucine and lysine, on a digestible basis, to perform similarly to pigs receiving a standard corn soybean meal diet. Further research is necessary to more accurately define the optimal dietary isoleucine level for finishing pigs when fed 20% DDGS.

Immunometabolism - The Role of Branched-Chain Amino Acids.

Immunometabolism has been the focus of extensive research over the last years, especially in terms of augmenting anti-tumor immune responses. Regulatory T cells (Tregs) are a subset of CD4+ T cells, which have been known for their immunosuppressive roles in various conditions including anti-tumor immune responses. Even though several studies aimed to target Tregs in the tumor microenvironment (TME), such approaches generally result in the inhibition of the Tregs non-specifically, which may cause immunopathologies such as autoimmunity. Therefore, specifically targeting the Tregs in the TME would be vital in terms of achieving a successful and specific treatment. Recently, an association between Tregs and isoleucine, which represents one type of branched-chain amino acids (BCAAs), has been demonstrated. The presence of isoleucine seems to affect majorly Tregs, rather than conventional T cells. Considering the fact that Tregs bear several distinct metabolic features in the TME, targeting their immunometabolic pathways may be a rational approach. In this Review, we provide a general overview on the potential distinct metabolic features of T cells, especially focusing on BCAAs in Tregs as well as in their subtypes.

Protective Mechanism of Leucine and Isoleucine against H2O2-Induced Oxidative Damage in Bovine Mammary Epithelial Cells.

Leucine and isoleucine possess antioxidative and anti-inflammatory properties. However, their underlying protective mechanisms against oxidative damage remain unknown. Therefore, in this study, the protective mechanism of leucine and isoleucine against H2O2-induced oxidative damage in a bovine mammary epithelial cell lines (MAC-T cells) were investigated. Briefly, MAC-T cells exposed or free to H2O2 were incubated with different combinations of leucine and isoleucine. The cellular relative proliferation rate and viability, oxidative stress indicators, and inflammatory factors were determined by specific commercial kits. The genes related to barrier functions was measured by real-time quantitative PCR. The protein expression differences were explored by 4D label-free quantitative proteomic analyses and validated by parallel reaction monitoring. The results revealed that leucine and isoleucine increased cell proliferation, total antioxidant status (TAS), and the relative mRNA expression of occludin, as well as decreased malondialdehyde (MDA), total oxidant status (TOS)/TAS, IL-6, IL-1ß, and TOS. When leucine and isoleucine were combined, MDA, TOS/TAS, and the relative mRNA expression levels of claudin-1, occludin, and zonula occludens-1 increased when compared to leucine or isoleucine alone. Proteomics analyses revealed that leucine significantly upregulated the propanoate metabolism; valine, leucine, and isoleucine degradation; and thermogenesis pathways, whereas isoleucine significantly upregulated the peroxisome and propanoate metabolism pathways. In conclusion, leucine protected MAC-T cells from H2O2-induced oxidative stress by generating more ATP to supplement energy demands, and isoleucine improved the deficit in peroxisome transport and promoted acetyl-CoA production. The findings of this study enhance our understanding of the protective mechanisms of leucine and isoleucine against oxidative damage.

A Mixture of Valine and Isoleucine Restores the Growth of Protein-Restricted Pigs Likely through Improved Gut Development, Hepatic IGF-1 Pathway, and Plasma Metabolomic Profile.

Valine (Val) alone or in combination with isoleucine (Ile) improves the growth under severe protein restriction; however, the underlying mechanisms remain unknown. In this study, we assessed whether Val/Ile-induced growth in protein-restricted pigs is associated with changes in gut development, hepatic insulin-like growth factor 1 (IGF-1) production, and blood metabolomics. Forty piglets were assigned to five dietary groups: positive control (PC) with standard protein content; low protein (LP) with very low protein content; and LP supplemented with Val (LPV), Ile (LPI), and Val and Ile (LPVI). LPVI reversed the negative effects of VLP diets on growth and gut morphology. Both LPV and LPVI restored the reduced transcript of IGF-1 while decreasing the transcript of insulin-like growth factor binding protein 1 (IGFBP1) in the liver. LPV and LPVI recovered the reduced plasma Val, glycine, and leucine concentrations, which were positively correlated with improved gut morphology and the hepatic IGF-1 gene expression and negatively correlated with hepatic IGFBP1 mRNA abundance. In conclusion, supplementation with a combination of Val and Ile into the VLP diets restored the decreased growth performance of pigs fed with these diets likely through improved gut development, hepatic IGF-1 expression and bioavailability, and plasma metabolomics profile.

The effect of Bacillus coagulans Unique IS-2 supplementation on plasma amino acid levels and muscle strength in resistance trained males consuming whey protein: a double-blind, placebo-controlled study.

PURPOSE: The aim of the present study was to assess the effect of Bacillus coagulans Unique IS-2 supplementation on absorption and utilization of protein in resistance-trained males. METHODS: In this double blind, placebo-control trial, resistance-trained males (21.08 ± 2.84 years) were randomized to consume, either 20 g of whey protein powder {80% whey protein concentrate (WPC80), amounting to 15.4 g protein} with 2 billion CFU Bacillus coagulans Unique IS-2 (supplemental group) or 20 g of whey protein powder and lactose instead of Bacillus coagulans (placebo group) once daily for 60 days with a controlled resistance exercise protocol. The whey protein concentrate (WPC-80) given to both groups had a lactose content of 6.8%. Plasma-free amino acids (PFAAs) were determined at baseline, at 30 and 60 days of supplementation. Muscle strength, hypertrophy, VO2 max, and body composition, and other biochemical parameters were assessed at baseline and end line. RESULTS: A positive effect of probiotic Bacillus coagulans Unique IS-2 supplementation was observed on protein absorption as evidenced by an increase in total PFAA by + 16.1% (p = 0.004). Branched chain amino acids (BCAA) comprising isoleucine (p = 0.016), leucine (p = 0.001), and valine (p = 0.002) were increased by + 33.1% in ITT analysis as compared to placebo after 60 days. At 30 days an increase in isoleucine by + 35% (p = 0.113), leucine by + 43% (p = 0.032), and valine by + 32% (p = 0.017) was observed in ITT analysis. Probiotic effect was shown on exercise performance as evidenced by an increase in one RM of leg press and vertical jump power by + 16.61% (p = 0.024) and + 7.86% (p = 0.007), respectively. CONCLUSION: Significantly increased absorption of BCAA with supplementation of B. coagulans Unique IS-2 along with whey protein and improvement in leg press and vertical jump power was noted indicating the positive effect of the probiotic on muscle power in the lower body. TRIAL REGISTRATION NUMBER: CTRI/2017/03/008117; Date:16.03.2017.

Assessment of antidiabetic effect of 4-HIL in type 2 diabetic and healthy Sprague Dawley rats.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia and insulin resistance. 4-hydroxyisoleucine (4-HIL) is a non-proteinogenic amino acid isolated from the fenugreek seeds and has enormous pharmacological activities. The present study was undertaken to investigate the antihyperglycemic effect of 4-HIL in streptozotocin (STZ)-induced diabetic rats. Moreover, its toxicity was evaluated in vitro and in vivo employing human embryonic kidney cells (HEK-293) and healthy rats, respectively. In experiment 1, STZ-induced diabetic male rats were subjected to an oral treatment of 4-HIL (100 mg/kg), while experiment 2 deals with the effects of 4-HIL on healthy male and female rats following oral administration. The treatment (experiment 1) declined the elevated blood glucose level, feed intake, and increased body weight(s). Additionally, blood glucose impairment was improved as observed by OGTT and IPGT tests. Pancreatic histopathology revealed mild changes in the 4-HIL group. Moreover, experiment 2 showed increased body weight, normal blood glucose levels (male-106.06 ± 7.49 mg/dl and female-100.06 ± 14.69 mg/dL), hematological parameters, and histopathological profiles in the treatment group. 4-HIL did not affect the viability of HEK-293 cells, and no signs of toxicity were observed in healthy rats. Therefore, the study concludes that 4-HIL has potential antihyperglycemic activity without any toxic effects.