Safety, Tolerability, and Biologic Activity of AXA1125 and AXA1957 in Subjects With Nonalcoholic Fatty Liver Disease.

INTRODUCTION: AXA1125 and AXA1957 are novel, orally administered endogenous metabolic modulator compositions, specifically designed to simultaneously support multiple metabolic and fibroinflammatory pathways associated with nonalcoholic fatty liver disease (NAFLD). This study assessed safety, tolerability, and biologic activity of AXA1125 and AXA1957 in NAFLD. METHODS: In this multicenter, 16-week, placebo-controlled, single-blind, randomized clinical study in subjects with NAFLD stratified by type 2 diabetes, AXA1125 24 g, AXA1957 13.5 g or 20.3 g, or placebo was administered twice daily. Key metabolism (MRI-proton density fat fraction [MRI-PDFF] and homeostasis model assessment of insulin resistance [HOMA-IR]) and fibroinflammation markers (alanine aminotransferase [ALT], corrected T1 [cT1], keratin-18 [K-18] M65, and N-terminal type III collagen propeptide [Pro-C3]) were evaluated. Safety outcomes included adverse events and standard laboratory assessments. RESULTS: Baseline characteristics of the 102 enrolled subjects, including 40 with type 2 diabetes, were consistent with presumed nonalcoholic steatohepatitis. AXA1125 showed consistently greater biologic activity than AXA1957 or placebo. Week 16 changes from baseline with AXA1125 vs placebo: MRI-PDFF -22.9% vs -5.7%, HOMA-IR -4.4 vs +0.7, ALT -21.9% vs -7.2%, K-18 M65 -13.6% vs +20.1%, cT1 -69.6 vs +18.3 ms (P < 0.05), and Pro-C3 -13.6% vs -3.6%. Week 16 changes from baseline with AXA1957 20.3 g: MRI-PDFF -8.1%, HOMA-IR +8.4, ALT -20.7%, K-18 M65 6.6%, cT1 -34.7 ms, and Pro-C3 -15.6%. A greater proportion of subjects treated with AXA1125 achieved clinically relevant thresholds: ≥30% MRI-PDFF, ≥17-IU/L ALT, and ≥80-ms cT1 reductions at week 16. Study products were safe and well tolerated with stable lipid and weight profiles. DISCUSSION: Both compositions showed multitargeted activity on relevant NAFLD pathways. AXA1125 demonstrated the greatest activity over 16 weeks, warranting continued clinical investigation in nonalcoholic steatohepatitis subjects.

Spray-Dried Inhalable Powder Formulations of Therapeutic Proteins and Peptides.

Respiratory diseases are among the leading causes of morbidity and mortality worldwide. Innovations in biochemical engineering and understanding of the pathophysiology of respiratory diseases resulted in the development of many therapeutic proteins and peptide drugs with high specificity and potency. Currently, protein and peptide drugs are mostly administered by injections due to their large molecular size, poor oral absorption, and labile physicochemical properties. However, parenteral administration has several limitations such as frequent dosing due to the short half-life of protein and peptide in blood, pain on administration, sterility requirement, and poor patient compliance. Among various noninvasive routes of administrations, the pulmonary route has received a great deal of attention and is a better alternative to deliver protein and peptide drugs for treating respiratory diseases and systemic diseases. Among the various aerosol dosage forms, dry powder inhaler (DPI) systems appear to be promising for inhalation delivery of proteins and peptides due to their improved stability in solid state. This review focuses on the development of DPI formulations of protein and peptide drugs using advanced spray drying. An overview of the challenges in maintaining protein stability during the drying process and stabilizing excipients used in spray drying of proteins and peptide drugs is discussed. Finally, a summary of spray-dried DPI formulations of protein and peptide drugs, their characterization, various DPI devices used to deliver protein and peptide drugs, and current clinical status are discussed.

Improving performance of finishing pigs with added valine, isoleucine, and tryptophan: validating a meta-analysis model.

Based on results of a recent meta-analysis, we hypothesized that increased dietary Val, Ile, or Trp could correct possible amino acid interactions because of excess Leu in diets containing high levels of corn protein, namely dried distiller's grains with solubles (DDGS). A total of 1,200 pigs (PIC TR4 × (Fast LW × PIC L02); initially 33.6 ± 0.6 kg) were used in a 103-d study. The 6 dietary treatments were corn-soybean meal (SBM)-DDGS-based as follows: (1) high SBM and low level of l-Lys HCl (HSBM), (2) high l-Lys HCl and moderate Ile, Val, Trp (AA above NRC 2012 estimates; NC), (3) moderate l-Lys HCl and high Ile, Val, and Trp (PC), and PC with either increased (4) L-Val (PC+Val), (5) L-Ile (PC+Ile), or (6) L-Trp (PC+Trp). Pigs fed the NC diet were predicted to have the poorest average daily gain (ADG), the PC diet to be intermediate, and pigs fed the HSBM, PC+Val, PC+Ile, and PC+Trp have the same and highest predicted ADG. In the grower period (34 to 90 kg), ADG was greater (Ρ < 0.05) for the pigs fed HSBM and PC+Val diets than the NC with pigs fed other diets intermediate. Pigs fed HSBM were more (Ρ < 0.05) efficient (G:F) than the NC and PC with pigs fed other diets intermediate. In the finisher period (90 to 136 kg), ADG was greater (Ρ < 0.05) for pigs fed PC+Ile than that of the NC with pigs fed other diets intermediate. Pigs fed PC+Val had greater (Ρ < 0.05) average daily feed intake (ADFI) than the NC with pigs fed other diets intermediate. However, PC+Ile pigs were more (Ρ < 0.05) efficient than PC+Val with pigs fed other diets intermediate. Overall, ADG was greater (Ρ < 0.05) for pigs fed HSBM, PC+Val, and PC+Ile diets than the NC with pigs fed other diets intermediate. Pigs fed the PC+Val diet had greater (Ρ < 0.05) ADFI than the NC with pigs fed other diets intermediate. No differences were detected between treatments for overall G:F or other carcass characteristics. In conclusion, increasing Val or Ile in high l-Lys-HCl-DDGS-based diets improved growth performance compared with pigs fed diets containing high levels of l-Lys HCl without added Val and Ile. These results present evidence that the recently developed meta-analysis can predict the relative differences in overall ADG for pigs fed the NC, PC, PC+Val, and PC+Ile diets; however, the predicted G:F was less accurate. The data demonstrate that the negative effects of high Leu concentrations in corn-DDGS-based diets can be reversed by increasing the ratios of Val and Ile relative to Lys.

l-Isoleucine Administration Alleviates DSS-Induced Colitis by Regulating TLR4/MyD88/NF-κB Pathway in Rats.

Inflammatory bowel disease (namely, colitis) severely impairs human health. Isoleucine is reported to regulate immune function (such as the production of immunoreactive substances). The aim of this study was to investigate whether l-isoleucine administration might alleviate dextran sulfate sodium (DSS)-induced colitis in rats. In the in vitro trial, IEC-18 cells were treated by 4 mmol/L l-isoleucine for 12 h, which relieved the decrease of cell viability that was induced by TNF-α (10 ng/ml) challenge for 24 h (P <0.05). Then, in the in vivo experiment, a total of 44 Wistar rats were allotted into 2 groups that were fed l-isoleucine-supplemented diet and control diet for 35 d. From 15 to 35 d, half of the rats in the 2 groups drank the 4% DSS-adding water. Average daily gain, average daily feed intake and feed conversion of rats were impaired by DSS challenge (P <0.05). Drinking the DSS-supplementing water also increased disease activity index (DAI) and serum urea nitrogen level (P <0.05), shortened colonic length (P <0.05), impaired colonic enterocyte apoptosis, cell cycle, and the ZO-1 mRNA expression (P <0.05), increased the ratio of CD11c-, CD64-, and CD169-positive cells in colon (P <0.05), and induced extensive ulcer, infiltration of inflammatory cells, and collagenous fiber hyperplasia in colon. However, dietary l-isoleucine supplementation attenuated the negative effect of DSS challenge on growth performance (P <0.05), DAI (P <0.05), colonic length and enterocyte apoptosis (P <0.05), and dysfunction of colonic histology, and downregulated the ratio of CD11c-, CD64-, and CD169-positive cells, pro-inflammation cytokines and the mRNA expression of TLR4, MyD88, and NF-κB in the colon of rats (P <0.05). These results suggest that supplementing l-isoleucine in diet improved the DSS-induced growth stunting and colonic damage in rats, which could be associated with the downregulation of inflammation via regulating TLR4/MyD88/NF-κB pathway in colon.

Isoleucine improved growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli × Leiocassis longirostris.

This study was performed to determine effects of dietary isoleucine (Ile) on growth performance, and intestinal immunological and physical barrier function of hybrid catfish Pelteobagrus vachelli × Leiocassis longirostris. Six hundred and thirty fish (33.11 ± 0.09 g) were randomly divided into seven experimental groups with three replicates each, and respectively fed seven diets with 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0 g Ile kg-1 diets for 8 weeks. The results showed improvement of growth performance, feed intake, feed utilization, relative gut length (RGL), and intestinal fold height and width by dietary Ile (P < 0.05). Meanwhile, dietary Ile (12.5 g kg-1 diet) improved the activities of lysozyme (LZM), acid phosphatase, alkaline phosphatase and the contents of complement 3 (C3), C4, and immunoglobulin M (IgM) (P < 0.05). The c-type-lectin, c-LZM, g-LZM, and hepcidin mRNA expressions in the intestine were up-regulated in fish fed diets with 10.0-20.0 g Ile kg-1 diet (P < 0.05). Dietary Ile (10.0-12.5 g Ile kg-1 diet) increased intestinal ß-defensin mRNA expression partially in association with Sirt1/ERK/90RSK signaling pathway. Dietary Ile (12.5-15.0 g Ile kg-1 diet) decreased oxidative damage and improved antioxidant ability by increasing activities and expressions of superoxide dismutase, glutathione peroxidase, and glutathione reductase, glutathione-S-transferase (P < 0.05). The occludin, ZO-1, ZO-2, claudin3, and claudin 7 mRNA expressions in the intestine were up-regulated in fish fed diets with 10.0 and 12.5 g Ile kg-1 diet (P < 0.05), whereas the myosin light chain kinase gene expression was decreased in fish fed diets with 7.5-17.5 g Ile kg-1 diet. Dietary Ile (10-12.5 g Ile kg-1 diet) decreased apoptotic responses by reducing the expression of caspase3 and caspase 9 via the AKT/TOR signaling pathway. Based on the quadratic regression analysis of PWG, the dietary Ile requirement of hybrid catfish was estimated to be 12.43 g Ile kg-1 diet, corresponding to 32.05 g Ile kg-1 dietary protein. Collectively, dietary Ile improved growth performance and immunological and physical barrier function of intestine in hybrid catfish.

High leucine levels affecting valine and isoleucine recommendations in low-protein diets for broiler chickens.

Four experiments were conducted to estimate the optimal standardized ileal digestible (SID) level of branched-chain amino acids in low-protein diets during the starter, grower, and finisher periods, using the response surface methodology, and to study their effects on performance and mRNA expression of genes involved in the mechanistic target of rapamycin (mTOR) pathway of broiler chickens from 8 to 21 D of age. In experiments 1, 2, and 3, a total of 1,500 Cobb male broiler chickens were assigned to 15 diets of a central composite rotatable design (CCD) of response surface methodology containing 5 levels of SID Leu, Val, and Ile with 5 replicate pens of 20 birds each. A 3-factor, 5-level CCD platform was used to fit the second-order polynomial equation of broiler performance. In experiment 4, a total of 540 8-day-old Cobb male broiler chickens were distributed in a completely randomized 2 x 3 x 3 factorial arrangement with 2 SID Leu levels (1.28 or 1.83%), 3 SID Val levels (0.65, 0.90, or 1.20%), and 3 SID Ile levels (0.54, 0.79, or 1.09%) for a total of 18 treatments with 5 replicate cages of 6 birds each. High Leu levels impaired (P < 0.05) gain:feed when birds were fed marginal Val or Ile diets. However, gain:feed was restored when both Val and Ile were supplemented to reach adequate or high levels. High Leu levels increased (P < 0.05) mRNA expression of S6K1 and eEF2 genes only in birds fed high Ile levels. Dietary SID Leu, Val, and Ile levels required for gain:feed optimization in low-protein diets were estimated at 1.37, 0.94, and 0.87% during the starter period; 1.23, 0.82, and 0.75% during the grower period; and 1.15, 0.77, and 0.70% during the finisher phase, respectively. Higher Val and Ile levels are required to optimize the effect of Leu supplementation on mRNA expression of mTOR pathway genes in the pectoralis major muscle of broilers from day 1 to 21 after hatch.

Ergogenic Effect of BCAAs and L-Alanine Supplementation: Proof-of-Concept Study in a Murine Model of Physiological Exercise.

BACKGROUND: Branched-chain amino acids (BCAAs: leucine, isoleucine, valine) account for 35% of skeletal muscle essential amino acids (AAs). As such, they must be provided in the diet to support peptide synthesis and inhibit protein breakdown. Although substantial evidence has been collected about the potential usefulness of BCAAs in supporting muscle function and structure, dietary supplements containing BCAAs alone may not be effective in controlling muscle protein turnover, due to the rate-limiting bioavailability of other AAs involved in BCAAs metabolism. METHODS: We aimed to evaluate the in vivo/ex vivo effects of a 4-week treatment with an oral formulation containing BCAAs alone (2:1:1) on muscle function, structure, and metabolism in a murine model of physiological exercise, which was compared to three modified formulations combining BCAAs with increasing concentrations of L-Alanine (ALA), an AA controlling BCAAs catabolism. RESULTS: A preliminary pharmacokinetic study confirmed the ability of ALA to boost up BCAAs bioavailability. After 4 weeks, mix 2 (BCAAs + 2ALA) had the best protective effect on mice force and fatigability, as well as on muscle morphology and metabolic indices. CONCLUSION: Our study corroborates the use of BCAAs + ALA to support muscle health during physiological exercise, underlining how the relative BCAAs/ALA ratio is important to control BCAAs distribution.

The Effect of Isoleucine Supplementation on Body Weight Gain and Blood Glucose Response in Lean and Obese Mice.

Chronic isoleucine supplementation prevents diet-induced weight gain in rodents. Acute-isoleucine administration improves glucose tolerance in rodents and reduces postprandial glucose levels in humans. However, the effect of chronic-isoleucine supplementation on body weight and glucose tolerance in obesity is unknown. This study aimed to investigate the impact of chronic isoleucine on body weight gain and glucose tolerance in lean and high-fat-diet (HFD) induced-obese mice. Male C57BL/6-mice, fed a standard-laboratory-diet (SLD) or HFD for 12 weeks, were randomly allocated to: (1) Control: Drinking water; (2) Acute: Drinking water with a gavage of isoleucine (300 mg/kg) prior to the oral-glucose-tolerance-test (OGTT) or gastric-emptying-breath-test (GEBT); (3) Chronic: Drinking water with 1.5% isoleucine, for a further six weeks. At 16 weeks, an OGTT and GEBT was performed and at 17 weeks metabolic monitoring. In SLD- and HFD-mice, there was no difference in body weight, fat mass, and plasma lipid profiles between isoleucine treatment groups. Acute-isoleucine did not improve glucose tolerance in SLD- or HFD-mice. Chronic-isoleucine impaired glucose tolerance in SLD-mice. There was no difference in gastric emptying between any groups. Chronic-isoleucine did not alter energy intake, energy expenditure, or respiratory quotient in SLD- or HFD-mice. In conclusion, chronic isoleucine supplementation may not be an effective treatment for obesity or glucose intolerance.

Effect of dietary isoleucine on skin mucus barrier and epithelial physical barrier functions of hybrid bagrid catfish Pelteobagrus vachelli × Leiocassis longirostris.

The study investigated the effects of dietary isoleucine (Ile) on skin mucus barrier and epithelial physical barrier functions of hybrid bagrid catfish Pelteobagrus vachelli × Leiocassis longirostris. A total of 630 fish (33.11 ± 0.09 g) were fed semi-purified isonitrogenous diets containing 5.0 (control), 7.5, 10.0, 12.5, 15.0, 17.5, and 20.0 g Ile kg -1 diet for 8 weeks. The results indicated that dietary Ile increased (P < 0.05) in skin (1) mucus protein content and antimicrobial activity against three gram-negative bacteria (Aeromonas hydrophila, Escherichia coli, and Yersinia ruckeri) and two gram-positive bacteria (Streptococcus agalactiae and Staphylococcus aureus), (2) mucus lysofew information is available about the influencezyme (LZM), acid phosphatase (ACP), and alkaline phosphatase (AKP) activities, and complement 3 and 4 (C3 and C4) and immunoglobulin M (IgM) contents, (3) intelectin 1 (intl1), intelectin 2 (intl2), c-type-lysozyme (c-LZM), g-type-lysozyme (g-LZM), and ß-defensin mRNA levels. Dietary Ile decreased (P < 0.05) reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl (PC) contents, and up-regulated (P < 0.05) CuZnSOD, GST, GPX1a, muc5ac, muc5b, zonula occludens-1 (ZO-1), zonula occludens-2 (ZO-2), occludin, and claudin 3 mRNA levels in skin. These results indicated that Ile improved skin mucus barrier function via increasing mucus protein, C3 and C4, and IgM contents and antibacterial factors activities, and promoted epithelial physical barrier function via decreasing skin antioxidant damage and improving tight junction structure in hybrid bagrid catfish.

Association between Dietary Habits and Physical Function in Brazilian and Italian Older Women.

The present study investigated and compared the patterns of dietary protein intake and physical function in Brazilian and Italian older women. Seventy-five Brazilian older women were recruited in a community senior center. Fifty-three age-matched Italian older women were selected from participants of the Longevity check-up (Lookup) 7+ study. In both samples, physical performance was evaluated by isometric handgrip strength (IHG) and five-time sit-to-stand (5 × STS) tests, while diet was assessed through 24-h recall. Results indicated that Brazilian women had a higher intake of plant-based protein (52.7% vs. 30.5% kcal), while Italian women consumed greater amounts of animal-derived protein (29.7% vs. 41.5% kcal). In Brazilian women, the binary logistic regression analysis indicated that body weight-adjusted protein consumption was associated with IHG adjusted by body mass index and with 5 × STS performance. In the Italian sample, the intake of isoleucine, leucine, and valine was significantly associated with 5 × STS performance. Our findings indicate that Brazilian and Italian community-dwelling older women show different patterns of protein intake, with higher consumption of plant-based protein in the Brazilian sample and higher ingestion of animal-derived protein in the Italian subgroup. These dietary patterns may differentially impact the relationship between physical function and protein intake observed in Brazilian and Italian older women.

Egg production and quality responses to increasing isoleucine supplementation in Shaver white hens fed a low crude protein corn-soybean meal diet fortified with synthetic amino acids between 20 and 46 weeks of age.

The present study evaluated production performance responses to Ile supplementation in laying hens fed low crude protein (LCP), amino acid (AA) balanced diets. A total of 179 Shaver white pullets were distributed into 30 battery cages (6 birds/cage, n = 6) and observed over the course of 27 wk in a 2-phase (20 to 27 and 28 to 46 wk of age) feeding program. Five isocaloric diets were formulated for standardized ileal digestible (SID) Lys intake of 750 and 710 mg/D in phase 1 and 2, respectively, and included a positive control with standard levels of crude protein (CP) (CON; 18 and 16% CP for phases 1 and 2), and 4 LCP diets (16 and 14% CP for phase 1 and 2, respectively) with graded levels of Ile to satisfy SID Ile:Lys ratios of 70 (Ile70), 80 (Ile80), 90 (Ile90), and 100% (Ile100). Based on analyzed dietary AA, the calculated SID Ile:Lys of LCP diets were 75, 84, 88, 99% and 66, 72, 82, 95% for phase 1 and 2, respectively. Dietary treatments significantly (P < 0.05) affected feed intake, hen-day egg production (HDEP), egg weight (EW), feed conversion ratio, and egg quality (Haugh unit) and composition (yolk to albumen). Lowering dietary CP negatively affected HDEP with a 3.3 and 1.5% reduction in phase 1 and 2, respectively, and this was restored with the addition of Ile (P < 0.001) suggesting that Ile was limiting in the LCP basal diet. Average EW was reduced in Ile100 only; however, the Ile:Lys appeared to influence egg size uniformity, with Ile90 producing a greater proportion of large (56 g ≤ EW > 63 g) eggs, suggesting that Ile may be used to manipulate EW at the expense of HDEP. Overall, the results indicated that CP in laying hen diets can be reduced by 2% units if fortified with synthetic AA (Met, Lys, Thr, Trp) + Ile, with optimal responses observed between 82 and 88% SID Ile:Lys.

Impact on Isoleucine and Valine Supplementation When Decreasing Use of Medical Food in the Nutritional Management of Methylmalonic Acidemia.

BACKGROUND: Methylmalonic acidemia (MMA) is an autosomal recessive disorder treated with precursor-free medical food while limiting natural protein. This retrospective chart review was to determine if there was a relationship between medical food, valine (VAL) and/or isoleucine (ILE) supplementation, total protein intake, and plasma amino acid profiles. Methods: A chart review, of patients aged 31 days or older with MMA treated with dietary intervention and supplementation of VAL and/or ILE and followed at the Children's Hospital Colorado Inherited Metabolic Diseases Clinic. Dietary prescriptions and plasma amino acid concentrations were obtained at multiple time points. RESULTS: Baseline mean total protein intake for five patients was 198% of Recommended Dietary Allowance (RDA) with 107% natural protein and 91% medical food. Following intervention, total protein intake (p = 0.0357), protein from medical food (p = 0.0142), and leucine (LEU) from medical food (p = 0.0276) were lower, with no significant change in natural protein intake (p = 0.2036). At baseline, 80% of patients received VAL supplementation and 100% received ILE supplementation. After intervention, only one of the cohort remained on supplementation. There was no statistically significant difference in plasma propiogenic amino acid concentrations. CONCLUSIONS: Decreased intake of LEU from medical food allowed for discontinuation of amino acid supplementation, while meeting the RDA for protein.

The Impact of a Large Bolus Dose of l-leucine and l-isoleucine on Enteroendocrine and Pancreatic Hormones, and Glycemia in Healthy, Inactive Adults.

BACKGROUND: The ingestion of whey protein and amino acids with carbohydrate (CHO) enhances the release of glucagon-like peptide-1 (GLP-1) and glucose-dependent-insulinotropic peptide (GIP) that promote insulin secretion. It is unknown if L-isoleucine (Ile) and L-leucine (Leu) have this same effect. The purpose of this study was to examine how Ile and Leu influence both GLP-1 and GIP, subsequent pancreatic hormones, and glycemia in healthy, inactive adults. METHODS: Twelve adults (6F/6M; age 27.4 ± 2 years; BMI 26.3 ± 2 kg/m2; lean body mass 53.2 ± 5 kg; body fat 34.1 ± 3%) completed four conditions in a randomized, cross-over fashion. Treatments standardized (0.3 g/kg·LBM-1) (1) Leu, (2) Ile, (3) Equal (1:1 g) of Leu + Ile, and (4) placebo (Pla, 3.5 g inert stevia) ingested 30 min prior to an oral glucose tolerance test (OGTT). Samples of plasma glucose, insulin, glucagon, GIPTotal, and GLP-1Active were assessed. RESULTS: A treatment (p = 0.01) effect comparing Ile vs. Leu (p = 0.02) in GIPTotal. Area under the curve showed an increase in GIPTotal from Ile compared to Leu and Pla (p = 0.03). No effect was found on GLP-1. The ingestion of Ile prior to CHO augmented GIP concentration greater than Leu or Pla. No correlation was found between GIP, insulin, and glucose between conditions. CONCLUSIONS: Ile impacts GIP concentration, which did not relate to either insulin or glucose concentrations. Neither Ile, nor Leu seem to have an effect on hyperglycemia ingested prior to a CHO drink.

Automated therapy preparation of isoleucine formulations using 3D printing for the treatment of MSUD: First single-centre, prospective, crossover study in patients.

Maple syrup urine disease (MSUD) is a rare metabolic disorder with a worldwide prevalence of 1 in every 185,000 live births. However, certain populations display a significant overexpression of the disorder where incidence is reported to be 1 in every 52,541 new-borns. The first-line therapy for MSUD involves a strict dietary leucine restriction and oral supplementation of isoleucine and valine. The dose administered to patients requires strict tailoring according to age, weight and blood levels. In current clinical practice, however, practitioners still have to prepare extemporaneous formulations due to the lack of suitable oral treatments for MSUD. Herein, we evaluate the first time use of 3D printing in a hospital setting for the preparation of personalised therapies with the aim of improving safety and acceptability to isoleucine supplementation in paediatric patients suffering from MSUD. This investigation was a single-centre, prospective crossover experimental study. Four paediatric patients with MSUD (aged 3-16 years) were treated at the Clinic University Hospital in Santiago de Compostela, Spain which is a MSUD reference hospital in Europe. The primary objective was to evaluate isoleucine blood levels after six months of treatment with two types of formulations; conventional capsules prepared by manual compounding and personalised chewable formulations prepared by automated 3D printing. A secondary investigation was to evaluate patient acceptability of 3D printed formulations prepared with different flavours and colours. Isoleucine blood levels in patients were well controlled using both types of formulations, however, the 3D printed therapy showed mean levels closer to the target value and with less variability (200-400 µM). The 3D printed formulations were well accepted by patients regarding flavour and colour. The study demonstrates for the first time that 3D printing offers a feasible, rapid and automated approach to prepare oral tailored-dose therapies in a hospital setting. 3D printing has shown to be an effective manufacturing technology in producing chewable isoleucine printlets as a treatment of MSUD with good acceptability.

Effects of jugular infusions of isoleucine, leucine, methionine, threonine, and other amino acids on insulin and glucagon concentrations, mammalian target of rapamycin (mTOR) signaling, and lactational performance in goats.

The supply and profile of absorbed AA may affect milk protein synthesis through hormonal changes and mammalian target of rapamycin (mTOR) signaling pathways; and Ile, Leu, Met, and Thr (ILMT) are the 4 AA that have been reported to have the greatest effect on mammary mTOR signaling. The extent to which ILMT and the other remaining AA (RAA) differ in their effects on milk protein synthesis needs to be systematically investigated. In this study, 5 lactating goats, averaging 120 ± 10 d in milk, fitted with jugular vein and carotid artery catheters, were fasted for 24 h, followed by intravenous infusions of a mixture containing AA and glucose for 8 h in a 5 × 5 Latin square design. The AA mixtures were formulated according to the profile of casein. The amounts of AA infused were calculated based on supplies of AA when metabolizable protein (MP) was at requirement (MR). Treatments were an infusate containing glucose without AA (NTAA); an infusate containing 3 × the MR of Ile, Leu, Met and Thr (3F0R); and infusates containing 3F0R plus 1, 2, or 3 × MR of RAA (3F1R, 3F2R, and 3F3R, respectively) according to amounts provided when fed to meet MP requirements for maintenance and lactation for each goat. Milk, arterial blood, and mammary tissue samples were collected immediately after halting the infusion. Relative to NTAA, supplementation of ILMT tended to increase milk protein production and plasma glucose concentrations, and increased milk and lactose production, but had no effects on production or content of milk fat. Graded supplementation of RAA tended to quadratically affect production of milk and lactose. Arterial glucose and glucagon concentrations decreased linearly, and plasma insulin concentrations decreased quadratically with increased RAA. Mammary p70-S6K1 phosphorylation was decreased by addition of ILMT compared with NTAA but increased linearly with increased RAA infusion. Furthermore, EIF4EBP1 gene expression was much lower for 3F-treated goats than for the NTAA treatment. Both MTOR and RPS6KB1 gene expressions were decreased quadratically with increased RAA supply. These results suggested that short-term milk protein yield tended to be increased by elevated ILMT availability, and this trend was not explained by variations in mammary mTOR signaling or pancreatic hormone secretions, whereas graded increase of RAA in combination with ILMT appeared to regulate the efficiency of conversion of glucose to lactose in a manner not involving milk protein production.

The influence of dietary leucine above recommendations and fixed ratios to isoleucine and valine on muscle protein synthesis and degradation pathways in broilers.

This study investigated the hypothesis that dietary supplementation of leucine (Leu) above actual recommendations activates protein synthesis and inhibits protein degradation pathways on the molecular level and supports higher muscle growth in broilers. Day-old male Cobb-500 broilers (n = 180) were allotted to 3 groups and phase-fed 3 different corn-wheat-soybean meal-based basal diets during periods 1 to 10, 11 to 21, and 22 to 35 D. The control group (L0) received the basal diet which met the broiler's requirements of nutrients and amino acids for maintenance and growth. Groups L1 and L2 received basal diets supplemented with Leu to exceed recommendations by 35 and 60%, respectively, and isoleucine (Ile) and valine (Val) were supplemented to keep Leu: Ile and Leu: Val ratios fixed. Samples of liver and breast muscle and pancreas were collected on days 10, 21, and 35. The gene expression and abundance of total and phosphorylated proteins involved in the mammalian target of rapamycin pathway of protein synthesis, in the ubiquitin-proteasome pathway and autophagy-lysosomal pathway of protein degradation, in the general control nonderepressible 2/eukaryotic translation initiation factor 2A pathway involved in the inhibition of protein synthesis, and in the myostatin-Smad2/3 pathway involved in myogenesis were evaluated in the muscle, as well as expression of genes involved in the growth hormone axis. Growth performance, feed intake, the feed conversion ratio, and carcass weights did not differ between the 3 groups (P > 0.05). Plasma concentrations of Leu, Ile, and Val and of their keto acids, and the activity of the branched-chain α-keto acid dehydrogenase in the pancreas increased dose dependently with increasing dietary Leu concentrations. In the breast muscle, relative mRNA abundances of genes and phosphorylation of selected proteins involved in all investigated pathways were largely uninfluenced by dietary Leu supplementation (P > 0.05). In summary, these data indicate that excess dietary Leu concentrations do not influence protein synthesis or degradation pathways, and subsequently do not increase muscle growth in broilers at fixed ratios to Ile and Val.

Isoleucine attenuates infection induced by E. coli challenge through the modulation of intestinal endogenous antimicrobial peptide expression and the inhibition of the increase in plasma endotoxin and IL-6 in weaned pigs.

Enteric infection is a major cause of morbidity and mortality in both humans and animals worldwide. Immunotherapy against intestinal infection is a well-known alternative to the antibiotic strategy. Herein, we demonstrated that isoleucine significantly suppressed the multiplication of E. coli in the presence of IPEC-J2 cells. Isoleucine supplementation enhanced the concentrations of total plasma protein and IgA in pigs compared to the alanine control diet, while inhibiting the increase in plasma endotoxin and IL-6 contents induced by E. coli challenge. A significant interaction between the E. coli challenge and the diet treatment was found in the red blood cell volume. Isoleucine improved the expression of porcine ß-defensin-1 (pBD-1), pBD-2, pBD-3, pBD-114 and pBD-129 in the jejunum and ileum of pigs with or without E. coli challenge. Conclusively, isoleucine attenuated the infection caused by the E. coli challenge possibly through increasing the intestinal ß-defensin expression and inhibiting the increase in plasma endotoxin and IL-6 in weaned pigs.

Factorial models to estimate isoleucine requirements for broilers.

The objective of this work was to determine the efficiency of utilization (EU) and produce factorial models for optimal isoleucine (Ile) intake. Six dose-response trials were carried out, three for males and three for females, with 640 Ross 308 in each studied phase. The initial (1-14 days), grower (15-28 days) and finisher (29-42 days) phases were evaluated to cover the growing phase of the broiler chicken. In total, eight treatments were randomly distributed to four replicates of 20 birds each. The treatments consisted of seven crescent levels of Ile and one counter proof to ensure that Ile was the first limiting amino acid in the diet. Dilution technique was applied to produce the levels of Ile and keep the amino acid ratio with lysine. The EU was determined to account for whole body or partitioned for feather-free body (Bff) and feather. Two distinct factorial models were adjusted, M1 and M2. The M2 model was evaluated for one or two EU, being denominated as M2 and M3. When the efficiency was partitioned, the values of 53% and 69% for feather and Bff were determined. The optimal Ile intake estimated for each model were of 275, 908, 1,412 mg of Ile/bird/day (M1); 258, 829, 1,321 mg of Ile/bird/day (M2); and 284, 835, 1,288 mg of Ile/bird/day (M3) for initial, grower and finisher phases respectively. The EU partitioned for feather-free body and feather reduced the biased of the model M3. Overall, higher values of Ile intake are estimated when model M1 is used, which may be the difference in account for body weight gain (M1) or only protein gain (M2 and M3) to estimate the amount of amino acid required for broiler.

Effects of leucine supplementation on muscle protein synthesis and degradation pathways in broilers at constant dietary concentrations of isoleucine and valine.

The present study investigated the hypothesis that dietary concentrations of leucine (Leu) in excess of the breeder´s recommendations activates protein synthesis and decreases protein degradation in muscle of broilers. Day-old male Ross 308 broilers (n = 450) were phase-fed corn-soybean meal-based diets during starter (d 1-10), grower (d 11-22), and finisher (d 23-34) period. The basal diets fed to the control group (L0) met the broilers' requirements for nutrients and amino acids, and contained Leu, Leu:isoleucine (Ile) and Leu:valine (Val) ratios, close to those recommended by the breeder (Leu:Ile: 100:54, 100:52, 100:51; Leu:Val 100:64, 100:61, 100:58; in starter, grower and finisher diet, resp.). Basal diets were supplemented with Leu to exceed the breeder's recommendations by 35% (group L35) and 60% (group L60). Growth performance during 34 d, and carcass weights, and breast and thigh muscle weights on d 34 were similar among groups. Hepatic and muscle mRNA levels of genes involved in the somatotropic axis [growth hormone receptor, insulin-like growth factor (IGF)-1, IGF binding protein 2, IGF receptor] on d 34 were not influenced by Leu. In the breast muscle, relative mRNA abundances of genes involved in the mammalian target of rapamycin (mTOR) pathway of protein synthesis (mTOR, ribosomal p70 S6 kinase) and the ubiquitin-proteasome pathway of protein degradation (F-box only protein 32, Forkhead box protein O1, Muscle RING-finger protein-1) on d 34 were largely similar among groups. Likewise, relative phosphorylation and thus activation of mTOR and ribosomal protein S6 involved in the mTOR pathway, and of eukaryotic translation initiation factor 2A (eIF2a) involved in the general control nonderepressible 2 (GCN2)/eIF2a pathway of protein synthesis inhibition, were not influenced. These data indicate that dietary Leu concentrations exceeding the broiler´s requirements up to 60% neither influence protein synthesis nor degradation pathways nor muscle growth in growing broilers.

Essential Amino Acid Profile in Parenteral Nutrition Mixtures: Does It Meet Needs?

BACKGROUND AND AIMS: The study compares the essential amino acid (EAA) composition of different parenteral nutrition (PN) mixtures with whey protein EAA profile and the theoretical daily EAA requirements (set by WHO/FAO/UNU or IAAO method). According to the individual EAA profile, the potential effect of several PN mixtures was evaluated on the skeletal muscle mass (SMM) of patients on home PN. METHODS: Eight AA solutions and fifteen complete PN mixtures were considered. Twenty-nine clinically stable patients with short bowel syndrome on home total PN were retrospectively evaluated. SMM was estimated by bioelectrical impedance analysis. RESULTS: The prescribed doses of EAA that showed a significant increase in home PN patients muscle mass were considerably greater than the theoretical ones, showing an EAA profile similar to whey protein. At the daily dose of 1 g of total AA s/kg body weight (BW), the considered PN mixtures mostly failed to improve SMM. Only prescribed doses which included more than 0.25 g/kg BW of total BCAA with at least 0.10 g/kg BW leucine, 0.08 g/kg BW isoleucine, and 0.06 g/kg BW methionine showed a significant increase in SMM. CONCLUSIONS: The theoretical daily requirement for each EAA was met by all considered PN solutions when the prescribed daily dose of total AAs was set at 1 g/kg BW. Nevertheless, our data suggest that only an increase in total BCAA, also richer in single AA leucine, isoleucine, and methionine, is associated with the maintenance and/or increase of SMM. According to these preliminary observations, we support the prescription of an EAA composition of PN mixtures close to that of whey protein for the preservation of SMM in patients on long-term total PN.