Metabolism of Bile Salts in Mice Influences Spore Germination in Clostridium difficile

Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.     

Reducing protein content in the diet of growing goats: implications for nitrogen balance,intestinal nutrient digestion and absorption,and rumen microbiota

Reducing dietary CP content is an effective approach to reduce animal nitrogen excretion and save protein feed resources. However, it is not clear how reducing dietary CP content affects the nutrient digestion and absorption in the gut of ruminants, therefore it is difficult to accurately determine how much reduction in dietary CP content is appropriate. This study was conducted to investigate the effects of reduced dietary CP content on N balance, intestinal nutrient digestion and absorption, and rumen microbiota in growing goats. To determine N balance, 18 growing wether goats (25.0 ± 0.5 kg) were randomly assigned to one of three diets: 13.0% (control), 11.5% and 10.0% CP. Another 18 growing wether goats (25.0 ± 0.5 kg) were surgically fitted with ruminal, proximate duodenal, and terminal ileal fistulae and were randomly assigned to one of the three diets to investigate intestinal amino acid (AA) absorption and rumen microbiota. The results showed that fecal and urinary N excretion of goats fed diets containing 11.5% and 10.0% CP were lower than those of goats fed the control diet (P < 0.05). When compared with goats fed the control diet, N retention was decreased and apparent N digestibility in the entire gastrointestinal tract was increased in goats fed the 10% CP diet (P < 0.05). When compared with goats fed the control diet, the duodenal flow of lysine, tryptophan and phenylalanine was decreased in goats fed the 11.5% CP diet (P < 0.05) and that of lysine, methionine, tryptophan, phenylalanine, leucine, glutamic acid, tyrosine, essential AAs (EAAs) and total AAs (TAAs) was decreased in goats fed the 10.0% CP diet (P < 0.05). When compared with goats fed the control diet, the apparent absorption of TAAs in the small intestine was increased in goats fed the 11.5% CP diet (P < 0.05) and that of isoleucine, serine, cysteine, EAAs, non-essential AAs, and TAAs in the small intestine was increased in goats fed the 10.0% CP diet (P < 0.05). When compared with goats fed the control diet, the relative richness of Bacteroidetes and Fibrobacteres was increased and that of Proteobacteria and Synergistetes was decreased in the rumen of goats fed a diet with 10.0% CP. In conclusion, reducing dietary CP content reduced N excretion and increased nutrient utilization by improving rumen fermentation, enhancing nutrient digestion and absorption, and altering rumen microbiota in growing goats.     

Combined or Individual Effects of Dietary Probiotic <Emphasis Type="Italic">Pedicoccus acidilactici</Emphasis> and Nucleotide on Growth Performance,Intestinal Microbiota,Hemato-biochemical Parameters,and Innate Immune Response in Goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

This study investigated the effect of dietary supplementation of probiotic Pedicoccus acidilactici and nucleotide (separately or combined) on growth performance, intestinal microbiota, hemato-immunological parameters, and immunity response in goldfish (Carassius auratus). Fish (average weight 5–6 g) were acclimatized and divided into eight experimental diets supplemented with P. acidilactici of different concentrations (0.1, 0.2, and 0.3% diet) and nucleotides (0 and 0.5% diet) for 6 months. Fish fed with experimental diets showed significant differences in terms of final weight, weight gain, feed conversion ratio, daily growth rate, and condition factor when compared to control diet (P < 0.05). Fish fed with probiotic (0.3%) separately and combined with nucleotide (0.5%) had highest RBC and WBC when compared to other diets (P < 0.05), while the highest values for Hb and Hct as well as total protein, glucose, albumin, and globulin were observed in probiotic (0.2%) and nucleotide (0.5%) combined diet. Serum lysozyme and anti-protease activities were significantly higher in probiotic (0.1 and 0.2%) and nucleotide (0.5%) combined diets. Similarly, these two diets combined showed the highest colonization of P. acidilactici when compared to other diets. In conclusion, combined dietary probiotic and nucleotide improve the growth performance, hemato-biochemical parameters, and intestine growth in C. auratus.     

Phylogenomics of 8,839 Clostridioides difficile genomes reveals recombination-driven evolution and diversification of toxin A and B

Clostridioides difficile is the major worldwide cause of antibiotic-associated gastrointestinal infection. A pathogenicity locus (PaLoc) encoding one or two homologous toxins, toxin A (TcdA) and toxin B (TcdB), is essential for C. difficile pathogenicity. However, toxin sequence variation poses major challenges for the development of diagnostic assays, therapeutics, and vaccines. Here, we present a comprehensive phylogenomic analysis of 8,839 C. difficile strains and their toxins including 6,492 genomes that we assembled from the NCBI short read archive. A total of 5,175 tcdA and 8,022 tcdB genes clustered into 7 (A1-A7) and 12 (B1-B12) distinct subtypes, which form the basis of a new method for toxin-based subtyping of C. difficile. We developed a haplotype coloring algorithm to visualize amino acid variation across all toxin sequences, which revealed that TcdB has diversified through extensive homologous recombination throughout its entire sequence, and formed new subtypes through distinct recombination events. In contrast, TcdA varies mainly in the number of repeats in its C-terminal repetitive region, suggesting that recombination-mediated diversification of TcdB provides a selective advantage in C. difficile evolution. The application of toxin subtyping is then validated by classifying 351 C. difficile clinical isolates from Brigham and Women’s Hospital in Boston, demonstrating its clinical utility. Subtyping partitions TcdB into binary functional and antigenic groups generated by intragenic recombinations, including two distinct cell-rounding phenotypes, whether recognizing frizzled proteins as receptors, and whether it can be efficiently neutralized by monoclonal antibody bezlotoxumab, the only FDA-approved therapeutic antibody. Our analysis also identifies eight universally conserved surface patches across the TcdB structure, representing ideal targets for developing broad-spectrum therapeutics. Finally, we established an open online database (DiffBase) as a central hub for collection and classification of C. difficile toxins, which will help clinicians decide on therapeutic strategies targeting specific toxin variants, and allow researchers to monitor the ongoing evolution and diversification of C. difficile.     

The effects of dietary supplementation with butyrate and polyhydroxybutyrate on the digestive capacity and intestinal morphology of Pacific White Shrimp (Litopenaeus vannamei)

This study evaluated dietary supplementation with sodium butyrate and polyhydroxybutyrate (PHB) on the enzymatic activity, in vitro nutrient digestibility, and intestinal morphology of Pacific white shrimp (Litopenaeus vannamei). The treatments were: shrimp fed diet supplemented with 2% butyrate, shrimp fed diet supplemented with 2% PHB, and shrimp fed unsupplemented diet. Shrimp fed with PHB-supplemented diet showed higher values of intestinal protease, trypsin, chymotrypsin, and amylase. Shrimp fed with butyrate-supplemented diet showed higher intestinal lipase than unsupplemented shrimp. Butyrate increased hepatopancreatic protease, trypsin, and chymotrypsin activity and in vitro protein digestibility. Shrimp fed with PHB-supplemented diet and butyrate had higher digestibility of polysaccharides and lipids. Shrimp fed with PHB-supplemented diet presented increase in the length, width, and perimeter of intestinal villi. Animals fed with both diets retained overall integrity of their intestinal mucous membrane. The findings show that dietary supplementation with PHB and butyrate can alter intestinal morphology and could improve the digestive capacity of L. vannamei.     

CD44 Promotes Intoxication by the Clostridial Iota-Family Toxins

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44⁺ melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.     

Tracing the Spread of Clostridium difficile Ribotype 027 in Germany Based on Bacterial Genome Sequences

We applied whole-genome sequencing to reconstruct the spatial and temporal dynamics underpinning the expansion of Clostridium difficile ribotype 027 in Germany. Based on re-sequencing of genomes from 57 clinical C. difficile isolates, which had been collected from hospitalized patients at 36 locations throughout Germany between 1990 and 2012, we demonstrate that C. difficile genomes have accumulated sequence variation sufficiently fast to document the pathogen''s spread at a regional scale. We detected both previously described lineages of fluoroquinolone-resistant C. difficile ribotype 027, FQR1 and FQR2. Using Bayesian phylogeographic analyses, we show that fluoroquinolone-resistant C. difficile 027 was imported into Germany at least four times, that it had been widely disseminated across multiple federal states even before the first outbreak was noted in 2007, and that it has continued to spread since.     

Identification of Toxemia in Patients with Clostridium difficile Infection

Toxemia can develop in Clostridium difficile-infected animals, and correlates with severe and fulminant disease outcomes. Circumstantial evidence suggests that toxemia may occur in patients with C. difficile infection (CDI), but positive diagnosis is extremely rare. We analyzed the potential for C. difficile toxemia in patients, determined its characteristics, and assessed challenges. C. difficile toxins in serum from patients were tested using an ultrasensitive cell-based assay and further confirmed by Rac1 glucosylation assay. The factors that hinder a diagnosis of toxemia were assessed, including investigation of toxin stability, the level of toxins-specific neutralizing antibodies in sera and its effect on diagnosis limits. CDI patients develop detectable toxemia in some cases (2.3%). Toxins were relatively stable in stored sera. Neutralizing anti-toxin antibodies were present during infection and positively correlated with the diagnosis limits. Thus, the masking effect of toxin-specific neutralizing antibodies is the major obstacle in diagnosing C. difficile toxemia using cell-based bioassays.     

Molecular Characterization of Clostridium difficile Isolates from Human Subjects and the Environment

Clostridium difficile is a spore-forming, gram-positive, anaerobic bacillus that can cause C. difficile infection (CDI). However, only a few studies on the prevalence and antibiotic resistance of C. difficile in healthy individuals in China have been reported. We employed a spore enrichment culture to screen for C. difficile in the stool samples of 3699 healthy Chinese individuals who were divided into 4 groups: infants younger than 2 years of age and living at home with their parents; children aged 1 to 8 years of age and attending three different kindergarten schools; community-dwelling healthy adult aged 23–60 years old; and healthcare workers aged 28–80 years old. The C. difficile isolates were analyzed for the presence of toxin genes and typed by PCR ribotyping and multilocus sequence typing (MLST). The minimum inhibitory concentration of 8 antimicrobial agents was determined for all of the isolates using the agar dilution method. The intestinal carriage rate in the healthy children was 13.6% and ranged from 0% to 21% depending on age. The carriage rates in the 1654 community-dwelling healthy adults and 348 healthcare workers were 5.5% and 6.3%, respectively. Among the isolates, 226 were toxigenic (225 tcdA+/tcdB+ and 1 tcdA+/tcdB+ ctdA+/ctdB+). Twenty-four ribotypes were found, with the dominant type accounting for 29.7% of the isolates. The toxigenic isolates were typed into 27 MLST genotypes. All of the strains were susceptible to vancomycin, metronidazole, fidaxomicin, and rifaximin. High resistance to levofloxacin and ciprofloxacin at rates of 39.8% and 98.3%, respectively, were observed. ST37 isolates were more resistant to levofloxacin than the other STs. The PCR ribotypes and sequence types from the healthy populations were similar to those from the adult patients.     

EhRho1, a RhoA-Like GTPase of Entamoeba histolytica, Is Modified by Clostridial Glucosylating Cytotoxins

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[¹⁴C]glucose.     

Development and reproduction of the predatory mite Amblyseius swirskii on artificial diets

Development, survival and reproduction of the predatory mite Amblyseius swirskii (Athias-Henriot) (Acari: Phytoseiidae) were assessed when fed on cattail pollen (Typha latifolia L.), dried fruit mite (Carpoglyphus lactis L.), or on two artificial diets. The basic artificial diet (AD1) was composed of honey, sucrose, tryptone, yeast extract, and egg yolk. This diet was enriched (AD2) by adding hemolymph from oak silkworm pupae (Antheraea pernyi (Guérin-Méneville)). Mites fed on C. lactis and AD2 had shorter immature and preoviposition periods than those fed on the other diets. The total number of deposited eggs was significantly higher for females fed on AD2 than for those maintained on the other diets. The intrinsic rate of increase (r_m) of A. swirskii was highest on AD2 and C. lactis, followed by T. latifolia pollen, and AD1. In conclusion, the artificial diet AD2 supported development and reproduction of A. swirskii to the same extent as a factitious prey which is routinely used in the mass rearing of the phytoseiid. Our findings indicate the potential of artificial diets for the mass production of this economically important predatory mite.     

High intake of chicken and pork proteins aggravates high-fat-diet-induced inflammation and disorder of hippocampal glutamatergic system

High-fat diets have been associated with neurodegenerative diseases, which are also largely related to the type and amount of dietary proteins. However, to our knowledge, it is little known how dietary proteins affect neurodegenerative changes. In this study, we investigated the effects of dietary proteins in a high-fat diet on hippocampus functions related to enteric glial cells (EGCs) in Wistar rats that were fed either 40% or 20% (calorie) casein, chicken protein or pork protein for 12 weeks (n=10 each group). Inflammatory factors, glutamatergic system, EGCs, astrocytes and nutrient transporters were measured. A high-chicken-protein diet significantly increased the levels of systemic inflammatory factors, Tau protein and amyloid precursor protein mRNA level in the rat hippocampus. The type and level of dietary proteins in high-fat diets did not affect the gene expression of glial fibrillary acidic protein and α-synuclein (P>.05), indicating a negligible effect on astrocyte activity. However, the high-protein diets up-regulated glutamate transporters compared with the low-protein diets (P<.05), while they reduced the γ-aminobutyric acid content in high-chicken and -pork-protein diets (P<.05). Thus, compared with a low-protein diet (20%), a high-chicken or -pork-protein diet (40%) under a high-fat background could alter the balance between glutamatergic system and neurotransmitter and have a stronger effect on the interactions between hippocampal glutamatergic system and EGCs.     

Bile acid-independent protection against Clostridioides difficile infection

Clostridioides difficile infections occur upon ecological / metabolic disruptions to the normal colonic microbiota, commonly due to broad-spectrum antibiotic use. Metabolism of bile acids through a 7α-dehydroxylation pathway found in select members of the healthy microbiota is regarded to be the protective mechanism by which C. difficile is excluded. These 7α-dehydroxylated secondary bile acids are highly toxic to C. difficile vegetative growth, and antibiotic treatment abolishes the bacteria that perform this metabolism. However, the data that supports the hypothesis that secondary bile acids protect against C. difficile infection is supported only by in vitro data and correlative studies. Here we show that bacteria that 7α-dehydroxylate primary bile acids protect against C. difficile infection in a bile acid-independent manner. We monoassociated germ-free, wildtype or Cyp8b1^-/- (cholic acid-deficient) mutant mice and infected them with C. difficile spores. We show that 7α-dehydroxylation (i.e., secondary bile acid generation) is dispensable for protection against C. difficile infection and provide evidence that Stickland metabolism by these organisms consumes nutrients essential for C. difficile growth. Our findings indicate secondary bile acid production by the microbiome is a useful biomarker for a C. difficile-resistant environment but the microbiome protects against C. difficile infection in bile acid-independent mechanisms.     

Nutritional profile of soaked Cajanus cajan (L.) Millsp. and its utilization as partial replacement for soybean meal in the diet of Clarias gariepinus (Burchell, 1822) fingerlings

This study aimed to investigate the efficacy of soaking as a cheap processing method in improving the nutritional utilization of Cajanus cajan for partial replacement of soybean meal in formulated diets of the African catfish Clarias gariepinus (Burchell, 1822). The nutrient profile of soaked C. cajan revealed a significant reduction (≤50%) in all anti‐nutritional factors. Proximate and amino acids were also affected by this processing method. Six practical diets (35 g/kg crude protein, 19.1 KJ/g diet) containing substitution levels of 0, 10, 20, 30, 40 and 50% C. cajan for soybean protein (included at 578 g/kg) were then formulated and fed to triplicate groups (n = 50 in each group) of C. gariepinus fingerlings (1.41 ± 0.06 g) in a hapa pond system (1 × 1 × 1 m³) for 56 days (mean temperature 25.3 ± 0.6). The results revealed that fish survival was not significantly affected until a 40% substitution of soybean was reached. However, beyond 20% soybean substitution, growth was significantly reduced (≥7%; p ≤ .05). Fish fed the highest substitution of soaked C. cajan meal (50% soybean replacement) had the poorest performance. The cost of the compound diets was substantially reduced with the inclusion of soaked C. cajan. Production costs of 1 kg fish using the diet with an inclusion of up to 20% soaked C. cajan meal replacement were comparable to using the control diet. It was concluded that soaking C. cajan seed for 24 hr improved the nutritional profile of the ingredients to replace 20% of the soybean (included at 57.8%) protein in the diet of C. gariepinus with no negative effect on growth and nutrient utilization.     

Combination of legume-based herbage and total mixed ration (TMR) maintains intake and nutrient utilization of TMR and improves nitrogen utilization of herbage in heifers

Diets combining herbage and total mixed rations (TMR) are increasingly used in temperate regions for feeding ruminants, but little information is available regarding the effects on nutrient intake and digestion of this feeding management in beef cattle. The aim of this study was to determine the effects of combining TMR (10% CP and 13% ADF), and legume-based herbage (14% CP and 27% ADF) on intake, nutrient digestion, ruminal fermentation, microbial N flow and glucose and nitrogen metabolism in heifers. The experiment was a 3×3 Latin square design replicated three times; each period lasted 18 days (10 adaptation days and 8 measurement days). Nine cross-bred (Aberdeen Angus×Hereford) heifers (214±18 kg) fitted with permanent rumen catheters and housed in individual metabolic cages were assigned to one of three treatments: 24 h access to TMR (T), 24 h access to herbage (H) or combined diets with 18 h access to TMR and 6 h access to herbage (T+H). Data were evaluated using a mixed model. Animals fed T+H (TMR 71% and herbage 29%) diets tended to have a higher dry matter intake as a proportion of their BW than animals fed T. The T+H diet did not change ruminal fermentation (pH, N–NH₃ and volatile fatty acids) or the N metabolism relative to the T diet, but increased the glucagon concentration and altered glucose metabolism. Conversely, animals fed T+H had increased purine derivatives excretion, increased N use efficiency for microbial protein synthesis and decreased plasma urea and urinary N excretion relative to animals fed H diet. The use of combined diets led to consumption of nutrients similar to a TMR diet, without reducing nutrient use and could improve N utilization compared with the herbage-only diet.     

Dietary nucleotides influence immune responses and intestinal morphology of red drum Sciaenops ocellatus

Dietary nucleotides have been shown to benefit many physiological and nutritional functions in higher vertebrates and fish. Therefore, a 6-week feeding trial was conducted to evaluate the effects of graded levels of a commercial nucleotide product on growth performance, immune responses and intestinal morphology of juvenile red drum (initial average weight of 7.1 g). The basal diet was formulated to contain 40% protein, 10% lipid and a digestible energy level of 3.5 kcal g^?1. Two levels of nucleotide (Ascogen P^®, 0.5% and 1% of diet) were added to the basal diet with menhaden fishmeal and menhaden oil adjusted to provide isonitrogenous and isolipidic diets. Nucleotide supplementation tended to improve weight gain and survival of red drum, but not at a significant level. Neutrophil oxidative radical anion production and serum lysozyme activity tended to be higher for fish fed diets supplemented with nucleotide, while extracellular superoxide anion production of head kidney macrophages from fish fed diets with 1% nucleotide was significantly (P < 0.05) increased, although no significant differences were observed between fish fed 0.5% nucleotide diet and the basal diet.Nucleotide supplementation significantly (P < 0.05) increased fold height in the proximal intestine, and enterocyte height in the pyloric caeca, proximal and distal enteric sections. A significantly (P < 0.05) higher microvilli height was observed in all evaluated enteric sections of fish fed with diets supplemented with nucleotides. It is therefore possible to use dietary nucleotides supplementation to significantly enhance the intestinal structure of red drum. Likewise, nucleotides in the diet may improve some components of the non-specific immune response of this sciaenid fish.     

Effects of feeding corn bran and soybean hulls on nutrient digestibility,rumen microbial protein synthesis,and growth performance of finishing lambs

Some grain processing by-products rich in digestible fiber are good feed resources for ruminants. This experiment was conducted to investigate the effects of replacing a portion of corn and corn stover with the combinations of corn bran and soybean hulls in the diet of fattening lambs on nutrient digestion, rumen microbial protein synthesis, and growth performance. A total of 36 Dorper × Small Thin-Tailed crossbred ram lambs (BW = 22.2 ± 0.92 kg; mean ± SD) were randomly divided into three groups, and each group was fed 1 of 3 treatment diets: 1) 0% corn bran and soybean hulls (control); 2) 9% corn bran and 9% soybean hulls (18MIX); and 3) 17% corn bran and 17% soybean hulls (34MIX). The feeding experiment was conducted for 70 days, with the first 10 days for adaption. The DM intake was higher for 34MIX (1635.3 g/d) than for control diet (1434.7 g/d; P = 0.001). Lambs fed 18MIX and 34MIX diets (230.2 and 263.6 g/d, respectively) had higher average daily gain and feed efficiency than those fed control diet (194.8 g/d; P < 0.01). Dry matter and NDF digestibility for 34MIX group (60.9 and 49.5%) were higher than for control (55.2 and 41.3%; P < 0.01). No difference was observed in nitrogen digestibility among treatment diets (P = 0.778). The lambs fed 34MIX diet excreted more urinary purine derivatives, indicating that more microbial protein was yielded than those fed control diet (P < 0.01), while 18MIX was not different from the other two diets (P > 0.05). The metabolizable protein supplies were improved with increasing co-products inclusion rate. The results indicated that corn bran and soybean hulls in combination can effectively replace a portion of corn and corn stover in the ration of finishing lambs with positive effect on nutrient digestion and growth performance.     

Prevention and reversal of hepatic steatosis with a high-protein diet in mice

The hallmark of NAFLD is steatosis of unknown etiology. We tested the effect of a high-protein (HP)² diet on diet-induced steatosis in male C57BL/6 mice with and without pre-existing fatty liver. Mice were fed all combinations of semisynthetic low-fat (LF) or high-fat (HF) and low-protein (LP) or HP diets for 3 weeks. To control for reduced energy intake by HF/HP-fed mice, a pair-fed HF/LP group was included. Reversibility of pre-existing steatosis was investigated by sequentially feeding HF/LP and HF/HP diets. HP-containing diets decreased hepatic lipids to ~ 40% of corresponding LP-containing diets, were more efficient in this respect than reducing energy intake to 80%, and reversed pre-existing diet-induced steatosis. Compared to LP-containing diets, mice fed HP-containing diets showed increased mitochondrial oxidative capacity (elevated Pgc1α, mAco, and Cpt1 mRNAs, complex-V protein, and decreased plasma free and short-chain acyl-carnitines, and [C₀]/[C₁₆ + C₁₈] carnitine ratio); increased gluconeogenesis and pyruvate cycling (increased PCK1 protein and fed plasma–glucose concentration without increased G6pase mRNA); reduced fatty-acid desaturation (decreased Scd1 expression and [C_16:1n ? 7]/[C_16:0] ratio) and increased long-chain PUFA elongation; a selective increase in plasma branched-chain amino acids; a decrease in cell stress (reduced phosphorylated eIF2α, and Fgf21 and Chop expression); and a trend toward less inflammation (lower Mcp1 and Cd11b expression and less phosphorylated NFκB). Conclusion: HP diets prevent and reverse steatosis independently of fat and carbohydrate intake more efficiently than a 20% reduction in energy intake. The effect appears to result from fuel-generated, highly distributed small, synergistic increases in lipid and BCAA catabolism, and a decrease in cell stress.