A prospective multicenter study of the efficacy of a fiber-supplemented dietary intervention in dogs with chronic large bowel diarrhea.

BACKGROUND: Chronic large bowel diarrhea is common in dogs and can have a significant impact on their overall health and well being. We evaluated the safety and efficacy of a therapeutic food with select dietary plant fibers known to contain antioxidant and polyphenol compounds on clinical signs in dogs with chronic diarrhea. METHODS: A prospective clinical study was conducted in 31 adult dogs currently experiencing chronic diarrhea from private veterinary practices in the United States. Enrolled dogs were switched to a complete and balanced dry therapeutic food containing whole grains and polyphenol-containing fiber sources for 56 days. Veterinarians evaluated changes from baseline in overall clinical signs, recurrence of clinical signs, and stool parameters at Days 2, 3, 4, 28, and 56. Dog owners evaluated stool consistency daily and nausea/vomiting, quality of life (QoL), and stooling behaviors at Days 1, 14, 28, and 56. Statistical analysis was performed using a mixed-effects model with Day as a fixed-effect. RESULTS: Assessments of overall clinical response and stool parameters indicated that diarrhea improved significantly within 1 day of initiating the therapeutic food. Veterinarians reported that 68% of dogs had complete resolution of their clinical signs by Day 56 and the remaining 32% experienced improvement (P < 0.05), with no cases of recurrence. Veterinarians also reported improvement in stool consistency (P < 0.001) and reductions of blood and mucus in stool (P < 0.001). Significant improvements in nausea/vomiting, stooling behaviors, and quality of life (QoL) were reported by dog owners after 28 days and were sustained through day 56 (P < 0.05). The therapeutic food was safe and well tolerated. CONCLUSIONS: In dogs with chronic large bowel diarrhea, the therapeutic food rapidly improved stool consistency, resolved clinical signs, and improved stooling behaviors and QoL. Therapeutic foods supplemented with fiber sources rich in antioxidant and anti-inflammatory compounds contribute to rapid resolution of chronic diarrhea without recurrence and may contribute to long term health.

Microbiome function underpins the efficacy of a fiber-supplemented dietary intervention in dogs with chronic large bowel diarrhea.

BACKGROUND: Chronic large bowel diarrhea is a common occurrence in pet dogs. While nutritional intervention is considered the primary therapy, the metabolic and gut microfloral effects of fiber and polyphenol-enriched therapeutic foods are poorly understood. METHODS: This prospective clinical study enrolled 31 adult dogs from private veterinary practices with chronic, active large bowel diarrhea. Enrolled dogs received a complete and balanced dry therapeutic food containing a proprietary fiber bundle for 56 days. Metagenomic and metabolomic profiling were performed on fecal samples at Days 1, 2, 3, 14, 28, and 56; metabolomic analysis was conducted on serum samples taken at Days 1, 2, 3, 28, and 56. RESULTS: The dietary intervention improved clinical signs and had a clear effect on the gut microfloral metabolic output of canines with chronic diarrhea, shifting gut metabolism from a predominantly proteolytic to saccharolytic fermentative state. Microbial metabolism of tryptophan to beneficial indole postbiotics and the conversion of plant-derived phenolics into bioavailable postbiotics were observed. The intervention altered the endocannabinoid, polyunsaturated fatty acid, and sphingolipid profiles, suggesting a modulation in gastrointestinal inflammation. Changes in membrane phospholipid and collagen signatures were indicative of improved gut function and possible alleviation of the pathophysiology related to chronic diarrhea. CONCLUSIONS: In dogs with chronic diarrhea, feeding specific dietary fibers increased gut saccharolysis and bioavailable phenolic and indole-related compounds, while suppressing putrefaction. These changes were associated with improved markers of gut inflammation and stool quality.

Dietary Protein and Carbohydrate Levels Affect the Gut Microbiota and Clinical Assessment in Healthy Adult Cats.

BACKGROUND: Relative levels of dietary protein and carbohydrate intake influence microbiota and their functional capabilities, but the effect has not been well documented in cats. OBJECTIVES: The impact of 3 foods with different protein:carbohydrate ratios on the gut microbiota and functional attributes in healthy adult cats was evaluated. METHODS: Male and female cats (n = 30; mean age: 5.1 y; mean body weight: 5.26 kg) were fed 1 of 3 foods [P28 (28.3% protein, dry matter basis), P35 (35.1%), and P55 (54.8%)] for 90 d in a Williams Latin Square design. Each food had a 1:1 ratio of animal (dried chicken) to plant (pea) protein; protein replaced carbohydrate as protein level increased. Fecal microbiota and their functional capability were assessed with 16S sequencing and the Kyoto Encyclopedia of Genes and Genomes database, respectively. RESULTS: Fecal pH, ammonia, and branched-chain fatty acids (BCFAs) were higher when cats consumed P55 food than when they consumed P28 and P35. Clear separation of samples between P28 and P55 based on bacterial genera was observed, with partitioning into saccharolytic and proteolytic functions, respectively. Significantly higher α diversity was seen with P55 than with P28 and P35. Amino acid metabolism, mucin foraging pathways, and urea metabolism were higher with P55 than with P28, whereas feces from cats fed P28 had higher concentrations of carbohydrate-active enzymes and enzymes involved in SCFA pathways than with P55. Bacterial genera that showed positive associations with amino acid catabolism also showed positive associations with mucin degradation. CONCLUSIONS: Despite higher protein digestibility and less protein arriving to the colon, when healthy adult cats consumed the highest level of protein (P55), their gut microbiota exhibited higher mucin glycan foraging and amino acid metabolism, leading to higher fecal pH, ammonia, and BCFAs. This is likely due to lower availability of carbohydrate substrates and dietary fiber as protein replaced carbohydrate in the food.

Consumption of identically formulated foods extruded under low and high shear force reveals that microbiome redox ratios accompany canine immunoglobulin A production.

Digestion-resistant starch (RS) can provide health benefits to the host via gut microbiome-mediated metabolism. This study tested the physiological effects on healthy dogs of identically formulated foods processed under high (n = 16) or low (n = 16) shear extrusion conditions resulting in respective lower and higher levels of RS. Faecal samples collected at weeks 3 and 6 were assayed for stool score, proximate analysis, short-chain fatty acids (SCFA), immunoglobulin A (IgA) and microbiome; faecal metabolome was characterized at week 6. Proximate and digestibility analyses of the foods and stool scores and stool proximate analysis showed few differences between the two shear methods except for increased apparent fibre digestibility in the low shear food. In contrast, levels of butyrate (p = .030) and total SCFA (p = .043) were significantly greater in faeces at week 6 from dogs who consumed the low versus high shear food. Faecal IgA levels were significantly higher at week 3 (p = .001) but not week 6 (p = .110) in the low shear food. Significant differences in 166 metabolites between consumption of the two foods were identified via faecal metabolomic analysis, with changes in sugars, bile acids, advanced glycation end products and few amino acids. Strikingly, consumption of the low shear food resulted in elevated levels of the reduced members of redox couples derived from metabolized sugars and branched-chain and phenyl amino acids. Alpha diversity of the microbiome showed significantly higher species richness in faeces from the low shear group at week 6, though other measures of diversity were similar for both foods. Twelve genus-level operational taxonomic units (OTU; half Firmicutes) significantly differed between the food types. Six OTU significantly correlated with RS-derived sugars and ratios of the redox couples. Taken together, these data show that RS impacts microbiome-mediated metabolism in the gut, resulting in changes in the reducing state.

Immune-Modulating Effects of Low-Carbohydrate Ketogenic Foods in Healthy Canines.

Background: Ketogenic foods limit digestible carbohydrates but contain high fat, and have antioxidant and anti-inflammatory effects as well as improving mitochondrial function. ß-Hydroxybutyrate (BHB), 1 of the ketone bodies, reduces the proinflammatory NLR family pyrin domain containing 3 inflammasomes, as well as chemokines in cultures. Objectives: We assessed the immune-modulating effects of 2 low-carbohydrate (LoCHO) foods varying in protein and fat and compared their effects with a food replete with high-carbohydrate (HiCHO) in healthy canines. Methods: Dogs were fed control food [HiCHO; ketogenic ratio (KR: 0.46) followed by LoCHO_PROT (KR: 0.97), then LoCHO_FAT (KR: 1.63) or LoCHO_FAT followed by LoCHO_PROT. Each food was fed for 5 wk, with collections in the 5th wk; 15 wk feeding total. Gene expression for circulating inflammatory cytokines from 10 dogs was assessed using the Canine RT2 Profiler polymerase chain reaction array, and fold changes were calculated using the ΔΔCt method. Results: LoCHO_FAT significantly increased circulating ß-hydroxybutyrate compared with both HiCHO and LoCHO_PROT. When compared with HiCHO, there was a significant decrease in several proinflammatory cytokines/chemokines in LoCHO_PROT and LoCHO_FAT groups, including chemokine (C-C motif) ligand (CCL)1, CCL8, CCL13, CCL17, CCL24, chemokine (C-X3-C motif) ligand 1, chemokine (C-X-C motif) receptor 1, Interleukin-10 receptor alpha ((IL)-10RA), IL-1 receptor antagonist, IL-5, and secreted phosphoprotein 1 (all P < 0.05). Interestingly, a subset of inflammatory proteins that decreased in LoCHO_PROT but not in LoCHO_FAT included IL-33, IL-6 receptor, IL-7, IL-8, Nicotinamide phosphoribosyltransferase, and tumor necrosis factor (TNF) receptor superfamily member 11B. In contrast, the decrease in inflammatory markers in LoCHO_FAT, but not in LoCHO_PROT, included complement component 5, granulocyte colony-stimulating factor or G-CSF, interferon-γ, IL-3, IL-10RB, IL-17C, Tumor necrosis factor superfamily (TNFSF)13, TNFSF13B, and TNFSF14. Decreased concentrations of selected cytokines indicate that both low-carbohydrate foods exert an anti-inflammatory effect and provide a strong rationale for testing their efficacy in dogs with inflammatory conditions. Conclusions: Both LoCHO_PROT and LoCHO_FAT foods might be important as part of immune-modulating therapeutic nutritional strategies to reduce inflammation to maintain health in canines. Our study identifies several inflammatory genes that are reduced when fed ketogenic food that were not previously reported.

S-adenosylmethionine-dependent protein methylation is required for expression of selenoprotein P and gluconeogenic enzymes in HepG2 human hepatocytes.

Cellular methylation processes enable expression of gluconeogenic enzymes and metabolism of the nutrient selenium. Selenium status has been proposed to relate to type II diabetes risk, and plasma levels of selenoprotein P (SEPP1) have been positively correlated with insulin resistance. Increased expression of gluconeogenic enzymes glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase 1 (PCK1) has negative consequences for blood glucose management in type II diabetics. Transcriptional regulation of SEPP1 is directed by the same transcription factors that control the expression of G6PC and PCK1, and these factors are activated by methylation of arginine residues. We sought to determine whether expression of SEPP1 and the aforementioned glucoconeogenic enzymes are regulated by protein methylation, the levels of which are reliant upon adequate S-adenosylmethionine (SAM) and inhibited by S-adenosylhomocysteine (SAH). We treated a human hepatocyte cell line, HepG2, with inhibitors of adenosylhomocysteine hydrolase (AHCY) known to increase concentration of SAH before analysis of G6PC, PCK1, and SEPP1 expression. Increasing SAH decreased 1) the SAM/SAH ratio, 2) protein-arginine methylation, and 3) expression of SEPP1, G6PC, and PCK1 transcripts. Furthermore, hormone-dependent induction of gluconeogenic enzymes was reduced by inhibition of protein methylation. When protein-arginine methyltransferase 1 expression was reduced by siRNA treatment, G6PC expression was inhibited. These findings demonstrate that hepatocellular SAM-dependent protein methylation is required for both SEPP1 and gluconeogenic enzyme expression and that inhibition of protein arginine methylation might provide a route to therapeutic interventions in type II diabetes.

The effects of nitroxyl (HNO) on H2O2 metabolism and possible mechanisms of HNO signaling.

Nitroxyl (HNO) possesses unique and potentially important biological/physiological activity that is currently mechanistically ill-defined. Previous work has shown that the likely biological targets for HNO are thiol proteins, oxidized metalloproteins (i.e. ferric heme proteins) and, most likely, selenoproteins. Interestingly, these are the same classes of proteins that interact with H2O2. In fact, these classes of proteins not only react with H2O2, and thus potentially responsible for the signaling actions of H2O2, but are also responsible for the degradation of H2O2. Therefore, it is not unreasonable to speculate that HNO can affect H2O2 degradation by interacting with H2O2-degrading proteins possibly leading to an increase in H2O2-mediated signaling. Moreover, considering the commonality between HNO and H2O2 biological targets, it also seems likely that HNO-mediated signaling can also be due to reactivity at otherwise H2O2-reactive sites. Herein, it is found that HNO does indeed inhibit H2O2 degradation via inhibition of H2O2-metaboilizing proteins. Also, it is found that in a system known to be regulated by H2O2 (T cell activation), HNO behaves similarly to H2O2, indicating that HNO- and H2O2-signaling may be similar and/or intimately related.

Fatty liver accompanies an increase in lactobacillus species in the hind gut of C57BL/6 mice fed a high-fat diet.

High-fat (HF) diets can produce obesity and have been linked to the development of nonalcoholic fatty liver disease and changes in the gut microbiome. To test the hypothesis that HF feeding increases certain predominant hind gut bacteria and development of steatohepatitis, C57BL/6 mice were fed an HF (45% energy) or low-fat (LF) (10% energy) diet for 10 wk. At the end of the feeding period, body weights in the HF group were 34% greater than those in the LF group (P < 0.05). These changes were associated with dramatic increases in lipid droplet number and size, inflammatory cell infiltration, and inducible nitric oxide (NO) synthase protein concentration in the livers of mice fed the HF diet. Consistent with the fatty liver phenotype, plasma leptin and tumor necrosis factor-α concentrations were also elevated in mice fed the HF diet, indicative of chronic inflammation. Eight of 12 pairs of polymerase chain reaction (PCR) primers for bacterial species that typically predominate hind gut microbial ecology generated specific PCR products from the fecal DNA samples. The amount of DNA from Lactobacillus gasseri and/or Lactobacillus taiwanensis in the HF group was 6900-fold greater than that in the LF group. Many of these bacteria are bile acid resistant and are capable of bile acid deconjugation. Because bile acids are regulators of hepatic lipid metabolism, the marked increase of gut L. gasseri and/or L. taiwanensis species bacteria with HF feeding may play a role in development of steatohepatitis in this model.

Feeding of fish oil and medium-chain triglycerides to canines impacts circulating structural and energetic lipids, endocannabinoids, and non-lipid metabolite profiles.

Introduction: The effect of medium-chain fatty acid-containing triglycerides (MCT), long-chain polyunsaturated fatty acid-containing triglycerides from fish oil (FO), and their combination (FO+MCT) on the serum metabolome of dogs (Canis familiaris) was evaluated. Methods: Dogs (N = 64) were randomized to either a control food, one with 7% MCT, one with FO (0.18% eicosapentaenoate and 1.3% docosahexaenoate), or one with FO+MCT for 28 days following a 14-day washout period on the control food. Serum metabolites were analyzed via chromatography followed by mass spectrometry. Results: Additive effects of serum metabolites were observed for a number of metabolite classes, including fatty acids, phospholipids, acylated amines including endocannabinoids, alpha-oxidized fatty acids, and methyl donors. Some effects of the addition of FO+MCT were different when the oils were combined compared with when each oil was fed separately, namely for acylcarnitines, omega-oxidized dicarboxylic acids, and amino acids. Several potentially beneficial effects on health were observed, including decreased circulating triglycerides and total cholesterol with the addition of FO (with or without MCT) and decreases in N-acyl taurines with the addition of MCT, FO, or FO+MCT. Discussion: Overall, the results of this study provide a phenotypic characterization of the serum lipidomic response to dietary supplementation of long-chain n3-polyunsaturated and medium-chain saturated fats in canines.

Predictive equations for dietary energy are improved when independently developed for dry and wet food which could benefit both the pet and the environment.

Introduction: Measuring energy availability through metabolizable energy feeding studies is the "gold standard" for establishing metabolizable energy concentration. However, predictive equations are often used to estimate metabolizable energy in dog and cat pet foods. The goal of this work was to evaluate the prediction of energy density and compare those predictions to each other and the energy needs of the individual pets. Methods: Feeding studies used 397 adult dogs and 527 adult cats on 1,028 canine and 847 feline foods. Individual pet results for the estimate of metabolizable energy density were used as outcome variables. Prediction equations were generated from the new data and compared to previously published equations. Results and discussion: On average the dogs consumed 747 kilocalories (kcals) per day (SD = 198.7) while cats consumed 234 kcals per day (SD = 53.6). The difference between the average prediction of energy density and the measured metabolizable energy varied from the modified Atwater prediction 4.5%, 3.4% (NRC equations), 1.2% (Hall equations) to the new equations calculated from these data at 0.5%. The average absolute values of the differences between measured and predicted estimates in pet foods (dry and canned, dog and cat) are: 6.7% (modified Atwater), 5.1% (NRC equations), 3.5% (Hall equations) and 3.2% (new equations). All of these estimates resulted in significantly less variation in the estimate of the food expected to be consumed than the observed variation associated with actual pet consumption to maintain body weight. When expressed as a ratio of energy consumed to metabolic body weight (weight in kilograms3/4) the within species variation in energy consumed to maintain weight was still high as compared to the energy density estimates variance from measured metabolizable energy. The amount of food offered as the central point in a feeding guide, based on the prediction equations, would on average result in an average variance between 8.2% error in the worst case estimate (feline dry using modified Atwater estimates) and approximately 2.7% (the new equation for dry dog food). All predictions had relatively small differences in calculating food consumed when compared to the differences associated with the variation in normal energy demand.