The impact of a complete and balanced dietary intervention containing select plant fibers was clinically evaluated in dogs with chronic large bowel diarrhea. Results from the clinical companion study demonstrated that the therapeutic food rapidly alleviated disease symptoms in the cohort [24]. Here, we consider the underlying therapeutic mechanisms of the dietary intervention through microbiome and biochemical profiling. Switching to the fiber-rich food resulted in immediate compositional changes in the stool, including significant reductions in moisture content and changes in several mineral components within 24 hours, consistent with an improved stool quality. These decreases were largely sustained over the 8-week study. A previous study evaluating an analogous food compared to a control lacking the fiber bundle observed similar reductions in moisture and ash content, which were attributed to the fiber increasing fecal organic dry matter content and improving inorganic mineral bioavailability, respectively [15]. While that study included both healthy dogs and dogs with enteritis, our study measured the impact on stool quality longitudinally in an intent-to-treat population of dogs with chronic diarrhea. Remarkably, the rapid and durable improvements in stool quality appeared to correlate with stool composition in this cohort, regardless of the subject’s pet food habits before the study and lifestyles during the study, which could not be strictly controlled.

The bacterial processes of proteolysis and saccharolysis occur in the canine hindgut and depend on the availability of substrate protein and carbohydrate, respectively, as well as the catabolic capacity of the resident microflora. Fecal metabolic profiling was employed to monitor the end products of these processes throughout the duration of the 8-week dietary intervention. Observed decreases in the metabolic products of protein degradation, including ammonia, most amino acids, several dipeptides, as well as the branched SCFAs strongly support the finding that high-fiber intervention reduced bacterial proteolytic and putrefactive processes. The polyamines encompass additional products of bacterial putrefaction, whereas decarboxylation of lysine and arginine yields cadaverine and putrescine, respectively. Subsequent addition of decarboxylated S-adenosyl methionine (SAM-dc) to putrescine generates spermine and spermidine, producing 5-methylthioadenosine (MTA) as a byproduct. Almost all measured polyamine parent and catabolic products decreased upon intervention. Intriguingly, serum metabolomics revealed increases in several circulating amino acids, suggesting the intervention improved protein digestibility and absorption in the small intestine. In contrast, pathway analysis of the microbial composition identified few changes in proteolytic capacity due to the dietary intervention, other than in tryptophan metabolic potential. Taken together, these results suggest that the intervention altered the protein-derived substrate availability for colonic microbiota, resulting in a decreased catabolic signature.

Our results also suggest an increase in saccharolytic activity by the gut microflora. An overall increase in fecal monosaccharides was observed, particularly the plant-derived compounds arabinose, mannose, xylose, and ribulose/xylulose, which may be construed as markers of microbial saccharolysis of the fiber bundle [15, 28]. Among the linear SCFAs, only butyrate showed an appreciable increase. Serum butyrate levels significantly increased over the study, suggesting that bacterially produced butyrate from fermented fiber that was not utilized locally by colonocytes was absorbed into circulation. Fecal microbiome pathway analysis supports the metabolomics differences, as only butyrate-related enzymes were altered after 24 hours of intervention. Additionally, levels of mannitol/sorbitol and ribose were decreased in stool but elevated in serum. Similarly, mannose levels increased in stool while decreasing in circulation. Taken together, these results suggest the dietary intervention differentially altered absorption of plant-derived monosaccharides by improving mannitol/sorbitol and ribose absorption, while leaving excess mannose to be eliminated. Furthermore, stool pH remained constant throughout the intervention. Given that the serum metabolomics analysis also indicated increased levels of circulating monosaccharides and showed few changes in fermentative capacity resulting from alterations in the fecal microbiome, the fiber-rich food positively impacted carbohydrate absorption before nutrients arrived in the colon, rather than altering the fermentative capacity of the colonic microbiome. Therefore, the dietary intervention likely altered the composition of the substrate available for microbial fermentation.

It is generally accepted that putrefactive postbiotics resulting from undigested protein reaching colonic bacteria can have deleterious health effects in both companion pets [29] and humans [30], including those with ulcerative colitis (UC) [31]. Conversely, a shift from proteolytic to saccharolytic microfloral catabolism is associated with positive gut health outcomes [30]. A recent human study found higher fecal levels of amino acids and peptides in patients with irritable bowel syndrome (IBS) compared to healthy controls, further demonstrating the poor outcomes associated with altered dietary protein utilization on gut health [32]. Finally, fermentative butyrate nourishes colonocytes and its serum abundance was recently shown to negatively correlate with dyslipidemia in humans [33]. In our study, increases in fecal and circulating butyrate levels may suggest that the dietary intervention may have metabolic health benefits beyond its impact on alleviating symptoms of large bowel disease.

Tryptophan catabolism in the gut relies heavily on host-microbiota interactions with several downstream functional implications. In humans, approximately 90% of serotonin is produced in the GI tract by colonic enterochromaffin (EC) cells, and resident microflora play an integral role in regulating serotonin levels [34]. Reductions in serotonin levels observed following the dietary intervention could be attributed to several factors, including reductions in tryptophan precursors, microbial-dependent attenuated production by EC cells, or possibly selection of serotonin-degrading microbes, given the significant increase in 5-hydroxyindoleacetate. Pro-inflammatory cytokines can activate indoleamine 2,3-dioxygenase and the subsequent rise in kynurenine pathway metabolites, which are implicated in controlling local and systemic inflammatory responses [35]. In our study, the high fiber food appeared to have minimal effect on kynurenine pathway stimulation (Fig. 2c); therefore, any immune-related amelioration of inflammation associated with large bowel diarrhea occurred through other mechanisms.

The presence of indole pathway metabolites in canines is attributed to microbial activity, given that mammals lack tryptophanase and other enzymes that generate these compounds. Indole pathway metabolites have been shown to be potent aryl hydrocarbon receptor (AHR) ligands in mice and humans, explaining their role in regulating intestinal barrier function and immune homeostasis [36, 37]. 3-indoxyl sulfate (3-IS) arises from a combination of host and microfloral metabolism and represents a chronic renal toxin among many mammals, including dogs [38]. The reductions in 3-IS and indole we observed in our study suggest that the intervention may have provided an additional gut-renal axis improvement.

The dietary intervention we evaluated resulted in significant enrichment of three related indole pathway metabolites in stool, suggesting that the food favored gut commensals that metabolized tryptophan to the postbiotics indoleacetate, indole-3-carboxylate, and 2-oxindole-3-acetate. Secreted by microbes in response to stress and a potent AHR agonist, indoleacetate is associated with suppression of pro-inflammatory cytokines and is often considered a beneficial postbiotic [39, 40]. Likewise, circulating indolepropionate is also suppressed in both active human UC and mouse models of colitis [41]. Restored serum levels of indolepropionate serves as a biomarker of human disease remission, which is attributed to anti-inflammatory IL-10 induction. In our study, the dietary intervention increased serum levels of indolepropionate while leaving fecal levels unchanged. We surmise that excess indolepropionate produced by gut microflora was readily absorbed and circulated, leading to its accumulation in serum. It is intriguing to speculate that circulating indolepropionate may also serve as a biomarker for the alleviation of chronic diarrhea.

A recent study investigating AHR activation identified indole-3-carboxylate as a novel human AHR ligand [42]. Interestingly, indoleacrylate, which was unchanged in the feces but significantly increased in serum in our study, was a more potent agonist than indole-3-carboxylate. Indeed, indoleacrylate production by commensal gut microbes has been shown to stimulate NRF2 gene expression, activating anti-inflammatory and antioxidant immune responses that promote intestinal epithelial barrier integrity [43]. Finally, 2-oxindole-3-acetate, as well as the serotonin catabolite 5-hydroxyindoleacetate, were significantly decreased in the stool of cats with CE [44], trends that appear to be reversed upon intervention in this study. Overall, the serum-plus-fecal signature of microbially-produced indoles may reflect an improvement in chronic large bowel diarrhea, possibly through increased synthesis of AHR ligands.

Changes in fecal levels of flavonoid species likely reflect their content in the interventional food, compared to baseline pet foods, while also providing insights into the metabolic capacities of the canine gut microbiota and their potential GI benefit. These plant-derived polyphenols are poorly absorbed in the GI tract yet are believed to be widely metabolized by resident gut microflora in both humans and pets in a manner that may influence health [45]. The anti-inflammatory properties of several of these compounds are attributed to their influence on intracellular signaling pathways, such as NF-κB and mitogen-activated protein kinases (MAPKs), and their contribution to reducing colitis severity in several rodent models has been documented in vivo [46].

The fiber bundle of the intervention contained citrus and pressed cranberries rich in flavanones, primarily hesperidin [15], a 7-O-glycoside containing the disaccharide rutinose. None of the flavanone-7-O-rutinosides measured in canine stool in our study—hesperidin, narirutin, and neoponcerin—changed appreciably in abundance with the dietary intervention. Conversely, their postbiotic flavonoid components hesperitin and naringenin significantly increased over the feeding period, as well as the disaccharide rutinose. It should be noted that ponciretin, which was also significantly elevated, is a stereoisomer of isosakuranetin, the flavonoid component of neoponcerin; therefore, neoponcerin and ponciretin may represent an additional hydrolyzed pair. Both ponciretin and poncirin were shown to suppress NF-κB and TNF-α-linked inflammation and ameliorate colitis in a chemically induced mouse model, with ponciretin demonstrating superior anti-inflammatory properties [47]. The anti-inflammatory and antioxidant properties of naringenin have been established in vitro, yet clinical data are largely lacking [48]. Hesperidin is readily hydrolyzed by rhamnosidase enzymes found in several probiotic formulations [49]. The observation that the parent glucosides from plants were unchanged while their hydrolyzed components were enriched may be indicative of increased microbial rhamnosidase activity converting flavanones to more bioavailable flavonoids in canine hindguts.

Evaluation of differences in the relative abundances of fecal phytoestrogen suggested that the dietary intervention may stimulate additional microfloral metabolic conversion. Primary dietary sources of phytoestrogens in pet foods include the flavonoid daidzein, as well as the lignans matairesinol and secoisolariciresinol, sourced from soy and flaxseed, respectively. The bioactive forms of equol, enterolactone, and enterodiol arise from colonic microbial metabolism [50]. No soy plant components were included in the intervention food, although several of the dogs were presumably consuming soy-containing food before enrollment in the study, as evident by the significant fecal depletion of daidzein throughout the feeding period. Since the intervention fiber bundle in our study contained flaxseed, matairesinol and secoisolariciresinol significantly increased in feces, as expected. Interestingly, the diglucoside of secoisolariciresinol, presumably a common form of the lignan in the flaxseed component of the food, only trended towards an increase, possibly implicating microbial activity enriching the hydrolyzed conformation. The lower levels of the phytoestrogen equol we observed were likely due to reduced availability of daidzein in the pet food. Enterolactone and enterodiol levels, on the other hand, did not correlate with changes in the concentrations of their parent lignans. Despite significant availability of the lignans secoisolariciresinol and matairesinol, we did not see increases in the microbial products enterolactone and enterodiol. These results point to suppressed microbial metabolic production of lignan-sourced bioactive phytoestrogens.

Mounting evidence implicates the endocannabinoid system, including cannabinoid receptor 1 (CB₁) and N-acyl amide agonists, as an intersection of several nodes of host-microbiota intestinal function, including inflammation, microbial metabolism, gut permeability, and gut motility. Several studies investigating obesity in murine models suggest interplay between alterations in gut microbiota, CB₁ receptor activation and its ligands (e.g., arachidonoyl ethanolamide) promote gut permeability, which along with obesity-induced inflammation can be readily ameliorated by CB₁ blockade [51–53].

The rapid and consistent decreases in fecal N-acyl amides we observed with this dietary intervention appear consistent with the reduction in inflammation and improved gut permeability reported in those studies. Furthermore, endocannabinoid lipid mediators inhibit GI motility through the activation of CB₁, which was demonstrated by exogenous addition of palmitoyl ethanolamide in a murine model of intestinal inflammation [54]. Given the improved clinical stool quality results in this study [24], we suspect the fiber intervention helped normalize colonic transit time. Since endogenous N-acyl amides are synthesized on-demand from membrane lipid precursors and are rapidly degraded after release, the slowed transit time associated with the test food may subsequently temper endogenous demand for endocannabinoids to accomplish the same physiological function, and thus reduce N-acyl amide levels relative to pre-treatment.

Initiation of the dietary intervention resulted in immediate and consistent reductions in fecal sphingolipid metabolites in our study. Sphingolipid metabolism, with ceramide at the core of this pathway, can be influenced by multiple stimuli including inflammatory responses and hypoxia [55]. Both dietary and endogenous sphingolipids are metabolized in the intestine [56], and dysregulation of sphingolipid metabolism has been implicated in several human inflammatory diseases, including UC [57]. In experimental murine models of colitis, dietary sphingomyelin exacerbated mucosal damage, colonic inflammation, and intestinal epithelial cell apoptosis in a manner suggestive of ceramide activation of the lysosomal cell death moderator cathepsin D [58]. The reduction in fecal sphingolipids observed in this study may reflect a diet-induced amelioration of inflammation and a reduction in epithelial apoptosis. Given that serum sphingomyelin levels were also largely increased, the intervention may have also mediated sphingolipid availability in the gut, whereby sphingomyelin components were more readily absorbed and increased in circulation, rather than being eliminated in the stool. While few individual sphingolipids were altered in circulation, as a class they were significantly reduced. Since elevated ceramide levels are associated with inflammatory stimuli, the reduced fecal and serum ceramide signature we observed is consistent with a modulated inflammatory state.

Alterations to both the fecal and serum PUFA profile provide additional evidence for the intervention’s alleviation of inflammation in our study. Typically, n3 and n6 PUFAs are associated with anti-inflammatory and pro-inflammatory properties, respectively. These findings are attributed to the physiology of effector lipids metabolized from either EPA (n3) or arachidonate (n6), as well as the local tissue inflammatory environment, including the types of cells present [59]. Overall, the fecal and serum n3 and n6 profiles of dogs consuming the high-fiber intervention reflected increases in the n3 PUFAs and decreases in n6 PUFAs. Since essential PUFAs are derived from food, changes from baseline in fecal PUFA levels likely reflect their relative abundances in the dietary intervention compared to pet foods the dogs were eating prior to the initiation of the intervention, while serum PUFA levels support their absorption. Indeed, the test food contains fish oil, a rich source of PUFAs, particularly n3 [15]. Additionally, the circulating eicosanoids, which are the bioactive, effector lipids metabolized by immune cell cyclooxygenase and lipoxygenase activity, largely reflected the abundances of their n3 and n6 precursors. Taken together, these profiles suggest the intervention altered the n3/n6 ratio and downstream effector abundances in a manner consistent with a reduction in pro-inflammatory precursors.

In addition to the flavonoids described above, increased abundances of additional classes of molecules found in the intervention food likely imparted nutritional benefit towards the relief of chronic large bowel diarrhea in our study. Components of vitamin E are considered potent antioxidants and radical scavengers with anti-inflammatory properties, particularly the CEHC derived from cytochrome P450-directed metabolism [60]. Systemic absorption and/or availability of these compounds was differentially impacted by the intervention, as we observed increases in circulating alpha-tocopherol and several alpha-CEHC metabolites, while gamma-tocopherol/beta-tocopherol was reduced in both stool and serum. The intervention largely increased terpenoid phytosterols, including beta-sitosterol, stigmasterol, stigmastadienone, and ergosterol. It is well documented that phytosterols can lower serum cholesterol in humans, which is attributed to their inhibition of intestinal cholesterol uptake, and potentially through regulation of cholesterol metabolism [61]. Dietary phytosterol was also shown to improve intestinal inflammation in an experimental colitis model among mice fed low-fat foods [62]. Finally, the terpenoid oleanolate has been shown to alleviate Salmonella typhimurium-induced diarrhea and enteritis in a mouse model through reduced NF-kB and MAPK-mediated intestinal inflammation [63]. Elevated fecal oleanolate upon intervention may also signal alleviation of chronic diarrhea in the study subjects.

While inflammatory enteropathy in the dogs in the trial was not confirmed by endoscopic biopsy, previous study has demonstrated that the underlying cause of the majority of cases of chronic diarrhea in dogs is chronic inflammatory enteropathy, including colitis and other forms of inflammatory bowel disease (IBD) [1]. IBD is associated with perturbations in host immune and microbial molecular interactions at the colonic epithelial barrier [64]. PC, ECM, and to a lesser extent sphingomyelin, are integral components of the colonic mucosal lining, providing the hydrophobic barrier that separates epithelial cells from the resident microbiota. Alterations to the phospholipid content of the colonic mucosa are implicated in the pathophysiology of UC in humans. Although the exact mechanism is unknown, patients with UC present with lower PC levels, which may be due to decreased secretion, reduced adhesion, and/or increased (host or microbe) lipase activity [65, 66]. Indeed, reintroduction of phosphatidylcholine (lecithin) may be clinically therapeutic [67]. In our study, the levels of most PC species were reduced in stool while they tended to increase in circulation. These results suggest the fiber intervention addressed PC depletion associated with inflammation, either by improved absorption in the upper GI tract, increased mucosal adhesion, and/or decreased colonic phospholipid turnover, resulting in additional systemic PC availability for the host, rather than eliminated in the feces. The latter function is further corroborated by the observed rapid increases in fecal phosphocholine, a fundamental constituent necessary for both PC and sphingomyelin biosynthesis, for which colonic depletion is associated with UC [68]. Thus, the significant and sustained increase in fecal phosphocholine with concomitant increases in circulating PCs may reflect an improvement in inflammatory enteropathy and chronic diarrhea.

Collagen fibrils constitute a significant proportion of the ECM and GI connective tissue associated with the colonic mucosa. Postranslational hydroxylation of lysine and proline residues, as well as glycosylation of the former, yields metabolic markers that reflect collagen turnover. The matrix metalloproteases (MMPs) comprise zinc-dependent endopeptidases that sculpt the ECM, including collagen. Upregulation of several secreted MMPs has been reported in the colonic mucosa of dogs with colitis, including MMP-1, MMP-3, and MMP-13 [69], as well as MMP-2 and MMP-9 [70]. Increased expression of the gelatinases MMP-2 and MMP-9 are associated with active human IBD, while their double knockout in murine models of UC is protective [71]. Collectively, these studies imply that elevated colonic mucosal collagen turnover is a consequence of colitis. Here, significant reductions from baseline in collagen markers prohydroxyproline, hydroxyproline, and 5-hydroxylysine were observed in feces, strongly suggesting that the fiber intervention modulated this presumed molecular signature of colitis pathophysiology. Moreover, human patients with IBD present with elevated serum C-terminal telopeptide, indicative of collagen fibril breakdown and typically interpreted as increased bone turnover [72]. In our study, serum hydroxyproline and collagen metabolites overall were reduced upon fiber intervention, which may be construed as suppressed collagen turnover. Further study is necessary to determine whether collagen turnover is systemic in dogs with IBD, and in turn whether the intervention ameliorated systemic collagen turnover.

A major limitation to this study was lack of a control arm; therefore, changes in microbiome composition, metabolic potential, and metabolite levels were discerned from comparisons to baseline. Additionally, not all biochemicals were detected in every patient, necessitating imputation to the observed minimum for missing metabolomic measurements. We, therefore, interpreted the data in the context of pathway changes, rather than individual biomarkers, to account for metabolites that were not uniformly detected. Controlled studies comparing the test food to a control food in healthy and diseased canines are described elsewhere [15]; however, this study was unique in its intent-to-treat objectives. Given the uncontrolled variables of the intent-to-treat population, including (but not limited to) baseline food diversity, breeds, care, and other environmental considerations, the molecular and microbiome baseline characteristics cannot be construed as a uniform disease phenotype. Another limitation is that the etiology of the dogs’ chronic diarrhea was not confirmed prior to enrollment. Although veterinarians in the trial thoroughly evaluated the dogs based on their medical history, clinical signs, physical examinations, and laboratory analyses, endoscopic biopsies were not conducted. Because of the high rates of response to dietary interventions in dogs with chronic diarrhea, dietary changes are typically recommended in dogs with chronic diarrhea before they undergo invasive diagnostic procedures. Consequently, conducting invasive diagnostic procedures may have subjected patients to unnecessary discomfort and would have violated the sponsor’s current animal welfare standards for clinical studies. However, the fact that we saw significant alterations in pathways associated with the pathophysiology of the primary causes of chronic large bowel diarrhea indicates the potential for a fiber-based intervention to drive the GI and systemic health of these canines.