Microbial Metabolites Determine Host Health and the Status of Some Diseases.

The gastrointestinal (GI) tract is a highly complex organ composed of the intestinal epithelium layer, intestinal microbiota, and local immune system. Intestinal microbiota residing in the GI tract engages in a mutualistic relationship with the host. Different sections of the GI tract contain distinct proportions of the intestinal microbiota, resulting in the presence of unique bacterial products in each GI section. The intestinal microbiota converts ingested nutrients into metabolites that target either the intestinal microbiota population or host cells. Metabolites act as messengers of information between the intestinal microbiota and host cells. The intestinal microbiota composition and resulting metabolites thus impact host development, health, and pathogenesis. Many recent studies have focused on modulation of the gut microbiota and their metabolites to improve host health and prevent or treat diseases. In this review, we focus on the production of microbial metabolites, their biological impact on the intestinal microbiota composition and host cells, and the effect of microbial metabolites that contribute to improvements in inflammatory bowel diseases and metabolic diseases. Understanding the role of microbial metabolites in protection against disease might offer an intriguing approach to regulate disease.

Pro- and anti-inflammatory effects of short chain fatty acids on immune and endothelial cells.

In the gastro-intestinal tract, short chain fatty acids (SCFAs) have protective effects on epithelial cells. However, their effects on inflammatory cytokine production by endothelial and immune cells and the recruitment of immune cells and their trans-migration across the endothelial layer remain controversial. Both cell types are associated with the initiation and development of inflammatory diseases, such as atherosclerosis and sepsis. SCFAs modulate immune and inflammatory responses via activation of free fatty acid (FFA) receptors type 2 and 3 (FFA2 and FFA3 receptors), G protein-coupled receptor 109A (GPR109A) and inhibition of histone deacetylases (HDACs). This review will focus on the effects of SCFAs on lipopolysaccharide (LPS)- or tumor necrosis factor-alpha (TNFα)-induced inflammatory response on endothelial and immune cells function, and an overview is presented on the underlying mechanisms of the effects of SCFAs on both immune and endothelial cells, including HDACs, FFA2 and FFA3 receptors and GPR109A regulation of nuclear factor-kappa B (NF-κB) activation and mitogen-activated protein kinase (MAPK) signaling pathways.

Glutamate excitotoxicity induced by orally administered propionic acid, a short chain fatty acid can be ameliorated by bee pollen.

BACKGROUND: Rodent models may guide investigations towards identifying either environmental neuro-toxicants or drugs with neuro-therapeutic effects. This work aims to study the therapeutic effects of bee pollen on brain glutamate excitotoxicity and the impaired glutamine-glutamate- gamma amino butyric acid (GABA) circuit induced by propionic acid (PPA), a short chain fatty acid, in rat pups. METHODS: Twenty-four young male Western Albino rats 3-4 weeks of age, and 45-60 g body weight were enrolled in the present study. They were grouped into four equal groups: Group 1, the control received phosphate buffered saline at the same time of PPA adminstration; Group 2, received 750 mg/kg body weight divided into 3 equal daily doses and served as acute neurotoxic dose of PPA; Group 3, received 750 mg/kg body weight divided in 10 equal doses of 75 mg/kg body weight/day, and served as the sub-acute group; and Group 4, the therapeutic group, was treated with bee pollen (50 mg/kg body weight) for 30 days after acute PPA intoxication. GABA, glutamate and glutamine were measured in the brain homogenates of the four groups. RESULTS: The results showed that PPA caused multiple signs of excitotoxicity, as measured by the elevation of glutamate and the glutamate/glutamine ratio and the decrease of GABA, glutamine and the GABA/glutamate ratio. Bee pollen was effective in counteracting the neurotoxic effects of PPA to a certain extent. CONCLUSION: In conclusion, bee pollen demonstrates ameliorating effects on glutamate excitotoxicity and the impaired glutamine-glutamate-GABA circuit as two etiological mechanisms in PPA-induced neurotoxicity.

Stability and inhibition of anaerobic processes caused by insufficiency or excess of ammonia nitrogen.

Ammonia increases buffer capacity of methanogenic medium in mesophilic anaerobic reactor thus increasing the stability of anaerobic digestion process. Optimal ammonia concentration ensures sufficient buffer capacity while not inhibiting the process. It was found out in this paper that this optimum depends on the quality of anaerobic sludge under investigation. The optimal concentrations for methanogens were 2.1, 2.6 and 3.1 g/L of ammonia nitrogen in dependence on inoculum origin. High ammonia nitrogen concentration (4.0 g/L) inhibited methane production, while low ammonia nitrogen concentration (0.5 g/L) caused low methane yield, loss of biomass (as VSS) and loss of the aceticlastic methanogenic activity. It was found out that negative effect of low ammonia nitrogen concentration on biomass is caused not only by low buffer capacity but also by insufficiency of nitrogen as nutrient. It was also found out that anaerobic sludge with higher ammonia nitrogen concentration (4.2 g/L) tolerates even concentration of volatile fatty acids (160 mmol/L) which causes inhibition of the process with low ammonia nitrogen concentration (0.2 g/L).

Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death.

Short-chain fatty acids (SCFAs) are the major by-products of bacterial fermentation of undigested dietary fibers in the large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development, and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKα phosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate-induced apoptosis through activation of caspase-7 and caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell apoptosis.

Comparative effects of cellulose and soluble fibers (pectin, konjac glucomannan, inulin) on fecal water toxicity toward Caco-2 cells, fecal bacteria enzymes, bile acid, and short-chain fatty acids.

The aim of this study was to compare the effects of cellulose and three soluble dietary fibers, pectin, konjac glucomannan (KGM), and inulin, on the cytotoxicity and DNA damage of fecal water-treated Caco-2 cells, a human colon adenocarcinoma cell line, and to investigate the fecal components that potentially modulate the fecal toxicity, that is, bacterial enzymes, bile acids, and short-chain fatty acids. Six-week-old BALB/cJ mice were randomly allocated to consume an AIN-93 diet that contained no dietary fiber (fiber-free) or 5% (w/w) cellulose, pectin, KGM, and inulin for 3 weeks. Feces were collected during days 18-21. Fecal waters were co-incubated with Caco-2 cells to determine the cytotoxicity and DNA damage. In addition, the fecal bacterial enzymes, bile acids, and short-chain fatty acids were determined. Results indicated that all fiber diets similarly increased the survival rate (%) of fecal water-treated Caco-2 cells as compared with the fiber-free diet. The inhibition of fecal water-induced DNA damage in Caco-2 cells was greater for the pectin and inulin diets than for the cellulose and KGM diets. In contrast, cellulose exerted the greatest inhibitory effect on the fecal ß-glucuronidase activity. Cellulose and all soluble dietary fibers reduced the secondary bile acid concentrations in the fecal water, but only soluble fibers increased the fecal concentrations of short-chain fatty acids, as compared with no fiber. Therefore, this study suggests that all dietary fibers substantially reduced the fecal water toxicity, which is associated with decreased secondary bile acid levels by all fibers, reduced fecal ß-glucuronidase activity by cellulose, and increased short-chain fatty acid levels by soluble dietary fibers.

Structure-activity-dependent regulation of cell communication by perfluorinated fatty acids using in vivo and in vitro model systems.

BACKGROUND: Perfluoroalkanoates, [e.g., perfluorooctanoate (PFOA)], are known peroxisome proliferators that induce hepatomegaly and hepatocarcinogenesis in rodents, and are classic non-genotoxic carcinogens that inhibit in vitro gap-junctional intercellular communication (GJIC). This inhibition of GJIC is known to be a function of perfluorinated carbon lengths ranging from 7 to 10. OBJECTIVES: The aim of this study was to determine if the inhibition of GJIC by PFOA but not perfluoropentanoate (PFPeA) observed in F344 rat liver cells in vitro also occurs in F344 rats in vivo and to determine mechanisms of PFOA dysregulation of GJIC using in vitro assay systems. METHODS: We used an incision load/dye transfer technique to assess GJIC in livers of rats exposed to PFOA and PFPeA. We used in vitro assays with inhibitors of cell signaling enzymes and antioxidants known to regulate GJIC to identify which enzymes regulated PFOA-induced inhibition of GJIC. RESULTS: PFOA inhibited GJIC and induced hepatomegaly in rat livers, whereas PFPeA had no effect on either end point. Serum biochemistry of liver enzymes indicated no cytotoxic response to these compounds. In vitro analysis of mitogen-activated protein kinase (MAPK) indicated that PFOA, but not PFPeA, can activate the extracellular receptor kinase (ERK). Inhibition of GJIC, in vitro, by PFOA depended on the activation of both ERK and phosphatidylcholine-specific phospholipase C (PC-PLC) in the dysregulation of GJIC in an oxidative-dependent mechanism. CONCLUSIONS: The in vitro analysis of GJIC, an epigenetic marker of tumor promoters, can also predict the in vivo activity of PFOA, which dysregulated GJIC via ERK and PC-PLC.

Neurobiological effects of intraventricular propionic acid in rats: possible role of short chain fatty acids on the pathogenesis and characteristics of autism spectrum disorders.

Clinical observations suggest that certain gut and dietary factors may transiently worsen symptoms in autism spectrum disorders (ASD), epilepsy and some inheritable metabolic disorders. Propionic acid (PPA) is a short chain fatty acid and an important intermediate of cellular metabolism. PPA is also a by-product of a subpopulation of human gut enterobacteria and is a common food preservative. We examined the behavioural, electrophysiological, neuropathological, and biochemical effects of treatment with PPA and related compounds in adult rats. Intraventricular infusions of PPA produced reversible repetitive dystonic behaviours, hyperactivity, turning behaviour, retropulsion, caudate spiking, and the progressive development of limbic kindled seizures, suggesting that this compound has central effects. Biochemical analyses of brain homogenates from PPA treated rats showed an increase in oxidative stress markers (e.g., lipid peroxidation and protein carbonylation) and glutathione S-transferase activity coupled with a decrease in glutathione and glutathione peroxidase activity. Neurohistological examinations of hippocampus and adjacent white matter (external capsule) of PPA treated rats revealed increased reactive astrogliosis (GFAP immunoreactivity) and activated microglia (CD68 immunoreactivity) suggestive of a neuroinflammatory process. This was coupled with a lack of cytotoxicity (cell counts, cleaved caspase 3' immunoreactivity), and an increase in phosphorylated CREB immunoreactivity. We propose that some types of autism may be partial forms of genetically inherited or acquired disorders involving altered PPA metabolism. Thus, intraventricular administration of PPA in rats may provide a means to model some aspects of human ASD in rats.

In vitro effects of hydrochloric acid and various concentrations of acetic, propionic, butyric, or valeric acids on bioelectric properties of equine gastric squamous mucosa.

OBJECTIVE: To compare the effects of hydrochloric acid (HCl) and various concentrations of volatile fatty acids (VFAs) on tissue bioelectric properties of equine stomach nonglandular (NG) mucosa. SAMPLE POPULATION: Gastric tissues obtained from 48 adult horses. PROCEDURES: NG gastric mucosa was studied by use of Ussing chambers. Short-circuit current (Isc) and potential difference (PD) were measured and electrical resistance (R) and conductance calculated for tissues after addition of HCl and VFAs (5, 10, 20, and 40 mM) in normal Ringer's solution (NRS). RESULTS: Mucosa exposed to HCl in NRS (pH of 1.5 and, to a lesser extent, 4.0) had a significant decrease in Isc, PD, and R, whereas tissues exposed to acetic acid at a pH of < 4.0, propionic and butyric acids at a pH of

Evaluation of the effects of short-chain fatty acids and extracellular pH on bovine neutrophil function in vitro.

OBJECTIVE: To investigate the effects of short-chain fatty acids (SCFAs) and pH on neutrophil oxidative burst, phagocytosis, and morphology after exposure to acetate, propionate, butyrate, or succinate at pH 5.5 and 6.7. SAMPLE POPULATION: Neutrophils isolated from bovine blood samples and Porphyromonas levii, Prevotella spp, and Bacteroides fragilis isolated from lesions of cattle with acute interdigital phlegmon (foot rot). PROCEDURES: Bacteria were cultured in strictly anaerobic conditions. Bacterial SCFA production was measured with high-performance liquid chromatography. Neutrophils were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ), and incubated with dihydroethidium or dichlorofluorescein diacetate to measure production of O(2)and H(2)O(2), respectively. Phagocytosis was assessed after exposure to serum-opsonized bacteria. Cellular morphology was assessed with differential staining. RESULTS: All bacteria produced at least 3 of the 4 SCFAs. Production of both O(2) and H(2)O(2) was markedly curtailed in PMA-stimulated neutrophils exposed to SCFA at pH 5.5, compared with production at pH 6.7. Succinate caused a significant dose-dependent decrease in O(2) production at pH 6.7 in OZ-stimulated neutrophils. Monoprotic SCFAs elicited a significant increase in H(2)O(2) production in OZ-stimulated neutrophils at pH 6.7 but a significant decrease at pH 5.5. Monoprotic SCFAs significantly increased phagocytosis at pH 6.7 but decreased phagocytic activity at pH 5.5. Cellular necrosis was observed in cells exposed to SCFAs at pH 5.5. CONCLUSIONS AND CLINICAL RELEVANCE: Establishment and persistence of anaerobic bacteria in cattle with foot rot infection may result in part from neutrophil dysfunction secondary to the effects of bacterially secreted SCFA in acidotic microenvironments.

Ranking the toxicity of fatty acids on Jurkat and Raji cells by flow cytometric analysis.

The fatty acids have an important role in the control of leukocyte metabolism and function. Higher concentrations of certain fatty acids, particularly polyunsaturated fatty acids (PUFAs) and volatile fatty acids, can cause cell death via apoptosis or, when concentrations are greater, necrosis. In this study, we determined the highest concentrations of various fatty acids that are non-toxic to two human leukemic cell lines, Jurkat (T-lymphocyte) and Raji (B-lymphocyte). Toxicity was evaluated by either loss of membrane integrity and/or DNA fragmentation using flow cytometric analysis. There were no remarkable differences for the toxicity of the fatty acids between B and T cell lines. The cytotoxicity of the fatty acids was related to the carbon chain length and number of double bonds: docosahexaenoic acid=eicosapentaenoic acid=arachidonic acid=gamma-linolenic acid=stearic acid=palmitic acid > linoleic acid=palmitoleic acid > vacenic acid=lauric acid > oleic acid > elaidic acid > capric acid > butyric acid > caprylic acid=caproic acid=propionic acid. The proportion of cells undergoing apoptosis or necrosis, induced by the fatty acids tested, remains to be investigated.

Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria.

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.