Gary Duncan

Divergent mechanisms of cis9, trans11-and trans10, cis12-conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach

Conjugated linoleic acids (CLA) affect atherogenesis, but mechanisms are not well understood. We explored how two isomers of CLA, cis9, trans11‐CLA and trans10, cis12‐CLA, affected lipid and glucose metabolism, as well as hepatic protein expression, in apolipoprotein E knockout mice. After 12 wk of intervention, plasma triglyceride, NEFA, and glucose concentrations were significantly higher in the trans10, cis12‐CLA group, whereas plasma triglyceride, NEFA, glucose, and insulin concentrations were significantly lower in the cis9, trans 11‐CLA group, compared with control mice consuming linoleic acid. Proteomics identified significant up‐ or down‐regulation of 113 liver cytosolic proteins by either CLA isomer. Principal component analysis revealed that the treatment effect of cis9, trans11‐CLA was mainly explained by the up‐regulation of different posttranslational forms of heat shock protein 70 kD. In contrast, the treatment effect of trans10, cis12‐CLA was mainly explained by up‐regulation of key enzymes in the gluconeogenic, β‐oxidation, and ketogenesic pathways. Correlation analysis again emphasized the divergent effects of both CLA isomers on different pathways, but also revealed a linkage between insulin resistance and increased levels of hepatic serotransferrin. Thus, our systems biology approach provided novel insights into the mechanisms by which individual CLA isomers differentially affect pathways related to atherogenesis, such as insulin resistance and inflammation. Baukje De Roos, Garry Rucklidge, Martin Reid, Karen Ross, Gary Duncan, Maria A. Navarro, Jose M. Arbones‐Mainar, Mario A. Guzman‐Garcia, Jesus Osada, John Browne, Christine E. Loscher, Helen M. Roche Divergent mechanisms of cis9, trans11‐ and trans10, cis12‐conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach. FASEB J. 19, 1–21 (2005)

The Human Fecal Microbiota Metabolizes Deoxynivalenol and Deoxynivalenol-3-Glucoside and May Be Responsible for Urinary Deepoxy-Deoxynivalenol

ABSTRACT Deoxynivalenol (DON) is a potent mycotoxin produced by Fusarium molds and affects intestinal nutrient absorption and barrier function in experimental and farm animals. Free DON and the plant metabolite DON-3-β-d-glucoside (D3G) are frequently found in wheat and maize. D3G is stable in the upper human gut, but some human intestinal bacteria release DON from D3G in vitro. Furthermore, some bacteria derived from animal digestive systems degrade DON to a less toxic metabolite, deepoxy-deoxynivalenol (DOM-1). The metabolism of D3G and DON by the human microbiota has not been fully assessed. We therefore conducted in vitro batch culture experiments assessing the activity of the human fecal microbiota to release DON from D3G. We also studied detoxification of DON to DOM-1 by the microbiota and its potential effect on urinary DON excretion in humans. Fecal slurry from five volunteers was spiked with DON or D3G and incubated anaerobically (from 1 h to 7 days), and mycotoxins were extracted into acetonitrile. Mycotoxins were detected in fecal extracts and urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The fecal microbiota released DON from D3G very efficiently, with hydrolysis peaking after 4 to 6 h. The fecal microbiota from one volunteer transformed DON to DOM-1. Urine from the same volunteer also contained DOM-1 (4.7% of DON), whereas DOM-1 was not detectable in urine from other volunteers. Our results confirm that the fecal microbiota releases DON from its glycosylated form, hence increasing the toxic burden in exposed individuals. Furthermore, this is first evidence that the human fecal microbiota of one volunteer detoxifies DON, resulting in the appearance of DOM-1 in urine.

Major phenylpropanoid‐derived metabolites in the human gut can arise from microbial fermentation of protein

SCOPE Plant secondary metabolites, such as phenolic acids are commonly associated with benefits for human health. Two of the most abundant phenylpropanoid-derived compounds detected in human faecal samples are phenylacetic acid (PAA) and 4-hydroxylphenylacetic acid (4-hydroxyPAA). Although they have the potential to be derived from diets rich in plant-based foods, evidence suggests that these compounds can be derived from the microbial fermentation of aromatic amino acids (AAAs) in the colon. METHODS AND RESULTS To identify the bacteria responsible, 26 strains representing 25 of the dominant human colonic species were screened for phenyl metabolite formation. Seven strains produced significant amounts of both PAA and 4-hydroxyPAA. These included five out of seven Bacteroidetes (Bacteroides thetaiotaomicron, Bacteroides eggerthii, Bacteroides ovatus, Bacteroides fragilis, Parabacteroides distasonis), and two out of 17 Firmicutes (Eubacterium hallii and Clostridium bartlettii). These species also produced indole-3-acetic acid (IAA), the corresponding tryptophan metabolite, but C. bartlettii showed 100 times higher IAA production than the other six strains. Four strains were further tested and PAA formation was substantially increased by phenylalanine, 4-hydroxyPAA by tyrosine and IAA by tryptophan. CONCLUSION This study demonstrates that certain microbial species have the ability to ferment all three AAAs and that protein fermentation is the likely source of major phenylpropanoid-derived metabolites in the colon.

Attenuation of inflammation and cellular stress-related pathways maintains insulin sensitivity in obese type I interleukin-1 receptor knockout mice on a high-fat diet.

The development of insulin resistance in the obese is associated with chronic, low‐grade inflammation. We aimed to identify novel links between obesity, insulin resistance and the inflammatory response by comparing C57BL/6 with type I interleukin‐1 receptor knockout (IL‐1RI−/−) mice, which are protected against diet‐induced insulin resistance. Mice were fed a high‐fat diet for 16 wk. Insulin sensitivity was measured and proteomic analysis was performed on adipose, hepatic and skeletal muscle tissues. Despite an equal weight gain, IL‐1RI−/− mice had lower plasma glucose, insulin and triacylglycerol concentrations, compared with controls, following dietary treatment. The higher insulin sensitivity in IL‐1RI−/− mice was associated with down‐regulation of antioxidant proteins and proteasomes in adipose tissue and hepatic soluble epoxide hydrolase, consistent with a compromised inflammatory response as well as increased glycolysis and decreased fatty acid β‐oxidation in their muscle. Their lower hepatic triacylglycerol concentrations may reflect decreased flux of free fatty acids to the liver, decreased hepatic fatty acid‐binding protein expression and decreased lipogenesis. Correlation analysis revealed down‐regulation of classical biomarkers of ER stress in their adipose tissue, suggesting that disruption of the IL‐1RI‐mediated inflammatory response may attenuate cellular stress, which was associated with significant protection from diet‐induced insulin resistance, independent of obesity.

Blood Folate Status and Expression of Proteins Involved in Immune Function, Inflammation, and Coagulation: Biochemical and Proteomic Changes in the Plasma of Humans in Response to Long-Term Synthetic Folic Acid Supplementation

We used plasma proteomics to identify human proteins responsive to folate status. Plasma was collected from subjects treated with placebo or 1.2 mg of folic acid daily for 12 weeks in a randomized controlled trial. Homocysteine and folate were measured by immunoassay and uracil misincorporation by electrophoresis. The plasma proteome was assessed by 2-D gel electrophoresis, and proteins were identified by LC MS/MS. 5-methylTHF increased 5-fold (P = 0.000003) in response to intervention. Red cell folate doubled (P = 0.013), and lymphocyte folate increased 44% (P = 0.0001). Hcy and uracil dropped 22% (P = 0.0005) and 25% (P = 0.05), respectively. ApoE A-1, alpha-1-antichymotrypsin, antithrombin, and serum amyloid P were downregulated, while albumin, IgM C, and complement C3 were upregulated (P < 0.05). More than 60 proteins were significantly associated with folate pre- and postintervention (P < 0.01). These were categorized into metabolic pathways related to complement fixation (e.g., C1, C3, C4, Factor H, Factor 1, Factor B, clusterin), coagulation (e.g., antithrombin, alpha-1-antitrypsin, kininogen) and mineral transport (e.g., transthyretin, haptoglobin, ceruloplasmin). Low folate status pre- and post-treatment were associated with lower levels of proteins involved in activation and regulation of immune function and coagulation. Supplementation with synthetic folic acid increased expression of these proteins but did not substantially disrupt the balance of these pathways.

The response of human colonocytes to folate deficiency in vitro: functional and proteomic analyses.

Low folate intake is associated with colon cancer. We combined a proteomics and biochemical approach to identify proteins and pathways affected by folate deficiency in human colonocytes. Folate differentially altered activity and expression of proteins involved in proliferation [e.g., PCNA], DNA repair [e.g., XRCC5, MSH2], apoptosis [e.g., BAG family chaperone protein, DIABLO and porin], cytoskeletal organization [e.g., actin, ezrin, elfin], and expression of proteins implicated in malignant transformation [COMT, Nit2].

Masked trichothecene and zearalenone mycotoxins withstand digestion and absorption in the upper GI tract but are efficiently hydrolyzed by human gut microbiota in vitro

SCOPE Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked metabolites. The fate of masked mycotoxins in the human gut is poorly understood. Here we assess the metabolism and transport of glucoside metabolites of common trichothecenes (deoxynivalenol, nivalenol, T-2 toxin) and zearalenone compounds (zearalenone, α- and β-zearalenol) in the human gut in vitro. METHODS AND RESULTS Masked mycotoxins were incubated with artificial digestive juices and absorption was assessed in differentiated Caco-2/TC7 cells. Colonic metabolism was studied using fecal batch cultures from five donors and mycotoxins were detected using LC-MS/MS. All masked mycotoxins were stable under upper GI tract conditions and no absorption was observed. Free trichothecenes were absorbed intact whereas free zearalenone compounds were absorbed and metabolized to undetected compounds by Caco-2/TC7 cells. Human gut microbiota efficiently hydrolyzed all masked mycotoxins. Trichothecenes were fully recovered as parent mycotoxins whereas 40-70% of zearalenone compounds were further metabolized to unknown metabolites. CONCLUSION Our results demonstrate that masked trichothecenes will reach the colon intact to be released as parent mycotoxins by gut microbiota, hence contributing to mycotoxin exposure. Masked zearalenone compounds are metabolized by gut microbiota and epithelial cells and the identity and toxicity of metabolites remain to be determined.

Annual variation of dietary deoxynivalenol exposure during years of different Fusarium prevalence: a pilot biomonitoring study

Dietary exposure to deoxynivalenol (DON) has been reported previously in the UK, but levels were low and most individuals are well protected by the maximum permitted levels in food set by the European Commission. However, no information is available on annual fluctuation in dietary DON exposure. We hypothesised that dietary DON exposure may vary when individuals consume cereals derived from harvests with low (2011) and high (2012) Fusarium prevalence. In this pilot study, spot urine samples were collected in years 1 and 2 from 15 volunteers following their habitual diet. Urinary DON was analysed by LC-MS/MS to estimate 24-h DON excretion and daily dietary DON intake. DON was detectable in all urine samples with an average excretion of 10.08 ± 9.13 µg/24-h urine in year 1 which significantly (p = 0.005) increased to 24.84 ± 13.83 µg/24-h urine in year 2. This resulted from an estimated dietary intake of 195.94 ± 166.44 ng DON kg–1 BW in year 1 and 518.64 ± 292.49 ng DON kg–1 BW in year 2. Based on these estimates, the tolerable daily intake for DON was exceeded in 13% of occasions in year 2 and none in year 1. This pilot study is based on estimates of DON intake derived from urinary DON excretion. Results suggest that DON exposure varies annually and that current maximum levels might not sufficiently protect consumers during years of high Fusarium prevalence.

Intervention with fish oil, but not with docosahexaenoic acid, results in lower levels of hepatic soluble epoxide hydrolase with time in apoE knockout mice.

Long-chain n-3 PUFA from fish oil protect against death from CHD but mechanisms are not well understood. Preliminary results indicate that fish oil may affect the enzyme soluble epoxide hydrolase (sEH) and influence inflammatory pathways in a time-dependent manner. In the present study male apoE knockout (Apoe-/-) mice were randomised to three dietary groups receiving a high-fat high-cholesterol diet supplemented with 2 % (w/w) high-oleic acid sunflower-seed (HOSF) oil, DHA oil or fish oil. Livers and proximal aortas were collected on day 2 and on weeks 1, 2, 4 and 10 to determine hepatic sEH levels, hepatic fatty acid composition, hepatic proteome and atherosclerotic plaque size in the aortic root. Intervention with fish oil, but not with DHA, resulted in significantly lower levels of hepatic sEH levels with time compared with HOSF oil. DHA and fish oil caused differential regulation of thirty-five hepatic proteins which were mainly involved in lipoprotein metabolism and oxidative stress. All mice developed atherosclerosis without differences in plaque size between the three groups. Thus EPA may be responsible for lowering levels of hepatic sEH and both fish oil and DHA could beneficially affect lipoprotein metabolism and oxidative stress.

Role of Pathogen-Derived Cell Wall Carbohydrates and Prostaglandin E2 in Immune Response and Suppression of Fish Immunity by the Oomycete Saprolegnia parasitica

ABSTRACT Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i.e., induction of interleukin-1β1 [IL-1β1], IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglanding E2 (PGE2) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE2 was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon [IFN-γ] and the IFN-γ-inducible protein [γ-IP]). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development.