Junhai Ou

Diet, microbiota, and microbial metabolites in colon cancer risk in rural Africans and African Americans

BACKGROUND Epidemiologic studies have suggested that most cases of sporadic colon cancer can be attributed to diet. The recognition that colonic microbiota have a major influence on colonic health suggests that they might mediate colonic carcinogenesis. OBJECTIVE To examine the hypothesis that the influence of diet on colon cancer risk is mediated by the microbiota through their metabolites, we measured differences in colonic microbes and their metabolites in African Americans with a high risk and in rural native Africans with a low risk of colon cancer. DESIGN Fresh fecal samples were collected from 12 healthy African Americans aged 50-65 y and from 12 age- and sex-matched native Africans. Microbiomes were analyzed with 16S ribosomal RNA gene pyrosequencing together with quantitative polymerase chain reaction of the major fermentative, butyrate-producing, and bile acid-deconjugating bacteria. Fecal short-chain fatty acids were measured by gas chromatography and bile acids by liquid chromatography-mass spectrometry. RESULTS Microbial composition was fundamentally different, with a predominance of Prevotella in native Africans (enterotype 2) and of Bacteroides in African Americans (enterotype 1). Total bacteria and major butyrate-producing groups were significantly more abundant in fecal samples from native Africans. Microbial genes encoding for secondary bile acid production were more abundant in African Americans, whereas those encoding for methanogenesis and hydrogen sulfide production were higher in native Africans. Fecal secondary bile acid concentrations were higher in African Americans, whereas short-chain fatty acids were higher in native Africans. CONCLUSION Our results support the hypothesis that colon cancer risk is influenced by the balance between microbial production of health-promoting metabolites such as butyrate and potentially carcinogenic metabolites such as secondary bile acids.

Fat, fibre and cancer risk in African Americans and rural Africans

Rates of colon cancer are much higher in African Americans (65:100,000) than in rural South Africans (<5:100,000). The higher rates are associated with higher animal protein and fat and lower fiber consumption, higher colonic secondary bile acids, lower colonic short chain fatty acid quantities and higher mucosal proliferative biomarkers of cancer risk in otherwise healthy middle aged volunteers. Here we investigate further the role of fat and fiber in this association. We performed two-week food exchanges in subjects from the same populations, where African Americans were fed a high-fiber, lowfat African-style diet, and rural Africans a high-fat low-fiber western-style diet under close supervision. In comparison to their usual diets, the food changes resulted in remarkable reciprocal changes in mucosal biomarkers of cancer risk and in aspects of the microbiota and metabolome known to affect cancer risk, best illustrated by increased saccharolytic fermentation and butyrogenesis and suppressed secondary bile acid synthesis in the African Americans.

Products of the Colonic Microbiota Mediate the Effects of Diet on Colon Cancer Risk

It is estimated that most colon cancers can be attributed to dietary causes. We have hypothesized that diet influences the health of the colonic mucosa through interaction with the microbiota and that it is the milieu interior that regulates mucosal proliferation and therefore cancer risk. To validate this further, we compared colonic contents from healthy 50- to 65-y-old people from populations with high and low risk, specifically low risk Native Africans (cancer incidence <1:100,000; n = 17), high risk African Americans (risk 65:100,000; n = 17), and Caucasian Americans (risk 50:100,000; n = 18). Americans typically consume a high-animal protein and -fat diet, whereas Africans consume a staple diet of maize meal, rich in resistant starch and low in animal products. Following overnight fasting, rapid colonic evacuation was performed with 2 L polyethylene glycol. Total colonic evacuants were analyzed for SCFA, vitamins, nitrogen, and minerals. Total SCFA and butyrate were significantly higher in Native Africans than in both American groups. Colonic folate and biotin content, measured by Lactobacillus rhamnoses and Lactobacillus plantarum ATCC 8014 bioassay, respectively, exceeded normal daily dietary intakes. Compared with Africans, calcium and iron contents were significantly higher in Caucasian Americans and zinc content was significantly higher in African Americans, but nitrogen content did not differ among the 3 groups. In conclusion, the results support our hypothesis that the microbiota mediates the effect diet has on colon cancer risk by their generation of butyrate, folate, and biotin, molecules known to play a key role in the regulation of epithelial proliferation.

Association between low colonic short-chain fatty acids and high bile acids in high colon cancer risk populations.

We propose that the influence of diet on colon cancer risk is mediated by the microbiota. To investigate how dietary fat influences risk, we compared the colonic contents of 12 adult high-risk African Americans (AAs) and 10 Caucasian Americans (CAs) who consumed a high-fat diet (123 ± 11 g/d and 129 ± 17 g/d, respectively) to 13 native Africans (NAs) who subsisted on a low-fat (38 ± 3.0 g/d) diet, all aged 50–60 yr. The colonic bile acids were measured by LC-MS and the short-chain fatty acids (SCFAs) by GC. The chief secondary colonic bile acids, deoxycholic acid and lithocholic acid, were correlated with fat intake and similar between AAs and CAs, but 3–4 times higher than in AAs (p < 0.05). The major SCFAs were lower in AAs (p < 0.001) and CAs (p < 0.001) compared to AAs, but conversely, the branched chain fatty acids (BFCA) were higher. Our results suggest that the higher risk of colon cancer in Americans may be partly explained by their high-fat and high-protein, low complex carbohydrate diet, which produces colonic residues that promote microbes to produce potentially carcinogenic secondary bile acids and less antineoplastic SCFAs. The role of BCFA in colonic carcinogenesis deserves further study.

Effect of fiber supplementation on the microbiota in critically ill patients

AIM To determine tolerance to fiber supplementation of semi-elemental tube feeds in critically ill patients and measure its effect on colonic microbiota and fermentation. METHODS Thirteen intensive care unit patients receiving jejunal feeding with a semi-elemental diet for predominantly necrotizing pancreatitis were studied. The study was divided into 2 parts: first, short-term (3-9 d) clinical tolerance and colonic fermentation as assessed by fecal short chain fatty acid (SCFA) concentrations and breath hydrogen and methane was measured in response to progressive fiber supplementation increasing from 4 g tid up to normal requirement levels of 8 g tid; second, 4 patients with diarrhea were studied for 2-5 wk with maximal supplementation to additionally assess its influence on fecal microbiota quantitated by quantitative polymerase chain reaction (qPCR) of microbial 16S rRNA genes and Human Intestinal Tract Chip (HITChip) microarray analysis. Nearly all patients were receiving antibiotics (10/13) and acid suppressants (11/13) at some stage during the studies. RESULTS In group 1, tolerance to progressive fiber supplementation was good with breath hydrogen and methane evidence (P = 0.008 and P < 0.0001, respectively) of increased fermentation with no exacerbation of abdominal symptoms and resolution of diarrhea in 2 of 4 patients. In group 2 before supplementation, fecal microbiota mass and their metabolites, SCFA, were dramatically lower in patients compared to healthy volunteers. From qPCR and HITChip analyses we calculated that there was a 97% reduction in the predominant potential butyrate producers and starch degraders. Following 2-5 wk of fiber supplementation there was a significant increase in fecal SCFA (acetate 28.4 ± 4.1 μmol/g to 42.5 ± 3.1 μmol/g dry weight, P = 0.01; propionate 1.6 ± 0.5 vs 6.22 ± 1.1, P = 0.006 and butyrate 2.5 ± 0.6 vs 5.9 ± 1.1, P = 0.04) and microbial counts of specific butyrate producers, with resolution of diarrhea in 3 of 4 patients. CONCLUSION Conventional management of critically ill patients, which includes the use of elemental diets and broad-spectrum antibiotics, was associated with gross suppression of the colonic microbiota and their production of essential colonic fuels, i.e., SCFA. Our investigations show that fiber supplementation of the feeds has the potential to improve microbiota mass and function, thereby reducing the risks of diarrhea due to dysbiosis.

Hydrogenotrophic microbiota distinguish native Africans from African and European Americans

Reduced susceptibility to sporadic colorectal cancer in native Africans (NA) is correlated with low consumption of animal products and greater microbial production of colonic methane. In this context, two hydrogenotrophic microbial groups are of interest, methanogenic Archaea (MA) utilizing H2 to produce methane and sulfate-reducing bacteria (SRB) generating hydrogen sulfide, which has been linked with chronic inflammatory disorders of the colon. In the present study, stool samples from NA, consuming a diet high in resistant starch and low in animal products, and from African Americans (AA) and European Americans (EA), both consuming a typical Western diet, were examined for genetic diversity and structure of Archaea, MA and SRB communities. In general, a greater proportion of NA than AA and EA harboured the full range of targeted hydrogenotrophic groups. Terminal restriction fragment length polymorphism analysis of 16S rRNA genes and specific functional genes, combined with multivariate statistical analyses, revealed that NA harboured more diverse and different Archaea and MA populations than AA and EA. Also, NA harboured significantly distinct SRB populations compared with AA and EA. Taken together, these data are consistent with diet selecting for distinct hydrogenotrophic microbiota.

Pancreatic and Intestinal Function Post Roux-en-Y Gastric Bypass Surgery for Obesity

OBJECTIVES: Despite the fact that the most effective treatment for morbid obesity today is gastric bypass surgery, some patients develop life‐threatening nutritional complications associated with their weight loss. METHODS: Here we examine the influence of the altered anatomy and digestive physiology on pancreatic secretion and fat absorption. Thirteen post Roux‐en‐Y gastric bypass (RYGB) patients who had lost >100 lbs in the first year following surgery and who gave variable histories of gastrointestinal (GI) dysfunction, were selected for study. Food‐stimulated pancreatic enzyme secretion and GI hormone responses were measured during 2 h perfusions of the Roux limb with a standard polymeric liquid formula diet and polyethylene glycol marker, with collections of secretions from the common channel distal to the anastomosis and blood testing. Fat absorption was then measured during a 72 h balance study when a normal diet was given containing ˜100 g fat/d. RESULTS: Result showed that all patients had some fat malabsorption, but eight had coefficients of fat absorption <80%, indicative of steatorrhea. This was associated with significantly lower feed‐stimulated secretion rates of trypsin, amylase, and lipase, and higher plasma peptide‐YY concentrations compared with healthy controls. Five steatorrhea patients were subsequently treated with low quantities of pancreatic enzyme supplements for 3 months, and then retested. The supplements were well tolerated, and fat absorption improved in four of five patients accompanied by an increase in lipase secretion, but body weight increased in only three. Postprandial breath hydrogen concentrations were elevated with some improvement following enzyme supplementation suggesting persistent bacterial overgrowth and decreased colonic fermentation. CONCLUSIONS: Our investigations revealed a wide spectrum of gastrointestinal abnormalities, including fat malabsorption, impaired food stimulated pancreatic secretion, ileal brake stimulation, and bacterial overgrowth, in patients following RYGB which could be attributed to the breakdown of the normally highly orchestrated digestive anatomy and physiology.

Tu1898 The Interactions Between Diet, Mucosal Proliferation and GI Cancer Risk in Rural Africans and African Americans

166 more tumors needed to be screened in the universal group compared to the reflex group. Conclusion: Reflex screening of CRC tumors for LS has similar outcomes as universal or referral strategies to identify LS. The universal group identified one additional case of LS that would have been missed by a reflex strategy, though 166 additional tumors needed to be tested. In clinical practice, a reflex testing strategy may be just as effective and less burdensome as compared to universal tumor screening to detect LS. The power to detect differences was a limitation to this study. Characteristics and outcomes of positively screened patients

Abstract SS01-03: Can variations in the intestinal microbiota explain differences in colon cancer risk?

Humans harbor a very complex and diverse collection of microbes, mostly bacteria, in their intestines that are collectively called the intestinal microbiota. It is known that we cannot live without the microbiota but on the other hand, the microbiota is associated to a number of intestinal and systemic diseases, including colorectal cancer (CRC). Nevertheless, the mechanisms underlying the key roles of the microbiota in health and disease are largely unknown. Compared to human populations in the Western countries, native Africans have a very low incidence and mortality of sporadic colorectal cancer (CRC). In contrast, African Americans, who are genetically similar to Africans, have a very high rate of CRC compared to other ethnic groups in the USA. Therefore, we hypothesize that differences in CRC risk between these groups is that the differential diets consumed by both groups (high fat and protein diets by African Americans; high starch and fibers diets by native Africans) impact the microbiota which as a result contributes to the differential CRC risk development. To test this hypothesis, we compared and contrasted the microbiota between African Americans, urbanized Africans (who live in a more Western semi-urban environment) and rural Africans at baseline, followed by a longitudinal study focusing on healthy middle aged African and African Americans volunteers who were subjected to reciprocal diet exchange under strict controlled in-house conditions over two weeks. Fresh fecal samples were collected and frozen immediately at baseline, and before and after diet exchange. DNA was isolated from these samples and subsequently used for microbiota profiling using the Human Intestinal Tract Chip (HITChip), which is a 16S ribosomal RNA-based phylogenetic microarray covering over 1,000 of the currently known bacterial species from the human intestine. HITChip profiling demonstrated that the microbiota composition is significantly different between the three groups at baseline with Prevotella (average abundance 22%), Oscillospira (average abundance 16%) and Bacteroides (28%) as key genera in urbanized Africans, rural Africans and African Americans, respectively. Co-occurrence analysis of microbial groups demonstrated that despite the differences in composition, the microbiota connectivity was similar between urban and rural Africans with potential butyrate producing and fiber consuming groups co-occurring at high correlation (p>0.8) in both groups. Remarkably, these groups did not show strong co-occurrences within African Americans. We speculate that these co-occurrences represent interacting bacterial groups that form the structural core Compared to differences at baseline, the microbiota compositional changes were subtle after the short dietary exchange with only ten and two bacterial groups changing significantly (p 0.8) co-occurring groups after consumption of the traditional African diet. We speculate that the decrease of co-occurring groups in the Western diet is associated to a weaker microbiota structure, which is more vulnerable for pertubations. In conclusion, our study demonstrated that long-term dietary patterns are likely responsible for the microbiota compositional differences observed between Africans and Americans, but that a short term dietary exchange can modify microbiota composition and structure. The fact that the microbiota structure changes reciprocally upon diet exchange holds promise for the prevention of diet-microbiota induced CRC risk. Citation Format: Erwin G. Zoetendal, Leo Lahti, Sebastian Tims, Philippe G.B. Puylaert, Junhai Ou, Kishore Vipperla, Willem M. de Vos, Stephen J. O9Keefe. Can variations in the intestinal microbiota explain differences in colon cancer risk?. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr SS01-03. doi:10.1158/1538-7755.DISP13-SS01-03

943 Gastric Bypass Surgery for Morbid Obesity Disrupts Entero-Pancreatic Physiology

G A A b st ra ct s synthesis, and third in terms of fatty acid synthesis (behind adipose and liver). Although the role of Insigs in regulating hepatic lipid metabolism is known, their role in the intestine is unknown. AIM: To study the role of Insig proteins on the regulation of lipid synthesis in the small intestine. METHODS: Mice with germline disruption of Insig2 and Villin-Cre mediated disruption of Insig1 in the intestine were generated (designated Vil-Insig mice). Tissues and plasma were harvested. Enterocytes were isolated and fractionated for immunoblotting, which was quantified by densitometry. mRNAs were quantified by qPCR. Plasma lipids were measured using a Vitros 250 chemistry analyzer. Jejuna were cryosectioned and stained with oil red O. RESULTS: The deletion of Insigs in intestine resulted in a loss of the normal feedback inhibition of SREBP processing and of HMGR proteolysis. Enterocyte nuclear SREBP-1 and SREBP-2 levels were increased in the Vil-Insig mice by 5-fold and 8fold, respectively. HMGR protein levels were increased by 18-fold. mRNA levels of SREBP lipogenic target genes were all increased; the greatest of which was stearoyl-CoA desaturase1 mRNA, which was increased 195-fold. As a consequence of this loss of SREBP-feedback inhibition, plasma cholesterol levels in the Vil-Insig mice were increased by 55% (123+/9 mg/dL vs 191+/-6 mg/dL, p,0.0001). Neutral lipid levels were increased in the jejunal crypts of the Vil-Insig mice as evidenced by oil red O staining. CONCLUSION: These studies indicate that the essential elements of the regulatory pathway for lipid synthesis function in the enterocyte as they do in the hepatocyte. Insig proteins provide essential negative feedback regulation on lipid biosynthesis in the intestine, and this regulation is required to maintain normal plasma cholesterol levels. Insigs mediate these effects by negatively regulating SREBPs and HMGR, to affect both cholesterol and fatty acid synthesis. Further studies will be required to determine if the increase in plasma cholesterol seen in the Vil-insig mice is related to changes in chylomicron synthesis/metabolism, and if Insig proteins affect enterocyte development, given the unique finding of lipid accumulation in the intestinal crypts.