ONE | DOI:10.1371/journal.pone.Lixisenatide supplier 0135758 August 18,2 /Obesity Alters the Gut Microbiome and Metabolomegut microbiome and intestinal tumorigenesis in Apc1638N mice. Because alterations in the stool metabolome likely play an important role in mediating these phenomena we also profiled the stool metabolome to identify elements that might contribute to the formation of a pro-tumorigeneic milieu in these fnins.2015.00094 two modes of obesity.Methods Animal StudyAnimal procedures were approved by the Institutional Animal Care and Use Committee of the Jean Mayer USDA HNRCA at Tufts University. To study tumorigenesis we utilized Apc1638N mice [17] (NCI Mouse Repository. Frederick, MD); heterozygosity for this Apc mutation (codon 1638) results in the formation of 1? small bowel BQ-123 cancer adenomas or carcinomas by 8 months of age. Although the predilection for developing small, rather than large, intestinal tumors is a common phenomenon in genetically-engineered models of CRC–such as the widely utilized Apcmin mouse–the small intestinal tumorigenesis in the Apc1638N animal appears to be highly relevant to colonic carcinogenesis since it responds to dietary modifications like obesity and 1-carbon nutrient depletion in the same fashion as to what occurs in the colon [18, 19]. To study genetically-induced obesity we utilized Leprdb/db mice, which lack a functional leptin receptor and become obese at 3? weeks of age [20] (Jackson Laboratory. Bar Harbor, Maine). Wildtype C57BL6/J (Charles jir.2012.0140 River, Wilmington, MA) were also utilized. The following three genotypes were generated: Apc+/+ Lepr+/+ (wildtype, Wt), Apc+/1638N Lepr+/+ (Apc) and Apc+/1638N Lepr db/db (DbDb). Starting at 8 weeks of age, Wt (n = 12) and DbDb (n = 10) mice were fed a low fat (LF) diet while Apc mice were randomized to receive LF (N = 10) or HF (N = 12) diet for 16 weeks. LF and HF diets provided 10 and 60 of calories from fat respectively (Table 1. BioServ, Frenchtown, NJ). Mice were individually housed on a 12 hr light-dark cycle at 23 and provided ad libitum access to water.Table 1. Diet composition. Ingredient (g/kg) Casein L-Cystine Corn Starch Maltodextrin Sucrose Lard Soybean Oil Cellulose Mineral Mix AIN-93G Calcium Phosphate Dibasic Vitamin Mix AIN-93 Choline Bitartrate Total Shikonin chemical information Energy ( kcal) Carbohydrate Protein Fat Total LF 210 3 280 50 325 20 20 37.2 35 2 15 2.8 1000 LFD 70 20 10 100 HF 265 4 0 160 90 310 30 65.5 48 3.4 21 3 1000 HFD 21 19 60Diets: LF, Low fat. HF, High fat. BioServ (custom) catalog numbers F6654, and F6653 respectively. doi:10.1371/journal.pone.0135758.tPLOS ONE | DOI:10.1371/journal.pone.0135758 August 18,3 /Obesity Alters the Gut Microbiome and MetabolomeMice were weighed weekly. After 15 weeks on diet, body composition was measured by MRI (EchoMRI, Houston, TX). After 16 weeks on diet, mice were euthanized by CO2 asphyxiation followed by cervical dislocation and exsanguination by cardiac puncture. The abdomen was then opened and the small intestine (SI) and large intestines removed onto ice-cold glass plates, opened longitudinally and buy Procyanidin B1 contents removed. Colon and cecum contents were combined, aliquots frozen in liquid N2 and then stored at -80 . Small and large intestines were then rinsed thoroughly with ice-cold PBS with protease inhibitors (Roche, Indianapolis, IN). The SI and colon were inspected for tumors by a blinded investigator under a dissecting microscope. Tumors were measured before being excised and fixed in formalin for later grading by a rodent pathologist. The re.ONE | DOI:10.1371/journal.pone.0135758 August 18,2 /Obesity Alters the Gut Microbiome and Metabolomegut microbiome and intestinal tumorigenesis in Apc1638N mice. Because alterations in the stool metabolome likely play an important role in mediating these phenomena we also profiled the stool metabolome to identify elements that might contribute to the formation of a pro-tumorigeneic milieu in these fnins.2015.00094 two modes of obesity.Methods Animal StudyAnimal procedures were approved by the Institutional Animal Care and Use Committee of the Jean Mayer USDA HNRCA at Tufts University. To study tumorigenesis we utilized Apc1638N mice [17] (NCI Mouse Repository. Frederick, MD); heterozygosity for this Apc mutation (codon 1638) results in the formation of 1? small bowel adenomas or carcinomas by 8 months of age. Although the predilection for developing small, rather than large, intestinal tumors is a common phenomenon in genetically-engineered models of CRC–such as the widely utilized Apcmin mouse–the small intestinal tumorigenesis in the Apc1638N animal appears to be highly relevant to colonic carcinogenesis since it responds to dietary modifications like obesity and 1-carbon nutrient depletion in the same fashion as to what occurs in the colon [18, 19]. To study genetically-induced obesity we utilized Leprdb/db mice, which lack a functional leptin receptor and become obese at 3? weeks of age [20] (Jackson Laboratory. Bar Harbor, Maine). Wildtype C57BL6/J (Charles jir.2012.0140 River, Wilmington, MA) were also utilized. The following three genotypes were generated: Apc+/+ Lepr+/+ (wildtype, Wt), Apc+/1638N Lepr+/+ (Apc) and Apc+/1638N Lepr db/db (DbDb). Starting at 8 weeks of age, Wt (n = 12) and DbDb (n = 10) mice were fed a low fat (LF) diet while Apc mice were randomized to receive LF (N = 10) or HF (N = 12) diet for 16 weeks. LF and HF diets provided 10 and 60 of calories from fat respectively (Table 1. BioServ, Frenchtown, NJ). Mice were individually housed on a 12 hr light-dark cycle at 23 and provided ad libitum access to water.Table 1. Diet composition. Ingredient (g/kg) Casein L-Cystine Corn Starch Maltodextrin Sucrose Lard Soybean Oil Cellulose Mineral Mix AIN-93G Calcium Phosphate Dibasic Vitamin Mix AIN-93 Choline Bitartrate Total Energy ( kcal) Carbohydrate Protein Fat Total LF 210 3 280 50 325 20 20 37.2 35 2 15 2.8 1000 LFD 70 20 10 100 HF 265 4 0 160 90 310 30 65.5 48 3.4 21 3 1000 HFD 21 19 60Diets: LF, Low fat. HF, High fat. BioServ (custom) catalog numbers F6654, and F6653 respectively. doi:10.1371/journal.pone.0135758.tPLOS ONE | DOI:10.1371/journal.pone.0135758 August 18,3 /Obesity Alters the Gut Microbiome and MetabolomeMice were weighed weekly. After 15 weeks on diet, body composition was measured by MRI (EchoMRI, Houston, TX). After 16 weeks on diet, mice were euthanized by CO2 asphyxiation followed by cervical dislocation and exsanguination by cardiac puncture. The abdomen was then opened and the small intestine (SI) and large intestines removed onto ice-cold glass plates, opened longitudinally and contents removed. Colon and cecum contents were combined, aliquots frozen in liquid N2 and then stored at -80 . Small and large intestines were then rinsed thoroughly with ice-cold PBS with protease inhibitors (Roche, Indianapolis, IN). The SI and colon were inspected for tumors by a blinded investigator under a dissecting microscope. Tumors were measured before being excised and fixed in formalin for later grading by a rodent pathologist. The re.ONE | DOI:10.1371/journal.pone.0135758 August 18,2 /Obesity Alters the Gut Microbiome and Metabolomegut microbiome and intestinal tumorigenesis in Apc1638N mice. Because alterations in the stool metabolome likely play an important role in mediating these phenomena we also profiled the stool metabolome to identify elements that might contribute to the formation of a pro-tumorigeneic milieu in these fnins.2015.00094 two modes of obesity.Methods Animal StudyAnimal procedures were approved by the Institutional Animal Care and Use Committee of the Jean Mayer USDA HNRCA at Tufts University. To study tumorigenesis we utilized Apc1638N mice [17] (NCI Mouse Repository. Frederick, MD); heterozygosity for this Apc mutation (codon 1638) results in the formation of 1? small bowel adenomas or carcinomas by 8 months of age. Although the predilection for developing small, rather than large, intestinal tumors is a common phenomenon in genetically-engineered models of CRC–such as the widely utilized Apcmin mouse–the small intestinal tumorigenesis in the Apc1638N animal appears to be highly relevant to colonic carcinogenesis since it responds to dietary modifications like obesity and 1-carbon nutrient depletion in the same fashion as to what occurs in the colon [18, 19]. To study genetically-induced obesity we utilized Leprdb/db mice, which lack a functional leptin receptor and become obese at 3? weeks of age [20] (Jackson Laboratory. Bar Harbor, Maine). Wildtype C57BL6/J (Charles jir.2012.0140 River, Wilmington, MA) were also utilized. The following three genotypes were generated: Apc+/+ Lepr+/+ (wildtype, Wt), Apc+/1638N Lepr+/+ (Apc) and Apc+/1638N Lepr db/db (DbDb). Starting at 8 weeks of age, Wt (n = 12) and DbDb (n = 10) mice were fed a low fat (LF) diet while Apc mice were randomized to receive LF (N = 10) or HF (N = 12) diet for 16 weeks. LF and HF diets provided 10 and 60 of calories from fat respectively (Table 1. BioServ, Frenchtown, NJ). Mice were individually housed on a 12 hr light-dark cycle at 23 and provided ad libitum access to water.Table 1. Diet composition. Ingredient (g/kg) Casein L-Cystine Corn Starch Maltodextrin Sucrose Lard Soybean Oil Cellulose Mineral Mix AIN-93G Calcium Phosphate Dibasic Vitamin Mix AIN-93 Choline Bitartrate Total Energy ( kcal) Carbohydrate Protein Fat Total LF 210 3 280 50 325 20 20 37.2 35 2 15 2.8 1000 LFD 70 20 10 100 HF 265 4 0 160 90 310 30 65.5 48 3.4 21 3 1000 HFD 21 19 60Diets: LF, Low fat. HF, High fat. BioServ (custom) catalog numbers F6654, and F6653 respectively. doi:10.1371/journal.pone.0135758.tPLOS ONE | DOI:10.1371/journal.pone.0135758 August 18,3 /Obesity Alters the Gut Microbiome and MetabolomeMice were weighed weekly. After 15 weeks on diet, body composition was measured by MRI (EchoMRI, Houston, TX). After 16 weeks on diet, mice were euthanized by CO2 asphyxiation followed by cervical dislocation and exsanguination by cardiac puncture. The abdomen was then opened and the small intestine (SI) and large intestines removed onto ice-cold glass plates, opened longitudinally and contents removed. Colon and cecum contents were combined, aliquots frozen in liquid N2 and then stored at -80 . Small and large intestines were then rinsed thoroughly with ice-cold PBS with protease inhibitors (Roche, Indianapolis, IN). The SI and colon were inspected for tumors by a blinded investigator under a dissecting microscope. Tumors were measured before being excised and fixed in formalin for later grading by a rodent pathologist. The re.ONE | DOI:10.1371/journal.pone.0135758 August 18,2 /Obesity Alters the Gut Microbiome and Metabolomegut microbiome and intestinal tumorigenesis in Apc1638N mice. Because alterations in the stool metabolome likely play an important role in mediating these phenomena we also profiled the stool metabolome to identify elements that might contribute to the formation of a pro-tumorigeneic milieu in these fnins.2015.00094 two modes of obesity.Methods Animal StudyAnimal procedures were approved by the Institutional Animal Care and Use Committee of the Jean Mayer USDA HNRCA at Tufts University. To study tumorigenesis we utilized Apc1638N mice [17] (NCI Mouse Repository. Frederick, MD); heterozygosity for this Apc mutation (codon 1638) results in the formation of 1? small bowel adenomas or carcinomas by 8 months of age. Although the predilection for developing small, rather than large, intestinal tumors is a common phenomenon in genetically-engineered models of CRC–such as the widely utilized Apcmin mouse–the small intestinal tumorigenesis in the Apc1638N animal appears to be highly relevant to colonic carcinogenesis since it responds to dietary modifications like obesity and 1-carbon nutrient depletion in the same fashion as to what occurs in the colon [18, 19]. To study genetically-induced obesity we utilized Leprdb/db mice, which lack a functional leptin receptor and become obese at 3? weeks of age [20] (Jackson Laboratory. Bar Harbor, Maine). Wildtype C57BL6/J (Charles jir.2012.0140 River, Wilmington, MA) were also utilized. The following three genotypes were generated: Apc+/+ Lepr+/+ (wildtype, Wt), Apc+/1638N Lepr+/+ (Apc) and Apc+/1638N Lepr db/db (DbDb). Starting at 8 weeks of age, Wt (n = 12) and DbDb (n = 10) mice were fed a low fat (LF) diet while Apc mice were randomized to receive LF (N = 10) or HF (N = 12) diet for 16 weeks. LF and HF diets provided 10 and 60 of calories from fat respectively (Table 1. BioServ, Frenchtown, NJ). Mice were individually housed on a 12 hr light-dark cycle at 23 and provided ad libitum access to water.Table 1. Diet composition. Ingredient (g/kg) Casein L-Cystine Corn Starch Maltodextrin Sucrose Lard Soybean Oil Cellulose Mineral Mix AIN-93G Calcium Phosphate Dibasic Vitamin Mix AIN-93 Choline Bitartrate Total Energy ( kcal) Carbohydrate Protein Fat Total LF 210 3 280 50 325 20 20 37.2 35 2 15 2.8 1000 LFD 70 20 10 100 HF 265 4 0 160 90 310 30 65.5 48 3.4 21 3 1000 HFD 21 19 60Diets: LF, Low fat. HF, High fat. BioServ (custom) catalog numbers F6654, and F6653 respectively. doi:10.1371/journal.pone.0135758.tPLOS ONE | DOI:10.1371/journal.pone.0135758 August 18,3 /Obesity Alters the Gut Microbiome and MetabolomeMice were weighed weekly. After 15 weeks on diet, body composition was measured by MRI (EchoMRI, Houston, TX). After 16 weeks on diet, mice were euthanized by CO2 asphyxiation followed by cervical dislocation and exsanguination by cardiac puncture. The abdomen was then opened and the small intestine (SI) and large intestines removed onto ice-cold glass plates, opened longitudinally and contents removed. Colon and cecum contents were combined, aliquots frozen in liquid N2 and then stored at -80 . Small and large intestines were then rinsed thoroughly with ice-cold PBS with protease inhibitors (Roche, Indianapolis, IN). The SI and colon were inspected for tumors by a blinded investigator under a dissecting microscope. Tumors were measured before being excised and fixed in formalin for later grading by a rodent pathologist. The re.
