Alpha-methylacyl-CoA racemase (Amacr) catalyzes the racemization of alpha-methyl-branched CoA esters. Sequence comparisons have shown that this enzyme is a member of the family III CoA transferases. The mammalian Amacr is involved in bile acid synthesis and branched-chain fatty acid degradation. In human, mutated variants of Amacr have been shown to be associated with disease states. Amino acid sequence alignment of Amacrs and its homologues from various species revealed 26 conserved protic residues, assumed to be potential candidates as catalytic residues. Amacr from Mycobacterium tuberculosis (MCR) was taken as a representative of the racemases. To determine their importance for efficient catalysis, each of these 26 protic residues of MCR was mutated into an alanine, respectively, and the mutated variants were overexpressed in Escherichia coli. It was found that four variants (R91A, H126A, D156A, and E241A) were properly folded but had much decreased catalytic efficiency. Apparently, Arg91, His126, Asp156, and Glu241 are important catalytic residues of MCR. The importance of these residues for catalysis can be rationalized by the 1.8 A resolution crystal structure of MCR, which shows that the catalytic site is at the interface between the large and small domain of two different subunits of the dimeric enzyme. This crystal structure is the first structure of a complete enzyme of the bile acid synthesis pathway. It shows that MCR has unique structural features, not seen in the structures of the sequence related formyl-CoA transferases, suggesting that the family III CoA transferases can be subdivided in at least two classes, being racemases and CoA transferases.
Sweden has one of the highest incidences of gallstone disease in the Western world. It is therefore important to characterize the mechanisms responsible for the formation of cholesterol gallstones in this population. In the present study, we have determined the kinetics of the two primary bile acids, cholic acid and chenodeoxycholic acid, and the hepatic secretion rates of the biliary lipids in 21 normolipidemic, nonobese gallstone patients (13 with functioning and 8 with nonfunctioning gallbladder) and in 23 healthy controls. The cholesterol saturation of fasting gallbladder bile averaged 110% in the gallstone patients with functioning gallbladder and 82% in the controls. The pool sizes of cholic acid and chenodeoxycholic acid were reduced by about 40% in the two groups of gallstone patients, whereas the rates of synthesis were close to normal. The fractional catabolic rate of both bile acids was increased in both groups of gallstone patients. The gallstone patients with functioning gallbladder had an increased (about 50%) cholesterol secretion but normal bile acid and phospholipid secretion rates. In the gallstone patients with nonfunctioning gallbladder the secretion rates of biliary lipids were not significantly different from those of the controls. The ratio between cholesterol and bile acids was about 50% higher in the gallstone patients with functioning gallbladder than in the controls or in those with nonfunctioning gallbladder. The results indicate that the hepatic secretion of cholesterol is an important determinant for the development of saturated gallbladder bile in Swedish gallstone patients.
In this study we investigated the effects of zymosan and prodigiozan, the macrophage stimulators, and GdCl3, a macrophage inhibitor, on blood lipoprotein composition, activities of liver cholesterly ester (CE) metabolising enzymes, incorporation of [14C]cholesterol (C) into bile acids and accumulation and synthesis of CE in peritoneal macrophages (PM) of rats fed with C-enriched diet for 7 days. The increase of number of phagocyte cells quantity in liver and blood colony-stimulating activity in rats pretreated with intravenous injection of zymosan and prodigiozan was accompanied by reduced C content in blood low density and very low density lipoproteins (LDL and VLDL), increase of liver lysosomal CE hydrolase activity (without change of acyl-CoA:C acyltransferase and cytoplasmatic CE hydrolase activities) and incorporation of labeled C into bile acids and decrease of CE formation and accumulation in PM in rats with hypercholesterolemia. In contrast, reduction of phagocyte population in liver caused by intravenous injection of GdCl3 was accompanied by enhancement of C and CE level in blood LDL and VLDL and decrease of lysosomal CE hydrolase activity and incorporation of C into bile acids in liver of C-feeding rats. The data obtained suggest that the stimulation of mononuclear phagocyte system may lead to a decrease of plasma C via activation of LDL and VLDL catabolism and induction of bile acid synthesis in liver.
Fructo-oligosaccharides (FOS) are a mixture of oligosaccharides consisting of glucose linked to fructose units. They are not digested in the human small intestine but fermented in the colon, where they could specifically promote the growth of some species of the indigenous microflora, especially bifidobacteria. We assessed in healthy humans the effects of FOS ingestion in fecal bifidobacteria and selected metabolic indexes potentially involved in colonic carcinogenesis. Twenty volunteers randomly divided into two groups were studied for three consecutive 12-day periods. During the ingestion period, they received 12.5 g/day FOS or placebo (saccharose) in three oral doses. Stools were regularly collected and analyzed. FOS ingestion led to an increase in fecal bifidobacterial counts [7.9 +/- 0.5 to 9.1 +/- 0.3 (SE) log colony-forming units/g wet wt, p
The dietary pattern, fecal bile acid and neutral sterol concentrations, and the bacterial beta-glucuronidase activity of 2 population groups with a varied risk for colon cancer development (i.e., a high-risk population in the metropolitan New York area and an intermediate-risk population in Umea, Sweden) were investigated. The average daily intake of dietary protein was the same in the 2 groups, but the fat intake was higher in Umea than in New York. The daily total fiber intake was also higher in Umea, as was the daily total stool output. The concentration of fecal secondary bile acids and beta-glucuronidase activity was lower in Umea than in New York, but the total daily excretion of these constituents was the same in both groups. The data suggest that one of the factors contributing to the lower risk of colon cancer in Umea, despite the high dietary fat intake, is the high intake of dietary whole grain and cereal fiber, which leads to an increase in stool bulk, thus diluting and/or binding promoters.
To review recent data on the role and interactions of fiber and fat as dietary risk factors associated with colorectal cancer (CRC) risk in humans.
Fiber intake shows convincing and linear dose-response negative correlation with CRC risk. Dietary fiber stimulates butyrogenic activity of the gut microbiota, providing high amounts of butyrate that shows extensive anti-neoplastic effects. A high-fat diet promotes CRC risk through stimulated bile acid metabolism, facilitating bile acid conversion by the gut microbiota to tumor-promoting deoxycholic acid. Comprehensive interactions of these microbial metabolites are likely to underlie mechanisms driving diet-dependent CRC risk in different populations, but require further experimental investigation. Dietary fiber and fat shape the composition and metabolic function of the gut microbiota, resulting in altered amounts of butyrate and deoxycholic acid in the colon. Fiber supplementation and restriction of fat intake represent promising strategies to reduce CRC risk in healthy individuals.
The major host defense mechanisms against bacterial overgrowth in the small bowel are the normal propulsive activity of the bowel itself and gastric acid secretion. Microbial interactions are a major factor in regulating the indigenous bacterial flora. Studies of the bacterial enzymes of the gut suggest that changes in diet may lead to marked changes in the colonic flora. Antibiotics affect the composition of the colonic microflora. The microflora also influence the degradation of mucin, the conversion of urobilin to urobilinogen, of cholesterol to coprostanol, and the production of short chain fatty acids. Current interests are focused on the bacterial flora of tropical sprue, the role of bacteria in colorectal cancer, and the involvement of intestinal microflora in the enterohepatic circulation of sex steroid hormones.
Wide-scale profiling technologies including metabolomics broaden the possibility of novel discoveries related to the pathogenesis of type 2 diabetes (T2D). By applying non-targeted metabolomics approach, we investigated here whether serum metabolite profile predicts T2D in a well-characterized study population with impaired glucose tolerance by examining two groups of individuals who took part in the Finnish Diabetes Prevention Study (DPS); those who either early developed T2D (n?=?96) or did not convert to T2D within the 15-year follow-up (n?=?104). Several novel metabolites were associated with lower likelihood of developing T2D, including indole and lipid related metabolites. Higher indolepropionic acid was associated with reduced likelihood of T2D in the DPS. Interestingly, in those who remained free of T2D, indolepropionic acid and various lipid species were associated with better insulin secretion and sensitivity, respectively. Furthermore, these metabolites were negatively correlated with low-grade inflammation. We replicated the association between indolepropionic acid and T2D risk in one Finnish and one Swedish population. We suggest that indolepropionic acid, a gut microbiota-produced metabolite, is a potential biomarker for the development of T2D that may mediate its protective effect by preservation of ß-cell function. Novel lipid metabolites associated with T2D may exert their effects partly through enhancing insulin sensitivity.
Antibiotic-associated infection with the bacterial pathogen Clostridium difficile is a major cause of morbidity and increased health care costs. C difficile infection follows disruption of the indigenous gut microbiota by antibiotics. Antibiotics create an environment within the intestine that promotes C difficile spore germination, vegetative growth, and toxin production, leading to epithelial damage and colitis. Studies of patients with C difficile infection and animal models have shown that the indigenous microbiota can inhibit expansion and persistence of C difficile. Although the specific mechanisms of these processes are not known, they are likely to interfere with key aspects of the pathogen's physiology, including spore germination and competitive growth. Increasing our understanding of how the intestinal microbiota manage C difficile could lead to better means of controlling this important nosocomial pathogen.