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In vitro effects of the intestinal microbiota and their prebiotic modulation on parameters regulating fat metabolism

Charlotte Grootaert UGent (2011)
abstract
Introduction. Obesity is a global problem that has reached epidemic proportions. Overweight is a risk factor for cardiovascular diseases, type II diabetes and certain types of cancer. Because obesity prevalence is continuously growing, it is clear that current methods to combat obesity are not effective enough to reduce the problem. Therefore, further investigation is needed to develop new strategies to prevent obesity. In 2004, the composition of the intestinal microbiota was hown to be involved in fat storage. It was found that obesity correlates with the ratio of the bacteria belonging to the Bacteroidetes over the Firmicutes phylum. In addition, pro-and prebiotics, which are living bacteria or substrates that stimulate the growth and activity of a specific group of intestinal bacteria in order to improve the host’s health, were shown to affect lipid metabolism of the host. The main mechanisms by which bacteria interfere with obesity were identified as the increase in (i) of liver lipogenesis and gluconeogenesis, (ii) gut peptides involved in satiety and (iii) a repression of gut peptides involved in fat storage. Objectives. This research focuses on the in depth investigation of two of these mechanisms. A first part includes the investigation of certain substrates that are able to shift the microbial fermentation towards more propionate and less acetate production, as propionate is involved in lipogenesis and satiety processes. A second part includes the investigation of the effect of specific bacteria and bacterial metabolites on the expression of the fasting induced adipose factor (FIAF), a multifunctional protein that is involved in the microbial regulation of fat storage through lipoprotein lipase inhibition. For both parts, an in vitro approach was chosen because this type of experimental setup facilitates the screening process and allows to study mechanisms under strictly controlled conditions. Results. In the propionate part, batch incubations were used for an initial screening of the prebiotic and propionate stimulating capacity of several substrates, including inulin, arabinoxylan oligosaccharides (AXOS), arabinogalactan, manno-oligosaccharides (MOS), guar gum substrates (GG) and glycerol. In addition, long term experiments with the simulator of the human intestinal microbial ecosystem (SHIME) were performed with inulin, AXOS-15-0.29, MOS and GG. From the batch experiments it was observed that (i) some of the results were dependent of the type of experimental setup, such as for inulin and AXOS, (ii) the bifidogenic and propionate stimulating effect of AXOS was dependent on the degree of polymerization and substitution, (iii) interesting consistent propionate stimulating properties were observed for arabinogalactan and (iv) the fermentation pattern of the other substrates was concentration dependent. The Twin-SHIME run with inulin and AXOS revealed that AXOS is mainly fermented in the distal parts of the colon, whereas inulin was mainly fermented in the proximal colon compartments. It was suggested that this was due to the inhibitory effect of glucose in the proximal parts on the AX degrading enzymes such as xylanases, arabinofuranosidases and xylosidases. AXOS was a more fermented to propionate than inulin. The main pathway of propionate production was identified as the acrylate pathway, and was probably the result of co-operation between lactate producing bacteria and propionate producing bacteria that consume lactate. The Twin-SHIME run with MOS and GG was less successfull, as no propionate stimulating effect of any of the substrates could be detected. In the second part of the research, in vitro epithelial cell line models, based on the colorectal cell lines Caco-2, HT-29 and HCT-116, were used to screen for epithelial FIAF modulation by specific bacteria and bacterial metabolites. In this part, a flow cytometric protocol was developed to determine the viability and adherence of the specific bacteria to the Caco-2 cell line, in order to investigate whether bacterial adhesion could be correlated with FIAF modulation. The major merit of this protocol is that it is applicable to study the adherence of bacteria towards differentiated epithelial-like cells, which display other adhesive factors than suspension or trypsinized cells. In addition, this protocol was suitable for the detection of both living and dead adhered microbial cells, and can be used in the combination with a mixed intestinal microbial culture. In future research, this protocol may be extended towards other cell lines. Although the method was working properly, we could not find any correlation between the adhesion of bacteria and their FIAF modulation capacity. Next, some bacteria and bacterial metabolites with FIAF modulating potential were identified. Using Western blot, it was seen that FIAF secretion was stimulated by several Enterococcus faecalis strains in Caco-2 and HT-29 cells independently of bacterial adhesion or metabolite (SCFA or H2O2) production. Clostridium perfringens was able to modify FIAF isoform structure in the cytoplasm protein fraction of the Caco-2 cells. E. coli decreased FIAF secretion in the HCT-116 cell line. No significant differences were detected for Bifidobacterium breve, Bacteroides thetaiotaomicron, Lactobacillus brevis. A second observation was that H2O2 and SCFA, such as propionate and butyrate, but not acetate, induced cleavage of the secreted FIAF protein. In addition, propionate and butyrate were also shown to increase the total FIAF production in colon and hepatic cancer cell lines. Conclusions. We conclude that inulin may be considered as the golden standard prebiotic, with propionate and butyrate stimulating effects, which has been thoroughly investigated in both in vitro and in vivo research and of which the lipid metabolism and gut peptide modulation was proven in many studies. However, the propionate stimulating effects of AXOS were even higher than those of inulin, which may be attributed to the arabinose substitution of these oligosaccharides. In addition, due to the shift of the sugar fermentation from proximal to distal parts of the colon, AXOS may be a candidate prebiotic against colon cancer by interference with protein fermentation. The batch experiments with arabinogalactan were promising, and further long term experiments are needed to confirm its propionate stimulating effect. Bacth and SHIME experiments could not reveal any propionate increasing effect of MOS and GG. We can also conclude that FIAF modulation in epithelial cell lines is bacterium and metabolite specific, and that changes are observed in both FIAF concentrations and isoform structures. Therefore, microbial induced FIAF effects can occur at the colon epithelial level itself, but because SCFA are generally absorbed through the colon to enter the blood stream, they may also target organs other than the colon. Therefore, investigation of microbial steered FIAF expression and cleavage in other organs such as the liver, adipose tissue, hypothalamus and blood vessels may be an interesting topic for further research, because in these organs, FIAF may also be involved in lipid, glucose and energy metabolism, angiogenesis, cancer and wound healing.
Please use this url to cite or link to this publication:
author
promoter
UGent and UGent
organization
alternative title
In vitro effecten van de darmmicrobiota en hun prebiotische modulatie op parameters die vetmetabolisme reguleren
year
type
dissertation (monograph)
subject
keyword
microbiota, obesity, intestine, fasting induced adipose factor, prebiotics
pages
VI, 197 pages
publisher
Ghent University. Faculty of Bioscience Engineering
place of publication
Ghent, Belgium
defense location
Gent : Faculteit Bio-ingenieurswetenschappen (A0.030)
defense date
2011-05-16 18:00
ISBN
9789059894402
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have retained and own the full copyright for this publication
id
1228945
handle
http://hdl.handle.net/1854/LU-1228945
date created
2011-05-20 16:57:46
date last changed
2011-05-23 12:46:59
@phdthesis{1228945,
  abstract     = {Introduction. Obesity is a global problem that has reached epidemic proportions. Overweight is a risk factor for cardiovascular diseases, type II diabetes and certain types of cancer. Because obesity prevalence is continuously growing, it is clear that current methods to combat obesity are not effective enough to reduce the problem. Therefore, further investigation is needed to develop new strategies to prevent obesity.
In 2004, the composition of the intestinal microbiota was hown to be involved in fat storage. It was found that obesity correlates with the ratio of the bacteria belonging to the Bacteroidetes over the Firmicutes phylum. In addition, pro-and prebiotics, which are living bacteria or substrates that stimulate the growth and activity of a specific group of intestinal bacteria in order to improve the host{\textquoteright}s health, were shown to affect lipid metabolism of the host. The main mechanisms by which bacteria interfere with obesity were identified as the increase in (i) of liver lipogenesis and gluconeogenesis, (ii) gut peptides involved in satiety and (iii) a repression of gut peptides involved in fat storage.
Objectives. This research focuses on the in depth investigation of two of these mechanisms. A first part includes the investigation of certain substrates that are able to shift the microbial fermentation towards more propionate and less acetate production, as propionate is involved in lipogenesis and satiety processes. A second part includes the investigation of the effect of specific bacteria and bacterial metabolites on the expression of the fasting induced adipose factor (FIAF), a multifunctional protein that is involved in the microbial regulation of fat storage through lipoprotein lipase inhibition. For both parts, an in vitro approach was chosen because this type of experimental setup facilitates the screening process and allows to study mechanisms under strictly controlled conditions.
Results. In the propionate part, batch incubations were used for an initial screening of the prebiotic and propionate stimulating capacity of several substrates, including inulin, arabinoxylan oligosaccharides (AXOS), arabinogalactan, manno-oligosaccharides (MOS), guar gum substrates (GG) and glycerol. In addition, long term experiments with the simulator of the human intestinal microbial ecosystem (SHIME) were performed with inulin, AXOS-15-0.29, MOS and GG. From the batch experiments it was observed that (i) some of the results were dependent of the type of experimental setup, such as for inulin and AXOS, (ii) the bifidogenic and propionate stimulating effect of AXOS was dependent on the degree of polymerization and substitution, (iii) interesting consistent propionate stimulating properties were observed for arabinogalactan and (iv) the fermentation pattern of the other substrates was concentration dependent. 
The Twin-SHIME run with inulin and AXOS revealed that AXOS is mainly fermented in the distal parts of the colon, whereas inulin was mainly fermented in the proximal colon compartments. It was suggested that this was due to the inhibitory effect of glucose in the proximal parts on the AX degrading enzymes such as xylanases, arabinofuranosidases and xylosidases. AXOS was a more fermented to propionate than inulin. The main pathway of propionate production was identified as the acrylate pathway, and was probably the result of co-operation between lactate producing bacteria and propionate producing bacteria that consume lactate. The Twin-SHIME run with MOS and GG was less successfull, as no propionate stimulating effect of any of the substrates could be detected.
In the second part of the research, in vitro epithelial cell line models, based on the colorectal cell lines Caco-2, HT-29 and HCT-116, were used to screen for epithelial FIAF modulation by specific bacteria and bacterial metabolites. In this part, a flow cytometric protocol was developed to determine the viability and adherence of the specific bacteria to the Caco-2 cell line, in order to investigate whether bacterial adhesion could be correlated with FIAF modulation. The major merit of this protocol is that it is applicable to study the adherence of bacteria towards differentiated epithelial-like cells, which display other adhesive factors than suspension or trypsinized cells. In addition, this protocol was suitable for the detection of both living and dead adhered microbial cells, and can be used in the combination with a mixed intestinal microbial culture. In future research, this protocol may be extended towards other cell lines. Although the method was working properly, we could not find any correlation between the adhesion of bacteria and their FIAF modulation capacity.
Next, some bacteria and bacterial metabolites with FIAF modulating potential were identified. Using Western blot, it was seen that FIAF secretion was stimulated by several Enterococcus faecalis strains in Caco-2 and HT-29 cells independently of bacterial adhesion or metabolite (SCFA or H2O2) production. Clostridium perfringens was able to modify FIAF isoform structure in the cytoplasm protein fraction of the Caco-2 cells. E. coli decreased FIAF secretion in the HCT-116 cell line. No significant differences were detected for Bifidobacterium breve, Bacteroides thetaiotaomicron, Lactobacillus brevis. A second observation was that H2O2 and SCFA, such as propionate and butyrate, but not acetate, induced cleavage of the secreted FIAF protein. In addition, propionate and butyrate were also shown to increase the total FIAF production in colon and hepatic cancer cell lines. 
Conclusions. We conclude that inulin may be considered as the golden standard prebiotic, with propionate and butyrate stimulating effects, which has been thoroughly investigated in both in vitro and in vivo research and of which the lipid metabolism and gut peptide modulation was proven in many studies. However, the propionate stimulating effects of AXOS were even higher than those of inulin, which may be attributed to the arabinose substitution of these oligosaccharides. In addition, due to the shift of the sugar fermentation from proximal to distal parts of the colon, AXOS may be a candidate prebiotic against colon cancer by interference with protein fermentation. The batch experiments with arabinogalactan were promising, and further long term experiments are needed to confirm its propionate stimulating effect. Bacth and SHIME experiments could not reveal any propionate increasing effect of MOS and GG. 
We can also conclude that FIAF modulation in epithelial cell lines is bacterium and metabolite specific, and that changes are observed in both FIAF concentrations and isoform structures. Therefore, microbial induced FIAF effects can occur at the colon epithelial level itself, but because SCFA are generally absorbed through the colon to enter the blood stream, they may also target organs other than the colon. Therefore, investigation of microbial steered FIAF expression and cleavage in other organs such as the liver, adipose tissue, hypothalamus and blood vessels may be an interesting topic for further research, because in these organs, FIAF may also be involved in lipid, glucose and energy metabolism, angiogenesis, cancer and wound healing.},
  author       = {Grootaert, Charlotte},
  isbn         = {9789059894402},
  keyword      = {microbiota,obesity,intestine,fasting induced adipose factor,prebiotics},
  language     = {eng},
  pages        = {VI, 197},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {In vitro effects of the intestinal microbiota and their prebiotic modulation on parameters regulating fat metabolism},
  year         = {2011},
}

Chicago
Grootaert, Charlotte. 2011. “In Vitro Effects of the Intestinal Microbiota and Their Prebiotic Modulation on Parameters Regulating Fat Metabolism”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
Grootaert, C. (2011). In vitro effects of the intestinal microbiota and their prebiotic modulation on parameters regulating fat metabolism. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
Grootaert C. In vitro effects of the intestinal microbiota and their prebiotic modulation on parameters regulating fat metabolism. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2011.
MLA
Grootaert, Charlotte. “In Vitro Effects of the Intestinal Microbiota and Their Prebiotic Modulation on Parameters Regulating Fat Metabolism.” 2011 : n. pag. Print.