In research funded by Saputo Dairy UK, a team suggest the prebiotics galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS) communicate with lectin molecules to determine cell differentiation, development and pathological states.
Results using Caco–2 cells treated with GOS or FOS products highlight their effects on transmembrane trafficking, differences in xenobiotic biotransformation, and the production of antimicrobial agents.
“A total of 89 significant differentially expressed genes were identified between GOS and mock-treated groups,” the research team says.
“For FOS treatment, a reduced number of 12 significant genes were observed to be differentially expressed relative to the control group.
“Genes up-regulated in the presence of GOS were involved in digestion and absorption processes, fatty acids and steroids metabolism, potential antimicrobial proteins, energy-dependent and -independent transmembrane trafficking of solutes and amino acids.”
Commercial GOS products typically contain residual lactose, glucose and galactose reactants with commercial FOS products similarly containing residual sucrose, fructose and glucose.
In vivo, GOS supplementation has been shown to increase iron absorption from micronutrient powder in infants or to reduce stress-induced gastrointestinal dysfunction and days of cold or flu symptoms in controlled trials of healthy university students.
However, one study suggested short-term intake of high-dose GOS and FOS prebiotics had an adverse effect on glucose metabolism despite increased Bifidobacterium in the faecal microbiota.
Caco–2 cells are a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells that produce tight junctions, microvilli, enzymes and transporters characteristic of enterocytes.
The Caco–2 monolayer is widely used in the pharmaceutical industry as an in vitro model of the human intestinal mucosa to predict the absorption of orally administered drugs.
Study details
Researchers from the University of Nottingham and Saputo Dairy UK began examining the effects of Nutrabiotic GOS and Beneo FOS on the integrity of the Caco–2 monolayers.
The GOS treatment media contained 2% v/v of Nutrabiotic GOS, equivalent to 1.4% w/v of DP 2–7+ galacto-oligosaccharides.
Meanwhile, the FOS treatment media contained 2% v/v of Orafti®L95 FOS, equivalent to 2% w/v of DP 2–8 fructo-oligosaccharides.
To account for the presence of mono- and digestible di-saccharides contained in Nutrabiotic GOS and OraftiL95 FOS syrups used for treatment, the research team tailored the mock-treatments accordingly for GOS.
This was achieved by incorporating galactose (0.03%), glucose (0.4%) and lactose (0.2%) in the GOS mock control, and by inclusion of fructose (0.06%), glucose (0.004%) and sucrose (0.04%) in the FOS mock control.
The human Caco–2 cells were cultured as a monolayer and exposed to treatment for 24 hours.
GOS and FOS treatment media were prepared by dissolving 2% (v/v) of the oligo-saccharides syrups in Foetal Bovine Serum (FBS)-free and antibiotic-free cell culture ‘Dulbecco's Modified Eagle's medium’ (DMEM).
All treatment culture media were free from serum and antibiotics to eliminate any interference from extraneous molecules, proteins or hormones.
TEER increase
Findings revealed that compared to the mock-exposed cells, treatment of Caco–2 cell monolayers with GOS generated a significant increase (+33.62%) of the trans-epithelial electrical resistance (TEER), a method used for quantifying barrier tissue integrity that measures the electrical resistance across the tissue.
Similarly, FOS also induced greater TEER when compared to the mock-exposed cells (+28.68%) suggesting an improvement of the monolayer integrity under the influence of the oligosaccharide treatments.
The distribution of differentially expressed genes following GOS and FOS exposure was also further investigated, which showed a limited number of genes modulated by FOS when compared to GOS.
Following exposure to GOS, the team found 89 genes were differentially expressed according to the criteria, of which 53 were up-regulated and 36 down-regulated when compared to control mock-treated cells.
Whereas for FOS, the total number of genes fulfilling the criteria for differential expression was limited to 12, comprising of eight up-regulated and four down-regulated genes
“Exposure to GOS (1.4% w/v) increased trans-epithelial electrical resistance of the monolayer suggesting GOS can improve the integrity of the tights junctions,” the team says.
“FOS (2% w/v) exposed cells similarly increased trans-epithelial electrical resistance despite marginally failing to meet significance.”
‘Changes in membrane sterols and phospholipids’
According to the research team, the FOS treatment linked with 12 differentially expressed genes may have resulted in compositional changes in membrane sterols and phospholipids that affected the physical properties of the cell membrane, altering rigidity/fluidity leading to stabilization or disruption.
In a similar vein, data collected on GOS treatment’s association with 53 up-regulated genes supported the hypothesis that GOS elicited energy-dependent transmembrane trafficking of solutes in Caco–2 cell monolayers.
This was accompanied by collagen and cytoplasmic membrane remodelling that may have been linked to the observed increases in TEER.
GOS-induced up-regulation was also observed for gene SERPINA5 encoding Protein C inhibitor (PCI), a serine protease inhibitor found in blood plasma and human urine.
“As an antimicrobial agent, PCI has the ability to disrupt the bacterial cell wall to cause death by interacting with lipid membranes leading to permeabilization of bacterial pathogens,” the study writes.
“PCI also inhibits proteases of the blood coagulation and fibrinolysis system, whilst in cancer cells it suppresses tumour invasion by inhibiting urokinase-type plasminogen activators and inhibits tumour growth and metastasis, which are independent of its protease-inhibitory activity.”
Source: Nutrients
Published online ahead of print: doi.org/10.3390/nu12051281
“In Vitro Evaluation of the Effects of Commercial Prebiotic GOS and FOS Products on Human Colonic Caco–2 Cells.”
Authors: Geraldine Flaujac Lafontaine, Neville Fish and Ian Connerton