Tal Assa Glazer1, Noa Sela3, Abraham Nyska2, Zecharia Madar*1
1Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture Food and Environment, the Hebrew University of Jerusalem, Rehovot, Israel
2Sackler School of Medicine, Tel Aviv University, and Consultant in Toxicologic Pathology. Timrat, Israel
3 Department of Plant Pathology and Weed Research, Agricultural Research Organization, the Volcani Center, Beit Dagan, Israel
*Corresponding Author: Zecharia Madar, Food Science and Nutrition department, Faculty of Agriculture, Food and Environmental Quality Science. Email: firstname.lastname@example.org.
Received: April 29, 2019; Accepted: May 06, 2019; Published: August 10, 2019
Non-alcoholic fatty liver disease (NAFLD) is an epidemic metabolic disease with limited therapeutic strategies. Cumulative data support the major role of gut microbiota in NAFLD. Here, we investigated the hypothesis whether intestinal content intubation (ICI) is effective in attenuating the effect of high trans-fat diet in mice. Donor mice were fed normal diet (ND) or high fiber diet (FIBER) and their intestinal content (IC) was removed. Recipient mice were fed high trans-fat diet and randomized into antibiotic and no antibiotic treated groups to induce dysbiosis. After 2 days, recipient mice were divided into three intubation treatment groups of PBS (control), or IC from ND or from FIBER donor mice. Body weight, gut microbiota structure, liver pathology, blood biochemistry parameters and glucose tolerance were examined. Our results showed that ICI treatment modulates the bacterial population and improves fasting glucose level regardless of donor source and with no effect on other metabolic parameters. These observations suggest that compositional alterations in the microbiome may be made by intubation of IC from donor mice. These changes are not accompanied by significant changes in the development of NAFLD in mice fed high trans-fat diet.
Keywords:Fecal transplantation, gut microbiota, antibiotics and NAFLD
IC: intestinal content; ICI: intestinal content intubation; ND: normal diet; NAFLD: non-alcoholic fatty liver disease; PBS: phosphate buffer saline; OGTT: oral glucose tolerance test.
Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide, affecting all racial, ethnic, and age groups without sex predilection. Thus, the very high prevalence of fatty liver means that this disorder will contribute significantly to an increased burden of ill-health including increased mortality due to liver damage and related cardiovascular events in the future .
It has become evident that progression of NAFLD is not just a consequence of free fatty acid derived triglyceride accumulation in hepatocytes but rather the inadequate adaptation of the cells to toxic lipid-derived metabolites. Trans-fatty acids are a kind of unsaturated fatty acid that are uncommon in nature, but commonly produced industrially from vegetable fats for use in margarines, snack foods, packaged baked goods and fried foods . Based on epidemiological data, trans-fatty acid consumption was associated with increased risk of cardiovascular disease and metabolic disorders . Recent studies have reported that high trans-fatty acid levels could induce NAFLD and that high trans-fatty acid diet induces more severe obesity, insulin resistance and hepatic steatosis compared to a high fat diet in mice .
Dietary fiber is known to have many health benefits. Functionally, fibers are known to increase volume and frequency of discharge, improve intestinal health, control blood glucose levels, and lower circulating cholesterol. Different dietary fibers have different effects on digestion of food and intestinal microbiota . Dietary fibers reach the large intestine where fermentation is carried out by the intestinal bacterial population and short chain fatty acids and gases are produced . Pectin originates mainly in the peel and core of apples and citrus fruit and is often used in fiber‐rich diets to treat constipation. Pectin is known to lower cholesterol and glucose, and leads to weight loss . The addition of apple pectin to a high‐fat diet in rats revealed changes in microbiota, and decreased inflammation and accumulation of fat in the liver .
The human gut microbiota contains more than 100 fold genes than its host , and has been suggested to be an important environmental factor involved in the pathogenesis of NAFLD . Recent evidence from animal and human models suggests microbiota transplantation could be used as a therapeutic intervention against obesity [11,12], Type 2 diabetes, and NAFLD, whereby the replacement of a microbial population by a new one that has been manipulated to include healthy factors should confer a beneficial health effect on the host . The gut microbial composition is distinctive in obese individuals, and tends to show reduced complexity . For example, obese mice have reduced numbers of Bacteroidetes and increased numbers of Firmicutes when compared to lean mice. These changes in gut microbial populations have significant implications for energy homeostasis . Furthermore, it is well known that antibiotic treatment reduces the diversity and abundance of gut microbiota. Indirectly, this destroys the community structure, thereby disturbing the interactions among microbial species and the complementary systems of nutrient metabolic pathways. A dysbiotic gut microbiota is one that contains an overt imbalance in microbial structure and function of the commensal microbiome, compared with that found in healthy individuals .
Replacement of a microbial population that promotes obesity with a healthy microbial population, may represent a possible treatment. This led us to investigate whether modification of gut microbiome by intubating with intestinal content (IC) from healthy mice might improve metabolic alterations caused by high trans fatty acid diet.
Materials and Methods
All animal experiments were performed within the guidelines of the Authority for Biological and Biomedical Models and were approved by the Institutional Animal Care Ethics Committee, both of the Hebrew University of Jerusalem.
Donor mice group:
Male C57BL/6J mice, 7-8 weeks old, were purchased from Harlan Laboratories (Jerusalem, Israel). Twenty mice (23.05 ± 0.21 gr) were randomly divided into two groups: 1) Mice fed with normal diet (ND, n = 10). 2) Mice fed with high citrus fruit pectin (FIBER, n = 10) for 8 weeks. The diet composition is presented in Table 1. Body weight and food consumption were monitored weekly. After 8 weeks on diet, mice were sacrificed and the IC was removed and treated as described.
Table 1. Diet composition per 100 gr
|Ingredients (gr)||Normal||Fiber Diet||High Trans|
|Diet (ND)||(FIBER)||Fat diet|
**CBR (Cocoa Butter Replacer) 88 is a partial hydrogenated fat made from palm oil fraction.
Collecting intestinal content from donor mice:
After 8 weeks of the donor mice experiment, mice fasted for 12h, their body weight was recorded, and they were sacrificed by isoflurane inhalation. Followed immediately by washing the mice with 70% ethanol under biological hood, and IC of each group was collected. Each test tube was mixed with 0.1% Cysteine/ PBS at a ratio of 0.5 gr/10 ml, filtered by a 0.45 μm filter, and was mixed with an equal volume of 30% glycerol in 0.1% Cysteine/PBS. The solution obtained was frozen at -80 ° C until use.
Recipient mice group:
Sixty mice (23.56 ± 0.244 gr) were fed high trans fatty acid diet and divided into two groups.
Diet composition is presented in Table 1. After two weeks, for inducing dysbiosis of the microbiome, the mice (n=30, (+)) were intubated with 0.3 ml mix of streptomycin (20 mg/mouse) ampicillin (10 mg/mouse) and vancomycin 10 mg/mouse) for 3 days. The corresponding group received PBS to eliminate the possible effect of the gavage (n = 30, (-)).
Two days after antibiotic treatment, each mouse in the “recipient” group was administered 200 microliters of IC or PBS once a day for ten days. The mice were divided into six groups as follow:
1). (+) ND, mice treated with antibiotics and intubated with IC of donor mice fed with ND.
2). (+) FIBER, mice treated with antibiotics and intubated with IC of donor mice fed with high fiber diet.
3). (+) PBS mice treated with antibiotics and intubated with PBS
4). (-) ND, mice not treated with antibiotics and intubated with IC of donor mice fed with ND.
5). (-) FIBER mice not treated with antibiotics and intubated with IC of donor mice fed with high fiber diet.
6). (-) PBS mice not treated with antibiotics and intubated with PBS (see scheme 1).
Scheme 1. Experiment design
Recipient mice sacrifice and tissues collection:
At the end of the experiment, the mice fasted for 12h, their body weight was recorded, and they were sacrificed by isoflurane inhalation. Blood was collected from the vena cava, and plasma was obtained by centrifugation at 5,000 xg at 4°C for 10 min, and stored at -20°C. Epididymal adipose tissue was removed, weighed, placed in liquid nitrogen, and stored at -80°C. Liver tissue was collected and weighed; a small sample from the right lobe was placed in 4% formaldehyde, and the remaining liver tissue was minced in liquid nitrogen and stored at -80°C. IC were collected, weighed and placed on ice. The cecum, large intestines and their content were collected for microbiota analysis. American Laboratories (Herzliya, Israel) performed analyses of serum lipid profiles.
Oral glucose tolerance test (OGTT):
A week before the end of the experiment, a glucose-loading test was conducted for the “recipients” group. Prior to the OGTT, mice were weighed, marked, and were given D-glucose (3 g/kg body weight) by gavage. Glucose levels were monitored in blood drawn from the tail tip, at 0, 30, 60, and 120 min after the glucose loading. Glucometer was used to measure glucose levels.
Patholab (Rehovot, Israel) prepared histological slides. The paraffin blocks were sectioned at approximately 3–5 µm and placed on glass slides, which then were stained with Hematoxylin and Eosin (H&E) and covered by an automatic apparatus. Prof. A. Nyska performed the histopathological examination (Website: http://www.nyska.net).
For histology, tissue samples were trimmed according to the RITA guide , dehydrated, embedded in paraffin wax, and sectioned at four to five-micron thickness. Histopathological changes were described and scored by a Board Certified toxicologic pathologist (AN), using semi-quantitative grading of five grades (0-4), taking into consideration the severity of the changes . The scoring reflects the predominant degree of the specific lesion seen in the entire field seen in the histology section.
A generic grading criteria which was used is as follows: Grade 0 – no lesion; Grade 1/Minimal: The first (lowest) level of severity in an ordered list based on a four-level scale of minimal, mild, moderate, and severe; Grade 2/Mild: The second level of severity in an ordered list based on a four-level scale of minimal, mild, moderate, and severe; Grade 3/Moderate: The third level of severity in an ordered list based on a four-level scale of minimal, mild, moderate, and severe; Grade 4/Severe: The fourth (highest) level of severity in an ordered list based on a four-level scale of minimal, mild, moderate, and severe.
The morphological criteria was bases according to the standards in the field, as specified in the in hand morphological criteria for the liver .
Preparation of 16S ribosomal RNA gene amplicons for the Illumina system:
In order to study the effect of each treatment on the gut microbiome, the prokaryotic 16S ribosomal RNA gene (16S rRNA) was analyzed; it is approximately 1,500 bp long and contains nine variable regions interspersed among conserved regions. These variable regions were subjected to phylogenetic classification according to genus or species, in diverse microbial populations. The following protocol describes a two-step PCR-based method for preparing samples for sequencing the variable V3 and V4 regions of the 16S rRNA gene. Bacterial DNA was extracted from the IC studied mice with the PureLink Genomic DNA Mini Kit (Invitrogen, Paisley, UK). Each sample then was quantified with a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and diluted to a final concentration of 5 ng/µL in 10 mM Tris at pH 8.5. The 16S library preparation was carried out as described in Illumina’s 16S sample preparation guide with minor modifications – the PrimeStar HS DNA polymerase premix (Takara-Clontech, Mountain View, CA, USA) was used instead of the PCR enzyme.
The Operational Taxonomic Units data were used to calculate the index of biodiversity Chao1 and to perform a beta diversity analysis with the Principal Coordinate Analysis (PCoA) method based on unweighted UniFrax.
The bioinformatics analysis was performed using MOTHUR software. For the taxonomic classification we used the SILVA  version 123.
Values are presented as means ± SEM. Analysis of variance (one-way ANOVA) and Tukey were used to compare means. The significance level was p < 0.05 for all analyses. The JMP 14 Pro software suites (SAS Institute, Cary, NC, USA) were used for the analyses.
Body weight: The effects of ICI and antibiotic treatment on body weight are shown in Figures 1 A; B. All groups gained weight during the experiment, although without statistical significance.
Figure 1. Effect of antibiotic treatment and ICI on weight gain. Male C57BL/6J recipient mice aged 7-8 weeks fed on high trans-fat diet and treated with antibiotics (+) or not (-). All recipient mice were intubated with IC of donor mice fed with normal diet (ND) or high fiber diet (FIBER). Control mice were intubated with PBS (PBS). A) Body weight measurements of groups receiving antibiotics and IC. B) Body weight measurements of groups receiving IC only. All values are mean ± SEM. n = 8-10.
Glucose tolerance test and blood biochemistry parameters:
The results showed fasting glucose levels were significantly lower in the groups receiving antibiotics and IC from ND and FIBER donors, compared to mice receiving antibiotics only (Figure 2 A, B). In non-antibiotics treated groups, mice receiving IC from high fiber diet donor mice showed lower glucose fasting levels than control mice. After 30 min of glucose, loading high levels were found in all groups and were lowered at 120 min. The total serum cholesterol levels in all groups were higher than the normal range (80-100 mg/dl), HDL cholesterol was above normal range (98-101 mg/dl) in all groups, Triglycerides levels were similar in all groups (Table 2). No statistical significance has been shown. Liver enzyme levels (ALK, SGOT and SGPT) were elevated above normal range in mice in all groups, an indication of liver injury with no statistical significance between the groups.
Figure 2. Effect of antibiotic treatment and ICI on Glucose tolerance. Male C57BL/6J recipient mice aged 7-8 weeks fed on high trans-fat diet and treated with antibiotics (+) or not (-). All recipient mice were intubated with IC of donor mice fed with normal diet (ND) or high fiber diet (FIBER). Control mice were intubated with PBS (PBS). Oral glucose tolerance test was performed at week 7 with blood sampled from mouse tail tips. A) Glucose levels between 0 to 120 during OGTT in groups receiving antibiotics and IC. B) Glucose levels between 0 to 120 during OGTT in groups receiving IC only. All values are mean ± SEM. n = 8-10. Data marked with different letters (a, b) are significantly different (p < 0.05). OGTT – oral glucose tolerance test.
|Liver weight (gr)||1.23±0.04||1.32±0.06||1.24±0.05||1.4±0.07||1.57±0.06||1.5±0.07|
|Adipose weight (gr)||0.56±0.05||0.8±0.11||0.75±0.08||0.94±0.13||0.97±0.06||1.05±0.14|
|ALK P (IU/L)||69.6 ± 7.7||79.29 ± 10.06||81.43 ± 11.18||88.17 ± 8.21||83.57 ± 16.56||79.43 ± 6.55|
|SGOT (IU/L)||47.6 ±8.26||46.43 ± 1.27||54 ± 8.85||54.83 ± 6.97||69 ± 33.46||58.57 ± 14.03|
|SGPT (IU/L)||29.6 ± 5.73||29.71 ± 1.5||35.29 ± 11.51||41.33 ± 9.91||72.86 ± 66.09||44.71 ± 27.57|
Table 2. Effect of antibiotic treatment and ICI on Blood biochemistry. Male C57BL/6J recipient mice aged 7-8 weeks fed on High Trans Fat Diet and treated with antibiotics (+) or not (-). All recipient mice were intubated IC of donor mice fed with normal diet (ND) or high fiber diet (FIBER). Control mice were intubated with PBS (PBS). All values are mean ± SEM. n = 8-10.
In all groups, hepatocytic vacuolation consistent with fatty liver pathology was noted (Table 3). Changes in fatty liver pathology either involved the lobular structure (defined as “diffuse”), or primarily involved the peri lobular regions of the lobular structure (defined as “perilobular”). The severity was scored as explained in the Methods section. In groups (-) ND, (+) FIBER and (+) PBS the most severe grade (i.e., grade 4, diffuse), was not reported.
Figure 3. Histopathology (H&E) of recipient mice A) treated with antibiotics and intubated with PBS. B) treated with antibiotics and intubated with IC of donor mice fed on normal diet. C) treated with antibiotics and intubated with IC of donor mice fed on high fiber diet. D) not treated with antibiotics and intubated with PBS. E) not treated with antibiotics and intubated with IC of donor mice fed on normal diet. F) not treated with antibiotics and intubated with IC of donor mice fed on high fiber diet.
Table 3. Histopathological changes were scored by the study pathologist, using semi-quantitative grading of five grades (0-4), taking into consideration the severity of the changes (0, no lesion; 1, minimal change; 2, mild change; 3, moderate change; 4, severe change)
Gut Microbiota Composition:
Microbiota composition following the treatments was evaluated (Figure 4). At the phylum levels, the population of Proteobacteria increased in group receiving IC from donor mice fed normal diet (ND) and high fiber diet (FIBER) regardless of antibiotic treatment. Firmicutes abundance decreased statistically only in antibiotic treated groups receiving IC from both donor ND and FIBER groups. There is a tendency towards an increase in abundance of Bacteroidetes in antibiotic treated groups that received IC from donors fed on a high fiber diet. Similar trends were noted in non-antibiotic treated groups.
Figure 4. Metagenomics applied to microbiota of recipient mice IC. Male C57BL/6J recipient mice aged 7-8 weeks fed on high trans-fat diet and treated with antibiotics (+) or not (-). All recipient mice were intubated with IC of donor mice fed with normal diet (ND) or high fiber diet (FIBER). Control mice were intubated with PBS (PBS). The effect of antibiotics and ICI on changing microbiota populations was evaluated. (A-D) Relative abundance of phyla. B) Columnar view of Figure A. D) Columnar view of Figure C. Columns marked with different letters (a, b, c) are significantly different (p < 0.05).
The ratio of Bacteroides to Firmicutes was evaluated (Figure 5 A, B). The ratio increased statistically in antibiotic treated groups, which received IC from donor FIBER mice. No such trend was seen in non-antibiotic treated mice.
Chao1 index (Figure 6 A, B) increased in response to ICI from both donor groups, ND and FIBER diets. This result was observed in all groups. Strikingly, as seen in PCoA (Figure 6 C) all IC treated groups are similar but very different from groups not receiving IC ((+) PBS and (-) PBS). Antibiotic treatment with no ICI ((+) PBS) changed the gut microbiota relative to non-antibiotic treated mice ((-) PBS) and all ICI treated mice.
Figure 5. The ratio of Firmicutes and Bacteroidetes in recipient mice. Male C57BL/6J recipient mice aged 7-8 weeks fed on high trans-fat diet and treated with antibiotics (+) or not (-). All recipient mice were intubated with IC of donor mice fed with normal diet (ND) or high fiber diet (FIBER). Control mice were intubated with PBS (PBS). n = 4-5. Columns marked with different letters (a, b) are significantly different (p < 0.05).
Figure 6. Altered biodiversity of gut microbiota in recipient mice. Male C57BL/6J recipient mice aged 7-8 weeks fed on high trans-fat diet and treated with antibiotics (+) or not (-). All recipient mice were intubated with IC of donor mice fed with normal diet (ND) or high fiber diet (FIBER). Control mice were intubated with PBS (PBS). The Alpha diversity, richness of gut microbiota, was determined by A, B) Chao1 index. C) Principal coordinates analysis (PCoA) plots were used to assess the variation between experimental groups. n = 3-5. Columns marked with different letters (a, b) are significantly different (p < 0.05).
The prevailing hypothesis that the replacement of a dysbiotic microbial population with one that has been manipulated to include healthy factors should confer a beneficial health effect on the host led us to investigate whether ICI from healthy donor mice will improve metabolic alterations in recipient mice fed high trans-fat diet.
All treatment groups exhibited higher than normal liver enzyme levels, a fatty liver and high cholesterol, suggesting that the experimental diet induced metabolic syndrome-like symptoms in recipient mice. Our findings are consistent with previously published results showing that trans-fatty acid diet triggers obesity, insulin resistance and hepatic steatosis in mice  and rats  . To test the effect of diet and ICI on metabolic parameters, we examined lipid and fasting glucose levels in recipient mice. We found that mice intubated with IC from donor mice fed on ND or FIBER exhibited a decrease in fasting glucose levels, with no change in other parameters studied.
Many studies point to a link between the use of antibiotics and changes in microbial composition in the gut. Two main families of bacteria, Firmicutes and Bacteroidetes are found in the mice and human intestine. When comparing the microbiota of obese human and mice to the microbiota of lean, healthy individuals, a lower ratio of Bacteroidetes/Firmicutes is observed . Our microbial analysis showed a high ratio of Bacteroidetes/Firmicutes in the group treated with antibiotics followed by ICI from donor mice fed with a high fiber diet.
This suggest that at least in the short term, a change in the microbial population can be observed in the absence of a physiological phenotype. The groups that did not receive antibiotics before ICI did not show any change in the Bacteroidetes/Firmicutes ratio. It is possible that antibiotics caused dysbiosis to the recipient mice microbiota, and the administration of healthy IC, i.e. IC from donor mice fed high fiber diet, corrected the ratio. It is known that high-fat diet shifts the intestinal microbiome very quickly to a decrease in Bacteroidetes and an increase in both Firmicutes and Proteobacteria.
Proteobacteria is proposed to be closely correlated to glucose homeostasis. Studies have shown that higher levels of Proteobacteria are associated with the inability to maintain a balanced microbiota population. An increase in the Proteobacteria population could serve as a potential diagnostic marker for dysbiosis and metabolic disease risk. The abundance of Proteobacteria increased in groups receiving IC regardless of antibiotic treatment. We suggest that the increase in beta diversity per se may contribute to the increase in the abundance of these bacteria. We found that fasting glucose levels were lower in antibiotic treated groups prior to ICI. In these groups, there was a statistically significant change in the composition of the intestinal bacteria, with a reduction in the levels of the Firmicutes and a higher ratio of Bacteroides to Firmicutes. We believe that this is associated with the improvement of glucose fasting levels in those groups. It is important to note that although there was an improvement in fasting glucose levels, all treatment groups presented a disturbed metabolic profile and obesity.
In relation to the richness and the diversity of the gut bacterial populations, our finding that all groups receiving ICI showed greater bacterial richness and similarity in bacterial composition (Figure 6 A-C) compared to the groups not receiving ICI, regardless of prior antibiotic treatment suggests that prior antibiotic treatment is not required in order to alter these parameters. The significance of these findings needs to be further studied.
The current study showed modulation of the gut bacterial population and improvement in fasting glucose levels following transplantation of IC by intubation, regardless of donor source. No effect has been shown on other metabolic parameters. There are two main limitations to the present study. First, IC contains, in addition to bacteria, metabolites, which might affect the benefits of transplantation. Second, the limited frequency and length of transplantation performed, daily ICI (10 days), which may not be sufficient to have a physiological effect.
Conflicts of interest
The authors declare no conflict of interest.
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