Investigating the role of dietary fat and gut microbiota on development of

obesity in mice

ABSTRACT
This study aims to investigate the causal interaction between high fat diet, gut microbiota and
obesity using mice as animal model. A total of 10 fecal samples from obese patients were
collected and 18 morphologically different strains were isolated. Biochemical
characterization of the purified strains was done. Out of 18 strains, 3 most biochemically
abundant strains were characterized genetically. 16srRNA gene sequencing identified the
strains as Lactobacillus acidophilus, Lactobacillus reuteri and Staphylococcus aureus. In
vivo study was performed using the strains alone and with high fat diet (HFD) in mice. For
this purpose mice were divided into four groups i.e., group 1 consist of mice fed with chow
diet, group 2 mice were fed with HFD, group 3 mice were treated with gut microbiota and
HFD and group 4 mice were given bacterial inoculum and fed with normal chow diet.
Increase in body weight (BMI; kg/m 2) was observed in group 3 as compared to control group.
Biochemical parameters including cholesterol, triglycerides, HDL, LDL and VLDL were
assessed after 3 months period. ANOVA revealed significant increase (p≤0.01) in values of
cholesterol, triglycerides, LDL and VLDL in group 2, group 3 and group 4 compared with
control (group 1), however HDL values among four groups showed a non-significant
difference. In conclusion, HFD diet induced significant changes in shaping gut microbiota
composition which led to development of obesity.

INTRODUCTION
Obesity is a condition in which excess fat accumulate in body to such an extent that it may
cause harmful effects on health (Raoult, 2008). Genetic and environmental factors play
important role in development of obesity. Together with environmental factors including
increased fat intake or decreased physical activities, obesity may result from changes in
energy balance i.e. energy storage and harvest (Kahn et al., 2006). Obesity may be linked to
development of several other serious ailments including hypertension, diabetes, dyslipidemia
and hypertriglyceridemia (Zimmet et al., 2005). Prevalence of obesity has increased more
than 75% in adult US population since 1980 (Ogden et al., 2007). According to WHO data,
globally 1 billion adults were overweight and 300 million were considered obese (Waxman &
World 2004).
Obesity may result due to change in gut microbiota, gut barrier disruption and inflammation.
Most of the symbiotic gut microbiota of human consists of fungi, bacteria, viruses and micro
eukaryotes. Approximately 1014 bacteria inhabits human gut and continuous changes in
diversity of the gut microbiota may increase the person’s exposure to diseases like obesity,
diabetes, IBDs , asthma etc (Blaser and Falkow, 2009). All humans and animals born germ
free and sterile but soon after birth, many colonies of microbes from the environment inhabit
the exposed surfaces of their body e.g. mouth, skin, gut and vagina etc. (Round and
Mazmanian, 2009). The new born gut is mainly inhabited Escherichia coli and Streptococcus
(Thompson-Chagoyán et al., 2007) while high abundance of Bifidobacterium and
Clostridium genera is present among the adolescent microbiota (Agans et al., 2011). Three
most prominent gastrointestinal phyla of adult human include Gram negative Bacteroidetes,
Gram positive Firmicutes and Actinobacteria. Gut of obese human shows higher populations
of Firmicutes and lesser Bacteroidetes while more Bacteroidetes and less Fermicutes were
found in lean individuals (Ley et al., 2005). Studies revealed that there is a significant link
between obesity and certain bacterial groups like S. aureus, Lactobacillus species,
Faecalibacterium prausnitzii and E.coli (Collado et al., 2008; Balamurugan et al.,
2010).While a difference is seen at specie level, such as Lactobacillus reuteri are abundantly
present in the gut microbiota of the obese individual and in lean individual Lactobacillus
plantarum excess is seen (Million et al., 2012).
Diet may be considered as one of the main factor involved in diversity of gut microbiota
(Backhed et al., 2004). High fat diet (HFD) produce high levels of chylomicrons in intestine
and increase LPS transportation in circulation, and is considered to be involved in developing
metabolic disorders (Cani et al., 2007). Gut microbiota may hydrolyze the indigestible
polysaccharides and change it to digestible monosaccharaides which activates lipoprotein
lipase causing increased blood glucose level and start lipogenesis and the fat excessively
deposit in adipose tissues by de novo synthesis of triglycerides, resulting in obesity (Samuel
et al., 2008).
There are still lots of pitfalls regarding the understanding of relationship between diet, gut
microbiota and obesity. In pursuit of this, we used culture dependent and independent
techniques to assess the impact of high fat diet aloine and coupled with gut microbiota on
obesity using mice as an animal model.
MATERIALS AND METHODS
Sample collection: Fecal samples from 10 lean and 10 obese volunteers from Mayo hospital
Lahore and general population were collected in properly labeled 50 ml sterile stool
collection vials. Fresh stool inoculum was prepared by mixing a sample in saline (0.85%) and
spread on MacConkey agar, Nutrient agar, Trypto soy agar (TSA) and Xylose lysine
deoxycholate agar (XLD).
Morphological, biochemical and genetic characterization of bacterial isolates: Isolated
strains were morphologically differentiated by Gram staining and different biochemical tests
such as catalase, citrate utilization, urease, H 2S production, Methyl red, Indole, Voges
Proskauer and denitrification were performed to identify isolated strains, following Gerhardt
et al. (1994). Genomic DNA was isolated using TIANamp bacterial DNA kit and PCR was
performed to amplify 16S rRNA gene under standard conditions in a thermal cycler using
Universal primers 16S-27-F (5′ AGAGTTTGATCCTGGCTCAG-3′) and 16S-1522-R (5′-
AAGGAGGTGATCCAGCCGCA-3′).The amplified product was sent for sequencing to Axil
scientific, Singapore. Sequenced data was obtained and examined using BLAST software.
Animal housing (weaning and adult study): Albino mice were mated and one week before
birth, ampicillin was added to drinking water of mother. 16 pups were selected and they were
treated with ampicillin (1g/L) water till the age of 3 weeks (Ellekilde et al., 2014). The mice
were randomized into following 4 groups.
 Control (Group 1) animals were given chow diet (Lamont et al., 2016).
 Group 2 animals were fed with HFD (Lamont et al., 2016).
 Group 3 animals were inoculated with gut micro biota+ HFD
 Group 4 animals were inoculated with gut micro biota + chow diet

BMI of the experimental and control mice were measured on weekly basis.

High fat feeding: Control and group 4 were fed with normal standard chow diet (20%
protein, 70% carbohydrate and 10% fat).While group 2 and 3 were fed HFD. The energy
content of high fat diet was consisted of 13% from protein, 6% from carbohydrate and 81%
from fats (Lamont et al., 2016).

Gut microbiota transfer: The bacterial inoculum was prepared by taking isolated bacteria
of obese human subjects and diluting it 1:10 in a 50% glycerol solution. The prepared
inoculum was divided into small aliquots and stored at −80°C. At the day of inoculation it
was diluted futhur in ratios of 1:5 and 0.15ml and given to each mouse of group 3 and 4
(Ellekilde et al., 2014).
Mice sacrifice and blood collection: Blood samples were collected in EDTA tubes by
cardiac puncture technique and lipid profile (cholesterol, LDL, HDL, TGs, and VLDL) was
done (Radilla-Vázquez et al., 2016). After blood collection the mice were sacrificed and
cecal content was collected and bacterial cultures were prepared.

Statistics: Statistical analysis was done using SPSS statistical software. All values have been
expressed as means ± SEM. Paired t-test and ANOVA was used to analyze date taking p≤
0.05 as statistically significant in all tests.
RESULTS
Gut bacterial characterization: Out of 30 isolated and purified strains, 18 morphologically
different bacterial strains were isolated and biochemically tested. These strains were found to
belong to genera Lactobacillus, Enterococcus, Prevotella, Escherichia, Staphylococcus,
Clostridia, Bacillus and Enterobacter.
16S rRNA gene sequencing: The bacterial strains identified by 16S rRNA were L.
acidophilus (ATCC 658270), L. reuteri (ATCC 658276) and S. aureus (ATCC (658278).
Demographic and biochemical characteristics of mice

Body Mass Index (BMI): In comparison to control (group 1), group 2, 3 and 4 showed
significance difference (p =0.04) in BMI values at the end of experiment (Table 1; Fig. 1).

Cholesterol: As compared to control (group 1), cholesterol values significantly increased (p<
0.01) in group 3 followed by group 2 (p= 0.007) whereas decrease in cholesterol was
observed in group 4 (Table 1; Fig. 2).

Triglycerides: In comparison to the control group, triglycerides values were observed

elevated in group 3 while group 4 showed slight differences with group 3, Group 2 values
were lesser than Group 4. Highly significant difference (p= 0.001) was observed in the values
of Ttriglycerides within the four groups under study (Table 1; Fig. 3)

High Density Lipoprotein (HDL): Group 1(control) mice showed highest values of HDL
cholesterol, followed by Group group 3 mice, then comes Group group 4 and lowest values
of HDL cholesterol were observed in mice of Group 2. A non-significant difference (p= 0.4)
was observed (Table 1; Fig. 4).
Low Density Lipoprotein (LDL): Highest values of LDL were observed in Group group 3
while Group group 4 showed a lesser values and Group 2 values were slightly low. ANOVA
showed significant increase of LDL (p< 0.01) among experimental groups as compared to
control (Table 1; Fig. 5).

Very Low Density Lipoprotein (VLDL): There was a significant increase (p= 0.001) in
VLDL values of experimental groups as compared to control. Highest mean VLDL values
were observed in mice of Group 4 while group 2 and 3 showed almost same similar results
(Table 1; Fig. 6).

Mice cecal bacterial characterization: Out of 23 isolated strains, 15 isolates were found to
be morphologically different on the basis of physical appearance and Gram staining. Some
colonies were smooth, entire, regular, and shiny some wereor elevated, while others were
small, round, mucoid or dry. Only four strains were found Gram negative while all others
appeared to be Gram positive. The biochemical characterization of cecal bacteria identified
that the ceca of obese mice mainly consisted of following genera; Staphylococcus,
Lactobacillus, Clostridia, Bacillus, Enterobacter, Escherichia and Shigella.
DISSCUSSION
Obesity is a worldwide epidemic, related to other disorders like hypertension, diabetes
and hypertriglyceridemia. Lower HDL and high blood LDL level is related to insulin
resistance and elevated triglyceride levels leading to obesity (Hoogeveen et al, 2014). High
fat feeding changes the gut microbiota which is involved in developing obesity and diabetes.
Gut microbes act as major environmental factor involved in obesity. Normal gut flora consist
of more gram negative bacteridetes and lesser gram positive Firmicutes. On the other hand
obese patients show fewer Bacteroidetes, more Firmicutes and Actinobacteraia (Ley et al.,
2005).

In order to investigate the effect of dietary fat and gut microbes in development of
obesity, fecal samples from 10 obese subjects were collected and 18 morphologically
different strains were isolated and purified while most of the strains were Gram positive.
Morphological and biochemical characterization identified the most of the strains belonged to
Lactobacillus, Staphyllococus, Enterobacter, Bacilli, Escherichia and Clostridium spp.
Similar results were shown by Santacruz et al., (2010) on obese human gut microbiota study,
showed that gut of obese mainly consist of increased Firmicutes, decreased Bacteroidetes,
Bacteroides and Bifidobacterium. Molecular characterization of 3 most abundantly occurring
bacteria was done by 16S rRNA gene sequencing identified the strains as Lactobacillus L.
acidophilus, Lactobacillus L. reuteri and Staphylococcus S. aureus. Our results were
supported by Million et al (2012) in a study which revealed that gut of obese patients consist
of mainly LcatobacillusLactobacillus including L. reuteri, L. ingluviei, L. acidophilus and L.
fermentum while some Lactobacillus species are were also involved in weight loss including
L. plantarum and L. casei.

Values are mean± SEM. P-value ≤0.05 = * indicate significant difference in BMI values of
mice groups, P-value < 0.01 = ** indicate highly significant difference in values of
cholesterol, triglycerides, LDL and VLDL while P-value >0.05= NS indicate non-significant
difference in HDL values of four groups.

To check the effect of gut microbiota on albino mice, the inoculum of genetically identified
strains was given to mice of group 3 and group 4 by oral gavage. Similar study was done by
Ellekilde et al (2014) in which they transplanted lean gut with obese microbiota to check
effect of gut microbiota in development of obesity. HFD (6% carbohydrates, 81% fat) was
given to mice of group 2 and 3. Bacterial inoculum and HFD treatment was given to mice till
the age of 18 weeks. To compare groups BMI and lipid profiling of mice was done.
BMI at start and at the end of experiment showed a non-significant difference
(p>0.05).Similar study was done by Shabbir et al., (2016) to check the effect of HFD on
different anthropometric parameters in mice, result showed no significant change in BMI.
In comparison to control there was a significant increase in cholesterol, triglycerides,
LDL and VLDL values of group 3. i.e. 167.5± 9.0, 120± 8.8, 165± 14.4, 18 ±1.2 respectively.
While only HFD treated group 2 showed 162± 12.9, 106.2± 3.9, 105.7± 3.4, 18.0 ± 0.9
respectively. Similarly inoculum treated group 4 showed slight changes i.e. 150 ±14.3, 110 ±
5.2, 118.2 ± 1.6 and 19.5 ± 0.95 respectively. HDL values showed a non-significant increase.
HDL was found highest in control i.e. 48.8 ± 3.1, while in group 2, 3 and 4 were 42.5± 1.7,
46± 4.4 and 43 ± 1.8 respectively. Similar study was performed by by Lamont et al., (2016),
where they checked for checking the effect of low carbohydrate HFD on weight gain. Their
findings indicated showed contrasting results that mice developed obesity with decreased
plasma triglycerides (P = 0.001) but showed a positive correlation of HFD with cholesterol,
HDL , LDL and VLDL. Our results proposed that both HFD+ coupled gut microbiota are
collectively involved in increasing serum triglycerides and cholesterol. High fat diet and
bacterial inoculum alone can cause alteration in plasma cholesterol but its effects are were not
much adverse as observed in group 3, which explains that HFD modulates the recipients gut
microbiota by decreasing Bacteriodetes and increasing Firmicutes (Corcos et al., 2003).

The cecal culture of obese mice was done and 15 morphologically and biochemically
tested species were isolated. Biochemical characterization at genus level showed that gut
microbiota of obese mainly consisted of species of phylum Firmicutes including
Lactobacillus species, Bacillus species, Staphylococcus, Clostridia and Enterococcus.
Previous study by Million et al., (2012) on dietary interventions to analyzed the changes in
gut microbiota and, supported our results by suggesting that gut microbiota of children and
adults mainly consisted of Staphylococcus and Lactobacillus species.

CONCLUSIONS

Obesity is a life threatening disease, costly to treat and results in morbidity and may
also result in other obesity related diseases including diabetes and hypertension. Lipid profile
of Group group 3 mice in whichfed with HFD+ coupled Bacterial bacterial inoculum given
to mice, showed synergistic effect and revealed a significant increase (p≤ 0.05) in serum
cholesterol and Triglycerides triglycerides as compared to control group and other
experimental groups. Hence, consumption of HFD diet might change alter gut microbiota
which leads to change in gut setting causing metabolic disorders specifically obesity. So,
strategies to modify gut microbiota could be a useful tool for reducing the impact of HFD on
obesity.

REFERENCES

Agans, R., Rigsbee, L., Kenche, H., Michail, S., Khamis, H. J., & Paliy, O. (2011). Distal gut
microbiota of adolescent children is different from that of adults. FEMS microbiology
ecology, 77(2), 404-412.

Backhed, F., Ding, H., Wang, T., Hooper, L. V., Koh, G. Y., Nagy, A., Semenkovich,C.F.
and Gordon, J. I. (2004). Gut microbiota as an environmental factor that regulates fat
storage. Proceedings of the National Academy of Sciences of the United States of
America.

Balamurugan, R., George, G., Kabeerdoss, J., Hepsiba, J., Chandragunasekaran, A. M., &
Ramakrishna, B. S. (2010). Quantitative differences in intestinal Faecalibacterium
prausnitzii in obese Indian children. British journal of nutrition, 103(3), 335-338.
Blaser, M. J., & Falkow, S. (2009). What are the consequences of the disappearing human
microbiota?. Nature Reviews Microbiology, 7(12), 887.
Cani, P. D., Amar, J., Iglesias, M. A., Poggi, M., Knauf, C., Bastelica, D., Neyrinck, A.M.,
F.,Tuohy, K.M., Chabo, C and Waget, A. (2007). Metabolic endotoxemia initiates
obesity and insulin resistance. Diabetes, 56(7), 1761-1772.
Collado, M. C., Isolauri, E., Laitinen, K., & Salminen, S. (2008). Distinct composition of gut
microbiota during pregnancy in overweight and normal-weight women–. The
American journal of clinical nutrition, 88(4), 894-899.

Corcos, M., Guilbaud, O., Paterniti, S., Moussa, M., Chambry, J., Chaouat, G., ... &
Jeammet, P. (2003). Involvement of cytokines in eating disorders: a critical review of
the human literature. Psychoneuroendocrinology, 28(3), 229-249.

Ellekilde, M., Selfjord, E., Larsen, C. S., Jakesevic, M., Rune, I., Tranberg, B.,
Vogensen,F.K., Nielsen, D.S., Bahl, M.I., Licht., T.R. & Hansen, A. K. (2014).
Transfer of gut microbiota from lean and obese mice to antibiotic-treated
mice. Scientific reports, 4, 5922.
Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R., 1994. Methods for general and
Molecular bacteriology. American Society for Microbiology, 1325 Massachusetts
Ave.,Washington, DC
Hoogeveen, R. C., Gaubatz, J. W., Sun, W., Dodge, R. C., Crosby, J. R., Jiang, J.,
Couper, D., Virani, S.S., Kathiresan, S., Boerwinkle, E.  & Ballantyne, C. M. (2014).
Small dense low-density lipoprotein-cholesterol concentrations predict risk for
coronary heart disease: the Atherosclerosis Risk In Communities (ARIC)
study. Arteriosclerosis, thrombosis, and vascular biology, ATVBAHA-114.

Kahn, S. E., Hull, R. L., & Utzschneider, K. M. (2006). Mechanisms linking obesity to
insulin resistance and type 2 diabetes. Nature, 444(7121), 840.

Lamont, B. J., Waters, M. F., & Andrikopoulos, S. (2016). A low-carbohydrate high-fat diet
increases weight gain and does not improve glucose tolerance, insulin secretion or β-
cell mass in NZO mice. Nutrition & diabetes, 6(2), e194.
Ley, R. E., Bäckhed, F., Turnbaugh, P., Lozupone, C. A., Knight, R. D., & Gordon, J. I.
(2005). Obesity alters gut microbial ecology. Proceedings of the National Academy of
Sciences, 102(31), 11070-11075.
Million, M., Maraninchi, M., Henry, M., Armougom, F., Richet, H., Carrieri, P., Valero, R.,
Raccah, D., Vialettes, B. & Raoult, D. (2012). Obesity-associated gut microbiota is
enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and
Methanobrevibacter smithii. International journal of obesity, 36(6), 817.
Ogden, C. L., Yanovski, S. Z., Carroll, M. D., & Flegal, K. M. (2007). The epidemiology of
obesity. Gastroenterology, 132(6), 2087-2102.

Radilla-Vázquez, R. B., Parra-Rojas, I., Martínez-Hernández, N. E., Márquez-Sandoval, Y.

F., Illades-Aguiar, B., & Castro-Alarcón, N. (2016). Gut microbiota and metabolic
endotoxemia in young obese Mexican subjects. Obesity facts, 9(1), 1-11.

Raoult, D. (2008). Obesity pandemics and the modification of digestive bacterial flora.
Redondo-Lopez, V., Cook, R. L., & Sobel, J. D. (1990). Emerging role of lactobacilli in the
control and maintenance of the vaginal bacterial microflora. Reviews of infectious
diseases, 12(5), 856-872.

Round, J. L., & Mazmanian, S. K. (2009). The gut microbiota shapes intestinal immune
responses during health and disease. Nature Reviews Immunology, 9(5), 313.
Samuel, B. S., Shaito, A., Motoike, T., Rey, F. E., Backhed, F., Manchester, J. K., Hammer,
R.E., Williams, S.C., Crowley, J., Yanagisawa, M.  & Gordon, J. I. (2008). Effects of
the gut microbiota on host adiposity are modulated by the short-chain fatty-acid
binding G protein-coupled receptor, Gpr41. Proceedings of the National Academy of
Sciences, 105(43), 16767-16772.
Santacruz, A., Collado, M. C., Garcia-Valdes, L., Segura, M. T., Martin-Lagos, J. A.,
Anjos, T., Marti-Romero, M., Lopez, R.M., Florido, J., Campoy, C. & Sanz, Y.
(2010). Gut microbiota composition is associated with body weight, weight gain and
biochemical parameters in pregnant women. British Journal of Nutrition, 104(1), 83-
92.
Shabbir, F., Khan, S., Yousaf, M. J., Khan, M. A., & Rajput, T. A. (2016). Comparison of
effect of high fat diet induced obesity and subsequent atorvastatin administration on
different anthropometric measures in sprague dawley rats. Pakistan Armed Forces
Medical Journal, (5), 699.
Thompson-Chagoyán, O. C., Maldonado, J., & Gil, A. (2007). Colonization and impact of
disease and other factors on intestinal microbiota. Digestive Diseases and
Sciences, 52(9), 2069-2077.

Waxman, A., & World, H. A. (2004). WHO global strategy on diet, physical activity and
health. Food and nutrition bulletin, 25(3), 292.
Zimmet, P., Alberti, K. G. M., & Ríos, M. S. (2005). A new International Diabetes
Federation (IDF) worldwide definition of the metabolic syndrome: the rationale and
the results.
Table I: Demographic characteristics and biochemical parameters of mice at the end of
experiment

Parameters Group 1 Group 2 Group 3 Group 4 p-values

n=4 n=4 n=4 n=4
BMI (kg/m²) 0.85 ± 0.02 1.1 ± 0.05 1.1 ±0.07 0.9 ± 0.09 0.04*

Cholesterol (mg/dl) 106.5 ± 2.7 162± 12.9 167.5± 9.0 150 ± 14.3 0.007 **

Triglycerides (mg/dl) 74.7±5.7 106.2± 3.9 120±8.8 110± 5.2 0.001**

HDL (mg/dl) 48.8 ± 3.1 42.5± 1.7 46± 4.4 43± 1.8 0.4NS
LDL (mg/dl) 44.5±0.68 105.7 ± 3.4 165± 14.4 118.2 ± 1.6 < 0.01**
VLDL (mg/dl) 11.1± 1.4 18.0 ± 0.9 18 ± 1.2 19.5 ± 0.95 0.001**
 1.2
1
BMI(kg/m²)

0.8
0.6
0.4
BMI
0.2
0
G1 G2 G3 G4
Mice groups
Fig 1: Mean BMI values of Mice. Group 3 and
Group 2 showed significant weight gain while group 4 showed slight variation in BMI
values.
Mean Cholesterol (mg/dl)

160
120
80
Cholesterol
40
0
G1 G2 G3 G4
Groups
Fig 2: Mean cholesterol values of mice.
Group 3 showed highest blood cholesterol levels while Group 2 and 4 exhibited significant
variation, as compared to control.
Mean Triglycerides(mg/dl)

140
120
100
80
60
40 Triglycerides
20
0
G1 G2 G3 G4
Mice groups
Fig 3: Mean triglycerides of mice. Group 3
showed highest triglycerides values while group 2 and 4 showed slight changes as compared
to control
 50
Mean HDL (mg/dl) 48
46
44
42 HDL
40
38
G1 G2 G3 G4
Mice groups
Fig 4: Mean HDL values of mice. Control group
showed highest HDL values. Group 3 showed slight changes as compared to other two
experimental groups

180
160
140
120
Mean LDL

100
80
60 LDL
40
20
0
G1 G2 G3 G4
Mice groups
Fig 5: Mean LDL values of mice. As compared to
control group 3 showed highest LDL cholesterol values while group 2 and 4 showed slight
changes.

25

20
Mean VLDL (mg/dl)

15

10
VLDL
5

0
G1 G2 G3 G4
Mice groups
Fig 6: Mean VLDL values of mice. As
compared to control, group 4 showed highest values of VLDL while group 2 and 3 showed
slight variation.