1. The effect of linolenic acid concentration on the expression of fatty acid
biosynthetic genes in the female C57BL/6j weaning mouse liver
Daniel Mikel
Introductory Biology 152, Department of Cell & Regenerative Biology, University of Wisconsin–Madison
Introduction
• Examined linolenic acid’s
relationship to gene
expression of fatty acid
biosynthetic genes
• Increased expression
with decrease in linolenic
acid concentration
• 35% of American adults
are currently obese
• Nearly 10% of American
adults are afflicted with
diabetes
• Obesity development:
1. Metabolic imprinting
2. Epigenetic
programming
3. Lifestyle choices
4. Imbalance of omega-3s
and omega-6s
ResultsAbstract
The effect of linolenic acid concentration on gene expression in C57BL/6j mice was
evaluated to ascertain if there is a direct link between linolenic acid concentration
during gestation and lactation and the levels of expression of fatty acid synthase
(Fasn), elongation of very long chain fatty acids family member 6 (Elovl6), and
stearoyl-CoA desaturase-1 (Scd1) in offspring. These genes play a major role in
adiposity levels and an individual’s disposition to metabolic diseases. To evaluate
this relationship, pregnant mice were place on one of two diets with differing
linolenic acid concentrations, their pups were sacrificed at 21 days of age and their
livers were examined for the levels of expression of each gene. The data showed a
higher relative expression of all three genes in the pups whose mothers were fed the
soybean diet of higher linolenic acid concentration. The statistical significance was
assessed using unpaired t-tests with a significant p-value = 0.05. The results were
statistically significant for Elovl6 but not significant for Fasn & Scd1.
Methods
Figure 1: Metabolic pathways leading to the synthesis
of triglycerides in the liver
This graphic shows multiple pathways for the synthesis
of triglycerides. It has been modified to show the the
location of Fasn, Elovl6, and Scd1 in the pathway.
Problems/Questions:
• What is linolenic acid’s effect on gene
expression in female C57BL/6j weaning
mouse liver?
Research Approach:
• Introduce dams to one of two diets with
different linolenic acid concentrations
and assess genetic expression of
offspring.
Goal:
• Understanding linolenic’s effect on
gene expression may lead to therapies
and cures for metabolic diseases with
further research.
Figure 2: Linolenic acid Linoleic acid
Figure 3. The average levels of normalized
gene expression of each gene (Fasn, Elovl6,
Scd1) between each individual mouse pup
in the soybean diet group.
A higher concentration of linolenic acid correlates to a
higher level of fatty acid biosynthetic gene expression
Figure 5. The overall average levels of
normalized gene expression of each gene
(Fasn, Elovl6, Scd1) between each diet
group (soybean, cottonseed).
Ethics Statement: Experimental protocols were approved by the University of
Wisconsin School of Medicine & Public Health and the College of Agriculture
& Life Sciences Animal Care and Use Committees (ACUC; protocol number:
M00682), in strict accordance to the EU Directive 2010/63/EU.
- Place mice on one of two diets at 4.5 days gestation: Soybean oil (8%
linolenic acid) or cottonseed oil (0.3% linolenic acid)
- Harvest mice: At 21 days of age six female pups were euthanized and liver
tissues were taken.
- Purify mRNA: Liver samples underwent the QIAGEN RNeasy mRNA
extraction protocol.
- Nanodrop & run gel: mRNA samples were quantitated and normalized to
obtain four μg of mRNA. A 1.5% agarose denaturing gel was run to determine
if the mRNA remained intact.
- Convert to cDNA: mRNA was converted to cDNA via reverse transcriptase
methodology by the addition of random hexamers.
- Quantitatively evaluate: cDNA samples were analyzed via the process of real-
time PCR. Gene specific primers were added at a concentration of 300 nM. A
small amount of SYBR Green was integrated into each PCR product. This
allowed for quantitation relative to the ribosomal 18S gene.
- Analyze data: The data was analyzed using unpaired parametric t-tests
without Welch’s correction. A p-value of .05 or less was considered to be
statistically significant.
Figure 4. The average levels of normalized
gene expression of each gene (Fasn, Elovl6,
Scd1) between each individual mouse pup
in the cottonseed diet group.
Average expression of each gene for each mouse
Mouse
ID
Sample
Number
Diet Avg. Fasn
Normalized
Expression
Avg. Elovl6
Normalized
Expression
Avg. Scd1
Normalized
Expression
112986-1 1 12% soy 5.98 5.22 7.33
112986-4 2 12% soy 4.29 5.60 7.16
112986-5 3 12% soy 1.51 2.16 2.38
Average 3.93 4.33 5.62
SEM 0.83 0.70 1.08
WT vas
wean
3.6
4 12%
cottonseed
3.81 2.42 6.01
WT vas
wean
4.3
5 12%
cottonseed
0.87 1.37 0.17
WT vas
wean
6.5
6 12%
cottonseed
2.86 3.76 2.56
Average 2.51 2.52 2.91
SEM 0.55 0.44 1.07
P-Values: Fasn: 0.19 Elovl6: 0.05 Scd1: 0.11
Figure 6. The mRNA gel showing
the presence of two bands which
indicated that the mRNA was intact
Acknowledgements: I would like to thank Dr. Michele Larsen and Dr. Colin Jefocate for their
guidance and support throughout this project.
References
1. Postic, C., & Girard, J. (2008). Contribution of de novo fatty acid synthesis to hepatic steatosis and insulin resistance: Lessons from
genetically engineered mice. Journal of Clinical Investigation J. Clin. Invest., 829-838.
2. 2. ALS Environmental. (n.d.). Retrieved December 6, 2015, from www.caslab.com/Linoleic-Acid.php5, www.caslab.com/Linolenic-
Acid.php5
Discussion & Conclusion
Findings:
• A higher concentration of linolenic acid resulted in a higher level of fatty
acid biosynthetic gene expression of Elovl6. Fasn and Scd1 expression is
inconclusive.
Challenges/Limitations:
• Very small sample size (6)
• Only female mice were used
• Inter-animal variability
• Changes in expression as maturity is reached
Future Directions:
• Expand sample size dramatically
• Include male mice as separate entities or in general construct
• Allow some mice from litter to reach maturity
• Determine mechanism for why increased gene expression occurs.
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