Effects of Estradiol Treatment on Lipoprotein Lipase and Carnitine Palmitoyltransferase I
mRNA Expression in Skeletal Muscle Myotubes
Ralston Lockett1, Jac’Quese Hargrove2, Katie Campbell-Jackson3 and Espen E. Spangenburg3:
1 Prairie View A&M University, Prairie View, Tx, 2Tuskegee University, Tuskegee, Al and 3University of Maryland, Department of Kinesiology, College Park, MD
Introduction
Fig 1.A
(C2C12
resuspended
from cryopreservation
C2C12
Decreases in circulating ovarian hormones (i.e. menopause) leads to increases in
visceral fat mass which is a risk factor for cardiovascular disease (CVD) and the
metabolic syndrome. Further, treatment for estrogen positive cancers also results
in increases in visceral fat mass. Thus, it is necessary to understand how
estrogens influence tissue metabolism.
Summary
Fig. 2.A 3well
plates treated
with E2
Since a common anatomical characteristic of post-menopausal women is
increased lipid accumulation it is not unreasonable to suggest estrogens affect
regulators of lipid metabolism.
Currently, it is unclear which metabolic pathways are affected by estrogen
deficiency, but
identifying putative biological markers would help to develop appropriate medical
interventions (Carr 2004).
100mL
100mL
100mL
Thus, we developed the following hypothesis and goal:
Goal
FUTURE STUDIES
Results
The long term goal is develop a reductionist approach that will allow us to identify
Due to the variability in mRNA
expression of our targets, it is
necessary to repeat these
measures to determine if our
model is valid. However, using the
120 hr time point does appear to
be useful due to potential toxic
effects.
novel estrogen sensitive targets in skeletal muscle.
Hypothesis
24hr
Exposing cultured muscle cells to a physiological dose of 17-beta-estradiol (E2) will
increase gene expression associated with lipid metabolism in a time dependent
manner.
Lipoprotein Lipase (LPL): hydrolyzes lipoproteins for energy utilization by the
muscle.
48hr.
120hr.
96hr.
10nM E2
0Hrs. No E2
• There was a significant amount of variability
within our mRNA targets across the time
points. Thus, it is difficult to make strong
scientific conclusions about our cell culture
approach.
• We did determine that 120 hrs of E2 exposure
resulted in myotubes that did not appear
healthy and a loss of 18S expression. This
suggests that this long of E2 exposure
resulted in potential toxic effects on the cells.
• With these experiments we became familiar
with cell culture, RNA isolation, and RT-PCR.
Treatment
(LE2)
Carnitine palmitoyltransferase I (CPT1): transports long-fatty chain fatty acids
through the
outer membrane of the mitochondria for β-oxidation.
50nM E2
Treatment
(HE2)
Methodology
1. C2C12 myoblasts, an immortalized muscle cell line was used in this study.
2. Cryo-preserved C2C12 cells were cultured from liquid-nitrogen suspension
and plated in 100mM plates until they were 85-100% confluent (Figure 1). The
cells were then passed and a 1-10 dilution was used to re-plate the cells in
dulbeccos minimum eagle’s media (DMEM) containing 10% fetal bovine
serum and 1% penicilin/streptomyocin.
3. (2) 6 well plates were constructed from the 100mM stock plates to
differentiate the C2C12 myoblasts into myotubes. Differentiation media
consisted of 2% horse serum and 1%penicillin/streptomyocin in DMEM. The
cells differentiated for 96hrs until myotubes were visible.
4. Each well was treated with 10nM (Low dose) or 50nM (High dose) of E2 for
either 24,48,72,96, or 120 hours (Figure 2). One 6 well plate was used as a
time matched control containing no estradiol. At either 0,24,48,72,96, or 120
hours cells were lysed using Trizol for RNA extraction.
• RNA was extracted using Trizol which permits dissociation of nucleoprotein
complexes. 0.2mL chloroform per 1ml of Trizol was added to the cells and the
solution was spun at 12,000 RCF for 8mins. The 80% upper aqueous phas
was transferred into a new tube and 0.5mL isopropyl alcohol was added. The
tubes were spun again for 8 mins. The subsequent pellet was dried and
suspended in TE.
4. Following RNA extraction, 3µL of RNA sample was diluted in 147µL of TE
buffer to determine the concentration of RNA in each sample using a
spectrophotometer.
5. cDNA was constructed using a reverse transcription kit and 1µg of RNA.
cDNA of LPL and CPT-1 was amplified using a polymerase chain reaction
protocol (PCR) consisting of:
LPL :6XPCR buffer (2.5µL), dNTP(4µL), MgCl2 (1.5µL), LPL Forward (5’
CCAATGGAG GCACTTTCCA 3”)(1µL), LPL Reverse (5’
TGGTCCACGTCTCCGAGT 3’) (1µL), Taq (.2µL), Dh20(13.8µL), cDNA (1uL).
There were 35 cycles and anneal at 53oC
CPT-1: 6XPCR buffer (2.5µL), dNTP(4µL), MgCl2 (1.5µL),0.4µL CPT-1 Forward
(5’GTCGCTTCTTCAAGGTCTGG3’),0.4µL Reverse
(5’AAGAAAGCACGTTCGAT3’), Taq (.2µL), Dh20(13.8µL), cDNA (1uL). There
were 35 cycles and anneal at 53oC.
6. CPT-1 and LPL were separated on 1% agarose gel for 30-45mins and
visualized using a UV computer driven documentation system.
References:
•Ogawa E, Kanazawa M, Yamamoto S, Ohtsuka S, Ogawa A, Ohtake A,
Takayanagi M, Kohno Y (2002). "Expression analysis of two mutations
in carnitine palmitoyltransferase IA deficiency". J. Hum. Genet. 47 (7):
342–7.
Control.
(No E2)
24hrs.
LE2
LPL
CPT-I
18S
HE2
•Carr, Molly (2003)”The Emergence of the Metabolic Syndrome with
Menopause”The Journal of Clinical Endocrinology & Metabolism Vol.
88, No. 6 2404-2411
•Zechner R (1997). "The tissue-specific expression of lipoprotein
lipase: implications for energy and lipoprotein metabolism". Curr.
Opin. Lipidol. 8 (2): 77–88.
•van der Leij FR, Huijkman NC, Boomsma C, Kuipers JR, Bartelds B
(2000). "Genomics of the human carnitine acyltransferase genes". Mol.
Genet. Metab. 71 (1-2): 139–53
48hrs.
Control
LE2
HE2
96hrs.
Control
LE2
HE2
120hrs.
Control
LE2
HE2
Control
Acknowledgements:
•UM-Stars program was
funded by a grant from the
NIH
•Ms Katie Jackson and
Espen’s Lab