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Original Studies |
Expression in Human Skeletal Muscle1
Metabolism and Diabetes Research Group, The Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia
Address all correspondence and requests for reprints to: Dr. Naras Lapsys, Metabolism and Diabetes Research Group, The Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia. E-mail: n.lapsys{at}garvan.unsw.edu.au
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Abstract |
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(PPAR-) activation in
adipose tissue is known to regulate genes involved in adipocyte
differentiation and lipid metabolism. However, the role of PPAR- in
muscle remains unclear. To examine the potential regulation of genes by
PPAR- in human skeletal muscle, we used semiquantitative RT-PCR to
determine the expression of PPAR-, lipoprotein lipase (LPL), muscle
carnitine palmitoyl transferase-1 (mCPT1), fatty acid-binding protein
(FABP), carnitine acylcarnitine transferase (CACT), and glucose
transporter-4 (GLUT4) in freeze-dried muscle samples from 14 male
subjects. These samples were dissected free of adipose and other tissue
contamination, as confirmed by minimal or absent adipsin expression.
Between individuals, the messenger ribonucleic acid concentration of
PPAR- varied up to 3-fold, whereas LPL varied up to 6.5-fold, mCPT1
13-fold, FABP 4-fold, CACT 4-fold, and GLUT4 up to 3-fold. The
expression of LPL (r2 = 0.54; P =
0.003), mCPT1 (r2 = 0.42; P =
0.012), and FABP (r2 = 0.324; P =
0.034) all correlated significantly with PPAR- expression in the
same samples. No significant correlation was observed between the
expression of CACT and PPAR- or between GLUT4 and PPAR-. These
findings demonstrate a relationship between PPAR- expression and the
expression of other genes of lipid metabolism in muscle and support the
hypothesis that PPAR- activators such as the antidiabetic
thiazolidinediones may regulate fatty acid metabolism in skeletal
muscle as well as in adipose tissue. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PPAR-) is a member of the nuclear hormone receptor superfamily
of ligand-dependent transcription factors. Three isoforms of PPAR-
(1, 2, and 3) have been identified in humans, and their
distribution is tissue specific (1, 2, 3). PPAR- receptors
are most abundantly expressed in adipose tissue and are thought to play
a role in adipocyte differentiation, lipid metabolism, and insulin
action through coordinate effects on gene transcription (2, 4, 5, 6).
Recent studies indicate that thiazolidinediones (TZDs), a new class of
antidiabetic drugs known to bind and activate PPAR-, enhance insulin
sensitivity in humans with insulin resistance (7, 8). Although the precise mechanism of action of these drugs
remains unclear, recent literature reports the ability of TZDs to
influence the expression of regulatory genes involved in adipocyte
differentiation and proliferation, presumably via PPAR- activation.
In rodent adipose tissue and adipocyte cell cultures, the
administration of TZDs results in increased expression of fatty acid
transport protein (FATP), acyl-coenzyme A synthetase (ACS)
(9), and lipoprotein lipase (LPL) (10), all
of which contain functional PPAR response elements. It has been
suggested that by stimulating the differentiation of adipocytes and
increasing the sequestration of lipid into fat cells, lipid is less
available in muscle and other tissues, where its accumulation and
metabolism might otherwise contribute to insulin resistance (11, 12). Although the expression of PPAR- has been reported in
skeletal muscle (2, 13, 14), little is known about the
potential effects of PPAR- on gene regulation in this tissue.
Furthermore, it has been suggested that accurate determination of
PPAR- expression in skeletal muscle is not possible due to
contamination of samples with adipose tissue, in which the expression
of PPAR- is manyfold greater (14, 15).
We have examined the messenger ribonucleic acid (mRNA) concentration of
PPAR- and other genes that might influence im lipids in freeze-dried
human skeletal muscle dissected free of adipose tissue contamination.
In this report we show that PPAR- is expressed in human skeletal
muscle and that the expression of three genes important in lipid
metabolism, LPL, muscle carnitine palmitoyl transferase-1 (mCPT1), and
fatty acid-binding protein (FABP) correlates with the expression of
PPAR- in these muscle samples. These data provide supportive
evidence that PPAR- might contribute to the antidiabetic effects of
TZDs by regulating lipid metabolism genes in skeletal muscle as well as
in adipose tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The protocol was approved by the St. Vincent’s Hospital human research ethics Committee. Fourteen healthy volunteers were selected on the basis that they were scheduled to undergo elective knee surgery. Volunteers were excluded if they had a history of cardiovascular disease or if they had clinically significant hepatic, renal, or hematological disease based on routine laboratory panels obtained during the screening visit.
Written informed consent was obtained after all procedures were
explained to each subject. The subjects were elderly men (aged
68.1 ± 1.3 yr) and, as a group, slightly overweight. They
represent the range of insulin sensitivities and body composition
expected from a normal aging population. The clinical characteristics
of the subjects are presented in Table 1
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Elective knee surgery was performed at St. Vincent’s Hospital
(Darlinghurst, Sydney, Australia). After an overnight fast, general
anesthesia was induced by a short-acting barbiturate and was maintained
by alfentanil-hydrochloride. A tissue biopsy (
1 g) of the vastus
lateralis muscle was taken at the beginning of the surgical procedure,
avoiding measures that might create ischemia. The biopsies were divided
into aliquots, snap-frozen in liquid nitrogen, and stored at -80 C
until required.
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Approximately 50 mg wet weight vastus lateralis skeletal muscle was freeze-dried under vacuum for 24 h. After freeze-drying, the muscle sample was viewed under a microscope (x6.3 magnification) at room temperature for careful dissection and removal of all traces of adipose tissue, connective tissue, and blood contaminants. On the average, this preparation yielded approximately 10 mg dry weight dissected skeletal muscle.
RNA isolation, extraction, and quantitation
Total RNA was extracted and purified from approximately 10 mg freeze-dried and dissected muscle using a guanidinium thiocyanate-phenol technique (Tri-Reagent, Sigma, St. Louis, MO), according to the manufacturer’s instructions. After extraction, RNA samples were treated with ribonuclease-free deoxyribonuclease (Promega Corp., Madison, WI). Total RNA yields were determined and standardized to 0.2 mg/mL by correlation to a standard RNA concentration curve, using a SYBR Green II (Molecular Probes, Inc., Eugene, OR) RNA standard assay (16).
Semiquantitative RT-PCR
First strand complementary DNA was generated from 0.2 µg RNA
in a 10-µL volume using random hexamers as primers (SuperScript,
Life Technologies, Inc., Gaithersburg, MD). One microliter
of the RT reaction mix was amplified with primers specific for human
PPAR-, LPL, mCPT1, FABP, carnitine acyl carnitine transferase
(CACT), glucose transporter-4 (GLUT4), glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), and adipsin. PPAR- mRNA expression in human
skeletal muscle was studied using PCR primer pairs designed by
Vidal-Puig et al. (13), which amplified a
region common to both PPAR-1 and PPAR-2. All primer sequences and
PCR conditions are shown in Table 2. The
linearity of the PCR was tested by amplification of 1 µL of the RT
reaction from 25–40 cycles. The linear range was between 25–38
cycles. The samples were amplified with AmpliTaq Gold
(Perkin-Elmer Corp.-Cetus, Palo Alto, CA) for 25–38
cycles after an initial activation of 93 C for 10 min, using the
following parameters: 93 C for 30 s, 50–60 C for 30 s (see
Table 2), and 72 C for 30 s. GAPDH was amplified as a control
gene. Controls for genomic DNA contamination were included. To test for
the presence of fat contamination in freeze-dried and dissected muscle
biopsy samples, the abundance of adipsin mRNA was determined by PCR
amplification using the same or a greater number of cycles required to
amplify target genes. The same cycle number detected adipsin mRNA
expression from control human adipose tissue preparations. The
amplified products were size fractionated on a 2.5% agarose gel and
stained with 0.5 µg/mL ethidium bromide. The agarose gel was
visualized under UV light, and the image was captured using the
Molecular Analyst software package on the Gel Doc 1000 gel
documentation image system (Bio-Rad Laboratories, Inc.,
Hercules, CA). The densities of the PCR products were measured using
NIH Image software (version 1.61). Levels of mRNA were expressed as the
ratio of signal intensity for the target genes relative to that for
GAPDH. Median densities from 4–8 separate PCR determinations from
duplicate RT reactions were used to construct the correlations.
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Subject characteristics are expressed as the mean ± SE (unless specifically noted). Coexpression of genes was assessed by simple linear regression analyses using StatView 4.51 (Abacus Concepts, Inc., Berkeley, CA); P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, LPL, mCPT1, FABP,
CACT, GLUT4, and GAPDH in human skeletal muscle. Figure 1 shows representative experiments from 6
of the 14 subjects, demonstrating the range of variation in mRNA
concentrations that were observed between individuals. Overall, we
observed up to 3-fold variation in PPAR- mRNA expression, 6.5-fold
variation in LPL, 13-fold variation in mCPT1, 3-fold variation in
GLUT4, and up to 4-fold variation in both FABP and CACT mRNA
expression. Figure 1 also demonstrates very low or no adipsin mRNA
expression in the muscle samples when amplified at the same or a
greater number of PCR cycles as that required to amplify the target
genes. This suggests that the PCR products we measured reflected mRNA
expression in skeletal muscle only.
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, which could
imply potential regulation by PPAR-, the expression of all genes of
interest was first corrected for the expression of GAPDH. We then
compared the mRNA expression of our target genes to the PPAR- mRNA
expression. The PPAR- mRNA expression was significantly correlated
with the mRNA expression of LPL (r2 = 0.54;
P = 0.003, Fig. 2a), mCPT1
(r2 = 0.42; P = 0.012; Fig. 2b),
and FABP (r2 = 0.324; P = 0.034;
Fig. 2c). No significant correlation was observed between the mRNA
expression of PPAR- and that of CACT (r2 =
0.182 P = 0.129), and no significant correlation was
observed between PPAR- and GLUT4 (r2 = 0.106;
P = 0.256), a gene expressed in muscle but not expected
to be regulated by PPAR-.
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expression in skeletal muscle, the relationship between mRNA expression
of PPAR- and the clinical characteristics of the subjects were
investigated. Simple linear regression analysis showed no significant
correlation between age, body mass index (BMI), plasma glucose, and
plasma insulin and plasma lipids (nonesterified fatty acids and
triglycerides) and PPAR- mRNA expression in the subject group (data
not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in low amounts under basal conditions, and PPAR-1 is
thought to be the only or the predominant isoform present in this
tissue (1, 13). Despite the low abundance of PPAR- in
human skeletal muscle (<15% of that in adipose tissue)
(17), there have been a number of studies focusing on the
expression of PPAR- in this tissue because of the central role of
skeletal muscle in the development of insulin resistance in obese and
type 2 diabetic subjects. Several recent reports suggest that the
evaluation of PPAR- expression in samples of human skeletal muscle
is compromised by possible contamination of the muscle by adipose
tissue (15). This problem can be overcome to some extent
by the use of primary human skeletal muscle myoblast cultures
(14) or by monitoring the extent of adipose tissue
contamination by measuring the expression of adipocyte-specific genes,
such as adipocyte protein 2 or adipsin (1, 13, 17). We
applied a different approach by freeze-drying skeletal muscle samples
and microscopically dissecting away any adipose tissue and connective
tissue contamination. Thus, the variations in mRNA expression we report
here for PPAR-, LPL, mCPT1, FABP, CACT, and GLUT4 probably reflect
differential expression in skeletal muscle only. The lack of adipsin
expression in dissected muscle highlights the lack of adipocyte
contamination in muscle biopsy samples treated in this way.
The observation that human skeletal muscle only expresses low amounts
of PPAR- raises the question of whether PPAR- in muscle has any
role in improving insulin sensitivity after TZD treatment. In rodents,
one study reported that TZD administration resulted in a small
induction of FATP mRNA expression and an induction of ACS mRNA
expression in skeletal muscle (9), suggesting a regulatory
role for PPAR- in this tissue. We have demonstrated in human
skeletal muscle a significant correlation of PPAR- expression with
LPL and FABP expression. Furthermore, we have demonstrated that
PPAR- expression correlated significantly with the expression of
mCPT1, an essential gene in the fatty acid ß-oxidation pathway.
Recently, a transcriptionally active PPAR response element has been
identified upstream of mCPT1 (18). This provides
supportive evidence that this gene may be a target for the action of
PPAR-. Overall, these findings imply that PPAR- may play a role
in the regulation of lipid metabolism genes in skeletal muscle by
up-regulating the amounts of LPL, FABP, and mCPT1 in a coordinated
fashion, which would serve to increase the oxidation of fatty acids in
muscle. Although an increase in fatty acid oxidation in this tissue
would seem to be contradictory to an improvement of insulin action
(19), it is possible that such a change in metabolism
would eventually lead to decreased lipid availability in muscle,
particularly if PPAR--mediated actions were partitioning lipid into
adipose tissue and reducing circulating lipid availability at the same
time. The time required for the beneficial action of the TZDs to
develop in humans (1–3 weeks) (8) suggests that there is
considerable adaptation in metabolism required for the PPAR-
activators to exert their effects.
In adipose tissue, regulation of PPAR- mRNA expression has shown to
be influenced by insulin and obesity (13, 20, 21). Similar
investigations with respect to skeletal muscle PPAR- expression have
been less conclusive. Significant positive correlations have been
reported between skeletal muscle PPAR- mRNA levels and BMI and
between PPAR- and fasting insulin concentrations using obese and
type 2 diabetic and lean nondiabetic control subjects
(14). However, other investigations have shown that
skeletal muscle PPAR- mRNA expression does not differ between normal
and type 2 diabetic subjects, and that PPAR- expression is not
induced by short-term hyperinsulinemia (17). In our study
skeletal muscle PPAR- mRNA concentrations varied up to 3-fold;
however, these variations did not correlate with BMI, plasma insulin,
or plasma fatty acids in our subject group. Thus, it could be argued
that in this population, these metabolic factors are not determinants
of increased PPAR- and other lipid metabolism gene mRNA levels.
However, it is possible that PPAR- and the other measured
transcripts are being influenced by another unknown or unmeasured
factor.
In summary, we identified variation in mRNA expression of PPAR-,
LPL, mCPT1, FABP, CACT, and GLUT4 in human skeletal muscle and report
that the expression of LPL, mCPT1, and FABP is positively correlated
with PPAR- mRNA expression in this tissue. These data would be
consistent with a hypothesis that TZDs may in part improve insulin
action directly in skeletal muscle by stimulating the expression and
action of PPAR- and modulating fatty acid metabolism in muscle
(22). The implications of these findings for therapeutic
agents in the treatment of diabetes and obesity warrant further
investigation.
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Footnotes |
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2 N.M.L. and A.D.K. were equally involved in this study and should be
regarded as dual first authors.
Received April 12, 2000.
Revised July 11, 2000.
Accepted August 9, 2000.
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References |
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2 versus PPAR1 and activation with retinoid X receptor
agonists and antagonists. J Biol Chem. 272:8071–8076.
gene. J Biol Chem. 272:18779–18789.3
mRNA: a distinct PPAR mRNA subtype transcribed from an independent
promoter. FEBS Lett. 438:55–60.[CrossRef][Medline]
and PPAR
activators. J Biol Chem. 272:28210–28217. and PPAR activators direct a distinct
tissue-specific transcriptional response via a PPRE in the lipoprotein
lipase gene. EMBO J. 15:5336–5348.[Medline]
gene expression is elevated
in skeletal muscle of obese and type II diabetic subjects. Diabetes. 46:1230–1234.[Abstract]
in human adipocytes. Diabetes. 48:699–705.[Abstract]
expression in obesity and non- insulin-dependent diabetes
mellitus. J Clin Invest. 101:543–548.[Medline]
gene expression by nutrition and obesity in
rodents. J Clin Invest. 97:2553–2561.[Medline]
. J Clin Endocrinol Metab. 83:2830–2835.This article has been cited by other articles:
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