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Original Studies |
First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan
Address all correspondence and requests for reprints to: Masami Murakami, M.D., First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan. E-mail: mmurakam{at}sb.gunma-u.ac.jp
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Abstract |
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decreased DII expression in those cells in a
dose-dependent manner. These data have demonstrated, for the first
time, that DII activity and DII mRNA are present in cultured human
skeletal muscle cells, and that the DII expression is stimulated by
ß-adrenergic mechanisms through a cAMP-mediated pathway and is
negatively regulated by thyroid hormones and tumor necrosis factor-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, a complementary DNA (cDNA) encoding DII was cloned from Rana catesbeiana tissues (3), and its mammalian counterpart was subsequently isolated from rat brown fat (4). In humans, DII messenger ribonucleic acid (mRNA) was unexpectedly detected in thyroid gland, skeletal muscle, and heart, suggesting previously unrecognized roles of DII in those tissues (4, 5). Although the expression of DII in human skeletal muscle tissue has been described (5), it is not known whether DII is expressed in cultured human skeletal muscle cells per se. In addition to the possible physiological roles for local intracellular T3 production by DII in skeletal muscle, T3 production by DII in human skeletal muscle has been suggested to contribute to the circulating T3 level, considering the large tissue volume of skeletal muscle (5). Therefore, it seems of importance to study the mechanisms involved in the regulation of DII in human skeletal muscle tissue.
In the present study, we have identified and characterized DII activity and DII mRNA in cultured human skeletal muscle cells and studied the mechanisms involved in the regulation of its expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[125I]T4,
[125I]rT3, and [-32P]UTP
were purchased from New England Nuclear Corp. (Boston, MA). LH-20 was
obtained from Pharmacia Biotech (Uppsala, Sweden). AG
50W-X2 resin and the protein assay kit were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Human tumor necrosis
factor- (TNF), human interleukin-1ß (IL-1ß), and human IL-6
were obtained from R\|[amp ]\|D Systems (Minneapolis, MN). All
other chemicals of the highest quality were obtained from Sigma Chemical Co. (St. Louis, MO) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless otherwise indicated.
Cell culture
Human skeletal muscle cells (HSkMCs) were obtained from Takara Shuzo Co. (Otsu, Japan). They were collected from nondiseased human skeletal muscle tissue, and their purity was tested by immunostaining with antisarcomeric myosin and morphological observation. HSkMCs were cultured in serum-free SkGM medium containing 10 ng/mL human epidermal growth factor, 0.1 mg/mL insulin, 0.5 mg/mL BSA, 0.5 mg/mL fetuin, 0.39 µg/mL dexamethasone, 50 µg/mL gentamicin, 50 ng/mL amphotericin-B (Takara Shuzo Co.). They were maintained at 37 C in the humidified atmosphere of 5% CO2 and 95% air, and the culture medium was changed every 2 days. HSkMCs at the second to the sixth passage were used for the following experiments. Cells were grown to semiconfluence in SkGM medium in 60-mm plastic culture dishes (Becton Dickinson and Co., Franklin Lakes, NJ) and then incubated in the same medium containing the compounds to be tested for the periods indicated. When adrenergic agonists were used, 15 µmol/L ascorbic acid was added to the incubation medium.
Measurement of iodothyronine deiodinase activity
Iodothyronine deiodinase activity was measured as previously
described (6) with minor modifications (7). Briefly, HSkMCs were washed
twice with phosphate-buffered saline, scraped off the dish, and
transferred into 1.5 mL ice-cold assay buffer [100 mmol/L potassium
phosphate, pH 7.0, containing 1 mmol/L ethylenediamine tetraacetate,
and 20 mmol/L dithiothreitol (DTT)]. After centrifugation at 3000 rpm
for 10 min at 4 C, the resultant precipitates were sonicated in 100
µL assay buffer/tube and incubated in a total volume of 50 µL with
2 nmol/L or the indicated amount of [125I]T4
or [125I]rT3, which were purified using LH-20
column chromatography on the day of experiment in the presence or
absence of 1 mmol/L 6-propyl-2-thiouracil (PTU) or in the presence
of 1 mmol/L iopanoic acid (IOP) for the indicated periods at the
indicated temperature in duplicate. After characterization of the
deiodinating activity in cultured HSkMCs, the sonicates were routinely
incubated with 2 nmol/L [125I]T4 in the
presence of 1 mmol/L PTU at 37 C for 1 h. The reaction was
terminated by adding 100 µL ice-cold 2% BSA and 800 µL ice-cold
10% trichloroacetic acid. After centrifugation at 3000 rpm for 10 min
at 4 C, the supernatant was applied to a small column packed with AG
50W-X2 resin (bed volume, 1 mL) and then eluted with 2 mL 10% glacial
acetic acid (column method). Separated 125I was counted
with a 
-counter. Nonenzymatic deiodination was corrected by
subtracting I- released in control tubes without cell
sonicates. The protein concentration was determined by Bradford’s
method using BSA as a standard (8). The deiodinating activity was
calculated as femtomoles of I- released per mg protein/h
after multiplication by a factor of 2 to correct random labelling at
the equivalent 3'- and 5'-positions. In some experiments, the
incubation mixtures were extracted with 2 vol absolute ethanol after
the addition of 5 µL 10 µmol/L T4 and T3
and were analyzed by descending paper chromatography (hexane-tertiary
amyl alcohol-2 N ammonia, 1:5:6) (9).
Preparation of complementary RNA (cRNA) probes
As the expression of DII mRNA has been demonstrated in human
thyroid (10), total RNA was extracted from the thyroid tissue obtained
from a patient with Graves’ disease at the time of surgery by a
modified acid guanidinium thiocyanate-phenol-chloroform method
according to Chomczynski and Sacchi (11). Total RNA was reverse
transcribed, and human DII cDNA fragment containing residues 110-1051
(numbering of residues as GenBank accession no. U53506) was amplified
by PCR using a forward 5'-GGAACTGACTCAGGAGGCAG-3' (nucleotides
110–129) and a reverse 5'-AGCCAATAGGGCTCTGTTGA-3' (nucleotides
1051–1032) primer (Perkin Elmer Corp. PE Applied Biosystems, Foster City, CA) (4). The PCR product was
fractionated on agarose gel and subsequently cloned into pCRII-TOPO TA
cloning vector (Invitrogen, San Diego, CA). Sequencing
analysis (Perkin Elmer Corp. PE Applied Biosystems) confirmed the identity of the amplified DNA. Human
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was cloned as
described above, except that its fragment containing residues 71–1053
was amplified by the PCR using a forward
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (nucleotides 71–96) and a reverse
5'-CATGTGGGCCATGAGGTCCACCAC-3' (nucleotides 1053–1030) primer (12).
The cRNA probes for human DII and human G3PDH were synthesized with
[-32P]UTP and SP6 RNA polymerase or T7 RNA polymerase,
respectively.
RNA preparation and Northern analysis
Total RNA was isolated from each dish, and Northern analysis was performed as previously described (13, 14). Ten micrograms of total RNA per lane were electrophoresed on a 1.4% agarose gel containing 0.66 mol/L formaldehyde and transferred overnight in 20 x SSC (1 x SSC = 150 mmol/L sodium chloride and 15 mmol/L trisodium citrate) to a nylon membrane (Biodyne, Pall BioSupport Corp., East Hills, NY). RNA was cross-linked to the nylon membrane with a UV Stratalinker (Stratagene, San Diego, CA). The membrane was prehybridized with the hybridization buffer (50% formamide, 0.2% SDS, 5% dextran sulfate, 50 mmol/L HEPES, 5 x SSC, 5 x Denhart’s solution, and 100 µg/mL denatured salmon sperm DNA) at 68 C for 2 h. Subsequently, the membrane was hybridized at 68 C overnight with the hybridization buffer containing a human DII cRNA probe. The membrane was washed twice in 2 x SSC-0.1% SDS at 25 C for 15 min and twice in 0.1 x SSC-0.1% SDS at 68 C for 1 h. Autoradiography was established by exposing the filters for 7–14 days to x-ray film (XAR-2, Eastman Kodak Co., Rochester, NY) at -70 C. After the detection of DII mRNA, the probe was stripped off, and blots were rehybridized with human G3PDH cRNA probe as a control. Hybridization and washing were performed as described above, and the membrane was exposed for 1 h. mRNA levels were quantitated by densitometry using NIH Image version 1.61, and the optical density of the DII band 7.5 kb in length was corrected for G3PDH RNA samples for comparison were analyzed on the same blot (15).
Statistics
Statistical differences were evaluated by Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the preliminary experiments, the basal deiodinating activity
was found to be low in the cultured HSkMCs used in the present study.
Because the DII activity in the rat astrocytes and pineal gland were
stimulated through a cAMP-mediated pathway (16, 17), and the presence
of adenylate cyclase systems was described in human skeletal muscle
tissue (18), the effects of forskolin and (Bu)2cAMP on the
deiodinating activity in HSkMCs were studied. The HSkMCs were incubated
with forskolin or (Bu)2cAMP for 6 h, and the
deiodinating activity was measured by the release of I-
from 2 nmol/L [125I]T4 in the presence of 20
mmol/L DTT and 1 mmol/L PTU using the column method. The deiodinating
activity in cultured HSkMCs was significantly stimulated by both
forskolin (10-5 mol/L) and (Bu)2cAMP
(10-3 mol/L) as shown in Fig. 1a
. From the double reciprocal plot shown
in Fig. 1b, kinetic constants were calculated to be:
Km = 1.43 nmol/L and Vmax =
71.4 fmol I- released/mg protein·h in
forskolin-stimulated HSkMCs, where Vmax is the maximum
velocity. When [125I]rT3 was used as the
substrate, the release of I- was approximately one third
of that from [125I]T4, indicating that
T4 is the preferred substrate for the iodothyronine
deiodinase in cultured HSkMCs. The T4 deiodination was
dependent on the protein concentration of HSkMCs and an incubation
period of up to 2 h. Incubation at 4 C or preheating the cell
sonicate at 56 C for 30 min completely abolished the deiodination. The
deiodinating activity was not influenced by 1 mmol/L PTU, but was
completely inhibited by 1 mmol/L IOP. When sonicates of HSkMCs were
incubated with 2 nmol/L [125I]T4 in the
presence of 20 mmol/L DTT and 1 mmol/L PTU, and the reaction products
were subsequently analyzed by descending paper chromatography, there
were only three definable peaks corresponding to I-,
T4, and T3, and radioactivity in the
I- peak was comparable to that in the T3 peak.
These results indicate that the characteristics of the deiodinating
activity in HSkMCs are compatible with DII and its activity is
stimulated through the cAMP-mediated pathway.
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Northern analysis of total RNA extracted from cultured HSkMCs
using human DII cRNA probe demonstrated the hybridization signal with
an approximately 7.5 kb in size, which is compatible with the size of
DII mRNA described in the previous studies (4, 5). DII mRNA was clearly
increased by treatment with forskolin or (Bu)2cAMP for
6 h, as shown in Fig. 2, a and b. In
the time-course study shown in Fig. 3, a
and b, both DII activity and DII mRNA were increased by forskolin
(10-5 mol/L) within 3 h and reached peak levels at
6 h. The rapid increase in DII mRNA by the cAMP-elevating agent is
in agreement with the results obtained using cultured rat astrocytes
and cultured rat pineal gland (14, 19). These results indicate that DII
mRNA as well as DII activity are stimulated through the cAMP-mediated
pathway in cultured HSkMCs.
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Addition of thyroid hormones to the incubation medium for 6 h
decreased the deiodinating activity in forskolin-stimulated HSkMCs, and
the potency of the inhibitory effect was
T4>rT3>T3, as shown in Fig. 4a. The inhibition of deiodinating
activity in HSkMCs by thyroid hormones further supports the
presence of authentic DII activity in cultured HSkMCs. Although DII
mRNA in HSkMCs was also inhibited by thyroid hormones, the potency of
the inhibitory effect was
T3>T4>rT3, as shown in Fig. 4, b
and c. These results suggest that both pretranslational and
posttranslational mechanisms are involved in the suppression of DII
expression in HSkMCs by thyroid hormones.
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Based on the evidence that ß-adrenergic agonists stimulate DII
activity in astrocytes (16), pineal gland (20), and Harderian gland
(15) in the rat, and ß-adrenergic receptors are present in human
skeletal muscle tissue (21), we studied the effects of ß-adrenergic
agonists on DII expression in cultured HSkMCs. Incubation with
isoproterenol (ISO) or norepinephrine (NE) for 6 h resulted in
stimulation of DII activity in HSkMCs in a dose-dependent manner, and
the potency of stimulation was ISO>NE, as shown in Fig. 5a. DII mRNA in HSkMCs was also
stimulated by ISO or NE in a dose-dependent manner, as shown in Fig. 5, b and c. In the time-course study shown in Fig. 6, a and b, ISO (10-5 mol/L)
rapidly stimulated both DII activity and DII mRNA in cultured HSkMCs.
NE (10-5 mol/L) also stimulated both DII activity and DII
mRNA in cultured HSkMCs with a similar time course (data not shown).
These results suggest that ß-adrenergic mechanisms are involved in
the regulation of DII expression in cultured HSkMCs.
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Because the modulation of thyroid hormone metabolism, including
the inhibition of DI expression by various cytokines, has been reported
(22, 23, 24), the possible effects of cytokines on DII expression in
HSkMCs were also examined in the present study. Forskolin-stimulated
DII activity in cultured HSkMCs was decreased by the addition of TNF
for 6 h in a dose-dependent manner as shown in Fig. 7a. However, IL-1ß and IL-6 did not
show significant effects on DII activity in cultured HSkMCs. TNF
also decreased DII mRNA in cultured HSkMCs in a dose-dependent manner,
as shown in Fig. 7, b and c. These results suggest that TNF
negatively regulates DII expression in cultured HSKMCs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, T4 and rT3 were demonstrated to be more potent than T3 to inhibit DII activity in HSkMCs, indicating that the posttranslational mechanisms are involved in the regulation of DII in HSkMCs by thyroid hormones. These results are compatible with the previous observations, suggesting that posttranslational mechanisms are involved in the regulation of cerebrocortical and adenohypophyseal DII activity by thyroid hormones in the rat (25, 26). However, T3 was more potent than T4 to inhibit DII mRNA in HSkMCs, suggesting that pretranslational mechanisms are also involved in the regulation of DII expression in HSkMCs. This speculation is supported by the recent observation (27) suggesting the presence of both pretranslational and posttranslational mechanisms in the regulation of DII by thyroid hormones in the rat cerebral cortex.
Forskolin and (Bu)2cAMP stimulated DII activity and DII mRNA in cultured HSkMCs in the present study, suggesting that cAMP-related mechanisms are involved in the regulation of DII expression in HSkMCs. In the rat, cAMP-related mechanisms have been reported to be involved in the regulation of DII activity in astrocytes, brown adipocytes, and pineal gland (16, 17, 28) and DII mRNA in astrocytes (19) and pineal gland (14). The rapid induction of DII mRNA in cultured HSkMCs by forskolin or (Bu)2cAMP suggests the pretranslational regulation of DII expression in HSkMCs by cAMP-mediated mechanisms.
Because DII in rat astrocytes (16), pineal gland (14, 17, 20), and Harderian gland (15) has been demonstrated to be regulated by ß-adrenergic mechanisms, and human skeletal muscle has been shown to express ß-adrenergic receptors (21), we studied the possible effects of ß-adrenergic agonists on DII expression in cultured HSkMCs. Both DII activity and DII mRNA in HSkMCs were significantly stimulated by ß-adrenergic agonists, presumably through a cAMP-mediated pathway as suggested for DII in the rat pineal gland (14, 17). These results indicate that DII expression in HSkMCs is regulated at least in part by ß-adrenergic mechanisms at the pretranslational level. It appears of interest to study whether ß-adrenergic regulation of DII expression in human skeletal muscle may contribute to the circulating T3 level, considering the large tissue volume of skeletal muscle (5).
In the present study, DII activity and DII mRNA in HSkMCs were
decreased by the addition of TNF to the incubation medium in a
dose-dependent manner. However, IL-1ß and IL-6 did not show
significant effects on DII expression in HSkMCs. Recently, it has came
to the attention of the investigators that TNF plays a pivotal role
in the mechanisms of insulin resistance in the skeletal muscle (29).
The physiological significance of the reduction of DII expression by
TNF, including the effect on insulin action on skeletal muscle,
requires further studies.
In summary, the present results have demonstrated, for the first time,
that DII is expressed in cultured HSkMCs, and that DII expression is
regulated by ß-adrenergic mechanisms, presumably through the
cAMP-mediated pathway, and is negatively regulated by thyroid hormones
and TNF. The physiological roles of the expression and the
regulation of DII in human skeletal muscle remain to be elucidated in
further studies.
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Acknowledgments |
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Footnotes |
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Received March 8, 1999.
Revised May 6, 1999.
Accepted May 21, 1999.
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References |
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-adrenergic agonists and that
its adrenergic-stimulated and spontaneous rhythmic nocturnal rise
require RNA and protein synthesis. Proc Soc Exp Biol Med. 190:190–194.[CrossRef][Medline]
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interleukin-6 on type I iodothyronine 5'-deiodination in rat thyroid
cell line, FRTL-5. J Interferon Cytokine Res. 15:367–375.[Medline]
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