Expression and Regulation of Type II Iodothyronine Deiodinase in ...

0021-972X/99/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 1999 by The Endocrine Society

Vol. 84, No. 9 Printed in U.S.A.

Expression and Regulation of Type II Iodothyronine Deiodinase in Cultured Human Skeletal Muscle Cells* YASUHIRO HOSOI, MASAMI MURAKAMI, HARUO MIZUMA, TAKAYUKI OGIWARA, MAKOTO IMAMURA, AND MASATOMO MORI First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371-8511, Japan ABSTRACT T4, which is a major secretory product of the thyroid gland, needs to be converted to T3 by iodothyronine deiodinase to exert its biological activity. After the molecular cloning of human type II iodothyronine deiodinase (DII) complementary DNA, DII expression was unexpectedly detected in human skeletal muscle tissue. In the present study, we have identified DII activity and DII messenger ribonucleic acid (mRNA) in cultured human skeletal muscle cells and studied the mechanisms involved in the regulation of DII expression in those cells. All of the characteristics of the deiodinating activity in cultured human skeletal muscle cells were compatible with those of DII. Northern analysis has demonstrated that DII mRNA, approximately 7.5 kb in size, was expressed in cultured human skeletal muscle cells. DII

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4, WHICH is a major secretory product of the thyroid gland, needs to be converted to T3 by iodothyronine deiodinase to exert its biological activity (1, 2). Two different isozymes have been demonstrated for the iodothyronine deiodinase to catalyze T4 activation (1, 2). Type I iodothyronine deiodinase (DI) is present in thyroid gland, liver, kidney, and many other tissues, whereas type II iodothyronine deiodinase (DII) is present in a limited number of tissues, including brain, anterior pituitary, brown fat, pineal gland, and Harderian gland in the rat (1, 2). DI activity is known to decrease in the hypothyroid state and is believed to have a primary role in maintaining the circulating T3 levels (1, 2). DII activity, in contrast, increases in the hypothyroid state and plays a critical role in providing the local intracellular T3 (1, 2). 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 Received March 8, 1999. Revision received May 6, 1999. Accepted May 21, 1999. 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: [email protected]. * This work was supported in part by Grant-in-Aid 09671024 (to M.Mu.) for scientific research from the Ministry of Education, Science, and Culture, Japan.

mRNA and DII activity were rapidly increased by (Bu)2cAMP, forskolin, or b-adrenergic agonists and were negatively regulated by thyroid hormones in cultured human skeletal muscle cells. Although interleukin-1b and interleukin-6 did not decrease DII expression in cultured human skeletal muscle cells, tumor necrosis factor-a 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 b-adrenergic mechanisms through a cAMP-mediated pathway and is negatively regulated by thyroid hormones and tumor necrosis factor-a. (J Clin Endocrinol Metab 84: 3293–3300, 1999)

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. Materials and Methods Materials [125I]T4, [125I]rT3, and [a-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-a (TNFa), human interleukin-1b (IL-1b), and human IL-6 were obtained from R&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 mg/mL dexamethasone, 50 mg/mL gentamicin, 50 ng/mL amphotericin-B (Takara Shuzo Co.). They were maintained at 37

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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 mmol/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 mL assay buffer/tube and incubated in a total volume of 50 mL 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 FIG. 1. Forskolin and (Bu)2cAMP stimulation of deiodinating activity in cultured HSkMCs. a, HSkMCs were incubated with SkGM medium only (control) or with medium containing forskolin (1025 mol/L) or (Bu)2cAMP (1023 mol/L) for 6 h. Deiodinating activity was measured in the cell sonicates as described in Materials and Methods. The deiodinating activity shown represents the mean 6 SE of three dishes. *, P , 0.05; **, P , 0.01 (compared with control). b, Double reciprocal plot of T4 deiodination by forskolin-stimulated HSkMCs. Incubations were performed for 1 h at 37 C with various concentrations of [125I]T4. Kinetic constants were calculated to be: Km 5 1.43 nmol/L, Vmax 5 71.4 fmol I2 released/mg proteinzh.

FIG. 2. Forskolin and (Bu)2cAMP stimulation of DII mRNA in cultured HSkMCs. a, Northern analysis of DII mRNA using human DII and G3PDH cRNA probe in HSkMCs incubated with SkGM medium only (control), forskolin (1025 mol/L), or (Bu)2cAMP (1023 mol/L) for 6 h. Each lane represents 10 mg total RNA obtained from cells in an individual dish. b, DII mRNA (DII mRNA/G3PDH mRNA ratio) in HSkMCs incubated with SkGM medium only (control), forskolin (1025 mol/ L), or (Bu)2 cAMP (1023 mol/L) for 6 h. The optical density of the DII mRNA band was corrected for G3PDH, and the results were expressed as a percentage of the value obtained for control HSkMCs.

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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 mL ice-cold 2% BSA and 800 mL 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 g-counter. Nonenzymatic deiodination was corrected by subtracting I2 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 I2 released per mg protein/h after multiplication by a factor of 2 to correct random labelling at the equivalent 39- and 59-positions. In some experiments, the incubation mixtures were extracted with 2 vol absolute ethanol after the addition of 5 mL 10 mmol/L T4 and T3 and were analyzed by descending paper chromatography (hexane-tertiary amyl alcohol-2 n ammonia, 1:5:6) (9).

DEIODINASE IN SKELETAL MUSCLE CELLS

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FIG. 3. Time course of stimulation of DII activity and DII mRNA in HSkMCs by forskolin. a, Northern analysis of DII mRNA in HSkMCs incubated with forskolin (1025 mol/L) for various hours. Each lane represents 10 mg total RNA obtained from cells in an individual dish. b, DII activity (closed circle) and DII mRNA (DII mRNA/G3PDH mRNA ratio; open circle) in HSkMCs. The DII activity shown represents the mean of two dishes. For DII mRNA, the optical density of the DII band was corrected for G3PDH, and the results were expressed as a percentage of the value obtained for control HSkMCs (0h).

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 59-GGAACTGACTCAGGAGGCAG-39 (nucleotides 110 –129) and a reverse 59-AGCCAATAGGGCTCTGTTGA-39 (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 59-TGAAGGTCGGAGTCAACGGATTTGGT-39 (nucleotides 71–96) and a reverse 59-CATGTGGGCCATGAGGTCCACCAC-39 (nucleotides 1053–1030) primer (12). The cRNA probes for human DII and human G3PDH were synthesized with [a-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 3 SSC (1 3 SSC 5 150 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 3 SSC, 5 3 Denhart’s solution, and 100 mg/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 3 SSC-0.1% SDS at 25 C for 15 min and twice in 0.1 3 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 270 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.

Results Characteristics and forskolin or (Bu)2cAMP stimulation of iodothyronine deiodinase in HSkMCs

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 cAMPmediated 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 I2 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 (1025 mol/L) and (Bu)2cAMP (1023 mol/L) as

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shown in Fig. 1a. From the double reciprocal plot shown in Fig. 1b, kinetic constants were calculated to be: Km 5 1.43 nmol/L and Vmax 5 71.4 fmol I2 released/mg proteinzh in forskolin-stimulated HSkMCs, where Vmax is the maximum velocity. When [125I]rT3 was used as the substrate, the release of I2 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 I2, T4, and T3, and radioactivity in the I2 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. Identification and forskolin or (Bu)2cAMP stimulation of DII mRNA in HSkMCs

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 (1025 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. Regulation of DII expression in cultured HSkMCs by thyroid hormones

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.

FIG. 4. Effects of thyroid hormones on DII activity and DII mRNA in HSkMCs. a, DII activity in HSkMCs incubated with forskolin (1025 mol/L) only (control) and with forskolin (1025 mol/L) and thyroid hormones for 6 h. Closed circle, T3; open circle, T4; closed square, rT3. The DII activity shown represents the mean of two dishes. b, Northern analysis of DII mRNA in HSkMCs incubated with forskolin (1025 mol/L) only (control) and with forskolin (1025 mol/L) and thyroid hormone for 6 h. Each lane represents 10 mg total RNA obtained from cells in individual dish. c, DII mRNA (DII mRNA/G3PDH mRNA ratio) in HSkMCs incubated with forskolin (1025 mol/L) only (control) and with forskolin (1025 mol/L) and thyroid hormone for 6 h. The optical density of the DII band was corrected for G3PDH, and the results were expressed as a percentage of the value obtained for control HSkMCs. Closed circle, T3; open circle, T4; closed square, rT3.


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Regulation of DII expression in cultured HSkMCs by b-adrenergic agonists

Based on the evidence that b-adrenergic agonists stimulate DII activity in astrocytes (16), pineal gland (20), and Harderian gland (15) in the rat, and b-adrenergic receptors are present in human skeletal muscle tissue (21), we studied the effects of b-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 timecourse study shown in Fig. 6, a and b, ISO (1025 mol/L) rapidly stimulated both DII activity and DII mRNA in cultured HSkMCs. NE (1025 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 b-adrenergic mechanisms are involved in the regulation of DII expression in cultured HSkMCs. Effects of cytokines on DII expression in cultured HSkMCs

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 TNFa for 6 h in a dose-dependent manner as shown in Fig. 7a. However, IL-1b and IL-6 did not show significant effects on DII activity in cultured HSkMCs. TNFa also decreased DII mRNA in cultured HSkMCs in a dose-dependent manner, as shown in Fig. 7, b and c. These results suggest that TNFa negatively regulates DII expression in cultured HSKMCs. Discussion

The present results have clearly demonstrated that iodothyronine 59-deiodinating activity is present in cultured HSkMCs. The characteristics of the deiodinating activity clearly indicate its enzymatic nature. T4 was a better substrate for the deiodinating activity in cultured HSkMCs than rT3. The deiodinating activity had low Km (1.43 nmol/L) for T4 and was not inhibited by 1 mmol/L PTU, but was completely inhibited by 1 mmol/L IOP. In addition, the activity was inhibited when the cells were incubated with thyroid hormones. The deiodinating activity in cultured HSkMCs, therefore, has characteristics compatible with DII (1, 2). Northern analysis using human DII cRNA probe demonstrated hybridization signals approximately 7.5 kb in size in cultured HSkMCs. The size of DII mRNA in HSkMCs is compatible with previous observations (4, 5) of transcripts for DII in human skeletal muscle tissue. Although the presence of DII activity and DII mRNA in human skeletal muscle tissue has been described in the previous study (5), the present results appear to be the first demonstration of the expression of DII in cultured HSkMCs per se.

FIG. 5. Effect of ISO or NE on DII activity and DII mRNA in HSkMCs. a, DII activity in HSkMCs incubated with various concentrations of ISO or NE for 6 h. The DII activity shown represents the mean 6 SE of three dishes. Closed circle, ISO; open circle, NE. *, P , 0.05; **, P , 0.01 (compared with control HSkMCs incubated with SkGM medium only). b, Northern analysis of DII mRNA in HSkMCs incubated with ISO or NE for 6 h. Each lane represents 10 mg total RNA obtained from cells in an individual dish. c, DII mRNA (DII mRNA/G3PDH mRNA ratio) in HSkMCs incubated with ISO or NE for 6 h. The optical density of the DII band was corrected for G3PDH, and the results were expressed as a percentage of the value obtained for control HSkMCs (incubated with SkGM medium only). Closed circle, ISO; open circle, NE.

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FIG. 6. Time course of stimulation of DII activity and DII mRNA in HSkMCs by ISO (1025 mol/L). a, Northern analysis of DII mRNA in HSkMCs incubated with ISO (1025 mol/L) for various hours. Each lane represents 10 mg total RNA obtained from cells in an individual dish. b, DII activity (closed circle) and DII mRNA (DII mRNA/G3PDH mRNA ratio; open circle) in HSkMCs incubated with ISO (1025 mol/L) for various time periods. DII activity shown represents the mean of two dishes. The optical density of the DII band was corrected for G3PDH, and the results were expressed as a percentage of the value obtained for control HSkMCs (0h).

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 b-adrenergic mechanisms, and human skeletal

muscle has been shown to express b-adrenergic receptors (21), we studied the possible effects of b-adrenergic agonists on DII expression in cultured HSkMCs. Both DII activity and DII mRNA in HSkMCs were significantly stimulated by b-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 b-adrenergic mechanisms at the pretranslational level. It appears of interest to study whether b-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 TNFa to the incubation medium in a dose-dependent manner. However, IL-1b and IL-6 did not show significant effects on DII expression in HSkMCs. Recently, it has came to the attention of the investigators that TNFa 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 TNFa, 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 b-adrenergic mechanisms, presumably through the cAMP-mediated pathway, and is negatively regulated by thyroid hormones and TNFa. The physiological roles of the expression and the regulation


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of DII in human skeletal muscle remain to be elucidated in further studies. Acknowledgments The authors are indebted to Drs. Tadashi Morimura, Osamu Araki, and Yuji Kamiya for useful discussion.

References

FIG. 7. Effects of cytokines on DII activity and DII mRNA in HSkMCs. a, DII activity in HSkMCs incubated with forskolin (1025 mol/L) only (control) and with forskolin (1025 mol/L) and cytokines for 6 h. The DII activity shown represents the mean 6 SE of three dishes. Closed circle, TNFa; open circle, IL-1b; closed square, IL-6. *, P , 0.05; **, P , 0.01 (compared with control). b, Northern analysis of DII mRNA in HSkMCs incubated with forskolin (1025 mol/L) and various concentrations of TNFa for 6 h. Each lane represents 10 mg total RNA obtained from cells in an individual dish. c, DII mRNA (DII mRNA/ G3PDH mRNA ratio) in HSkMCs incubated with forskolin (1025 mol/L) and various concentrations of TNFa for 6 h. The optical density of the DII band was corrected for G3PDH, and the results were expressed as a percentage of the value obtained for control HSkMCs.

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