THIAZOLIDINEDIONE INDUCES THE ADIPOSE DIFFERENTIATION OF FIBROBLAST-LIKE CELLS RESIDENT WITHIN BOVINE SKELETAL MUSCLE

THIAZOLIDINEDIONE INDUCES THE ADIPOSE DIFFERENTIATION OF FIBROBLAST-LIKE CELLS RESIDENT WITHIN BOVINE SKELETAL MUSCLE

Cell Biology International 1998, Vol. 22, No. 6, 421–427 Article No. cb980270, available online at http://www.idealibrary.com on THIAZOLIDINEDIONE IN...

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Cell Biology International 1998, Vol. 22, No. 6, 421–427 Article No. cb980270, available online at http://www.idealibrary.com on

THIAZOLIDINEDIONE INDUCES THE ADIPOSE DIFFERENTIATION OF FIBROBLAST-LIKE CELLS RESIDENT WITHIN BOVINE SKELETAL MUSCLE SHIN-ICHIRO TORII1*, TERUO KAWADA2, KYOKO MATSUDA1, TOHRU MATSUI1, TOMOMI ISHIHARA3 and HIDEO YANO1 Laboratory of Animal Nutrition and 2Laboratory of Nutrition Chemistry, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 Japan; 3Lead Optimization Research Laboratory, Tanabe Seiyaku Co. Ltd, Toda, Saitama 335-0015, Japan 1

Received 4 February 1998; accepted 3 June 1998

To investigate the role of peroxisome proliferator-activated receptor ã (PPARã) in adipocyte formation within the skeletal muscle of beef cattle, fibroblast-like cells were isolated from the longissimus muscle of cattle and cultured with activators of murine PPARã. A thiazolidinedione T-174, which is a specific ligand for PPARã, stimulated adipose differentiation (evaluated by counting differentiated adipocytes under microscopic observation) in a dose-dependent fashion. A peroxisome proliferator Wy14,643 which strongly activates the á isoform of murine PPAR also stimulated differentiation but its potency was weaker than that of T-174. Unexpectedly, 15-deoxy-Ä12,14-prostaglandin J2, which is believed to be an endogenous ligand for PPARã, could not induce adipose differentiation in doses which have been found to be effective on rodent cells. Immunoblotting analysis confirmed the significant expression of PPARã protein in fibroblast-like cell cultures prepared from bovine skeletal muscle. In conclusion, bovine skeletal muscle contains adipose precursor cells expressing functionally active PPARã.  1998 Academic Press

K: peroxisome proliferator-activated receptor; thiazolidinedione; preadipocyte; adipocyte differentiation; skeletal muscle

INTRODUCTION Beef marbling is characterized by adipocyte formation within the skeletal muscle of cattle (Kawada et al., 1996), and is one of the important factors that influences beef flavor. The development of beef marbling in cattle is closely associated with an increase in adipocyte number within muscle (Cianzio et al., 1985), suggesting that the proliferation and differentiation of adipose precursor cells could occur within the muscle during the formation of beef marbling. Recent studies using mouse preadipose cell lines have revealed that adipose differentiation is transcriptionally regulated by the expression and the activation of the ã isoform of peroxisome proliferator-activated receptor (PPARã), which is a ligand-dependent transcriptional regulator *To whom correspondence should be addressed. 1065–6995/98/060421+07 $25.00/0

(Tontonoz et al., 1994b). Natural ligands for PPARã are poorly understood, although 15-deoxyÄ12,14-prostaglandin J2 (15d-Ä12,14-PGJ2), a metabolite of arachidonic acid, has been shown to bind and activate PPARã, and has been proposed as a candidate (Forman et al., 1995; Kliewer et al., 1995). Thiazolidinediones, which have potential as potent oral antidiabetic agents, are known to be PPARã-selective synthetic ligands and have marked adipogenic effects on mouse preadipocytes (Kletzien et al., 1992) and mesenchymal stem cells (Lehmann et al., 1995) in vitro. These observations suggest a role for PPARã in the increase of adipocyte number in the skeletal muscle of beef cattle. However, the role of PPARs in meat animals has not been described. In the present study, we investigated the effects of activators of murine PPARs (a thiazolidinedione T-174, a peroxisome proliferator Wy14,643 and  1998 Academic Press

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15d-Ä12,14-PGJ2) on the adipose differentiation of fibroblast-like cells isolated from the skeletal muscle of cattle. MATERIALS AND METHODS Chemicals A thiazolidinedione, T-174, was synthesized by Tanabe Seiyaku (Osaka, Japan). Wy14,643 and 15d-Ä12,14-PGJ2 were products of Cayman Chemical Company (Ann Arbor, MI, U.S.A.). Type I collagenase was purchased from Sigma Chemical Co. (St Louis, MO, U.S.A.). Fetal bovine serum (FBS) was obtained from Bio Whittaker (Walkersville, MD, U.S.A.). All other chemicals were guaranteed reagent grade or tissue culture grade. Animals and tissue collection Japanese Black (Wagyu) steers aged 14 to 18 months, with body weights of 401 to 594 kg, were used. All animals received humane care as outlined in the Guide for the Care and Use of Laboratory Animals (Kyoto University Animal Care Committee according to NIH #86-23; revised 1985). Animals were fed according to typical Japanese standards. Muscle biopsy was performed of the longissimus lumborum under local anesthesia, and approximately 50 mg (per animal) of muscle tissue with intramuscular adipose tissue was collected. The tissues were immediately placed in Hanks’ balanced salt solution at approximately 35C. Preparation of fibroblast-like cells from muscle tissue Collagenase digestion was initiated within an hour of tissue collection. Tissues were incubated in Hanks’ balanced salt solution containing Type I collagenase (1 mg/ml) at 38C for 60 min with reciprocal shaking at 120 cycles per min. The cell suspension was filtered through a 250-ìm nylon mesh in order to remove undigested material. After centrifugation (600g for 5 min), mature adipocytes were discarded by decantation, and the remaining cell pellet was resuspended and washed three times with Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS (growth medium). Cell culture Cells were inoculated into a 25-cm2 tissue culture flask and propagated in the growth medium at

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37C under humidified 5% CO2/95% air atmosphere. At subconfluence, the cells were trypsinized and reinoculated to Corning 12 well plates at the density of 1104 cells per cm2, and were grown in the growth medium. After confluence, the medium was replaced by DMEM:HamF12 (1:1) supplemented with 10 ìg/ml of bovine insulin, 10 ìg/ml of bovine transferrin, 33 ì of biotin, 17 ì of sodium pantothenate and 10% FBS or adult cattle serum (differentiation medium), and the cultures were maintained for 10 days in the differentiation medium. T-174, Wy14,643 or 15d-Ä12,14-PGJ2 was added into the differentiation medium at concentrations shown in Table 1 and the Figures. T-174 and Wy14,643 were stored at 20C in dimethyl sulfoxide and diluted with the differentiation medium. 15d-Ä12,14-PGJ2 was stored as supplied by the manufacturer, in methyl acetate at 85C and diluted with the differentiation medium just prior to use. The final concentrations of dimethyl sulfoxide and methyl acetate are 0.1% for both and they did not affect cell viability. In all experiments, culture media were changed every second day. Morphological evaluation of adipose conversion Differentiated adipocytes were identified by the presence of lipid droplets in the cytoplasm, which were positive of Oil Red O staining. The morphological assessment of adipocyte differentiation was quantified by counting these differentiated adipocytes under a phase-contrast microscope in five randomly selected fields. Western blotting for PPARã protein Fibroblast-like cells, prepared from bovine longissimus lumborum muscle and cultured using the same protocol as described above, were harvested at confluence. The cells were stripped from culture well and collected into 1.0 ml (per culture well) of Tris (25 m)/EDTA (1 m) buffer (pH 7.5) and lyzed by ultrasonication. Cell lysates were run on 11% SDS-PAGE gels. The proteins were then transferred to polyvinylidine difluoride (PVDF) membranes by electroblotting at 2.5 mA/cm2 for 30 min. The membranes were then blocked in Trisbuffered saline containing 1% skim milk and 0.1% Tween 20. The blots were incubated for 1 h with polyclonal rabbit anti-PPARã2 peptide antibody (Affinity BioReagents, Neshanic Station, NJ, U.S.A.). The secondary antibody used was goat anti-rabbit IgG conjugated with horseradish peroxidase (Biosys, Compiegne, France). The immunocomplexes were visualized using the

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Table 1. Induction of adipose differentiation of the fibroblast-like cells from skeletal muscle of adult beef cattle by a thiazolidinedione T-174 Animal No.

1

2

3

4

5

6

Number of cell with lipid droplets (cells/well) Control (DMSO) T-174 (25 ìM)

0 0 9118

0 0 1410

00 22

0 0 12845

0 0 48431

0 0 3110

Experiments were performed using six animals (No. 1–6). Values are expressed as means.. for three replicated cultures. DMSO, dimethylsulfoxide.

enhanced chemiluminescence system (Amersham, Buckinghamshire, U.K.). Prestained molecular weight markers (Bio-Rad, Hercules, CA, U.S.A.) were used as molecular weight standards. For the Western blotting, the cell lyzates of differentiated mouse 3T3-L1 adipocytes and fibroblast-like cells obtained from bovine adipose tissue were also used. 3T3-L1 cells at confluence were treated for 40 h with 0.25 µ dexamethasone, 0.5 m 3-isobutyl-1-methylxanthine and 10 ìg/ml insulin to induce adipose differentiation, and were harvested 8 days later (Kawada et al., 1990). The protocols for the preparation and culture of the fibroblast-like cells obtained from bovine perirenal adipose tissue were similar to those used for the culture of muscle-derived cells. The bovine perirenal adipose tissue was collected from a Japanese Black steer of unknown age at a local slaughterhouse. RESULTS The exposure of the fibroblast-like cells obtained from bovine skeletal muscle to T-174 at final concentration of 25 ì for 10 days led to the appearance of spherical lipid-containing cells which are the characteristics of differentiated adipocytes in culture (Table 1 and Fig. 1). We also determined that withdrawal of T-174 from the culture medium 3 days after commencing the treatment resulted in no acquisition of differentiated adipocytes (data not shown), indicating that long-term exposure (more than 3 days) is required for eliciting the adipogenic effect of T-174 on these cells. This contrasts with the observation that only 48 h of exposure to T-174 was sufficient to stimulate terminal differentiation of 3T3-L1 mouse preadipose cell line (Kawada, unpublished results). The potency of T-174, Wy14,643 and 15d-Ä12,14PGJ2 for inducing adipose differentiation was

investigated using two cell populations prepared from two distinct donors (Fig. 2). The adipogenic effect of T-174 was dose-dependent. Wy14,643 also stimulated production of mature adipocytes but ten times higher dose than T-174 was required, on a molar basis, to obtain a comparable number of differentiated adipocytes. The number of differentiated adipocytes appeared to be variable among donor animals (Table 1 and Fig. 2). It is unlikely that such in vitro variation is associated with the difference of marbling performance of each animal since the marbling scores were similar among the animals used in the present study. Therefore, the highly variable extent of differentiation among the experiments may result from sampling site variation in the density of adipogenic cells within the isolated cell populations or in the sensitivity to adipogenic agents. Unexpectedly, 15d-Ä12,14-PGJ2 did not induce adipocyte differentiation of the muscle-derived fibroblast-like cells even in doses which have been found to be effective on certain cell lines (Forman et al., 1995; Kliewer et al., 1995). It is unlikely that this was due to spontaneous degradation of 15dÄ12,14-PGJ2 during handling, since we have observed that this compound, from the same lot, at a similar concentration was sufficiently effective on a primary culture of preadipocytes from brown adipose tissue of mouse, to induce differentiation (unpublished data). The immunoreactive PPARã proteins, which exhibited mobilities by SDS-PAGE according to the reported molecular weights (Tontonoz et al., 1994a) of mouse PPARã2 (56 kDa) and ã1 (52 kDa), were detected in the cultures of musclederived cells, as well as perirenal fat-derived cells, of cattle (Fig. 3). The intensity of PPARã protein expression in the culture of cells originating from skeletal muscle appeared somewhat low compared with that from perirenal fat and differentiated

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3T3-L1 adipocytes. Significant expressions of PPARã were detected in the cell cultures prepared from all of cattle investigated (five individual animals).

300 (a) 250

Adipocyte number (cells/well)

200 150 100 50 0

None

–7 –6 –5 Concentration (log M)

–4

–5 –7 –6 Concentration (log M)

–4

150 (b) 100 50 0 None

Fig. 2. Effects of different PPARã activators on the adipose differentiation of fibroblast-like cells from skeletal muscle of beef cattle. The cells were isolated from the muscle of two animals (a) No. 7 and (b) No. 8. Values are expressed as meansSD for three replicated cultures. (), T-174; ( ), Wy14,643; (), 15d-Ä12,14-PGJ2.

DISCUSSION The present study demonstrated that fibroblast-like cells, prepared from skeletal muscle of beef cattle, can differentiate into adipocytes in the presence of a thiazolidinedione T-174, a PPARã-specific ligand. The concentrations of T-174 required to stimulate adipose differentiation in the present study (1–10 ì) were near the ED50 values of this compound for the adipogenic effects on mouse 3T3-L1 preadipocytes (approximately 0.7 ì) and the activation of human PPARã (1 ì) in transient transfection assays (Mizukami and Taniguchi, 1997). Our data provide evidence that bovine skeletal muscle contains cells which possess functionally active PPARã and can undergo adipose differentiation by the action of exogenous activators of PPARã. The frequency of adipose differentiation among cells in the entire cell population was much lower compared with our previous report in which the fibroblast-like cells prepared from perirenal fat of Fig. 1. Phase-contrast photomicrographs of the fibroblast-like cells from skeletal muscle of beef cattle at day 10 of culture in the differentiation medium. (a) control; (b) culture with 25 ì of T-174.

425

M

Pe

ri

us c

le

re na 3T lf at 3L1

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kDa 1

2

3

4

5

6

7

103 77 PPARγ2 PPARγ1 48

Fig. 3. Immunoblot of extracts of fibroblast-like cells isolated from bovine skeletal muscles with anti-PPARã antibody. Lane 1–5: Muscle-derived cell culture from five different cattle; lane 6: perirenal fat-derived cell culture; lane 7: differentiated mouse 3T3-L1 adipocytes (used as positive control). The amount of total protein loaded onto each lane was 10 ìg.

beef cattle were cultured using the same culture protocol (Ohyama et al., 1998). In agreement with this observation, cellular glycerol-3-phosphate dehydrogenase (GPDH) activity, which is a biochemical index of adipocyte differentiation, was too low to be detected (data not shown). These results raise the possibility that only a minority of the mononucleated cell population in skeletal muscle is committed to adipose lineage. However, our immunoblot analysis data indicated significant expression of PPARã protein in muscle-derived cell populations and there was only a slight difference in expression between muscle-derived and fatderived cultures. These data suggest that the ratios of PPARã-positive cells in these two cell populations are comparable. Therefore, it is unlikely that the level of PPARã expression is the primary factor for the low frequency of adipose differentiation in the muscle-derived cell cultures. It is possible that muscle-derived cells may be more resistant to T-174 or require additional (but unidentified) agent(s) for maximal acquisition of adipose differentiation. These PPARã-expressing adipogenic cells in skeletal muscle probably contribute to the intramuscular fat deposition in beef cattle in vivo. It is possible that the existence of these cells within the skeletal muscle is confined to livestock because other mammals such as humans and rodents in good health appear to possess much less adipose content than livestock. However, minor expression of PPARã mRNA is detected in the skeletal muscle of humans (Elbrecht et al., 1996; Mukherjee et al., 1997) and rodents (Vidal-Puig et al., 1997). The localization of PPARã expression to a certain cell type has not been specified. Immunohistochemistry or in situ hybridization analysis may be required for

precise identification of PPARã-positive cells in skeletal muscle. The endogenous ligand for PPARã involved in in vivo adipogenesis has not been well defined. It was reported that 15d-Ä12,14-PGJ2 binds to the human or mouse PPARã ligand binding domain and induces adipogenesis in C3H10T1/2 fibroblast (Kliewer et al., 1995) and NIH 3T3 cells expressing retroviral PPARã (Forman et al., 1995). In the present study, however, 15d-Ä12,14-PGJ2 failed to induce adipose differentiation of muscle-derived fibroblast-like cells while T-174 was effective. This discrepancy between different cell types may be explained by the following. (1) Bovine preadipose cells or the other coexisting cell type(s) in our culture system such as endothelial cells may have destroyed exogenous 15d-Ä12,14-PGJ2 more vigorously than C3H10T1/2 and NIH 3T3 cells. (2) Alternatively, bovine PPARã may not be activated by 15d-Ä12,14-PGJ2. A ligand-binding assay or transient cotransfection assays using bovine PPARã could verify this hypothesis. Wy14,643 induced adipose differentiation but it required 10 times higher dose than T-174 to exert an equivalent effect. In transient transfection assays, Wy14,643 strongly activates the á isoform of mouse PPAR but the activation of ã isoform by this compound is undetectable (Kliewer et al., 1994). A role for PPARá in adipocyte differentiation has been suggested by the observation that a PPARá-selective activator, 8(S)-hydroxyeicosatetraenoic acid, stimulated terminal differentiation of 3T3-L1 preadipocytes (Yu et al., 1995). The present results support such a putative role of PPARá in adipogenesis. Our findings that T-174 was a more potent stimulator of adipose differentiation of muscle-derived fibroblastic cells than Wy14,643 suggests a greater contribution by PPARã than PPARá to adipogenesis in skeletal muscles. However, it is possible that PPARá is also an active transcription factor for in vivo adipogenesis, as PPARá, not PPARã, is easily activated by linoleic acid (Kliewer et al., 1994; Yu et al., 1995) which is abundant in muscle and fat tissues. The origin of adipose cells appearing in the skeletal muscle of adult beef cattle or in muscular diseases of man is not fully elucidated. One of the putative sources is a population of mesenchymal stem cells persisting in adult muscle. Pluripotent stem cells have been found to exist within the skeletal muscle of neonatal and even adult animals (Pate et al., 1993; Lucas et al., 1995). It is possible that the adipocytes in muscle originate from such stem cells through the facilitated commitment to adipogenic lineage by unknown mechanisms.

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Another possible explanation is that transdifferentiation of satellite cells into adipocytes may occur in muscle. Satellite cells are quiescent myoblasts located between the sarcolemma and basal lamina of postnatal myofibers. They provide myonuclei to support skeletal muscle hypertrophy and are principal cells responsible for myofiber repair and regeneration (Dodson et al., 1996). The transdifferentiation of satellite cells, isolated from newborn mice, into adipogenic lineage has been observed in vitro through exposure to a thiazolidinedione BRL49653 or polyunsaturated fatty acids (Teboul et al., 1995). It is to be further elucidated whether committed satellite cells in the muscle of adult animals express PPARã and can differentiate into adipocytes in vitro or in vivo. In summary, fibroblast-like cell populations, which were prepared from skeletal muscle of beef cattle, contained cells expressing functionally active PPARã. Our findings provide evidence that skeletal muscle of adult cattle contains PPARã-positive cells which can undergo adipose differentiation in the presence of endogenous or exogenous ligands for PPARã and are responsible for the beef marbling.

ACKNOWLEDGEMENTS We are indebted to Oita Prefecture Animal Experiment Station for offering muscle samples. This work was supported in part by a Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and by a grant from the Japan Society for the Promotion of Science (JSPS-RFTF97L00905) and the Ito Memorial Foundation. REFERENCES B O, F F, S C, D M, W W, 1996. Differential expression of peroxisome proliferatoractivated receptors (PPARs): tissue distribution of PPARalpha, -beta, and -gamma in the adult rat. Endocrinology 137: 354–366. C DS, T DG, W GB, B DC, S HL, 1985. Adipose tissue growth and cellularity: changes in bovine adipocyte size and number. J Anim Sci 60: 970–976. D MV, MF DC, G AL, D ME, V SG, 1996. Extrinsic regulation of domestic animal-derived satellite cells. Domestic Anim Endocrinol 13: 107–126. E A, C Y, C CA, H N, L MD, M DE, B J, 1996. Molecular cloning, expression and characterization of human peroxisome pro-

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