Ronald E. Allen, Lynda S. Luiten and Michael V. Dodson ...

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Ronald E. Allen 2, Lynda S. Luiten and Michael V. Dodson. University of Arizona 3, Tucson 85721. Summary. Differentiation of rat skeletal muscle satel- lite cellsĀ ...
Effect of Insulin and Linoleic Acid on Satellite Cell Differentiation Ronald E. Allen, Lynda S. Luiten and Michael V. Dodson J ANIM SCI 1985, 60:1571-1579.

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EFFECT OF INSULIN AND LINOLEIC ACID CELL DIFFERENTIATION 1

ON SATELLITE

Ronald E. Allen 2, Lynda S. Luiten and Michael V. Dodson University of Arizona 3, Tucson 85721

Summary Differentiation of rat skeletal muscle satellite cells was studied in vitro. Linoleic acid and insulin, two unrelated compounds that reportedly stimulate differentiation of other types of myogenic cells, were used to examine the regulation of differentiation in satellite cell cultures. As in cultures of chick embryo muscle cells, linoleic acid stimulated fusion but only at low serum concentrations or in defined medium without fibroblast growth factor (FGF). The effects of insulin on differentiation were quite variable, however; at very low cell densities no stimulatory effect was observed. In intermediate and, to a lesser extent, high density satellite cell cultures, the addition of insulin at concentrations between .01 and 1.0 /IM stimulated satellite cell fusion. Whenever increases in fusion were observed, however, a parallel increase in cell number was also found. A closer examination of the relationship between differentiation and the presence or absence of mitogenic agents in the medium suggested that a mitogenic signal and the resultant proliferation of cells prevented differentiation. Subsequent experiments indicated that fusion could be induced by lower serum concentration or by removal of FGF, as long as linoleic acid was present in the medium. Therefore, proliferation and differentiation appear to be antagonistic processes in cultured satellite cells. If the rate of proliferation is depressed, either by mitogen removal or by increasing cell densi-

ty, differentiation is favored. Differentiation can, therefore, be regulated and appIied to in vitro studies of satellite cell activity. (Key Words: Satellite Cell, Differentiation, Insulin, Linoleic Acid, Fibroblast Growth Factor, Muscle.)

Introduction Postnatal muscle growth and fiber hypertrophy are accompanied by large increases in DNA accretion (Cheek et al., 1965; Winick and Noble, 1966) and the addition of nuclei to fibers is intimately associated with the rate and extent of muscle growth (Moss, 1968). The source of these new nuclei is generally assumed to be the small population of progenitor cells in muscle referred to as satellite cells (Mauro, 1961). Although the population dynamics of these cells during growth has been described (Schultz, 1974; Schmalbruch and Hellhammer, 1976; Campion et al., 1981), the regulation of satellite cell proliferation, differentiation and fusion is poorly understood. Satellite cells have been studied to a limited extent in vitro and have many characteristics in common with myogenic cells of neonatal or embryonic origin. They proliferate and fuse to form myotubes (Bischoff, 1974; Konigsberg et al., 1975) as do other myogenic cells, and they also accumulate muscle specific proteins (Cossu et al., 1980; Allen et al., 1982). However, recent data indicate that satellite ceils are not identical to myogenic cells isolated from embryonic or neonatal muscle (Cossu et al., 1980, 1983; Allen et al., 1982). The objectives of these experiments were to examine the influence of mitogens, differentiation promoters and fusion-supporting agents on satellite cell differentiation and to devise strategies for regulating this event in vitro. The specific agents selected for this study were linoleic acid, known to enhance fusion of chick embryo myoblasts (Horowitz et al., 1978), and insulin, a protein that reportedly stimulates differentiation (de la Haba et al., 1966; Mandel and

~This communication is Arizona Agr. Exp. Sta. Journal Paper No. 3818. This research was supported by grants from the National Institutes of Health, AG03393, Lilly Research Laboratories. Merck Institute for Therapeutic Research and the Arizona Agr. Exp. Sta. 2To whom correspondence should be addressed: Dept. of Anita. Sci., Univ. of Arizona, Tucson, AZ 85721. 3Dept. of Anita. Sci. and of Nutr. and Food Sci. Received February 16, 1984. Accepted February 13, 1985. 1571 JOURNAL OF ANIMAL SCIENCE, Vol. 60, No. 6, 1985

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ALLEN ET AL.

Pearson, 1974; Ewton and Florini, 1981). Either fibroblast growth factor (FGF) or high concentrations of serum were used as sources of mitogens to trigger proliferation. Materials and Methods

Materials. Medium, serum, penicillin, streptomycin, fungizone and Deutsch fetuin were purchased from Gibco 4. Fibroblast growth factor, bovine serum albumin (BSA), linoleic acid, fatty acid-free BSA, bovine insulin, selenium, transferrin and fibronectin were purchased from Collaborative Research, Inc. s Dexamethasone was purchased from Sigma Chemical Co. 6 , gentamycin was purchased from Schering Corp. 7 and pronase was purchased from Calbiochem s. Male Sprague Dawley rats (200 to 300 g) were obtained from the Division of Animal Resou rces, University o f Arizona. Satellite Cell Cultures. Primary cultures of rat satellite cells were established by the procedures of Bischoff (19.74), as modified by Allen et al. (1980). In brief, hind limb and back muscles were exised and subjected to pronase digestion. Satellite cells were separated from cell debris and tissue fragments by differential centrifugation and then subjected to a 2-h preplanting treatment in untreated cell culture dishes. After the preplanting period, cells were seeded in fibronectin-coated, 16-mm cell culture wells at a density equivalent to 1 to 2 g of tissue/well. All cultures were maintained in a humidified atmosphere of 95% air and 5% carbon dioxide. Initially, cells were plated in Dulbecco's Modified Eagle Medium (DMEM) containing 10% horse serum (HS), 100 units penicillin, 100 units streptomycin, 3 /2g fungizone and 85 /2g gentamycin per ml. From 24 to 96 h, cells received medium containing only 5% HS, and after 96 h, cultures were switched to treatment media for 48 h. Fresh medium was provided daily throughout the experiment. Treatment media were prepared with DMEM plus HS or with a serum-free defined medium (DM) consisting of DMEM, 1.0 nM insulin, .1 //M dexa-

4Grand Island, NY. s Waltham, MA. 6St. Louis, MO. Union, NJ. aLaJolla, CA.

methasone, 5 /lg transferrin/ml, 30 nM selenium, 10/~M fetuin and 1 #g linoleic acid/ml. After 48 h in treatment medium, cultures were fixed and stained with Giemsa. Ten random fields from each of three culture wells per treatment were counted to determine the number of myotube nuclei per m m 2 , the total number of nuclei per m m 2 and the fusion percentage. Results and Discussion

The purpose of these initial experiments was to explore the regulation of skeletal muscle satellite cell differentiation. Differentiation of myogenic cells derived from embryonic or neonatal muscle has been extensively studied in vitro for the past two decades (Konigsberg, 1961; Stockdale and Holtzer, 1961). A variety of treatments have been employed to modulate differentiation, including bromodeoxyuridine treatment (Stockdale et al., 1964), exposure to medium conditioned b y cultures of fusing muscle cells (Konigsberg, 1971) and the addition of HS in place of fetal calf serum (Yaffe and Dym, 1973). The differentiation of satellite cells, however, has not been so extensively studied. For these initial experiments, two very different kinds of compounds were selected, both of which have been reported to stimulate the differentiation of other types of myogenic cells. The first compound selected was the unsaturated fatty acid, linoleic acid. Linoleic acid was selected based upon the demonstration by Horowitz et al. (1978) of an enhancement of chick embryo myoblast fusion when either oleic or linoleic acid was added to the medium. The second, compound was the protein hormone, insulin. Several investigators have reported that the addition of the supraphysiological concentrations of insulin can stimulate differentiation and fusion of myoblasts (Mandel and Pearson, 1974; Ball and Sanwall, 1980; Kumegawa et al., 1980; Ewton and Florin/, 1981). however, there is not total agreement on the reason for this stimulation. Some reports suggest that stimulation by insulin is the indirect result of the mitogenic effect of insulin; as cell density is increased, fusion is promoted (Kumegawa et al., 1980). Others propose a direct stimulatory role for insulin on myoblast differentiation and fusion (Mandel and Pearson, 1974; Ewton and Florini, 1981). Because satellite cells have many characteristics in common with other myogenic cells, we

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% HORSE SERUM Figure 1. Differentiation of satellite cells in the presence of 1 #g linoleic acid/ml and various concentrations of HS. Mean fusion percentage (open bars) and mean total nuclei density (stippled bars) are presented for each treatment (+SE). Increasing the level of HS promoted a subsequent increase in mean nuclei/ mm 2 (r=.96).

sought to d e t e r m i n e w h e t h e r or n o t satellite cells w o u l d also respond to insulin and linoleic acid and, if so, to use insulin and linoleic acid as tools for controlling satellite cell differentiation in culture. In the first series of experiments, 1 #g linoleic acid/ml was administered to satellite cell cultures t h a t had been maintained in mediu m containing 1, 10 or 15% HS and, 48 h later, the cells were fixed, stained and microscopically evaluated to d e t e r m i n e nuclei density and percentage fusion. As illustrated in figure 1, fusion was n o t enhanced by the addition o f linoleic acid to m e d i u m containing 10 or 15% HS, although nuclei density increased as the c o n c e n t r a t i o n o f serum in the m e d i u m increased. In m e d i u m containing I% HS, however, linoleic acid dramatically increased fusion (P