Aria Physiol Scand 1992, 146, 79-86
IGF-I binding and IGF-I expression in regenerating muscle of normal and hypophysectomized rats E. J E N N I S C H E and G . L. M A T E J K A Department of Histology, University of Goteborg, Gotebort, Sweden
JENNISCHE, E. & MATEJKA, G. L. 1992. IGF-I binding and IGF-I expression in regenerating muscle of normal and hypophysectomized rats. Acta Physiol Scand 146, 79-86. Received 2 December 1991, accepted 30 March 1992. ISSN 0001-6752. Department of Histology, University of Goteborg, Goteborg, Sweden. Binding of iodinated IGF-I to tissue sections from regenerating muscle was studied by autoradiography in normal and in hypophysectomized rats. Binding of IGF-I was low in control muscle in both groups of animals, but increased transiently about 10-fold during regeneration after injury. Maximal binding occurred later in hypophysectomized rats than in control rats, and there was also a slower regeneration process in these animals. IGF-I, as demonstrated by immunohistochemistry, and IGF-I mRNA, as demonstrated by in situ hybridization, were expressed by the regenerating muscle cells in both groups of animals. It is concluded that locally produced IGF-I is the most likely ligand for IGF-I receptors during muscle regeneration. Key words : Autoradiography, digoxigenin labelling, IGF-I binding, in zation, insulin-like growth factor I, muscle regeneration.
T h e expression of insulin-like growth factor I (IGF-I) is low in normal rat muscle, but increases significantly during regeneration after muscle injury (Jennische et al. 1987). An increased expression of IGF-I is seen also in regenerating muscle of hypophysectomized rats (Edwall et al. 1989), indicating that IGF-I can be produced in the absence of growth hormone. I n the normal rat there is a rather high level of circulating IGF-I mainly produced by the liver in a GH-dependent manner (Guler et al. 1988). T h e blood concentration of IGF-I decreases significantly after hypophysectomy (Guler et al. 1988). T h e function of IGF-I during muscle regeneration is not known. I t is difficult to study since there are no specific inhibitors of IGF-I, and as a further complicating factor, a regenerating muscle may use IGF-I from two independent sources, from the blood and from the muscle tissue itself. Correspondence : Eva Jennische, Department of Histology, University of Goteborg, Medicinaregatan 5 , S-413 90 Goteborg, Sweden.
S Z ~ Uhybridi-
I n the present study we have tried to evaluate the relative importance of these two sources of IGF-I. T h e binding and expression of IGF-I were studied in skeletal muscle of normal rats and hypophysectomized rats during regeneration after injury. Binding of IGF-I was studied on tissue sections by autoradiography and expression of IGF-I was studied by immunohistochemistry and by in situ hybridization. In order to follow the progress of the regeneration, tissue sections were also stained to demonstrate the intermediate filament vimentin; a filament which is only expressed in immature muscle fibres (Thornell 1990).
METHODS Animals. Male SpragueDawley rats were used. Hypophysectomized rats were purchased from Mollegaard (Skensved, Denmark). The rats were hypophysectomized at 6 weeks of age and were used for experiments 2-4 weeks after hypophysectomy. No substitution therapy was given. Normal age-matched rats were used as controls. The animals were allowed free access to food and water throughout the experiments.
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'The animals were anaesthetized xith pentobarbital AIuscle i n j u r ~was induced in the extensor digitorum longus muscle of one hindlimb, b! ischaemia preceded h! gl!cogen depletion (Jennische 1986). In this model one fraction of the muscle fibres undergoes necrosis ing number and subsequent regeneration, while a w-! of muscle fibres is unaffectcd b! the ischaemia and sur\-i\-es.'lhe blood vessels are not injured. aIlo\ving for a r'ipid and ordered regenerati\-e response. 'l'he animals v c r c examined in groups of 4-6, 2. 3. 4.5 and 7 days after the ischaeniic injur! . The animals were killed b! an ox-erdose of pentobarbital and the regenerating extensor digitoruni longus muscles \\ere rapidl! remox-ed and frozen in liquid nitrogen. . ~ r r l o t l c i i r c i : r . i c ~ ~ ~12 ) , . jrm crpsections were cut and fastened to gelatin-coared microscope slidcs and allowed to dr! a t room teniper'iture. [""I]I(iF-I, nith A specific acti\-it! of 2000 Ci/niniol-', \\as purchased from _linersham (.1mersham, L K ) . ' I h c labelled IGF-I was dissolved in a Hepes bufer ( 2 5 mxt Hepes, 140 m.ci NaCI, .j m\i KCI. 1.S ni\l (:a(.;l2) at p H 7.4and containing 1 h i n e serum albumin (BS.1). 'The concentration of [""I]IC;F-I was 0.1 n u . Sonspecific binding \\as determined b!adding 100 11x1 of unlabelled IGF-I. '1'0 further characterize the specificit!. of the [ I ' ,1] IGl:-l binding, the abilit! of porcine insulin to disp!,ice labelled IGF-I from its binding site was in\-eitipted, In this set of experiments the labelled IGF-I competed with 100 /t\< of insulin. Each section n a s incubated with 100 //I [lL"I]IGFI solution or with 100 ,ctl of tracer IGF-I, compered with unlabelled IGF-I or insulin, respecti\el!. Incubation was performed for 20 h a t + 4 "C. The sections were then rinsed 2 x 5 min in ice-cold Hepes buffer nithour BS.l, followed by washes 2 x 1 min in cold, deionized \rater. 'The sections nere then air dried at room temperature and exposed to Hybond3H-film (.hersham) for 4 d a ~ at s - 80 "C. The films &-ere developed in D-19 developer (Kodak. Hemel Henipstead, LK) for 4 min at room temperature. Some sections were dipped in LlI-1 photoemulsion (.4mershani). ?'he!- were developed after 5 da!s exposure and were counterstained with haematox! lin and eosin to \ isualize the cellular distribution of the binding signal. I3ensitometric analyses were performed on the films. In addition photographs nere taken using the films as negatives. The optical absorbenc! was measured for sections incubated with labelled ItiF-I .\lone, and with labelled IGF-I competed nith unlabelled I(iF-I or insulin. Optical densities n ere calculared for labelled IGF-1 minus unlabelled IGF1 to determine the specific binding. I n order to ensure that binding of IGF-1 was not due to binding of IGF-I to the insulin receptor, optical densities were calculated for sections incubated "()
with labelled IGF-I minus optical densities for sections incubated with insulin. Probu mid probe labrlling. A 153 bp fragment of a genomic subclone of mouse IGF-I (exon 3 by analogy to human IGF-I) subcloned into a pSP64 plasmid in opposite directions (&lathews et al. 1986) was used as '1 template for probe synthesis. T h e plasmid was linearized with EtoR I and used as a template for IGF-I cKN.4 synthesis. Antisense and sense strands were labelled 1)) digoxigenin-labelled U T P using a D I G RY.4 labelling kit (Boehringer, Mannheim, German!) according to the manuficturers' instructions. One /rg of linearized plasmid was used in each labelling reaction and the yield of labelled probe was approximately 10 / r g as judged by a dot blot dilution series tonards the standard supplied by the manufacturer. In situ h.ybrrdrzlrtion. Cryostat sections, cut at 10 p m , ere prepared from the fresh frozen tissue and adhered to slides coated with 3-aminopropyltriethoxysilane (Sigma? Sr Louis, MO, USA) (Rentrop et ol. 1986). The sections were fixed for 5 min at room temperature \rith 0.6", glutaraldehyde in 0.1 M Trisbuffer, pH 8.0. .1fter rinsing in the same buffer, containing 50 mxr EDT-4, the sections were covered vith 20 / t l of the h!-bridization solution, containing 50°,1 formamide, 3.3 x SSC (10 x SSC being 1.5 M sodium citrate and 0.15 11 sodium citrate) and tRNA (Boehringer), 20 p g 100 ,u-' of hybridization solution. The probe concentration was about 25 ng section-'. Finall!, to avoid evaporation, the sections were covered n ith small glass coverslips. Control sections were incubated either with the labelled sense probe at the same concentration as the probe or with only the h!-hridization solution. Hybridization was performed at 45 "C for 16 h. -1fter h!-bridization the coverslips were removed, and after a short rinse in 4 x SSC, the sections were treated with RNase -4(Boehringer), 20 pg mi-', in a buffer containing 10 mM Tris, 0.5 M NaCl and 1 mM EDT.1 (Morel1 1989), at 37 "C for 30 min. 'The sections were then rinsed in the same buffer without RNase, for 30 min a t room temperature, followed by 1 x SSC for 30 min at 45 "C and 0.1 x S S C for 30 min at 45 "C, each with two changes. The sections were then incubated with a monoclonal antibod!. directed against digoxigenin (Boehringer). The sections were covered with 100 pl of the diluted antibodies (10 p g ml-' in phosphate-buffered saline (PBS), pH 7.39, and were incubated in a micro-oven (HP 2.500, BioRad, Hemel Hempstead, UK) for 2 x 3 min. After rinsing 3 times with PBS the sections nere incubated for 30 min with rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark) diluted 1/50 in PBS. T h e antibody solution had been preadsorbed to rat serum, 100 p1ml-' antibody solution. After rinsing the sections were incubated for 30 min with an alkaline-anti-alkaline phosphatase
IGF-I binding in regenerating muscle 0.3
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Fig. 1. Specific binding of iodinated IGF-I to muscle secretions from normal and hypophysectomized rats. The absorbency was measured for sections incubated with labelled IGF-I. From this measurement the absorbency from sections incubated with excess of unlabelled IGF-I was subtracted and the absorbency calculated. Each point represents the mean _+ SEM for 4-6 individual measurements. Normal aniHypophysectomized animals. mals.
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-a-
(APAAP) complex (Dakopatts, Denmark) diluted 1/50 in PBS. To enhance the reaction the sections were then exposed to a second sequence of rabbit antimouse immunoglobulin, followed by the APAAP complex.
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The reaction was visualized by incubating the sections with a substrate solution containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate tolodium salt in a Tris-buffer, pH 9.5 and containing levamisole, 1 mM, to inhibit endogenous alkaline phosphatases. Finally the sections were rinsed in water and mounted in glycerol-gelatine. Immunohzstochemistry. For the IGF-I staining thin slices were cut from the fresh frozen muscles and were fixed in 4 yh PBS-buffered formaldehyde containing 7.5 yosucrose for 30 min. After rinsing in PBS/sucrose cyrostat sections were prepared and incubated with a polyclonal antibody raised in rabbits against human IGF-I [K 1792, (Nilsson et af. 1986)l. Control sections were incubated without the antiserum or with the antiserum pre-adsorbed with IGF-I. FITCconjugated secondary antibodies (donkey anti-rabbit immunoglobulin, Amersham) were used to visualize the immunoreaction. For the vimentin staining fresh cryostat sections were produced from the same blocks as used for in situ hybridization. The sections were, without previous fixation, covered with 100 pl of diluted monoclonal antibodies against vimentin, (Dakopatts, Denmark; 1/100 in PBS) and were incubated in the micro-oven for 2 x 3 min at 50% effect. After rinsing, the sections were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (anti-mouse Ig F(ab')2-fragments; Amersham), diluted 1/100 in So;" fat-free dried milk in PBS. The reaction was visualized
Fig. 2 Photographs from muscle sections from a normal rat incubated with iodinated IGF-I alone (a) or with iodinated IGF-I competed with 1000-fold excess of unlabelled IGF-I ( b ) . The upper tissue section in each fig is a control muscle, the lower a regenerating muscle 3 days after injury. Bar = 1 mm. Micrographs showing tissue sections from control muscle (c) and 3 days regenerating muscle ( d ) incubated with iodinated IGF-I and dipped in photographic emulsion. Silver grains are sparse in control muscle but increase significantly in the regenerating muscle. Bars = 50 pm.
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Fig. 3. Sections from regenerating muscles from normal rats. Bars = 50 p. &IF = normal sur\-iving muscle fibre. (0) Inmunohistochemical staining for IGF-I, 3 days after injury. Regenerating myoblasts/m!-otubes (arrows) show distinct staining for IGF-I. PITC. ( h ) In situ h!-bridization 3 days after injury. The section a a s incubated with the control probe, the sense sequence of IGF-I mRN.4. There is only a ~ e a kback-ground staining. (c) Itz situ hybridization ?. da!s after injury, using the anti-sense probe. There is distinct expression of IGF-I mRNA in regenerating muscle cells, shoning up as a dark colour. Some of the regenerating muscle cells are multinuclear (arrows), i.e. postmitotic myotubes. Normal muscle fibres (hIF) show hackground
IGF-I binding in regenerating muscle by incubating the sections with diaminobenzidine and hydrogen peroxide. The sections were then rinsed, dehydrated, cleared and mounted.
RESULTS After the type of muscle injury induced in the present study, regeneration occurs in a unique sequence resulting in an almost complete restoration to the pre-injured state (Schmalbruch 1986). In short, when a muscle fibre is injured dormant satellite cells, which are positioned beneath the basal membrane, are activated and differentiate to myoblasts. After repeated divisions the myoblasts fuse to form multinuclear myotubes. These eventually mature into muscle fibres. Figure 1 shows specific binding of IGF-I expressed as absorbency units, measured by densitometry and calculated as described in Methods. Binding was low in control muscle of both groups of animals, but increased transiently during regeneration after the ischaemic insult. In both groups maximal binding was about 10 times higher than that of control muscle. I n the normal rate maximal bind was observed 3 days after the injury, while in the hypophysectomized rats the increase in IGF-I binding was slower and reached a maximum after 4 days. Binding was still high after 5 days in these animals. Figure 2a shows IGF-I binding in sections from control muscle and 3 days regenerating muscle from a normal animal. T h e IGF-I binding was effectively competed by unlabelled IGF-I (Fig. 2b). A 1 ,UM concentration of insulin was much less effective than 100 nM of unlabelled IGF-I in the competition binding of the iodinated IGF-I to the tissue sections. At 3 days the specific binding, i.e. binding of labelled IGF-I minus binding of labelled IGF-I competed with unlabelled IGF-I, was 0.244 0.028 (mean & SEM, n = 4), while binding of labelled IGF-I minus binding of labelled IGF-I competed with insulin was 0.055 0.003 (n = 4). This finding suggests that the IGF-I binding is
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specific and was not due to cross-reactivity with the insulin receptor. On tissue sections dipped in photographic emulsion, a low number of silver grains was found in control muscle (Fig. 2c). I n regenerating muscle the number of silver grains was increased (Fig. 2d). On the time point of maximal binding, i.e. day 3 in normal animals and day 4 in hypophysectomized animals, tissue sections were processed to demonstrate IGF-I mRNA expression by in situ hybridization. Immunostaining for the IGFI peptide was performed 3,4 and 7 days after the injury in both groups. I n the normal rats distinct expression of both IGF-I (Fig. 3 a) and IGF-I mRNA (Fig. 3 c) was evident in the regenerating muscle cells 3 days after injury. Many cells expressing IGF-I mRNA were multinuclear myotubes, i.e. postmitotic cells (Fig. 3 c). Neither IGF-I nor IGF-I mRNA could be demonstrated in the normal surviving muscle fibres (Fig. 3a, c). in control sections for the in situ hybridization, incubated with the labelled sense probe, only weak background staining could be demonstrated (Fig. 3 b). Four days after injury the immunostaining for IGF-I was less intense than after 3 days (Fig. 3 d) and after 7 days there was no staining above background (Fig. 3e). Staining for vimentin showed a slightly higher level in regenerated muscle fibres than in the normal ones (Fig. 30. I n hypophysectomized animals the regeneration process was slower and 3 days after injury myoblasts were sparse and IGF-I immunoreactivity was evident only in satellite cells (Fig. 4a). After 4 days distinct expression of IGF-I (Fig. 4b) as well as of IGF-I mRNA (Fig. 4c) was evident in the regenerating cells. No staining for either IGF-I peptide or IGF-I mRNA could be demonstrated in the normal muscle fibres (Fig. 4b,c). After 7 days the regenerated cells still showed IGF-I immunoreactivity (Fig. 4d) and persistent staining for vimentin indicated that the cells were immature (Fig. 4e).
staining. Most inflammatory cells in the connective tissue are negative (arrow heads). ( d ) Immunostaining for IGF-I, 4 days after injury. Moderate staining for IGF-I is evident in the immature muscle fibres (arrows). FITC. (e) Immunostaining for IGF-I, 7 days after injury. The regenerated muscle fibres no longer show staining for IGF-I. FITC. (f)Immunostaining for the intermediate filament vimentin, 7 days after injury. The regenerated muscle fibres show a slightly increased staining compared to the normal fibres (arrow). The stained cells between muscle fibres are fibroblasts; a cell type which always express vimentin. HRP/DAB.
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Fig. 4. Sections from regenerating muscle of hypophysectomized rats. Bars = 50 pm. M F = normal suri-iving muscle fibre. ( a ) Immunostaining for IGF-I 3 days after iniury. The regeneration process is dela! ed, remnants of dead muscle fibres (arrow heads) are not removed. Some of the stained cells probabl!. represent satellite cells. FITC:. ( h ) Immunostaining for IGFI 4 days after injurl-. There is distinct staining in the regenerating mj-ohlasts/myotubes (arrows). Undamaged muscle fibres (MF) are negative. FITC. (c) Zn situ hybridization 4 days after injury. Regenerating myoblasts/myotubes show distinct expression of IGF-I mRNA, showing up as a dark staining (arrows). Undamaged muscle fibres (MF) are negative. ( d ) Immunostaining for IGF-I 7 days after injury. 'The regenerating muscle fibres are still small and show staining for IGF-I (arrows). FITC. ( P ) Immunostaining for vimentin 7 days after injury. The regenerating muscle fibres express vimentin (arrows), indicating immaturity. HRP/DiZB.
DISCLSSION 'The present data show that there is a low number of binding sites for IGF-I in normal rat muscle, v, hile there is a significant and transient
increase in such binding during regeneration after muscle injury in both normal and in hypoph>sectomized rats. The time point for masimal binding occurred later in the hypoph!-sectomized animals, but a similar level of
IGF-I binding in regenerating muscle binding of IGF-I was found in both groups of animals at the day of maximal binding. Furthermore, IGF-I and IGF-I mRNA were expressed in the regenerating muscle cells in both groups of animals. The maturation of the regenerating muscle cells was slower in hypophysectomized animals, as evidenced by the morphology as well as by staining for IGF-I and vimentin. The low binding of IGF-I in control muscle indicates that circulating IGF-I is not utilized by adult skeletal muscle to a major degree. Furthermore, it can explain the moderate effect of IGF-I infusions on skeletal muscle growth in hypophysectomized animals (Guler et al. 1988). The present method for studying TGF-I binding does not separate between binding to receptors and binding to binding proteins (Rutanen et al. 1988). However, several in vitro and in vivo studies have shown that the number of IGF-I receptors is related to the maturation of muscle cells. In BC3H-1 muscle cells the number of IGF-I receptors decreases 60% when the cells differentiate from myoblasts to myocytes (Rosenthal et al. 1991). Furthermore, in foetal rat muscle the number of IGF-I receptors is about 10 times higher than that in adult muscle (Alexandrides et al. 1988), a ratio very similar to that obtained between binding in regenerating and in control muscle in the present study. This indicates that the measured IGF-I binding is mainly due to binding to receptors. However, although it is known that IGF-I and binding proteins are co-expressed in foetal muscle (Hill et al. 1989) nothing is known about changes in the expression of binding proteins in skeletal muscle during repair after injury. A recent study has demonstrated a rapid induction of mRNA for one IGF-I binding protein, BNP1, in regenerating rat liver (Mohn et al. 1991), suggesting that changes in binding proteins may be of importance for the regeneration process. Whether or not this also occurs in other tissues remains to be elucidated. Our previous data indicate that IGF-I mRNA expression, as measured by solution hybridization, is similar in regenerating muscle in normal and hppophysectomized rats (Edwall et al. 1989). The present study shows that IGF-I and IGF-I mRNA are co-expressed in regenerating muscle cells in both groups of animals. Normal animals have access to circulating IGFI in addition to that locally produced, while in
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hypophysectomized animals the level of circulating IGF-I is low (Guler et al. 1988). However, the maximal binding of IGF-I was similar in both groups of animals. This indicates that both groups had a similar local access to the ligand, since any large difference in accessibility to the ligand would be expected to be reflected in differences in receptor density and consequently in IGF-I binding (Dardevet et al. 1991). Furthermore the time curve for IGF-I binding found in the present study very closely resembles that obtained for IGF-I mRNA expression in normal rats, using the same experimental model (Edwall et al. 1989). Taken together the data indicate that during muscle regeneration, the muscle cells preferentially use locally produced IGF-I in an autocrine or paracrine mode. The morphological data of this and previous studies (Jennische et al. 1987; Jennische 1990) show that IGF-I is produced by regenerating muscle cells at certain stages of maturation, indicating that expression of IGF-I is developmentally regulated. A large number of in vitro studies indicate that both IGF-I and IGF-I1 have important effects on muscle cells (Florini 1987). Most in vitro studies have focused on mitogenic actions of IGF-I, but there is no good proof that these effects are important also in vivo. Indeed, since, as shown in this study, IGF-I mRNA is still expressed in postmitotic myotubes, it is more likely that IGF-I has metabolic effects. It might function as locally produced insulin. Furthermore, since IGF-I is expressed at similar stages of cell maturation in normal and in hypophysectomized animals, the delay in regeneration observed in hypophysectomized animals does not appear to be due to a lack of IGFI as such, but rather to a lack of other factors initiating the regeneration process in an adequate way. This study was supported by grants from the Swedish Medical Council (no 7120); the Nordic Insulin Fund and Magnus Bergvall’s fund. The gift of antiserum against IGF-I from KABI, Sweden is gratefully
acknowledged. REFERENCES ALEXANDRIDES, T., MOSES, A.C. & SMITH, R.J. 1989.
Developmental expression of receptors for insulin, insulin-like growth factor I (IGF-I), and IGF-I1 in rat skeletal muscle. Endocrinology 124, 1064-1 076.
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K.L., kIEl.RY, A.E., TEWARI, D.S., 1 , . 4 ~ , T.31. & TAL~B, R. 1991. The gene encoding rat insulin like growth factor-binding protein 1 is protein diets on insulin and insulin-like gron th rapidly and highly induced in regenerating liver. factor-I binding to skeletal muscle and liver in the .W CeII B d 11, 1393-1401. growing rat. B r 3 .\:utr 65, 47-60. J .L I . , 1989. Application of in situ hybridisation EDWALL, D., SCHALLING, &I JENNISCHE, ., E. & ~ I O R E L with radioactive nucleotide probes to detection of NORSTEDT, G. 1989. Induction of insulin-like mRNL4inthecentral nervous system. In: G. R. Rulgrowth factor I messenger ribonucleic acid during lock & P. Petrusz (eds), Techniques in Imrnunoregeneration of rat skeletal muscle. Errdocrrnolog), histochemistry rol. 4 pp. 127F146. Academic Press, 124, 820-825. London. F L O R I N I , J.R. 1987. Hormonal control of muscle NILSSOV, A,, ISGAARD. J , , I,INDAHL, A, DAHLSTR~M, growth. .Muscle .Yerise 10. .577-398. I, SKOTTNER, A. & ISAKSSON,O.G.P. 1986. GULER, H.-P., ZAPF,J., SCHEIWII.LER, E. & FRO~SC:H, Regulation b!- growth hormone of number of E. R. 1988. Recombinant human insulin-like gron th chondroc!-tes containing IGF-I in rat growth plate. factor I stimulates growth and has distinct effect on S&we 233, .571-574. organ size in h!-pophysectomized rats. Pro(. .\at/ RESTROP, A l . , KSAPP,B., WINTER, H. & SCHWEIZER, .4rud Scz (L.S-4)85, 4889-4893. J. 1986. .4minoalkplsilane-treated glass slides as H I L L , D.J., CLEMMOSS,D.R., W I L S O N , S., H ~ N , support for in situ hybridisation of keratin cDNA to V.K.M., S T R A I N , A.J. & ~ I I L S E RR.D.G. , 1989. frozen tissue sections under varying fixation and Immunological distribution of one form of insulinpretreatment conditions. Histol.hom 3 8, 271--276. like growth factor (1GF)-binding protein and IGF ROSENTHAL, S.M., BRITNETTI,A., BRON-N,E.J., peptides in human fetal tissues. ,j’.l*fol Endoc.rinol2. R~~x~LT P.IV. L . ~& , GOLDFINE, I.D. 1991. Regulation 31-38. of insulin-like growth factor (IGF) I receptor JENXISCHE, E. 1986. Rapid regeneration in postexpression during muscle cell differentiation. Poischaemic skeletal muscle with undisturbed microtential autocrine role of IGF-IT. J Clzn Inzvsf 87, 12 12-1 2 19. circulation. .4ctu P h ~ ~ i Scarzd ol 128, 409411. RL.T.ANES~ E.A1., PEKONEN, F. & MAKINEN,T. 1988. ISCHE, E. 1990. Sequential immunohistochemical Soluble 34 K binding protein inhibits the binding expression of IGF-I and the transferrin receptor in of insulin-like growth factor to its cell receptor in regenerating rat muscle in rizo. .-lrta Enr/oc.rrno/ human secretory phase endometrium : evidence for (Cph) 121, i33-738. autocrine/paracrine regulation of growth factor JENVSCHE, E., SKOTTNER, -1.8; H A N N ~ O N , H.-A. action. 3 Clin Endor.rino/ Metnh 66, 173-179. 1987. Satellite cells express the trophic factor IGFSCHM4LBRUCH, H. 1986. Muscle regeneration : fetal I in regenerating skeletal muscle. .-frta P h y i o l m!-ogenesis in a new setting. Bihlthcu .4nat 29, St.and 129, 9-1.7. 1 2 61.53. >1.4’I’HF\VS, L.S., NORSTEDT, G. 8; PALXiITER. R.D. THORSELL, L.-E. 1990. Immunohistocheniistry of 1986. Regulation of insulin-like growth factor I intermediate filaments. (Abstract) 3 Nezirol Scz 98 gene expression b!- growth hormone. Pro1 .\ar/ (Suppl), 58. -4c.d S c i (I’SA) 83. 9343-9347.
DARDEVET,
D., I\IANINA, l f . , BALAGE, hl., SORNET,
& G R I Z A R D ,J. 1991. Influence of low- and high-
%IOIiN,
IGF-I binding and IGF-I expression in regenerating muscle of normal and hypophysectomized rats E. J E N N I S C H E and G . L. M A T E J K A Department of Histology, University of Goteborg, Gotebort, Sweden
JENNISCHE, E. & MATEJKA, G. L. 1992. IGF-I binding and IGF-I expression in regenerating muscle of normal and hypophysectomized rats. Acta Physiol Scand 146, 79-86. Received 2 December 1991, accepted 30 March 1992. ISSN 0001-6752. Department of Histology, University of Goteborg, Goteborg, Sweden. Binding of iodinated IGF-I to tissue sections from regenerating muscle was studied by autoradiography in normal and in hypophysectomized rats. Binding of IGF-I was low in control muscle in both groups of animals, but increased transiently about 10-fold during regeneration after injury. Maximal binding occurred later in hypophysectomized rats than in control rats, and there was also a slower regeneration process in these animals. IGF-I, as demonstrated by immunohistochemistry, and IGF-I mRNA, as demonstrated by in situ hybridization, were expressed by the regenerating muscle cells in both groups of animals. It is concluded that locally produced IGF-I is the most likely ligand for IGF-I receptors during muscle regeneration. Key words : Autoradiography, digoxigenin labelling, IGF-I binding, in zation, insulin-like growth factor I, muscle regeneration.
T h e expression of insulin-like growth factor I (IGF-I) is low in normal rat muscle, but increases significantly during regeneration after muscle injury (Jennische et al. 1987). An increased expression of IGF-I is seen also in regenerating muscle of hypophysectomized rats (Edwall et al. 1989), indicating that IGF-I can be produced in the absence of growth hormone. I n the normal rat there is a rather high level of circulating IGF-I mainly produced by the liver in a GH-dependent manner (Guler et al. 1988). T h e blood concentration of IGF-I decreases significantly after hypophysectomy (Guler et al. 1988). T h e function of IGF-I during muscle regeneration is not known. I t is difficult to study since there are no specific inhibitors of IGF-I, and as a further complicating factor, a regenerating muscle may use IGF-I from two independent sources, from the blood and from the muscle tissue itself. Correspondence : Eva Jennische, Department of Histology, University of Goteborg, Medicinaregatan 5 , S-413 90 Goteborg, Sweden.
S Z ~ Uhybridi-
I n the present study we have tried to evaluate the relative importance of these two sources of IGF-I. T h e binding and expression of IGF-I were studied in skeletal muscle of normal rats and hypophysectomized rats during regeneration after injury. Binding of IGF-I was studied on tissue sections by autoradiography and expression of IGF-I was studied by immunohistochemistry and by in situ hybridization. In order to follow the progress of the regeneration, tissue sections were also stained to demonstrate the intermediate filament vimentin; a filament which is only expressed in immature muscle fibres (Thornell 1990).
METHODS Animals. Male SpragueDawley rats were used. Hypophysectomized rats were purchased from Mollegaard (Skensved, Denmark). The rats were hypophysectomized at 6 weeks of age and were used for experiments 2-4 weeks after hypophysectomy. No substitution therapy was given. Normal age-matched rats were used as controls. The animals were allowed free access to food and water throughout the experiments.
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r i d
G. L. M i i t e j k l i
'The animals were anaesthetized xith pentobarbital AIuscle i n j u r ~was induced in the extensor digitorum longus muscle of one hindlimb, b! ischaemia preceded h! gl!cogen depletion (Jennische 1986). In this model one fraction of the muscle fibres undergoes necrosis ing number and subsequent regeneration, while a w-! of muscle fibres is unaffectcd b! the ischaemia and sur\-i\-es.'lhe blood vessels are not injured. aIlo\ving for a r'ipid and ordered regenerati\-e response. 'l'he animals v c r c examined in groups of 4-6, 2. 3. 4.5 and 7 days after the ischaeniic injur! . The animals were killed b! an ox-erdose of pentobarbital and the regenerating extensor digitoruni longus muscles \\ere rapidl! remox-ed and frozen in liquid nitrogen. . ~ r r l o t l c i i r c i : r . i c ~ ~ ~12 ) , . jrm crpsections were cut and fastened to gelatin-coared microscope slidcs and allowed to dr! a t room teniper'iture. [""I]I(iF-I, nith A specific acti\-it! of 2000 Ci/niniol-', \\as purchased from _linersham (.1mersham, L K ) . ' I h c labelled IGF-I was dissolved in a Hepes bufer ( 2 5 mxt Hepes, 140 m.ci NaCI, .j m\i KCI. 1.S ni\l (:a(.;l2) at p H 7.4and containing 1 h i n e serum albumin (BS.1). 'The concentration of [""I]IC;F-I was 0.1 n u . Sonspecific binding \\as determined b!adding 100 11x1 of unlabelled IGF-I. '1'0 further characterize the specificit!. of the [ I ' ,1] IGl:-l binding, the abilit! of porcine insulin to disp!,ice labelled IGF-I from its binding site was in\-eitipted, In this set of experiments the labelled IGF-I competed with 100 /t\< of insulin. Each section n a s incubated with 100 //I [lL"I]IGFI solution or with 100 ,ctl of tracer IGF-I, compered with unlabelled IGF-I or insulin, respecti\el!. Incubation was performed for 20 h a t + 4 "C. The sections were then rinsed 2 x 5 min in ice-cold Hepes buffer nithour BS.l, followed by washes 2 x 1 min in cold, deionized \rater. 'The sections nere then air dried at room temperature and exposed to Hybond3H-film (.hersham) for 4 d a ~ at s - 80 "C. The films &-ere developed in D-19 developer (Kodak. Hemel Henipstead, LK) for 4 min at room temperature. Some sections were dipped in LlI-1 photoemulsion (.4mershani). ?'he!- were developed after 5 da!s exposure and were counterstained with haematox! lin and eosin to \ isualize the cellular distribution of the binding signal. I3ensitometric analyses were performed on the films. In addition photographs nere taken using the films as negatives. The optical absorbenc! was measured for sections incubated with labelled ItiF-I .\lone, and with labelled IGF-I competed nith unlabelled I(iF-I or insulin. Optical densities n ere calculared for labelled IGF-1 minus unlabelled IGF1 to determine the specific binding. I n order to ensure that binding of IGF-1 was not due to binding of IGF-I to the insulin receptor, optical densities were calculated for sections incubated "()
with labelled IGF-I minus optical densities for sections incubated with insulin. Probu mid probe labrlling. A 153 bp fragment of a genomic subclone of mouse IGF-I (exon 3 by analogy to human IGF-I) subcloned into a pSP64 plasmid in opposite directions (&lathews et al. 1986) was used as '1 template for probe synthesis. T h e plasmid was linearized with EtoR I and used as a template for IGF-I cKN.4 synthesis. Antisense and sense strands were labelled 1)) digoxigenin-labelled U T P using a D I G RY.4 labelling kit (Boehringer, Mannheim, German!) according to the manuficturers' instructions. One /rg of linearized plasmid was used in each labelling reaction and the yield of labelled probe was approximately 10 / r g as judged by a dot blot dilution series tonards the standard supplied by the manufacturer. In situ h.ybrrdrzlrtion. Cryostat sections, cut at 10 p m , ere prepared from the fresh frozen tissue and adhered to slides coated with 3-aminopropyltriethoxysilane (Sigma? Sr Louis, MO, USA) (Rentrop et ol. 1986). The sections were fixed for 5 min at room temperature \rith 0.6", glutaraldehyde in 0.1 M Trisbuffer, pH 8.0. .1fter rinsing in the same buffer, containing 50 mxr EDT-4, the sections were covered vith 20 / t l of the h!-bridization solution, containing 50°,1 formamide, 3.3 x SSC (10 x SSC being 1.5 M sodium citrate and 0.15 11 sodium citrate) and tRNA (Boehringer), 20 p g 100 ,u-' of hybridization solution. The probe concentration was about 25 ng section-'. Finall!, to avoid evaporation, the sections were covered n ith small glass coverslips. Control sections were incubated either with the labelled sense probe at the same concentration as the probe or with only the h!-hridization solution. Hybridization was performed at 45 "C for 16 h. -1fter h!-bridization the coverslips were removed, and after a short rinse in 4 x SSC, the sections were treated with RNase -4(Boehringer), 20 pg mi-', in a buffer containing 10 mM Tris, 0.5 M NaCl and 1 mM EDT.1 (Morel1 1989), at 37 "C for 30 min. 'The sections were then rinsed in the same buffer without RNase, for 30 min a t room temperature, followed by 1 x SSC for 30 min at 45 "C and 0.1 x S S C for 30 min at 45 "C, each with two changes. The sections were then incubated with a monoclonal antibod!. directed against digoxigenin (Boehringer). The sections were covered with 100 pl of the diluted antibodies (10 p g ml-' in phosphate-buffered saline (PBS), pH 7.39, and were incubated in a micro-oven (HP 2.500, BioRad, Hemel Hempstead, UK) for 2 x 3 min. After rinsing 3 times with PBS the sections nere incubated for 30 min with rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark) diluted 1/50 in PBS. T h e antibody solution had been preadsorbed to rat serum, 100 p1ml-' antibody solution. After rinsing the sections were incubated for 30 min with an alkaline-anti-alkaline phosphatase
IGF-I binding in regenerating muscle 0.3
r
Days
Fig. 1. Specific binding of iodinated IGF-I to muscle secretions from normal and hypophysectomized rats. The absorbency was measured for sections incubated with labelled IGF-I. From this measurement the absorbency from sections incubated with excess of unlabelled IGF-I was subtracted and the absorbency calculated. Each point represents the mean _+ SEM for 4-6 individual measurements. Normal aniHypophysectomized animals. mals.
-+-
-a-
(APAAP) complex (Dakopatts, Denmark) diluted 1/50 in PBS. To enhance the reaction the sections were then exposed to a second sequence of rabbit antimouse immunoglobulin, followed by the APAAP complex.
81
The reaction was visualized by incubating the sections with a substrate solution containing nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate tolodium salt in a Tris-buffer, pH 9.5 and containing levamisole, 1 mM, to inhibit endogenous alkaline phosphatases. Finally the sections were rinsed in water and mounted in glycerol-gelatine. Immunohzstochemistry. For the IGF-I staining thin slices were cut from the fresh frozen muscles and were fixed in 4 yh PBS-buffered formaldehyde containing 7.5 yosucrose for 30 min. After rinsing in PBS/sucrose cyrostat sections were prepared and incubated with a polyclonal antibody raised in rabbits against human IGF-I [K 1792, (Nilsson et af. 1986)l. Control sections were incubated without the antiserum or with the antiserum pre-adsorbed with IGF-I. FITCconjugated secondary antibodies (donkey anti-rabbit immunoglobulin, Amersham) were used to visualize the immunoreaction. For the vimentin staining fresh cryostat sections were produced from the same blocks as used for in situ hybridization. The sections were, without previous fixation, covered with 100 pl of diluted monoclonal antibodies against vimentin, (Dakopatts, Denmark; 1/100 in PBS) and were incubated in the micro-oven for 2 x 3 min at 50% effect. After rinsing, the sections were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (anti-mouse Ig F(ab')2-fragments; Amersham), diluted 1/100 in So;" fat-free dried milk in PBS. The reaction was visualized
Fig. 2 Photographs from muscle sections from a normal rat incubated with iodinated IGF-I alone (a) or with iodinated IGF-I competed with 1000-fold excess of unlabelled IGF-I ( b ) . The upper tissue section in each fig is a control muscle, the lower a regenerating muscle 3 days after injury. Bar = 1 mm. Micrographs showing tissue sections from control muscle (c) and 3 days regenerating muscle ( d ) incubated with iodinated IGF-I and dipped in photographic emulsion. Silver grains are sparse in control muscle but increase significantly in the regenerating muscle. Bars = 50 pm.
82
E.Jrnnische lrnd G. L. Matejka
Fig. 3. Sections from regenerating muscles from normal rats. Bars = 50 p. &IF = normal sur\-iving muscle fibre. (0) Inmunohistochemical staining for IGF-I, 3 days after injury. Regenerating myoblasts/m!-otubes (arrows) show distinct staining for IGF-I. PITC. ( h ) In situ h!-bridization 3 days after injury. The section a a s incubated with the control probe, the sense sequence of IGF-I mRN.4. There is only a ~ e a kback-ground staining. (c) Itz situ hybridization ?. da!s after injury, using the anti-sense probe. There is distinct expression of IGF-I mRNA in regenerating muscle cells, shoning up as a dark colour. Some of the regenerating muscle cells are multinuclear (arrows), i.e. postmitotic myotubes. Normal muscle fibres (hIF) show hackground
IGF-I binding in regenerating muscle by incubating the sections with diaminobenzidine and hydrogen peroxide. The sections were then rinsed, dehydrated, cleared and mounted.
RESULTS After the type of muscle injury induced in the present study, regeneration occurs in a unique sequence resulting in an almost complete restoration to the pre-injured state (Schmalbruch 1986). In short, when a muscle fibre is injured dormant satellite cells, which are positioned beneath the basal membrane, are activated and differentiate to myoblasts. After repeated divisions the myoblasts fuse to form multinuclear myotubes. These eventually mature into muscle fibres. Figure 1 shows specific binding of IGF-I expressed as absorbency units, measured by densitometry and calculated as described in Methods. Binding was low in control muscle of both groups of animals, but increased transiently during regeneration after the ischaemic insult. In both groups maximal binding was about 10 times higher than that of control muscle. I n the normal rate maximal bind was observed 3 days after the injury, while in the hypophysectomized rats the increase in IGF-I binding was slower and reached a maximum after 4 days. Binding was still high after 5 days in these animals. Figure 2a shows IGF-I binding in sections from control muscle and 3 days regenerating muscle from a normal animal. T h e IGF-I binding was effectively competed by unlabelled IGF-I (Fig. 2b). A 1 ,UM concentration of insulin was much less effective than 100 nM of unlabelled IGF-I in the competition binding of the iodinated IGF-I to the tissue sections. At 3 days the specific binding, i.e. binding of labelled IGF-I minus binding of labelled IGF-I competed with unlabelled IGF-I, was 0.244 0.028 (mean & SEM, n = 4), while binding of labelled IGF-I minus binding of labelled IGF-I competed with insulin was 0.055 0.003 (n = 4). This finding suggests that the IGF-I binding is
83
specific and was not due to cross-reactivity with the insulin receptor. On tissue sections dipped in photographic emulsion, a low number of silver grains was found in control muscle (Fig. 2c). I n regenerating muscle the number of silver grains was increased (Fig. 2d). On the time point of maximal binding, i.e. day 3 in normal animals and day 4 in hypophysectomized animals, tissue sections were processed to demonstrate IGF-I mRNA expression by in situ hybridization. Immunostaining for the IGFI peptide was performed 3,4 and 7 days after the injury in both groups. I n the normal rats distinct expression of both IGF-I (Fig. 3 a) and IGF-I mRNA (Fig. 3 c) was evident in the regenerating muscle cells 3 days after injury. Many cells expressing IGF-I mRNA were multinuclear myotubes, i.e. postmitotic cells (Fig. 3 c). Neither IGF-I nor IGF-I mRNA could be demonstrated in the normal surviving muscle fibres (Fig. 3a, c). in control sections for the in situ hybridization, incubated with the labelled sense probe, only weak background staining could be demonstrated (Fig. 3 b). Four days after injury the immunostaining for IGF-I was less intense than after 3 days (Fig. 3 d) and after 7 days there was no staining above background (Fig. 3e). Staining for vimentin showed a slightly higher level in regenerated muscle fibres than in the normal ones (Fig. 30. I n hypophysectomized animals the regeneration process was slower and 3 days after injury myoblasts were sparse and IGF-I immunoreactivity was evident only in satellite cells (Fig. 4a). After 4 days distinct expression of IGF-I (Fig. 4b) as well as of IGF-I mRNA (Fig. 4c) was evident in the regenerating cells. No staining for either IGF-I peptide or IGF-I mRNA could be demonstrated in the normal muscle fibres (Fig. 4b,c). After 7 days the regenerated cells still showed IGF-I immunoreactivity (Fig. 4d) and persistent staining for vimentin indicated that the cells were immature (Fig. 4e).
staining. Most inflammatory cells in the connective tissue are negative (arrow heads). ( d ) Immunostaining for IGF-I, 4 days after injury. Moderate staining for IGF-I is evident in the immature muscle fibres (arrows). FITC. (e) Immunostaining for IGF-I, 7 days after injury. The regenerated muscle fibres no longer show staining for IGF-I. FITC. (f)Immunostaining for the intermediate filament vimentin, 7 days after injury. The regenerated muscle fibres show a slightly increased staining compared to the normal fibres (arrow). The stained cells between muscle fibres are fibroblasts; a cell type which always express vimentin. HRP/DAB.
84
E. Jennische and G. L . Mutejka
Fig. 4. Sections from regenerating muscle of hypophysectomized rats. Bars = 50 pm. M F = normal suri-iving muscle fibre. ( a ) Immunostaining for IGF-I 3 days after iniury. The regeneration process is dela! ed, remnants of dead muscle fibres (arrow heads) are not removed. Some of the stained cells probabl!. represent satellite cells. FITC:. ( h ) Immunostaining for IGFI 4 days after injurl-. There is distinct staining in the regenerating mj-ohlasts/myotubes (arrows). Undamaged muscle fibres (MF) are negative. FITC. (c) Zn situ hybridization 4 days after injury. Regenerating myoblasts/myotubes show distinct expression of IGF-I mRNA, showing up as a dark staining (arrows). Undamaged muscle fibres (MF) are negative. ( d ) Immunostaining for IGF-I 7 days after injury. 'The regenerating muscle fibres are still small and show staining for IGF-I (arrows). FITC. ( P ) Immunostaining for vimentin 7 days after injury. The regenerating muscle fibres express vimentin (arrows), indicating immaturity. HRP/DiZB.
DISCLSSION 'The present data show that there is a low number of binding sites for IGF-I in normal rat muscle, v, hile there is a significant and transient
increase in such binding during regeneration after muscle injury in both normal and in hypoph>sectomized rats. The time point for masimal binding occurred later in the hypoph!-sectomized animals, but a similar level of
IGF-I binding in regenerating muscle binding of IGF-I was found in both groups of animals at the day of maximal binding. Furthermore, IGF-I and IGF-I mRNA were expressed in the regenerating muscle cells in both groups of animals. The maturation of the regenerating muscle cells was slower in hypophysectomized animals, as evidenced by the morphology as well as by staining for IGF-I and vimentin. The low binding of IGF-I in control muscle indicates that circulating IGF-I is not utilized by adult skeletal muscle to a major degree. Furthermore, it can explain the moderate effect of IGF-I infusions on skeletal muscle growth in hypophysectomized animals (Guler et al. 1988). The present method for studying TGF-I binding does not separate between binding to receptors and binding to binding proteins (Rutanen et al. 1988). However, several in vitro and in vivo studies have shown that the number of IGF-I receptors is related to the maturation of muscle cells. In BC3H-1 muscle cells the number of IGF-I receptors decreases 60% when the cells differentiate from myoblasts to myocytes (Rosenthal et al. 1991). Furthermore, in foetal rat muscle the number of IGF-I receptors is about 10 times higher than that in adult muscle (Alexandrides et al. 1988), a ratio very similar to that obtained between binding in regenerating and in control muscle in the present study. This indicates that the measured IGF-I binding is mainly due to binding to receptors. However, although it is known that IGF-I and binding proteins are co-expressed in foetal muscle (Hill et al. 1989) nothing is known about changes in the expression of binding proteins in skeletal muscle during repair after injury. A recent study has demonstrated a rapid induction of mRNA for one IGF-I binding protein, BNP1, in regenerating rat liver (Mohn et al. 1991), suggesting that changes in binding proteins may be of importance for the regeneration process. Whether or not this also occurs in other tissues remains to be elucidated. Our previous data indicate that IGF-I mRNA expression, as measured by solution hybridization, is similar in regenerating muscle in normal and hppophysectomized rats (Edwall et al. 1989). The present study shows that IGF-I and IGF-I mRNA are co-expressed in regenerating muscle cells in both groups of animals. Normal animals have access to circulating IGFI in addition to that locally produced, while in
85
hypophysectomized animals the level of circulating IGF-I is low (Guler et al. 1988). However, the maximal binding of IGF-I was similar in both groups of animals. This indicates that both groups had a similar local access to the ligand, since any large difference in accessibility to the ligand would be expected to be reflected in differences in receptor density and consequently in IGF-I binding (Dardevet et al. 1991). Furthermore the time curve for IGF-I binding found in the present study very closely resembles that obtained for IGF-I mRNA expression in normal rats, using the same experimental model (Edwall et al. 1989). Taken together the data indicate that during muscle regeneration, the muscle cells preferentially use locally produced IGF-I in an autocrine or paracrine mode. The morphological data of this and previous studies (Jennische et al. 1987; Jennische 1990) show that IGF-I is produced by regenerating muscle cells at certain stages of maturation, indicating that expression of IGF-I is developmentally regulated. A large number of in vitro studies indicate that both IGF-I and IGF-I1 have important effects on muscle cells (Florini 1987). Most in vitro studies have focused on mitogenic actions of IGF-I, but there is no good proof that these effects are important also in vivo. Indeed, since, as shown in this study, IGF-I mRNA is still expressed in postmitotic myotubes, it is more likely that IGF-I has metabolic effects. It might function as locally produced insulin. Furthermore, since IGF-I is expressed at similar stages of cell maturation in normal and in hypophysectomized animals, the delay in regeneration observed in hypophysectomized animals does not appear to be due to a lack of IGFI as such, but rather to a lack of other factors initiating the regeneration process in an adequate way. This study was supported by grants from the Swedish Medical Council (no 7120); the Nordic Insulin Fund and Magnus Bergvall’s fund. The gift of antiserum against IGF-I from KABI, Sweden is gratefully
acknowledged. REFERENCES ALEXANDRIDES, T., MOSES, A.C. & SMITH, R.J. 1989.
Developmental expression of receptors for insulin, insulin-like growth factor I (IGF-I), and IGF-I1 in rat skeletal muscle. Endocrinology 124, 1064-1 076.
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c.
K.L., kIEl.RY, A.E., TEWARI, D.S., 1 , . 4 ~ , T.31. & TAL~B, R. 1991. The gene encoding rat insulin like growth factor-binding protein 1 is protein diets on insulin and insulin-like gron th rapidly and highly induced in regenerating liver. factor-I binding to skeletal muscle and liver in the .W CeII B d 11, 1393-1401. growing rat. B r 3 .\:utr 65, 47-60. J .L I . , 1989. Application of in situ hybridisation EDWALL, D., SCHALLING, &I JENNISCHE, ., E. & ~ I O R E L with radioactive nucleotide probes to detection of NORSTEDT, G. 1989. Induction of insulin-like mRNL4inthecentral nervous system. In: G. R. Rulgrowth factor I messenger ribonucleic acid during lock & P. Petrusz (eds), Techniques in Imrnunoregeneration of rat skeletal muscle. Errdocrrnolog), histochemistry rol. 4 pp. 127F146. Academic Press, 124, 820-825. London. F L O R I N I , J.R. 1987. Hormonal control of muscle NILSSOV, A,, ISGAARD. J , , I,INDAHL, A, DAHLSTR~M, growth. .Muscle .Yerise 10. .577-398. I, SKOTTNER, A. & ISAKSSON,O.G.P. 1986. GULER, H.-P., ZAPF,J., SCHEIWII.LER, E. & FRO~SC:H, Regulation b!- growth hormone of number of E. R. 1988. Recombinant human insulin-like gron th chondroc!-tes containing IGF-I in rat growth plate. factor I stimulates growth and has distinct effect on S&we 233, .571-574. organ size in h!-pophysectomized rats. Pro(. .\at/ RESTROP, A l . , KSAPP,B., WINTER, H. & SCHWEIZER, .4rud Scz (L.S-4)85, 4889-4893. J. 1986. .4minoalkplsilane-treated glass slides as H I L L , D.J., CLEMMOSS,D.R., W I L S O N , S., H ~ N , support for in situ hybridisation of keratin cDNA to V.K.M., S T R A I N , A.J. & ~ I I L S E RR.D.G. , 1989. frozen tissue sections under varying fixation and Immunological distribution of one form of insulinpretreatment conditions. Histol.hom 3 8, 271--276. like growth factor (1GF)-binding protein and IGF ROSENTHAL, S.M., BRITNETTI,A., BRON-N,E.J., peptides in human fetal tissues. ,j’.l*fol Endoc.rinol2. R~~x~LT P.IV. L . ~& , GOLDFINE, I.D. 1991. Regulation 31-38. of insulin-like growth factor (IGF) I receptor JENXISCHE, E. 1986. Rapid regeneration in postexpression during muscle cell differentiation. Poischaemic skeletal muscle with undisturbed microtential autocrine role of IGF-IT. J Clzn Inzvsf 87, 12 12-1 2 19. circulation. .4ctu P h ~ ~ i Scarzd ol 128, 409411. RL.T.ANES~ E.A1., PEKONEN, F. & MAKINEN,T. 1988. ISCHE, E. 1990. Sequential immunohistochemical Soluble 34 K binding protein inhibits the binding expression of IGF-I and the transferrin receptor in of insulin-like growth factor to its cell receptor in regenerating rat muscle in rizo. .-lrta Enr/oc.rrno/ human secretory phase endometrium : evidence for (Cph) 121, i33-738. autocrine/paracrine regulation of growth factor JENVSCHE, E., SKOTTNER, -1.8; H A N N ~ O N , H.-A. action. 3 Clin Endor.rino/ Metnh 66, 173-179. 1987. Satellite cells express the trophic factor IGFSCHM4LBRUCH, H. 1986. Muscle regeneration : fetal I in regenerating skeletal muscle. .-frta P h y i o l m!-ogenesis in a new setting. Bihlthcu .4nat 29, St.and 129, 9-1.7. 1 2 61.53. >1.4’I’HF\VS, L.S., NORSTEDT, G. 8; PALXiITER. R.D. THORSELL, L.-E. 1990. Immunohistocheniistry of 1986. Regulation of insulin-like growth factor I intermediate filaments. (Abstract) 3 Nezirol Scz 98 gene expression b!- growth hormone. Pro1 .\ar/ (Suppl), 58. -4c.d S c i (I’SA) 83. 9343-9347.
DARDEVET,
D., I\IANINA, l f . , BALAGE, hl., SORNET,
& G R I Z A R D ,J. 1991. Influence of low- and high-
%IOIiN,
