Myoblast Cell Proliferation Research Articles

Administration of insulin to newly hatched chicks improves growth performance via impairment of MyoD gene expression and enhancement of cell proliferation in chicken myoblasts

The insulin/PI3K/Akt signaling pathway is strongly involved in the differentiation of C2C12 cells, as has been demonstrated by the addition of IGFs and insulin to culture media. In this study, we have characterized the role of insulin in chick myoblast proliferation and differentiation in vitro and in vivo, and have revealed novel details of how this exogenous hormone influences myogenic genes during differentiation. Chick myoblast cells cultured in differentiation medium (DMEM containing 2% FBS) supplemented with insulin exhibited a significant decrease in MyoD and myogenin mRNA expression after 12 h of culture compared to cells cultured in differentiation media alone. MyoD and myogenin immunoreactive proteins in cells cultured in differentiation medium supplemented with insulin were quite low compared to those in control culture. Supplementation of the differentiation media containing insulin with LY294002 (a PI3K inhibitor) induced myoblast differentiation. A significant increase in MyoD and myogenin mRNA expression was observed in these cells after incubation for 12 h, and the level of expression was similar to that of control cells incubated with differentiation media alone. The DNA content and the phosphor-Erk1/2 protein level were increased by the addition of insulin to the differentiation medium. These results suggest that insulin and its signaling pathway play an inhibitory role in chick myoblast differentiation. A high level of Pax7 mRNA was observed in the skeletal muscle of 3-day-old chicks administered insulin or tolbutamide at 1-day-of-age. In addition, body weight at 21 and 50 days-of-age was significantly greater for chickens administered insulin or tolbutamide at 1-day-of-age than for control chickens. These results detail not only species-specific differences in insulin action for myoblasts but also provide novel information that may be used for the improvement of chicken meat production.

Abstract 17550: PPAR-δ Activation Induces Angiomyogenesis Through the Potentiation of Endothelial Progenitor Cell Paracrine Network

Background Endothelial progenitor cells (EPCs) paracrine network has been known to play a key role in neovascularization. We focused on skeletal muscle regeneration as well as new vessel formation by EPCs and their modulation by PPAR-δ. Methods and Results GW501516 , a PPAR-δ agonist enhanced MMP-9 mRNA expression, protein excretion and enzyme activity among several paracrine factors in EPCs. MMP-9 in conditioned media (CM) from GW501516 treated EPCs degraded IGFBP-3 in Western blot. The CM treatment phosphorylated IGF-1 receptor (R) in HUVECs, enhancing BrdU incorporation, Matrigel tube formation and migration. It also induced proliferation of C2C12 myoblast cells and protected them from hypoxia. Systemic treatment of GW501516 in mice increased MMP-9 expression in EPCs and degraded IGFBP-3 in serum Western. In mice which received bone marrow transplantation (BMT) from WT mice, continuous infusion of GW501516 phosphorylated IGF-1R in ECs and skeletal muscle. It increased capillary density and regenerating myotubes with central nuclei, and decreased muscle fibrosis in MT stain. IF for Ki67 and TUNEL stain demonstrated the in vivo proliferation and protection from apoptosis of ECs and skeletal muscle cells by GW501516. Limb perfusion recovery was also enhanced in LDPI. BMT from MMP-9 KO mice or IGFBP-3 systemic infusion abolished these effects. Intramuscular injection of ex vivo GW501516 treated EPCs to ischemic limbs showed the same effects. But EPCs from MMP-9 KO mice reversed the results. In skin punch wound model, GW501516 systemic administration enhanced wound healing with increased angiogenesis in WT mice, not in MMP-9 KO mice. Conclusion EPC paracrine network through MMP-9 mediated IGF-1 signaling pathway is important in angiomyogenesis, and is potentiated by PPAR-δ activation. Our results suggest PPAR-δ agonist as a good candidate drug for patients with peripheral vascular diseases who need angiomyogenesis for recovery.

Apoptosis-Inducing Factor Regulates Skeletal Muscle Progenitor Cell Number and Muscle Phenotype

Apoptosis Inducing Factor (AIF) is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal and cardiomyocyte apoptosis induced by oxidative stress. Conversely in vitro, AIF has been demonstrated to have a pro-apoptotic role upon induction of the mitochondrial death pathway, once AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. Given that the aif hypomorphic harlequin (Hq) mutant mouse model displays severe sarcopenia, we examined skeletal muscle from the aif hypomorphic mice in more detail. Adult AIF-deficient skeletal myofibers display oxidative stress and a severe form of atrophy, associated with a loss of myonuclei and a fast to slow fiber type switch, both in “slow” muscles such as soleus, as well as in “fast” muscles such as extensor digitorum longus, most likely resulting from an increase of MEF2 activity. This fiber type switch was conserved in regenerated soleus and EDL muscles of Hq mice subjected to cardiotoxin injection. In addition, muscle regeneration in soleus and EDL muscles of Hq mice was severely delayed. Freshly cultured myofibers, soleus and EDL muscle sections from Hq mice displayed a decreased satellite cell pool, which could be rescued by pretreating aif hypomorphic mice with the manganese-salen free radical scavenger EUK-8. Satellite cell activation seems to be abnormally long in Hq primary culture compared to controls. However, AIF deficiency did not affect myoblast cell proliferation and differentiation. Thus, AIF protects skeletal muscles against oxidative stress-induced damage probably by protecting satellite cells against oxidative stress and maintaining skeletal muscle stem cell number and activation.

1,25(OH)2Vitamin D3 Stimulates Myogenic Differentiation by Inhibiting Cell Proliferation and Modulating the Expression of Promyogenic Growth Factors and Myostatin in C2C12 Skeletal Muscle Cells

Skeletal muscle wasting is an important public health problem associated with aging, chronic disease, cancer, kidney dialysis, and HIV/AIDS. 1,25-Dihydroxyvitamin D (1,25-D3), the active form of vitamin D, is widely recognized for its regulation of calcium and phosphate homeostasis in relation to bone development and maintenance and for its calcemic effects on target organs, such as intestine, kidney, and parathyroid glands. Emerging evidence has shown that vitamin D administration improves muscle performance and reduces falls in vitamin D-deficient older adults. However, little is known of the underlying mechanism or the role 1,25-D3 plays in promoting myogenic differentiation at the cellular and/or molecular level. In this study, we examined the effect of 1,25-D3 on myoblast cell proliferation, progression, and differentiation into myotubes. C(2)C(12) myoblasts were treated with 1,25-D3 or placebo for 1, 3, 4, 7, and 10 d. Vitamin D receptor expression was analyzed by quantitative RT-PCR, Western blottings and immunofluorescence. Expression of muscle lineage, pro- and antimyogenic, and proliferation markers was assessed by immunocytochemistry, PCR arrays, quantitative RT-PCR, and Western blottings. Addition of 1,25-D3 to C(2)C(12) myoblasts 1) increased expression and nuclear translocation of the vitamin D receptor, 2) decreased cell proliferation, 3) decreased IGF-I expression, and 4) promoted myogenic differentiation by increasing IGF-II and follistatin expression and decreasing the expression of myostatin, the only known negative regulator of muscle mass, without changing growth differentiation factor 11 expression. This study identifies key vitamin D-related molecular pathways for muscle regulation and supports the rationale for vitamin D intervention studies in select muscle disorder conditions.

Mesenchymal stromal cells stimulate C2C12 myoblast cell proliferation: potential relevance in skeletal muscle regeneration

Skeletal muscle tissue harbors of a population of resident myoblastic stem cells which are capable of regenerating the damaged tissue. However, in case of extended injury, their myogenic potential is compromised by excessive inflammatory response and collagen deposition which hamper the environmental conducivity to muscle regeneration. Therefore, the identification of new strategies aimed at potentiating the proliferative attitude of myoblasts may have potential therapeutic application for muscle repair. In this line, we have previously demonstrated in a co-culture system, that adult mouse bone marrow-derived mesenchymal stromal/stem cells (MSCs) stimulate mouse neonatal cardiomyocyte proliferation. On these premises, in the present study, we searched whether MSCs were also able to influence C2C12 myoblast cells proliferation. We found that myoblast proliferation was significantly enhanced in co-culture as compared with the single culture, and that this response involved the activation of Notch-1 signalling in the myoblastic cells. These data were confirmed by the finding that Hes1 transcriptional regulator, the major downstream effector of Notch-1, was also upregulated in C2C12 cells cultured in the presence of MSCs or their derived conditioned medium. In particular, MSCs were able to release growth factors, including FGF and VEGF, thus underscoring potential paracrine mechanisms involved in the regulation of myoblast proliferation and Notch-1 expression by MSCs. In conclusion, the results of the present study provide strong evidence for a role of MSCs in stimulating myoblast cell proliferation and suggest that the functional interaction betweeen the two cell types may be exploited to the development of new and more efficient cell-based skeletal muscle repair strategies.

Production of a Rat Monoclonal Antibody Specific for Myf5

Myogenic regulatory factors (MRFs) are transcription factors that possess a characteristic basic helix-loop-helix domain. Myf5, MyoD, MRF4, and myogenin are well-known MRF family members that activate muscle-specific genes during differentiation. Myf5 is expressed first among MRFs at the very early phase and plays an important role in myoblast specificity and cell proliferation. Myf5 shares high homology with MyoD, and therefore some commercial Myf5 antibodies are cross-reactive for Myf5 and MyoD. To allow for detailed studies of the function of Myf5, we generated a monoclonal antibody specific for Myf5 utilizing a rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody enabled us to identify Myf5 protein from rat myoblast L6E9 cell extract. Moreover, cell immunostaining revealed the nuclear localization of Myf5 in the L6E9 cells. This monoclonal antibody against Myf5 will allow us to perform further detailed studies of Myf5 and Myf5 function.

Knockdown of the myostatin gene by RNA interference in caprine fibroblast cells

Myostatin (MSTN), a member of transforming growth factor-β superfamily is a negative regulator of the skeletal muscle growth. It suppresses the proliferation and differentiation of myoblast cells. Dysfunction of MSTN gene either by natural mutation or induced through genetic manipulation (knockout or knockdown) has been reported to increase the muscle mass in mammalian species. RNA interference (RNAi) is the most promising method for inhibition of gene expression that can be utilized for MSTN gene knockdown by developing short hairpin RNA (shRNA) construct against it. In the present investigation silencing of MSTN gene in caprine fibroblast cell line was evaluated using four different shRNA expressing constructs. Variation in the efficiency of silencing (22–92%) was obtained among different constructs. It was observed that sh1 and sh4 constructs downregulated the MSTN gene expression by reducing 92.4 and 80.5% (P<0.05) level of downregulation MSTN mRNA, respectively. On the contrary, the sh3 construct significantly upregulated the MSTN mRNA level (P<0.05). These two promising constructs (sh1 and sh4) need to be further tested for interferon (IFN) response before their use in long term stable expression of anti-MSTN shRNA in muscle cells to improve chevon production.

Muscle Development in the Embryo and Hatchling

Muscle cell proliferation, migration, adhesion, and fusion are processes involved with the formation of multinucleated myotubes that will further differentiate into mature muscle fibers. The process of muscle fiber development is nearly complete at the time of hatch. Muscle growth during the embryonic period of development is characterized by an increase in myoblast cell number through hyperplasia. Posthatch muscle fiber growth occurs through muscle fiber enlargement by the process of hypertrophy, which results from the recruitment of satellite cell nuclei. Hyperplasia and hypertrophy are regulated by factors extrinsic to the cell. These extrinsic elements include growth factors and the extracellular matrix. The growth factors, hepatocyte growth factor, fibroblast growth factor 2, transforming growth factor-β, insulin-like growth factor, and myostatin stimulate or inhibit myoblast and satellite cell proliferation and differentiation. Some of these growth factors like fibroblast growth factor 2 must interact with a low affinity extracellular matrix macromolecule to bind to their high affinity receptor necessary for cell signaling. It is probable that the expression of extracellular matrix proteins involved in growth factor signaling will affect muscle growth properties during hyperplasia and hypertrophy. As signaling pathways associated with muscle growth mechanisms are further understood, the poultry industry may find it beneficial to include the expression of key genes in their selection strategies.

Morlin, an inhibitor of cortical microtubule dynamics and cellulose synthase movement

Morlin (7-ethoxy-4-methyl chromen-2-one) was discovered in a screen of 20,000 compounds for small molecules that cause altered cell morphology resulting in swollen root phenotype in Arabidopsis. Live-cell imaging of fluorescently labeled cellulose synthase (CESA) and microtubules showed that morlin acts on the cortical microtubules and alters the movement of CESA. Morlin caused a novel syndrome of cytoskeletal defects, characterized by cortical array reorientation and compromised rates of both microtubule elongation and shrinking. Formation of shorter and more bundled microtubules and detachment from the cell membrane resulted when GFP::MAP4-MBP was used to visualize microtubules during morlin treatment. Cytoskeletal effects were accompanied by a reduction in the velocity and redistribution of CESA complexes labeled with YFP::CESA6 at the cell cortex. Morlin caused no inhibition of mouse myoblast, bacterial or fungal cell proliferation at concentrations that inhibit plant cell growth. By contrast, morlin stimulated microtubule disassembly in cultured hippocampal neurons but had no significant effect on cell viability. Thus, morlin appears to be a useful new probe of the mechanisms that regulate microtubule cortical array organization and its functional interaction with CESA.

Atrophy of S6K1−/− skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control

The mammalian target of rapamycin (mTOR) and Akt proteins regulate various steps of muscle development and growth, but the physiological relevance and the downstream effectors are under investigation. Here we show that S6 kinase 1 (S6K1), a protein kinase activated by nutrients and insulin-like growth factors (IGFs), is essential for the control of muscle cytoplasmic volume by Akt and mTOR. Deletion of S6K1 does not affect myoblast cell proliferation but reduces myoblast size to the same extent as that observed with mTOR inhibition by rapamycin. In the differentiated state, S6K1(-/-) myotubes have a normal number of nuclei but are smaller, and their hypertrophic response to IGF1, nutrients and membrane-targeted Akt is blunted. These growth defects reveal that mTOR requires distinct effectors for the control of muscle cell cycle and size, potentially opening new avenues of therapeutic intervention against neoplasia or muscle atrophy.

Two distal Sp1-binding cis-elements regulate fibroblast growth factor receptor 1 (FGFR1) gene expression in myoblasts

Skeletal myoblast cell proliferation and subsequent differentiation are dependent on developmentally regulated expression of the fibroblast growth factor receptor 1 (FGFR1) gene. We have previously reported the isolation and initial characterization of the chicken FGFR1 gene ( cek1) promoter. Both distal and proximal regions of the promoter were identified as necessary for developmentally regulated transcriptional activity in proliferating myoblasts, including its down-regulation in differentiated muscle fibers in vitro. Here we report detailed characterization of the molecular mechanism regulating FGFR1 promoter activity via the distal promoter region in proliferating myoblasts. This region was identified as a 242 base pair segment located greater than 1 kilobase upstream from the start of transcription that conferred increased transcriptional activity to a minimal thymidine kinase promoter. This segment contains two Sp1 binding sites. Site directed mutagenesis and transfection studies indicated that both Sp1 sites are functional and both are required for FGFR1 promoter activity. Furthermore, Sp1 binding to the two sites was synergistic enhancing FGFR1 promoter activity. The specificity of Sp1 binding to the two distal promoter cis-elements was verified by electromobility shift and transfection assays employing an Sp1 expression construct. Differences in myoblast versus fibroblast-specific protein-DNA complex formation at these sites correlated with high promoter activity in myoblasts and significantly reduced promoter activity in fibroblasts. These studies for the first time establish a molecular mechanism regulating FGFR1 gene expression during myoblast proliferation.

Messenger ribonucleic acid expression of the MyoD gene family in muscle tissue at slaughter in relation to selection for porcine growth rate.

Livestock meat production capacity is related to muscle fiber numbers and growth. Muscle fibers develop during early embryonic development from proliferating and differentiating myoblasts. Post-natal muscle growth requires satellite cell proliferation and differentiation. Myoblast and satellite cell proliferation and differentiation is regulated by the genes of the MyoD gene family (myogenin, myf-5, myf-6, and MyoD1). Our aim was to study the mRNA expression of these genes in postnatal muscle tissue in relation to porcine selection for growth rate or leanness. Five boars from a line selected for fast growth (F-line) and five boars from a line selected against backfat thickness (L-line) were slaughtered, and biopsies were taken from 12 muscles. Between-line effects, within-line effects in relation to the performance of the pigs, and muscle-specific effects were studied. Comparing the F-line with the L-line revealed significantly greater myogenin, myf-5, and MyoD1 mRNA expression in some muscles of the F-line. The expression of myf-6 showed a tendency for the opposite effect in some muscles. Muscles were ordered by their muscle-specific growth rate (b-value). Within-line evaluation of the data revealed a systematic muscle effect for the myf-6 expression level in the F-line because higher b-values correlated with increased myf-6 expression level. Backfat thickness was negatively related to myogenin expression in the F-line. A relationship was found between myogenin:MyoD1 mRNA expression ratio and meat color/muscle fiber type composition in the L-line. Furthermore, the myogenin:MyoD1 ratio was greater in muscles from F-line boars than in muscles from L-line boars, which relates to the difference between the lines in muscle fiber type. We conclude that the mRNA levels of the MyoD genes in porcine muscle tissue at slaughter showed different relationships to selection for growth rate when evaluated between selection lines and within selection lines.

1,25(OH) 2-Vitamin D 3 Affects the Subcellular Distribution of Protein Kinase C Isoenzymes in Muscle Cells

Previous studies have shown the involvement of protein kinase C (PKC) in 1,25-dihydroxy-vitamin D 3 [1,25(OH) 2D 3] regulation of DNA synthesis (long-term effect) and Ca 2+ channel activity (short-term effect) in cultured myoblasts. Both events mediate stimulation of myoblast cell proliferation and growth by 1,25(OH) 2D 3. To characterise further the role of PKC in the hormone mode of action in muscle cells, the presence of PKC isoenzymes in chicken embryo myoblasts and changes in their total cell and subcellular levels after treatment (72 h and 5 min) with 1,25(OH) 2D 3 (1 nM), 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) and 1,2-dioctanoyl-rac-glycerol (DOG; 50 μM) were investigated. Western blot analysis provided evidence on the expression of PKC α, β and δ isoforms in avian myoblasts. Two immunoreactive bands of 80 kDa (intact molecule) and 50 kDa (catalytic fragment) were detected for each isoenzyme. 1,25(OH) 2D 3 and DOG, which increased myoblast PKC activity parallel with the stimulation of DNA synthesis and culture growth and the phorbol ester TPA which induced the opposite changes, exerted differential effects on PKC isoenzymes. Long-term (72 h) treatment with 1,25(OH) 2D 3 and DOG did not change total PKC isoform levels but decreased the 80 kDa species and increased the release of the catalytic fragment of PKC δ and β, whereas TPA augmented the total amounts of the three PKC isoforms, increasing the band of 80 kDa of PKC β and δ and the 50 kDa species for PKC α. Subcellular distribution studies showed that the 80 kDa molecule is only present in the cytosolic fraction whereas in the particulate fractions the 50 kDa fragments are detected. Increased amounts of the catalytic fragments of PKC β and δ both in the nucleus and membranes were observed after 72 h treatment with DOG while 1,25(OH) 2D 3 increases PKC β in the nucleus and PKC δ in membranes. TPA induced the appearance of the 50 kDa species of PKC α in the nuclear and membrane fractions. The phorbol ester also decreased the catalytic fragments of PKC β and δ in membranes. Increased levels of PKC β, and to a lesser extent of PKC δ, in membranes and cytosol could be detected after short exposure (5 min) of myoblasts to 1,25(OH) 2D 3, DOG and TPA. In conclusion, the data indicate the operation in myoblasts of PKC signal transduction pathways mediated by the Ca 2+-dependent PKCs α and β and the Ca 2+-independent PKC δ. Moreover, the results suggest that the β and δ isoforms of PKC could play a role in the regulation of muscle cell metabolism by 1,25(OH) 2D 3.

Differential Effects of 3,5,3′-Triiodothyronine on Control andmdxMyoblasts and Fibroblasts: Analysis by Flow Cytometry

The possibility of differential effects of triiodothyronine (T3) treatmentin vivoon myoblast and fibroblast cell proliferation was examined in control andmdxmuscle cultures. Cell isolates were purified in a Percoll gradient, sorted by flow cytometry (light scatter), and characterized as myoblasts and fibroblasts using anti-skeletal muscle myosin fluorescence. The two cell types were grown separately or remixed (1:1). Cultures were incubated with or without T3 (10−9M) for 19 h. Cells were either exposed to [3H]thymidine for 1 h and DNA prepared for scintillation counts or stained with propidium iodide for cell cycle analysis by flow cytometry. Overall [3H]thymidine uptake per cell was greater inmdxthan control cells (mainly fibroblasts and mixed cells) and was decreased by T3 only in myoblast and mixed cultures. Cell cycle data showed that the effects of T3 originated primarily at the G0/G1phase. There were moremdxthan control myoblasts at G0/G1without T3. After T3 treatment, more control fibroblasts than myoblasts were at G0/G1, but moremdxmyoblasts than fibroblasts were at G0/G1. In the absence of T3, there were also fewermdxthan control myoblasts in S. After T3, only the proportion ofmdxmyoblasts in S phase was reduced. Results are consistent with distinct T3 effects on muscle regenerationin vivoand support the hypothesis that cycling and proliferation ofmdxand control myoblasts are differentially modulated by T3. As control andmdxfibroblasts also showed distinct responses to T3, muscle regeneration likely occurs by a complex regulation of gene expression endogenous to specific cell types as well as interactions between cells of different lineage.

Neurokinin a induces C- fos, C- jun, and C- myc expression in L6 rat myoblasts

Neurokinin A (NKA), a neuropeptide belonging to the tachykinin family, induced c-fos proto-oncogene mRNA expression in serum-deprived L6J1 rat skeletal myoblasts in vitro. The marked increase reached maximal levels after 15 to 30 min. In contrast to this, c-jun and c-myc proto-oncogene expression were only slightly induced, with peak levels after 30 min. NKA did not stimulate DNA synthesis or cell proliferation in serum-deprived L6J1 myoblasts. We demonstrate a relationship between NKA treatment and induction of c-fos, c-jun and c-myc mRNA expression in serum-deprived L6J1 rat myoblasts. The results on DNA synthesis and cell proliferation indicate that the induced proto-oncogene expression alone is not enough to induce a cellular response to NKA. Possible mechanisms of action are discussed.

Effects of glycosaminoglycans and extracellular matrix components on metastatic rat rhabdomyosarcoma tumor and myoblast cell proliferation

Experiments were performed to determine the relative effects of glycosaminoglycans and extracellular matrix components alone or in association with various substrates, including extracellular matrix, on the proliferation of rat rhabdomyosarcoma (RMS) cell lines of different metastatic potential and nontumorigenic rat myoblast L6 cells. The assays used various substrates: tissue culture plastic, type I and IV collagen, fibronectin, laminin and extracellular matrix deposited by corneal endothelial cells. In control experiments, tumor cells grew faster on fibronectin and extracellular matrix than on the other substrates, and their proliferation rate was decreased slightly by laminin. Collagens were growth-inhibitory only for the highly metastatic line. The proliferation rate of L6 myoblasts was not greatly affected by the different substrates. The addition of exogenous glycosaminoglycans to the culture medium modified cell proliferation on the various substrates. Heparin inhibited the growth of all the cell lines tested, independent of the substrate. When cultured on laminin substrate the proliferation rates of the cell lines were depressed by addition of heparan sulfate to the medium, and this effect was more pronounced in the metastatic RMS lines. Chondroitin sulfate and dermatan sulfate enhanced the growth rates of the tumorigenic cells when cultured on collagen type I surfaces. Hydrocortisone, which induces myogenic differentiation, decreased the cell proliferation rates of all the cell lines tested and intensified the inhibitory effects of heparin when added simultaneously to the culture medium. The results showed that glycosaminoglycans and other matrix components can affect the proliferation rates of rhabdomyosarcoma cell lines.

Fragments of bovine insulin-like growth factors I and II stimulate proliferation of rat L6 myoblast cells

The active sites of bovine insulin-like growth factor (IGF) I and II fragments were studied. Overlapping fragments of IGF I (residues 1-25, 11-35, 21-45, 31-55, and 41-70) and of IGF II (residues 1-24, 10-34, 20-44, 30-54, and 40-67) were chemically synthesized. The activity of the fragments was measured by stimulating the proliferation of rat L6 myoblast cells. Two fragments of IGF I (residues 21-45 and 31-55) and two fragments of IGF II (residues 20-44 and 30-54) were active while the other fragments were inactive in stimulating cell proliferation. Although the activity of these fragments was observed only at a high concentration of 0.1 mM, the results imply that the active site is located around residues 31-45 for IGF I fragments and residues 30-44 for IGF II fragments. Consequently, an IGF I fragment (residues 26-50) having a five-residue extension to both the N- and C-terminal sites of residues 31-45 also stimulated the proliferation of L6 myoblast cells. Furthermore, the substitution of Ile-35 in two IGF II fragments (residues 21-45 and 31-55) by Ser inactivated these fragments. This suggests that Ile-35 is an essential residue for IGF II fragment activity. Ser-35, which was reported in the original sequencing of bovine IGF II, is incorrect in the sequence and furthermore has been consistently found to be an Ile-35 in our hands.(ABSTRACT TRUNCATED AT 250 WORDS)