James R. Florini

The Mitogenic and Myogenic Actions of Insulin-like Growth Factors Utilize Distinct Signaling Pathways

It is well established that mitogens inhibit differentiation of skeletal muscle cells, but the insulin-like growth factors (IGFs), acting through a single receptor, stimulate both proliferation and differentiation of myoblasts. Although the IGF-I mitogenic signaling pathway has been extensively studied in other cell types, little is known about the signaling pathway leading to differentiation in skeletal muscle. By using specific inhibitors of the IGF signal transduction pathway, we have begun to define the signaling intermediates mediating the two responses to IGFs. We found that PD098059, an inhibitor of mitogen-activated protein (MAP) kinase kinase activation, inhibited IGF-stimulated proliferation of L6A1 myoblasts and the events associated with it, such as phosphorylation of the MAP kinases and elevation of c-fos mRNA and cyclin D protein. Surprisingly, PD098059 caused a dramatic enhancement of differentiation, evident both at a morphological (fusion of myoblasts into myotubes) and biochemical level (elevation of myogenin and p21 cyclin-dependent kinase inhibitor expression, as well as creatine kinase activity). In sharp contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase, and rapamycin, an inhibitor of the activation of p70 S6 kinase (p70S6k), completely abolished IGF stimulation of L6A1 differentiation. We found that p70S6k activity increased substantially during differentiation, and this increase was further enhanced by PD098059. Our results demonstrate that the MAP kinase pathway plays a primary role in the mitogenic response and is inhibitory to the myogenic response in L6A1 myoblasts, while activation of the phosphatidylinositol 3-kinase/p70S6k pathway is essential for IGF-stimulated differentiation. Thus, it appears that signaling from the IGF-I receptor utilizes two distinct pathways leading either to proliferation or differentiation.

Effects of the somatomedins and insulin on myoblast differentiation in vitro

Abstract The effects of insulin and the somatomedins on differentiation of rat myoblasts were investigated in experiments on cells cloned from Yaffes L6 line. Incubation for 48 hr with either insulin or Temins multiplication stimulating activity (MSA), a member of the somatomedin family, caused a dramatic increase in myoblast fusion. This stimulation of differentiation is not a simple consequence of the increased cell density resulting from the effects of these hormones on myoblast proliferation, and the increase in fusion is not an effect common to all mitogens (FGF inhibits the process). Other somatomedins (human somatomedin C and insulin-like growth factor I), were as effective as MSA in stimulating differentiation. The somatomedins were active at concentrations in the range of their levels in fetal blood, in contrast to insulin, which was inactive at concentrations below 10−7, M. Growth hormone (GH) had no effect on muscle differentiation. In serum-free medium MM-1 (in which myoblasts maintain apparently normal morphology and metabolic activity), the very high levels of insulin required to stimulate differentiation could be replaced entirely by physiological levels (1.0 μg/ml) of MSA, further supporting our view that insulin at high concentrations serves primarily as an analogue of the somatomedins in stimulating the growth and development of muscle cells.

Modulation of insulin-like growth factor actions in L6A1 myoblasts by insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5: A dual role for IGFBP-5

We have previously shown that the insulin‐like growth factors (IGFs) stimulate both proliferation and differentiation of skeletal muscle cells in culture, and that these actions in L6A1 muscle cells may be modulated by three secreted IGF binding proteins (IGFBPs), IGFBP‐4, ‐5, and ‐6. Since we found that the temporal expression pattern of IGFBP‐4 and IGFBP‐5 differed dramatically during the transition from proliferating myoblasts to differentiated myotubes, we undertook the current study to examine the effects of purified IGFBP‐4 and IGFBP‐5 on IGF‐ stimulated actions in L6A1 muscle cells. As has been shown for other cell types, we found that IGFBP‐4 had only inhibitory actions, inhibiting IGF‐I and IGF‐II‐ stimulated proliferation and differentiation. In contrast, IGFBP‐5 exhibited both inhibitory and stimulatory actions. When added in the presence of 30 ng/ml IGF‐I, IGFBP‐5 (250 ng/ml) inhibited all markers of the early proliferative response: the tyrosine phosphorylation of the cytoplasmic signaling molecules IRS‐1 and Shc, the activation of the MAP kinases, ERK1 and 2, the elevation of c‐fos mRNA, the early inhibition of the elevation in myogenin mRNA, and the increase in cell number. In contrast, IGFBP‐5 stimulated all aspects of the myogenic response to IGF‐I: the later rise in myogenin mRNA, the elevation of creatine kinase activity, and the fusion of myoblasts into myotubes. This dual response to IGFBP‐5 was greatest when it was added at a molar ratio of IGFBP‐5 to IGF‐I of 2:1. In contrast, when IGFBP‐5 was added in the presence of IGF‐II, it inhibited both proliferation and differentiation. Neither IGFBP had any effect when added in the presence of R3 IGF‐I, an analog with substantially reduced affinity for IGFBPs. Our results suggest that the role of IGFBP‐4 is mainly to sequester excess IGFs, and thus inhibit all actions. IGFBP‐5, however, is capable of eliciting a dual response, possibly due to its unique ability to associate with the cell membrane. J. Cell. Physiol. 177:47–57, 1998.

A rapid fluorometric method for the estimation of DNA in cultured cells

Abstract We present here a procedure for the rapid measurement of both DNA and protein from the same aliquot of cell lysate. DNA estimates obtained by this method were compared to replicate determinations using the method of Kissane and Robins (1). The optimal range for the estimation of DNA (1 to 20 μg) is well suited for use with portions of extracts from individual cell cultures; the remainder of the extract remains available for enzyme assays or other parallel determinations.

A serum-free medium for the growth of muscle cells in culture

SummaryRates of cell proliferation essentially equal to those in 10% serum were obtained when Yaffes L6 myoblasts were incubated in Hams F-12 medium containing 10−5m fetuin, 10−6m insulin, and 10−7m dexamethasone; we have designated this mixture muscle medium-1 (MM-1). Addition of other growth factors and hormones in various combinations did not increase the proliferation of myoblasts above the rate in MM-1, and neither fetuin nor insulin could be replaced by other growth factors. All glucocorticoids tested (but no other steroid hormones) were active. Fetuins prepared by the rather different procedures of Pedersen, Deutsch, and Spiro were all active, and the active material was heat labile and nondialyzable; this is the first cell culture system in which highly purified Spiro fetuin has been found active. Primary rat myoblasts proliferated more rapidly than fibroblasts in parallel cultures when incubated in MM-1. This simple medium, composed of relatively inexpensive and readily available components, should be useful for the study of muscle cell growth and differentiation.

Effect of rat age on serum levels of growth hormone and somatomedins.

The somatomedins are a family of hormones which appear to mediate many of the anabolic actions of growth hormone; these processes often exhibit an age-associated deterioration in intact animals. We have demonstrated the validity of a radioreceptor assay for the determination of somatomedin levels in rat serum. In this assay, we measure displacement of 125I-labeled Multiplication Stimulating Activity (MSA) from receptors prepared from human placental membranes. Results with this procedure confirm and extend a previous report from this laboratory indicating a significant decrease in somatomedin levels during the latter part of the lifespan. Data are presented to eliminate possible artifactual explanations for the observed age-related changes. Furthermore, we find that the decrease in somatomedin levels can not be a simple result of an age-related decrease in basal levels of growth hormone in serum. We conclude that the decrease with age in circulating levels of the somatomedins is most probably attributable to a decrease in the activity of responsiveness of the tissues (most probably liver) which secrete somatomedins in response to stimulation by growth hormone.

Assay of creatine kinase in microtiter plates using thio-NAD to allow monitoring at 405 nM.

An assay system for creatine kinase using microtiter plates and a plate reader that records absorbancies at 405 nM has been devised. The system is an adaptation of well-established assays that couple creatine kinase with the reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase (G6PDH), to give a measurable increase in reduced pyridine nucleotide quantitated by absorbance at 340 nM. Two features of this system are modified for reading at 405 nM: (i) The thioamido derivative of NAD is used because its reduced form exhibits a substantial increase in absorbance at 405 nM, the most commonly available wavelength on microplate readers; and (ii) glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is used because it can reduce either NAD or NADP (unlike most other G6PDH enzymes, which require NADP), thus making it unnecessary to use the more expensive thio-NADP. The rate of thio-NAD reduction is linear with enzyme concentration and time over a 20-fold range of concentrations of purified creatine kinase, and the assay also works well with myogenic cells allowed to grow and differentiate in the 96-well plate in which the assay is performed. This system offers considerable savings in cells, time, and material in studies of muscle cell differentiation, for which creatine kinase levels are frequently measured. It also provides a potential method for the convenient and economical measurement of activities of many other enzymes that can be coupled to reduction of thio-NAD.

INSULIN ACTS AS A SOMATOMEDIN ANALOG IN STIMULATING MYOBLAST GROWTH IN SERUM-FREE MEDIUM

SummaryA serum-free medium that supports the proliferation of myoblasts (but not of fibroblasts) has been developed recently in this laboratory. It is composed of 10−6M insulin, 10−7M dexamethasone, and 10−5M fetuin, and is designated medium MM-1. The latter two components gave optimal stimulation at or near “physiological” concentrations, but insulin was required at levels far in excess of those found in serum. Accordingly, we have now investigated the possibility that insulin acts as a week analog of the somatomedins, as has been suggested in other systems. We found that maximal growth rates were observed when 10−6M insulin was replaced by 0.5 to 1.0 μg/ml multiplication stimulating activity (MSA), indicating that insulin serves a somatomedinlike function of MM-1. We also investigated the possibility that a contaminant of fetuin is responsible for its action in MM-1 but found no evidence to support this suggestion. We conclude that MM-1 is suitable for the study of muscle cell growth and differentiation under rather well-defined conditions, and that insulin probably is serving as a somatomedin analog in this medium.

Insulin-like Growth Factor I, Somatomedin C, Induces the Synthesis of Tropoelastin in Aortic Tissue

The action of IGF-I (insulin-like growth factor I) on the synthesis of tropoelastin in chick embryonic aortae was examined. Maximal and selective stimulation of the relative (40%) and absolute (145%) rates of tropoelastin synthesis over control occurred at an IGF-I concentration of 100 ng/ml of medium. Parallel to the increase in synthesis was a 92% increase in the amount of tropoelastin activity per 100 ng of poly (A)+ RNA translated in a cell-free system. The relative rate of tropoelastin synthesis achieved at maximal stimulation is greater than that observed during normal aortic embryogenesis. The stimulatory action of the hormone on elastin synthesis appears to be at a pretransitional level perhaps involving increased transcription or stabilization of the tropoelastin mRNAs. These results suggest that IGF-I may play a key role in the regulation of elastogenesis in arterial tissue.

Polyamine depletion inhibits the differentiation of L6 myoblast cells.

Exposure to alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, inhibited the insulin induced differentiation of L6 myoblast cells. Differentiation was assessed by measuring creatine kinase activity and by determining the percentage of nuclei in myotubes. The levels of putrescine and spermidine increased in stimulated cultures prior to their differentiation and these increases were blocked by alpha-difluoromethylornithine. Provision of exogenous putrescine was able to reverse the inhibitory effect of the drug. The anti-differentiative effect is observed only if alpha-difluoromethylornithine is added within twenty-four hours of insulin stimulation. In the experimental protocol used, alpha-difluoromethylornithine was added as the cultures approached confluence and had no effect on their ultimate DNA content. Therefore, the effect of alpha-difluoromethylornithine on myoblast differentiation is not secondary to an effect on cellular proliferation. These results indicate that polyamines may be involved in the mediation of muscle cell differentiation.