Acceleration of somitic myogenesis in embryos of myogenin promoter-MRF4 transgenic mice.

The four muscle regulatory factors (MRFs) of the MyoD family are expressed in distinct temporal and spatial patterns in developing somites. To examine MRF function and regulation in somites, we generated myogenin promoter-MRF4 transgenic mice in which MRF4 was expressed in rostral somites about a half day earlier than normal. We found that the transgene, which was expressed at about the same level as endogenous MRFs, did not noticeably alter developing or adult mice, whereas the rostral somites of transgenic embryos showed accelerated myocyte formation, as well as precocious expression of the endogenous MRF4 gene. In an individual transgenic somite, MRF4 was expressed in both presumptive myotomal (mesenchymal) and dermatomal (epithelial) cells. Transgenic dermatomal cells also contained myogenin, which is expressed early in myogenesis, but did not contain myosin, which is expressed late in myogenesis. In transgenic myotomal cells, in contrast, precocious expression of MRF4 accelerated late events in myogenesis, including myosin expression and striated myofibril formation. MRF function, therefore, appears to be differentially regulated in dermatomal and myotomal cells.

Cellular and molecular diversity in skeletal muscle development: news from in vitro and in vivo.

Skeletal muscle formation is studied in vitro with myogenic cell lines and primary muscle cell cultures, and in vivo with embryos of several species. We review several of the notable advances obtained from studies of cultured cells, including the recognition of myoblast diversity, isolation of the MyoD family of muscle regulatory factors, and identification of promoter elements required for muscle-specific gene expression. These studies have led to the ideas that myoblast diversity underlies the formation of the multiple types of fast and slow muscle fibers, and that myogenesis is controlled by a combination of ubiquitous and muscle-specific transcriptional regulators that may be different for each gene. We further review some unexpected results that have been obtained when ideas from work in culture have been tested in developing animals. The studies in vivo point to additional molecular and cellular mechanisms that regulate muscle formation in the animal.

A unique pattern of expression of the four muscle regulatory factor proteins distinguishes somitic from embryonic, fetal and newborn mouse myogenic cells.

A unique pattern of expression of the four muscle regulatory factor (MRF) proteins was found to distinguish early somitic from embryonic, fetal and newborn limb myogenic cells in vitro. Expression of the myosin heavy chain (MHC), MyoD, myogenin, Myf-5, and MRF4 proteins was examined by immunocytochemistry in cultures of four distinct types of mouse myogenic cells: somitic (E8.5), embryonic (E11.5), fetal (E16.5) and newborn limb. In embryonic, fetal and newborn cultures, the MRF proteins were expressed in generally similar patterns: MyoD was the first MRF expressed; MyoD and myogenin were expressed by more cells than Myf-5 or MRF4; and each of the four MRFs was found both in cells that expressed MHC and in cells that did not express MHC. In cultures of somitic cells, in contrast, Myf-5 was expressed first and by more cells than MyoD or myogenin; MRF4 was not detected; and the MRFs were never found to be coexpressed with MHC in the same cell. Thus, some somitic cells had the unexpected ability to maintain MHC expression in the absence of detectable MRF protein expression. The different myogenic programs of embryonic, fetal and newborn myogenic cells are not, therefore, a simple result of qualitatively different MRF expression patterns, whereas myogenesis by somitic cells does include a unique pattern of MRF expression.

Phosphorylation of the rodent negative acute-phase protein alpha 1-inhibitor-III by the insulin receptor tyrosine kinase.

alpha 1-Inhibitor-III (alpha 1I3), a broad range proteinase inhibitor, member of the alpha-macroglobulin family, is abundant in normal rat plasma. Insulin-dependent tyrosine phosphorylation of a monomeric 195K glycoprotein (pp195) was observed in wheatgerm agglutinin (WGA)-Sepharose-purified insulin receptor preparations from rat liver and muscle. Phosphorylation of pp195 in vitro required a basic poly-amino acid, i.e. poly-L-lysine. We present evidence identifying pp195 as alpha 1I3. In situ perfusion with saline essentially removed pp195 from rat livers. Addition of normal rat plasma to liver homogenates or to WGA eluates restored insulin-stimulated phosphorylation of pp195; plasma from streptozotocin-diabetic rats was much less effective. Liver-derived pp195 copurified with an abundant plasma protein, with the characteristics of alpha 1I3, on size exclusion and ion-exchange chromatography. An approximately 195K protein, comigrating with alpha 1I3, was markedly diminished in plasma from diabetic rats, and alpha 1I3 concentration was decreased by approximately 70% upon immunoblot analysis. Highly purified alpha 1I3 was phosphorylated by muscle- or liver-derived insulin receptors in the presence of 1 microM poly-L-lysine and comigrated with pp195 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 1I3 phosphorylation was half-maximal at approximately 70 nM and was stimulated by insulin 7-fold. Hindlimb perfusion removed more than 90% plasma albumin but only approximately 20% pp195 from muscles. alpha 1I3 messenger RNA was identified in liver but not in muscle. A specific antibody against alpha 1I3 immunoprecipitated phosphorylated pp195 in WGA-purified insulin receptor preparations from nonperfused liver and from saline perfused and nonperfused muscle. alpha 1I3 is bound and internalized by alpha-macroglobulin receptors; whether it is phosphorylated in vivo is unknown. Hepatic alpha 1I3 synthesis may diminish in diabetic rats.

Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells.

Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes.

Skeletal muscle insulin-receptor kinase. Effects of substrate inhibition and diabetes.

Insulin receptor tyrosine kinase activity solubilized from hind limb muscle of control and streptozocin-induced diabetic (STZ-D) rats (2-3 wk) was studied with the substrates histone H2B and poly glutamic acid-tyrosine (glu-tyr) (4:1). Basal and insulin-stimulated kinase activities were inhibited when high concentrations of either substrate were added before initiation of phosphorylation with ATP. Under these conditions, insulin-stimulated activities of diabetic- and control-derived receptor kinase toward H2B were similar at 0.008 mg/ml H2B. However, higher concentrations of H2B (0.04-1 mg/ml) progressively reduced the ratios of diabetic-derived to control-derived receptor kinase activities to approximately 0.5. When inhibition of receptor kinase activities was prevented by allowing maximal autophosphorylation of insulin receptors before addition of H2B, kinase activity of diabetic- and control-derived receptors was similar at all H2B concentrations. Diabetic-derived insulin-receptor tyrosine kinase activity toward poly glu-tyr (4:1) was not significantly different from that of control rats. Under conditions of substrate inhibition (0.4 mg/ml H2B), insulin receptor H2B kinase activity from muscles of rats with severe diabetes (85 mg/kg STZ, 7 days) was significantly decreased, whereas the same activity from rats with moderate diabetes (50 mg/kg STZ, 7 days) was not significantly different from control rats. Insulin receptor alpha,beta dimers were not detectable in muscle preparations from control or diabetic rats. The data suggest that the impairment of muscle-derived insulin-receptor tyrosine kinase activity associated with insulinopenic diabetes reflects, in part, enhanced inhibition by some substrates. If solubilized insulin receptors and the exogenous substrates studied model in vivo events, impaired signaling of the muscle insulin receptor in insulinopenic diabetes may depend on the type and concentration of intracellular tyrosine kinase substrates and the severity of the metabolic derangements.

Effect of denervation on the expression of two glucose transporter isoforms in rat hindlimb muscle.

Denervation rapidly (within 24 h) induces insulin resistance of several insulin-responsive pathways in skeletal muscle, including glucose transport; resistance is usually maximal by 3 d. We examined the effect of denervation on the expression of two glucose transporter isoforms (GLUT-1 and GLUT-4) in rat hindlimb muscle; GLUT-4 is the predominant species in muscle. 1 d postdenervation, GLUT-1 and GLUT-4 mRNA and protein concentrations were unchanged. 3 and 7 d postdenervation, GLUT-4 mRNA and protein (per microgram DNA) were decreased by 50%. The minor isoform, GLUT-1 mRNA increased by approximately 500 and approximately 100%, respectively, on days 3 and 7 while GLUT-1 protein increased by approximately 60 and approximately 100%. The data suggest that the insulin resistance of glucose transport early after denervation does not reflect a decrease in total glucose transporter number; however, decreased GLUT-4 expression may contribute to its increased severity after 3 d. Parallel decreases in GLUT-4 mRNA and GLUT-4 protein postdenervation are consistent with pretranslational regulation; GLUT-1 expression may be regulated pre- and posttranslationally. The cell type(s) which overexpress GLUT-1 postdenervation need to be identified. Nervous stimuli and/or contractile activity may modulate the expression of GLUT-1 and GLUT-4 in skeletal muscle tissue.

Diabetes-associated impairment of hepatic insulin receptor tyrosine kinase activity: a study of mechanisms.

Insulin receptor tyrosine kinase activity solubilized from liver of control and streptozotocin diabetic rats was studied using histone H2b and poly-Glu-Tyr (4:1) as phosphoacceptors. Both substrates inhibited autophosphorylation and exogenous kinase activity when added before, but not after, receptor activation with ATP. When H2b was added before ATP, insulin stimulated exogenous kinase activity of diabetic-derived receptors was significantly higher (approximately 50%) than control values at low H2b concentrations, but significantly lower (approximately 50%) than control values at high H2b concentrations, suggesting a decrease in the apparent Km and maximal velocity of the diabetic receptor tyrosine kinase toward H2b. When receptors were allowed to maximally autophosphorylate before the addition of H2b, the maximal H2b kinase activity of diabetic-derived receptors was only approximately 25% lower than that of controls. These effects were not attributable to altered ATP kinetics. Insulin receptor kinase activity toward the substrate poly-Glu-Tyr (4:1) was unaltered by insulinopenic diabetes. Insulin receptor alpha-beta dimers were not detectable in either control or diabetic-derived preparations. We conclude that the impairment of hepatic insulin receptor kinase activity associated with insulinopenic diabetes reflects a decreased ability to maximally activate, which is enhanced when the receptor is activated in the presence of some substrates, e.g. H2b. Impaired signalling by the diabetic-derived receptor appears to be dependent on the type of substrate and its concentration.

Insulin-stimulated phosphorylation of a 195K protein from muscle and liver in the presence of poly-L-lysine.

Substrates of the insulin receptor tyrosine kinase have not been identified in skeletal muscle, a major target organ of insulin action. We observed the insulin-stimulated phosphorylation of a 195K protein (pp195) in extracts prepared from rat skeletal muscle and liver. pp195 copurifies with the insulin receptor on wheat germ agglutinin affinity chromatography. pp195 is not related to the insulin receptor, as assessed by lack of recognition by antinsulin receptor antibodies and by phosphopeptide mapping. Reduction of sulfhydryl bonds does not affect its apparent mol wt. Phosphorylation of pp195 has an absolute requirement in vitro for Mn2+ or Mg2+ and for certain basic poly-amino acids, i.e. poly-L-lysine or poly-L-ornithine. In the presence of 1 microM poly-L-lysine insulin stimulates pp195 phosphorylation in a dose-dependent manner (k0.5, approximately 5 x 10(-10) M; maximum approximately 10(-8) M insulin); pp195 phosphorylation by insulin-like growth factor-I requires about 100-fold higher doses. By phosphoamino acid analysis, pp195 is predominantly phosphorylated on tyrosine, and it is recognized by antiphosphotyrosine antibodies. Insulin receptors isolated from rat muscles 5 min after insulin injection induce about 2-fold greater phosphorylation of pp195 in vitro than receptors isolated from saline-injected controls. Streptozotocin-induced diabetes results in marked diminution of insulin-stimulated pp195 phosphorylation in extracts of muscle and liver (approximately 50% when normalized to protein content of wheat germ agglutinin eluates or approximately 80% reduction when normalized to equal receptor number). The defect is reversible by insulin therapy in vivo.

Effects of hypercortisolemia and diabetes on skeletal muscle insulin receptor function in vitro and in vivo.

Activation of skeletal muscle insulin receptor tyrosine kinase in vitro and in vivo was studied in two rat models of insulin resistance: insulinopenic diabetes and hypercortisolemia. In control rats, intravenous insulin administration resulted in dose-dependent in vivo activation of the muscle insulin receptor kinase towards histone H2b. Half-maximal and maximal activation were observed 5 min after injecting 0.1 and 0.5 U insulin/100 g, respectively. Diabetes (7 days) induced with streptozotocin did not affect insulin binding affinity of solubilized muscle receptors but depressed receptor kinase activation in vivo by 52 or 40% after intravenous insulin administration (0.1 or 2 U/100 g, respectively). Cortisone treatment (5 days) resulting in weight loss, hyperglycemia, and hyperinsulinemia did not affect the number, insulin binding affinity, or kinase activity of solubilized receptors activated with insulin in vitro or in vivo. It is concluded that impaired insulin receptor tyrosine kinase activation was demonstrated in vivo in rats with insulinopenic diabetes and that glucocorticoid-induced insulin resistance probably reflects postreceptor defect(s) in muscle.

Structural differences between liver- and muscle-derived insulin receptors in rats.

The structure of insulin receptors, solubilized from rat skeletal muscle and liver, was studied. The alpha subunit was identified by specific cross-linking to A14 125I-insulin with disuccinimidyl suberate. Muscle- and liver-derived alpha subunits migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a Mr of 131,000 and 135,000, respectively. There was no significant difference in insulin binding affinity. Treatment of cross-linked, immunoprecipitated receptors with either neuraminidase or endoglycosidase H decreased the Mr of muscle- and liver-derived alpha subunits but did not affect the difference in Mr. Autophosphorylated beta subunits migrated with a Mr of 98,000 for muscle and 101,000 for liver. After partial V8 digestion of autophosphorylated, immunoprecipitated receptors the major phosphopeptide fragment migrated on SDS-PAGE at Mr 57,000 from muscle and 60,000 from liver. Glycosidase digestion of autophosphorylated receptors suggested that Mr heterogeneity was due in part to differences in the sialic acid content of beta subunits. Muscle and liver are the major target organs of insulin; the apparent heterogeneity of insulin receptor structure may be relevant to tissue-specific differences in insulin action.