Sulfation of the isoflavones genistein and daidzein in human and rat liver and gastrointestinal tract

Phytoestrogens, in particular the isoflavone aglycones genistein and daidzein, are thought to be the bioactive components of soy. Like estrogens, isoflavones can be sulfur-conjugated. However, although isoflavones in the serum are found largely in the form of glucuronide and sulfur conjugates following soy consumption, little is known regarding the relative contributions of sulfotransferases in the liver and small intestine to isoflavone sulfation. Since the sulfates may be deconjugated in target tissues, circulating isoflavone sulfates may act as a source of tissue aglycones. In the current study genistein and daidzein sulfotransferase activities were measured in cytosol from human and rat liver and gastrointestinal tract. Isoflavone sulfation in the human gastrointestinal (GI) tract was correlated with activities towards substrates for previously characterized human sulfotransferases. Western blots of human cytosols were also conducted using antisera towards human sulfotransferases SULT1E1 and SULT2A1. Whereas rat liver was almost fourfold more active than small intestine in sulfation of genistein, in the human, activities in the two tissues were comparable. In contrast, intestinal sulfation of daidzein was comparable to hepatic sulfation in the rat and significantly greater in the human. Genistein and daidzein sulfation occurred throughout the human GI tract, but with a different distribution and different interindividual variability. Whereas genistein sulfation in the human GI tract correlated significantly with sulfation of the prototypical human phenolic sulfotransferase SULT1A family substrate 2-naphthol (r2 = 0.71), daidzein sulfotransferase activity did not correlate with activities towards any prototypical sulfotransferase substrate or with genistein sulfation. Our results suggest that metabolism in the human GI tract has an important role in the generation of potentially bioactive isoflavone sulfates and a major role for the human phenolic sulfotransferase SULT1A family in metabolism of genistein in the gut. However, human intestinal daidzein sulfation appears to be catalyzed by a separate enzyme.     

Soybean protein isolate and soybean lectin inhibit iron absorption in rats.

Inhibitory effects of soybean protein isolate (SPI) and soybean lectin on the intestinal absorption of nonheme iron were investigated by in vivo studies in rats. Rats fed the SPI-based diet absorbed significantly less iron than did control rats fed the casein-based diet. Supplementing the SPI diets with 8% D-galactose significantly increased the incorporation of iron into liver ferritin, although D-galactose did not significantly increase iron absorption. Heat treatment of SPI significantly increased iron absorption. Ascorbate did not enhance iron absorption in rats fed the SPI-based diet. The presence of lectin in an aqueous extract of SPI was suggested by hemagglutination activity as well as by immunoreactivity with soybean lectin antibody. Soybean lectin introduced into ligated segments of the upper small intestine of rats inhibited ferrous iron absorption. This inhibitory effect, especially in the mucosal uptake, was significantly improved by addition of N-acetyl-D-galactosamine to soybean lectin. Soybean lectin had no effect on ferric iron absorption. Our results suggest that a portion of the reduction in iron absorption in rats fed SPI may be due to lectins.     

Lycopene and lutein inhibit proliferation in rat prostate carcinoma cells

Consumption of lycopene, a carotenoid without provitamin A activity, has been associated with a lower risk of prostate and breast cancer. Lutein is another carotenoid that may be associated with a reduced risk of age-related macular degeneration, the leading cause of blindness in adults 65 years of age and older. Bioactive compounds such as lycopene and lutein, derived from natural plant sources, have been shown to act at low substrate levels through the action of intrinsic cytokines and growth factors and their receptors within tissues, particularly those of the fibroblast growth factor and transforming growth factor beta families. The effects of grapefruit-derived and commercial lycopene and lutein preparations on androgen independent cultured malignant type II tumor cells [Dunning R3327AT3 or AT3 cells (androgen-responsive, slow-growing tumor cells with well developed epithelium and stroma)] were compared to their benign parent type I tumor epithelial cells (DTE). Results demonstrated that both lycopene, in an alpha -cyclodextrin water soluble carrier, and lutein inhibited malignant AT3 cells in a concentration and time-dependent manner. No such effect was observed when benign DTE cells were examined, demonstrating selective inhibition of extremely malignant AT3 prostate cancer cells relative to their benign parent. Lutein demonstrated a similar but slightly diminished response as lycopene. When cells were treated with cocktails of lycopene and lutein, no synergistic or additive effect occurred. These studies are consistent with epidemiological studies that show inverse relationships of these carotenoids with prostate cancer.     

染料木黄酮对乳鼠颅骨成骨细胞增殖分化的影响及其与c-jun的关系

目的研究染料木黄酮对成骨细胞活性的影响及其相关机制。方法胰蛋白酶和Ⅱ型胶原酶分步消化法获得乳鼠盖骨成骨细胞,Ⅱ代细胞用于实验。MTT和3H-TdR测定成骨细胞增殖和DNA合成,原位杂交的方法测定c-jun表达。结果成骨细胞培养基中加入(10-5、10-6和10-7mol/L)染料木黄酮或10-9mol/L和10-10mol/L雌激素培养48h和72h后,MTT的吸光度值与对照组相比均明显升高,48h和72h后对照组、染料木黄酮组和雌激素组MTT值分别为0.19、0.15;0.39、0.45、0.46;0.29、0.32、0.37和0.35、0.38;0.50、0.49。3H-TdR掺入量均显著增加,对照组、染料木黄酮组和雌激素组3H-TdR掺入量分别为68.47;101、844、512和1108.8、1204.2。c-jun表达各组差异无显著性。结论染料木黄酮不是通过促进c-jun表达来促进成骨细胞增殖与分化。     

Intestinal cellular proliferation and protein synthesis in zinc-deficient rats

Immature male Wistar rats were given a low-zinc semi-synthetic diet (2 mg Zn/kg) for 28 d. Control groups received a similar diet supplemented with 58 mg Zn/kg either ad lib. or in amounts matched to the consumption of the Zn-deficient group. Rates of growth, food consumption and small intestinal length were significantly reduced in the Zn-depleted rats. Zn deficiency in the rat was associated with a reduction in the ratio, crypt: villus and a lower rate of crypt cell division in the jejunum. This resulted in a substantial decrease in the net influx of new cells into the villi of the Zn-deficient animals compared with controls. The fractional rates of protein synthesis in jejunal mucosa were measured by a technique based on the determination of L-[4-3H]phenylalanine incorporation. There was no evidence of a decline in the protein synthetic rate in total mucosa from Zn-deficient rats. It is suggested that a reduction in cell influx into the villi may be responsible for the morphological and functional changes observed in the small intestine of rats fed on a low-Zn diet.     

A high isoflavone soy protein diet and intravenous genistein delay rejection of rat cardiac allografts

Genistein, a soy isoflavone, has in vitro immunosuppressive properties. We investigated whether genistein or dietary soy protein containing isoflavones could influence the outcome of rat cardiac allografts. Lewis rats were fed a diet with protein from high isoflavone soy protein fraction (HIS), casein (CAS) or casein with isoflavones added (CI) starting 1 wk before heart transplants from Wistar Furth donors, and continuing throughout the study. HIS-fed rats had significantly prolonged time to rejection compared with CAS- and CI-fed recipients (10.8 +/- 2.62 vs. 7.18 +/- 0.75 and 7.22 +/- 0.44 d, P < 0.001). Intravenous genistein [20mg/(kg. d) for 14 d] significantly prolonged heart survival compared with controls and dissolvent-treated recipients (23.2 +/- 7.4 vs. 8.4 +/- 1.3 and 11.4+/3.6 d, P < 0.0005), and had an additive effect when given to heart recipients also receiving low dose cyclosporine for 7 d (30.8 +/- 2.3 vs. 23.4 +/- 2.4 d, P < 0.005). Concanavalin A-stimulated lymphocytes, isolated from Lewis rats given intraperitoneal genistein for 7 d, had decreased production of interferon gamma compared with controls or dimethyl sulfoxide-treated groups (22.6 +/- 9.9 vs 149 +/- 105 and 154 +/- 103 micro g/L, P < 0.05). In conclusion, a high isoflavone soy diet and intravenous genistein, but not isoflavone extract alone, delay rejection of rat cardiac allografts, with an additive effect in cyclosporine-treated rats. In addition, intraperitoneal genistein has immunosuppressive properties in vivo.     

Soybean saponins inhibit cell proliferation by suppressing PKC activation and induce differentiation of HT-29 human colon adenocarcinoma cells

Soybeans are major dietary sources of saponins, which have been suggested as possible anticarcinogens. This study was performed to determine the effect of soybean saponins on cell proliferation, differentiation, and apoptosis in human colon cancer cells. HT-29 cells were incubated in various concentrations of saponins for 24, 48, and 72 hours. Cell growth and whole cell protein kinase C (PKC) activity were determined. Alkaline phosphatase activity and carcinoembryonic antigen level were measured as markers for cell differentiation. Apoptotic cells were quantified. Study results indicated that soybean saponin treatment decreased cell growth in a concentration-dependent manner, and pre-treatment of the cells with saponins significantly suppressed the 12-O-tetradecanoyl phorbol 13-acetate-stimulated PKC activity. Cells treated with 300 and 600 ppm of saponins significantly increased alkaline phosphatase activity by 146% and 242% of the control, respectively. Also, 4-10 times more carcinoembryonic antigen was produced in cells treated with saponins. However, at all the concentrations used, saponins did not induce apoptosis, although there were slight decreases in apoptotic activity in cells treated with 240 and 600 ppm of soybean saponins. These results suggest that crude soybean saponin extract effectively suppresses PKC activation and induces differentiation, which possibly mediate the growth inhibition of tumor cells. Further experiments, including preclinical efficacy studies, are required to fully evaluate soybean saponins for their chemopreventive properties.     

Urinary disposition of the soybean isoflavones daidzein, genistein and glycitein differs among humans with moderate fecal isoflavone degradation activity

Glycitein metabolism was compared with other isoflavones to begin to understand the effect of this compound. Total isoflavones of 4.5 micromol/kg body weight from soymilk (high in genistein and daidzein) and soygerm (high in daidzein and glycitein) was fed to seven women and seven men. To minimize interindividual variation, only subjects with moderate fecal isoflavone degradation rates (half-lives of daidzein and genistein were 15.7 and 8.9 h, respectively) were included. The average 48-h urinary excretion of glycitein, daidzein and genistein was approximately 55, 46 and 29% of the dose ingested, respectively, which was significantly different from each other in men and women (P < 0.001). Plasma isoflavone concentrations at 6 and 24 h after soymilk feeding paralleled relative amounts of isoflavones in soymilk (genistein > daidzein > glycitein) (P < 0.05) in men and women, but plasma isoflavone concentrations after soygerm feeding did not parallel soygerm isoflavone concentrations in women because genistein and glycitein did not differ from each other at 6 h after feeding. Six hours after soygerm dosing, plasma isoflavone concentrations paralleled soygerm isoflavone levels in men. Based on plasma isoflavone concentrations at 6 h after dosing, the bioavailabilities of daidzein and genistein were similar in men and women. At the high glycitein dose (soygerm), plasma concentration at 24 h after dosing suggested a modest gender difference in glycitein bioavailability.     

Lack of significant genotoxicity of purified soy isoflavones (genistein,daidzein, and glycitein) in 20 patients with prostate cancer

BACKGROUND: Genistein may be useful in the prevention or treatment of prostate cancer; however, it causes genetic damage in cultured human cells. OBJECTIVE: The objective was to assess the potential genotoxicity of a purified soy unconjugated isoflavone mixture in men with prostate cancer. DESIGN: Twenty patients with prostate cancer were treated with 300 mg genistein/d for 28 d and then with 600 mg/d for another 56 d. In peripheral lymphocytes, DNA strand breaks were assessed as nuclear tail moment, chromosomal damage was assessed as micronucleus frequency (MF), and translocations of the MLL gene (11q23) were assessed by using fluorescence in situ hybridization. Values are also reported for 6 healthy men. The studies were performed under Investigational New Drug application no. 54 137 at a tertiary referral academic medical center. RESULTS: No changes in group average or individual nuclear tail moment and MF were observed. We observed a single elevated MF value in one subject that exceeded a clinical threshold set before we initiated the study. A significant decrease in average COMET tail moment was observed on day 28 relative to day 0. We detected no genistein-induced rearrangements of the MLL gene in the 3 subjects we studied with this technique. MF increased significantly in lymphocytes exposed in vitro to unconjugated genistein at concentrations > or = 100 micromol/L. Total genistein never exceeded a peak concentration of 27.1 micro mol/L, and unconjugated genistein never exceeded a peak concentration of 0.32 micromol/L. CONCLUSION: Although isoflavones are capable of inducing genetic damage in vitro, a similar effect was not observed in subjects treated with a purified soy unconjugated isoflavone mixture.     

Effects of cadmium on ribosomal protein synthesis in rat liver.

Cadmium administered to rats at a dosage level of 2 mg Cd/kg body weight per week for 2–3 weeks caused inhibition of protein synthesis in vitro by isolated ribosomes in an amino-acid-incorporating system prepared from liver. The rate of translation on treated ribosomes was lowered by 25% when normal pH 5 enzyme was used in the assay system, and by 35% when cell sap was used as an enzyme source. The rate of incorporation was further diminished when enzymes were prepared from Cd-poisoned animals. Significant quantities of cadmium were found to be present in purified ribosomes and enzyme preparations, indicating attachment of cadmium to isolated ribosomes and proteins.     

Effect of starvation on protein synthesis in neonatal rat liver.

Hepatic protein synthesis was measured in vivo and in vitro (liver slice) in 6- and 19-day-old rats. Fed and 15-hour starved rats were compared in each individual experiment. The study demonstrated that in the 6-day-old rat, hepatic protein synthesis is very sensitive to restrictions in dietary influx. In contrast, no significant differences in protein synthesis were detected when 19-day-old rats were fasted for 15 hours. In the fed state, the protein synthetic activity of the tissue unit was the same irrespective of age. Thus, it appears that it is not the maximal capacity for protein synthesis that changes with age, but rather the extent to which the system can adapt to the changing nutritional environment.     

Role of estrogen receptors and aromatase on brain protein synthesis rates in ovariectomized female rats fed genistein

Abstract

We have reported that the dietary addition of genistein, a phytoestrogen found abundantly in soy products, stimulates brain protein synthesis rates of ovariectomized female rats. In the present study, we determine whether stimulation of brain protein synthesis rates in ovariectomized female rats by the dietary addition of genistein was conducted via estrogen receptors and aromatase-mediating actions. After ovariectomy, Wistar female rats were treated with genistein, the estrogen receptor antagonist ICI 182,780, and/or fadrozole a systemic aromatase inhibitor. In the cerebral cortex, the cerebellum and the hypothalamus, the fractional (Ks) rates of protein synthesis were increased by the dietary addition of genistein. These effects of genistein were inhibited by the administration of ICI 182,780 and fadrozole. However, the degrees to which ICI 182,780 and fadrozole inhibited the effects of genistein differed depending on the brain region. This result suggests that dietary genistein elevates the rate of protein synthesis in the brain of ovariectomized female rats. In addition, the estrogen receptors of the brain and the aromatase of the peripheral tissue and brain are, at least partly, related to the rate of brain protein synthesis caused by genistein.     

Role of estrogen receptors and aromatase on brain protein synthesis rates in ovariectomized female rats fed genistein

We have reported that the dietary addition of genistein, a phytoestrogen found abundantly in soy products, stimulates brain protein synthesis rates of ovariectomized female rats. In the present study, we determine whether stimulation of brain protein synthesis rates in ovariectomized female rats by the dietary addition of genistein was conducted via estrogen receptors and aromatase-mediating actions. After ovariectomy, Wistar female rats were treated with genistein, the estrogen receptor antagonist ICI 182,780, and/or fadrozole a systemic aromatase inhibitor. In the cerebral cortex, the cerebellum and the hypothalamus, the fractional (Ks) rates of protein synthesis were increased by the dietary addition of genistein. These effects of genistein were inhibited by the administration of ICI 182,780 and fadrozole. However, the degrees to which ICI 182,780 and fadrozole inhibited the effects of genistein differed depending on the brain region. This result suggests that dietary genistein elevates the rate of protein synthesis in the brain of ovariectomized female rats. In addition, the estrogen receptors of the brain and the aromatase of the peripheral tissue and brain are, at least partly, related to the rate of brain protein synthesis caused by genistein.     

White button mushroom phytochemicals inhibit aromatase activity and breast cancer cell proliferation.

Estrogen is a major factor in the development of breast cancer. In situ estrogen production by aromatase/estrogen synthetase in breast cancer plays a dominant role in tumor proliferation. Because natural compounds such as flavones and isoflavones have been shown to be inhibitors of aromatase, it is thought that vegetables that contain these phytochemicals can inhibit aromatase activity and suppress breast cancer cell proliferation. Heat-stable extracts were prepared from vegetables and screened for their ability to inhibit aromatase activity in a human placental microsome assay. The white button mushroom (species Agaricus bisporus) suppressed aromatase activity dose dependently. Enzyme kinetics demonstrated mixed inhibition, suggesting the presence of multiple inhibitors or more than one inhibitory mechanism. "In cell" aromatase activity and cell proliferation were measured using MCF-7aro, an aromatase-transfected breast cancer cell line. Phytochemicals in the mushroom aqueous extract inhibited aromatase activity and proliferation of MCF-7aro cells. These results suggest that diets high in mushrooms may modulate the aromatase activity and function in chemoprevention in postmenopausal women by reducing the in situ production of estrogen.     

Glutamine depletion disrupts mitochondrial integrity and impairs C2C12 myoblast proliferation,differentiation, and the heat-shock response

Glutamine and glucose are both oxidized in the mitochondria to supply the majority of usable energy for processes of cellular function. Low levels of plasma and skeletal muscle glutamine are associated with severe illness. We hypothesized that glutamine deficiency would disrupt mitochondrial integrity and impair cell function. C2C12 mouse myoblasts were cultured in control media supplemented with 5.6 mmol/L glucose and 2 mmol/L glutamine, glutamine depletion (Gln⁻) or glucose depletion (Glc⁻) media. We compared mitochondrial morphology and function, as well as cell proliferation, myogenic differentiation, and heat-shock response in these cells. Glc⁻ cells exhibited slightly elongated mitochondrial networks and increased mitochondrial mass, with normal membrane potential (ΔΨm). Mitochondria in Gln⁻ cells became hyperfused and swollen, which were accompanied by severe disruption of cristae and decreases in ΔΨm, mitochondrial mass, the inner mitochondrial membrane remodeling protein OPA1, electron transport chain complex IV protein expression, and markers of mitochondrial biogenesis and bioenergetics. In addition, Gln⁻ increased the autophagy marker LC3B-II on the mitochondrial membrane. Notably, basal mitochondrial respiration was increased in Glc⁻ cells as compared to control cells, whereas maximal respiration remained unchanged. In contrast, basal respiration, maximal respiration and reserve capacity were all decreased in Gln⁻ cells. Consistent with the aforementioned mitochondrial deficits, Gln⁻ cells had lower growth rates and myogenic differentiation, as well as a higher rate of cell death under heat stress conditions than Glc⁻ and control cells. We conclude that glutamine is essential for mitochondrial integrity and function; glutamine depletion impairs myoblast proliferation, differentiation, and the heat-shock response.     

Bovine milk antibodies against cell surface protein antigen PAc-glucosyltransferase fusion protein suppress cell adhesion and alter glucan synthesis of Streptococcus mutans.

Cell surface protein antigen (PAc) and glucosyltransferases (GTF) produced by Streptococcus mutans are considered major colonization factors of the organism, and the inhibition of these factors is thought to prevent dental caries. In this study, 8-mo-old pregnant Holstein cows were immunized with fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc with the glucan binding (GB) domain of GTF-I, an enzyme catalyzing the synthesis of water-insoluble glucan from sucrose. High titers of immunoglobulin antibodies specific for the fusion protein were found in normal milk after reimmunization, and they persisted for approximately 3 mo. The immunoglobulin G (IgG) antibodies against PAcA-GB were purified from immunized milk. The antibodies significantly inhibited the adhesion of S. mutans cells to saliva-coated hydroxyapatite beads. IgG antibodies purified from immunized milk also inhibited total glucan synthesis by cell-associated GTF preparation and GTF-I from S. mutans. The immunized milk may be useful as a means of passive immunization for the prevention of dental caries in humans.     

Soybean phytochemicals inhibit the growth of transplantable human prostate carcinoma and tumor angiogenesis in mice.

The objectives of our studies are to characterize the ability of dietary soybean components to inhibit the growth of prostate cancer in mice and alter tumor biomarkers associated with angiogenesis. Soy isoflavones (genistein or daidzein) or soy phytochemical concentrate inhibit the growth of prostate cancer cells LNCaP, DU 145 and PC-3 in vitro, but only at supraphysiologic concentrations, i.e., 50% inhibitory concentration (IC(50)) > 50 micromol/L. G2-M arrest and DNA fragmentation consistent with apoptosis of prostate cancer cells are also observed at concentrations causing growth inhibition. In contrast, the in vitro proliferation of vascular endothelial cells was inhibited by soy phytochemcials at much lower concentrations. We evaluated the ability of dietary soy phytochemical concentrate and soy protein isolate to inhibit the growth of the LNCaP human prostate cancer in severe combined immune-deficient mice. Mice inoculated subcutaneously with LNCaP cells (2 x 10(6)) were randomly assigned to one of the six dietary groups based on the AIN-76A formulation for 3 wk. A 2 x 3 factorial design was employed with two protein sources (20%, casein vs. soy protein) and three levels of soy phytochemical concentrate (0, 0.2 and 1.0% of the diet). Soy components did not alter body weight gain or food intake. Compared with casein-fed controls, the tumor volumes after 3 wk were reduced by 11% (P = 0.45) by soy protein, 19% (P = 0.17) by 0.2% soy phytochemical concentrate, 28% by soy protein with 0.2% soy phytochemical concentrate (P < 0.05), 30% by 1.0% soy phytochemical concentrate (P < 0.05) and 40% by soy protein with 1.0% soy phytochemical concentrate (P < 0.005). Histologic examination of tumor tissue showed that consumption of soy products significantly reduced tumor cell proliferation, increased apoptosis and reduced microvessel density. The angiogenic protein insulin-like growth factor-I was reduced in the circulation of mice fed soy protein and phytochemical concentrate. Our data suggest that dietary soy products may inhibit experimental prostate tumor growth through a combination of direct effects on tumor cells and indirect effects on tumor neovasculature.     

Soyfoods, soybean isoflavones, and bone health: a brief overview.

Soyfoods have received considerable attention during the past 5 years for their role in disease prevention, especially in relation to heart disease, osteoporosis, and cancer. However, limited research also suggests that soy protein favorably affects renal function. Much of the research interest in soy is aimed at establishing the physiological effects of isoflavones. Isoflavones are diphenolic compounds that have a very limited distribution in nature. Soybeans and soyfoods are, for practical purposes, the only nutritionally relevant dietary sources of isoflavones. Isoflavones are weak estrogens in that they bind to estrogen receptors, but they also have important nonhormonal properties as well. Initial speculation that soyfoods, and in particular isoflavones, might promote bone health was based on the estrogenic properties of isoflavones and the similarity in structure between isoflavones and the osteoporosis drug, ipriflavone, which is a synthetic isoflavone. In ovariectomized rodents, isoflavones retard bone loss almost as effectively as estrogen. Most research, but not all, also indicates that soyfoods rich in isoflavones favorably affect bone turnover and spinal bone mineral density in perimenopausal and postmenopausal women. However, studies conducted thus far have been of short duration and involved small numbers of subjects. Furthermore, no studies have actually examined the effect of soy feeding on fracture risk. Thus, although the data in general are encouraging, no firm conclusions can be drawn about the relationship between soy consumption and bone health. In addition to a possible direct effect of isoflavones on bone tissue, soy protein when substituted for animal protein may indirectly enhance bone strength. Several studies have found that in comparison with animal protein, soy protein decreases calcium excretion, a result of the lower sulfur amino acid content of soy protein. Although the high potassium content of soy is a consideration, the evidence clearly indicates that clinicians should consider recommending that their renal patients incorporate soyfoods into their diet.     

Ractopamine increases total and myofibrillar protein synthesis in cultured rat myotubes

The ability of the phenethanolamine (beta-adrenergic agonist) ractopamine to stimulate cellular protein accretion and protein synthesis in cultured muscle cells was evaluated. ELC5 myoblasts (a subclone of rat L6 cells) were proliferated in culture (Dulbecco's Modified Eagle Medium plus 10% fetal bovine serum at 37 degrees C) to confluency and then allowed to differentiate to form myotubes. Myotubes were then further incubated in the presence of 10(-9), 10(-8), 10(-7), 10(-6) or 10(-5) mol/L ractopamine. A significant (p less than 0.05) response in cellular protein accretion was observed for the 10(-6) and 10(-5) concentrations when compared to 10(-8) and 10(-9) mol/L ractopamine. Ractopamine at 0 and 10(-6) mol/L was used to examine the effect of the beta agonist on [35S]methionine incorporation (protein synthesis) into total cellular protein, 43-kDa proteins and myosin heavy-chain (200 kDa) protein. Protein synthesis in response to beta agonist treatment was measured at 4, 24, 48, 72 and 96 h after ractopamine addition to the ELC5 myotubes in culture. Ractopamine (10(-6) mol/L) increased [35S]methionine incorporation (apparent protein synthesis) at 24 h (p less than 0.01), 48 h (p less than 0.05), 72 h (p less than 0.01) and 96 h (p less than 0.05) in cultured ELC5 muscle cells. Ractopamine also increased apparent protein synthesis rate of the 43-kDa proteins (p less than 0.05) and myosin heavy-chain protein (200 kDa) (p less than 0.05). These results indicate that ractopamine-enhanced ELC5 myotube protein accretion is mediated, at least in part, by stimulating cellular protein synthesis.