Anabolic effect of plant brassinosteroid

Abstract

Brassinosteroids are plant-derived polyhydroxylated derivatives of 5a-cholestane, structurally similar to cholesterol-derived animal steroid hormones and insect ecdysteroids, with no known function in mammals. 28-Homobrassinolide (HB), a steroidal lactone with potent plant growth-promoting property, stimulated protein synthesis and inhibited protein degradation in L6 rat skeletal muscle cells (EC₅₀ 4 μM) mediated in part by PI3K/Akt signaling pathway. Oral administration of HB (20 or 60 mg/kg/d for 24 d) to healthy rats fed normal diet (protein content 23.9%) increased food intake, body weight gain, lean body mass, and gastrocnemius muscle mass as compared with vehicle-treated controls. The effect of HB administration increased slightly in animals fed a high-protein diet (protein content 39.4%). Both oral (up to 60 mg/kg) and subcutaneous (up to 4 mg/kg) administration of HB showed low androgenic activity when tested in the Hershberger assay. Moreover, HB showed no direct binding to the androgen receptor in vitro. HB treatment was also associated with an improved physical fitness of untrained healthy rats, as evident from a 6.7% increase in lower extremity strength, measured by grip test. In the gastrocnemius muscle of castrated animals, HB treatment significantly increased the number of type IIa and IIb fibers and the cross-sectional area of type I and type IIa fibers. These findings suggest that oral application of HB triggers selective anabolic response with minimal or no androgenic side-effects and begin to elucidate the putative cellular targets for plant brassinosteroids in mammals.—Esposito, D., Komarnytsky, S., Shapses, S., Raskin, I. Anabolic effect of plant brassinosteroid.

Brassinosteroids are plant-specific polyhydroxylated derivatives of 5α-cholestane, structurally similar to cholesterol-derived animal steroid hormones and ecdysteroids from insects. They are found at low levels in pollen, seeds, leaves, and young vegetative tissues throughout the plant kingdom (1). The first biologically active plant brassinosteroid was isolated from the pollen of rapeseed Brassica napus in 1979 (2). The natural occurrence of >50 compounds of this group has been reported following the initial discovery (3). The brassinosteroids function in cell elongation and cell division and have been particularly studied in relation to processes such as germination and plant photomorphogenesis (4).

Similar to animal steroid hormones (5), brassinosteroids regulate the expression of specific plant genes and complex physiological responses involved in growth (6), partly via interactions with other hormones setting the frame for brassinosteroid responses (7). While animal steroid hormones are perceived by nuclear receptor family of transcription factors, brassinosteroids signal through a cell surface receptor kinase-mediated signal transduction pathway (8, 9) that includes inactivation of glycogen synthetase kinase 3 (GSK3)-like kinase BIN2 by dephosphorylation at a conserved phospho-tyrosine residue pTyr 200, therefore allowing for accumulation of transcriptional factors BZR1 and BES1 in the nucleus (10). This signal transduction pathway shares a striking parallel with animal Wnt signaling. In mammals, Wnt binds to its receptor, Frizzled, causing inhibition of GSK-3β kinase activity and subsequent accumulation of β-catenin in the nucleus, where it directly affects the transcription of target genes (11). Although BIN2 is homologous to GSK-3β, transcriptional factors BZR1 and BES1 are unique plant proteins that do not share sequence similarity with β-catenin (12).

Akt is a serine/threonine kinase that signals downstream of growth factor receptors and phosphoinositide-3 kinase (PI3K); therefore, growth factor receptors, nutrients, and even muscle contraction increase Akt activity (13). Akt stimulates glucose uptake, glycogen synthesis, and protein synthesis via Akt/mTOR and Akt/GSK-3β signaling networks (14) and inhibits apoptosis and protein degradation in skeletal muscle by inactivating FoxO transcription factors (15). Akt is therefore situated at a critical juncture in muscle signaling where it responds to diverse anabolic and catabolic stimuli.

Very little is known about the effects of brassinosteroids in animals. A natural brassinosteroid and its synthetic derivatives were found to inhibit herpes simplex virus type 1 (HSV-1) and arenavirus (16), measles (17), Junin (18), and vesicular stomatitis virus (19) replication in cell culture. A synthetic brassinosteroid analog prevented HSV-1 multiplication and viral spreading in a human conjunctival cell line with no cytotoxicity and reduced the incidence of herpetic stromal keratitis in mice when administered topically (20), possibly by the modulation of the response of epithelial and immune cells to HSV-1 infection (21). Natural brassinosteroids also inhibited growth of several human cancer cell lines without affecting the growth of normal cells (22). 24-Epibrassinolide, the most widely used brassinosteroid in agriculture, has a favorable safety profile. The median lethal dose (LD₅₀) of this compound is >1000 mg/kg in mice and >2000 mg/kg in rats when applied orally or subcutaneously (23).

28-Homobrassinolide (HB; Fig. 1A) is almost as active as 24-epibrassinolide in inducing plant growth in various bioassay systems (24). HB is a steroidal lactone initially isolated from pollen of Chinese cabbage Brassica campestris var pekinensis (25) and anthers of Japanese cedar Cryptomeria japonica (26). It is readily available through chemical synthesis, as its concentration in plants is very low (27). The plant growth-promoting effect of HB is associated with the increased synthesis of nucleic acids and proteins (28, 29). In addition, HB activated total protein synthesis, induced de novo polypeptide synthesis, and increased thermotolerance of total protein synthesis in plants subjected to heat shock (30).

Figure 1.

Chemical structure of HB (A) in comparison with testosterone (B) and 20-hydroxyecdysone (C).

Almost no data exist on the in vivo effects of brassinosteroids in animals. Given the importance of identifying novel agents that influence muscle growth, development, and/or regeneration with possible therapeutic application for the age- or disease-related skeletal muscle atrophy (sarcopenia), we sought to explore the effects of HB on protein synthesis and degradation in animals and to study the signal transduction pathways it interacts with. Furthermore, we compared the activity of HB to that of insulin-like growth factor-1 (IGF-1) in vitro and showed that the PI3K/Akt pathway is up-regulated in skeletal muscle cells treated with HB. We also found that HB treatment produced anabolic effects and improved physical fitness in healthy animals without detrimental androgenic effects. These data establish selective anabolic action of plant brassinolides in animals and provide important insight into the role of Akt signaling in mediating this activity.

MATERIALS AND METHODS

Chemicals

HB [(22S, 23S, 24S)-2α,3α,22,23-tetrahydroxy-24 ethyl-β-homo-7-oxo-5α-cholestane-6-one; Fig. 1] was purchased from Waterstone Technology (Carmel, IN, USA), and its structure was confirmed by ESI-LC/MS and NMR. l-[2,3,4,5,6-³H]phenylalanine was obtained from GE Healthcare (Piscataway, NJ, USA). Reagents and enzymes used for quantitative PCR were obtained from Stratagene (La Jolla, CA, USA) and Applied Biosystems (Foster City, CA, USA). SB203580 and PD98059 were from EMD Chemicals (Gibbstown, NJ, USA). All other chemicals and cell culture media were obtained from Invitrogen (Carlsbad, CA, USA) and Sigma (St. Louis, MO, USA) unless specified otherwise.

Cell culture

Rat L6 skeletal muscle cell line CRL-1458 was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Myoblasts were routinely maintained in DMEM containing 10% FBS and 0.1% penicillin-streptomycin at 37°C and 5% CO₂. Cells were subcultured into 24-well plates for protein synthesis, degradation, and cell viability studies and 6-well plates for Western blot analysis (Greiner Bio One, Monroe, NC, USA). Once cells reached 90% confluence, differentiation was induced by lowering the serum concentration to 2%, and medium was changed every 2 d. After 7–9 d of culture, the myoblasts had fused into multinucleated myotubes (31).

Cell viability assay and dose range determination

Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in triplicate, essentially as described previously (32), and quantified spectrophotometrically at 550 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The concentrations of test reagents that showed no changes in cell viability compared with that of the vehicle (0.1% ethanol) were selected for further studies.

Measurement of protein synthesis

For the HB dose response, fully differentiated myotubes were washed with serum-free DMEM and treated in triplicate with vehicle (0.1% ethanol), increasing concentrations of HB, or 6.5 nM of IGF-1 as positive control. Compounds were added to serum-free medium containing 0.5 μCi/ml [³H]phenylalanine and incubated for 4 h. For HB time course study, fully differentiated myotubes were treated with 3 μM HB for 1–24 h using the same culture conditions. The incubation was stopped by placing cells on ice, discarding the medium, and washing the cells extensively with ice-cold PBS to remove the nonincorporated trace. Proteins were precipitated with 5% trichloroacetic acid and dissolved in 0.5 N NaOH (33). Specific radioactivity of protein-bound phenylalanine was quantified using liquid scintillation counter LS 6500 (Beckman Coulter, Fullerton, CA, USA) and normalized to milligrams of total protein, determined by BCA protein assay (Pierce Biotechnology, Rockford, IL, USA).

Measurement of protein degradation

The effect of HB on protein degradation was investigated in fully differentiated myotubes as described previously (34), with slight modifications. For the HB dose response, fully differentiated myotubes were incubated for 16 h to allow labeling of cellular proteins with 1.5 μCi/ml [³H]phenylalanine. Cells were washed twice with PBS to remove the nonincorporated trace and treated for 4 h with vehicle (0.1% ethanol), increasing concentrations of HB, or 10 nM of insulin in serum-free medium. The incubation was stopped by placing the cells on ice, and protein in the medium was precipitated with 5% trichloroacetic acid. Specific radioactivity of protein-free phenylalanine was quantified using liquid scintillation counter LS 6500 (Beckman Coulter) and normalized to milligrams of total cell protein, determined by BCA protein assay (Pierce Biotechnology).

Western blot analysis

Fully differentiated L6 myotubes were cultured as described above, and whole-cell extracts were prepared in ice-cold RIPA buffer supplemented with 10 mM sodium fluoride, 2 mM sodium orthovanadate, 1 mM PMSF, and protease inhibitor cocktail (Sigma) and centrifuged at 12,000 g for 20 min at 4°C. Equal amounts of protein (50 μg) from the supernatants were separated on 10% SDS-polyacrylamide gels and blotted onto the nitrocellulose membrane. Western blot detection was performed with monoclonal phospho-Akt (Ser473) antibodies according to the manufacturer's instructions (Cell Signaling Technology, Danvers, MA, USA). After being washed, the blots were incubated with an anti-rabbit peroxidase-labeled secondary antibody and visualized using ECL Western blotting detection reagent (GE Healthcare). After being stripped, the same blots were probed with total Akt antibodies to serve as loading controls.

Androgen receptor binding

Rat androgen receptor binding assays were performed by MDS Pharma Services (Taipei, Taiwan; Study No. 1019130 and 1096057) as described elsewhere (35). Vehicle (1% DMSO), increasing concentrations of HB, or methandrostenolone were incubated in presence of specific binding ligand [³H]mibolerone for 4 h at 4°C, and disintegrations per minute of the incubation buffer were measured to quantify displacement of the ligand. Each treatment was repeated 2–3 times, and the results were averaged.

Animal studies

All animal experiments were performed according to procedures approved by the Rutgers Institutional Animal Care and Use Committee in an Association for Assessment and Accreditation of Laboratory Animal Care accredited animal care facility. Six-week-old male Wistar rats (180–220 g; Charles River Laboratories, Wilmington, MA, USA) were housed in individual chambers in a room maintained at a constant temperature with 12-h light-dark cycle and had free access to food and water. Animals were allowed to adapt to new conditions for 7 d, and animals were handled daily during this time to reduce the stress of physical manipulation. Animals were randomly distributed into groups according to body weight 1 d before dosing.

Protocol 1

Three groups of Wistar rats (n=6) fed normal diet containing 23.9% protein, 10.7% fat, 5.1% fiber, and 58.7% carbohydrates, resulting in 4.61 kcal/g energy value (Rodent Chow Diet 5001; Purina, St. Louis, MO, USA) were gavaged daily for 24 d with 1 ml of vehicle (5% DMSO in corn oil) or 20 or 60 mg/kg body weight of HB. The body weight of each animal and the total amount of food consumed (accounting for spillage) were recorded every 2 d for the duration of the experiment. At the end of experiment, blood was collected by heart puncture after CO₂ inhalation, and animal body composition was assessed before necropsy. At necropsy, tissue weights were recorded, then tissue samples were collected by snap-freezing in the liquid nitrogen and stored at −80°C for further studies.

Protocol 2

Three groups of Wistar rats (n=8) fed high-protein diet containing 39.4% protein, 10.0% fat, 4.3% fiber, and 37.0% carbohydrates, resulting in 3.93 kcal/g energy value (diet 5779; Testdiet/Purina, Richmond, IN, USA) were gavaged daily for 24 d with 1 ml of vehicle (5% DMSO in corn oil) or 20 or 60 mg/kg of HB. All procedures and measurements followed the protocol 1 setup.

Protocol 3

Four-week-old sham-operated (sham; n=6) or orchiectomized (ORX; n=24) Wistar rats (Charles River Laboratories) were subject to 10 d Hershberger assay (surgically castrated peripubertal adult model) under the following experimental conditions: sham, ORX (vehicle), ORX (20 mg/kg of HB orally), ORX (60 mg/kg of HB orally), and ORX (0.4 mg/kg of testosterone propionate subcutaneously, serving as a positive control for the assay). All procedures and measurements followed the protocol 1 setup except no body composition measurements were taken. Limb grip strength was measured for control animals and animals receiving 60 mg/kg of HB using a digital force gauge (Wagner Instruments Model FDV5; Product Safety Laboratories, Dayton, NJ, USA). After the rats were allowed to grip the screen with paws, the animals were quickly pulled until the paws released from the screen, and the required release force was recorded. Three trials on each animal were performed in triplicate, and significance was determined using Student's t test (P<0.05). In addition to gastrocnemius muscle, androgen-sensitive tissues (ventral prostate, seminal vesicles, bulbocavernosus/levator ani muscle complex, glans penis, and Cowper's gland) were dissected out and weighed.

Protocol 4

Four-week-old sham-operated (n=6) or ORX (n=24) Wistar rats (Charles River Laboratories) were subject to 10 d Hershberger assay (surgically castrated peripubertal adult model) under following experimental conditions: sham, ORX (vehicle), ORX (0.4 mg/kg of HB subcutaneously), ORX (4 mg/kg of HB subcutaneously), and ORX (0.4 mg/kg of testosterone propionate subcutaneously, serving as a positive control for the assay). All procedures and measurement followed the protocol 4 setup.

Body composition

Body composition was assessed by dual-energy X-ray absorptiometry (DEXA) using Lunar Prodigy total body scanner with a total body scanner (Prodigy Advanced; GE-Lunar Corp., Milwaukee, WI, USA) that uses Encore small animal body software. Quality control was performed daily. The coefficient of variation for total body, lean mass, and bone mineral content was 1.93, 3.43, and 2.10%, respectively, as measured in 5 rats scanned 3 times each.

Muscle histology

The muscle samples for histochemical analysis were taken from the middle section of the mixed-fiber gastrocnemius muscle of the castracted animals according to the protocol 4 setup to allow us to observe differences in fiber type distribution and cross section area associated with ORX and HB treatments. Serial transverse cryosections (10 μm) were prepared from each muscle and were analyzed for myofibrillar adenosine triphosphatase (mATPase) histochemistry after alkaline (pH 9.5) preincubation. Fiber cross section area and enzyme activity levels were determined from digitized images of the muscle cross-sections that were stored as gray-level pictures using ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA).

Assays of plasma samples

Blood samples were taken from animals by heart puncture after overnight food deprivation, collected in EDTA-coated tubes, and centrifuged at 1500 g for 20 min, and separated plasma was stored at −80°C until analysis. Glucose in blood samples was measured with a Lifescan glucometer (Johnson and Johnson, New Brunswick, NJ, USA). Plasma concentrations of insulin were determined by rat/mouse insulin ELISA kit (Millipore, Billerica, MA, USA; assay sensitivity, 0.2 ng/ml; intra-assay and interassay coefficients of variation, 0.9–8.4 and 6.0–17.9%, respectively; accuracy 83–102%). Plasma triglycerides and total cholesterol were measured by enzymatic colorimetric assays (Wako Diagnostics, Richmond, VA, USA; intra-assay and interassay coefficients of variation, 9–10%). Total testosterone in plasma samples was quantified by ELISA assay (DRG Diagnostics, Marburg, Germany; assay sensitivity, 0.066 ng/ml; intra-assay and interassay coefficients of variation, 6.5–11.1 and 9.3–11.3%, respectively; accuracy, 84–123%).

Statistics

Statistical analyses were performed using Prism 4.0 (GraphPad Software, San Diego, CA, USA). Unless otherwise noted, data were analyzed by 1-way ANOVA with treatment as a factor. Post hoc analyses of differences between individual experimental groups were made using the Dunnett's multiple comparison test. Body weight gain was analyzed by 2-factor repeated-measures ANOVA, with time and treatment as independent variables. Significance was set at P < 0.05. Values are reported as means ± se.

RESULTS

Effect of HB on protein synthesis

HB showed no toxicity to fully differentiated L6 rat skeletal myotubes up to 25 μM, as established by the MTT assay and cytological observations (data not shown). To determine whether HB induces protein synthesis, the incorporation of the radiolabeled [³H]phenylalanine was assessed. Cells were treated with several concentrations of HB (0.3–20 μM) for 4 h. At the lower concentration, 1 μM HB increased protein synthesis by 12.4 ± 2.3% above control levels (P<0.05). A response approached saturation between 10 and 20 μM of HB (EC₅₀ 4 μM), with increases of 34.9 ± 3.1 and 36.9 ± 2.9%, respectively (Fig. 2A). IGF-1 at 6.5 nM served as positive control in this assay; it increased protein synthesis by 42.5 ± 4.5%. Higher concentrations of HB were less effective (not shown). To investigate the kinetics of HB effect on protein synthesis, a 1- to 24-h study was performed with 3 μM HB, selected as 50% effective dose to treat myotubes. A time-dependent increase in protein synthesis in response to HB was observed (Fig. 2B). HB-stimulated protein synthesis peaked at 3 h and started to decrease after 4 h. We observed similar kinetics of protein synthesis increase in response to IGF-1 treatment, although of a greater magnitude.

Figure 2.

Concentration- and time-dependent effects of HB on protein synthesis and degradation in L6 rat myotubes. A) Cells were incubated for 4 h with [³H]phenylalanine and treated in triplicate with vehicle (0.1% ethanol), increasing concentrations of HB, or 6.5 nM of IGF-1 as a positive control, and protein synthesis was measured as incorporation of [³H]phenylalanine into protein normalized by total protein. B) To measure time-dependent effect of HB treatment on protein synthesis, cells were treated with 3 μM HB or 6.5 nM IGF-1 for 1–24 h. C) Dose-dependent effect of HB on protein degradation was observed in cells labeled overnight with [³H]phenylalanine and subsequently treated for 4 h with increasing concentrations of HB or 10 nM of insulin as a positive control, and then protein degradation was measured as release of acid-soluble [³H]phenylalanine into medium. D) For HB time-course study of protein degradation, fully differentiated myotubes were treated with 3 μM HB or 10 nM of insulin for 1–4 h. Results are expressed as means ± se of determinations performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; 1-way ANOVA and Dunnett's posttest.

Effect of HB on protein degradation

IGF-1 has both an anabolic effect on protein synthesis and an anticatabolic effect on protein degradation in skeletal muscle, similar to insulin (36). Protein synthesis is more sensitive to IGF-1 infusion than to insulin infusion and is not mediated by insulin receptors (37). On the contrary, insulin affects protein turnover by inhibiting protein degradation (38). To assess whether HB affects protein degradation, we monitored degradation of proteins labeled with [³H]phenylalanine by the release of acid-soluble radioactivity into the medium. HB at concentrations of 0.3–20 μM inhibited protein degradation dose dependently, and its activity reached a plateau between 3 and 10 μM (Fig. 2C). At the lower concentration, 1 μM HB decreased protein degradation by 8.2 ± 0.6% above control levels (P<0.05). At the higher concentration, 10 μM HB decreased protein degradation by 9.5 ± 0.9% above control levels (P<0.05). Insulin at 10 nM served as positive control in this assay; it reduced protein degradation by 13.0 ± 1.6%. To investigate the kinetics of protein degradation in response to HB, a 1- to 4-h study was performed with 3 μM HB. Suppression of protein degradation occurred time dependently and reached a plateau at 3 h for both HB and insulin (Fig. 2D).

HB stimulates phosphorylation of Akt

IGF-1 inhibits protein degradation in myotubes through PI3K/Akt/GSK-3β- and PI3K/Akt/mTOR-dependent mechanisms (39). Therefore, Akt is the key intermediate in the IGF-1 signaling pathway that modulates downstream targets known to regulate protein synthesis and degradation (40). To characterize more closely the transduction pathway through which HB signals to induce positive net protein balance, we next investigated the phosphorylation level of Akt in L6 myotubes. Consistent with the results obtained with the [³H]phenylalanine incorporation assay, HB stimulated phosphorylation of Akt in dose- and time-dependent manner (Fig. 3). Increasing concentrations of HB stimulated Ser473 phosphorylation of Akt up to 3-fold with 3 μM after 1 h of treatment (Fig. 3A). Akt stimulation was detected at 30 min after addition of HB and phosphorylation was maintained up to 1 h, whereas total Akt protein levels were also increased at some time points. The ratio of phospho-Akt to total Akt normalized to control values increased up to 3-fold following the treatment (Fig. 3B). Although the effect of HB on Akt phosphorylation is not as robust as that described for IGF-1 (14), these data support a role for the PI3K/Akt pathways in HB stimulation of anabolic signaling in L6 myotubes.

Figure 3.

HB increases Akt (Ser473) phosphorylation in L6 myotubes. A) Representative immunoblot of Akt phosphorylation stimulated with the increasing doses of HB or 6.5 nM IGF-1 as a positive control. B) Representative immunoblot of time-dependent Akt phosphorylation in response to 3 μM HB or 15 min exposure to 6.5 nM IGF-1 as a positive control. Cells were treated with the indicated doses of HB and cell lysates were then analyzed by immunobloting with phospho- and non-phospho-specific antibodies. Phospho-Akt (pAkt) bands were quantified by ImageJ software, normalized to unphosphorylated protein, and depicted as relative increase in band intensity compared with control lysate.

Next, we wanted to investigate the molecular mechanism responsible for the anabolic effect of HB in mammalian cells. To test whether PI3K signaling is responsible for HB-mediated protein synthesis, we used the specific Akt inhibitor triciribin and the PI3K inhibitor LY294002. The anabolic response of L6 cells to HB treatment was abolished by the addition of both inhibitors from 50% over control to only 4%. A similar effect was observed with PKC inhibitor GO6976, while MEK1 inhibitor PD98059 and p38 MAPK inhibitor SB203580 had no effect (Fig. 4).

Figure 4.

Stimulation of protein synthesis by HB depends on PI3K/Akt and PKC but not on MAPK signaling. Pretreatment with Akt inhibitor triciribine (20 μM), PI3K inhibitor LY294002 (25 μM), and PKC inhibitor GO6976 (10 nM) inhibited HB-mediated (3 μM) protein synthesis in L6 rat skeletal muscle cells, while MEK1 inhibitor PD98059 (2 μM) and p38 MAPK inhibitor SB203580 (50 nM) had no effect. IGF-1 (6.5 nM) served as a positive control. Data represent an averages ± se of 3 independent experiments. *P < 0.05, **P < 0.01 vs. control; 1-way ANOVA and Dunnett's posttest.

Anabolic effects of HB on body composition

Anabolic is defined as any state in which nitrogen is differentially retained in lean body mass, either through stimulation of protein synthesis and/or decreased breakdown of protein anywhere in the body (41). To evaluate the potential anabolic effects of plant brassinosteroids in animals, we orally administered 20 and 60 mg/kg body weight of HB (HB20 and HB60 treatment, respectively) daily to healthy rats fed normal diet for 24 d. By the end of the treatment, the total body weight gain relative to initial body weight in rats treated with HB20 or HB60 was 18.3 and 26.8% more compared with vehicle-treated controls (Fig. 5A). A slight but statistically significant increase in total daily food intake (20.8±0.4 g for controls; 22.2±0.8 g for HB20 group; and 23.6±0.5 g for HB60 group) was associated with HB administration, but when adjusted for body weight, food intake did not differ among all groups (Fig. 5B). Therefore, the increase in body weight gain in the HB-treated groups could not be attributed to changes in animal feeding habits. Body composition, determined by DEXA analysis showed that increase in lean body mass was significantly higher in HB20 (7.0%) and HB60 animals (14.2%). Fat mass was slightly lower in HB20 (−3.9%) and HB60 groups (−4.9%) vs. their control counterparts. Thus, the greater body weight gain in the HB-treated rats was predominantly due to increased lean mass (Table 1). Administration of HB increased gastrocnemius muscle mass by 15.6 and 19.0% in HB20 and HB60 animals, respectively. Total body bone mineral content was slightly higher in HB-treated animals, but the difference did not reach significance. Supplementation with HB had no effect on basal plasma cholesterol or triglycerides. A higher dose of HB was associated with slightly lower plasma glucose levels (4.5±0.3 mM) vs. controls (5.0±0.3 mM), but the difference did not reach statistical significance. Insulin levels were slightly elevated (Table 1).

Figure 5.

Effect of HB on body weight gain and food intake in rats fed normal (A, B) and high-protein diet (C, D). Animals received 20 (HB20) or 60 (HB60) mg/kg body weight of HB daily for 24 d. Food intake (FI) was recorded daily, and cumulative food intake was normalized for 350 g body weight. Results are expressed as means ± se. Body weight gain was analyzed by 2-factor repeated-measures ANOVA, with time and treatment as independent variables. *P < 0.05 vs. control; 1-way ANOVA and Dunnett's posttest.

Table 1.

Body composition and blood biochemistry of rats treated with HB

Parameter	Normal diet			High-protein diet
	Control	HB20	HB60	Control	HB20	HB60
Body weight (g)	308.8 ± 4.2	324.3 ± 11.5	342.3 ± 4.9*	316.9 ± 6.5	337.6 ± 6.4	335.8 ± 5.1
Body weight gain (g)	107.0 ± 2.9	126.6 ± 7.1	135.7 ± 9.4*	76.2 ± 5.7	98.3 ± 5.2*	91.1 ± 3.2*
Lean mass (g)	250.8 ± 9.1	268.3 ± 9.0	286.5 ± 4.3*	232.8 ± 6.9	246.8 ± 7.4	258.6 ± 2.7*
Fat mass (g)	51.0 ± 5.9	49.0 ± 4.3	48.5 ± 3.8	62.7 ± 1.6	68.6 ± 3.1	61.2 ± 3.8
Bone mineral content (g)	7.1 ± 0.2	7.0 ± 0.3	7.4 ± 0.1	7.0 ± 0.1	7.5 ± 0.2	7.4 ± 0.1
Gastrocnemius muscle (g)	1.79 ± 0.06	2.07 ± 0.06	2.13 ± 0.12*	1.90 ± 0.04	1.98 ± 0.01	2.17 ± 0.03***
Glucose (mM)	5.0 ± 0.2	5.1 ± 0.5	4.5 ± 0.3	5.2 ± 0.1	5.1 ± 0.2	4.8 ± 0.2
Insulin (ng/ml)	1.92 ± 0.16	2.39 ± 0.58	2.65 ± 0.45	1.80 ± 0.27	2.39 ± 0.34	2.38 ± 0.41
Cholesterol (mg/dl)	69.0 ± 3.2	69.9 ± 6.0	84.2 ± 5.3	89.2 ± 4.2	97.5 ± 6.0	87.4 ± 2.9
Triglycerides (mM)	1.9 ± 0.2	1.8 ± 0.2	1.8 ± 0.5	1.4 ± 0.1	1.2 ± 0.1	1.3 ± 0.1

Rats were fed either normal (23.9% protein content) or high-protein (39.4% protein content) diet and gavaged daily with 20 or 60 mg/kg body weight of HB for 24 d. Body composition was measured by DEXA. Results are expressed as means ± se.

^*P < 0.05, **P < 0.01, ***P < 0.001 vs. appropriate control; 1-way ANOVA and Dunnett's posttest.

Anabolic effects of HB in rats fed high-protein diet

It has been shown that short-term increase in dietary protein can favor lean body mass and reduce body fat in rats, possibly due to initial decrease in food intake that gradually returns to normal with time (42). To investigate whether high-protein diet can further enhance HB-associated effect on lean mass and muscle mass, rats fed high-protein diet (39.4% protein) were orally administered with 20 and 60 mg/kg body weight of HB daily for 24 d. Indeed, control animals fed high-protein diet consumed less food and gained less weight than control animals fed normal diet (Table 1). The stimulatory effects of HB on body weight and food consumption were apparent on the background of both normal and high-protein diet. The high-protein diet possibly enhanced the stimulatory effect of the lower dose of HB (20 mg/kg) on the body weight gain (Fig. 5C). No HB-associated increase in food intake was observed in these animals (Fig. 5D). There were no additional differences in body composition or blood biochemistry that could be attributed to a high-protein diet (Table 1).

Androgen receptor binding

Since administration of classical mammalian steroids often causes both anabolic and androgenic effects, we performed a study to rule out the possibility that HB activates androgen receptor. Competitive binding assay to the rat nuclear androgen receptor in the presence of the labeled [³H]mibolerone was used to compare HB with methandrostenolone, an androgen analog used therapeutically as an anabolic agent (43). Methandrostenolone produced specific binding to the androgen receptor with an IC₅₀ of 24 nM and a binding curve similar to the endogenous ligand testosterone. However, HB showed no significant binding from concentrations of 0.01 μM up to 10 μM (Fig. 6A).

Figure 6.

HB has low androgenic activity. A) Increasing concentrations of HB or methandrostenolone (positive control, IC₅₀ 24 nM) were incubated in presence of specific androgen receptor binding ligand [³H]mibolerone for 4 h at 4°C, and disintegrations per minute of the incubation buffer were measured to quantify displacement of the ligand. B) Oral or subcutaneous administration of HB to intact or ORX rats did not affect plasma testosterone levels in animals. Sham-operated or ORX animals received either 20 or 60 mg HB/kg daily for 10 d orally, or 0.4 and 4 mg/kg of HB daily for 10 d via subcutaneous injection. No plasma testosterone was detected (ND) in ORX animals and ORX animals treated with HB as compared with a positive control, a subcutaneous injection of 0.4 mg/kg testosterone propionate daily for 10 d. Results are expressed as means ± se. *P < 0.05 vs. vehicle-treated control; 1-way ANOVA and Dunnett's posttest.

Selective effects of HB in ORX rats

All anabolic steroids are also androgenic, as they stimulate growth and function of the reproductive system. Individual drugs vary in their balance of anabolic/androgenic activity, but none of the currently available drugs are purely anabolic (41). Therefore, we examined the ability of HB vs. injected testosterone propionate (positive control) to restore androgen-dependent tissues after androgen deprivation in a surgically castrated peripubertal rat model (44). Oral and subcutaneous treatments at appropriate dose ranges were initiated 2 wk after ORX and continued for 10 d. As expected, androgen deprivation caused significant decrease in the size of the prostate, seminal vesicles, bulbocavernosus/levator ani muscle complex, glans penis, and Cowper's gland, with these organs shrinking to 8.6, 6.5, 23.9, 54.6, and 40.5%, respectively, of those observed in sham-operated animals (Table 2). Injection of testosterone propionate at 0.4 mg/kg increased the weight of androgen-sensitive organs 3- to 8-fold; however, it failed to restore ventral prostate, seminal vesicles, and bulbocavernosus/levator ani muscle complex to their original size as compared with sham controls. After 10 d of treatment, oral administration of HB at 20 and 60 mg/kg failed to prevent the loss of androgen sensitive tissue weight associated with ORX, although a slight but significant dose-dependent increase in glans penis was associated with HB treatment (55.3±1.7 mg for H₂O and 59.6±1.6 mg for H60 vs. 45.7±1.5 mg for control animals). In contrast, HB increased the weight of bulbocavernosus/levator ani muscle complex (the skeletal muscle biomarker of anabolic activity), although the change was not statistically significant. When HB was injected subcutaneously at 1- and 10-fold doses relative to positive control in the Hershberger assay (testosterone propionate at 0.4 mg/kg), androgen-sensitive tissue weights did not differ from those of ORX controls with the exception of glans penis and bulbocavernosus/levator ani muscle complex, for which a significant increase was observed at 4 mg/kg HB (Table 2).

Table 2.

Weights of androgen-sensitive tissues from sham-treated and ORX rats treated with HB

Admini-stration and treatment group	Tissue
	Ventral prostate (mg)	Seminal vesicles (mg)	Bulbocavernosus/levator ani (mg)	Glans penis (mg)	Cowper's gland (mg)
Oral
Sham	222.5 ± 15.2***	515.0 ± 12.9***	517.0 ± 10.5***	83.7 ± 0.8***	26.7 ± 1.8***
ORX	19.2 ± 2.5	33.3 ± 3.0	123.7 ± 6.8	45.7 ± 1.5	10.8 ± 1.3
ORX + HB20	26.0 ± 2.5	37.3 ± 3.5	109.2 ± 8.6	55.3 ± 1.7*	11.2 ± 0.7
ORX + HB60	23.0 ± 2.4	34.7 ± 3.3	137.7 ± 9.9	59.6 ± 1.6**	12.5 ± 1.1
ORX + TP0.4a	110.50 ± 9.5***	262.5 ± 12.5***	382.0 ± 22.0***	93.8 ± 4.2***	34.5 ± 4.7***
Subcutaneous
ORX	22.2 ± 3.1	27.8 ± 1.3	109.2 ± 6.4	41.3 ± 2.6	11.3 ± 0.7
ORX + HB0.4	18.3 ± 1.2	29.0 ± 3.1	131.3 ± 7.1	50.7 ± 1.9	11.3 ± 0.5
RX + HB4	23.2 ± 1.2	30.2 ± 1.2	147.5 ± 6.9*	56.3 ± 3.3*	10.5 ± 0.3
ORX + TP0.4	92.8 ± 6.3***	228.7 ± 35.3	293.8 ± 19.0***	72.3 ± 4.5***	34.3 ± 1.9***

Rats were fed normal diet (23.9% protein content) and gavaged daily with 20 or 60 mg/kg body weight of HB or subcutaneously injected with 0.4 and 4 mg/kg body weight of HB for 10 d. Results are expressed as means ± se.

^*P < 0.05,

^**P < 0.01,

^***P < 0.001 vs. ORX; 1-way ANOVA and Dunnett's posttest.

^aTeststerone propionate (TP) was given as subcutaneous injection at 0.4 mg/kg and served as positive control.

In sham-treated animals, oral administration of 20 or 60 mg/kg HB did not modify plasma testosterone levels. As expected, no plasma testosterone was detected following an oral or subcutaneous administration of HB to ORX animals that have virtually not detectable levels of testosterone due to orchiectomy, while a 0.4 mg/kg injection of testosterone propionate partially restored plasma testosterone levels in ORX rats to 20.5 of their original level (Fig. 6B).

Physical performance and muscle fiber distribution in ORX rats

Change in grip strength of lower extremities was significantly larger in ORX animals receiving oral administration of 60 mg/kg HB for 10 d (0.0851±0.0197 vs. −0.0143±0.0392 kg for controls). The change in grip strength for front limbs was also greater in HB-treated animals (Fig. 7A) but did not reach significance (0.2711±0.0660 vs. 0.1631±0.0405 kg for controls).

Figure 7.

HB increases physical fitness of untrained ORX rats (A), increases mass of mixed-fiber gastrocnemius muscle (B), and induces favorable changes in myofiber type distribution and cross-section area (C–E). ORX rats received vehicle or 20 or 60 mg/kg of HB daily for 10 d orally. At the end of the study, the grip strength of hind- and forelimbs of the castrated animal was measured using digital force gauge. Gastrocnemius muscle was excised and weighed, and the serial transverse cryosections of the middle section of the muscle of vehicle-treated animals (C, top panel) or animals receiving 20 mg/kg (C, middle panel) or 60 mg/kg (C, bottom panel) of HB were stained for mATPeease activity to analyze myofiber type distribution (D) and cross section area (E). Results are expressed as means ± se. *P < 0.05, ***P < 0.001 vs. vehicle-treated control; 1-way ANOVA and Dunnett's posttest.

As expected, androgen deprivation caused significant decrease in the gastrocnemius muscle mass to 85.8% of that observed in sham-operated animals (Fig. 7B). Oral administration of HB to ORX rats increased gastrocnemius muscle mass by 13.8% and 10.3% in HB20 and HB60 animals, respectively, therefore almost restoring the muscle to its original size. At the same time, subcutaneous administration of HB at doses of 1- and 10-fold of the positive control increased gastrocnemius muscle mass by 2.8 and 9.1%, respectively.

To determine the structural events underlying alterations in muscle mass and function, we analyzed changes in fiber distribution (Fig. 7C, top panel) and cross section area (Fig. 7C, middle panel) using ATPase stain that differentiates between muscle fiber types based on their oxidative and glycolytic activity. Under these conditions, type I fibers stain black, while type IIb fibers stain dark gray, and type IIa fibers remain pale gray. HB treatment in castrated mice prevented gastrocnemius fiber atrophy and increased median fiber area of type I and type IIa fibers above castrated control levels (P<0.001). Compared with control animals, fiber type distribution was significantly affected in HB-treated animals (20 or 60 mg/kg/d for 10 d). While the total number of type IIa and type IIb fibers increased by ∼60% independent of HB dose, the significant increase in number of type I fibers was observed only with higher dose of HB.

DISCUSSION

Brassinosteroids are present in small quantities in foods and plants (1). They are similar in many respects to animal steroids but appear to function very differently at the cellular level. While animal steroid hormones act through nuclear receptor family of transcription factors, plant brassinosteroids signal through a cell surface receptor kinase-mediated signal transduction pathway (8, 9). In plants, brassinosteroids play an essential role in plant growth and development (4–7), as well as thermotolerance (30). For example, 24-epibrassinolide limited the loss and increased expression level of some of the components of the translational apparatus during the heat stress, which was correlated with a more rapid resumption of cellular protein synthesis (45). The plant growth-promoting effects of HB, the brassinosteroid used in this study (Fig. 1A), are associated with increased synthesis of nucleic acids and proteins (28, 29). At the same time, brassinosteroids share some similarities with ecdysteroids (Fig. 1C) that have a wide array of physiological and pharmacological effects in animals and insects (46), including modulation of protein synthesis (47) and carbohydrate metabolism (48).

The present study demonstrates a selective anabolic effect of plant brassinosteroid in animals. Our findings suggest that HB dose and time dependently stimulated protein synthesis and inhibited protein degradation in L6 rat skeletal muscle cells (EC₅₀ 4 μM), in part by inducing Akt phosphorylation (Figs. 2 and 3). The effective HB concentrations that produced Akt activation were comparable with the concentration required to modulate protein synthesis, suggesting that HB involves Akt activation in the stimulation of protein synthesis and suppression of protein degradation. Akt is a serine/threonine kinase that signals downstream of growth factor receptors and phosphoinositide-3 kinase PI3K. Therefore, growth factor receptors, nutrients, and even muscle contraction all increase Akt activity (13). Akt stimulates glucose uptake, glycogen synthesis, and protein synthesis via Akt/mTOR and Akt/GSK-3β signaling networks (14) and inhibits apoptosis and protein degradation in skeletal muscle by inactivating FoxO transcription factors (15). Akt is therefore situated at a critical juncture in muscle signaling where it responds to diverse anabolic and catabolic stimuli. Moreover, both FOXO3a and GSK-3β modulate transcription of androgen receptor (AR); while the former promotes transcriptional activity of AR (49), the latter acts as its inhibitor (50). At the same time, GSK-3β also controls cell survival through regulation of β-catenin, 1 of the key molecules in Wnt signaling (11), and inhibition of Akt suppresses the Wnt pathway by activation of GSK-3β and degradation of ß-catenin (51). Curiously, brassinosteroid signaling in plants resembles the Wnt pathway and is mediated by GSK3-like kinase (10).

Since our initial data suggested that brassinosteroid might modulate cell survival and growth via Akt activation similar to plant ecdysteroids (52), we performed a set of experiments with known antagonists to further elucidate the signal transduction pathways involved in mediating brassinosteroid effects in mammalian cells. Specifically, we analyzed effects of triciribine (selective direct inhibitor of the cellular phosphorylation/activation of Akt1/2/3) and LY294002 (competitive inhibitor of the PI3K kinase upstream of Akt). Both treatments abolished anabolic effect of brassionosteroid treatment on protein synthesis in muscle cells. Interestingly, when the same experiment was repeated with GO6976 (specific inhibitor of another serine/threonine kinase family, the calcium-dependent protein kinase C isoforms), effect of brassinosteroid treatment on protein synthesis was also reduced to great extent, suggesting that PKC isozymes may also act as a link between an unknown receptor and Akt activation induced by brassinosteroids. However, neither PD98059 (a specific inhibitor of MEK1 kinase that functions in a mitogen activated protein kinase cascade) nor SB203580 (a specific inhibitor of p38 MAP kinase homologues) had any measurable effect on brassinosteroid-driven modulation of protein synthesis in muscle cells (Fig. 4). Taken together, we demonstrated that brassinosteroid-mediated protein synthesis in muscle cells is positively influenced by the PI3K/Akt and PKC but not by MAPK pathways.

This raises an interesting possibility of direct activation of unidentified mammalian steroid hormone receptor by the brassinosteroid. In addition to classical nuclear receptor responses, mammalian steroid hormones have been shown to elicit rapid nongenomic signaling events that mediate cell proliferation and survival. For example, cell membrane-localized estrogen receptor has been demonstrated to interact with the regulatory subunit of PI3K and thereby to increase Akt activity (53). In addition, putative progesterone G-protein coupled receptor has been cloned, and a putative membrane-bound AR has been suggested (54). A similar receptor may be responsible for the rapid effects of HB in mammalian cells.

Oral 24-d administration of HB to healthy rats selectively increased body weight gain, lean body mass, and gastrocnemius muscle mass as compared with vehicle-treated controls (Fig. 5 and Table 1). In vivo action of HB on body composition and bone could not be attributed to endogenous testosterone (Fig. 1B) action, as plasma testosterone levels did not differ in response to HB treatment (Fig. 6B). Supplementation of HB-treated animals with high-protein diet enhanced the effect of the lower dose of HB (Table 1). As expected (42), control animals fed a high-protein diet (Fig. 5C, D) exhibited decreased body weight gain, food intake, and other body composition parameters compared with control animals fed normal diet (Fig. 5A, B). Their plasma tryglicerides were also decreased (Table 1). Experiments comparing diets with different protein contents demonstrated favorable changes in body composition for high-protein diets, with part of these alterations being attributed to an increased intake of leucine (55). Since leucine and other branched amino acids do not affect the phosphorylation status of Akt but rather stimulate anabolic signaling in skeletal muscle cells through modulation of mTOR and 4EBP1 phosphorylation (56), we proposed that high-protein diet may potentiate anabolic effects of HB. Treatment with HB did not modify blood biochemistry in animals fed either normal or high-protein diet, with the exception of fasting glucose that was slightly lower in cohorts receiving higher dose of HB.

HB showed very low androgenic activity when tested in the Hershberger assay (Table 2) and improved physical fitness of untrained ORX rats (Fig. 7). Although HB produced anabolic effects in animals similar to androgens, they seemed to be pharmacologically different, as HB administration (oral or subcutaneous) produced only minimal androgenic side effects, in sharp contrast to powerful androgenic effects of anabolic steroids. The additional observation that HB has low or no significant binding to the androgen receptor and did not modulate plasma testosterone levels (Fig. 6A) suggests that HB may exert its anabolic effect through an androgen-independent mechanism. Even though both HB and androgens contain similar steroid backbone, there are major structural differences that distinguish the 2 classes of compounds, including the lactone function at C6/C7, the 2 hydroxyls at C2 and C3, and the methyl substitution at C24. These chemical differences may restrict HB from activating the nuclear androgen receptor and explain the difference in pharmacological responses. It cannot be excluded that HB administration could change the abundance or phosphorylation status of the androgen receptor by modulating FOXO3a and GSK-3β signaling networks downstream of Akt (49, 50). However, the differential effect of HB on physical fitness of front and hindlimbs of untrained rats (Fig. 7) seems to indicate that a stronger pharmacological response was observed in hindlimb area where the abundance of androgenic receptor is typically lower in males (57).

Skeletal muscle shows plasticity and can undergo conversion between different fiber types in response to exercise training, modulation of motoneuron activity, or castration (55). In this study, we show that 10 d oral administration of HB to castrated animals led to substantial increases in the total number of myofibers and the cross-sectional area of oxidative type I and type IIa muscle fibers important for increased physical performance and endurance.

HB was safe in rats when tested at doses up to 1000 mg/kg (58). At the same time, brassinosteroids caused cell cycle arrest and apoptosis of human breast cancer cells when tested at doses >30 μM, without affecting the normal nontumor cell growth of BJ fibroblasts (22). This effect is similar to classical mammalian steroid hormones that either inhibit or induce apoptosis in the concentration- or tissue-specific manner (59).

In summary, we hypothesize that HB may exert its anabolic effect by stimulating protein synthesis and inhibited protein degradation in muscle cells, in part by inducing PI3K/Akt signaling. The stimulatory effect of HB on protein synthesis in muscle cells subsequently translates into whole body anabolic effects, such as increases in lean body mass, muscle mass, and physical performance. Moreover, our data demonstrate that oral application of HB triggers an anabolic response with minimal or no androgenic side effects. This property may pharmacologically differentiate HB from anabolic steroids.