Androgen receptor

By upregulating Fst mRNA via AR at 500 nM, YK11, a partial agonist of androgen acquisition, causes myogenic secretion in C2C12 cells. Although it needs to be at high doses (100 nM or 500 nM), YK11 increases the expression of Myf5 and Myogenin mRNA [1]. YK11 (0.5 μM) stimulates osteoblast MC3T3-E1 cell proliferation and amplifies ALP activity via AR. Additionally, YK11 boosts the quantitative expression of osteocalcin mRNA (0.1-1.0 μM) and osteoprotegerin mRNA (0.5 μM). Furthermore, YK11 uses quick non-genomic signaling to increase the phosphorylation of the Akt protein [2].

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YK11 and DHT Induce Myogenic Differentiation of C2C12 Cells [1]
Firstly, we examined whether AR is expressed in C2C12 myoblast cells. AR expression was detected in C2C12 cells during cell differentiation by immunoblot assay (Fig. 1A). To investigate the effect of YK11 on C2C12 cells, the expression of the differentiation marker, myosin heavy chain (MyHC), was examined. C2C12 cells were cultured with YK11, DHT or solvent in differentiation medium. The MyHC protein level on Day 7 was enhanced by both YK11 and DHT treatments (Fig. 1B), suggesting that like DHT, YK11 can induce myogenic differentiation of C2C12 cells.

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YK11 and DHT Upregulate the mRNA Expression of MRFs [1]
Next, we analyzed the mRNA expression of the MRFs, Myf5, MyoD and myogenin. Cells were treated with YK11, DHT or solvent, and the expression of Myf5, MyoD and myogenin was analyzed by qRT-PCR on Day 2 and 4. Myf5 and myogenin mRNA expression on Day 4 was more significantly enhanced by YK11 treatment than by DHT treatment (Figs. 1C, E). Interestingly, upregulation of Myf5 and myogenin mRNA on Day 4 by YK11 treatment required high YK11 concentrations (100 nM or 500 nM; Figs. 1F–H), whereas DHT increased the expression of these genes at lower concentrations. Co-treatment with the AR antagonist, FLU, suppressed this upregulation, suggesting that YK11-induced MRFs expression may be mediated by AR (Figs. 2A–C).

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YK11 Induces Myf5 mRNA Expression via Fst mRNA Upregulation by AR [1]
It has been reported that androgen-induced myogenic differentiation is regulated by Fst, which stimulates Myf5 expression.23,24) Fst modulates the function of a number of TGF-β family members, such as myostatin, which is a negative regulator of myogenic differentiation, by directly binding to them. We speculated that the AR-dependent Fst induction may be responsible for the functional difference between YK11 and DHT. To verify this hypothesis, Fst expression was measured on Day 2 and Day 4 after the addition of YK11 or DHT. Fst mRNA expression was significantly induced by YK11 treatment, whereas it was not affected by DHT treatment (Fig. 3A). The YK11-induced upregulation of Fst mRNA was significantly reduced by co-treatment with FLU, supporting the AR-dependence of the YK11-mediated upregulation of Fst expression (Fig. 3B). Furthermore, we carried out an AR knockdown experiment. Similarly to the FLU treatment, the YK11-induced upregulation of Fst mRNA was significantly reduced by knockdown of AR (Fig. 3C).

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Effect of YK11 on Osteoblast Proliferation [2]
The first step of osteoblast differentiation is known to be cell proliferation. Thus, to investigate whether YK11 accelerates osteoblast cell growth, we performed the MTS assay. Osteoblastic MC3T3-E1 cells were treated with YK11 0.5 µM and DHT 0.01 µM for 96 h. The result showed that the cell growth were increased by YK11 and DHT treatments. Further, these observations were reversed by co-treatment with the AR antagonist, HF (Fig. 1). These results indicate that YK11 accelerates osteoblast cell proliferation via AR similar to DHT.

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Effect of YK11 on Osteoblast ALP Activity and Mineralization [2]
Increase in the activity of the membrane bound ecto-enzyme ALP is a key indicator of early stage in osteoblast differentiation. MC3T3-E1 cells were cultured in differentiation medium and treated with YK11 or DHT for 10 d. ALP activity of YK11 and DHT-treated cells increased as compared to that in solvent-treated cells (Fig. 2A). These increases in ALP activity were inhibited by co-treatment with HF (Fig. 2A).

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Effect of YK11 on the Expression of Osteogenic Markers [2]
Osteoprotegerin (OPG) and osteocalcin (OC) are early and late markers of osteoblast differentiation, respectively. To test the expression of these osteogenic markers in YK11-treated cells, their mRNA expression were measured by RT-qPCR. OPG mRNA expression was increased by YK11 similar to by DHT at day 4 (Fig. 3A) and day 14 (Fig. 3B). OC mRNA expression was increased by YK11 similar to by DHT at day 14 (Fig. 3D), but not at day 4 (Fig. 3C). Furthermore, YK11 increased OC mRNA expression in a dose dependent manner at day 14 (Fig. 3E).

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Effect of YK11 on Non-genomic Signaling [2]
AR was reported to directly activate the phosphoinositide 3-kinase (PI3K)/Akt pathway, which is a key regulator of osteoblast proliferation and differentiation.26) In this study, we demonstrated that YK11 promotes osteoblast cell proliferation and differentiation via AR (Figs. 1–3). To define the molecular mechanism underlying YK11 activity, we investigated whether YK11 activated Akt signaling in a manner similar to DHT. Cells were treated with YK11 or DHT for 15 min and the levels of phosphorylated Akt protein were investigated. Similar to DHT, phosphorylation of Akt protein increased upon YK11-treatment. This observation suggests that YK11 can activate the Akt signaling pathway via rapid non-genomic signaling similar to DHT (Fig. 4).

The myostatin inhibitor YK11 impedes muscle atrophy triggered by gram-negative bacteria Although muscle protein degradation in septic mice is well observed, myostatin expression and activity are not well understood. Therefore, we analyzed muscle atrophy and its relation to myostatin. To address this, we administered the myostatin inhibitor YK11 orally at the same checkpoint time for 10 days (6 h/3 times/day) into mice. Subsequently, we induced sepsis via intraperitoneal (i.p.) injection of E. coli K1 on the last day (Fig. 1A). On the 10th day of the experiment, the total body weight and ratio of recovered muscles and fats (as percentage of total body weight) were estimated. Control groups of mice were also maintained without any YK11 administration. YK11 was found to decrease weight loss in the mice in a concentration-dependent manner, i.e., total body weight of the mice increased or recovered better with increasing concentration of YK11 (Fig. 1B). However, the ratio of fats (as percentage of total body weight) tended to decrease in the YK11 treatment group. These results are consistent with research papers that myostatin deficiency increases skeletal muscle mass and reduces body fat mass [15]. Moreover, YK11 uptake helped recover the muscle mass loss in the backs and thighs of mice, which was triggered by E. coli K1 (Fig. 1C). In addition, H&E staining analysis confirmed that YK11 reduced muscle cell atrophy (Fig. 1D). YK11 potential efficiency was also shown against CRAB (Fig. S1). YK11 increased total body weight (Fig. S1B), reverted the muscle mass loss (Fig. S1C), and muscle cell atrophy (Fig. S1D). This suggests that inhibition of myostatin through YK11 can contribute to the reversal of muscle atrophy in septic mice triggered by gram-negative bacteria in both, standard and resistant strains. [3]

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YK11 attenuates the pathophysiology of gram-negative bacteria-inoculated septic mice Previous studies have clearly established that YK11 holistically affects the innate immune response. Therefore, we sought to test its effect on the pathophysiology of sepsis. Sepsis was induced by i.p. injection of E. coli K1 at 1 × 108 CFU/mouse into mice that had a daily uptake of YK11 (6 h/3 times/day). YK11 injection at 350 and 700 mg/kg increased the survival rate of septic mice by 20% and 40%, respectively, within 72 h (Fig. 2A). YK11 also significantly reduced endotoxin levels in the serum (Fig. 2B), and the levels of inflammatory cytokines TNF-α, IL-1β, IL-6, IL-12p70, and IL-10 in the lungs of mice (Fig. 2C). Additionally, the levels of organ damage biomarkers—aspartate aminotransferase (AST), alanine aminotransferase (ALT) in the liver and blood urea nitrogen (BUN) in the kidneys—were alleviated by YK11 (Fig. 2D). Furthermore, YK11 promoted bacterial clearance from the organs of septic mice (Fig. 2E). It is YK11 effect was also shown against CRAB (Fig. S2). These data suggest that YK11 prevent septic mice caused by gram-negative bacteria by regulating inflammatory cytokine levels. [3]

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Myostatin inhibition improves muscle protein metabolism in gram-negative bacteria-inoculated septic mice The above results (Fig. 2) confirmed that YK11 prevent severe septic shock in mice. To further investigate the potential role of YK11 in septic mice, we attempted to identify the mechanisms through bacterial LPS-induced NF-κB activation and TGF-β signaling. Myostatin inhibition improves muscle protein metabolism in E. coli K1-inoculated septic mice We later investigated the expression of FST, which has a natural inhibitory function against myostatin, in the same animal model. Interestingly, the level of FST, which was reduced by E. coli K1, was fully recovered in the YK11-treated group (Fig. 3A). YK11 oral uptake repressed the protein levels of the muscle regulators myostatin, myogenin, and MyoD that were increased by E. coli K1 bacteria induction (Fig. 3B). Besides myostatin, several members of the TGF-β superfamily can also signal through myostatin-like pathways, which affects the downstream phosphorylation of FOXO3a and Smad2 transcription factors. Both factors were found to decrease in the YK11-treated group (Fig. 3B). It is YK11 effect was also shown against CRAB (Fig. S3). These results suggest that YK11 can alleviate gram-negative bacteria-induced sepsis by interfering with myostatin expression to stop muscle atrophy. Based on these collective data, we propose a mechanistic model for the inhibition myogenesis by gram-negative bacteria in vivo [3].

Alizarin Red S Staining [2]
Mineralization (calcium deposit (ion)) in osteoblast differentiation was investigated by alizarin red S staining. For alizarin red S staining, cells were fixed with cold methanol at 4°C for 20 min after rinsed three times with phosphate buffered saline (PBS), and were then rinsed three times with H2O to remove methanol completely at day 10. The cells were then stained with 40 mM alizarin red S stain solution (pH 6.38) for 15 min to stain the calcium deposits. Next, the stained cells were rinsed five times with H2O to remove the unbound alizarin red S and the stained cultures were imaged.

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ALP Activity [2]
ALP activity was measured by using Lab Assay™ ALP. On the indicated experimental endpoints, after the medium was removed, the cell layers were rinsed thrice with PBS, and lysed by 0.05% Triton X-100. The cell lysate were mixed with 0.1 M carbonate buffer (pH 9.8) containing 2 mM MgCl2, and 6.7 mM of p-nitrophenyl phosphate. The reaction mixture was incubated at 37°C for 15 min and the reaction was stopped by adding NaOH. Absorbance was measured at 405 nm in a multimode detector.

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Real-Time RT-Quantitative (q)PCR [2]
Total RNA was isolated using ISOGEN II. The cDNA was synthesized using the ReverTra Ace® qPCR RT Kit. Real-time qPCR was conducted using KOD SYBR qPCR Mix in a final volume of 25 µL according to the manufacturer’s protocol, and the results were analyzed using Applied Biosystems 7500 Fast System SDS software. The primer pairs used are shown in Table 1.

Cell Culture [1]
Mouse myoblast C2C12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2. C2C12 cells were seeded on plates and maintained in culture medium for 24 h. To induce myogenic differentiation, YK11 or DHT in DMEM supplemented with 2% horse serum (differentiation medium) was added to the cells on day 0. For the neutralization assay of Fst (also known as activin-binding protein), C2C12 cells were maintained in differentiation medium in the presence of anti-Fst antibody.

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Immunoblotting [1]
Cells were harvested and lysed in sodium dodecyl sulfate (SDS) sample buffer containing 125 mM Tris–HCI, pH 6.8, 4% SDS, 10% sucrose, 10 mM dithiothreitol and 0.01% bromophenol blue. Whole-cell lysates were resolved by SDS-polyachylamide gel electrophoresis (PAGE) and immunoblotting was performed using anti-myosin heavy chain, anti-androgen receptor and anti-tubulin antibodies as primary antibodies. Horseradish peroxidase-conjugated anti-mouse or rabbit immunoglobulin G (IgG) antibody was used as secondary antibody.

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Cell Culture [2]
Mouse osteoblast cells, MC3T3-E1, were cultured in Minimum Essential Medium (MEM) α supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2. MC3T3-E1 cells were seeded on plates and maintained in MEM α supplemented with 10% charcoal-Stripped FBS (csFBS) for 24 h. To induce osteoblast differentiation, YK11 or DHT in MEM α supplemented with 10% csFBS, 50 µg/mL ascorbic acid, and 5 mM β-glycerol phosphate (differentiation medium) were added to the cells on day 0. The medium replacement was performed every 3 or 4 d.

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MTS Cell Proliferation Assay [2]
MC3T3-E1 cells were seeded in 96-well microplates at a density of 5000 cells per well for 24 h with csFBS and were treated with solvent control, DHT, or YK11 as indicated. After 96 h of incubation, cell viability was evaluated using the MTS assay kit according to the manufacturer’s instructions. The number of living cells is directly proportional to the absorbance at 490 nm of a formazan product reduced from MTS by living cells. Absorbance at 490 nm was measured in a multimode detector

Bacteria-induced sepsis mouse model [3]
Eight-week-old male BALB/c mice fed with YK11 (350, 700 mg/kg) for 10 days were assessed daily. Another group of WT (wild type) mice were also fed with YK11 at 350 and 700 mg/kg/day. Body weights and food eaten were assessed daily for 10 days. After 10 days, some WT mice were intraperitoneally injected with E. coli K1 (1 × 108 CFU/mouse) and CRAB (1 × 107 CFU/mouse) to induce sepsis. Body weight and thigh muscle weight were measured, and one of the thigh muscles was fixed with PFA. For survival rate analysis, after 30 min, YK11 -injected mice were intraperitoneally injected with 1 × 109 CFU/mouse of E. coli K1 (5 mice/group) and 1 × 109 CFU/mouse of CRAB (5 mice/group). The survival rate was observed for 72 h. For other analyses, after 30 min, YK11 -injected mice were intraperitoneally injected with 1 × 108 CFU/mouse of E. coli K1 (5 mice/group) and 1 × 107 CFU/mouse of CRAB (5 mice/group). These mice were utilized following animal ethics protocols. The lungs, livers, kidneys, and spleens were homogenized using a Bullet Blender homogenizer. The pro-inflammatory cytokines in lung lysates were measured by ELISA. The remaining bacteria in the tissue lysates were diluted with PBS and incubated overnight on LB agar plates. AST, ALT, and BUN levels in the serum were measured using total laboratory automation (Hitachi, Japan) and TBA-200FR NEO systems. Serum endotoxin levels were measured using the LAL assay method, and the color of the reaction was measured at 405 nm.

[1]. Selective androgen receptor modulator, YK11, regulates myogenic differentiation of C2C12 myoblasts by follistatin expression. Biol Pharm Bull. 2013;36(9):1460-5.

[2]. Selective Androgen Receptor Modulator, YK11, Up-Regulates Osteoblastic Proliferation and Differentiation in MC3T3-E1 Cells. Biol Pharm Bull. 2018;41(3):394-398.

[3]. Myostatin inhibitor YK11 as a preventative health supplement for bacterial sepsis. Biochem Biophys Res Commun. 2021 Mar 5:543:1-7.

The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11. [1]

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Androgens are key regulators that play a critical role in the male reproductive system and have anabolic effects on bone mineral density and skeletal muscle mass. We have previously reported that YK11 is a novel selective androgen receptor modulator (SARM) and induces myogenic differentiation and selective gene regulation. In this study, we show that treatment of YK11 and dihydrotestosterone (DHT) accelerated cell proliferation and mineralization in MC3T3-E1 mouse osteoblast cells. Further, YK11-treated cells increased osteoblast specific differentiation markers, such as osteoprotegerin and osteocalcin, compared to untreated cells. These observations were attenuated by androgen receptor (AR) antagonist treatment. To clarify the effect of YK11, we investigated rapid non-genomic signaling by AR. The phosphorylated Akt protein level was increased by YK11 and DHT treatment, suggesting that YK11 activates Akt-signaling via non-genomic signaling of AR. Because it is known Akt-signaling is a key regulator of androgen-mediated osteoblast differentiation, YK11 has osteogenic activity as well as androgen. [2]

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Muscle wasting caused by catabolic reactions in skeletal muscle is commonly observed in patients with sepsis. Myostatin, a negative regulator of muscle mass, has been reported to be upregulated in diseases associated with muscle atrophy. However, the behavior of myostatin during sepsis is not well understood. Herein, we sought to investigate the expression and regulation of myostatin in skeletal muscle in mice inoculated with gram-negative bacteria. Interestingly, the protein level of myostatin was found to increase in the muscle of septic mice simultaneously with an increase in the levels of follistatin, NF-κΒ, myogenin, MyoD, p- FOXO3a, and p-Smad2. Furthermore, the inhibition of myostatin by YK11 repressed the levels of pro-inflammatory cytokines and organ damage markers in the bloodstream and in the major organs of mice, which originally increased in sepsis; thus, myostatin inhibition by YK11 decreased the mortality rate due to sepsis. The results of this study suggest that YK11 may help revert muscle wasting during sepsis and subdue the inflammatory environment, thereby highlighting its potential as a preventive agent for sepsis-related muscle wasting.[3]

C25H34O6

430.54

430.236

C, 69.74; H, 7.96; O, 22.30

1370003-76-1

119058028

White to off-white solid powder

3.1

0

6

3

31

860

6

O1C(C)(OC)O/C(=C/C(=O)OC)/[C@]21CC[C@H]1[C@@H]3CCC4=CC(CC[C@@H]4[C@H]3CC[C@@]12C)=O

KCQHQCDHFVGNMK-PQUNLUOYSA-N

InChI=1S/C25H34O6/c1-23-11-9-18-17-8-6-16(26)13-15(17)5-7-19(18)20(23)10-12-25(23)21(14-22(27)28-3)30-24(2,29-4)31-25/h13-14,17-20H,5-12H2,1-4H3/b21-14+/t17-,18+,19+,20-,23-,24?,25+/m0/s1

methyl (2E)-2-[(8R,9S,10R,13S,14S,17S)-2'-methoxy-2',13-dimethyl-3-oxospiro[1,2,6,7,8,9,10,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthrene-17,5'-1,3-dioxolane]-4'-ylidene]acetate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.81 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)