Main Research Findings

1) YK11 is capable of improving proliferation and differentiation of osteoblasts in MC3T3-E1 mouse cells.

2) Through its ability to inhibit myostatin, YK11 can potentially act as a preventative treatment for bacterial sepsis.

 

Selected Data

1) The study conducted by Yatsu et. Al examined the potential of YK11 to improve osteoblast proliferation in mouse cells. The chemicals utilized in the experiment included: YK11, DHT, hydroxyflutamide (HF), ascorbic acid, and beta-glycerol phosphate; all obtained from Wako Pure Chemical Industries, Ltd. in Osaka, Japan. The mouse cells used were labeled as MC3T3-E1, and cultured in a mixture of Minimum Essential Medium (MEM) alpha, obtained from Wako Pure Chemical Industries Ltd., and 10% fetal bovine serum (FBS) in a humidified 5% CO2 climate. After the cells were cultured, they were seeded on plates and maintained for 24 hours in a solution of MEM alpha and 10% charcoal-stripped FBS. In order to induce osteoblast differentiation, either YK11 or DHT were added to the solution of MEM alpha, 10% csFBS, 50 ug/mL ascorbic acid, and 5 mM beta-glycerol phosphate [1].

MC3T3-E1 cells were then seeded in 96-well microplates at a density of 5000 cells per well. After seeding, the cells were maintained with csFBS for 24 hours, followed by treatment with YK11, DHT, or a control. The cells were incubated for 96 hours after treatment while an MTS assay kit evaluated the level of cell viability. The researchers thought it was important to note that the number of living cells is directly proportional to absorbance at 490 nm of the product reduced from MTS by living cells. That being said, for the sake of this study absorbance at 490 nm was measured by a multimode detector.

Following treatment, the research team investigated any changes in calcium deposition through the use of alizarin red S staining. For the staining, the cells were fixed with cold methanol for 20 min and rinsed three times with phosphate buffered saline (PBS). On day 10 the cells were rinsed three times with water in order to remove all traces of methanol. After the final rinse the cells were stained with 40 mM concentration of pH 6.38 alizarin red S stain solution for 15 minutes. In order to remove all unbound alizarin red S, the stained cells were rinsed with water 5 times followed by culture imaging [1].

Next, the researchers recorded levels of alkaline phosphatase (ALP) activity through the use of Lab Assay ALP. The medium was removed at the experimental endpoints and the cell layers were rinsed with PBS three times and lysed by 0.05% TritonX-100. The remaining cell lysates were mixed with a 0.1 mM carbonate buffer composed of a solution of 2 nM MgCl2 and 6.7 mM of p-nitrophenyl phosphate, followed by 15 minutes of incubation. NaOH was added to the mixture by the researchers in order to stop the reaction, and a multimode detector measured the absorbance at 405 nm. Additionally, a real-time quantitative PCR (RT-qPCR) was completed by isolating total RNA. RT-qPRC was conducted according to the manufacturer’s protocol using KOD SYBR qPCR Mix in a volume of 25 uL, while results were analyzed using SDS software.

For further analysis, cells were harvested and lysed in an SDS sample buffer composed of 123 mM Tris-HCl, pH 6.8, 4% SDS, 10% sucrose, 10 mM dithiothreitol, and 0.01% bromophenol blue. Whole cell lysates were resolved by SDS-PAGE technology. Immunoblotting was completed by using anti-Akt, anti-phospho Ser 473 Akt , and horseradish peroxidase-conjugated anti-tubulin antibody, as the primary antibodies. Researchers utilized horseradish peroxidase-conjugated anti-mouse or rabbit immunoglobulin G (IgG) antibody as the secondary antibody in the study. Band density was measured through the use of Image J software. All statistical differences were recorded by one-way ANOVA testing and Dunnett’s multiple comparison post-hoc test [1].

2) The research team of Lee. Et Al conducted a study examining the relationship between muscle atrophy and myostatin and how the two variables respond to treatment with YK11. The study began by obtaining 8-week old male BALB/c experimental mice from Orient Bio as well as a control group composed of wild-type (WT) mice. 350 or 750 mg/kg of YK11 were administered to the subjects over a 10 day treatment period. After 10 days of SARM treatment, the E. coli strain, RS218, was administered to both experimental and WT mice in order to induce sepsis. The body weight, amount of food eaten, and weight of the thigh muscle were continuously monitored and recorded throughout the experiment [2].

Figure 1: Visual representation of the treatment period and experimental procedures

In order to analyze the survival rate of the test subjects, following YK11 treatment, sepsis was induced through the administration of varying doses of different E. coli strains as well as Carbapenem-resistant Acinetobacter baumannii (CRAB). The researchers observed a 72 hour survival rate after administration of the septic compounds. After sepsis was induced, the lungs, liver, and kidneys were all extracted from the mice for various analytical purposes. The lungs were extracted and fixed in a 4% paraformaldehyde (PFA) solution prior to dehydration and preparation of the samples. Following extraction, organ samples were placed in 1 mL sterile PBS solution and then homogenized on ice. The homogenates were further diluted with PBS while samples were prepared in order to accurately assess the bacterial cultures.

The samples gathered from the test subjects allowed the research team to efficiently and effectively identify the various proteins and endotoxins present in the samples. The research team utilized an ELISA assay kit in order to measure and record follistatin protein levels and pro-inflammatory cytokines in tissue lysates from total muscle extracts. SDS-PAGE technology was used in order to separate the samples prior to transfer to fluoride membranes blocked with 5% skim milk in Tris-buffered saline containing 0.05% Tween-20 (TBS-T). After the samples were separated they were incubated with the primary antibodies, washed with TBS-T solution, and incubated for a second time with the secondary antibody [2].

After the samples were prepared Western blot analysis as well as an enhanced chemiluminescence kit was used to record any changes in response to the treatment. Furthermore, limulus amebocyte lysate (LAL) chromogenic end-point assay was used to measure endotoxin levels in the mice. Following dilution of the mouse sera with and endotoxin- free PBS, the research team completed the LAL chromogenic end-point assay to reveal the approximate amounts of endotoxins present in each sample. It is important to mention that the researchers conducted all of the experiments at least three times in order to ensure consistent results. For statistical analysis purposes, Tukey’s multiple comparison test and the Student’s t-tests were used to compare the experimental and control groups. Survival rates were analyzed through the completion of a log-rant text and the development of the Kaplan-Meier curves [2].

 

Discussion

1) Researchers Yatsu et. Al completed an MTS assay in order to investigate how YK11 affects osteoblast differentiation and cell proliferation. The MC3T3-E1 cells were treated with 0.5 uM of YK11 and 0.01 uM of DHT, over the course of 96 hours. Results of the treatment reported that both YK11 and DHT increased the rate of cell growth in the MC3T3-E1 cells. It was also determined that these effects could be reversed by co-treatment with the AR antagonist, HF. These findings suggest that YK11 successfully accelerated the proliferation of osteoblasts in a manner similar to DHT.

Figure 2: Effects of YK11 treatment on osteoblast proliferation

As the selected data mentioned, ALP activity was recorded since the enzyme acts as an early indicator of osteoblast differentiation. The cells were cultured in the standard differentiation medium and treated with either YK11 or DHT over a treatment period of 10 days. Both treatments led to an increase in ALP activity in comparison to the cells treated with a control solvent. However, when the MC3T3-E1 cells were co-treated with AR antagonist, HF, ALP activity levels exhibited no significant increases. Furthermore, mineralization in osteoblasts was quantified through alizarin red S staining. The cells were cultured in the standard differentiation medium and treated with YK11 or DHT over a 3 week period of time. In comparison to the cells treated with a control solvent, calcium deposits were observed in the cells administered both the YK11 and DHT treatments. These results allowed the researchers to conclude that YK11 promotes differentiation of the osteoblasts with increased ALP activity and calcium deposition. The effects elicited by YK11 are similar to those elicited by DHT [1].

Figure 3: (A) Effects of YK11 treatment on osteoblast ALP activity (B) Effects of YK11 treatment on mineralization shown through alizarin red S staining

In addition to ALP activity, the osteogenic marker osteoprotegerin (OPG) is an early indicator of osteoblast differentiation. Levels of the osteogenic marker, osteocalcin (OC), also suggest the occurrence of osteoblast differentiation, however, this signaling typically comes much later in the differentiation process. RT-qPCR was utilized in YK11-treated cells in order to measure mRNA expressions. By day 4 of treatment, YK11 was shown to increase OPG mRNA expression in a manner similar to DHT, the similar effects elicited by the two treatments were still observed at day 14 of treatment.

Figure 4: OPG expression in response to control, YK11, and DHT treatments

In terms of OC mRNA expression, there were no similarities observed between treatment with YK11 versus DHT on day 4, however the two treatments elicited similar results by day 14 of treatment. The researchers also observed that at day 14 YK11 was found to have increased OC mRNA expression in a dose-dependent manner. Similar to the previous differentiation tests, co-treatment with HF led to the inhibition of both YK11 and DHT. Overall, the reported findings allowed the research team to conclude that YK11 was able to successfully promote osteoblast differentiation at a level similar to DHT in the MC3T3-E1 cells [1].

Figure 5: OC expression in response to control, YK11, and DHT treatments

The final experimental step was to evaluate how treatment with YK11 affects non-genomic signaling. Results of the examination reported that the androgen receptor was able to directly activate the PI3K/Akt pathway. It is important to mention that the PI3K/Akt pathway plays a crucial role in the regulation of osteoblast proliferation and differentiation. After cells were treated with YK11 or DHT for 15 minutes, the research team was able to determine the effects of YK11 on the activation of the Akt signaling pathway. In a manner similar to DHT, phosphorylation of the Akt protein significantly increased in response to YK11 treatment. These findings indicated that YK11 activates the Akt signaling pathway through non-genomic signaling in a manner similar to DHT [1].

2) The research team of Lee et. Al examined the ability of YK11 to treat muscle atrophy that occurred as a side effect of bacterial sepsis. The SARM was administered to the test subjects at the same time for 10 days. On day 10 the subjects were intraperitoneally injected with E. coli or CRAB in order to induce sepsis. Results of the study reported that YK11 decreased muscle atrophy in a dose-dependent manner. This suggests that the higher the dose of YK11 administered, the more effective the compound was at treating atrophy. While total body weight was found to increase as a result of YK11 treatment, the ratio of fats as a percentage of total body weight decreased following treatment. The researchers believed these findings were relevant considering that prior research indicates that myostatin inhibition leads to increased skeletal muscle mass and decreased body fat mass. YK11 was also shown to recover muscle mass lost from the back and thighs of the animals that were administered the K1 E. coli strain. Additionally, results collected from staining the cell samples revealed that the muscle cells in animals treated with YK11 experienced reduced atrophy. The SARM was also effective against the resistant bacteria strand, CRAB; data collected during this portion of the study indicates that YK11 is able to revert muscle mass loss and increase total body weight. This suggests that YK11 is capable of treating muscle atrophy triggered by standard and resistant gram-negative bacteria strains [2].

 

Figure 6: Changes in body weight, muscle weight, and fat weight in response to experimental treatment.

In addition to the effects of YK11 on muscle atrophy, the research team attempted to identify how YK11 responds to the pathophysiology of sepsis. After treatment with YK11 and an injection of E. coli or CARB, the survival rate of the mice was estimated to be 20% for the 350 mg/kg dose of YK11 and 40% for the 700 mg/kg dose. Serum endotoxin levels were also significantly reduced in the animals treated with YK11. Reduced levels of various inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-10. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) were also reported after administration of YK11. The researcher team thought it was important to include changes in plasma levels of ALT, AST, and BUN, as these compounds act as biomarkers for organ damage. Treatment with YK11 also had the potential to promote the clearance of bacteria out of the organs of septic mice. Overall, these results indicate that by regulating inflammatory cytokines, YK11 is able to prevent severe septic shock induced by gram-negative bacteria [2].

Figure 7: Changes observed in A) survival rate, B) endotoxin levels, C) inflammatory cytokines, and D) biomarkers for muscle damage in response to different treatment methods.

The research team focused on bacterial LPS-induced NF-KB activation and TGF-beta signaling when attempting to identify the mechanism of action through which YK11 prevents sepsis. Results reported that myostatin was successfully inhibited and muscle protein metabolism was significantly improved following treatment with YK11 and injection of the K1 E. coli strain. The inhibitory effects were also seen in cases of increased follistatin expression; the animal model demonstrated the natural inhibitory effect follistatin elicits on myostatin. The researchers thought it was interesting to note that the E. coli K1 group experienced diminished levels of follistatin. Following treatment with YK11, follistatin was recovered to baseline levels, leading to enhanced inhibitory effects on myostatin.

Figure 8: Follistatin levels in response to different experimental treatment methods.

Injection of the E. coli K1 strain of bacteria lead to an increase in not only myostatin, but myogenin, and MyoD as well. Oral administration of YK11 prevented increased levels of these proteins. Furthermore, several compounds belonging to the TGF-beta category are capable of signaling through pathways similar to myostatin. This type of signaling ultimately results in altered phosphorylation of FOXO3a and Sma2 transcription factors downstream; mice administered YK11 treatment experienced a reduced rate of phosphorylation of both transcription factors. The research team concluded that administration of YK11 is capable of alleviating cases of bacteria-induced sepsis by combating muscle atrophy through the inhibition of myostatin activity [2].

Figure 9: Change in the expression of myostatin, myogenin, MyoD, and various transcription factors

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Yatsu T, Kusakabe T, Kato K, Inouye Y, Nemoto K, Kanno Y. Selective Androgen Receptor Modulator, YK11, Up-Regulates Osteoblastic Proliferation and Differentiation in MC3T3-E1 Cells. Biol Pharm Bull. 2018;41(3):394-398. doi: 10.1248/bpb.b17-00748. PMID: 29491216.

[2] Lee SJ, Gharbi A, Shin JE, Jung ID, Park YM. Myostatin inhibitor YK11 as a preventative health supplement for bacterial sepsis. Biochem Biophys Res Commun. 2021 Mar 5;543:1-7. doi: 10.1016/j.bbrc.2021.01.030. Epub 2021 Feb 12. PMID: 33588136.

YK-11 SARM is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.