recpetor downgrade/oversaturation/diminishing returns

[candidizzle]

 what do you guys thinkk ? real or not? to what degree ?   and if you think its real to a large degree, then explain why you think that; and what you think the underlying mechanisms for that are ...

[Big_Tymer]

diminishing returns exist in virtually all forms of life and economics on earth, gear use is no different imo.  Take a guy who uses 250mg/week test, and adds another amp making it 500 total.  He will see a much bigger  response increase than a guy who goes from 1250 to 1500/week.   If the 1250 guy doubled his dose to 2500 (which is what the 250 to 500 guy did, double the dose), I still seriously doubt he would see the same response.

Im not sure about receptor downgrade or oversaturation, although if you look at guys like kovacs/kamili especially and dorian/ronnie to an extent there is definitely something going on there, they clearly arent responding like they did when they were younger.  What causes it I dont have a clue.

[candidizzle]

aaahhhh

my bad, yeah diminishing returns is not what i was talking abut

you guys can forge about that one

its the "oversaturation" and "receptor down grade" issue id like to discuss


personally, with the knowledge i currently have, i tend to believe there is no such thing as over saturation or receptor downgrade.  but again thats just with what i currently know; im not saying its fact just what i tend to believe at the moment.

[candidizzle]

the reason i dont think i necessarily believe in this "oversaturation"...    WELL, i can see a problem with too high of doses and side effects; BUT, side effects aside....growth and body composition only thing considered...     i dont believe in over saturation

heres why (this just dealing with AR mediated growth and body fat reduction..  )

lets say you have X number of muscular androgen receptors.  lets say X androgen receptors requires Y milligrams per week of androgens to "fully saturate" (fully activate) all the receptors.  lets say you take Y+300mg per week.    300 mg surplus? yes. 300mg left over after activating all muscular receptors. BUT, its been show that EXTRA anrdrogens create EXTRA receptors. so once your "fully saturated", you will make space for more androgens and more growth.  so by taking Y+300mg. you now have x+300mg worth of muscular receptors. so, next time you inject, youll be filiing up those extra receptors.   increase the dose again? more receptors created. then you fill those recetors. and so on. ect ect ect.. 

and THEN, also you have androgen related lypolysis...   i guess fat cells have androgen receptors ! and extra androgens mean youll have more fat cell AR gettinjg activated meaning a further increase in reduction of body fat levels.


so i guess this is why i dont think this "over saturaton" exists


and as for "receptor down grade"...   well from what i have read and been told and seen in our sport...   androgen receptors do not get tired and they do not need to be "cleaned out" or anything of the sort..   

[Vet]

I find it very hard to believe that there isn't a limit to the total amount and saturation abilities of androgen receptors.  At some point they all have to be being utilized, which to me seems like it'd be a prime point for the body to say "well, looks like we got enough of this, no need for so many test receptors) and will downregulate the number of receptors. 

[bigdarksnake]

Maybe high levels of synthetic hormones damage the receptors since there is a surplus of androgens. Would naturally occurring testosterone ever be produced in excess of the amount of available receptors?

Can diminishing returns be separated from receptor downgrade?

Like in the use of opiates is it just diminishing returns that forces the user to increase the level of opiates? (of course androgen receptors don't come into play here) the μ-opioid receptors do suffer some sort of downgrade (they are actually decreased after heavy opiate use)....

[Alex23]

It's not about saturation, it's about "damage" to the receptors themselves; like "puzzle pieces", the molecule binds to the receptor. A synthetic form tends to be slightly "deformed" and damage the receptor.

Which is why older "drugs" like methandrostelone and methyl test downgrade have poor afinity and downgrade more...   

[Vet]

It's not about saturation, it's about "damage" to the receptors themselves; like "puzzle pieces", the molecule binds to the receptor. A synthetic form tends to be slightly "deformed" and damage the receptor.

Which is why older "drugs" like methandrostelone and methyl test downgrade have poor afinity and downgrade more...   

That doesn't make sense to me.   


Hormone receptors like we are talking about basically have 4 types of actions (this is really simplified and some people will say there are 5 types of reactions):

1) the molecule in question doesn't bind to the receptor, so the receptor remains "turned off" or "turned on"
2) the molecule in question binds "loosely" to the receptor, activating the receptor causing a physiological response, but because its bound "loosely" it can be easily displaced by different structures
3) the molecule in question binds "tightly" to the receptor, activating the receptor causing a physiological responce.  Because it is bound tightly, it cannot be easily displaced, thus causing a longer/greater effect
4) the molecule in question binds "irreversably".  This means it binds permenantly to the receptor causing an irreversable response---either activating or permenantly deactivating the receptor and the receptors actions.  This effectively removes that receptor from action if its a permenant deactivating molecule. 

[Van_Bilderass]

It's not about saturation, it's about "damage" to the receptors themselves; like "puzzle pieces", the molecule binds to the receptor. A synthetic form tends to be slightly "deformed" and damage the receptor.

Which is why older "drugs" like methandrostelone and methyl test downgrade have poor afinity and downgrade more...   

Where did you pick up this piece of info? 

Where did you read or hear that drugs with poor affinity "downgrade" androgen receptors?

We know that at least in the short term androgens increase AR density (AR are made constantly).

Poor affinity doesn't mean a steroid is weak in its effects either. Depends on which genes the drug activates. A drug like Anadrol barely binds yet is powerful (the effects still mediated through the AR most likely).

[candidizzle]

yup acccording to wiki, (i was readin it last night..."androgen receptors", "androgens", "steroid hormones" ect ect ) anavar binds super tiht to the AR. while dbol binds very loosely.


van what do YOU think about "over saturation" and/or "tired receptors"

[Van_Bilderass]

yup acccording to wiki, (i was readin it last night..."androgen receptors", "androgens", "steroid hormones" ect ect ) anavar binds super tiht to the AR. while dbol binds very loosely.


van what do YOU think about "over saturation" and/or "tired receptors"

Actually from what I remember Anavar hasn't ever been shown to bind to the AR at all. But it must since the effects it has must be mediated through the AR.

I haven't read anything about ARs becoming desensitized or "tired". But the mechanisms involved are very complex I'm sure. The scientists don't know everything yet. There probably are regulatory mechanisms involved, even if AR count isn't reduced, etc.

[candidizzle]

i just went back and checked on wiki and it says oxymetholone binds tightly.... which now that i look it up ... oxymetholone = anadrol .... i always get the chemical names of drol and var mixed up since they sound so similar

[Van_Bilderass]

i just went back and checked on wiki and it says oxymetholone binds tightly.... which now that i look it up ... oxymetholone = anadrol .... i always get the chemical names of drol and var mixed up since they sound so similar

Anadrol binds very weakly too, almost not at all. Let me see if I can find a reference.

In any case, receptor affinity is not a good way to determine if a steroid is "effective". It's the genes it activates that determines the effects.

[candidizzle]

from wiki

Anabolic steroids such as methandrostenolone bind weakly to this receptor and instead directly affect protein synthesis or glycogenolysis.[51] On the other hand, steroids such as oxandrolone bind tightly to the receptor and act mostly on gene expression.


http://en.wikipedia.org/wiki/Anabolic_steroid

(last portion of 'mechanism of action')

[Van_Bilderass]

Let me see if I can find a reference.

1: Endocrinology. 1984 Jun;114(6):2100-6.Links
    Relative binding affinity of anabolic-androgenic steroids: comparison of the binding to the androgen receptors in skeletal muscle and in prostate, as well as to sex hormone-binding globulin.
    Saartok T, Dahlberg E, Gustafsson JA.

    It is unclear whether anabolic steroids act on skeletal muscle via the androgen receptor (AR) in this tissue, or whether there is a separate anabolic receptor. When several anabolic steroids were tested as competitors for the binding of [3H]methyltrienolone (MT; 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one) to the AR in rat and rabbit skeletal muscle and rat prostate, respectively, MT itself was the most efficient competitor. 1 alpha-Methyl-5 alpha-dihydrotestosterone (1 alpha-methyl-DHT; mesterolone) bound most avidly to sex hormone-binding globulin (SHBG) [relative binding affinity (RBA) about 4 times that of DHT]. Some anabolic-androgenic steroids bound strongly to the AR in skeletal muscle and prostate [ RBAs relative to that of MT: MT greater than 19-nortestosterone ( NorT ; nandrolone) greater than methenolone (17 beta-hydroxy-1-methyl-5 alpha-androst-1-en-3-one) greater than testosterone (T) greater than 1 alpha-methyl-DHT]. In other cases, AR binding was weak (RBA values less than 0.05): stanozolol (17 alpha-methyl-5 alpha- androstano [3,2-c]pyrazol-17 beta-ol), methanedienone (17 beta-hydroxy-17 alpha-methyl-1,4-androstadien-3-one), and fluoxymesterolone (9 alpha-fluoro-11 beta-hydroxy-17 alpha-methyl-T). Other compounds had RBAs too low to be determined (e.g. oxymetholone (17 beta-hydroxy-2-hydroxymethylene-17 alpha-methyl-5 alpha-androstan-3-one) and ethylestrenol (17 alpha-ethyl-4- estren -17 beta-ol). The competition pattern was similar in muscle and prostate, except for a higher RBA of DHT in the prostate. The low RBA of DHT in muscle was probably due to the previously reported rapid reduction of its 3-keto function to metabolites, which did not bind to the AR [5 alpha-androstane-3 alpha, 17 beta-diol and its 3 beta-isomer (3 alpha- and 3 beta-adiol, respectively)]. Some anabolic-androgenic steroids (only a few synthetic) bound to SHBG (1 alpha-methyl-DHT much greater than DHT greater than T greater than 3 beta-adiol greater than 3 alpha-adiol = 17 alpha-methyl-T greater than methenolone greater than methanedienone greater than stanozolol). The ratio of the RBA in rat muscle to that in the prostate (an estimate of the myotrophic potency of the compounds) was close to unity, varying only between about 0.4 and 1.7 in most cases.(ABSTRACT TRUNCATED AT 400 WORDS)

Relative binding affinity too low to be determined.

[candidizzle]

haha

fuck i am retarded

it WAS anavar after all

damn those names are so close

[Van_Bilderass]

from wiki

Anabolic steroids such as methandrostenolone bind weakly to this receptor and instead directly affect protein synthesis or glycogenolysis.[51] On the other hand, steroids such as oxandrolone bind tightly to the receptor and act mostly on gene expression.


http://en.wikipedia.org/wiki/Anabolic_steroid

(last portion of 'mechanism of action')

Ah ok. Well, from what I've gathered the anabolic effects of anabolic steroids are basically all mediated through the AR.  There is no "separate anabolic receptor" like the old abstract I posted asked nor is there a lot of support for the supposed non-ar mediated anabolic mechanisms - that was the basis of Bill Roberts Class I/Class II steroid classification theory. He didn't understand that the genes a steroid activates can have powerful effects = you don't need a high binding affinity for good effects necessarily. I don't know what the Wiki entry means by "directly affecting protein synthesis".

Anavar binding hasn't been determined as far as I know so that's wrong.

[Van_Bilderass]

Anavar binding hasn't been determined as far as I know so that's wrong.

Remembered this sentence from this study:

"In addition, new data from this study of androgen-deficient men indicate that ARs are significantly decreased in response to severe hypogonadism (28). Although there is no direct evidence that OX binds to the ARs, the findings of the present study and those reported by Hayes et al. (28) suggest that androgens may work directly through the androgen receptor to exert their effects on protein metabolism. Nevertheless, we do not know from the present study the physiological importance of the increased expression of mRNA for AR."

http://jcem.endojournals.org/cgi/content/full/84/8/2705?ijkey=1c18e10cb501555f36c9cd0781adbaeac590bbaf

[candidizzle]

yeah i remember when i asked about non ar mediated effects and you told me how you dont need tight binding for strong activation

i am wondering how one andro receptor can be activated by one compound and do one thing, and be activated by another and do a total different thing. AR are either ON or OFF i though ? it seems they have more than one "ON" setting... ?

[Van_Bilderass]

yeah i remember when i asked about non ar mediated effects and you told me how you dont need tight binding for strong activation

i am wondering how one andro receptor can be activated by one compound and do one thing, and be activated by another and do a total different thing. AR are either ON or OFF i though ? it seems they have more than one "ON" setting... ?

I remember I posted that one article by Karl Hoffman that you though was a bit too complex. He explained it pretty well though.

[ChinoXL]

Candidizzle, I can appreciate your intuitive nature regarding the use of the chemicals while you are experimenting with them.  It's the right thing to do.  Never go in blind,  but subjecting yourself to this requires above all else EXTREME ATTENTION TO DETAIL.  Hypothetical question:  What if your dealer only knew chemical names?  For some reason I can see you taking ten tabs of oxymethylone50 a day and wondering why your 50mgs of anavar is causing headaches, swollen hands, feet, ankles and jaundice.  Your whole take on this subject is leading me to believe that your five month cycle is no longer supporting the 3lbs that you gained and need an excuse to up the dosage.  Look kid, the nature of this fucking beast is this, either you have it or you don't.  Sorry.  Hope this helps you wake up.






PS:  Stop giving advice.




PSS:  This is constructive criticism.  Use it.

[candidizzle]

I remember I posted that one article by Karl Hoffman that you though was a bit too complex. He explained it pretty well though.

maybe now i will understand it...   still have it ?



chino, thanks but no thanks, buddy. you trying to mask your negative-ism and hatred in a guise of "good natured advcie" but it fails.

please keep that shit to forums like the G&O
(on a side note i dont know why you have such a strong dislike for me. it seems the ONLY time i ever see you posting is in response to some post i make and its all negative and hate ridden. )

[Van_Bilderass]

maybe now i will understand it...   still have it ?

Fantastic articles if you have the patience to read them. 

This guy was The God of AAS info on the forums but unfortunately passed away. 

Understanding Androgen Actions
by Karl Hoffman

   
Researchers as well as athletes and bodybuilders know that besides the two principal physiological androgens, testosterone and dihydrotestosterone, there exist a number of synthetic anabolic-androgenic steroids (AAS) that exhibit diverse biological actions. For example, dihydrotestosterone (DHT) is considered androgenic relative to testosterone since it is essential for the virilization of the external genital organs. On the other hand, DHT is not considered anabolic because it is not active in skeletal muscle (it is enzymatically deactivated); testosterone is anabolic in this regard, being responsible (along with other hormones and growth factors) for the development and maintenance of skeletal muscle. The array of synthetic AAS were developed to meet differing needs and like the physiological androgens differ in their relative anabolic/androgen potency. Some, like methyltestosterone and fluoxymesterone are relatively androgenic (although not as much so as DHT) and are indicated for androgen replacement while others like oxandrolone and stanozolol are relatively more anabolic. Yet despite the wide range of effects and potencies of both the natural and synthetic androgens, to date only one androgen receptor has been identified. What accounts for the diversity of effects of the different AAS?

Surprisingly, despite the number of synthetic AAS that have been developed, their modes of action are poorly understood. This holds for the naturally occurring androgens as well. There is some evidence (which we will discuss below) that androgens are able to exert some of their actions independently of the androgen receptor (AR). Antagonism of the glucocorticoid receptor is one possible way androgens may exert an anabolic effect.

Binding affinity to the androgen receptor has also been invoked to explain the differences in potencies and effects of the natural and synthetic androgens. For example, dihydrotestosterone binds the androgen receptor much more strongly than does testosterone at the same concentration, yielding a higher degree of ligand-receptor stability. When the concentration of testosterone is increased however, the receptor stability increases to a level similar to that seen with dihydrotestosterone (1). This has led to the proposal that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone in target tissues such as the prostate resides in testosterone’s weaker interaction with the androgen receptor. Yet it is well known that some steroids which are very potent anabolic agents, such as stanozolol or oxymetholone, bind the AR only very weakly (2). If we assume that AR binding affinity is the sole determinant of an agent’s ability to act via the AR to promote anabolic or androgenic actions, then we are forced into the conclusion that certain potent AAS that bind the AR with negligible affinity must be exerting their anabolic effects via some other routes that do not involve AR binding. Indeed, this has become to a large degree dogma in the bodybuilding literature.

Some interesting recent research has shed light on this problem by showing that AR binding affinity is only partly responsible for the androgen receptor mediated effects of both physiologic androgens and synthetic AAS. In the study I would like to discuss, the authors present evidence for the existence of distinct steroid specific target gene transcription profiles following AR activation (3). In other words, the structures of androgen responsive genes vary in such a way that some genes are more readily activated by certain androgens than by others. The set of genes readily switched on by a given androgen determines the net physiological effect of that androgen. This theory readily explains how an anabolic steroid like oxandrolone, whose AR binding affinity is quite low, can be so anabolic: it happens to preferentially turn on genes whose products promote skeletal muscle anabolism, while failing to activate genes which promote virilization.

Before looking at this research in detail, a brief review of how androgens activate genes is in order. The AR is generally thought to reside primarily in the cytoplasm of the target cell, bound to so-called heat shock proteins. The androgen (ligand) diffuses into the cytoplasm and binds to part of the receptor called, appropriately enough, the ligand binding domain. The heat shock proteins dissociate from the ligand-receptor complex, the complex dimerizes (binds to another ligand-receptor complex), and then translocates to the nucleus where the target gene(s) is located. The stretch of chromosomal DNA comprising the target gene acts as a template for the synthesis of RNA in a process called transcription.

Part of the gene (depicted below) consists of a promoter region that contains a subsection called the androgen response element (ARE). When the ligand binds to the AR, it induces a conformation change in the ligand-receptor complex that allows the complex to recognize and bind to the specific nucleotide sequence comprising the ARE. There it proceeds to recruit coactivators, which act as “power boosters” that amplify transcription, as well as other transcription factors, which are proteins that are required to initiate transcription of the target gene via RNA polymerase II. The receptor/ligand and coactivators along with perhaps other transcription factors would form a large complex that serves as a sort of platform for RNA polymerase to dock with, allowing the polymerase to begin transcribing the gene. The messenger RNA (mRNA) created from the DNA template of the gene then leaves the nucleus and enters the cytoplasm, where in the process known as translation, the mRNA in turn serves as a template for the construction of a specific protein.

The exons in the gene depicted below contain the segments of DNA that actually code for the protein that will ultimately be transcribed.

Fig 1. Generic gene structure showing exon (protein coding region), RNA polymerase II bound to gene; TATA box; and promoter with bound transcription factors. The androgen/AR complex would bind to a specific region within the promoter, the Androgen Response Element (ARE). From (4)

Upstream from the exon is the region of the promoter called the TATA box. It contains a sequence of seven bases TATAAAA and is a common feature of promoters found in all genes. The base sequences in the remaining upstream portion of the promoter vary from gene to gene.

The authors of the paper under discussion wanted to see how the promoter base sequence affected steroid hormone binding and action. In order to do this, they first constructed a set of so called artificial reporter genes; these consisted first of an exon coding for the enzyme luciferase. Luciferase is found in fireflies and produces luminescence when it acts on its substrate luciferin. To the luciferase exon they then spliced 3 different well-characterized promoters whose base sequences varied greatly. The three resulting artificial genes were designated GRE-OCT-luc; (ARE)2TATA-luc; and MMTV-luc.

The idea here is that if a given androgen is exposed to one of these genes and is able to bind to the promoter and induce transcription of the luciferase gene, detectable light will be emitted in proportion to the effectiveness of that androgen to activate the gene. What are the possible outcomes and interpretations of the experiment? If for example all androgens induce transcription to the same extent in all three genes, then it could be assumed that the structure of a given gene’s promoter would probably not be a determinant in the biological profiles of differing androgens. If on the other hand stanozolol, say, activated only one of the genes, while testosterone activated another, then the different biologic profiles of the two steroids (e.g. their different anabolic/androgenic ratio) could be due in part to the possibility that the two steroids activate different sets of genes in the body, depending on the promoter structure of the gene. If the latter is the case, a particular AAS that only binds the AR weakly could still be quite potent if it turned out to be a strong activator of anabolism promoting genes in skeletal muscle. This obviates the need to invoke non-AR mediated actions for weak androgen receptor agonists (the dubious class I/class II theory of steroid action). Receptor binding may be only part of the picture; promoter binding and the strength of the transcription signal could be equally if not more important than AR affinity in determining the biological effects of a given agent.

Chinese hamster ovary cells (which do not express the androgen receptor or any androgen responsive genes) were transfected with the three genes described above, as well as with a vector expressing the androgen receptor. The cells were treated with varying concentrations of a number of different androgens, including R1881 (methyltrienolone), testosterone, DHT, nandrolone, oxandrolone, androstenedione, and DHEA.

The main result of the study was that the androgens could be divided into two main subgroups based on reporter gene activation. DHT, nandrolone, R1881, and testosterone grouped together statistically based on their activation profile, while the precursor hormones together with the anabolic steroids oxandrolone and stanozolol fell into a separate subgroup based on the reporters they preferentially activated.

There were some interesting individual results. Testosterone showed twice the ability of DHT to activate the GRE-OCT-luc reporter at all concentrations, suggesting that AR binding affinity is certainly not the determinant of gene transcription with this reporter. DHT on the other hand maximally stimulated the (ARE)2TATA-luc construct at 10nM concentration.

The anabolic steroids oxandrolone, nandrolone, and stanozolol were potent activators of the MMTV-luc construct. Remarkably, at 10nM, stanozolol, which has a very weak AR binding affinity exceeded R-1881 induced activity for this reporter despite the fact that R-1881 has one of the highest AR binding affinities of any androgen. Here, once again, we see binding affinity is not the sole determinant of androgen activity.

Another interesting result was the fact that the androgen precursors DHEA and androstenedione were potent AR ligands leading to differential target gene expression. The authors concluded their data potentially support a relevant contribution of testosterone-precursor hormones to mechanisms of in vivo androgen action.

These findings are in accord with earlier work (5) where two different androgen response elements were discovered that showed different T- vs. DHT-induced AR transactivation. In vivo work supports in vitro findings that different androgens are capable of differentially regulating AR responsive genes. In castrated rats, DHT proved more potent at maintaining prostate epithelial cell function, whereas testosterone and DHT were equipotent at inhibiting prostatic apoptosis (programmed cell death) (6). In another study that looked at the effects of testosterone and DHT on prostatic regrowth in castrated rats, testosterone proved to be more potent than DHT in activating genes governing cellular differentiation than those responsible for proliferation. (Differentiation is the process whereby immature cells activate genes that commit them to the path to becoming fully functioning mature cells, whereas proliferation is the process of multiple cell division that leads to an increase in cell number) (7).

Now that we see that steroid receptor agonists activate transcription in part by recruiting coactivators to aid in transcription it is relatively easy to understand how receptor antagonists might block transcription: by inhibiting coactivator binding. This has been well studied for the interaction between the estrogen receptor (ER) and tamoxifen, which acts as an antiestrogen in some tissues. The ligand binding domain of the estrogen receptor consists of a number of amino acid sequences folded into a series of helixes. Different ER ligands can relatively easily change the conformation of one helix in particular, helix 12. When an agonist like estradiol binds the ER, helix 12 takes on a conformation that forms part of the coactivator binding pocket once the ligand/receptor binds to the gene to be transcribed. In contrast, when an estrogen antagonist binds to the ER, the antagonist changes the shape of the ligand binding domain in such a way that helix 12 now bends so as to occupy part of the coactivator binding pocket, blocking coactivator binding. Without a coactivator present, transcription of the gene cannot proceed. It turns out the estrogen receptor contains two regions that can bind coactivators, so called AF-1 and AF-2. Tamoxifen inactivates AF-2, but AF-1 still retains the ability to bind coactivators. Tamoxifen is a Selective Estrogen Receptor Modulator, or SERM; it has the ability to act as an antiestrogen with regard to certain genes, and an estrogen with respect to others, blocking transcription of the former and initiating transcription of the latter.. It is believed that in the case where tamoxifen acts as an antiestrogen, the promoter of the gene in question depends on AF-2 to hold the coactivator in place, and we have seen that tamoxifen renders AF-2 incapable of doing so. With other genes where tamoxifen acts as an agonist, it is believed AF-1, which is unaffected by tamoxifen, functions as the important coactivator binding site.

Pure antiestrogens, such as faslodex, block transcription of all estrogen responsive genes by blocking both coactivator binding sites, AF-1 and AF-2. In this case it is impossible for any coactivator to bind the target gene once faslodex has attached, so transcription cannot proceed.

INDIRECT MECHANISMS OF ANDROGEN ACTION

While the results described above may obviate the need to invoke non-AR mediated mechanisms to explain some of the biological activity of various AAS, such mechanisms nevertheless do exist. For example, androgens undergo differential metabolism in target tissues. DHT is inactive in skeletal muscle because the enzyme 3 alpha-hydroxysteroid dehydrogenase, present in large quantities in skeletal muscle, rapidly metabolizes it. On the other hand, androgen target tissues such as the prostate, skin, and scalp are relatively rich in the 5 alpha reductase enzymes that convert testosterone to DHT, so DHT is considered the active androgen in those tissues.

We also mentioned above the possibility that androgens may exert anabolic activity by binding to and antagonizing the glucocorticoid receptor. Endogenous glucocorticoids such as cortisol exert a catabolic effect on skeletal muscle by activating the ubiquitin proteasome proteolytic pathway and to a lesser extent calcium-dependent protein breakdown. Testosterone seems to be a particularly potent glucocorticoid antagonist (8,9), more so than the anabolic steroid trenbolone (10). Speculating a bit, and using some “contrarian endocrinology”, this may explain the observation commonly made by bodybuilders that trenbolone is a more effective lipolytic agent than is testosterone, since research indicates that cortisol is a predominantly lipolytic hormone:

Cortisol's effects on lipid metabolism are controversial and may involve stimulation of both lipolysis and lipogenesis...In conclusion, the present study unmistakably shows that cortisol in physiological concentrations is a potent stimulus of lipolysis and that this effect prevails equally in both femoral and abdominal adipose tissue. (11)

So by antagonizing the glucocorticoid receptor and blocking the lipolytic effects of cortisol, testosterone could possibly be losing some of its lipolytic power. It has also been proposed that glucocorticoid activity at the gene level is inhibited via androgen interference with the glucocorticoid response element in genes targeted by cortisol (11).

Androgens are capable of stimulating both the production of hepatic insulin like growth factor (IGF-1), as well as local IGF-1 production within skeletal muscle. One often reads in the bodybuilding literature that the former is an attribute only of oral 17-alpha alkylated steroids and occurs by direct action of these steroids on the liver. In fact, testosterone as well as oxandrolone (12) and methandrostenelone (Dianabol) (13, 14) all elevate hepatically derived IGF-1, but secondary to an increase in growth hormone secretion. So these agents are not acting directly on the liver to elevate IGF-1; rather they stimulate pituitary growth hormone secretion, and this GH in turn is responsible for inducing hepatic IGF-1 secretion.

The second effect mentioned above, the local production of IGF-1 within skeletal muscle, may be more important than hepatic IGF-1 production for growth, while hepatically derived IGF-1 may play a more important role in carbohydrate and lipid metabolism (15). The locally produced IGF-1 acts back on the muscle tissue that produced it in an autocrine manner to stimulate growth. Based on their research on testosterone suppression in normal men, Mauras et al concluded that:

[During androgen suppression] [t]here were, however, significant decreases in [intramuscular] mRNA concentrations for IGF-I and a trend toward increased IGFBP-4 gene expression, the main inhibitory binding protein for IGF-I in muscle. The gene expression for actin and myosin in muscle was not altered by the systemic decrease in testosterone concentrations. These observations are congruent with the observation made in elderly men treated with testosterone and suggest that, within skeletal muscle tissue, androgens are necessary for local IGF-I production, independent of GH production and systemic IGF-I concentrations. IGF-I and its type I receptor are ubiquitously expressed in skeletal muscle and appear to be important in both the proliferation and differentiation of skeletal myocytes. Even though the gene expression of actin and myosin, the main contractile proteins of skeletal muscle, were not altered during severe hypogonadism, testosterone deficiency was associated with a marked decrease in measures of muscle strength, indicating that other mechanisms besides changes in muscle protein expression are affected by this severe degree of androgen deficiency (16).

So one of primary anabolic effects of androgens may be their ability to stimulate IGF-1 production in skeletal muscle.

It may be somewhat misleading to call the production of GH and IGF-1, either local or hepatic, an indirect action of androgens in the same sense that glucocorticoid receptor antagonism is. It is likely that the genes for GH and IGF-1 are direct targets for androgens and are activated by the androgen/AR complex, just as any other androgen responsive genes would be.

References

(1) Grino PB, Griffin JE, Wilson JD Endocrinology 1990 Feb;126(2):1165-72

(2) Saartok T, Dahlberg E, Gustafsson JA Endocrinology 1984 Jun;114(6):2100-6

(3) Holterhus PM, Piefke S, Hiort O J Steroid Biochem Mol Biol 2002 Nov;82(4-5):269-75.

(4) Biology. John W. Kimball 1994 by Wm. C. Brown,

(5) Hsiao PW, Thin TH, Lin DL, Chang C. Mol Cell Biochem 2000 Mar;206(1-2):169-75

(6) Wright AS, Thomas LN, Douglas RC, Lazier CB, Rittmaster RS.
J Clin Invest 1996 Dec 1;98(11):2558-63

(7) Dadras SS, Cai X, Abasolo I, Wang Z.Gene Expr 2001;9(4-5):183-

( Danhaive PA, Rousseau GG. J Steroid Biochem 1988 Jun;29(6):575-81

(9) Danhaive PA, Rousseau GG J Steroid Biochem 1986 Feb;24(2):481-7

(10) Djurhuus CB, Gravholt CH, Nielsen S, Mengel A, Christiansen JS, Schmitz OE, Moller N.
Am J Physiol Endocrinol Metab 2002 Jul;283(1):E172-7

(11) Hickson RC, Czerwinski SM, Falduto MT, Young AP. Med Sci Sports Exerc 1990 Jun;22(3):331-40 .

(12) Ulloa-Aguirre A, Blizzard RM, Garcia-Rubi E, Rogol AD, Link K, Christie CM, Johnson ML, Veldhuis JD. J Clin Endocrinol Metab 1990 Oct;71(4):846-54

(13) Hochman IH, Laron Z Horm Metab Res 1970 Sep;2(5):260-4

(14) Steinetz BG, Giannina T, Butler M, Popick F.Endocrinology 1972 May;90(5):1396-8

(15) Isaksson OG, Jansson JO, Sjogren K, Ohlsson C.Horm Res 2001;55 Suppl 2:18-21

(16) Mauras N, Hayes V, Welch S, Rini A, Helgeson K, Dokler M, Veldhuis JD, Urban RJ.
J Clin Endocrinol Metab 1998 Jun;83(6):1886-92

[Van_Bilderass]

Anabolic Steroid Action In Vitro and In Vivo
by Karl Hoffman

    The traditional view of anabolic steroids is that their anabolic-androgenic potency is to a large extent dependent on the relative binding affinity of a given steroid to the androgen receptor. In an earlier edition of Mind and Muscle Magazine I reviewed some recent research suggesting that while binding affinity is still a factor controlling the action of anabolic steroids, the particular genes activated by any given steroid may be just as important or even more so.

There I made the comments that:

Surprisingly, despite the number of synthetic AAS that have been developed, their modes of action are poorly understood. This holds for the naturally occurring androgens as well. There is some evidence (which we will discuss below) that androgens are able to exert some of their actions independently of the androgen receptor (AR). Antagonism of the glucocorticoid receptor is one possible way androgens may exert an anabolic effect.
Binding affinity to the androgen receptor has also been invoked to explain the differences in potencies and effects of the natural and synthetic androgens. For example, dihydrotestosterone binds the androgen receptor much more strongly than does testosterone at the same concentration, yielding a higher degree of ligand-receptor stability. When the concentration of testosterone is increased however, the receptor stability increases to a level similar to that seen with dihydrotestosterone (1). This has led to the proposal that the weaker androgenic potency of testosterone compared to that of dihydrotestosterone in target tissues such as the prostate resides in testosterone’s weaker interaction with the androgen receptor. Yet it is well known that some steroids which are very potent anabolic agents, such as stanozolol or oxymetholone, bind the AR only very weakly (2). If we assume that AR binding affinity is the sole determinant of an agent’s ability to act via the AR to promote anabolic or androgenic actions, then we are forced into the conclusion that certain potent AAS that bind the AR with negligible affinity must be exerting their anabolic effects via some other routes that do not involve AR binding. Indeed, this has become to a large degree dogma in the bodybuilding literature.
Some interesting recent research has shed light on this problem by showing that AR binding affinity is only partly responsible for the androgen receptor mediated effects of both physiologic androgens and synthetic AAS. In the study I would like to discuss, the authors present evidence for the existence of distinct steroid specific target gene transcription profiles following AR activation (3). In other words, the structures of androgen responsive genes vary in such a way that some genes are more readily activated by certain androgens than by others. The set of genes readily switched on by a given androgen determines the net physiological effect of that androgen. This theory readily explains how an anabolic steroid like oxandrolone, whose AR binding affinity is quite low, can be so anabolic: it happens to preferentially turn on genes whose products promote skeletal muscle anabolism, while failing to activate genes which promote virilization.

In this paper I would like to discuss a different but not necessarily contradictory explanation of why some steroids that appear to bind only weakly to the AR still manage to exert potent anabolic and androgenic effects. The solution to the apparent paradox is a simple one: virtually all binding affinity studies to date have been carried out in vitro. Here we will look at the recently published research by Feldkoren and Andersson [1] who compared the in vivo and in vitro actions of the anabolic steroids stanozolol (Winstrol) and methanedienone (Dianabol). We will see that the interactions of these anabolic steroids with the AR are much different when measured in vitro when compared to measurements carried out in vivo.

In [1] the authors examined the interaction of the above mentioned steroids with the AR by using three different systems: (1) a recombinant AR ligand binding in vitro assay (the modern standard method of expressing binding affinities); (2) a cell based AR-dependent transactivation assay; and (3) an in vivo assay based on steroid induced cytosolic AR depletion in skeletal muscle. The logic behind system (3) is that normally the unbound AR resides in the cytosol of the cell. Upon ligand (steroid) binding the ligand-receptor complex translocates to the nucleus. So the degree of cytosolic depletion of the AR when exposed to a particular steroid serves as a measure of the degree of binding of the steroid to its receptor. The binding affinities of testosterone and 17-alpha methyltestosterone were examined as well.

System (1) measured the in vitro binding strengths of the given steroids by determining how effectively they displaced radiolabeled methyltrienolone (MT) from the recombinant AR. Methyltrienolone binds extremely strongly to the AR and to what degree it can be displaced from the AR serves as the standard assay for measuring the binding strength of any given androgen receptor ligand.

By observing the amount of displaced radiolabeled MT as a function of the concentration of the competing steroid, it’s possible to calculate the binding affinity of the competitor. The affinity is usually quantified as the equilibrium dissociation constant, Ki. The subscript i is used to indicate that the competitor inhibited radioligand binding. The Ki is the concentration of the competing ligand that will bind to half the binding sites at equilibrium, in the absence of radioligand or other competitors. If the Ki is low, the affinity of the receptor for the inhibitor is high, and vice versa.

Not surprisingly MT possessed the highest affinity with a Ki of 0.20 nM, the Ki for testosterone and 17alpha-methyltestosterone were 0.80 and 0.90 nM, respectively. Both stanozolol and methanedienone were the least effective competitors with a Ki for stanozolol of 4.5nM and methanedienone of 5.0 nM. The calculated in vitro binding affinities of the various agents are depicted below in Figure 1, excerpted from [1].


Fig. 1. Binding strength of various steroids to the recombinant rat AR. The data presented in Fig. 1 were analyzed to calculate the equilibrium dissociation constant Ki for each steroid mentioned above. T, testosterone; MT, methyltrienolone; 17alpha-MeT, 17alpha-methyltestosterone; S, stanozolol; MA, methanedienone.

So here we see the expected picture from all we have read about various anabolic steroids; namely, certain oral steroids like Winstrol and Dianabol only bind relatively weakly (compared to testosterone, for example) to the AR yet are well known to be quite potent.

System (2) employed a full length recombinant AR and a section of DNA consisting of the androgen response element (the portion of a gene to which the AR-ligand complex binds) spliced to a luciferase reporter gene. These are then inserted into a cell. Luciferase emits light when activated, and the idea here is that when the cell containing this artificial gene complex is exposed to a given steroid, the steroid will bind to the AR, attach to the artificial gene construct, and activate the luciferase. The amount of light emitted is a measure of the binding of the AR-ligand complex to the reporter gene, which in turn is a function of the strength of binding of the ligand to the AR.

Transcriptional activation is usually expressed in terms of so called EC50 of the ligand. EC50 is defined as the molar concentration of a ligand, which produces 50% of the maximum possible response for that ligand. Methyltrienolone was found to be the most effective transcriptional activator with an EC50 of 5 pM (picomoles), in agreement with its high affinity in vitro. The calculated EC50 values for the other steroids were 44pM for 17alpha-methyltestosterone, 52pM for stanozolol, and 79pM for methanedienone; all four steroids induced the same level of maximum transactivation. The researchers did not measure the EC50 of testosterone because it metabolized to the relatively inactive androgen androstenedione by the enzyme 17beta-hydroxysteroid dehydrogenase present in the type of cells used in the experiment. Hence, in intact cells, both stanozolol and methanedienone are potent activators of the AR, of the same order of magnitude as 17alpha-methyltestosterone.

Finally, to test the in vivo strength of the various steroids, rats were injected with each steroid at a dose 0.3 mg/kg of body weight. On hour after treatment, muscle cells were removed from the animals and the cytosolic depletion analysis of the AR was carried out. Methyltrienolone resulted in a 67% reduction of androgen binding sites in muscle cytosol. Stanozolol possessed almost as much activity (44% depletion) as testosterone (55%), followed by methanedienone, which caused a 33% reduction in binding sites. 17alpha-Methyltestosterone demonstrated the lowest degree of cytosolic AR depletion (11%) of all of the AS.

We see then a clear discrepancy between the typically published in vitro binding affinities of various anabolic steroids, and the ability of these steroids to evoke biological responses via classical androgen receptor mediated transcription in both cell based systems and in vivo.

It is interesting to compare the in vivo binding affinities obtained above with previously published binding data. Saartok et.al.[2] measured the binding affinities of a number of anabolic steroids relative to methyltrienolone (MT) using a system based on the binding of steroids to the AR in the cytosols obtained by grinding rat muscle and prostate tissue. Their relative binding affinities (RBA) were calculated as the ratio between the molar concentrations of unlabeled MT and of the competitor required to displace 50% of the radiolabeled MT from cytosolic binding sites (i.e. androgen receptors). If MT is arbitrarily given an RBA of 1, testosterone exhibited an RBA of 0.7; 17alpha-methyltestosterone’s was 0.10; stanozolol 0.03; and methanedienone 0.02.

We immediately see large differences in the results obtained in vivo in [1] compared to the in vitro data published in [2]. For example in [1] the binding affinities between MT and testosterone differed by only 18%. In [2] the difference was 30%. The disparity between studies for stanozolol and methanedienone is even greater. Looking at stanozolol, in [1] we see an affinity difference from MT of 34%. In [2] the difference is enormous, almost 2 orders of magnitude.

References

1. Feldkoren BI, Andersson S. Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study. J Steroid Biochem Mol Biol. 2005 Apr;94(5):481-7.

2. Saartok T, Dahlberg E, Gustafsson JA. Relative binding affinity of anabolic-androgenic steroids: comparison of the binding to the androgen receptors in skeletal muscle and in prostate, as well as to sex hormone-binding globulin. Endocrinology. 1984 Jun;114 (6):2100-6.

[Alex23]

Where did you pick up this piece of info? 

Where did you read or hear that drugs with poor affinity "downgrade" androgen receptors?

We know that at least in the short term androgens increase AR density (AR are made constantly).

Poor affinity doesn't mean a steroid is weak in its effects either. Depends on which genes the drug activates. A drug like Anadrol barely binds yet is powerful (the effects still mediated through the AR most likely).

I read this on here a couple of years ago... make what you want of that...

Train Natural.