Understanding Androgen Actions - Mind And Muslce

muscle mapby: Karl Hoffman
Understanding Androgen Actions

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.


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.


(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-

(8) 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

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