When they talk about the "action" of testosterone enanthate, most often they mean the visible result - more muscles and strength. But between the injection and the growth of the muscle fiber lies a long chain of molecular events. The editors went through this chain step by step: from ester cleavage to gene activation and protein synthesis.
From prodrugs to active hormone
Testosterone enanthate itself does not interact with receptors. This is a prodrug — a molecule that becomes active only after chemical transformation in the body. The enanthic acid ester attached to the 17-beta-hydroxyl group of the steroid blocks exactly that part of the molecule that is required for tight binding to the androgen receptor.
After entering the blood and tissues from the oil depot, non-specific esterase enzymes hydrolyze the ester bond. Free testosterone is released, a molecule identical to the endogenous hormone. The speed of this stage is so high that almost all circulating active hormone is represented by testosterone, not its ester.
In the blood, most of the testosterone is bound to proteins. About half or more is strongly bound to sex hormone-binding globulin (SGH), a significant fraction is loosely bound to albumin, and only about 1–3% remains free. It is the fractions that are free and weakly bound to albumin that are considered bioavailable, that is, able to penetrate into cells.
Testosterone is a lipophilic molecule, so it passes through the cell membrane mainly by passive diffusion. Three possible "routes" await him inside the cell: direct binding to the androgen receptor, conversion to dihydrotestosterone or aromatization into estradiol. The ratio of these routes depends on the tissue, and this explains why testosterone acts differently on muscle, skin, prostate or brain.
Androgen receptor: structure and function
Androgen receptor (AR) belongs to the superfamily of nuclear receptors — proteins that are both hormone sensors and gene transcription regulators. The receptor gene is located on the X-chromosome, so it is present in one copy in men. This is of practical importance: gene variations directly affect a person's sensitivity to androgens.
In an inactive state, the receptor is in the cytoplasm in a complex with heat shock proteins. When testosterone or DHT binds to the ligand-binding domain, the receptor changes spatial configuration, is freed from chaperone proteins, forms a dimer, and moves to the cell nucleus.
- The N-terminal domain is responsible for the activation of transcription and contains a polymorphic CAG repeat.
- DNA-binding domain — recognizes specific regions of DNA, so-called androgen-sensitive elements.
- Hinge region — participates in the movement of the receptor into the nucleus.
- The ligand-binding domain is a "pocket" into which the hormone molecule enters.
In the nucleus, the receptor dimer binds to androgen-sensitive elements in the promoter regions of target genes and recruits coactivators. This triggers transcription: messenger RNA is synthesized on the DNA matrix, which is then translated into proteins. Thus, the hormonal signal is transformed into a change in the "protein composition" of the cell.
The length of the CAG repeat in the first exon of the gene is inversely related to receptor activity: shorter repeats are associated with higher sensitivity to androgens. This polymorphism partly explains why people with the same testosterone level can have different muscle mass, different propensities for acne or baldness, and different responses to therapy.
Protein synthesis and satellite cells
In skeletal muscles, androgen receptors are found both in the muscle fibers themselves and in satellite cells — stem cells located under the basal membrane of the fiber. Testosterone affects both populations, and it is this combination that provides a pronounced anabolic effect.
The first level of action is an increase in the fractional rate of muscle protein synthesis. As early as the 1990s, studies with isotopic labels (in particular, the group of Ferrando and Urban) showed that the administration of testosterone increases protein synthesis in muscle and improves the reutilization of amino acids. The end result is a positive protein balance where synthesis outweighs breakdown.
The second level is hypertrophy through satellite cells. Research by Sinha-Hikim and co-authors demonstrated that the dose-dependent increase in muscle fibers on the background of testosterone is accompanied by an increase in the number of satellite cells and the number of nuclei in the fiber. New nuclei allow the fiber to maintain a larger volume of cytoplasm, that is, to grow further.
The third level is the influence on the differentiation of progenitor cells. Experimental work shows that androgens direct mesenchymal pluripotent cells mainly in the myogenic direction and inhibit their transformation into adipocytes. This is consistent with clinical observation: against the background of testosterone, fat-free mass increases and the proportion of fat usually decreases.
Finally, testosterone has an anti-catabolic component. It partially counteracts the effects of glucocorticoids in the muscle and reduces the activity of some pathways of protein breakdown. However, the editors emphasize: most of these data were obtained on experimental models, and it is difficult to quantify the contribution of each mechanism in humans.
Metabolites: DHT and estradiol
The mechanism of action of testosterone cannot be described without its two key metabolites. The enzyme 5-alpha reductase converts testosterone to dihydrotestosterone, and aromatase (CYP19A1) to estradiol. Both reactions occur locally in tissues, so the effect of metabolites is often manifested exactly where they are formed.
| Metabolite | Enzyme | The main tissues of the formation | Key effects |
|---|---|---|---|
| Dihydrotestosterone (DHT) | 5-alpha-reductase type 1 and 2 | Prostate, skin, hair follicles | Stronger AR agonist; prostate growth, acne, androgenetic alopecia |
| Estradiol | Aromatase | Adipose tissue, brain, bones, testicles | Bone health, libido, pituitary feedback, excess gynecomastia |
| Testosterone (unchanged) | â | Skeletal muscles | The main active androgen in muscles |
DHT binds to the androgen receptor more strongly and dissociates more slowly than testosterone. However, in skeletal muscles, the activity of 5-alpha-reductase is low, so the anabolic effect in the muscle is mainly provided by testosterone itself. This is confirmed by studies in which blocking 5-alpha-reductase with finasteride did not cancel the increase in muscle mass on the background of testosterone.
Estradiol acts through estrogen receptors alpha and beta. In men, it is critically important for closing growth zones, mineralization of bones, and partly for libido and regulation of fat mass. The work of Finkelstein et al. (2013) in the NEJM is illustrative: when aromatization was blocked in men, some of the effects attributed to testosterone, particularly on fat mass and sexual function, appeared to be dependent on estradiol.
Thus, testosterone enanthate actually works as a "source" of three active hormones at once. The ratio between them depends on the dose, amount of adipose tissue, genetic variants of enzymes and receptors. That is why the same dose can give different people a markedly different profile of effects and adverse reactions.
Non-genomic effects and limits of the mechanism
The classical genomic pathway unfolds over hours and days. However, rapid, so-called non-genomic effects of androgens, which manifest themselves in seconds or minutes, have also been described: changes in intracellular calcium, activation of kinase cascades, influence on vascular tone. Some of them are probably mediated by membrane receptors or the extranuclear fraction of AR.
The practical significance of these rapid effects for humans is still debated. They are associated, in particular, with the influence of testosterone on the central nervous system — motivation, aggressiveness, perception of fatigue. However, the editors warn against popular claims that testosterone "instantly" increases strength: there is no convincing clinical data on such an effect.
An important aspect of the mechanism is saturation. The number of androgen receptors in tissue is limited, but clinical data from Bhasin et al (2001) showed that lean mass gains continued to increase with doses up to 600 mg per week in a study. This suggests that the simple "receptors are saturated at physiological level" model is not enough: higher concentrations can increase the number of receptors and involve additional pathways.
However, the same dose-dependent nature also applies to undesirable effects — a decrease in HDL, an increase in hematocrit, suppression of the hypothalamic-pituitary axis. The mechanism providing anabolism simultaneously operates in the liver, bone marrow and pituitary gland. It is impossible to separate the "beneficial" and "harmful" effects of testosterone within one molecule.
Editorial conclusion
Testosterone enanthate acts as a prodrug: after cleavage of the ester, normal testosterone works, which changes the activity of hundreds of genes through the androgen receptor. The main anabolic consequences are increased synthesis of muscle protein, involvement of satellite cells and a shift in the differentiation of progenitor cells towards muscle tissue.
A significant part of the effects of the drug is realized through metabolites. DHT is responsible for the effect on the skin, hair and prostate, estradiol - for bones, part of sexual functions and feedback with the pituitary gland. This explains why the effect of testosterone cannot be reduced only to "muscle growth".
The individual response to the same dose depends on the genetics of the receptor, the level of SHBG, enzyme activity and body composition. Therefore, it is incorrect to predict the result and risks "on average", and any therapy requires laboratory control.
If you want to understand the topic in more depth, the editors recommend our materials on the pharmacokinetics of testosterone enanthate, on its estrogenic activity and on which tests should be monitored during testosterone therapy.
References
- Bhasin S, Storer TW, Berman N, et al. The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. N Engl J Med. 1996;335(1):1â7.
- Bhasin S, Woodhouse L, Casaburi R, et al. Testosterone dose-response relationships in healthy young men. Am J Physiol Endocrinol Metab. 2001;281(6):E1172âE1181.
- Sinha-Hikim I, Artaza J, Woodhouse L, et al. Testosterone-induced increase in muscle size in healthy young men is associated with muscle fiber hypertrophy. Am J Physiol Endocrinol Metab. 2002;283(1):E154âE164.
- Finkelstein JS, Lee H, Burnett-Bowie SA, et al. Gonadal steroids and body composition, strength, and sexual function in men. N Engl J Med. 2013;369(11):1011â1022.
- Kicman AT. Pharmacology of anabolic steroids. Br J Pharmacol. 2008;154(3):502â521.
- Nieschlag E, Behre HM, Nieschlag S (eds). Testosterone: Action, Deficiency, Substitution. 4th ed. Cambridge University Press; 2012.
- Pope HG Jr, Wood RI, Rogol A, et al. Adverse health consequences of performance-enhancing drugs: an Endocrine Society scientific statement. Endocr Rev. 2014;35(3):341â375.
