Putting to bed the myth of AR downregulation Part1

[WCP]

Its long but one hell of a read!

Enjoy!

Androgen receptors down-regulate….Don’t they?

One misunderstood principle of steroid physiology is the concept of androgen receptors (AR), sometimes called "steroid receptors", and the effects of steroid use on their regulation. It is commonly believed that taking androgens for extended periods of time will lead to what is called AR "down regulation". The premise for this argument is; when using steroids during an extended cycle, you eventually stop growing even though the dose has not decreased. This belief has persisted despite the fact that there is no scientific evidence to date that shows that increased levels of androgens down regulates the androgen receptor in muscle tissue.

The argument for AR down-regulation sounds pretty straightforward on the surface. After all, we know that receptor down-regulation happens with other messenger-mediated systems in the body such as adrenergic receptors. It has been shown that when taking a beta agonist such as Clenbuterol, the number of beta-receptors on target cells begins to decrease. (This is due to a decrease in the half-life of receptor proteins without a decrease in the rate that the cell is making new receptors.) This leads to a decrease in the potency of a given dose. Subsequently, with fewer receptors you get a smaller, or diminished, physiological response. This is a natural way for your body to maintain equilibrium in the face of an unusually high level of beta-agonism.

In reality this example using Clenbuterol is not an appropriate one. Androgen receptors and adrenergic receptors are quite different. Nevertheless, this is the argument for androgen receptor down-regulation and the reasoning behind it. The differences in the regulation of ARs and adrenergic receptors in part show the error in the view that AR down-regulate when you take steroids. Where adrenergic receptor half-life is decreased in most target cells with increased catecholamines, AR receptors half-live’s are actually increased in many tissues in the presence of androgens.1

Let me present a different argument against AR down-regulation in muscle tissue. I feel that once you consider all of the effects of testosterone on muscle cells you come to realize that when you eventually stop growing (or grow more slowly) it is not because there is a reduction in the number of androgen receptors.

Testosterone: A multifaceted anabolic

Consider the question, "How do anabolic steroids produce muscle growth?" If you were to ask the average bodybuilding enthusiast I think you would hear, "steroids increase protein synthesis." This is true, however there is more to it than simple increases in protein synthesis. In fact, the answer to the question of how steroids work must include virtually every mechanism involved in skeletal muscle hypertrophy. These mechanisms include:

· Enhanced protein synthesis

· Enhanced growth factor activity (e.g. GH, IGF-1, etc.)

· Enhanced activation of myogenic stem cells (i.e. satellite cells)

· Enhanced myonuclear number (to maintain nuclear to cytoplasmic ratio)

· New myofiber formation

Starting with enhanced growth factor activity, we know that testosterone increases GH and IGF-1 levels. In a study by Fryburg the effects of testosterone and stanozolol were compared for their effects on stimulating GH release.2 Testosterone enanthate (only 3 mg per kg per week) increased GH levels by 22% and IGF-1 levels by 21% whereas oral stanozolol (0.1mg per kg per day) had no effect whatsoever on GH or IGF-1 levels. This study was only 2-3 weeks long, and although stanozolol did not effect GH or IGF-1 levels, it had a similar effect on urinary nitrogen levels.

What does this difference in the effects of testosterone and stanozolol mean? It means that stanozolol may increase protein synthesis by binding to AR receptors in existing myonuclei, however, because it does not increase growth factor levels it is much less effective at activating satellite cells and therefore may not increase satellite cell activity nor myonuclear number directly when compared to testosterone esters. I will explain the importance of increasing myonuclear number in a moment, first lets look at how increases in GH and IGF-1 subsequent to testosterone use effects satellite cells…

In part 2 we will discuss the role of satellite cells and myonuclei and how testosterone (androgens) activates these systems to create muscle growth far beyond what simple activation of the androgen receptor can produce.


WCP

 

[diamonddiceclay]

If I understood that correctly, I should be getting great results by using igf-1,gh,test at the same time. Am I correct?

 

[WCP]

Those drugs together yeah...but it was pointing out the increase in both IGF-1 and GH levels in males subjected to sythetic testosterone therapy.

I posted part 2 as well.

peace,
WCP

 

[Dr.Evil]

i'm so tired of people talking about "cleaning out" their receptors when they take time off...


another point i'd like to add is that the androgen receptor is in fact a promotor region on the mRNA within each cell. mRNAs do not decrease in number, but are pretty much constant within each cell. no amount of AS used for any duration will change that. steroids will either bind to this promotor region or they won't. how strongly they bind depends on each steroid.


good to see you back WCP. i hope your recovery from the hospital last year has gone smoothly and that you and your wife are well.

 

[panerai]

Changes in testosterone muscle receptors: effects of an androgen treatment on physically trained rats.
Cell Mol Biol (Noisy-le-grand) 1994 May;40(3):291-4 (ISSN: 0145-5680)

Bricout VA; Germain PS; Serrurier BD; Guezennec CY [Find other articles with these Authors]
IMASSA-CERMA, Departement de Physiologie Systemique, Bretigny sur Orge, France.

From results obtained in physiological investigations carried out on various tissues sensitive to androgens, it seems that the hormonal receptivity can reflect changes in the endocrine status and specific response of a tissue. The purpose of the present investigation was to test whether an androgen treatment could modify the receptivity to testosterone of the skeletal muscle and myocardium of endurance trained rats. The experiment extended over 8 weeks, and animals received injections of delayed testosterone heptylate every seven days. The myocardium and two skeletal muscles with opposed functions and typology were examined: the extensorum digitorum longus (EDL), and the soleus (SOL). Results obtained using techniques based upon the radio-competition principles provided information on the testosterone-receptor binding. The binding curves were plotted up to the saturating concentration of tritiated mibolerone, a synthetic androgen specific of androgen receptors. The quantity of receptors, calculated at the specific saturation plateau is expressed in fmol/mg protein. Results show that contractile muscular activity always increased the quantity of receptors whereas the steroid treatment decreased it. Thus for EDL and SOL of control trained rats the quantity of receptors was 0.78 and 0.82 fmol/mg protein, respectively, compared to 0.23 and 0.43 fmol/mg protein for sedentary testosterone-treated rats. The same "contractile activity" effect was observed on the myocardium but enhanced with values of 1.63 fmol/mg protein for control trained rats versus 0.30 fmol/mg protein for sedentary testosterone-treated rats. The receptivity to testosterone of the skeletal muscle and myocardium changes under the effect of an androgen treatment.(ABSTRACT TRUNCATED AT 250 WORDS).

 

[panerai]

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.
Mol Endocrinol 1993 Jul;7(7):924-36 (ISSN: 0888-8809)

Wolf DA; Herzinger T; Hermeking H; Blaschke D; Horz W [Find other articles with these Authors]
Institute of Physiological Chemistry, University of Munich, Germany.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work, the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nM of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease S1 analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcription 96 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to cells results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in vivo, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression.

 

[ultragainz]

 

[Shaved Ape]

Actually, mRNA do not contain promoters. Promoters are regulatory regions of genomic DNA located within the loci of the genes whose expression they regulate. Once the androgen binds the AR at the nucleus, the resulting complex can translocate to regulatory regions (including promoters and enhancers) on genomic DNA and activate the transcription of various genes. Transcriptional activation involves recruitment of the basal transcription machinery to the locus, followed by mRNA synthesis. These mRNA are then processed, spliced, and used as coding templates for protein synthesis.

 

[Bicho]

BUMP

 

[ebear]

WCP, good information. The studies panerai posted seem to offer quite a different perspective. Any thoughts?

 

[WCP]

Very interesting Panerai..

Where did you get those studies?...I would like to read more on them before I offer up any thoughts..

peace,
WCP

 

[Raffaz]

good post

 

[panerai]

Pubmed

 

[FlexManning]

There's also some obvious anecdotal evidence for the fact that receptor down-
regulation is a myth: A professional bodybuilder stops juicing for a few months, loses
an assload of muscle, then hops back on and gains it all back. How could he do that?
He doesn't really have those "fresh receptors" that everbody used to talk about.
Of course he's been off for awhile, but many of the pros are quite capable of reducing
themselves to nearly the size of a normal man, and then building themselves back up
into the top ranks in the space of a year. The guy
who will build muscle the fastest is almost always the guy who lost the most muscle in
the first place, not the guy with the freshest receptors.

 

[Ercole]

Excellent point, FlexManning! Food for thought.

 

[quenepo]

Good post,WCP

 

[Dr.Evil]

for the full article go to: http://mend.endojournals.org/cgi/content/full/13/11/1896

Mol Endocrinol 1999 Nov;13(11):1896-911


Multiple androgen response elements and a Myc consensus site in the androgen receptor (AR) coding region are involved in androgen-mediated up-regulation of AR messenger RNA.

Grad JM, Dai JL, Wu S, Burnstein KL.

Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101, USA.

The androgen receptor (AR) gene is transcriptionally regulated by AR (autoregulation); however, the androgen response elements (AREs) required for this process have not been found in the AR promoter or in the 5'-flanking region. We previously showed that the AR cDNA contains AREs involved in AR mRNA autoregulation and that auto(up)regulation is reproduced in PC3 cells (a human prostate cancer cell line) expressing the human AR cDNA driven by a heterologous promoter. A 350-bp fragment of the AR cDNA contains the requisite AREs (ARE-1 and ARE-2) and, when linked upstream of a reporter gene, confers androgen inducibility in a cell-specific manner. Here we report that, although an AR cDNA harboring silent mutations of ARE-1 and ARE-2 produces a transcriptionally active AR, AR mRNA encoded by this mutant cDNA is not up-regulated in androgen-treated PC3 cells. Thus, ARE-1 and ARE-2 are essential for androgen-mediated up-regulation of AR mRNA in this model. Since ARE-1 and ARE-2 are located on separate exons (exons D and E) in the AR gene, we evaluated these AREs in their native context, a 6.5-kb AR genomic fragment. Androgen regulated the 6.5-kb AR genomic fragment and the 350-bp region of the AR cDNA at comparable levels, suggesting that sequences in exons D and E are likely to be involved in androgen-mediated up-regulation of the native AR gene. Furthermore, androgen regulated both responsive regions in U2OS cells, a human osteoblastic cell line that exhibits androgen-mediated up-regulation of native AR mRNA. DNAse I footprinting of the 350-bp region with recombinant AR (DNA- and ligand-binding domains) suggested the presence of additional AREs. Gel shift analyses and mutational studies showed that maximal androgen regulation and AR binding were dependent on the integrity of four AREs (ARE-1, ARE-1A, IVSARE, and ARE-2). While the presence of multiple, nonconsensus AREs is common among other androgen-regulated enhancers, the androgen-responsive region of the AR gene is unique because it contains exonic AREs. DNA binding studies with nuclear extracts were performed to determine whether non-AR transcription factors contribute to androgen regulation of the 350-bp region. These studies, in conjunction with mutational analysis and reporter gene assays with dominant negative Myc and Max expression vectors, showed that Myc and Max interaction with a Myc consensus site is required for androgen regulation of the 350-bp fragment. These results represent a novel interaction between AR and the Myc family of proteins and support a model of androgenic control of AR mRNA via AR and Myc family interaction with a unique internal androgen-responsive region harboring multiple exonic regulatory sequences.

 

[Dr.Evil]

here's a study that somewhat contradicts the last one:

Mol Cell Endocrinol 1991 Apr;76(1-3):79-88 Related Articles, Books, LinkOut


Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCaP cells.

Krongrad A, Wilson CM, Wilson JD, Allman DR, McPhaul MJ.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8857.

We have examined the effect of androgen treatment on androgen receptor mRNA and protein expression in the LNCaP human prostate carcinoma cell line. Incubation with androgen caused a decrease in cellular androgen receptor mRNA content that was concentration and time dependent. Maximal suppression to approximately 35% of control level was observed after 49 h of exposure to androgen. By contrast, incubation of LNCaP cells with androgen resulted in a 2-fold increase in the cellular content of androgen receptor protein at 24 h. At 49 h androgen receptor protein increased 30% as assayed by immunoblots and 79% as assayed by ligand binding. These results suggest that ligand-induced changes in androgen receptor stability and/or the translational efficiency of androgen receptor mRNA account for the phenomenon of androgen receptor upregulation observed in cultured LNCaP cells. Furthermore, the suppression of androgen mRNA and protein that is caused by prolonged incubation with androgen is incomplete and is reversible upon removal of ligand.

 

[Dr.Evil]

Mol Endocrinol 1996 Dec;10(12):1582-94

Two androgen response elements in the androgen receptor coding region are required for cell-specific up-regulation of receptor messenger RNA.

Dai JL, Burnstein KL.

Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101, USA.

In most cells and tissues containing androgen receptors (ARs), androgen regulates the levels of AR messenger RNA (mRNA). As the AR concentration is correlated with androgen responsiveness, this autoregulation of AR mRNA may affect cellular sensitivity to androgens. Androgens decrease levels of AR mRNA in many cell lines and tissues; however, in some tissues and possibly also at certain developmental stages, AR mRNA is up-regulated by androgens. Sequences within the 5'-flanking region and AR promoter do not appear to be sufficient for androgen regulation of AR mRNA. We have previously shown that both down- and up-regulation of AR mRNA by androgen can be reproduced in cell lines expressing a transfected human AR complementary DNA (cDNA). Sequences within the AR cDNA confer this autoregulation in transfected cells, suggesting that sequences within the transcribed region of the AR gene are sufficient for autoregulation. In this study we have determined the mechanism of androgenic up-regulation of AR mRNA encoded by the human AR cDNA in the prostate cancer cell line, PC3, and have identified the cis-acting sequences of the AR cDNA that are required. The observations that actinomycin D blocked androgenic up-regulation of AR mRNA but cycloheximide had no effect are consistent with a model in which AR is directly involved in transcriptional up-regulation of AR cDNA expression. Nuclear run-on assays showed that androgen treatment resulted in increased transcription of the AR cDNA. Furthermore, a 350-bp AR cDNA fragment inserted 5' of a thymidine kinase promoter-chloramphenicol acetyltransferase gene conferred androgen induction of chloramphenicol acetyltransferase activity in PC3 cells. This 350-bp fragment, which is located in the AR coding region, contains two putative androgen response elements (AREs) separated by 182 bp. The 5'-most ARE (ARE-1, 5'-TGTCCT-3') resembles a half-site of the palindromic consensus hormone response element, recognized by several steroid receptors, including AR, and the 3'-sequence (ARE-2, 5'-AGTACTCC-3') is identical to a portion of an androgen-responsive region found in the rat probasin gene promoter. Analysis of either ARE-1 or ARE-2 mutants revealed that these elements function synergistically. AR protein binds to the 350-bp fragment, as demonstrated by electrophoretic mobility shift assays using a glutathione-S-transferase-AR fusion protein containing the DNA- and steroid-binding domains of AR. These results indicate that the AR coding region contains an androgen-responsive region that is involved in cell line-specific up-regulation of AR mRNA.

 

[davbrucas]

i'm so tired of people talking about "cleaning out" their receptors when they take time off...


another point i'd like to add is that the androgen receptor is in fact a promotor region on the mRNA within each cell. mRNAs do not decrease in number, but are pretty much constant within each cell. no amount of AS used for any duration will change that. steroids will either bind to this promotor region or they won't. how strongly they bind depends on each steroid.


good to see you back WCP. i hope your recovery from the hospital last year has gone smoothly and that you and your wife are well.

shaved ape is correct...there is no set number of mRNA molecules in a given cell. only when protein synthesis is needed (ligand binding to receptor) will DNA replication occur and an mRNA transcribed. the enhancer and promoter regions are on the DNA being transcribed. mRNA in eukaryotes has a 5' cap region, a pol-A tail, and introns and exons that are spliced to make the final molecule that the ribosomes translate to proteins.
high concentrations of a ligand (ie AS) causes less receptor production, and more turnover. the body tries to maintain homeostasis. so when you pump iron, you are basically putting a demand on the muscles to produce more mass. one way it does this is by increasing protein (receptor) synthesis and the excess AS present allows it to continue to grow as long as the demand and supply are there. in reality it is really not this simple, but this is a good overview of how things work, but i am sure that most of you know this because if i am putting things in my body, then i would research it fully! when i was getting my biochem degree at LSU, i worked in a prof's lab who was doing research on this very subject. interesting stuff!

 

[drveejay11]

Hell of a debate here! Anything else to add?