Androgen Receptors Downregulate Don’t They? Part 2

In part 1 of this article we discussed the mistake of thinking about androgen receptors (testosterone receptors) in the same way we think of other receptors such as beta-receptors. Beta-receptors down regulate in response to beta-adrenergic stimulation whereas there is good evidence that androgen receptors increase in numbers in response to androgens. We also discussed the various affects of testosterone on muscle growth. Testosterone does far more than simply increase the rate of protein synthesis!

Now in part 2 we will finish our discussion of androgen receptor regulation as it pertains to the way muscle cells grow. The very mechanism of real muscle growth opens the door for increased androgen receptor number in response to testosterone treatment.

Don’t forget Satellite cells!

Satellite cells are myogenic stem cells, or pre-muscle cells, that serve to assist regeneration of adult skeletal muscle. Following proliferation (reproduction) and subsequent differentiation (to become a specific type of cell), satellite cells will fuse with one another or with the adjacent damaged muscle fiber, thereby increasing the number of myonuclei for fiber growth and repair. Proliferation of satellite cells is necessary in order to meet the needs of thousands of muscle cells all potentially requiring additional nuclei. Differentiation is necessary in order for the new nucleus to behave as a nucleus of muscle origin. The number of myonuclei directly determines the capacity of a muscle cell to manufacture proteins, including androgen receptors.

In order to better understand what is physically happening between satellite cells and muscle cells, try to picture 2 oil droplets floating on water. The two droplets represent a muscle cell and a satellite cell. Because the lipid bilayer of cells are hydrophobic just like common oil droplets, when brought into proximity to one another in an aqueous environment, they will come into contact for a moment and then fuse together to form one larger oil droplet. Now whatever was dissolved within one droplet (i.e. nuclei) will then mix with the contents of the other droplet. This is a simplified model of how satellite cells donate nuclei, and thus protein-synthesizing capacity, to existing muscle cells.

Enhanced activation of satellite cells by testosterone requires IGF-1. Those androgens that aromatize are effective at not only increasing IGF-1 levels but also the sensitivity of satellite cells to growth factors.3 This action has no direct effect on protein synthesis, but it does lead to a greater capacity for protein synthesis by increasing fusion of satellite cells to existing fibers. This increases the number of myonuclei and therefore the capacity of the cell to produce proteins. That is why large bodybuilders will benefit significantly more from high levels of androgens compared to a relatively new user.

Testosterone would be much less effective if it were not able to increase myonucleation. There is finite limit placed on the cytoplasmic/nuclear ratio, or the size of a muscle cell in relation to the number of nuclei it contains.4 Whenever a muscle grows in response to training there is a coordinated increase in the number of myonuclei and the increase in fiber cross sectional area (CSA). When satellite cells are prohibited from donating viable nuclei, overloaded muscle will not grow.5,6 Clearly, satellite cell activity is a required step, or prerequisite, in compensatory muscle hypertrophy, for without it, a muscle simply cannot significantly increase total protein content or CSA.

More myonuclei mean more receptors

So it is not only true that testosterone increases protein synthesis by activating genetic expression, it also increases the capacity of the muscle to grow in the future by leading to the accumulation of myonuclei which are required for protein synthesis. There is good reason to believe that testosterone in high enough doses may even encourage new fiber formation. To quote the authors of a recent study on the effects of steroids on muscle cells:

“Intake of anabolic steroids and strength-training induce an increase in muscle size by both hypertrophy and the formation of new muscle fibers. We propose that activation of satellite cells is a key process and is enhanced by the steroid use.”7

Simply stated, supraphysiological levels of testosterone give rise to increased numbers of myonuclei and thereby an increase in the number of total androgen receptors per muscle fiber. Keep in mind that I am referring to testosterone and testosterone esters. Not the neutered designer androgens that people take to avoid side effects.

Another group of researchers are quoted as saying:

“…it is intriguing to speculate that the upregulation of AR levels via the administration of pharmacological amounts of androgens might convert some muscles that normally have a minor or no response to muscles with enhanced androgen responsiveness”(8)

This is not an argument to rapidly increase the dosages you use. It takes time for these changes to occur and the benefits of higher testosterone levels will not be immediately realized. It does shed some light however on the proportional differences between natural and androgen assisted bodybuilders physiques.

Maintenance of the kind of muscle mass seen in top-level bodybuilders today requires a given level of androgens in the body. That level will vary from individual to individual depending on their genetics. Nevertheless, if the androgen level drops, or if they were to “cycle off” the absolute level of lean mass will also drop. Likewise, as the level of androgens goes up, so will the level of lean mass that individual will be able to maintain. All of this happens without any evidence of AR down regulation. More accurately it demonstrates a relationship between the amount of androgens in the blood stream and the amount of lean mass that you can maintain. This does not mean that all you need is massive doses to get huge. Recruitment of satellite cells and increased myonucleation requires consistent “effective” training, massive amounts of food, and most importantly, time. Start out with reasonable doses. Then, as you get bigger you can adjust your doses upwards.

References:

1. Kemppainen JA, Lane MV, Sar M, Wilson EM. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J Biol Chem 1992 Jan 15;267(2):968-74

2. Fryburg DA., Weltman A., Jahn LA., et al: Short-term modulation of the androgen milieu alters pulsatile, but not exercise- or growth hormone releasing hormone-stimulated GH secretion in healthy men: Impact of gonadal steroid and GH secretory changes on metabolic outcomes. J Clin Endocrinol. Metab. 82(11):3710-37-19, 1997

3. Thompson SH., Boxhorn LK., Kong W., and Allen RE. Trenbolone alters the responsiveness of skeletal muscle satellite cells to fibroblast growth factor and insulin-like growth factor-I. Endocrinology. 124:2110-2117, 1989

4. Rosenblatt JD, Yong D, Parry DJ., Satellite cell activity is required for hypertrophy of overloaded adult rat muscle. Muscle Nerve 17:608-613, 1994

5. Rosenblatt JD, Parry DJ., Gamma irradiation prevents compensatory hypertrophy of overloaded extensor digitorum longus muscle. J. Appl. Physiol. 73:2538-2543, 1992

6. Phelan JN, Gonyea WJ. Effect of radiation on satellite cell activity and protein expression in overloaded mammalian skeletal muscle. Anat. Rec. 247:179-188, 1997

7. Kadi F, Eriksson A, Holmner S, Thornell LE. Effects of anabolic steroids on the muscle cells of strength-trained athletes. Med Sci Sports Exerc 1999 Nov;31(11):1528-34

8. Antonio J, Wilson JD, George FW. Effects of castration and androgen treatment on androgen-receptor levels in rat skeletal muscles. J Appl Physiol. 1999 Dec;87(6):2016-9.

Androgen Receptors Downregulate Don’t They? Part 1

There is as much misinformation about steroids as there is good information had among bodybuilding enthusiasts. Go to any gym and you will hear some kid spouting off to his buddies about how steroids do this, or how they do that, or whatever. This soon starts somewhat of a pissing contest (excuse the expression) as to who knows more about steroids. It’s the same kind of titillating and infectious banter that adolescent boys get into about girls and sex. With steroid banter you hear all the popular terms like Deca, Test, GH, gyno, zits, raisins, “h-u-u-u-ge”, roid, freak, monster, roid-rage, “I knew this guy once”, etc., etc.. If by some rare chance they are smart and have been reading this or some other high quality bodybuilding site on the net, they may actually get a few details right. More often than not they know just enough to be dangerous. Fortunately steroids haven’t proven to be all that dangerous. Not only that, but most of these guys who are infatuated with steroids won’t ever use or even see them except in magazines.

This kind of ego driven gym talk doesn’t really bother me until they begin giving advice to other clueless people who actually have access to them. Spewing out steroid lingo gives other less experienced kids the impression that these kids actually know what they are talking about. That’s how all of the psuedo-science folklore about steroids perpetuates. This is also why most people who actually use steroids know little about them. This last fact should bother anyone who cares about bodybuilding and/or bodybuilders.

I started out with this article planning on giving some textbook style explanation as to why using steroids doesn’t down regulate androgen receptors (AR). Then after considering some of my critics views that I tend to write articles that hardly anyone can read, I decided to write an easy to read, yet informative explanation about what androgens actually do and how this precludes androgen receptor down regulation. I still have a few references but not so many that it looks like a review paper.

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.

Testosterone – The Good, the Bad, the Ugly

One of the most infamous hormones around is Testosterone. You hear Clueless news anchors about it on the evening news. You hear about it in the gym. You even read about it in the “growing older with style” magazines. Depending on who you talk to, it is both the good, the bad, and the ugly of hormones.

In bodybuilding it is hailed as the king of muscle builders. Among forward-thinking baby boomers it is considered the fountain of youth. In other circles it is pointed to as the cause of all men’s shortcomings including violence and sexual promiscuity. Finally, it has even been associated with potentially lethal diseases that threaten the lives of thousands of men each year. So how can one hormone be so many different things to so many different people? Taking a closer look at this complex hormone may shed some light on this question.

First, what exactly is testosterone? Testosterone is the principle male hormone and belongs to a class of steroid chemicals called androgens (andro = man, gen = to make). It is produced primarily in the testes but can also be made by enzymatically converting other androgens (e.g. androstenediol) secreted from the adrenal gland into testosterone. Testosterone plays a role in everything from growth and maintenance of the male sexual organs during puberty, to male pattern baldness in the later years. It also plays an important role in bone growth, sexual behavior, male fertility, muscle protein synthesis, as well as inducing the appearance of secondary male sexual characteristics such as facial hair, body hair, and deepening of the voice.

Research has shown that resistance exercise can significantly raise testosterone levels. (1) This is good news if you’re looking to build a more muscular body. When in comes to muscle growth, testosterone production is the key to success. Testosterone literally turns on the genetic machinery leading to bigger and stronger muscles. It works like this. Testosterone binds to receptors inside your muscle cells. These receptors then transport the testosterone molecule to the nucleus. The nucleus is where your DNA is located. Your DNA contains blue prints for every protein found in your body. This androgen receptor, once bound to testosterone, acts as a messenger that tells the DNA which proteins to make from the blue prints. In muscle tissue the whole process results in the production of contractile proteins, which are used to make your muscle contract more forcefully, as well as structural proteins that are used to make the cell larger to accommodate the new contractile proteins. In plain and simple terms, testosterone is a messenger that tells your muscles to grow! Still, this barely touches the surface of the many secondary roles testosterone plays in muscle tissue as well as in the brain.

Clearly, testosterone is important to both mind and body. Among the anti-aging crowd, testosterone stands as a symbol of youth and vitality. One of the signs of aging is a reduction in the circulating levels of testosterone. This in turn has been associated with a decrease in muscle mass and strength as the years go by. Doctors are now calling this “andropause”. (2) Through testosterone replacement therapy, many older patients express a sense of psychological well-being and vitality they haven’t experienced since they were 30 years younger. (3,4) If men desire it, in the near future hormone replacement for men will be just as common as it is for women today.

Unfortunately, testosterone is not free from negative effects on the body. One common undesirable effect of testosterone, which could be considered minor, is alopecia or male pattern baldness. The drug Propecia, a 5-alpha reductase inhibitor, prevents the conversion of testosterone into a more potent androgen called dihydrotestosterone (DHT). DHT, and a set of your parent’s genes, is responsible for male pattern baldness. In many men Propecia is effective at preventing further hair loss and even allowing some to grow back. (5) On a more serious note, DHT may also be a serious risk factor for some cancers such as prostate cancer. (6) Treatment of prostate cancer often involves a total elimination of circulating testosterone. Although this helps to reduce the growth rate of tumors, removing a man’s testosterone leaves him feeling emotionally disoriented, there is a complete loss of sex drive and sexual function, muscle is lost and fat patterning takes on a feminine characteristic, even hot flashes, usually associated with female menopause, are experienced.

All in all testosterone plays a very important role in a man’s sense of health and well-being. It is the major muscle-building hormone; it increases the strength of both muscles and bones, and even affects our brains. Certainly a man’s interest in keeping his testosterone levels optimized is justified despite the unavoidable risks and negative effects it may impart. A healthy lifestyle including proper diet and regular resistance exercise will ensure that you are getting all the benefits testosterone has to offer.

Don’t think for a minute that testosterone is only important for men. For more information on how testosterone effects women, check out Contrarian Endocrinology Part I: Testosterone for Women by Karlis Ullis and Josh Shackman .

References:

1) Kremer WJ., Marchitelli L., Gordon SE., et al: Hormonal and growth factor responses to heavy resistance exercise. J Appl Physiol 69(4): 1442-1450, 1990

2) Tserotas K, Merino G. Andropause and the aging male. Arch Androl 1998 Mar-Apr;40(2):87-93

3) Lund BC, Bever-Stille KA, Perry PJ. Testosterone and andropause: the feasibility of testosterone replacement therapy in elderly men. Pharmacotherapy 1999 Aug;19(8):951-6

4) Tenover JL. Male hormone replacement therapy including “andropause”. Endocrinol Metab Clin North Am 1998 Dec;27(4):969-87

5) Baumann LS, Kelso EB. Selections from current literature: androgenetic alopecia: the science behind a new oral treatment. Fam Pract 1998 Oct;15(5):493-6

6) Sandow J, von Rechenberg W, Engelbart K. Pharmacological studies on androgen suppression in therapy of prostate carcinoma. Am J Clin Oncol 1988;11 Suppl 1:S6-10

Obesity, Health, and Metabolic Fitness

Fat. F-a-t. Perhaps no other word in our language is despised as much, nor focused on so intensely. Americans are obsessed about fat–body fat–and how to get rid of it. We have been conditioned to view health and fitness in strictly black (fat) and white (fit) terms: A “fat” body cannot possibly be fit and healthy. This fat-versus-fit dichotomy, made popular in the 1970s with the publication of fitness guru Covert Bailey’s “Fit or Fat?” (5), has become the mantra of many a fitness and health professional. You don’t have to read any more than the title to grasp the fundamental message of this perennial best-selling fitness bible: A person is either fit, or fat–but not both. [Read more…]

Connective Tissue Part 4: Glycosaminoglycans

This entry is part 4 of 4 in the series Connective Tissue

What are glycosaminoglycans?

Proteoglycans are very large molecules consisting of proteins with attached chains of polysaccharides called glycosaminoglycans (GAGs)(see Part 1). GAG chains contain repeating units of modified sugars: one of two amino sugars (glucosamine or galactosamine) and a uronic acid. Many of these chains attach to a protein core and are collectively referred to as a proteoglycan (PG) monomer. Imagine, if you will, a bottlebrush with the bristles as GAGs. The molecular weight of a PG monomer may be one million. In articular cartilage, up to a hundred of these monomers can link to a hyaluronic acid chain to form a PG aggregate. The molecular weight of the aggregate may be as much as 100,000,000. [Read more…]

Connective Tissue Part 3: The Good and the Bad Continued…

This entry is part 3 of 4 in the series Connective Tissue

As was discussed in Part II of this series, the major impact of diet upon connective tissue integrity is a deficiency in energy intake, usually associated with inadequate protein and carbohydrates. Aside from its role in mediation of inflammation, there is little research in effects of dietary fats on connective tissue. Micronutrients (minerals and vitamins) have many documented roles in cellular function and thus are critical in the wound healing process. Of nearly any population, athletes generally maintain an adequate diet specifically designed to meet the demands of their sport. Most athletes eat a balanced diet that adequately supplies both macro and micronutrients. Therefore, defects in collagen, elastin and proteoglycan metabolism are generally only seen as a result of deficiencies or excess. As well, successful healing of connective tissue injuries will rely on the presence of adequate nutritional stores. The roles of micronutrients in connective tissue injury healing are discussed in Part III of this series. [Read more…]

Connective Tissue Part 2: The Good, The Bad and the Ugly

This entry is part 2 of 4 in the series Connective Tissue

In Part I of this series, readers were introduced to basic histology and physiology of connective tissue. We learned that all connective tissue has similar components, although the proportions of these components vary. These variations impart the mechanical and biochemical attributes to specific connective tissue. To illustrate, mechanical properties of articular cartilage that allow it to absorb impact and resist wear are partially due to the large proteoglycan aggregates. [Read more…]

Connective Tissue Part 1: Tissue in Action

This entry is part 1 of 4 in the series Connective Tissue

Athletes are mostly concerned with increasing strength or speed in specific activities. Increasing muscle size and power, endurance abilities, fuel utilization efficiency: these are often the primary concerns in training. We spend much time, effort and money in maximizing our muscle capabilities. However, an integral part of our anatomy often takes a back seat: connective tissue. [Read more…]

The Myostatin Gene

Belgian Blue cow - myostatin

Super Cows and Mighty Mice

In 1997, scientists McPherron and Lee revealed to the public the ‘secret’ of an anomaly that livestock breeders have capitalized since the late 1800’s: the gene responsible for big beefy cows (1). More than a century ago, livestock breeders in Europe observed that some of their cattle were more muscled than others. Being dabblers in genetics, they selectively bred these cattle to increase the progeny displaying this trait. Thus two breeds of cattle (Belgian Blue and Piedmontese) were developed that typically exhibit an increase in muscle mass relative to other conventional cattle breeds. Little did they know that many years later Mighty Mouse would be more than merely a cartoon.

A team of scientists led by McPherron and Lee at John Hopkins University was investigating a group of proteins that regulate cell growth and differentiation. During their investigations they discovered the gene that may be responsible for the phenomenon of increased muscle mass, also called ‘double-muscling’ (1, 2). Myostatin, the protein that the gene encodes, is a member of a superfamily of related molecules called transforming growth factors beta (TGF-b ). It is also referred to as growth and differentiation factor-8 (GDF-8). By knocking out the gene for myostatin in mice, they were able to show that the transgenic mice developed two to three times more muscle than mice that contained the same gene intact. Lee commented that the myostatin gene knockout mice “look like Schwarzenegger mice.” (3).

Schwarzenegger Mice

Schwarzenegger Mice

Further exploration of genes present in skeletal muscle in the two breeds of double-muscled cattle revealed mutations in the gene that codes for myostatin. The double-muscling trait of the myostatin gene knockout mice and the double-muscled cattle demonstrates that myostatin performs the same biological function in these two species. Apparently, myostatin may inhibit the growth of skeletal muscle. Knocking out the gene in transgenic mice or mutations in the gene such as in the double-muscled cattle result in larger muscle mass. This discovery has paved the way for a plethora of futuristic implications from breeding super-muscled livestock to treatment of human muscle wasting diseases.

Researchers are developing methods to interfere with expression and function of myostatin and its gene to produce commercial livestock that have more muscle mass and less fat content. Myostatin inhibitors may be developed to treat muscle wasting in human disorders such as muscular dystrophy. However, several public media sources immediately raised the issue of abusing myostatin inhibitors by athletes. In addition, a hypothesis has been put forth that a genetic propensity for high levels of myostatin is responsible for the lack of muscle gain in weight trainees. Accordingly, this article presents a look at the science of myostatin and its implications for the athletic arena.

What is Myostatin?

Growth Factors

Before we can understand the implications of tampering with myostatin and its gene, we must learn what myostatin is and what it does. Higher organisms are comprised of many different types of cells whose growth, development and function must be coordinated for the function of individual tissues and the entire organism. This is attainable by specific intercellular signals, which control tissue growth, development and function. These molecular signals elicit a cascade of events in the target cells, referred to as cell signaling, leading to an ultimate response in or by the cell.

Classical hormones are long-range signaling molecules (called endocrine). These substances are produced and secreted by cells or tissues and circulated through the blood supply and other bodily fluids to influence the activity of cells or tissues elsewhere in the body. However, growth factors are typically synthesized by cells and affect cellular function of the same cell (autocrine) or another cell nearby (paracrine). These molecules are the determinants of cell differentiation, growth, motility, gene expression, and how a group of cells function as a tissue or organ.

Growth factors (GF) are normally effective in very low concentrations and have high affinity for their corresponding receptors on target cells. For each type of GF there is a specific receptor in the cell membrane or nucleus. When bound to their ligand, the receptor-ligand complex initiates an intracellular signal inside of the cell (or nucleus) and modifies the cell’s function.

A GF may have different biological effects depending on the type of cell with which it interacts. The response of a target cell depends greatly on the receptors that cell expresses. Some GFs, such as insulin-like growth factor-I, have broad specificity and affect many classes of cells. Others act only on one cell type and elicit a specific response.

Many growth factors promote or inhibit cellular function and may be multifactoral. In other words, two or more substances may be required to induce a specific cellular response. Proliferation, growth and development of most cells require a specific combination of GFs rather than a single GF. Growth promoting substances may be counterbalanced by growth inhibiting substances (and vice versa) much like a feedback system. The point where many of these substances coincide to produce a specific response depends on other regulatory factors, such as environmental or otherwise.

Transforming Growth Factors

Some GFs stimulate cell proliferation and others inhibit it, while others may stimulate at one concentration and inhibit at another. Based on their biological function, GFs are a large set of proteins. They are usually grouped together on the basis of amino acid sequence and tertiary structure. A large group of GFs is the transforming growth factor beta (TGFb ) superfamily of which there are several subtypes. They exert multiple effects on cell function and are extensively expressed.

A common feature of TGFb s is that they are secreted by cells in an inactive complex form. Consequently, they have little or no biological activity until the latent complex is broken down. The exact mechanism(s) involved in activating these latent complexes is not completely understood, but it may involve specific enzymes. This further exemplifies how growth factors are involved in a complex system of interaction.

Another common feature of TGFb s is that their biological activity is often exhibited in the presence of other growth factors. Hence, we can see that the bioactivity of TGFb s is complex, as they are dependent upon the physiological state of the target cell and the presence of other growth factors.

Myostatin

There are several TGFb s subtypes which are based on their related structure. One such member is called growth and differentiation factors (GDF) and specifically regulates growth and differentiation. GDF-8, also called myostatin, is the skeletal muscle protein associated with the double muscling in mice and cattle.

McPherron et al detected myostatin expression in later stages of development of mouse embryos and in a number of developing skeletal muscles (1). Myostatin was also detected in adult animals. Although myostatin mRNA was almost exclusively detected in skeletal muscle, lower concentrations were also found in adipose tissue.

To determine the biological role of myostatin in skeletal muscle, McPherron and associates disrupted the gene that encodes myostatin protein in rats, leading to a loss its function. The resulting transgenic animals had a gene that was rendered non-functional for producing myostatin. The breeding of these transgenic mice resulted in offspring that were either homozygous for both mutated genes (i.e. carried both mutated genes), homozygous for both wild-type genes (i.e. carried both genes with normal function) or heterozygous and carrying one mutated and one normal gene. The main difference in resulting phenotypes manifested in muscle mass. Otherwise, they were apparently healthy. They all grew to adulthood and were fertile.

Homozygous mutant mice (often called gene knockout mice) were 30% larger than their heterozygous and wild-type (normal) littermates irregardless of sex and age. Adult mutant mice had abnormal body shapes with very large hips and shoulders and the fat content was similar to the wild-type counterparts. Individual muscles from mutant mice weighed 2-3 times more than those from wild-type mice. Histological analysis revealed that increased muscle mass in the mutant mice was resultant of both hyperplasia (increased number of muscle fibers) and hypertrophy (increased size of individual muscle fibers).

Since this discovery, McPherron and other researchers investigated the presence of myostatin and possible gene mutations in other animal species. Scientists have reported the sequences for myostatin in 9 other vertebrate animals, including pigs, chickens and humans (2, 4). Research teams separately discovered two independent mutations of the myostatin gene in two breeds of double-muscled cattle: the Belgian Blue and Piedmontese (2, 5). A deletion in the myostatin gene of the Belgian Blue eliminates the entire active region of the molecule and is non-functional; and this mutation causes hypertrophy and increased muscle mass. The Piedmontese coding sequence for myostatin contains a missense mutation. That is, a point in the sequence encodes for a different amino acid. This mutation likely leads to a complete or nearly completes loss of myostatin function.

McPherron et al analyzed DNA from other purebred cattle (16 breeds) normally not considered as double-muscled and found only one similar mutation in the myostatin gene (2). The mutation was detected in one allele a single animal which was non-double-muscled. Other mutations were detected but these did not affect protein function.

Earlier studies reported high levels of myostatin in developing cattle and rodent skeletal muscles (2, 7). Furthermore, mRNA expression varied in individual muscles. Consequently, it was thought that myostatin was relegated to skeletal muscle and that the gene’s role was restricted to the development of skeletal muscle. However, A New Zealand team of researchers recently reported the detection of myostatin mRNA and protein in cardiac muscle (8).

TGF-b superfamily members are found in a wide variety of cell types, including developing and adult heart muscle cells. Three known isoforms of TGF-b (TGF-b 1, -b 2, and -b 3) are expressed differentially at both the mRNA and protein levels during development of the heart (9). This suggests that these isoforms have different roles in regulating tissue development and growth. Therefore, Sharma and colleagues investigated distribution of the myostatin gene in other organ tissues using more sensitive detection techniques than that used by earlier researchers (8).

They found a DNA sequence in sheep and cow heart tissue that was identical to the respective skeletal muscle myostatin protein sequence, indicating the presence of myostatin gene in these tissues. In heart tissue from a Belgian Blue fetus, the myostatin gene deletion present in skeletal tissue was detected. They detected the unprocessed precursor and processed myostatin protein in normal sheep and cattle skeletal muscle, but not in that of the Belgian Blue. As well, only the unprocessed myostatin protein was found in adult heart tissue.

Animals with induced myocardial infarction (causing death of cells in heart tissue) displayed high levels of myostatin protein, even at 30 days postinfarct, in cells immediately surrounding the dead lesion. However, undamaged cells bordering the infarcted area contained very low levels of myostatin protein similar to control tissue. Considering the increase in other TGF-b levels in experimentally infarcted heart tissue (10), these growth factors may be involved in promotion of tissue healing.

Shaoquan and colleagues at Purdue University detected myostatin mRNA in the lactating mammary glands of pigs, possibly serving a regulatory role in the neonatal pig (12). They also detected similar mRNA is porcine skeletal tissue, but not in connective tissue. Most studies, in addition to this one, confirm that high levels of myostatin mRNA in prenatal animals and reduced levels postnatal at birth and postnatal reflect a regulatory role of myostatin in myoblast (muscle cell precursors) growth, differentiation and fusion.

A mutation in the myostatin gene in the two cattle breeds is not as advantageous as in mice. The cattle have only modest increases in muscle mass compared to the myostatin knockout mice (20-25% in the Belgian Blue and 200-300% in the null mice). Also, the cattle with myostatin mutations have reduced size of internal organs, reductions in female fertility, delay in sexual maturation, and lower viability of offspring (6). Although no heart abnormalities in myostatin-null mice were reported, the hearts in adult Belgian Blue cattle are smaller (11). Although the reduction in organ weight has been attributed to skeletal muscle mass increases, this has yet to be confirmed. Since there is evidence that the effects of myostatin mutation on heart tissue are variable in different species, there may be other possible tissue variabilities as well. Additionally, research detected myostatin mRNA in tissues other than skeletal muscle, demonstrating its expression is not relegated to skeletal muscle tissue as originally thought. Only further research will elucidate these possibilities.

Although several TGF-b superfamily members are found in skeletal and cardiac muscle tissue, their exact roles in development is not yet clear. Apparently, based on the early studies, the myostatin protein may have diverse roles in developmental and adult stage tissues. Sharma et al proposes that “myostatin has different functions at different stages of heart development” (8). As we shall see, the same can conceivably apply to skeletal muscle as well.

Myostatin and regulation of skeletal muscle

While many of the studies demonstrate that myostatin is involved with prenatal muscle growth, we know little of its association with muscle regeneration. Muscle regeneration of injured skeletal muscle tissue is a complex system and ability for regeneration changes during an animal’s lifetime. Exposure of tissues to various growth factors is altered during a lifetime. In embryos and young animals, hormones and growth factors favor muscle growth. However, many of these factors are downregulated in adults. Alteration in growth factors inside and outside of the muscle cells may diminish their capacity to maintain protein expression. Although protein mRNA may be detected within the cell, there are many sites of protein regulation beyond mRNA levels. As mentioned above, myostatin protein occurs in an unprocessed (inactive) and processed (active) form. Therefore, bioactivity of myostatin may be regulated at any point of its synthesis and secretion.

Keep in mind that nearly all regulatory systems in the body are under positive and negative control. This includes cardiac and skeletal muscle tissues. Myoblasts in developing animal embryos respond to different signals that control proliferation and cell migration. In contrast, differentiated muscle cells respond to another set of different signals. Distinct ratios of signals regulate the transition from undetermined cells to differentiated cells and ensure normal formation and differentiation in cellular tissues. However, many of the factors that regulate the various development pathways in muscle tissue are still poorly understood.

MyoD, IGF-I and myogenin (growth promoters in muscle cells) gene products are associated with muscle cell differentiation and activation of muscle-specific gene expression (14). Muscle-regulatory factor-4 (MRF-4) mRNA expression increases after birth and is the dominant factor in adult muscle. This growth factor is thought to play an important role in the maintenance of muscle cells. In addition to myostatin, there are other inhibitory gene products, such as Id (inhibitor of DNA binding). Although in vitro experiments are revealing the mechanisms of these specific proteins, we know less regarding their roles in vivo.

Although we know that lack of myostatin protein is associated with skeletal muscle hypertrophy in McPherron’s gene knockout mice and in double-muscled cattle, we know little about the physiological expression of myostatin in normal skeletal muscle. Recent studies in animal and human models indicate a paradox in myostatin’s role on growth of muscle tissue.

For example, evidence shows that myostatin may be fiber-type specific. Runt piglets, which have lower birth weights than their normal littermates, had lower proportions of Type I skeletal muscle fibers in specific muscles (12). Similar observations were made in rats where undetectable levels of myostatin mRNA in atrophied mice soleus (Type I fibers) (13). Transient upregulation of myostatin mRNA was detected in atrophied fast twitch muscles but not in slow twitch muscles. Thus, myostatin may modulate gene expression controlling muscle fiber type.

Studies also demonstrated lack of metabolic effects on myostatin expression in piglets and mice (12, 13). Food restriction in both piglets and mice did not affect myostatin mRNA levels in skeletal muscle. Neither dietary polyunsaturated fatty acids nor exogenous growth hormone administration in growing piglets altered myostatin expression (12). These and other studies strongly suggest that the physiological role of myostatin is mostly associated with prenatal muscle growth where myoblasts are proliferating, differentiating and fusing to form muscle fibers.

Although authors postulate that myostatin exerts its effect in an autocrine/paracrine fashion, serum myostatin has been detected demonstrating that it is also secreted into the circulation (8, 4). It is believed that the protein detected in human serum is of processed (active form) myostatin rather than the unprocessed form. High levels of this protein have been associated with muscle wasting in HIV-infected men compared to healthy normal men (4). However, this association does not necessarily verify that myostatin directly contributes to muscle wasting. We do not know if myostatin acts directly on muscle or on other regulatory systems that regulate muscle growth. Although several authors postulate that myostatin may present a larger role in muscle regeneration after injury, this has yet to be confirmed.

Myostatin and athletes

Further complicating the issue of myostatin’s role in regulation of muscle growth is the report by a team of scientists that mutations in the human myostatin gene had little impact on responses in muscle mass to strength training (15, unpublished data). Based on the report that muscle size is a heritable trait in humans (16), Ferrell and colleagues investigated the variations in the human myostatin gene sequence. They also examined the influence of myostatin variations in response of muscle mass to strength training.

Study subjects represented various ethnic groups and were classified by the degree of muscle mass increases they experienced after strength training. Included were competitive bodybuilders ranking in the top 10 world-wide and in lower ranks. Also included were football players, powerlifters and previously untrained subjects. Quadricep muscle volume of all subjects was measured by magnetic resonance imaging before and after nine weeks of heavy weight training of the knee extensors. Subjects were grouped and compared by degree of response and by ethnicity.

There were several genetic coding sequence variations detected in DNA samples from subjects. Two changes were detected in a single subject and another two were observed in two other individuals. They were heterozygous with the wild-type allele, meaning they had one allele with the mutation and the other allele was normal. The other variations were present in the general population of subjects and determined common. One of the variations was common in the group of mixed Caucasian and African-American subjects. However, the less frequent allele had a higher frequency in African-Americans. Although, as the authors comment, “these variable sites [in the gene sequence] have the potential to alter the function of the myostatin gene product and alter nutrient partitioning in individuals heterozygous for the variant allele”, the data from this and other studies so far show that this may not occur. This study did not demonstrate any significant response between genotypes and response to weight training. Nor were there any significant differences between African-American responders to strength training and non-responders or between Caucasian responders and non-responders.

Further research will be necessary to determine whether myostatin has an active role in muscle growth after birth and in adult tissues. To ascertain benefit to human health, we also need to discover its role in muscle atrophy and regeneration after injury. Only extended research will reveal any such benefits.

The future of myostatin

Now that we have reviewed some of the biology of the myostatin protein, its gene, and the relevant scientific literature, what are the implications for its application?

Many authors of the myostatin studies have speculated that interfering with the activity of myostatin in humans may reverse muscle wasting disease associated with muscular dystrophy, AIDS and cancer. Some predict that manipulation of this gene could produce heavily muscled food animals. Indeed, current research is underway to investigate and develop these potentialities. Sure enough, a large pharmaceutical company has recently applied for a patent on an antibody vaccination for the myostatin protein.

A medical doctor and author of weight training articles asserts that overexpression of myostatin is to blame for weight lifters that have trouble gaining muscle mass. The spokesperson for a supplement and testing lab erroneously implied that the “rarest” form of mutation in the myostatin gene is responsible for a top competitive bodybuilder’s massive muscle gains, not taking into account the performance-enhancement substances the bodybuilder may be using. The public media has, of course, predicted that “steroid-popping” athletes will take advantage of myostatin inhibitors to gain competitive edge (3).

Many of these assertions are unfounded or they misrepresent the science. Granted, the possibility exists that manipulation of the myostatin gene in humans may be a key to reversing muscle-wasting conditions. However, too little is still yet unknown regarding myostatin’s role in muscle growth regulation. It is imperative that research demonstrates that the loss of myostatin activity in adults can cause muscle tissue growth. Likewise, research must also prove that overexpression or administration of myostatin causes loss of muscle mass. Also important is to know if manipulation of myostatin will interfere with other growth systems, especially in other tissues, and result in abnormal pathologies. Although McPherron’s gene knockout mice did not experience any other gross abnormalities, mice are not humans.

We do not fully understand the roles of myostatin in exercise-induced muscle hypertrophy or regeneration following muscle injury. Until we do, it may be premature to blame the lack of hypertrophy in weightlifters on overexpression of myostatin. Nor does the research support the claim that a top bodybuilder’s muscle mass gains are resultant of a detected mutation in the myostatin gene. The research simply does not advocate blaming genetic myostatin variations as a source of significant differences in human phenotypes.

Considering the history of the athlete’s propensity, in the public eye, to abuse performance-enhancement substances, the media’s prediction of myostatin-inhibitor may or may not be warranted. We all know that today’s athletic arena demands gaining the competitive edge to maintain top level competition. For many athletes, that is accomplished by supplementing hard training with substances that enhance growth or performance. Whether or not myostatin inhibitors will be added to the arsenal of substances is difficult to predict. Until science reveals the full nature of this growth factor and its role in the complex regulation of muscle tissue, and researchers determine its therapeutic implications, we can only surmise. Despite attempts to tightly control any pharmaceutical uses of myostatin protein manipulation, they will likely surface at some point in the black market world of bodybuilding supplements. Let us hope that science has determined the side effects and the benefits by that point.

References

McPherron, AC, AM Lawler, SJ Lee. Regulation of skeletal muscle mass in mice by a new TGF-b superfamily member. Nature 1997, 387:83.

McPherron, AC, SJ Lee. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA 1997, 94:12457

John Hopkins Magazine, June 1997. URL: http://www.jhu.edu./~jhumag/0697/web/science.html

Gonzalez-Cadavid, NF, WE Taylor, K Yarasheski, et al. Organization of the human myostatin gene and expression in healthy and HIV-infected men with muscle wasting. Proc Natl Acad Sci 1998, 95:14938.

Grobet, L, LJR Martin, D Poncelet, et al. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nature Genet 1997, 17:71.

Menissier, F. In: Muscle Hypertrophy of Genetic Origin and its Use to Improve Beef Production, eds. King, JWB and F Mennissier. Nijhoff, The Haugue, The Netherlands, pp. 23-53.

Kambadur R, M Sharma, TPL Smith, JJ Bass. Mutations in myostatin (GDF8) in double-muscled Belgian Bllue and Piedmontese cattle. Genome Res 1997, 7:910.

Sharma M, R Kambadur, KG Matthews, et al. Myostatin, a transforming growth factor-b superfamily member, is expressed in heart muscle and is upregulated in cardiomycetes after infarct. J Cell Physiol 1999, 180:1.

Millan FA, F Denhes, P Kondaiah, et al. Embryonic gene expression pattern of TGF-b 1, b 2, and b 3 suggest different developmental function in vivo. Development 1991, 111:131.

Sharma, HS, M Wunsch, T Brand, et al. Molecular biology of the coronary vascular and myocardial responses to ischemia. J Cardiovas Pharmacol 1992, 20:S23.

Bocard R. 1981. Facts and reflections on muscular hypertrophy in cattle: double muscling or culard. In: Developments in Meat Science, Vol. 2. Lawrie R, ed. Applied Science Publishers, London, pp. 1-28.

Shaoquan, J, RL Losinski, SG Cornelius, et al. Myostatin expression in porcine tissues: tissue specificity and developmental and postnatal regulation. Am J Physiol 1998, 275:R1265.

Carlson, JC, FW Booth, SE Gordon. Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol 1999, 277:R601.

Marsh, DR, DS Criswell, JA Carson, FW Booth. Myogenic regulatory factors during regeneration of skeletal muscle in young, adult and old rats. J Appl Physiol 1997, 83:1270.

Ferrell, RE, V Conte, EC Lawrence, et al. Frequent sequence variation in the human myostatin (GDF8) gene as a marker for analysis of muscle related phenotypes. Genomics, in press.

Loos, R, M Thomis, HH Maes, et al. Gender-specific regional changes in genetic structure of muscularity in early adolescence. J Appl Physiol 1997, 82:1602.

Pharmacological Approaches to Fat Loss – Targeting Beta-Adrenergic Receptors

There are dozens of products on the market that claim to be “fat burners”. There is such a demand for effective supplements and drugs to “burn fat” it has driven diet drugs and supplements into a big money industry. Amidst the clamor to try the latest drug to meet FDA approval an ancient and common remedy has been widely overlooked by the general public. Bodybuilders, on the other hand, have been using it widely for some time. What is this “ancient Chinese secret”? Ephedra of course.

Ephedra has been used in China for at least two thousand years. The most familiar form of Ephedra is the Chinese herb ma huang. It’s active ingredient is ephedrine. Ephedrine is an alkaloid that acts as a sympathomimetic and has thermogenic and anorectic properties. It is commonly used as a smooth muscle dilator in the treatment of asthma, bronchitis and nasal congestion. So what does this have to do with fat loss you ask? In order to properly use ephedrine as a tool for fat loss, it’s mechanism of action needs to be understood. Hereafter we will explore the possible mechanism of ephedrine’s thermogenic/lipolytic effects and it’s potential as a fat loss agent. Then we’ll take a look at human studies involving the use of ephedrine and a couple of additional compounds that seem to enhance ephedrine’s fat reducing properties.

As a sympathomimetic, ephedrine acts to stimulate the sympathetic nervous system. It does this by causing pre-synaptic nerve terminals to release norepinephrine, or what is commonly called noradrenaline (NA), into the synaptic space. It also has the effect of increasing circulating adrenaline (Adr), the body’s chief beta-2 agonist. Noradrenaline, once released into the synaptic space, interacts with adrenergic receptors on the surface of adipocytes (also known as plain old fat cells). This initiates a sequence of events within the adipocyte that increases lipolysis.


The Process of Lipolysis

Lipolysis is the process of breaking down triglycerides into glycerol and fatty acids. This process is dependent on an enzyme called hormone sensitive lipase (HSL). Activating HSL is the last step of a chain of intracellular reactions that make up the second messenger system. It is called a second messenger system because NA acts as the first messenger and Cyclic Adenosine Monophosphate (cAMP) acts as the second.

The entire chain of events that occurs after administration of ephedrine goes as follows: 1) Ephedrine stimulates the release of NA from sympathetic nerve endings. 2) NA then binds to adrenergic receptors on the surface of all tissues that contain these receptors. Adipose tissue and skeletal muscle have abundant adrenoreceptors on their surface. 3) As NA binds to beta-adrenergic receptors, stimulatory guanine nucleotide regulatory proteins(Gs-proteins) within the cell membrane activate the enzyme adenylate cyclase. 4) Adenylate cyclase then converts ATP into 3′-5′ cAMP. 5) cAMP then binds to the regulatory subunit of protein kinase A. 6) Once bound by cAMP, protein kinase A releases its catalytic subunit. 7) The catalytic subunit phosphorylates HSL, thus transforming it into the active form, HSL-P. 8) HSL-P then catalyzes a three step hydrolysis reaction to reduce triglycerides into glycerol and fatty acids.

Step One

Lets go back and take a closer look at these steps. In step one it is important to realize that ephedrine does not interact directly with adrenergic receptors. It is through it’s effects on the release of NA that ephedrine increases adrenergic activity.1This was determined by examining the effects of ephedrine on adipose tissue with intact sympathetic nerves or without. Once the nerves had been removed, the ephedrine had little, if any, effect at concentrations typical of oral administration. This has a number of disadvantages as well as some advantages. First the disadvantages. Ephedrine is called a non-specific adrenergic agonist because through the release of NA, it has an effect on more than one class of adrenergic receptor. NA can bind with alpha and beta receptors alike. This produces a generalized effect because alpha receptors, particularly alpha-2 receptors, decrease lipolysis and beta receptors increase lipolysis. The overall lipolytic effect of ephedrine is determined by the ratio of alpha and beta receptors on each particular adipocyte.

Another disadvantage is potency. Non-specific agonists have a far weaker effect on beta-receptors than specific beta agonists such as epinephrine, Albuterol or Clenbuterol. This is obvious once you look at the mechanism. Clenbuterol will interact directly with the beta-receptor with or without sympathetic activity in a dose dependent manner. Ephedrine is dependent on the release of noradrenaline to do the job and is only dose dependent up to a point. Continuing with the problem of potency, it is well known that the selective beta-agonist Clenbuterol, has potent anabolic activities in animal studies when used in dosages equal to about 4 mg per kg body weight for a period lasting approximately 10 days. This effect is dependent on long and steady activation of the receptor by the agonist.27Clenbuterol is the most effective anabolic beta-agonist by virtue of it’s long half-life (34-35 hours). In contrast, the half-life of ephedrine is only about 3-4 hours. Contributing to Clenbuterol’s long duration of action is the fact that it does not undergo first-pass metabolism like most other beta-agonists. The exception being the structurally related beta-agonist Mabuterol which has a half-life of 20-30 hours.28  The vast majority of beta-agonists have half-lives of only up to 6 hours.29It should be noted however that ephedrine does show some anabolic action even with such a short half-life.14This relationship between anabolic activity and half-life of beta agonists would indicate that all beta-agonists have the potential for anabolic activity, whether or not this translates into noticeable gains in muscle size depends on how long the active form of the drug interacts with the beta receptor on muscle tissue. The truthfulness of this statement was demonstrated in a study by Choo27which took the beta agonist Salbutamol which has not been shown to produce anabolic effects and compared it with Clenbuterol during continuous infusion in animals. Under these conditions the half-life of the substance is not a factor and the drug can bypass the liver, avoiding first pass degradation. During continuous infusion Salbutamol produced equal anabolic effects in muscle tissue as clenbuterol.

The assertion that beta-agonists such as clenbuterol and ephedrine have no anabolic effects in humans is premature. There is a large difference in the dosages normally given to animals (4 mg/kg) as compared to humans (up to 40 µg/day). Slow release Salbutamol has been shown to increase voluntary muscle strength in healthy men.30,31Research showing preservation of lean tissue and significantly improved protein deposition in response to treatment with ephedrine during caloric restriction indicates that beta-agonists are exerting an anabolic effect in humans.14More research is needed to determine the extent and most efficacious way to administer these compounds to elicit an anabolic effect in man.

The advantages to using a non-specific beta agonist are two fold. First, although ephedrine binds to other adrenergic receptors, it seems that the most beneficial adrenergic effects, such as thermogenesis, are actually enhanced after chronic use.2,3This may be explained by chronic stimulation of alpha receptors by NA and Adr. This chronic alpha-adrenergic stimulation may activate thyroxin deiodinases leading to the peripheral conversion of T4 to T3. In fact, significant increases in the ratio of T3 to T4 have been shown to occur after 4 weeks of chronic treatment of ephedrine.2Increased levels of T3 can sensitize adrenergic sensitivity to NA and Adr. It should be noted that the same study showed that this ratio decreased below initial values after week 12 of treatment.

Another explanation of its increased efficacy after chronic treatment is it’s interaction with the beta-3 receptor. Although the exact structure and function of this receptor is still being explored, it is almost certain that at least 40% of ephedrine’s actions are due to it’s effect on beta-3 receptors.6A study done to explore this used a beta-1 and beta-2 antagonist called nadolol. Nadolol was administered concomitantly with ephedrine to healthy volunteers. Nadolol completely inhibited changes in heart rate and plasma glucose due to it’s blockade of beta-1 and beta-2 receptors. However, the thermogenic effect of ephedrine was still at about 43%. This means that at least 40% of ephedrine’s thermogenic effects are due to beta-3 activation. This alone does not explain ephedrine’s effects after long term use. What does explain this is the desensitization properties of the beta-3 receptor. Beta-3 receptors lack most of the structural properties that are responsible for beta-2 receptor desensitization.7So even after ephedrine fails to have significant effects on the beta-2 receptor, it would potentially continue to stimulate adenylate cyclase activity by virtue of its effect on the beta-3 receptor.

There seems to be an impression that research with “selective” beta 3-agonists has not been promising. This may stem from earlier research done before second generation beta3-agonists were available. Early research showing questionable results was done by Rosenbaum32using a non-selective beta agonist, isoproterenol. Newer more potent compounds such as CL316,243 and CGP12177A (beta 1- and beta 2-AR antagonist, beta 3-AR agonist) are currently undergoing safety and efficacy testing in primates. Initial indicators appear to be promising with these newer “selective” beta 3-adrenergic agonists.33,34,35This avenue of pharmacologic treatment of obesity and diabetes which includes some benefit to bodybuilders, has great potential keeping in mind that activation of the beta 3-adrenergic receptor not only increases the production of thermogenic uncoupling proteins in fat and muscle, increases brown adipose tissue differentiation and perhaps proliferation, but also increases non-oxidative glucose disposal in peripheral tissues including muscle.36,37

Concerning beta-2 receptor desensitization, the fact that ephedrine is less potent than specific beta-2 agonists decreases the amount of beta receptor down regulation subsequent to chronic treatment. Chronic stimulation of beta2-adrenergic receptors causes a decrease in the sensitivity of tissues to beta agonists. This decrease in sensitivity involves either homologous desensitization, where the receptor’s active site is translocated within the cell membrane so that the binding site is no longer positioned extra cellularly, or it involves heterologous desensitization, where the receptor is phosphorylated rendering it incapable of participating in the second messenger system.4Receptor desensitization is a complex process with several different mechanisms. This complexity allows for more control of hormone signaling. As a general rule, the more potent the stimulus, the greater and more rapid the desensitization. Clenbuterol elicits a strong thermogenic effect but only for a short period of about two weeks. The usual practice is to increase the dosage at this time. Unfortunately, this only increases desensitization further and makes catecholamine stimulated lipolysis virtually come to a halt. Ephedrine, on the other hand, elicits a more mild response but it’s thermogenic effects can be seen up to 20 weeks.5

In summary, the advantages of using a non-selective beta agonist such as ephedrine are, it has beneficial effects on thyroxin deiodinase activity thereby increasing the T3/T4 ratio, it’s effect on beta-3 receptors, and it’s tendency not to cause extreme desensitization of beta-2 receptors. All of this lends to the fact that the thermogenic affects of ephedrine are enhanced after chronic treatment.

Step Two

Going back to the steps of ephedrine stimulated lipolysis, in step two we see that NA binds to the adrenergic receptors on the surface of tissues that contain them. By looking at all of ephedrine’s side effects you get an idea of what tissues contain adrenergic receptors. In the beginning you get an increase in heart rate. This is because beta adrenergic stimulation of the heart increases it’s rate, force and frequency of contraction. It also causes vasoconstriction which leads to a decrease in blood flow to most organs including skin, eyes, kidney and gastrointestinal tract. In most all of these tissues, the sympathetic response is designed to help the body respond to perceived emergencies. You may have heard of the “flight or flight” response. By reducing blood flow to the viscera, more blood is available to be diverted to working muscles. The heart pumps more blood, the eyes dilate, the GI tract slows motility (no time for potty breaks), and attention, or alertness, is enhanced allowing the animal (or person) to be focused on escape or capture and less focused or perceptive of pain.

Muscle and fat tissue also contain adrenergic receptors. In fat tissue there are gender specific ratios of beta to alpha receptors on various parts of the body. This gives rise to the familiar male (android) and female (gynoid) fat patterning. Females tend to resist lipolysis on their hips, buttocks and thighs, whereas men tend to resist lipolysis on their abdomen and oblique region. This is due to a preponderance of antilipolytic alpha receptors on the cells in these regions. Our goal in using beta agonists is to increase lipolysis in fat tissue. The effect of both alpha and beta receptors being located on fat tissue allows for more control of lipolysis. In essence it gives the body both an accelerator and a brake.

In muscle tissue, beta adrenergic activation seems to stimulate protein synthesis rates through the same second messenger system that stimulates lipolysis in fat tissue. In studies measuring body composition as well as weight loss, ephedrine has shown the ability to prevent lean tissue loss.8In a small double blind study lasting only eight weeks, two groups of obese women were given either 20 mg ephedrine with 200 mg caffeine (E+C) or placebo (P). After eight weeks weight-loss was not significantly different between the groups, but the E + C group lost 4.5 kg more body fat and 2.8 kg less fat-free mass (FFM). That is a difference of more than six pounds in eight weeks. The expected decrease in 24-hour energy expenditure (EE) seen in the P group was 10% at day 1 and 13% at day 56, but was only 7% and 8% in the treated group. The higher EE in the E +C group was entirely covered by fat oxidation. People have speculated that beta-agonists were anticatabolic. This is disproved somewhat by the fact that 3-methylhistidine, which is used as an index for skeletal muscle breakdown, was not altered by ephedrine administration yet nitrogen balance was significantly improved during very low calorie dieting.9So although muscle tissue was being broken down at the same rate, more lean tissue was being produced.

Step Three

In step three we see that G proteins play a key role in regulating fat metabolism in adipocytes. In this step NA binds to the adrenergic beta-receptor activating stimulatory G proteins (Gs). These G proteins then go on to activate adenylate cyclase. When alpha receptors are activated, inhibitory G proteins (Gi) are activated and adenylate cyclase is not activated. This puts a halt to cAMP formation and hence a halt to lipolysis. G proteins are also involved, at least in part, in receptor desensitization. If you recall it was briefly mentioned that one of the ways beta-receptors are desensitized is called heterologous desensitization. This involves uncoupling of the receptor from stimulatory G proteins. This has the effect of stopping the message from getting any further than the receptor itself. Other mechanisms are also involved in heterologous desensitization but we will not go into it any further in this article.

Step Four

In step four, ATP is converted into cAMP and inorganic phosphate (PPi) by the enzyme adenylate cyclase. cAMP contains a single phosphate group that is attached both to the 3′ carbon and the 5′ carbon of the sugar ribose. This is why it is called “cyclic” AMP. Neither PPi nor cAMP is allowed to exist in these forms for very long. The PPi that is formed when ATP is converted to cAMP is hydrolyzed by inorganic pyrophosphatase to form two Pi. 3′-5′-cAMP is also quickly rendered inactive by the enzyme known as cyclic AMP phosphodiesterase (PDE). PDE breaks the bond between the 3′ carbon of ribose and the phosphate group thus making 5′-cAMP. 5′-cAMP is inactive and does not bind to protein kinase A and does not lead to the activation of HSL. This is the first feedback inhibition mechanism we’ve discussed so far but there are others that act to shut off the original signal and slow lipolysis. As we will see later, feed back inhibition is one area we can target to increase the effectiveness of ephedrine.

Steps 5-8

In steps five, six, seven and eight cAMP binds to the regulatory subunit of protein kinase A. This binding releases the catalytic subunit of protein kinase A which then phosphorylates HSL. Once HSL is phosphorylated it can then participate in the actual process of lipolysis. This brings us to the final step. HSL-P catalyzes the breakdown of triglycerides in three steps. Each of the three steps removes one fatty acid until all that is left is glycerol and three fatty acids. Now, just because you have disassembled your stored fat does not mean it is gone for good. If you do not burn this fat it will simply be re-esterified and turned back into triacylgylcerol (storable triglycerides). This process of lipolysis and lipogenesis using the same fatty acids is called a “futile cycle” for obvious reasons.


Feedback Inhibition

The process of lipolysis is under feedback control which attenuates lipolysis at several levels. The chemicals involved in attenuating the effectiveness of ephedrine are phosphodiesterases, prostaglandins, and adenosine. As you might expect, these are the chemicals that we will try to minimize while using ephedrine as a fat loss agent.

Phosphodiesterases (PDE’s)

As with most biologically active molecules, cAMP must be rapidly inactivated in order to serve as a controllable second messenger in response to hormone activation. In target cells, phosphodiesterases (PDE’s) act to hydrolyze cAMP into inactive fragments. Because of PDE’s, the stimulatory effect of norepinephrine and epinephrine, which use cAMP as a second messenger, depends on continuous regeneration of cAMP and thus depends on the level of secretion of norepinephrine and epinephrine. Although it is our goal to overcome this feedback mechanism, I must emphasize its importance in physiological systems. A second messenger system with no PDE’s is a messenger system that cannot be shut off. It would be like not being able to let off of the accelerator on your car once you pushed it to the floor. Feedback inhibition is about control. A switch is worthless if it cannot be turned off as well as on.

Prostaglandins

Prostaglandins are produced in virtually all tissues of the body. Prostaglandins are abbreviated PG, with additional letters and numbers indicating their structure. For example, PGE2 means that it is a prostaglandin of the “E” type which designates it as a beta-hydroxyketone. The number indicates how many double bounds it has, in our case, two. Prostaglandins (PG) come in many varieties and participate in a number of physiological responses. The ones you are probably most familiar with are pain sensitivity and inflammation. Prostaglandins are made from 20 carbon fatty acids such as arachidonic acid. In the first step of the conversion of arachidonic acid into prostaglandins, the enzyme cyclooxygenase oxygenates arachidonic acid producing the prostaglandin PGG2. Most all of your non-steroidal anti-inflammatory agents like aspirin, ibuprofen, naproxin sodium and others, work by inhibiting cyclooxygenase activity which then diminishes prostaglandin synthesis. In response to beta-adrenergic stimulation, E2 prostaglandins are released into the synaptic space. These prostaglandins have receptors coupled to inhibitory G proteins (Gi). These Gi’s then decrease adenylate cyclase activity and thus decrease cAMP concentrations in the cell.10So the idea of using prostaglandin inhibitors seems a logical adjunct to ephedrine treatment for fat loss.

Adenosine

Adenosine is somewhat more of a complicated feedback molecule that has dual roles as both an activator as well an inhibitor. Adenosine is a purine nucleoside and our concern with it is its ability to inhibit cAMP accumulation. When a fat cell is stimulated by a beta adrenergic agonist such as norepinephrine, the cell produces adenosine. Adenosine then interacts with its receptor coupled to regulatory G proteins (Gi) which inhibits adenylate cyclase activity, and thus prevent the accumulation of cAMP.11It’s effects in the synaptic space are similar to those of alpha-2 agonists due to receptors coupled to inhibitory G proteins. Got all that? The bottom line is that when using sympathomimetics such as ephedrine, you activate regulatory mechanisms involving adenosine.

It should be apparent by now that the reason we are talking so much about negative feedback in response to adrenergic stimulation is because we have a plan to attenuate these responses. This is where we introduce methylxanthines and prostaglandin inhibitors.

Caffeine is a methylxanthine. Caffeine possesses the ability to inhibit phosphodiesterases within the cell and has even been shown to have the ability to prevent some re-uptake of norepinephrine.12Another property of caffeine is adenosine receptor blockade. There is some question as to weather oral caffeine ingestion actually inhibits phosphodiesterases but it does seem to inhibit adenosine action in vivo. Both of these properties make it potentially useful as an adjunct to ephedrine to enhance fat loss.

Aspirin, as discussed earlier, is a prostaglandin inhibitor. It works by inhibiting cyclooxygenase activity. Because certain prostaglandins act to inhibit lipolysis and are produced in response to adrenergic stimulation, prostaglandin inhibitors have the potential to enhance ephedrine’s actions on fat loss.

Now that we have discussed the mechanism of lipolysis and how ephedrine ,caffeine, and aspirin might enhance lipolytic activity in fat cells, let us take a look at what studies have shown concerning the effectiveness of these compounds in combination.


The ResearchLooking at Ephedrine / Caffeine / Aspirin and Weight Loss

In a double blind, placebo controlled study, caffeine alone was found to produce thermogenic and lipolytic effects in humans in a dose dependent manner.13These researchers found that the thermic effect was significantly correlated to plasma triglyceride levels, plasma lactate concentrations and vascular tone. The authors attribute the increase in lactate, triglycerides and enhanced vascular tone to the increased metabolic rate. In a study using caffeine and ephedrine researchers found no difference in the total amount of body weight that was lost over 8 weeks.14However, they did find significant differences in the source of the weight that was lost. Fourteen obese women were treated with a ~1000 kcal diet and either E + C (20mg E + 200mg C) or placebo three times per day for 8 weeks in a double-blind study. The total weight-lost was not different between groups, but the E + C group lost ~10 lbs. more body fat and ~6 pounds less fat-free mass. This is encouraging news for any bodybuilder. You must bear in mind, however, that these were obese women. Studies have shown that nutrient partitioning is determined in part by your % fat before you diet or before you over eat.15,16Nevertheless, that is a tremendous effect on fat loss and muscle retention.

Some research has shown that the anti obesity effects of ephedrine are not significant unless caffeine is used in conjunction with ephedrine.17In fact, most studies exploring the thermogenic effects of ephedrine also look at caffeine as a synergist. In a randomized, placebo-controlled, double blind study, 180 obese patients were treated by a calorie restricted diet and either an ephedrine/caffeine combination (20mg/200mg), ephedrine (20 mg), caffeine (200 mg) or placebo three times a day for 24 weeks. Average weight loss was significantly greater with the combination than with placebo from week 8 to week 24. Weight loss in both the ephedrine only and the caffeine only groups was similar to that of the placebo group. The authors conclude that the effect of either caffeine or ephedrine alone is ineffective in inducing significant weight loss.18,19Not only is it necessary to combine ephedrine and caffeine to elicit a significant fat burning effect, the two compounds exhibit synergistic effects in certain ratios. By comparing different ratios of ephedrine and caffeine, it was found that 20 mg of ephedrine and 200 mg of caffeine exhibited a supra additive or synergistic effect while no other ratio did.20This means that ephedrine and caffeine taken in a 1:10 ratio (20 mg ephedrine : 200 mg caffeine) creates effects greater than the sum of the two drugs added together. In other words, 2 + 2 = 5 in this ratio!

So what about aspirin? There has not been as much research done on aspirin in this “stack”. Looking first at animals, chronic administration of aspirin to obese mice had no effect on weight loss. Ephedrine given to these mice increased energy expenditure by 9% and reduced body weight and body fat by 18% and 50%, respectively: obesity however, was reduced but the mice still were not comparable to normal controls. When given both ephedrine and aspirin, increase in energy expenditure found during treatment with ephedrine alone was doubled, and the obese group lost greater than 75% of body fat, and obesity essentially was reversed.21The research done on humans has also been somewhat promising. The effect of ephedrine (30 mg) and aspirin (300 mg) on the acute thermogenic response to a liquid meal (250 kcal) was investigated in lean and obese women (n = 10 each group). Resting metabolic rate (RMR) was measured prior to each of the following treatments: meal only (M), meal plus ephedrine (ME) or meal plus ephedrine and aspirin (MEA). The postprandial rise in metabolic rate, following the MEA treatment compared to the ME, was significantly greater for the obese group but not the lean. It was concluded that aspirin potentiates the stimulatory effect of ephedrine on the thermogenic response to a meal in obese but not lean women.22One weakness of this study was the small number of subjects. Nevertheless, these findings are not all that surprising considering the fact that decreased thermic effect of food is often seen in the obese.23In another study24, a mixture of ephedrine (75-150mg), caffeine (150mg) and aspirin (330mg), in divided premeal doses, were investigated in 24 obese participants in a randomized double blind placebo-controlled trial. Energy intake was not restricted. Overall weight loss over 8 weeks was 2.2kg for ECA vs. 0.7 kg for placebo. Eight of 13 placebo subjects returned 5 months later and received ECA in an unblinded crossover. After 8 weeks, mean weight loss with ECA was 3.2 kg vs 1.3 kg for placebo. Six subjects continued on ECA for 7 to 26 months. Notice that there is no concern about receptor down regulation or trying useless dosing schedules like “2 weeks on and 2 weeks off”. Anyway, after 5 months on ECA, average weight loss in five of these was 5.2 kg compared to 0.03 kg gained during 5 months between studies with no intervention. The sixth subject lost 66 kg over 13 months by self-imposed caloric restriction. This sixth subject lost an amazing 150 lbs. By exercising and cutting calories! Can you believe they didn’t encourage the other subjects to diet and exercise? In all studies, no significant changes in heart rate, blood pressure, blood glucose, insulin, and cholesterol levels, and no differences in the frequency of side effects were found. ECA in these participants caused significant weight loss even without caloric restriction. The authors of this study go on to comment that the ECA combination might be more effective with caloric restriction. That kind of conservatism cracks me up!

In one study that really got my attention they compared the effects of ephedrine against the popular prescription drug dexfenfluramine that goes under the brand name Redux.25In order to compare the efficacy and safety of these two anorectic drugs, 103 patients with 20-80% overweight were included in a 15-week double-blind study in general practice. Patients were randomized to either 15 mg DF twice daily (n = 53), or 20 mg/200 mg ephedrine/caffeine three times a day (n = 50). Subjects went on a 1200 kcal/day diet during the treatment period. After 15 weeks of treatment, the DF group (n = 43) had lost ~15 +/- 9.46 lbs. and the EC group (n = 38) had lost ~18.3 +/- 11.5 lbs. In the subgroup of patients with BMI > or = 30 kg/m2 (n = 59), the mean weight loss was 7.0 +/- 4.2 kg in the DF group (n = 29) and 9.0 +/- 5.3 kg in the EC group (n = 30), P < 0.05. Both systolic and diastolic blood pressures were reduced similarly during both treatments. Central nervous system side-effects, especially agitation, were more pronounced in the EC group, whereas gastro-intestinal symptoms were more frequent in the DF group. The side-effects declined markedly during the first month of treatment in both groups. Not only was the weight loss with ephedrine and caffeine comparable to Redux, it was probably greater! This study did not look at body composition but I bet it would have shown the E/C combination as superior in retaining lean mass.

You may have noticed that most of the studies I have cited have used obese subjects. This is understandable considering it is the obese population that are targeted for drug therapy. It should be noted however, that the thermogenic properties of an ephedrine/caffeine mixture are also demonstrated in lean subjects as well.26You should expect increased effectiveness in obese people because of underlying problems with metabolic rate. Anytime you increase the metabolic rate in obese individuals you will see large changes in energy expenditure because the relative increase in metabolic rate is greater than in lean individuals.


In Conclusion

Now let us put all of this together. First, what do we know; Ephedrine stimulates lipolysis by increasing noradrenaline (NA) release from sympathetic nerve terminals. This increase in noradrenaline activates adrenergic receptors which increases cAMP levels in fat cells and muscle cells. This has the effect of increasing lipolysis in fat cells and increasing protein synthesis in muscle tissue. Negative feedback mechanisms are activated as well, and involve the production of phosphodiesterases, adenosine, and prostaglandins. Caffeine has the ability to inhibit phosphodiesterase activity and interfere with the adenosine receptor. This combined with its ability to prevent some NA re-uptake12 increase the effectiveness of ephedrine in a synergistic fashion. Aspirin has been shown to increase the effectiveness of ephedrine in some individuals presumably by its actions as a prostaglandin inhibitor.

Maximum effectiveness is achieved when taking 20 mg ephedrine with 200 mg caffeine and 300 mg aspirin three times a day about one half hour before meals. Common side effects are associated with its sympathetic activity namely, anorexia, initial rise in blood pressure, initial tachycardia, slowed GI motility (constipation), insomnia, agitation, anxiety, nervousness and depression- like withdrawal symptoms. Most all of these symptoms exhibit tachyphylaxis after about 4-6 weeks. Thermogenic activity seems to last upwards of 20 weeks due to its low desensitization properties and beta-3 affinity. About 75% of ephedrine’s effects on weight loss in the obese are due to appetite control.

Anyone considering taking ephedrine, caffeine and aspirin should educated themselves first about the potential side effects. Individuals with pre-existing high blood pressure should not use sympathomimetics such as ephedrine. When taking herbal forms of ephedrine, be sure you understand just how much is in each serving. Be aware that herbal preparations are standardized but you still can not be sure exactly how much you are taking with each capsule.

The future of fat loss for the bodybuilder will not, or should not, focus on appetite alone. It should focus on enhancing lipolysis and overcoming the regulatory mechanisms designed to prevent rapid and substantial fat loss. Ephedrine, caffeine and aspirin are effective but are still limited by inhibitory mechanisms built into our physiology. Gaining better understanding of the mechanisms involved in lipolysis and gaining funding for appropriate research is critical. The pharmaceutical industry already recognizes the profitability of weight loss agents unfortunately they are focusing at present on appetite control. Perhaps as these strategies continue to fail they will focus more on body composition instead of just “body weight”. When this happens you can be sure that adrenergic receptors and the second messenger system will be the focus of attention.


References

1. Dulloo AG, Seydoux J, Girardier L., Peripheral mechanisms of thermogenesis induced by ephedrine and caffeine in brown adipose tissue. Int J Obes.15(5):317-326. 1991.

2. Astrup A, Lundsgaard C, Madsen J, Christensen NJ. Enhanced thermogenic responsiveness during chronic ephedrine treatment in man. Am J Clin Nutr. 1985 Jul; 42(1): 83-94.

3. Astrup A, Madsen J, Holst JJ, Christensen NJ The effect of chronic ephedrine treatment on substrate utilization, the sympathoadrenal activity, and energy expenditure during glucose-induced thermogenesis in man. Metabolism 1986 Mar;35(3):260-265

4. Sibley DR, Daniel K, Strader CD, Lefkowitz RJ Phosphorylation of the beta-adrenergic receptor in intact cells: relationship to heterologous and homologous mechanisms of adenylate cyclase desensitization. Arch Biochem Biophys. 1987 Oct; 258(1): 24-32.

5. Toubro S, Astrup AV, Breum L, Quaade F. Safety and efficacy of long-term treatment with ephedrine, caffeine and an ephedrine/caffeine mixture. Int J Obes Relat Metab Disord Feb;17 Suppl 1:S69-S72 1993

6. Liu YL, Toubro S, Astrup A, Stock MJ. Contribution of beta 3-adrenoceptor activation to ephedrine-induced thermogenesis in humans. Int J Obes Relat Metab Disord 1995 Sep;19(9):678-685

7. Nantel F, Bonin H, Emorine LJ, Zilberfarb V, Strosberg AD, Bouvier M, Marullo S The human beta 3-adrenergic receptor is resistant to short term agonist-promoted desensitization. Mol Pharmacol 1993 Apr;43(4):548-555

8. Astrup A, Buemann B, Christensen NJ, Toubro S, Thorbek G, Victor OJ, Quaade F. The effect of ephedrine/caffeine mixture on energy expenditure and body composition in obese women. Metabolism Jul;41(7):686-688. 1992

9. Pasquali R, Casimirri F Clinical aspects of ephedrine in the treatment of obesity. Int J Obes Relat Metab Disord;17 Suppl 1:S65-S68 1993

10. Kather H, Simon B., Biphasic effects of prostaglandin E2 on the human fat cell adenylate cyclase. J Clin Invest 64(2):609-612. 1979

11. Lonnqvist F, Arner P, Interactions between adenylate cyclase inhibitors and beta-adrenoceptors in isolated human fat cells. Biochem Biophys Res Commun 1989 Jun 15;161(2):654-660

12. Kalsner S, Frew RD, Smith GM., Mechanism of methylxanthine sensitization of norepinephrine responses in a coronary artery. Am J Physiol 1975 Jun;228(6):1702-1707

13. Astrup A, Toubro S, Cannon S, et al. Caffeine: a double-blind, placebo-controlled study of its thermogenic, metabolic, and cardiovascular effects in healthy volunteers. Am J Clin Nutr 1990 May;51(5):759-767

14. Astrup A, Buemann B, Christensen NJ, Toubro S, et al. The effect of ephedrine/caffeine mixture on energy expenditure and body composition in obese women. Metabolism 1992 Jul;41(7):686-688

15. Girardier L, Control systems in the defense of body fat stores. Int J Obes Relat Metab Disord 1993 Feb;17 Suppl 1:S3-S8

16. Dulloo AG, Jacquet J, Girardier L, Autoregulation of body composition during weight recovery in human: the Minnesota Experiment revisited. Int J Obes Relat Metab Disord 1996 May;20(5):393-405

17. Dulloo AG, Miller DS, The thermogenic properties of ephedrine / methylxanthine mixtures: Human studies. Int J Obes 1986; 10: 467-81

18. Astrup A, Breum L, Toubro S, Hein P, Quaade F, The effect and safety of an ephedrine/caffeine compound compared to ephedrine, caffeine and placebo in obese subjects on an energy restricted diet. A double blind trial. Int J Obes Relat Metab Disord 1992 Apr;16(4):269-277

19. Toubro S, Astrup AV, Breum L, Quaade F. Safety and efficacy of long-term treatment with ephedrine, caffeine and an ephedrine/caffeine mixture. Int J Obes Relat Metab Disord 1993 Feb;17 Suppl 1:S69-S72

20. Astrup A, Toubro S, Cannon S, Hein P, Madsen J. Thermogenic synergism between ephedrine and caffeine in healthy volunteers: a double-blind, placebo-controlled study. Metabolism 1991 Mar;40(3):323-329

21. Dulloo AG, Miller DS., Aspirin as a promoter of ephedrine-induced thermogenesis: potential use in the treatment of obesity. Am J Clin Nutr 1987 Mar;45(3):564-569

22. Horton TJ, Geissler CA., Aspirin potentiates the effect of ephedrine on the thermogenic response to a meal in obese but not lean women. Int J Obes 1991 May;15(5):359-366

23. de Jonge L, Bray GA, The thermic effect of food and obesity: a critical review. Obes Res 1997 Nov;5(6):622-631

24. Daly PA, Krieger DR, Dulloo AG, Young JB, Landsberg L Ephedrine, caffeine and aspirin: safety and efficacy for treatment of human obesity. Int J Obes Relat Metab Disord 1993 Feb;17 Suppl 1:S73-S78

25. Breum L, Pedersen JK, Ahlstrom F, Frimodt-Moller J, Comparison of an ephedrine/caffeine combination and dexfenfluramine in the treatment of obesity. A double-blind multi-center trial in general practice. Int J Obes Relat Metab Disord 1994 Feb;18(2):99-103

26. Astrup A; Toubro S; Cannon S; Hein P; Madsen J Thermogenic synergism between ephedrine and caffeine in healthy volunteers: a double-blind, placebo-controlled study. Metabolism 1991 Mar; 40(3):323-9

27. Choo J, Horan M, Litlle R, and Rothwell N. Anabolic effects of Clenbuterol on skeletal muscle are mediated by beta2-adrenoreceptor activation. Am J Physiol 1992; 263:E50-E56.

28. Guentert TW, Buskin JN, Galeazzi RL Single dose pharmacokinetics of mabuterol in man. Arzneimittelforschung 1984;34(11A):1691-1696

29. Morgan J. Clinical Pharmacokinetics of Beta-Agonists. Clin Pharmacokinet 1990 Apr;18(4):270-294

30. Martineau L, Horan MA, Rothwell NJ, Little RA. Salbutamol, a beta 2-adrenoceptor agonist, increases skeletal muscle strength in young men. Clin Sci (Colch) 1992 Nov;83(5):615-621

31. Caruso J, Signorile J, Perry A, et al. Time Course Changes in Contractile Strength Resulting From Isokinetic Exercise and Beta2 Agonist Administration. J. Strength Condition. Res. 1997; 11(1):8-13

32. Rosenbaum M, Malbon CC, Hirsch J, Leibel RL. Lack of beta 3-adrenergic effect on lipolysis in human subcutaneous adipose tissue. J Clin Endocrinol Metab 1993 Aug;77(2):352-355

33. MacIntyre DE. Human Beta-adrenergic receptor agonists: identification and in vivo evaluation in the rhesus monkey. Proceedings of the 1996 International Congress on Anti-Obesity Drug Targets, Cambridge, MA, 9-11 December. Little Falls, NJ: International Quality and Productivity Center.

34. Meyers DS, Skwish S, Dickinson KE, et al. Beta 3-adrenergic receptor-mediated lipolysis and oxygen consumption in brown adipocytes from cynomolgus monkeys. J Clin Endocrinol Metab 1997 Feb;82(2):395-401

35. Danforth E, Himms-Hagen J. Obesity and diabetes and the beta-3 adrenergic receptor. Euro J Endocrin 1997; 136:362-365

36. Yoshida T, Umekawa T, Kumamoto K, et al. Beta 3-Adrenergic agonist induces a functionally active uncoupling protein in fat and slow-twitch muscle fibers. Am J Physiol 1998 Mar;274(3 Pt 1):E469-E475

37. Abe H, Minokoshi Y, Shimazu T. Effect of a beta 3-adrenergic agonist, BRL35135A, on glucose uptake in rat skeletal muscle in vivo and in vitro. J Endocrinol 1993 Dec;139(3):479-486.

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