Intermittent Thoughts on Building Muscle: IGF-1 and its Splice Variants MGF, IGF-IEa & Co - Master Regulators or a Bunch of Cogs in the Wheel of Muscle Hypertrophy?

Image 1: With regard to IGF-1 and its splice-variants like MGF, there is probably 10x-100x more bro- than pro-scientific data out there - this does not help us, though, since you never know which of the bro-reports is bogus and which is not.

In view of the fact that we have not covered much ground with the last installment (we did build a pretty solid foundation, though ;-), I will try my very best to steer a middle course between presenting impressive amounts of facts and explaining the complex and in part not even completely elucidated physiological underpinnings of skeletal muscle hypertrophy, or, as the bros would say, getting big and buffed! A pros pros Bro, you will unquestionably have read on one of the myriads of bodybuilding-related bulletin boards how the injection of X amounts of IGF-1 right into the muscle made BigGuns, or whatever the poster's pseudonym may have been, grow "3 inches in 2 weeks"... ok, his profile picture looks impressive, but is that credible? Does IGF-1 really have such profound effects on muscle growth? And about what type of growth are we talking here? The myostatin-negative "ballooning up" of the muscle, which leaves you with overblown myogenic domains and dysfunctional muscles?

IGF-1: Insulin, growth hormone, or what?

To be able to answer these and related question we will first have to understand what exactly this "insulin-like growth factor 1" actually is. From a (bio-)chemical perspective it is nothing but a bond of 70 amino acids which are entangled into a specific peptide structure that is characteristic for somatomedin C, as IGF-1 is also called. Both the "growth" in IGF-1, as well as the "somato" in its old-fashioned appellation already suggest that what we are dealing with, here, is a "growth hormone related" polypeptide. And in fact, the synthesis of IFG-1, which, in the case of the systemically available fraction, takes place primarily in the liver, and is triggered by systemic growth hormone (somatotropin) levels.

Figure 1: Changes in systemic IGF-1 levels after 5-weeks on either a "normal" (=55:15:30 carbs:protein:fats) or a low carb "high protein" (=20:30:50) diet in 8 men with untreated type II diabetes (data adapted from Nuttal. 2006)

The "insulin" in its name, however, is pretty misleading... or I should say people mislead themselves, by not reading  the name correctly: It's not "insulin-growth factor", but "insulin-like growth factor" and the "like" refers to the structure of the molecule and does not imply that it is released in response to insulin spikes, as you may have read it on one of the aforementioned bulletin boards. If you do take a look at the growth hormone and IGF-1 levels of eight male subjects in a 2006 study on the metabolic of 5-weeks on what the scientists call a "high protein, low carbohydrate diet" (Nuttall. 2006), you will see that an increase in protein and fat from 15% to 30% and 30% to 50%, respectively elicited an 34% increase in serum IGF-1 levels over the treatment period, a finding that is corroborated by the recently published results of Matthew B. Cooke and his colleages from the Department of Health, Human Recreation and Performance at Baylor University.

Figure 2: Serum IGF-1 levels in response to whey vs. maltodextrin supplementation and subsequent lower body resistance training (data adapted from Cooke. 2011)

In their randomized double-blinded cross-over study, Cooke et al. had a group of 10 recreationally active men (2-3 non-resistance training exercise sessions per week) perform a lower body exercise program (leg presses and knee extensions, 4 sets, 8-10 reps at 80% of the individual 1RM) with either 10g of maltodextrose or 10g of whey 30 minutes before the exercise bout (Cooke. 2011). The results of the study (equal IGF-1 response regardless of whey or carbohydrate supplementation) imply that even in the short term, in healthy subjects and in conjunction with exercise the ingestion of carbohydrates is not superior to the provision of fast acting protein sources as a means to either increase or maintain systemic IGF-1 levels.

On a side note: The insulin-mediated induction of Akt, which subsequently triggers the phosphorylation of the mammalian target of rapamycin (mTOR) and thusly does its bit to elevate protein synthesis, has no direct relation to IGF-1, which - I cannot emphasize that enough - has a structure resemblance to insulin, nothing more, nothing less. And what's more, the insulin response in the aforementioned study by Cooke et al. was identical in the whey vs. maltodextrin arm of the study.

Systemic vs. local IGF-1 expression: A crucial distinction

If you have been following the daily research updates here at the SuppVersity over the last months, you may now be wondering why I am even caring about those growth hormones (after all you should, after reading the first paragraph, realize that IGF-1 is something like the active incarnation of somatotropin), when Stuart Phillips lab has quite conclusively shown that even the exercise induced elevation of testosterone does not correlate with subsequent increases in muscle protein synthesis. Certainly a good question, but nevertheless not difficult to answer:

1. The previous installments of the Hypertrophy 101 (Part 1, Part 2) should have made it quite clear that protein synthesis alone is not sufficient to grow. Without intra-muscular restructuring / reorganization and the recruitement of new myonuclei from satellite cells, you would sooner or later grow beyond the maximally allowed myonuclear domain sizes (assuming that by whatever means you block the healthy upregulation of mystatin that will prevent that) and end up as an over-muscled but completely dysfunctional wrack.
2. In a very recently published study, the results of which I have actually been holding back, because I thought I would get to them much earlier in this series, the very same Stuart Phillips whose studies are "responsible" (in fact it is the way they are discussed by the lay-press and abused by the supp-companies that is actually "responsible") for the current over-emphasis on acute increases in the protein synthetic response to exercise and/or supplements, reports that there actually was a statistically significant correlation between exercise induced growth hormone release and increases in mean type I fiber (p<0.06) and type II (p<0.04) cross-sectional area (CSA) in 56 healthy previously non-resistance trained healthy young men in response to a 12-week, 5-day per week resistance training regimen (West & Phillips. 2011).
3. While we have hitherto been talking about systemic IGF-1, it has become evident in the course of the last decade that the hepatic IGF-1 output, which is the main determinant of circulating IGF-1 levels, has little to no impact on the IGF-1 induced increases in skeletal muscle mass and remodeling of muscle tissue that has been previously studies in Petri dishes. In fact, recent research suggests that, just like the liver produces IGF-1 for "the whole body", muscles produce their own IGF-1, or I should say, their own IGFs-1, whenever they are challenged to grow and/or repair (Velloso. 2010), and that the decline of muscle mass with age is at least in parts attributable to a defect / reduction in the expression of local IGF-1 splice variants (for an explanation of what this is, see red box below).

If we now count 2. and 3. together the result is not 5. but rather that it is the growth hormone mediated, exercised-induced local expression of IGF-1 splice variants, which drives the repair and restructuring process that allows for continuous (healthy) muscle growth.

Did you know that the intra-muscular (=autocrine, meaning directly in the tissue where it is supposed to work) "construction process" of the mature 70 amino acid polypeptide IGF-1 gives rise to three different splice variants of insulin-like growth factor (note: the structure of IGF-1 gene does theoretically allow for 6 variants)? And though we are just beginning to understand the physiological roles of IGF-IEa, IGF-IEb and IGF-IEc, also known as MGF (mechano-growth factor), their distinctly timed expression in response to physical overload appears to constitute one of the major driving forces of myocellular hypertophy.

In order to fully understand the role "the" insulin-like growth factor 1 plays in the physiology of muscle growth, it is thusly important to realize that the common perception of IGF-1 as a systemic hormone is, at best, incomplete - I would even venture to say that it is totally flawed.

MGF?! Yeah, I have heard of that one!

Figure 3: Stained myocyte migration (top) and infiltration (bottom) essays for IGF-1 and MGF; more stains = greater effect (taken from Mills. 2007).

Of the three primary splice variants that are expressed in skeletal muscle, IGF-IEc, or MGF (Mechano-Growth Factor) has probably received the greatest attention - so much attention that even the aforementioned bros, will probably have grasped the notion that this is somewhat of a local isoform of IGF-1 which is expressed in response to exercise induced muscle damage and could potentially be the magic bullet to grow beyond what we have hitherto believed to be possible... and, guess what, in essence this appears to be correct.

In one of the earlier studies on the cellular effect of MGF, Yang et al. were able to show that MGF stops the IGF-1 mediated cell differentiation process (in practice this means that it stops the satellite cells from differentiating = specializing and becoming muscle cells) and increases their proliferation. Or put more simply: While in vitro exposition to IGF would suffice to build muscle, as long as there are enough progenitor cells (satellite cells) available, MGF is necessary to replenishes the satellite cell pool of which you have learned in the previous installments that it is necessary to a) repair damaged muscle tissue and b) increase the number of myonuclei in order to grow beyond the physiological growth limit that arises due to the muscle-type-specific upper limit to the myonuclear domain size (cf. previous installments).

Figure 4: Cell proliferation data in response to MGF treatment after blocking the IGF-I receptor.

As the data in figure 4 goes to show the effects of the complete polypeptide IGF-1 and its splice variant MGF appear to be mediated, at least partly via distinct receptors. And while recent research suggest that MGF also exerts similar effects on tendon (Olesen. 2006), brain (Dluzniewska. 2005) and nervous tissue (Aperghis. 2004), our primary concern here, is its pivotal role in muscle repair, which involves the activation of satellite cells, their proliferation (Yang. 2002) and migration (Mills. 2007).

A series of studies by Hammad et al., which was originally intended to investigate the effects of age on the expression of the different IGF splice variants, goes to show that the "muscle (re-)building effects" of MGF are not restricted to the test tube. In their 2002 study (Hamed. 2002), the researchers were able to show profound increases in the MGF expression in the quadriceps muscles of 8 healthy young men (age 29.5 ± 1.5 years, body mass 81.1 ± 2.4 kg, height 179.3 ± 1.8 cm) 2.5h after a single muscle-damaging leg-extension exercise (10 sets of 6 repetitions at 80% 1-RM, 2 min rest between sets):

Figure 5: MGF (ng mRNA / 10^8 µg RNA) and IGF-IEa ng mRNA / 10^5 µg RNA) expresion in quadriceps muscle of young subjects before and 2.5h after 10 sets of 6 repetitions at 80% 1-RM on a leg-extension machine with 2min rest between sets (data adapted from Hamed. 2002)

If you take a closer look at the data in figure, you will probably notice that there was one subject with an extreme MGF response, the scientists explain by a particularly high type-IIx fiber content of the quadriceps of this individual. If you remember the mouse studies and the analysis of the muscle composition of bodybuilders from the previous installments, you will be aware that the shift from type IIb to type IIx muscle fibers is one of the main characteristics of "getting real big". The extreme MGF response (>10x higher than the mean MGF expression across the other subjects) in this subject thusly suggests the increased growth capacity of type IIx muscle fibers is in part due to their ability to release MGF in response to strenuous exercise and thusly multiply / replenish their satellite cell pool to prepare for future growth.

Figure 6: MGF (ng mRNA / 10^8 µg RNA) and IGF-IEa ng mRNA / 10^5 µg RNA) expresion in quadriceps muscle of young subjects after eccentric HIIT exercise on cycle ergometer (data adapted from Hamed. 2008)

Interestingly, a 2008 follow up study (this time involving nine healthy young men aged 20–27 years, cf. Hamed. 2008) with a completely different training protocol that consisted of

60min of opposing the rotation of the pedals down to 60 r.p.m. Subjects performed the following program of six working intervals: six working intervals: 0–6min at 50%, 6–12min at 75%, 12–20min at 100%, 20–25min at 130%, 25–40min at 100% and 40–60min at 75% of the load  eliciting concentric VO2max

illicited surprisingly similar results (cf. figure 6). And in both cases, it appears to be the MGF splice variant not the IGF-IEa variety that drives the short term (hours to days) response to strenuous exercise.

HIIT and resistance training a dynamic duo for MGF expression

Assuming that you are following each and every post here at the SuppVersity (you know you should be ;-), this should remind you of a previous blogpost of mine (cf. "HIT Your Satellite Cells to Increase Your Gains!"), in which I explained that one of the many advantages of high intensity training (not even interval) over classic "cardio" training is that it can increase satellite cell proliferation. Now, with this installment of the Intermittent Thoughts you finally understand, why this is the case.

Image 2: This is not the kind of muscle damage you should be aiming for in the gym.

Now, while protein synthesis and increases in domain size are partly mediated via nutrition, the intra-muscular expression of the IGF-IE splice variants appears (at least based on the current research) to depend solely on exercise, or I should say the wear and tear that goes hand in hand with heavy exercise. In that it seems to be less important, whether you are "pumping away" or "cycling like maniac", as long as its "hard" - to put that into perspective, in the 2008 study by Hamed et al. the subjects underwent ~3600 eccentric muscle contractions in only 1 h, their creatine kinase (CK) levels (marker of muscle damage) increased by +183% and all subjects reported profound muscle soreness.

This controlled amount of muscle damage ties in nicely with the topic of next week's installment which will center around the the intricate relation of the inflammatory response to exercise, the expression of the well-known and less known inflammatory cytokines, TNF-alpha, IL-6 and IL-15 (sorry, Trevor, I have already gone overtime, so your question will have to wait till next week ;-) and the muscle (re-)building effects of IGF-1 and its intra-muscular children.

The Intracrine Effects of Anabolic Steroids - Metanolone Promotes Stretch-Induced Intramuscular MGF Expression

Arnold's workout regimen are known to generate a hell lot of wear and tear and actually this could be part of his success formula.

I have to admit that the increase in intra-cellular MGF production is probably not the only, but certainly a new and very important pathway by which anabolic steroids "actively" promote muscle growth. According to a recent study from the Department of Rehabilitation and Physical Medicine, Graduate School of Medical and Dental Sciences at the Kagoshima University in Japan (Ikeda. 2013) anabolic agents such as metenolone which is a naturally occuring, WADA-listed long-acting anabolic steroid with weak androgenic (testosterone or androsterone-like) properties. It is isolated from the glands of pregnant domesticated felines, and is supplied as the acetate ester for oral administration and as the enanthate ester for intramuscular injection. Adult doses for the treatment of aplastic anemia are usually in a range of 1–3 mg/kg per day (Wikipedia).

Stretch-induced muscle growth

For the rodents in the study at hand the scientists did escalate the dosage and pumped roughly 10mg/kg (this is already the human equivalent) into the critters.

Then, the right gastrocnemius muscles were stretched repeatedly by manual ankle dorsiflexion 15 times per minute for 15 min. The contralateral muscles were not stretched as a control. In the control rats (n=6), the gastrocnemius was stretched as for the treatment group, but no metenolone was injected. Twenty-four hours after the procedure, the rats were sacrificed by injection of a lethal dose of sodium pentobarbital and their medial gastrocnemius muscles removed on both sides.

Actually, I suspect the sacrifice would not have been necessary as the extraction of the MGF, or as the scientists call it the "the specific autocrine IGF-I splicing variant mechano-growth factor" is something you can measure from a muscle biopsy. So, the only argument against a human study, is probably the dosage and the general administration of anabolic steroids to human subjects.

Figure 1: Treatment effects on MGF, MyoD, Myogenin (a.u.) in rats w/w/out metenolone injection (Ikeda. 2013)

With the highly significant effects on MGF and the non-significant effects on myoD and myogenin, both of which are involved in the recruitement of new muscle nuclei from the stem cell (satellite cell) pool in the musculature, the result of the study is yet of generic nature and will almost certainly apply to humans as well.

Sketch of a mammalian skeletal muscle fiber - myonucleus (turqouis), mitochondria (blue),  sarcoplasmic rectilium (buff), tubules (orange), myofibrils (pinkish)

Artist: Lesley Skeates. Originally from Gray's Anatomy 29th ed. Elsevier. 2008

Stretch to grow? It's not the act of stretching, but rather the wear and tear to the cells that is interpreted as being worked out heavily that induces muscle growth and most importantly structural adaptation (in other words a "stretch during a lift will do just as well as deliberate stretching). The downstream effects of these intracrine (=confined within the cell itself) reactions go way beyond simply pumping more protein into the existing muscular structure.  In a previous study, Ikeda et al. have already shown that "[e]ither continuous or repetitive short duration stretching of muscles for 1 week increased the mRNA expression levels of MyoD, myogenin, and embryonic MyHC relative to those of unstretched  muscles." (Ikeda. 2003)

The latter are markers for the often hailed activation, recruitment and replenishment of muscle "stem-", or "satellite cells", of which you know from the Muscle Hypertrophy 101 (read it) that they are what allows you to grow beyond the natural limit, a limit that makes the muscle of myostatin negative animals huge, but dysfunctional - a direct result of the structural changes not being able to keep pace with the constant influx of protein (learn more).

And with the effects of MGF being related to the important strength facilitating effects of exercise and the underlying cause of the changes being a simple stretch of the musculature the results put another emphasis on the necessity of the "wear and tear" for your body to make the necessary adaptations to exercise

---

Figure 2: Illustration of what you should have learned, if you read all installments of the Intermittent Thoughts on Building Muscle (read summary)

So what exactly is he result of the study then? I guess the elevator pitch is: Study confirms the facilitative role in skeletal muscle restructuring / growth of anabolic steroids. If you want more details, I suggest you go back to the "Intermittent Thoughts on Building Muscle Series" and educate yourself about IGF-1, MGF, GH, testosterone, myostatin and co and their specific roles in skeletal muscle hypetrophy (see "reading assignment).

Figure 2, to the right delivers a sneak peak of what you can expect and if that's not attractive enough, I'd suggest you use the "Preliminary Conclusion - Exercise, mTOR/AKT/MAPK, IGF-1, Testosterone, Estrogen, DHT, Nutrition, Supps & Sleep" (read it) of the series as a cognitive anabolic to promote your interest ;-)

I already hinted at that in a previous paragraph, but I think it's still worth repeating in the bottom line that despite being derived in a rodent study with an "exotic" anabolic agent, there is no question that the results of the study at hand will also be relevant for chemical athletes and little evidence they would not apply to

Your reading assignment for today:

1. IGF-1 and its Splice Variants MGF, IGF-IEa & Co - Are the growths hormones master regulators of muscle growth or just a bunch of cogs in the wheel of skeletal muscle hypertrophy?
   
   
2. IGF-1, TNF-α, IL-15 & Co and the Emerging Role of an Auto-/Endocrine-Immune Axis in Skeletal Muscle Hypertrophy  - How does inflammation factor in, is it beneficial or detrimental?
    
3. Zoning in on "The Big T" - Does testosterone (alone) build muscle?
    
4. Quantifying "The Big T" - Do increases of testosterone, which are well within the physiological range matter?
    
5. Understanding the "Big T" - How does testosterone work? What is its effect on stem cells and how come it makes you leaner?
    
6. Estrogen, Friend or Foe of Skeletal Muscle Hypertrophy? - Which role does estrogen play and why could you SERM away your growth potential, when you (ab-)use tamoxifen or potent aromatase inhibitors?
    
7. Dihydrotestosterone (DHT), The All Things Male Hormone - Will it make you bigger, stronger and faster or just balder, fatter and unhealthier?

References:

• Ikeda S, Yoshida A, Matayoshi S, Tanaka N. Repetitive stretch induces c-fos and myogenin mRNA within several hours in skeletal muscle removed from rats. Arch Phys Med Rehabil. 2003 Mar;84(3):419-23.
• Ikeda S et al. The Effect of Anabolic Steroid Administration on Passive Stretching-Induced Expression of Mechano-Growth Factor in Skeletal Muscle. The Scientific World Journal. 2013: Article ID 313605.

"Go Hard or Go Home?" Study Reveals Different Anabolic Signalling in Response to "Heavy" vs. "Medium" Intensity Leg Extensions at Different Times Under Tension

Working out ain't child's play, right? "Go Heavy or Go Home!" this is the mantra of true champions, but is it also the mantra of skeptical scientists?

I guess, it's important to say this first: The results of the study Daniil V. Popov et al. conducted at the Institute of Biomedical problems of Russian Academy of Sciences in Moscow need to be interpreted to have any relevance in terms of the question all of you keep asking yourselves: "What is the best training intensity to make fabulous gains?"

The data the Russian researchers offer is acute, not chronic. It's not based on muscle size measurements, but on the measurement of anabolic signalling proteins and the expression of MyoD, IGF-1, myostatin & co. We know that all of them are involved in the process of skeletal muscle hypertrophy, but even if all of them are elevated, this is not identical to muscle size increases as you would measure them in a long(er)-term study.

Learn more about effective training techniques at the SuppVersity

Optimizing Rest for Size and Strength Gains

When Rodents Squat, We Can Learn A Lot!

Farmer's Walk or Squat? Is Strong- men T. For You?

Full ROM ➯ Full Gains - Form Counts!

Battle the Rope to Get Ripped & Strong

Up Your Squat by 25% With Sodium Bicarbonate

I hope that the previous elaborations were detailed and convincing enough to increase your awareness of the limitation of the data I am about to cite.

Figure 1: Toque and angle during knee extensions in the high intensity (HI), medium intensity (MI) and medium intensity continuous tension trial (MIR) - the subjects did bilateral leg extensions (Popov. 2014)

Data that was recorded with 10 strength-trained athletes who performed high-intensity [HI, 74% of 1 repetition maximum (RM)], middle-intensity (MI, 54%1RM), or middle-intensity (54% 1RM) no-relaxation exercise (MIR; continous tension on the trained muscle).

"High intensity" vs. HIGH intensity: I am pretty sure most of you read my articles closely, for the rest it's important to note that the high intensity group trained at ~74% of the 1-RM - that's about 10 Reps to failure and actually not as heavy as some people may expect, when they hear "high intensity". Just remember one thing: Heavy ≠ intense - I see guys at my gym training with maximal weights and minimal intensity.

Figure 2: Changes in p21, MyoD, myostatin and MGF in response to each of the three training regimen (Popov. 2014)

The previously mentioned parameters were measured before, 45 min, 5h, and 20 h after exercise - you can see the results in Figure 2. The lactate concentration, which is not shown in Figure 2 was approximately 2-fold lower in the MI vs. the MIR & HI. It was the highest in the MIR session, and would thus coincide with the expression of the pro-anabolic protein p21 and the maximal early reduction in myostatin.

Over the course of the 24 study period, however, the high intensity trial yielded distinctively more pronounced anabolic effects.

A more pronounced and sustained elevation in p21, a maximal increase in MyoD, a marker of satellite cell (muscle precursor cell) activity, a significantly more pronounced increase in the intramuscular isform of IGF, i.e. MGF (learn more) and, last but not least, a sustained reduction of the muscle growth break myostatin.

Learn more about the fundamental signals that trigger and sustain muscle hypertrophy at the SuppVersity | more

Bottom line: The 24h+ reduction in myostatin in response to the high intensity trial alone would support the statement "Go Hard or Go Go Home!" In conjunction with the significant increase in MGF, the data Popov et al. present in their latest paper provide compelling evidence in favor of heavy resistance training as a means to trigger a maximal hypertrophy response - and that despite the fact that another often-measured indicator of skeletal muscle hypertrophy, i.e. ERK-1/2 (linked to protein synthesis) did not depend on exercise intensity and was thus identical for the similarly stressing MIR and HI trials.

As mentioned in the introduction, though, the acute changes in any of these measures are indicative of muscle growth, they are not identical to muscle growth. Thus, only a 6-12-week study with a training regimen that mimics the different training intensities could prove that high intensity is required to maximize muscle growth, when the basal stress is identical (i.e. HI would build more muscle than MIR).

References:

• Popov, Daniil V., et al. "The Influence of Resistance Exercise Intensity and Metabolic Stress on Anabolic Signaling and the Expression of Myogenic Genes in Skeletal Muscle." Muscle & nerve (2014).

Intermittent Thoughts on Building Muscle: A Preliminary Conclusion - Exercise, mTOR/AKT/MAPK, IGF-1, Testosterone, Estrogen, DHT, Nutrition, Supps & Sleep

Image 1: Arnold obviously is flabbergast that neither of the factors mentioned in the title of this blogpost was necessary to build the impressive physique of the Arnold statue which now stands in front of the Schwarzenegger Museum.

I don't know if some of you have seen it, but on January 3, 2012, ScienceDaily published a brief news-item titled "How work tells muscles to grow". It relates to a recent study by Guerci et al. who have found that the so-called serum response factor, which basically another "gene switch", is responsible, or I should say required for exercise induced satellite-cell mediated hypertrophy (Guerci. 2012). If you have been following all of the past installments of the Intermittent Thoughts from the early "Hypertrophy 101" (Part 1, Part 2), in which I explained the difference between the increase in myonuclear domain sizes as a consequence of increased protein synthesis, on the one hand and the formation of new myonuclear domains via satellite cell recruitement, on the other hand.

Do you remember myostatin?

You will probably also remember that myostatin, the legendary TGF beta protein that acts as an inhibitor of muscle growth, is something like a watchdog, who suppresses further increases in myonuclear domain size, when the latter begins to exceed a presumably fiber-type dependent maximum - with highly oxidative type I (slow twitch fibers) exhibiting less growth potential than their glycolytic (fast twitch) cousins. From analyses of muscle fibers from different athletes, we do yet know that the skeletal muscle of bodybuilders, the epitome of maximal skeletal muscle hypertrophy, are not - as some people argue - characterized by an abundance of ultra-fast highly glycolytic type II fibers (cf. figure 1).

Figure 1: Fiber composition of bodybuilders, recreational lifters, endurance rowers and sedentary control; determined via myosin heavy chain (MHC) isoform content of the triceps brachii muscle (data adapted from Jurimäe. 1997)

As you can see in figure 1, quite the opposite is the case, probably as a result of the high training volume, the additional endurance exercise (in the 1990s, when the study was done, no bodybuilder did HIIT ;-) and the use of performance enhancing "supplements", the dominant fiber type in the skeletal muscle of the bodybuilders in the Jurimäe study from 1997 is the intermediate fast-twitch, but still highly oxidative type IIa fiber.

Protein synthesis + satellite cell recruitment = growth!?

You probably did not realize it, but with the brief reference to the recently published study on the effects of the exercise induced release of serum response factor (Srf), the subsequent reminder of the difference in the increase in myonuclear domain sizes and the increase in their number and the allusion to training (and drug) induced changes in fiber-composition, we have covered the fundamental mechanisms by which the scrawny boy on the left side of image 2 turned into a, if not the, figurehead of physical culture and bodybuilding.

Image 2: The fundamentals of skeletal muscle hypertrophy apply to us all.

Yet, although we do have a broad understanding of what happens when we train, eat, sleep, train, eat, ... the underlying physiological processes are more complex than the ostensibly straightforward interplay of protein synthesis and satellite cell recruitment would suggest. Within the follow-ups on the "Hypertrophy 101", I have thusly done my very best to tackle each of the complex physiological processes (most of which are not even fully elucidated, yet) which allegedly or effectively trigger, sustain, facilitate or drive skeletal muscle hypertrophy in separate installments:

1. IGF-1 and its Splice Variants MGF, IGF-IEa & Co - Are the growths hormones master regulators of muscle growth or just a bunch of cogs in the wheel of skeletal muscle hypertrophy?
   
   
2. IGF-1, TNF-α, IL-15 & Co and the Emerging Role of an Auto-/Endocrine-Immune Axis in Skeletal Muscle Hypertrophy  - How does inflammation factor in, is it beneficial or detrimental?
    
3. Zoning in on "The Big T" - Does testosterone (alone) build muscle?
    
4. Quantifying "The Big T" - Do increases of testosterone, which are well within the physiological range matter?
    
5. Understanding the "Big T" - How does testosterone work? What is its effect on stem cells and how come it makes you leaner?
    
6. Estrogen, Friend or Foe of Skeletal Muscle Hypertrophy? - Which role does estrogen play and why could you SERM away your growth potential, when you (ab-)use tamoxifen or potent aromatase inhibitors?
    
7. Dihydrotestosterone (DHT), The All Things Male Hormone - Will it make you bigger, stronger and faster or just balder, fatter and unhealthier? 

In case you have forgotten about the way the local and not the systemic growth hormone response is responsible for the intricate restructuring process, in the course of which the increase in myonuclei (which must not be confused with an increase in muscle cells, i.e. hyperplasia) and motor proteins keeps the growing muscle strong and functional, about how IGF acts as a door opener for the macrophages (immune cells), which then "install" the satellite cells in the inflamed muscle and about the function of testosterone and its metabolites estrogen and dehydrotestosterone in this complex interplay of intra-, para- and endocrine processes, you can read up on all that in the previous installments of this series (see list above).

A picture is worth a thousand words - tying the knots graphically 

For those of you who either remember all that or have just returned from their virtual tour through the archives of the SuppVersity, I have come up with a graphical illustration which is an attempt to sum up "all" the major players and their interactions.

Figure 2: Graphical illustration of what you should have learned by now, if you read the previous installments of the Intermittent Thoughts; note: while I have used the arrows rather indiscriminately (they do not necessary mean "causes"), the stops at the end of other lines indicate an inhibition, eg. the line from exercise to myostatin indicates that exercise inhibits myostatin, which would inhibit increases in myonuclear domain sizes, if it was not "switched off" by exercise...

I bet there are still a few arrows missing here and there, but the overall message is clear: If there was one unifying factor that impacts all important aspects of (natural) skeletal muscle hypertrophy, it would be exercise. Exercise acts directly on the mTOR/p-AKT and the MAPK pathway and thusly increases muscle protein synthesis (assuming the respective substrates are present) and the accrual of motor proteins. It increases mitochondrial biogenesis via PGC-1a and induces adaptive changes in the fiber type composition of the trained muscle. It inhibits myostatin and thusly allows for further expansion of the myonuclear domain. The latter is possible because it will promote the proliferation and the recruitment of satellite cells in response to the exercise induced expression of inflammatory cytokines and local growth factors.

If you can show me another player in this orchestrate with a similarly or even more important function than physical activity with a focus on skeletal muscle overload, let me know... if not, you better make sure you do not miss your next workout ;-)

Three is More Than One: Higher Volume Increases Strength Gains in Legs, and Satellite Cell Recruitment and Fiber Size in Legs & Traps. Plus: Data on Myostatin, IGF1, MGF & Co.

Image 1: The green dots that are crowding left and right from the blue myonucleus are the satellite cells (Hanssen. 2012)

In case you are not really sure what a "satellite cell" is and why you should care abouts it's "recruitment", you have probably missed the Intermittent Thoughts on Building Muscle series and should get into detention. Otherwise, here is the news: In a study that has been published in the latest issue of the Scandinavian Journal of Medicine in Sports Science T.S. Hanssen et al. published a paper with the auspicious title "The effect of strength training volume on satellite cells, myogenic regulatory factors, and growth factors" (Hanssen. 2012). Exactly that kind of study that would have the potential to take the mostly common or bro-sensical reasoning behind the current recommendations on training volume to the next, a scientific level, if it the scientists would finally realize that strength training noobs are hardly a better model for advanced trainees than rodents :o(

Full body training 3x /week: How many sets are optimal?

For their study, Hanssen et al. recruited twenty two healthy untrained men (age 26.5y; height: 181.8cm; weight: 81kg) and assigned them to one out of two full-body workouts, with identical exercises (leg press, leg extension, leg curl, seated chest press, seated rowing, latissimus pull-down, biceps curl, and shoulder press), but different amounts of sets for a given body part:

• 3x Legs & 1x Upper Body: Trainees in this group performed 3 sets for each of the leg exercises and 1 set for the upper body exercises
   
• 1x Legs & 3x Upper Body: Trainees in this group performed 1 set for each of the leg exercises and 3 sets for each of the upper body exercises

All subjects started out with 10 reps per set in the first two week. In weeks 3-6, the intensity was increased to 8RM and in the last 5 weeks (7-11) all participants trained at their "7-rep max":

Participants were encouraged to continuously increase their RM loads during the intervention and they were allowed assistance on the last repetition.

In addition to the three strength training sessions per week, the participants were allowed to perform one (not more!) additional steady-state cardio session. And while this may help with standardization, my mathematical skills tell me that the 3L-1UB group performed only 14 sets per workout, while the 1L-3UB group performed 18 sets, simply because the number of lower and upper body exercises was not identical. Now, everyone who does not claim that he or she does not need to train legs (for whatever stupid reason) will know leg training is much more draining than upper body workouts, so "intensity-wise" the workouts were probably still identical.

Single vs. multiple-set training? Nothing new or exciting if it was not for the specific data

We have seen similar studies before - most of them with the same, very unfortunate limitation of being performed on strength training novices, by the way - and the results of this study would hardly be exciting, if it were not for the sheer amount of data Hansson et al. recorded:

Image 2: If those acronyms don't ring a bell, click here to learn more.

• muscle strength and muscle fiber size,
  
  
• the number of satellite cells and number of satellite cells positive for myogenin and MyoD,
   
• the number of myonuclei, myogenin and MyoD content of muscle samples, and
  
  
• myostatin and a whole host of growth factors, namely IGF-1, MGF, HGF, FGF2 and VEGF

This is what I call a "comprehensive" analysis ;-) And one which brought about some pretty interesting results, of which the increases in muscle strength and fiber size are unquestionably the most straight-forward and least surprising ones (cf. figure 1):

Figure 1: Changes in 1RM strength and fiber area after 11 weeks on the training regimen with different set schemes (data calculated based on Hanssen. 2012)

As you can see in figure 1 strength-wise, only the leg (m. vastus lateralis), yet not the back (trapezius) muscle benefited from the increased training volume. When you do take a look at the fiber size, however, it becomes obvious that as long as we compare 1 vs. 3 sets more is actually more +24%, to be precise (the 4% difference for the fiber size in the legs is statistically non-significant).

Figure 2: Relative changes in myonuclei number per muscle fiber and the number of satellite cells after 2 and 11 (post) weeks of the training intervention (data calculated based on Hanssen. 2012)

If you also take into account that the data in figure 2 clearly shows that an increase in training volume leads to increased satellite cell recruitment (indicated by the increases in the number of myonuclei per muscle fiber) and number, it becomes obvious that you are missing out on the growth promoting effects of strength training. The decrease in myonuclei per muscle fiber in the latter 9 weeks of the training intervention in the low volume upper body group (dark red bars in figure 2) would also suggest that this is particularly true for the experience strength trainee in whom the initial 2-week growth spurt of which Hansson et al. state that it was one of their two "main observations":

The novel finding in our study was the early increase in the proportion of activated satellite cells, the early increase in total number of satellite cells and the dependence on training volume in the leg muscle. The number of activated satellite cells, indicated by myoD and myogenin expression, increased from ~2% before training to 6–10% 2 weeks into the intervention.

So as "novel" as this finding may be, it is still somewhat unsatisfying for experienced strength trainees, who will have to rely on the hypothesis that their response will be an ameliorated version of the "late" growth response in the study at hand, which would support my personal observation that 3x3 i.e. three exercises à three sets per body part is at the lower end of the "optimal" volume continuum for advanced trainees. A volume continuum, by the way, that is capped at 12-15 sets for the largest body parst, i.e. legs and back and does by no means extent into the "insanity realm", where people perform 20 sets for biceps and 20 working  sets for triceps on a 5-day body-part split.

Surprise: No statistical significant differences in growth factors

What I found quite surprising though, is that this advantage of 3- vs. 1-set training was not reflected by greater increases in myostatin and/or MGF (want to know more about MGF, click here for the pertinent installment of the Intermittent Thoughts). MGF was even higher in the vastus of the 1 set group and the smaller incline in myostatin levels in the 3-set group (1.1x vs. 1.8x) did not reach statistical significance.

Figure 3: Changes in mRNA levels of myostatin, IGF-1, MGF, HGF, FGF2, and VEGF in the 10 subjects with the largest satellite cell response (seven from 3L-1UB and three from 1L-3UB; data adapted from Hanssen. 2012)

If we do yet take a look at the changes in mRNA levels of myostatin, IGF-1, MGF, HGF (hepatocyte growth factor; growth factor for satellite cells, cf. Sheehan. 2000), FGF2 (fibroblast growth factor; promotes cell adhesion and proliferation, as well as the build up of callege, cf. Yun. 2012) and VEGF (cf. figure 3) in the 10 subjects with the largest satellite cell response (seven from 3L-1UB and three from 1L-3UB), we do see that all but myostatin and VEGF were statistically significantly elevated above baseline. These results stand in line with the main message of the Intermittent Thoughts which was that it is the interaction of a whole host of "hormones" (and peptides) that "builds muscle" - and not as the producers of a certain category of 100% useless "all-natural" supplements want to make you believe, testosterone (or any other growth factor) alone.

And while it was to be expected that the expression of those growth factors would decline over the course of the 11-week intervention period, I am honestly wondering, whether the decrease in reps from 10 (initial two weeks) to 8 and subsequently 7 reps per set had anything to do with it... but I guess, this is an issue for the next study, about which, you will read nowhere else than right here, at the SuppVersity, the place where bro- and pro-science unite in the spirit of true wisdom - but I guess you know that by now, don't you? ;-)

BPA & Phtalate News for the Plasti-Nation. The Endocrine Contribution to Muscle Growth. Magnesium & Testosterone Increase in Parallel. Anabolic Vibes on the Lat Pull & More

You still have a couple of milli- or centimeter too much on your waistline? Let's hope this spring will provide ample time to work out in the sun, after all that's the energizing way to work out, something even virtual reality indoor exercise cannot compete with (Plante. 2006)

The SuppVersity figure of the week is "90". That was the average total amount of sunshine hours we got here in North-Rhine-Westphalia within the winter months (DWD. 2013; and there were places with only 40h!). A pretty depressing figure, in the literal sense. If you listened to the last installment of the Science Round Up (click here to download the podcast), you may remember that I was actually somewhat surprised to hear that this was the darkest Winter ever since the sunshine hours have been recorded - after all, I got my 45min of "artificial sunshine" in, every morning. So, just in case you are still wondering how it's possible to write a blogpost everyday, the data from a 2012 series of studies in Dutch schools would support that it's all about the right lighting (Sleeges. 2012).

Apropos news, here is your weekly serving of short news on all sorts of things... you are missing the exercise related news? Well, I saved those for a round-up and one or two individual posts in the course of the next week ;-)

• Fiber renders mammalian guts colon proof (Nagy-Szakal. 2013) -- As a recent rodent study from the Baylor University suggest, it can hardly be too early to keep an eye on fiber intake. The mice in the pertinent study that had been randomly assigned to a low-cellulose (indigestible fiber that's present in fruits, veggies, whole grains etc.) diet had little to no protection against experimentally induced cholitis.
  
  

  

  Semisynthetic non-fermentable viscous fiber Hydroxpropyl-Methylcellulose is imported pound-wise from China and ends up as E464 from in all sorts of food - obviously at too low dosages to do the anti-obesity trick, though (learn more).

  Murine pediatric cellulose supplementation, on other hand, induces transient trophic and  anticolitic effects, which is - and that's important (!) - dependent on a continuous supply of adequate amounts of dietary fiber. The same goes for the benefifical changes in the diversity of the gut microbiome which did largely  decline after only 10 days.
  
  The protective effect on the epithelial cell lining and the increase in surface area in response to cellulose supplementation should also facilitate the uptake of those vitally important nutrients, people with Crohn's etc. are lacking - a result that may well be significant for those of you who are trying to make the most of the nutrients and not just the energy in their diets.
  
  Edit: As George Henderson rightly said, the study at hand does not talk about cancer protection, I simply inferred that from previous human studies such as Mendez (2007), Terry (2001) or Roth (2001), without mentioning that, my mistake, sorry for that. I still changed the headline to reflect the contents of the study and want to point out that George also makes a valid point stating that (a) fiber often replaces other less healthy nutrients in the diet and (b) that (my addition) "once the baby has been thrown out with the bathtub" and you already have Crohn's and co different rules may apply.

• The diabesity syndrome - Is it the soda, or the bottle it's packaged in? (Indumathi. 2013) I am well aware that the doses of BPA we are supposed to get from our "diets" is way below those that are uses in rodent studies like the one by Indumathi et al., but don't you think that it is still remarkable that the same plastic poison aka BPA that leaches so readily into the acidic brown soup 50% of the US citizens guzzle on a daily basis, induces exactly the same nasty reductions in insulin receptor and GLUT-4 transporter expression, as well as glucose oxidation and the phosphorylation of Akt which are currently still ascribed exclusively to the soda itself (see figure 1).

  

  Figure 2: Effects of 20 or 200mg/kg body weight of BPA on insulin receptor and Akt phosphorylation, GLUT-4 expression in skeletal muscle and systemic insulin and testosterone levels (Indumathi. 2013)

  Now what's worst is that these pathological changes are not likely to be observed, because it happens "silently"; i.e. in the absence of elevated blood glucose levels.
  Did you know that the amount of BPA that leaches from plastics increases by x40, when you dishwash and reuse them (Brede. 2003)? And worst of all, these figures were observed in baby bottles and thus objects those of us get in contact for whom the otherwise probably harmless exposure could actually be dangerous (DiVall. 2013). Exposure to higher temperatures of liquids in plastic containers in the summer (>40°C) is another factor which contributes to an increased leakage of BPA from the container into the liquid (Makris.2013)
  With the latter being the only blood parameters that are evaluated on a regular basis within at least a reasonably large cross section of the population it's no wonder that many of us don't see how the diabetic beast and the BPA castrator are sneaking up on them before it's already too late. Obviously no reason for the FDA or any other governmental body to even bother. Aside from the U.S. Environmental Protection Agency which has published a reference dose of 0.05 mg/kg/day for BPA in 1993 (IRIS. 1993) an official acceptable daily reference intake for BPA is still lacking. Even if the current research does not suggest that our daily exposure is high enough to actually do any harm, I would expect the reference levels to reflect that - what about you?

• 

  In 1998 the Consumer Union wrote a letter to the FDA compaining about "endocrine disrupting chemicals" in cheese and dairy in plastics wrappings. I wonder if they knew about the masked phtalates in hygiene products, as well.

  More news for the"Plasti-Nations" (various): A couple of days ago Wu et el. published a paper on the TSH and estrogen suppressive effects of phtalates in Taiwanese children. With >40% lower TSH levels and estrogen levels the changes, in the 4-5 year olds with the highest (500ppm) phtalate exposure from food stuff is alarming. The only good news is that it these changes appear to be reversible, when further exposure is avoided (Wu.2013).
  
  Apropos good news, in December 2012, Fierens et al. investigated the effects of cooking at home on the phtalate content of foods and found that, except for veggies, the phtalate content of almost all foods decline after cooking (Fierens. 2012). I guess, I don't have to mention that this is not going to happen in a crock pot unless you pour away the brew.
  
  What you should also keep in mind is that your diet is by far not the only way you are exposed to phtalates, as a group of scientists working at a University right around my corner elucidated (Koch. 2013). The "pro-breast cancer" monoethyl carboxylpentyl phthalates (MEPs) for example usually come from personal hygiene products (it's the stuff thats called "fragrance"). The dosage, by the way, was high enough for the scientists to be able to actually measure rises in MEP concentrations after people (esp. men, by the way) took a shower. Ah, and not to forget a non-negligible amount of the low molecular weight phtalates is simply with ubiquitous sources including dust and indoor air. Up to now convincing evidence for the role of this constant assault in any of the ailments our society is suffering from is not conclusive.

• Don't like oats? Buckwheat has similar benefits (Stringer. 2013) -- In a recently published paper in the scientific journal Metabolism, a group of researchers from the University of Manitoba in Winnipeg, Canada, reports that the consumption of buckwheat cracker either instead of rice crackers or simply on their own could help both diabetics and non-diabetics maintain a healthy weight.
  
  

  

  4x more iron, 3x more calcium, >9 times more magnesium, ~6x more potassium and about twice as much zinc, copper and manganese that's what buckwheat flour has to offer compared to wheat (de Francischi. 1994). If you like those figures, what about havin' one of the pancakes you see above (recipe)

  Yet, despite an increased satiety effect and improvements (increases / decreases) in the corresponding hormones glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP) and pancreatic polypeptide (PPY) the glucose response to the buckwheat crackers was not significantly different from the one the scientists observed after the consumption of rice crackers.
  
  What's intriguing, though is that it appears as if these effects were not exclusively fiber-specific. After all, the rice crackers had almost as much fiber as the buckwheat. Moreover, a 2003 study Kawa et al. confirmed that even fiber-free buckwheat extracts exert potent anti-diabetic effects, the researchers ascribed to the presence of d-chiro-inositol in the extracts (Kawa. 2003).

• Anabolic vibes - Vibrate your way to higher testosterone levels (Couto.2013) -- If you still believe vibrators were for women only you got to check out the latest issue of the International Journal of Sports Medicine, in which a group of Brazilian scientists reports that the use of one of those fancy vibration devices (20-Hz and 12-mm) during the lat pulldown induced greater increases in testosterone and lactate concentrations. Dunno if that will also work if you just swipe your girlfriends vibrator and honestly, I don't even want to know that ;-)

• Does the testosterone  and overall hormonal response to workouts even count, or are we still chasing a hormonal ghost? (Schoenfeld. 2013) -- In his latest review of the literature, Brad Schoenfeld who has been busy writing reviews on everything muscle heads are interested in within the last couple of weeks, picks the role of the endocrine response to exercise apart. In that, Schoenfeld points out that it is important to look close before you can tell whether or not a certain "hormone" will effect or even drive skeletal muscle hypertrophy.
  
  

  

  Check out this previous analysis of post-workout anabolism here at the SuppVersity (read more)

  There is for example ample evidence for the involvement of the muscle specific IGF-1 isoform IGF-1Ec, or its locally expressed splice-variant MGF and the presence of high and low responders (cf. West. 2012). Studies using its parent growth hormone, which used injections of the artificially produced recombinant growth hormone are of questionable value, since they lack the natural diversity of "growth hormones" (different GH peptides). Schoenfeld also points out that the evidence that testosterone is anabolic "is inconvertible" (Schoenfeld. 2013) and that the debate would thus have to center around its influence in the vicinity of a workout, where - and this is interesting - we actually know that the effects should occur only after the initial repression of androgen receptors is reversed.
  
  In accordance with what I have pointed out in the Intermittent Thoughts on Building Muscle Series Schoenfeld comes to the conclusion that it would be inappropriate to totally discard the importance of the endocrine response to workouts (IGF-1 & Co, in particular), just because it does not promote the (imho totally overrated) short term protein synthetic response to exercise:

  "Based on limited cellular signaling data, it is conceivable that the primary effect of post-exercise hormonal elevations is to increase satellite cell activity as opposed to mediating acute increases in muscle protein synthesis. If so, this could favor greater long-term increases in muscle hypertrophy without significantly impacting short-term gains." (Schoenfeld. 2013)

  Obviously (and rightly so) Schoenfeld concludes this paragraph of the discussion of this excellent review with the sentence "This hypothesis requires further study." -- And you bet, I will keep an eye on the topic in order to let you know whether the 8% of which West and Phillips say that it's the upper limit of the contribution the transient increases in endocrine hormones have on skeletal muscle hypertrophy (West. 2012) is an over- or underestimation.

• Excess magnesium is good for your testosterone levels, bad for your prostate and could potentially become fatal for your heart (Chandra. 2013) -- According to the latest results from a rodent study, it appears as if an excess intake of magnesium, ingested not as supplement but in form of magnesium sulfate in the chow, can boost testosterone levels by almost 30% (figure 1):

  

  Figure 1: Testosterone  levels prostate weight, luteinizing hormone and serum magnesium levels (all data expressed relative to baseline) after 15 or 29 days on diets with different magnesium content.
  

  The increase in testosterone does yet not come without downsides in the form of slight, but significant increases in prostate size as well as a profound increase in magnesium levels, which would - if they were observed in human beings already mark the early stages of hypermagnesemia. So, as important an adequate amount of magnesium in your diet may be, it should be obvious that 1.5% of the diet or ~350mg/kg magnesium sulfate and thus 35mg elemental magnesium per kilogramm body weight (the HED of the dosage that was administered to the rodents; ) is simply too much - honestly, I am surprised that the rodents did not get chronic diarrhea, anyway ;-)

Since a wise man facebooked me earlier today to keep the SuppVersity posts short on this weekend, spending some time idling around, in order to avoid ending up totally burned out (I wonder if that really was an all altruistic advice of him ;-), I will now do just that - cut it short; yet not without pointing you towards the SuppVersity Facebook Wall, which does - as every day - hold 6-10 new short news for you to read up on.
 

+++ Have a nice weekend, everyone! +++

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• Chandra AK, Sengupta P, Goswami H, Sarkar M. Effects of dietary magnesium on testicular histology, steroidogenesis, spermatogenesis and oxidative stress markers in adult rats. Indian J Exp Biol. 2013 Jan;51(1):37-47.
• Couto BP, Silva HR, Filho AG, da Silveira Neves SR, Ramos MG, Szmuchrowski LA, Barbosa MP. Acute Effects of Resistance Training with Local Vibration. Int J Sports Med. 2013 Feb 26.
• de Francischi ML, Salgado JM, Leitão RF. Chemical, nutritional and technological characteristics of buckwheat and non-prolamine buckwheat flours in comparison of wheat flour. Plant Foods Hum Nutr. 1994 Dec;46(4):323-9.
• DiVall SA. The influence of endocrine disruptors on growth and development of children. Curr Opin Endocrinol Diabetes Obes. 2013 Feb;20(1):50-5.
• DWD. Press Release from February 27, 2013. < http://www.dwd.de/presse > 
• Fierens T, Vanermen G, Van Holderbeke M, De Henauw S, Sioen I. Effect of cooking at home on the levels of eight phthalates in foods. Food Chem Toxicol. 2012 Dec;50(12):4428-35.
• Indumathi D, Jayashree S, Selvaraj J, Sathish S, Mayilvanan C, Akilavalli N, Balasubramanian K. Effect of bisphenol-A on insulin signal transduction and glucose oxidation in skeletal muscle of adult male albino rat. Hum Exp Toxicol. 2013 Feb 19.
• Integrated Risk Information System (IRIS). Reference Dose (RfD): Description and use in health risk assessments. 1993 < http://www.epa.gov/ncea/iris/subst/0356.htm > 
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• Koch HM, Lorber M, Christensen KL, Pälmke C, Koslitz S, Brüning T. Identifying sources of phthalate exposure with human biomonitoring: Results of a 48h fasting study with urine collection and personal activity patterns. Int J Hyg Environ Health. 2013 Jan 17. 
• Makris Kc, Andra S, Jia A, Herrick L, Christophi C, Snyder Sa, Hauser R. The Association Between Water Consumption From Polycarbonate Containers And Bisphenol-A Intake During Harsh Environmental Conditions in Summer. Environ Sci Technol. 2013 Feb 28.
• Mendez, A M, Pera G, Agudo A, Bueno-de-Mesquita HB, Palli D, Boeing H, Carneiro F, Berrino F, Sacerdote C, Tumino R, Panico S, Berglund G, Manjer J, Johansson I, Stenling R, Martinez C, Dorronsoro M, Barricarte A, Tormo MJ, Quiros JR, Allen N, Key TJ, Bingham S, Linseisen J, Kaaks R, Overvad K, Jensen M, Olsen A, Tjønneland A, Peeters PH, Numans ME, Ocké MC, Clavel-Chapelon F, Boutron-Ruault MC, Trichopoulou A, Lund E, Slimani N, Jenab M, Ferrari P, Riboli E, González CA. Cereal fiber intake may reduce risk of gastric adenocarcinomas: the EPIC-EURGAST study. Int J Cancer. 2007 Oct 1;121(7):1618-23.
• Nagy-Szakal D, Hollister EB, Luna RA, Szigeti R, Tatevian N, Smith CW, Versalovic J, Kellermayer R. Cellulose supplementation early in life ameliorates colitis in adult mice. PLoS One. 2013;8(2):e56685. 
• Plante TG, Cage C, Clements S, Stover A. Psychological benefits of exercise paired with virtual reality: Outdoor exercise energizes whereas indoor virtual exercise relaxes. International Journal of Stress Management, Vol 13(1), Feb 2006, 108-117
• Roth J, Mobarhan S. Preventive role of dietary fiber in gastric cardia cancers. Nutr Rev. 2001 Nov;59(11):372-4.
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• Stringer DM, Taylor CG, Appah P, Blewett H, Zahradka P. Consumption of buckwheat modulates the post-prandial response of selected gastrointestinal satiety hormones in individuals with type 2 diabetes mellitus. Metabolism. Available online 26 February 2013.
• Terry P, Lagergren J, Ye W, Wolk A, Nyrén O. Inverse association between intake of cereal fiber and risk of gastric cardia cancer. Gastroenterology. 2001 Feb;120(2):387-91
• West DW, Phillips SM. Associations of exercise-induced hormone profiles and gains in strength and hypertrophy in a large cohort after weight training. Eur J Appl Physiol. 2012 Jul;112(7):2693-702.  
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