Trimethylglycine aka Betaine Sets the Anabolic Stage for Increased Muscle Growth: Higher IGF-1 & Lower Cortisol - Statistically Significant, but Physiologically (Ir-)Relevant?

Figure 1: Betaine content (in mg/100g) in some common food items (data based on Craig. 2004). Makes me wonder if Popeye ate wheat germ as well or whether he was celiac and stuck to spinach to get his daily dose of pro-anabolic betaine?

Trimethylglycine (TMG) the sciency name for a molecule most of you probably know by the name "betaine" is actually no longer a new-comer to the supplement scene (please note that this is not betaine HCL(!), the stuff you will find in digestive aids). I have already written about its purported ergogenic effects several times and there are actually quite a handful of proprietary blends with mostly undisclosed, but judged based on the total serving size and amount of ingredients in them, hilariously underdosed amounts of the zwitterionic compound and a methyl derivative of glycine in it on the market.

Friends and followers of the SuppVersity will also be aware that betaine is also found naturally in a variety of food sources such as sugar beets, wheat bran, spinach, shrimp, and many others (see figure 1) and that it can be synthesized from choline in your body, when dietary intake exceeds your current metabolic demands (Ueland 2011).

What you probably don't know, however, is...

... that the latest study from the Human Performance Laboratory at the Department of Kinesiology,
of the University of Connecticut, shows that "betaine (vs. placebo) supplementation enhanced
both the anabolic endocrine profile and the corresponding anabolic signaling environment, suggesting increased protein synthesis" (Apicella. 2013).

Figure 2: Effects of a standardized full-body workout (see text for details) on growth hormone (µmol/L), IGF-1 (nmol/L) and cortisol (µmol/L) levels in 12 recreationally trained young men after 2 weeks supplementation with betaine (2x 1.5g/day) or placebo (data based on Apicella. 2013)

And if we temporarily lose sight of the fact that the devil is in the detail, the data in figure 2 certainly looks as if you should run to the next best store with fishing equipment and get yourself a huge pot of trimethylglycine, of which the shop assistant will probably tell you that "This is a good choice Sir! The carps love the sweet taste!" But I am digressing, so let's get back to what's really sweet, namely ...

• stable growth hormone levels (vs. -17% in the placebo group)
• an 18% increase in IGF-1 (vs. a -10% decrease in the placebo group), and
• a -5% reduction in cortisol (vs. a 6% increase in the placebo group)

- they all sound pretty sweet, as well. Especially in conjunction with the stable p-AKT levels the scientists observed, when they analyzed the tissue samples. Unfortunately (but earnestly), Apicella et al.'s conclusion, still contains one word, too many people who read the abstract, are probably going to ignore:

"Betaine (vs. placebo) supplementation enhanced both the anabolic endocrine profile and the corresponding anabolic signaling environment, suggesting increased protein synthesis." (Apicella. 2013; my emphases)

Which one is it? A tip: It is none of the words I emphasized in bold. After all, that would make it way too easy for you... ha? Yeah! I see you've done your homework. Suggest(-ing) is in fact the most important word in this and the conclusions of many objectively written scientific papers.

So, the study "suggests increased protein synthesis"...

... and this means it does not even prove that the protein synthetic response in the immediate vicinity of the workout was increased in response to the to the two weeks of BID (=twice daily) supplementation with 1.25 g of betaine. In other words, all we know is that the funky gene essays for p-AKT and serum tests for growth hormone, IGF-1 and cortisol "suggest" that it could be the case, if we assume that marginally higher IGF-1 levels, stable growth hormone levels and lower cortisol levels (rememeber we are not talking about increasing any of them into the supraphysiological range, here) would

1. result in increased protein synthesis and ultimately
2. greater lean mass accrual,

because, if we are honest, no one is interested in a number you can measure, when you infuse a marked amino acid into the circulation and check how much of it goes into the muscle, but doesn't come out of it, afterwards.

Something to remember: What I find remarkable - and this is by no means something you will see only in this study, neither is it "fraud" or whatever, is how by simply adding a break into the Y-axes of the graph and thus omitting the lower 80% of the bar Apicella et al. give the impression that the effects on IGF-1 were more than twice as large than they actually are... remember that, because you will encounter that in many studies, and reproductions of graphs from scientific papers, especially if they are used to market certain products.

What we want is to get bigger, stronger and all that faster, and whether the 12 recreationally trained men (age 19.7±1.2 years; lean body mass 65.2±8.8 kg; fat mass 15.6±8.5 kg; body fat percentage 18.7±7.0 %; BMI 28.2±4.0) would have gained even a single inch of muscle more on whatever body part, if they had performed a real workout instead of the funky "AES" (=acute exercise session) that consisted of

• 10x maximal vertical jumps without pause, 
• 1x 10-s isometric squat, 
• 1x 10-s isometric bench press on a smith machine, and
• 1x 10 min of repeated box lifting (RBL)

is more or less guesswork. In view of the previously discussed results from the researchers at the McMasters University in Ontario (see "Anabolic Workouts Revisited"; a brief reminder. the systemic hormonal response to an acute exercise bout is irrelevant, if anything higher cortisol levels correlate with greater increases in lean muscle mass) and the "statistically significant", but physiologically probably irrelevant increases and decreases in IGF-1 and cortisol at least highly questionably (please take a look at the additional information in the red box to the right, as well).

... but there are still way too many "ifs" in here!

The sheer number of "suggests", "is touted", "is likely", "also possible", "as we presume", etc. is honest and speaks in favor of the quality of the study, but against the reliability of the statement that followeed the initially cited "suggests" in the conclusion. Moreover, the researchers freely admit that...

"[...] the mechanisms by which betaine may have affected the hormones measured in this study are still unclear and require further research" (Apicella. 2013)

so that even the fact that betaine is an organic osmolyte and could thus help stabilize skeletal muscle protein, promote / maintain optimal hydration and protect against

• hypertonic stress (Alfier. 2006), 
• urea-induced inactivation of muscle myosin ATPases (Ortiz-Costa. 2002), and 
• structural changes in myosin due to urea accumulation (Ortiz-Costa. 2002)

does lend credibility to the hypothesis that betaine could help you build muscle, but it does not prove it. In conjunction with the results of previous trials, like...

Betaine does not increase nitric oxide While I have no idea why everyone is so keen about those nitrate supplements, one thing is for sure: Betaine has no effects on serum nitrate or nitrite levels. The vasolidation effect of beet roots / beet root juice is simply a result of the nitrate that's in there along with the betaine (+ the sugar and the insulin spike, which will also trigger an increased NO-response).

At least this is what a study by Bloomer et al. which consisted of three independent experiments using 1.25 and 5.00g B, acutely, 2.5g per day for 14 days, chronically, and a combination of chronic (6g for 7 days) + acute (6g acutely before the test) betaine supplementation (Bloomer. 2011).

• Hoffman. 2009 - 2.5/day for 14 days; jump squat, squat, bench press; "Two-weeks of betaine supplementation in active, college males appeared to improve muscle endurance of the squat exercise, and increase the quality of repetitions performed." 
• Lee. 2010 - 2x 1.25g/day for 14 days; bench squat and jump tests; "[Betaine] supplementation increased power, force and maintenance of these measures in selected performance measures, and these were more apparent in the smaller upper-body muscle groups."
• Hoffman. 2011 -  2.5g/day for 15 days; 5 training + testing sessions; "15 days of betaine supplementation did not increase peak CON or ECC force outputs during an isokinetic chest press but did appear to reduce subjective measures of fatigue to the exercise protocol"
• Trepanoswki. 2011 - 2.5g/day for 14 days; resistance training; "moderate increase in total repetitions and volume load in the bench press exercise, without favorably impacting other performance measures."
• del Favero. 2013 - 2g/day for 10 days; muscle strength and power, muscle PCr content, and body composition, three "familiarization sessions" preparing the participants only to perform the tests; "we showed that betaine supplementation combined or not with creatine supplementation does not affect strength and power performance in untrained subjects."
• Pryor. 2013 - 2.5g/day for 7 days; cycling performance; "betaine ingestion significantly increased average peak power (3.4%; p = 0.026), maximum peak power max (3.8%; p = 0.007), average mean power (3.3%; p = 0.034), and maximum mean power (3.5%; p = 0.011) in recreationally active males and females"

... there is still room for long-term improvements in muscle gains as a consequence of the general ergogenic effects of betaine (every rep more counts!), but it appears unlikely that the "anabolic" hormonal milieu observed in the study at hand are the fundamental cause of the latter.

Reminder: If you want to try it, you got to get yourself "trimethylglycine" (TMG) not "betaine HCL" and you better don't buy it in capped form if you don't have lots of money to burn. I just checked with the next best bulk supplier - they got 1kg for $33.50. Even if you double dose, i.e. take 2x 2.5g per day (most studies mixed it with Gatorade) this will last you for 200days(!), which is probably the time it will take until you can actually see and not just measure any potential, possible, suggested, etc. anabolic effects ;-)

References:

• Alfieri RR, Bonelli MA, Cavazzoni A et al (2006) Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress. J Physiol 576:391–401.
• Bloomer RJ, Farney TM, Trepanowski JF, McCarthy CG, Canale RE. Effect of betaine supplementation on plasma nitrate/nitrite in exercise-trained men. J Int Soc Sports Nutr. 2011 Mar 18;8:5.
• Craig SA. Betaine in human nutrition. Am J Clin Nutr. 2004; 80: 539–549.
• del Favero S, Roschel H, Artioli G, Ugrinowitsch C, Tricoli V, Costa A, Barroso R, Negrelli AL, Otaduy MC, da Costa Leite C, Lancha-Junior AH, Gualano B. Creatine but not betaine supplementation increases muscle phosphorylcreatine content and strength performance. Amino Acids. 2013 Jun;42(6):2299-305.
• Hoffman JR, Ratamess NA, Kang J, Rashti SL, Faigenbaum AD. Effect of betaine supplementation on power performance and fatigue. J Int Soc Sports Nutr. 2009 Feb 27;6:7.
• Hoffman JR, Ratamess NA, Kang J, Gonzalez AM, Beller NA, Craig SA. Effect of 15 days of betaine ingestion on concentric and eccentric force outputs during isokinetic exercise. J Strength Cond Res. 2011 Aug;25(8):2235-41.
• Lee EC, Maresh CM, Kraemer WJ, Yamamoto LM, Hatfield DL, Bailey BL, Armstrong LE, Volek JS, McDermott BP, Craig SA. Ergogenic effects of betaine supplementation on strength and power performance. J Int Soc Sports Nutr. 2010 Jul 19;7:27. 
• Ortiz-Costa S, Sorenson MM, Sola-Penna M (2002) Counteracting effects of urea and methylamines in function and structure of skeletal muscle myosin. Arch Biochem Biophys 408:272–278
• Pryor JL, Craig SA, Swensen T. Effect of betaine supplementation on cycling sprint performance. J Int Soc Sports Nutr. 2013 Apr 3;9(1):12.
• Trepanowski JF, Farney TM, McCarthy CG, Schilling BK, Craig SA, Bloomer RJ. The effects of chronic betaine supplementation on exercise performance, skeletal muscle oxygen saturation and associated biochemical parameters in resistance trained men. J Strength Cond Res. 2011 Dec;25(12):3461-71.
• Ueland PM. Choline and betaine in health and disease. J Inherit Metab Dis. 2011;34:3–15.

Vitamin D, PPAR-γ & the Growth of Healthy Body Fat Stores; Betaine & Fat Inflammation; Free HMB & Muscle Protection; DHA Induced Forgetfulness & Statistical Shenanigan

"Is it already 2013? Must be the DHA, I totally forgot."

The SuppVersity or rather my personal figure of the week was and still is 2013. I actually managed to write 2013 instead of 2013 with every damn study in today's installment of On Short Notice that has been published online in the last couple of days. I wonder how long it will take me to get used to it  - that's like a "new year's date jetlag" ;-)

Now, usually the figure of the week has at least something to do with the topics I am writing about, so I decided to add not another figure, but rather some stats, namely the TOP3 SuppVersity posts (by visitors) from the year that still appears to be programed into my system - 2013:

1. "Particle Size & LCAT Analysis Shows: Three Whole Eggs per Day Improve Lipid Profile in Men & Women W/ MetSyn" (read more)
2. "22g Baking Soda 60min Before a Old-School 4 x 12RM Leg Workout Allow for a 22 Rep Volume Increase on Hypertrophy Oriented Squat + Leg Press + Leg Extension Quads Routine" (read more)
3. "Before, After or In-Between? Study Puts Another '?' Behind the Widely Accepted 'Cardio After Weights' Paradigm" (read more)

That's enough for the -12, let's get to the -13 now and take a look at this week's four On Short Notice items on Vitamin D, Betaine, HMB and DHA.

Study shows: Vitamin D helps your body with fat cell differentiation / maturation 

(Nimitphong. 2013) -- Those of you who have been around for a while will probably remember that I had the cheek to actually say that vitamin D3 could actually make you fat, not lean (suggested reads "Vitamin D3 a 'Fat Synthesizer'!?" "Stronger & Leaner of Fatter & Less Muscular w/ Vitamin D"). So in essence the results of the latest study by Nimitphong, MF Holick  et al. are not new (by the way, yep, this is the MF Holick whose brainchild 25-OHD actually is; cf. Holick. 1972):

Figure 1: 25(OH)D₃ promotes the differentiation of human preadipocytes via PPAR-gamma and reduces the release of stored fat by blunting the lipoprotein lipase levels (Nimitphong. 2013)

"In contrast to its inhibitory effects in a mouse preadipocyte cell line, 3T3-L1, 1,25(OH)₂D₃ promoted adipogenesis in primary human preadipocytes as evidenced by the increased expression of adipogenic markers and lipid filling. In addition, we show that 25(OH)D₃ can also promote the differentiation of human adipocytes, most likely via its activation to 1,25(OH)₂D₃. Furthermore, 1,25(OH)₂D₃ also had stimulatory effects on the differentiation of primary mouse preadipocytes. These results suggest that the local metabolism of vitamin D in adipose tissue may regulate the conversion of preadipocytes to adipocytes and hence support the healthy remodeling of human adipose tissue." (Nimitphong. 2013)

And while Holick is clever enough to bring the benefits of this "remodeling" to the forefront, we are dealing here with the exact same effects PPAR-gamma agonists have - not very surprising after all vitamin D3 turned out to be one (see figure 1). They allow the adipose tissue to expand "healthily", to allow to store more fat (and glucose after being converted to fat) without the negative health effects that occur, when the abundant energy is stored in existing adipocytes, until they burst from the seams.

Since I wanted to make this "short news", I will refer you to the red box in the Seabuckthorn post for further insights into the ambiguous effects of PPAR-gamma.

Betaine reduces inflammatory markers expressed by hypoxia (~suffocating) adipocytes

(Olli. 2013) -- You have read about the potential ergogenic effects of betaine aka trimethylglycine (TMG) at the SuppVersity, before. A a recent study from Finnish researchers does now suggest that it can also reduce the release of inflammatory cytokines and adipokines from hypoxic human adipocytes:

Betaine content (in mg/100g) of some common food items (based on Craig. 2004). You should also check some of your ready made supplements. More and more of the preworkout blends for example contain betaine.

"We show that betaine may reduce inflammation in adipose tissue in hypoxia and may therefore potentially help to reduce systemic inflammation and the risk for other obesity-related diseases." (Olli. 2013)

Usually this would be the point to say "Let's see what the real-world results are", but in this case previous epidemiological data has shown that free living healthy individuals

"[...] who consumed >360 mg/d of betaine had, on average, 10% lower concentrations of homocysteine, 19% lower concentrations of C-reactive protein, and 12% lower concentrations of tumor necrosis factor-α [TNF-alpha] than did those who consumed <260 mg/d." (Detopoulou. 2008)

Reason enough to take a look at the different TMG content of various foodstuffs in figure 1 don' you think so? And by the way, I will still keep you up-to-date as far as future developments in betaine research  are concerned (suggested reads: previous posts on betaine).

Free form HMB reduces acute muscle damage in previously resistant trained men

(Wilson. 2013) -- As far as the scientific background of the anti-catabolic effects of beta-Hydroxy-beta-methylbutyrate (HMB) is concerned There are a couple of promising long(er)term studies mostly in elderly individuals, there is the acute response study by Wilson et al. (result only a minor reduction in lactate dehydrogenase activity; Wilson. 2009) and since a couple of days, there is another acute supplementation study from Jacob M. Wilson et al. which differs from the previous one in terms of

Do you remember Volcan's recently published Master Thesis (read more)? In his experiment he analyzed the effects of eccentric leg extensions + HMB supplementation. I wonder if the results would likewise have been statistically significant, if he had chosen the free acid form of HMB.
• the subjects, now previously strength trained young men (21 years) instead of untrained subjects,
• the measured parameters, now serum creatine kinase (CK), urinary 3-methylhistadine (3-MH),
  testosterone, cortisol and perceived recovery status (PRS), and
• the form of HMB, now a free acid instead of the common calcium bound version of HMB

In this acute supplementation scenario, the replacement of the more common, yet slow absorbing 3g of CA-HMB (60-120min to peak plasma levels) with the faster absorbing free acid form (~30 min to peak plasma levels), was probably also the reason that the differences in post exercise creatine kinase levels (marker of skeletal muscle breakdown) did actually reach statistical significance in this "follow up study" (+329% in placebo vs. 104% in FA-HMB).

The notion that the consumption of 3g of FA-HMB can reduce skeletal muscle damage in trained individuals is supported by trend towards lower 3-MH / CK ratios (marker of protein breakdown) a significantly higher perceived recovery status (measured by questionnaires). There were yet no statistical significant differences in the post-exercise cortisol and testosterone responses. 

Does DHA make young women dumb? Or is it too much statistics that does?

(Beton. 2013) -- It sounds strange, but after receiving a 400mg DHA supplement (no EPA) for 50 days the 126 healthy young women who participated in a study that was conducted at the Department of Psychology of the University of Wales Swansea in the UK, were more likely to forget recently acquired information than their 133 peers who had received a soy + maize oil placebo. The scientists are not able to explain this phenomenon:

Figure 2:Took a hell lot of statistical shenanigan to "prove" the significance of the the results ;-)

"That there was a trend for more to be forgotten in both halves of the study supported the view that it was a genuine rather than a chance result, but naturally such a finding requires replication. The failure to find any difference in the ability to recall the names of capitals suggested that there was no problem in retrieving information from long-term memory stores; and so the finding must reflect some other aspect of the memory process. There is, however, in principle, no reason why n-3 fatty acid supplementation may not have both positive and negative influences. " (Benton. 2013)

In this particular study, the only "positive" thing about the DHA supplement, which contained DHASCO(R), a deodorised and bleached oil derived from the microalgae Crypthecodinium cohnii, was however that it did not have any (negative) effects on the other parameters the scientists measured, i.e. differences in mood, reaction times, vigilance or visual acuity. And if I am honest, I am pretty sure the "cognitive decline" is a null-result, as well - if it had real-world significance you would not need to "reanalyze" and "remerge" the data to finally get the one significant effect you'd like to have in any paper.

---

That's it for today, but as you all should know by now, there is always more on the SupppVersity Facebook Wall. Recent examples are

• Thank god, scientists stop thinking like tortoises: "Generalisations such as 'live fast, die young' may be too simple to explain the complex processes affecting ageing and lifespan." (read more)
• Korean cabbage kimchi increases nitric oxide production and suppresses inflammation in the aorta of apoE knockout mice, an atherosclerosis model (read more)
• Sports concussions make you dumb: "A total of 27.7% of concussed student-athletes who were symptom-free and returned to baseline on ImPACT at rest exhibited cognitive decline following moderate physical exertion." (read more)

...and since it's (a) Saturday and (b) there are and will soon be more news, I hope you won't get bored within the next 24 hours it may well take for the next "official" SuppVersity post to be written and published.

References:

• Benton D, Donohoe RT, Clayton DE, Long SJ. Supplementation with DHA and the psychological functioning of young adults. Br J Nutr. 2013 Jan;109(1):155-61.
• Detopoulou P, Panagiotakos DB, Antonopoulou S, Pitsavos C, Stefanadis C. Dietary choline and betaine intakes in relation to concentrations of inflammatory markers in healthy adults: the ATTICA study. Am J Clin Nutr. 2008 Feb;87(2):424-30.
• Holick MF, DeLuca HF, Avioli LV. Isolation and identification of 25-hydroxycholecalciferol from human plasma. Arch Intern Med. 1972 Jan;129(1):56-61.
• Nimitphong H, Holick MF, Fried SK, Lee MJ. 25-hydroxyvitamin d(3) and 1,25-dihydroxyvitamin d(3) promote the differentiation of human subcutaneous preadipocytes. PLoS One. 2013; 7(12):e52171.
• Olli K, Lahtinen S, Rautonen N, Tiihonen K. Betaine reduces the expression of inflammatory adipokines caused by hypoxia in human adipocytes. Br J Nutr. 2013 Jan;109(1):43-9.
• Wilson JM, Kim JS, Lee SR, Rathmacher JA, Dalmau B, Kingsley JD, Koch H, Manninen AH, Saadat R, Panton LB. Acute and timing effects of beta-hydroxy-beta-methylbutyrate (HMB) on indirect markers of skeletal muscle damage. Nutr Metab (Lond). 2009 Feb 4;6:6. doi: 10.1186/1743-7075-6-6.
• Wilson JM, Lowery RP, Joy JM, Walters JA, Baier SM, Fuller JC, Stout JR, Norton LE, Sikorski EM, Wilson SM, Duncan NM, Zanchi NE, Rathmacher J. β-Hydroxy-β-methylbutyrate free acid reduces markers of exercise-induced muscle damage and improves recovery in resistance-trained men. Br J Nutr. 2013 Jan 3:1-7.