Leucine, Insulin & Vitamin D*: A Hypertrophy Boosting Triplet That Does Not Make It From the Dish to the Gym? Evidence & Counter-Evidence from Human Trials

If you could simulate a workout at the beach in the petri dish, its beneficial health effect would be all the rage ;-)

I am pretty sure all of you still remember the recent post about the literal muscle building effect of vitamin D, right (see "Vitamin D Builds Muscle")? Me too, and so I was almost tricked to believe I was looking at the Girgis study, when I scanned the contents of the latest issue of Molecular Nutrition & Food Research, in which you'll with the telling title: "1,25(OH)2-vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes". Ah, ok, I see that's not exactly easy to understand. Well, in plain English this means as much as...

Calcitriol boosts the anabolic effect of leucine & insulin

If your read the "Vitamin D Builds Muscle" article, you should actually remember the dichotomous nature of the effects vitamin D had on muscle cell hypertrophy (which goes up) and proliferation (which goes down). This was after all the most intriguing result of the Girgis study (go back). The new data from the paper at hand, which is likewise dealing with in-vitro effects of vitamin D, does now provide us with some additional information on the underlying mechanisms of the hypertrophy effects.

Figure 1: Protein synthesis, insulin receptor expression and the levels of p-AKT, p-mTOR, and p-70S6K, all regulators of skeletal muscle protein synthesis in muscle cells with and without additional 1,25(OH)2D3 in a leucine + insulin filled Petri dish (Salles. 2013)

If we go by the data in Figure 1 it's obvious that the latter is driven by a direct amplifying effect of 1,25(OH)2D3, the active form of vitamin D, aka calcitriol, on the leucine and insulin induced increase in muscle protein synthesis. Unfortunately, this does not tell us a word about the potential consequences of the anti-proliferative effects of vitamin D on long-term increases in muscle size and the repair of damaged muscle fibers.

Vitamin D and health - What the latest systematic review says: As long as you look at things at the population level or sit in your well-climatized lab next to the Petri dishes, vitamin D is king. When you look at the real world, of which I still believe that it is populated by individuals, the excitement appears to be unwarranted. The most recent systematic review that has been published today in the (most) prestigious medical journal The Lancet says: "The discrepancy between observational and intervention studies suggests that low 25(OH)D is a marker of ill health. Inflammatory processes involved in disease occurrence and clinical course would reduce 25(OH)D, which would explain why low vitamin D status is reported in a wide range of disorders." (Autier. 2013) -- in other words: Being sick will lead to reduction ins 25(OH)D and not vice versa.

It is nevertheless intriguing to see, how the myotubes that had been cultured in 1,25(OH)2D3 solutions at 0, 1, or 10 nM for 72 h reacted to the leucine and insulin challenges. 14–16% increases in fractional protein synthesis rates (FSR) and an increased expression of insulin receptors, of which we can hope that it would also translate into increased glucose uptake into the cells. Assuming that similar effects occur in vivo and in a normal vs. high vitamin D environment, these benefits would be more than just statistically significant.

Ok, that's an in vitro study, so why do you discuss it at all?

If the above is what you've just been thinking, you know me quite well by now. I would indeed not have wasted a whole SuppVersity article on this vitamin D paper, if it would not contradict the real-world results of a recent randomized, double-blind, placebo-controlled study from the University of Oslo so "nicely".  In this paper, a group of Swedish researchers probed the effects of provision of 1,000IU and 400IU of vitamin D3 per day on 251 healthy adult men and women (age 18-50 years; Knutsen. 2013). 

Despite the fact that the vitamin D levels of the subjects almost doubled, none of the strength and performance parameters, i.e. jump height, handgrip strength and the chair-rising test, showed pre vs. post differences that differed from those in the control group.

Figure 2: Relative pre vs. post changes in jump height, grip strength and the time it took the subjects to complete the chair test; no statistically significant inter-group differences were observed (Knutsen. 2013) vs. relative increase in strength (no inter-group differences) in obese, vit D deficient resistance trained individuals (Carillo. 2013)

This in turn raises the question, whether all our new enthusiasm about 'vitamin D' (in the broad sense, i.e. D3, 25(OH)D3 and 1,25(OH)2D3) was misplaced. That's unquestionably a tricky question and if those "enthusiasm" was triggered by the idea that vitamin D would have independent anabolic or ergogenic effects, the answer is probably "Yes". If we are yet talking about the general role of vitamin D in the complex concert of muscular health, the answer must be "No!" There are, after all exceptions to the "vitamin D does not build muscle rule" that applies so nicely to the Knutsen study - there aren't many, but they are there.

The study Carillo et al., for example (Figure 2, right). If you take a look at the results , it's easy to see that for the twenty-three overweight and obese (age: 26.1±4.7 y; BMI: 31.3±3.2 kg/m², body fat: 43%) subjects with insufficient vitamin D levels (25-hydroxyvitamin D: 19.3±7.2 ng/ml) the researchers from the Purdue University recruited for their experiment the 4000IU of supplemental vitamin D each of them received on a daily basis had the expected effect on the resistance training induced increase in peak power and reduction in waist-to-hip ratio (not shown). The effect size is however is pathetic and the only thing that was "significant" - imho statistically, only - was the peak power in the vitamin D group.

A brief note on 25(OH)D vs. 1,25(OD)2D3: I know that it may sound as if it sucks that taking D3 supplements won't increase the amount of calcitriol in your veins, but if it did, I know more than a handful of people whose trust in some gurus would already have cost them a kidney or even their lives. There is a good reason that calcitriol is a prescription drug, because a dysregulation of the 1,25(OH)2D3 levels in the blood will increase the calcium deposition in the organs and vasculature (Bas. 2006) and would thus have the opposite effects most people expect from their high dose vitamin D3 supplementation regimen.

Bottom line: On paper, the real world-evidence from vitamin D deficient obese individuals in Carillo's recent study does supports the notion that vitamin D is an important facilitator of skeletal muscle protein synthesis, what it does not do, though is provide the missing conclusive evidence that being in the upper tertile of the physiological range (not restoring deficiencies as in Ceglia. 2013, for example) has beneficial real-world effects on muscle strength or size.

If you take another look at the doses the vitamin D enthusiasts from the Girgis study bathed their cells in, that the Swedes used active vitamin D, i.e. 1,25(OH)2D3 and that there is no direct relation between vitamin D3 intake, the serum levels of 25(OH)D3 and the amount of calcitriol (1,25(OH)2D3) your cells are exposed to, it's actually not surprising that the muscle building effects don't translate from Jérôme Salles' calcitriol saturated Petri dishes into the real world of the 251 participants of the Knutsen and the majority of the other vitamin D3 supplementation studies, is it?

References:

• Autier, P. et al. (2013) Vitamin D status and ill health: a systematic review. The Lancet Diabetes & Endocrinolog, Available online 6 Decembee
• Bas, A., Lopez, I., Perez, J., Rodriguez, M., & Aguilera‐Tejero, E. (2006). Reversibility of Calcitriol‐Induced Medial Artery Calcification in Rats With Intact Renal Function. Journal of Bone and Mineral Research, 21(3), 484-490.
• Ceglia, L., Niramitmahapanya, S., Morais, M. D. S., Rivas, D. A., Harris, S. S., Bischoff-Ferrari, H., ... & Dawson-Hughes, B. (2013). A randomized study on the effect of vitamin D3 supplementation on skeletal muscle morphology and vitamin D receptor concentration in older women. Journal of Clinical Endocrinology & Metabolism, jc-2013.
• Knutsen, K. V., Madar, A. A., Lagerløv, P., Brekke, M., Raastad, T., Stene, L. C., & Meyer, H. E. (2013). Does Vitamin D Improve Muscle Strength in Adults? A Randomized, Double-blind, Placebo-controlled Trial Among Ethnic Minorities in Norway. Journal of Clinical Endocrinology & Metabolism, jc-2013.
• Salles, J., Chanet, A., Giraudet, C., Patrac, V., Pierre, P., Jourdan, M., ... & Walrand, S. (2013). 1, 25 (OH) 2‐vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes. Molecular nutrition & food research.

Vitamin D Builds Muscle: 70% Reduction in Myostatin, 45% Increase in Myotube Size in 10 Days - So, What's the Catch? Plus: Where Could Retinoic Acid (Vitamin A) Figure In?

If rely on the results of the most recent study from Australia, the answer to the above question probably reads "Yes, to a certain degree it does".

It has been a while that a vitamin D study has made it into the SuppVersity news (see previous articles). The reason for that is simple. I am not interested in study no. 9235235 that discusses random associations of low vitamin D with whatever ailment is plaguing us or review no. 89359252 that presents a selection of papers and concludes: "Man, there are vitamin D receptors everywhere, so it must be the f*** most important vitamin in your body!" The upcoming publication of a paper in the scientific journal Endocrinology did yet appear to be a good reason to stop the vitamin D radio silence. It's an in vitro study, I know, but it could answer a question many of will be interested in.

Does vitamin D build muscle?

I guess all of you will tell me that, in view of the results of pertinent studies (cf. Girgis. 2013a), the answer is "no, it doesn't, but deficiency seems to hamper muscle growth and impair skeletal muscle function". This conclusion is hard to debate, especially in view of the fact that we don't even know what exactly vitamin D does in human muscle cells.

Exactly this, i.e. the question "what exactly happens, when muscle cells are exposed to vitamin D" must have been bother Girgis et al., too. Therefore they devised a very simple yet interesting in-vitro study in the course of which they treated C2C12 cells, which are a commonly used model (see bottom line for a comment on this) for human skeletal muscle with both, the active 1,25(OH)2D and inactive 25(OH)D form of 'vitamin D' and observed the effects on cell proliferation and growth.

Figure 1: Number of live cells (10^4/dish; middle) and images of the cells w/out & w/ 25(OH)D2 (Girgis. 2013b)

The first intriguing finding the scientists present in their paper is yet not related to the growth or proliferation of the cells, but to their ability to convert active into inactive vitamin D and vice versa. What we are talking about here, specifically, is the increased expression of CYP24A1. This enzyme is responsible for the 'deactivation' of active vitamin D into calcitriotic acid. This supposedly inactive metabolite (you never know with these vitamin Ds ;-) is then excreted in the urine. The reason that I mention this ostensibly unimportant observation is that the expression of CYP24A1 and CYP27B1, which will convert 25OHD into the active 1,25(OH)2D is evidence of the presence of an auto-regulatory vitamin D-endocrine system in muscle cells.

Ok, enough of the enzymes what about "getting big"?

Let's briefly forget about the mechanisms and return to the actual effects on growth and proliferation. Effects such as the 30-50% increases in G0/G1, a gene that's responsible for arresting the cell cycle, and the 30% and 20% decreases in Myc and Cyclin-D1 the scientists observed in response to both 25(OH)D and 1,25(OH)2D.

In view of the fact that these genes are necessary for the progression of the cell cycle, it is not surprising that the exposition to both forms of vitamin D brought the cycle to a screeching halt. In the end, this is yet a long-known phenomenon. The antiproliferative effects of 1,25(OH)2 D in muscle cells were first described in 1985 and are, as Girgis et al. point out, ...

"[...] they are consistent with antiproliferative effects of 1,25(OH)2 D in a number of other cells and tissues including skin, cancer cells and immune cells ." (Girgis. 2013b)

What's news though, is that the researchers were able to confirm that even 25OHD, the "prohormone" (Girgis. 2013b) to 25(OH)D2, displays antiproliferative effects in C2C12 cells.

Don't forget the "Underestimated Vitamin D Sources: Especially Eggs, But Also Chicken, Pork, Fish & Dairy Contain an Overlooked, Physiologically Relevant Amount of Ready-Made 25OHD" | read more

In that, it's important to acknowledge that these effects are not necessarily brought about by direct receptor interaction. They could also be mediated by the 'activation' of 25OHD via the previously mentioned CYP27B1. With CYP27B1 and its counterpart CYP24A1 the cells would thus be able to produce and clear active vitamin D on demand - and in this case the muscle cells were using it for anti-proliferative purposes.

"What? Vitamin D kills muscle growth?"

At first sight the cell-cycle arrest really suggest that high, and not the often cited low vitamin D levels should have anti-anabolic effects. Since muscle does not necessarily depend on proliferation, or more specifically cell devision, to grow this is yet not the case. At least up to a volume time-point your muscle cells and with them your total muscle volume can grow by simply taking up more protein. This process is called hypertrophy and it works quite nicely until a certain threshold is reached and myostatin pulls the emergency break (if you read my previous article "Getting Big Means Growing Beyond Temporary Physiological Limits" you will know that this is the point, when the activation and incorporation of satellite cells becomes important; learn more)

Figure 2: There may be less live cells, but once the cell cycle arrests, the cells that are bathed in serum with high amounts of acvite vitamin D 1,25(OH)2D grow like crazy, but probably only until they are 'ready to burst' (Girgis. 2013b)

Irrespective of all growth limits, it is thus no irreconcilable contradiction that the data in Figure 2 confirms that 'vitamin D builds muscle. Since proliferation and hypertrophy are independent (or rather mutually exclusive processes) the individual cell growth, while the total cell mass remains the same (remember: cell cycle arrest does not mean that the cell dies).

So, is this good or bad news? Whether the cell cycle arrest is a problem that could haunt you, in the long term, i.e. whence the limits of natural growth are reached (learn more) is something this study can't tell us, because...

... firstly, the cells the researchers used cells express proteins necessary for muscle contraction and display the morphology of individual fiber unit, but C2C12 cells are not adult muscle cells. With a varying degree of maturation, and mode (Langelaan. 2011) of glucose transport (Kotliar. 1992), even Girgis et al. have to admit that "effects in C2C12 cells do not always translate to adult muscle." (Girgis. 2013b) and ...

Figure 3: Primary C2 cells (chicken & mouse) were either untreated (A,C,E) or treated w/ 10 µM RA (B,D,F). A + B panels display satellite cells incubated w/ or w/out RA for 24 hr. C,D and E,F panels show satellite cells and C2 cells, respectively, after 48hr of incubation.

... sedondly, as with every in-vitro study, we cannot tell if the effects that are observed under direct exposition of cells to pharmacological doses of 1,25(OH)D will correspond with those of physiological levels of vitamin D - even high ones.

In the end, we are thus as clueless as before. Even if everything works as it does in the  model, the data in Figure 2 would suggest that after a couple of days of increased hypertrophy, the myostatin levels are identical and the D-advantage disappears.

When this 'growth limit' is reached it would require proliferative effects and new cells, or rather myonuclei, to grow further (learn more). With vitamin D alone, that's not going to happen. What could help though, is the villain of the average vitamin D enthusiast: Retinoic acid (RA) aka vitamin A. The latter has after all been shown to "induces adult muscle cell differentiation mediated by the retinoic acid receptor‐α", ten years ago (see Figure 2 from Halevy. 1993).

Now you tell me: Isn't it funny how we always end up with vitamin A (learn more), whenever we realize that 'vitamin D, without vitamin A' sucks? That cannot be mere coincidence, can it?

References:

• Girgis, Christian M., et al. "The roles of vitamin D in skeletal muscle: form, function, and metabolism." Endocrine reviews 34.1 (2013a): 33-83. 
• Girgis, Christian M., et al. "Vitamin D Signaling Regulates Proliferation, Differentiation and Myotube Size in C2C12 Skeletal Muscle Cells." Endocrinology (2013b): en-2013.
• Halevy, Orna, and Orna Lerman. "Retinoic acid induces adult muscle cell differentiation mediated by the retinoic acid receptor‐α." Journal of cellular physiology 154.3 (1993): 566-572.
• Kotliar, N., and P. F. Pilch. "Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells." Molecular Endocrinology 6.3 (1992): 337-345. 
• Langelaan, Marloes LP, et al. "Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells." Journal of tissue engineering and regenerative medicine 5.7 (2011): 529-539.