Monday, December 2, 2013

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?

  • 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.