Study Probes Muscle Building Effects of Vitamin D in Young and Old and Finds None, but Relative Strength in Old and Fiber Composition & Myostatin in Young Muscle Respond

Old or young, who is going to benefit and who is going to benefit most from vitamin D supplementation during a 12-week resistance training regimen. That was the question the study at hand sought to answer and this was a question it didn't find an unambiguous answer to.

Ok, I have to admit, I could have kept up the suspense by not giving away the main result of Jakob Agergaard's and colleagues' latest study in the headline, already. On the other hand, by giving away the most relevant information in the headline, I can make sure that future google searchers will immediately refute the claim that "vitamin D is a powerful muscle builder" - it is not. What it may very well be, is a vitamin that is necessary for your long-term success.

This is still much different from what you may conclude solely based on the associations that exist between low vitamin D and all sorts of ailments, though. Evidence that vitamin D(3) supplements are able to reduce the risk of bone fractures, diabetes, cardiovascular diseases, cancer, depression, osteoarthritis, multiple sclerosis, and other immune related diseases is still preliminary. Very unfortunate in view of big research dollars that have been spend without yielding D-finite results and hundreds of more or less practically useless observational studies.

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I was thus very happy to see that the scientists from the University of Copenhagen did not content themselves with correlating the individual gains of their young and old subjects with the corresponding vitamin D levels. Instead, they designed a randomized controlled trial in which they "investigate[d] whether vitamin-D intake during 12 weeks of resistance training has an additive effect on muscle hypertrophy and strength"  (Agergaard. 2015) in Healthy, sedentary young (aged 20–30 years) and elderly (aged 60–75 years) Caucasian men living within the local community in Copenhagen:

"We hypothesized that intake of vitamin-D plus calcium would improve the outcome of three months of resistance training in healthy untrained individuals resulting in greater muscle strength and hypertrophy compared to a training control-group supplemented with calcium alone (placebo). Moreover, we hypothesized that resistance exercise would increase the mRNA expression of VDR and CYP27B1. The study included a group of young and a group of elderly individuals to elucidate a possible blunted hypertrophic response in the aging muscle"  (Agergaard. 2015).

The study took place at Bispebjerg Hospital, Copenhagen, Denmark (latitude of 56°N). Inclusion was continuous from November 17, 2010 to December 21, 2010 and the last subject completed the study on April 25, 2011. Thus, the study was conducted in a period of low UVB irradiation from sunlight. The risk of interference by uncontrolled sun-exposure was thus low. About as low as I suppose some of you will say the supplementation dose was. The latter consisted of either

• placebo supplementation with 800mg of calcium per day, or
• vitamin D + calcium at a dosage of 48µg (1920 IU) vitamin-D 3 + 800 mg calcium/day

that was administered in two servings, with one tablet containing 10 μg vitamin-D 3  + 400 mg calcium and one tablet containing 38 μg vitamin-D 3  + 400 mg calcium and had to be taken with meals (this increases absorption | learn more).

You're too lazy to read and want some extra-information, also on the topic of fat cell cellularity, obesity and body weight regain (yoyo effect?) - Download yesterday's installment of Super Human Radio and listen to my interview an add-free version right here!

The scientists probably would have dosed higher, but since the maximum advisable daily dose according to the Danish Health and Medicines Authority is 50 μg, i.e. 2000 IU, they probably felt that their hands were tied.

Figure 1: Flowchart showing a young and b elderly subjects from first contact to end of study (Agergaard. 2015)

All subjects who had been randomly (double-blind) assigned to the respective group had to follow the same standardized workout routine consisting of a total of 36 training sessions (12 weeks with 3 sessions/week) with 5–10 min warm-up on cycle ergometers followed by resistance training exercises of the lower extremities (only!) performed in commercial knee extension and leg press devices (Technogym, Super Executive Line, Gambettola, Italy) in each session. All sessions were supervised. Progressive loading levels were monitored continuously and adjusted throughout the entire training period to maintain muscle loading at the intended values.

• During the first 6 training sessions, participants completed 3 sets of 12–15 repetitions at 65–70 % of 1RM. 
• During session 7–12, participants performed 3 sets of 10–12 repetitions at 70–75 % of 1RM, increasing to 4 sets at 70–75 % of 1RM during session 13–18. 
• From session 19 and onwards, participants performed 5 sets with training load progressing from 8–10 repetitions at 75–80 % of 1RM in session 19–27 to 6–8 repetitions at 80–85 % of 1RM in session 28–36 [38]. 

The exercises were performed in a moderate slow, controlled manner with 1–2 s in the concentric- and eccentric phase with a rest of 1–3 min between sets. Exercise compliance (sets, repetitions, and load) was calculated from daily exercise records completed by the instructors at each training session. Participants were informed that a mean attendance of less than 2 training sessions per week resulted in exclusion. All adverse events associated with the training intervention were recorded.

The complex ways in which vitamin D supplements interact with both the levels of the active form of vitamin D 1,25(OH)2D and the binding proteins vitamin D binding protein and serum album has yet not been considered in any of the "vitamin D and gains" studies - epic fail ? (data from Glendenning. 2015)

Vitamin D Binding Protein, Bioavailable Vitamin D & Receptor Polymorphisms - Although it has been known for decades that only 0.1% of the vitamin D in our body and only ~10% of the metabolites in our blood are free, the effects of being bound to its specific binding protein (VDBP) or albumin are still largely unknown. One of the reasons is that studies still rely on unreliable measurements of total vitamin D that are then run through algorithms to elucidate if there's a difference between the effects of free and bound vitamin D (Chun. 2014). This is not only problematic because it assumes that we'd all have the same / similar amounts of vitamin D binding protein, but also because it ignores already established genetic polymorphisms (e.g. inter-racial / whites are more likely to have a low binding affinity than blacks) in how VDBP works and how it affects our health and is affected by supplementing with vitamin D (sign. increases are seen w/ vit D2 or D3 | see Fig.).

A similar negligence can be observed with regard to the role of the vitamin D receptors on its various target organs. While we know that their expression increases with resistance training (no added increase was observed with vitamin D supplementation in the study at hand in contrast to a recent study by Makanae et al. (2015) in rodents), we still have almost no clue how they interact with free and bound vitamin D; and only recently researchers like Jia et al. (2015) have begun to investigate how certain vitamin D receptor polymorphisms (gene types) like the rs739837 gene are associated with increased risk of T2DM. In conjunction with the role of genetic polymorphisms of the binding proteins, the whole system is at the moment thus way too too complex for us to make predictions on a population or even sub-population levels (like the elderly, men and women at an increased risk of cancer, or patients with autoimmune diseases, or athletes). 

The outcome variables the scientists choose were skeletal muscle hypertrophy, isometric muscle strength, serum vitamin D levels, and a muscle biopsy that was used (a) to analyze several markers of muscle hypertrophy, metabolism & co, as well as (b) to determine whether training or treatment had triggered measurable or even significant changes in the fiber type composition of the subjects.

Figure 2: Serum vitamin D levels at all time-points during the study (I added the markups for the zones to the original figure from Agergaard to make it easier for you to interpret the data).

Of these, I deliberately chose the 25OHD serum levels to start with. Why? Well if you look at the small increase in the young subjects and the still existing gap between their 25OHD (=serum vitamin D) levels and the allegedly "optimal" zone for lower body strength gains (cf. Bischoff-Ferrari. 2006), you may feel reassured that the dosages were too low. This is yet only the case, if the goal was to get the levels into the "magic" 90-100nmol/L of which Bischoff-Ferrari estimated in 2006 that it was optimal for muscle function and health. Whether the effects would have been more pronounced if the subjects had reached this level is yet mere speculation and, if you look at the correlation analysis further down, even highly unlikely (see Figure 5 and respective explanations).

Figure 3: Cross sectional area (CSA), Isometric strength and strength/CSA of Quadriceps muscle. Change in a CSA, b isometric strength and c strength/CSA of quadriceps muscle for young and elderly vitamin-D and placebo groups, respectively. Data shown as mean percentage change from week 0 ± SEM. * different from week 0 (p < 0.05)

Now, as arbitrary as these ranges may be (things like the influence of the vitamin D binding protein levels and genotypes for example, are taken into account, at all | Chun. 2014; Koplin. 2015), we must not and will not ignore the fact that the young, unlike the old(er) subjects, didn't make it into 90-100 nmol/L zone of "magic gains" when we are looking at the data in Figure 3:

• No group effects - The first thing you should realize is that there were no significant inter-group differences and thus no group effects in response to the provision of vitamin D3 vs. placebo. This does imply that neither the increased size gains (A) in the vitamin D group in the young nor the decreased gains in the vitamin D group in the old subjects was statistically significant. The same can be said, albeit in the opposite directions for the strength increases (B) and the relative strength increases (C) in the young subjects.
• Significant time effects - Since subjects in both groups still gained significant amounts of muscle and strength, the one thing the study does confirm is the efficacy of resistance training as strength and mass builder in young and old.
• Significant group effect on relative strength in the elderly - Due to the reversal of the observations compared to the young group (lower size gains + higher strength gains in the older, higher size + lower strength gains in the younger subjects), the relative strength of the older subjects has improved by vitamin D supplementation (p = 0.008, not correctly indicated in Figure 3) - a result that stands in line with previous research like Moreira-Pfrimer et al. (2009) where the provision of 150,000 IU once a month during the first 2 months, followed by 90,000 IU once a month for another 4 months enhanced both, the 25(OH)D levels and the lower limb muscle strength of the > or =60 year old subjects, even in the absence of any regular physical exercise practice.

Now, I would be inclined to ignore the lack of statistical significance for the initially mentioned parameters and jump on the significant increase in the older subjects and the trends we may extrapolate from the rest of the data, if it were not for the results of the extra correlation analysis the scientists did. If higher levels of vitamin D3 (90-100nmol/L as they were achieved in the older subjects) could, as Bischoff-Ferrari et al. assume based on observations Guralnik, et al. (1995) and Seeman et al. made in elderly individuals, ameliorate exercise-induced strength gains in the young subjects, there should at least be a correlation between vitamin 25OHD levels and muscle size and strength similar to the one Bischoff-Ferrari et al. report for the 8-foot-walk and sit-to-stand test:

Figure 4: The optimal ranges, Bischoff-Ferrari et al. estimated are based on the above observational data from a 8-foot-walk and sit-to-stand test done in the elderly. That's super reliable and just like you, right? No? Well, that's why I believe those "optimal values" have no relevance for the young and low relevance for the old subjects (Bischoff-Ferrari. 2006)

If the trends you may believe to see in Figure 3 a-c remained trends, because the 25OHD levels didn't rise high enough, the graphs in Figure 5 would look much different: They would firstly show increasing, not no or decreasing slopes and would second of all provide evidence for a practically relevant correlation between the 25OHD levels, the muscle size, strength and relative strength.

Figure 5: Correlation between Quadriceps ΔCSA, ΔIsometric strength, Δstrength/CSA and 25(OH)D (Agergaard. 2015)

In practice, however, the correlation analysis yielded nothing: No correlation between 25OHD and size gains (A), no correlation between 25OHD and strength gains (B), and no correlation between 25OHD and relative strength gains (C). While this does not neglect the possibility that the vitamin D supplement still affected the increase in strength/size ratio of the elderly, the result warrants the conclusion that there was "[n]o additive effect of vitamin-D intake during 12 weeks of resistance training [...] on either whole muscle hypertrophy or muscle strength" (Agergaard. 2015).

So vitamin D supplementation is finally disproven? It is not just the specific study population (unhealthy individuals or athletes may benefit more, men and women may differ (Ko. 2015) etc.) that precludes making overgeneralized conclusions such as "vitamin D supplementation doesn't do anything for your gains". There is more! Firstly, there is the increase in what the scientists call "muscle quality", i.e. the ratio of strength/size increases in the elderly. Now, the data in Figure 5 indicates that this is clearly not a function of the serum 25OHD levels. If that's not the case, however, it could only be mediated by vitamin D3 directly or metabolites that haven't been tested in the study at hand (most prominently active vitamin D, i.e. 1,25-dihydroxycholecalciferol aka calcitriol). If that's the case, age may explain that the older subjects did not see the same changes in fiber type morphology (greater increase in type IIa) and myostatin expression the young ones did.

Figure 6: Significant treatment specific changes in fiber type (%), i.e. increases in fast-twitch type IIa fibers and decreases of the protein synthesis inhibitor myostatin were observed only in younger subjects (Agergaard. 2015).

I highlighted these changes with arrows in Figure 6 and would like to point out that they are the most interesting reason to still supplement w/ vitamin D. Eventually, both effects could affect your gains in the long-term: (I) lower myostatin = higher protein synthesis; (II) more type IIa fibers = higher growth potential. In only 12-weeks, however, newbies don't reach a level where myostatin and / or the fiber composition of their muscle is holding them back, significantly. For athlete and after longer training periods, however, the scientifically proven (albeit in vitro | Garcia. 2013, 2014)  ability of active vitamin D aka calcitriol (and / or vitamin D3 directly - not proven in human muscle) to increase the myogenic differentiation (would explain myofiber changes) and suppress myostatin in human myoblasts could turn out to be game changers.

To find out whether these purported long-term effects exist and / or if similar effects can be seen in non-sedentary adults, like athletes who would benefit the most of reduced myostatin levels and further changes in the muscle architecture, we do yet need more studies. Randomized controlled studies, maybe with different dosing schemes (the ~2,000 IU are not exactly much, if we consider potential direct effects) and no more observational bogus on vitamin D | Comment on Facebook!

References:

• Agergaard, Jakob, et al. "Does vitamin-D intake during resistance training improve the skeletal muscle hypertrophic and strength response in young and elderly men?–a randomized controlled trial." Nutrition & metabolism 12.1 (2015): 32.
• Bischoff-Ferrari, Heike A., et al. "Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes." The American journal of clinical nutrition 84.1 (2006): 18-28.
• Chun, Rene F., et al. "Vitamin D and DBP: the free hormone hypothesis revisited." The Journal of steroid biochemistry and molecular biology 144 (2014): 132-137.
• Garcia, Leah A., et al. "1, 25 (OH) 2 vitamin D 3 enhances myogenic differentiation by modulating the expression of key angiogenic growth factors and angiogenic inhibitors in C 2 C 12 skeletal muscle cells." The Journal of steroid biochemistry and molecular biology 133 (2013): 1-11.
• Garcia, Leah A., et al. "1, 25 (OH) 2vitamin D3 stimulates myogenic differentiation by inhibiting cell proliferation and modulating the expression of promyogenic growth factors and myostatin in C2C12 skeletal muscle cells." Endocrinology 152.8 (2011): 2976-2986.
• Glendenning, Paul, et al. "Calculated free and bioavailable vitamin D metabolite concentrations in vitamin D-deficient hip fracture patients after supplementation with cholecalciferol and ergocalciferol." Bone 56.2 (2013): 271-275.
• Guralnik, Jack M., et al. "Lower-extremity function in persons over the age of 70 years as a predictor of subsequent disability." New England Journal of Medicine 332.9 (1995): 556-562.
• Jia et al. "Vitamin D Receptor Genetic Polymorphism Is Significantly Associated with Risk of Type 2 Diabetes Mellitus in Chinese Han Population." Arch Med Res. (2015): Ahead of print. 
• Ko, Min Jung, et al. "Relation of serum 25-hydroxyvitamin D status with skeletal muscle mass by sex and age group among Korean adults." British Journal of Nutrition (2015): 1-7.
• Koplin, Jennifer J., et al. "Polymorphisms affecting vitamin D–binding protein modify the relationship between serum vitamin D (25 [OH] D 3) and food allergy." Journal of Allergy and Clinical Immunology (2015).
• Makanae, Yuhei, et al. "Acute bout of resistance exercise increases vitamin D receptor protein expression in rat skeletal muscle." Experimental physiology 100.10 (2015): 1168-1176.
• Moreira-Pfrimer, Linda DF, et al. "Treatment of vitamin D deficiency increases lower limb muscle strength in institutionalized older people independently of regular physical activity: a randomized double-blind controlled trial." Annals of Nutrition and Metabolism 54.4 (2009): 291-300.

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