High dose dietary vitamin D allocates surplus calories to muscle and growth instead of fat via modulation of myostatin and leptin signaling

Obesity occurs because the body stores surplus calories as fat rather than as muscle. Fat secretes a hormone, leptin, that modulates energy balance at the brain. Changes in fat mass are mirrored by changes in serum leptin. Elevated leptin prompts the brain to decrease appetite and increase energy expenditure. In obesity, however, impaired leptin sensitivity mutes these leptin-mediated changes. We have limited understanding of what controls leptin production by fat or leptin sensitivity in the brain. Muscle produces a hormone, myostatin, that plays a role in muscle analogous to the one that leptin plays in fat. Absent myostatin leads to increased muscle mass and strength. As with leptin, we also do not know what controls myostatin production or sensitivity. Although fat mass and muscle mass are closely linked, the interplay between leptin and myostatin remains obscure. Here we describe an interplay linked thru vitamin D. Conventionally, it is thought that vitamin D improves strength via trophic effects at the muscle. However, we find here that high dose dietary vitamin D allocates excess calories to muscle and linear growth instead of storage as fat. Vitamin D mediates this allocation by decreasing myostatin production and increasing leptin production and sensitivity. That is, high dose vitamin D improves integration of organismal energy balance. Obesity, aging and other chronic inflammatory diseases are associated with increased fat mass and decreased muscle mass and function (e.g. sarcopenia). Our work provides a physiologic framework for how high-dose vitamin D would increase allocation of calories to muscle instead of fat in these pathologies. Additionally, our work reveals a novel link between the myostatin and leptin signaling whereby myostatin conveys energy needs to modulate leptin effects on calorie allocation. This result provides evidence to update the conventional model of energy stores sensing to a new model of energy balance sensing. In our proposed model, integration of leptin and myostatin signaling allows control of body composition independent of weight. Furthermore, our work reveals how physiologic seasonal variation in vitamin D may be important in controlling season-specific metabolism and calorie allocation to fat in winter and muscle and growth in summer.


INTRODUCTION
Vitamin D signaling has been implicated in conveying nutrient status to the brain 1 .Vitamin D signaling is mediated primarily by activation of the vitamin D receptor (VDR) 2 .VDR knockout mice have a complex "failure-to-thrive" phenotype with abnormal serum calcium and phosphate, poor growth and nearlyabsent white fat 3 .Their failure to thrive occurs, in part, because they have decreased storage of lipids into white fat [3][4][5][6] .White fat produces the majority of circulating leptin.Thus, the lack of white fat in VDR knockout mice causes them to have persistently low leptin [3][4][5][6] .
In addition to modulation of leptin secretion and nutrient sensing, vitamin D signaling also plays a role in muscle function.Low vitamin D has long been known to cause muscle weakness that is relieved by replenishment of vitamin D. Animal models to date have primarily examined vitamin D signaling and effects through experimental models of de ciency.Speci cally, investigators have used approaches focusing on limitation of dietary vitamin D, or conventional or tissue speci c knockout of the VDR [7][8][9][10][11] .A number of clinical studies, however, suggest that increasing vitamin D within the normal range may have additional bene cial effects on muscle function [12][13][14][15][16] .
Scientists have largely examined these functions of vitamin D, regulation of fat metabolism and regulation of muscle mass and function, separately.However, fat mass and muscle mass are closely linked.Interventions that increase muscle mass increase both weight and fat mass.Conversely, when weight loss exceeds 10% of baseline weight, muscle mass loss increases proportionally to fat mass loss suggesting a complex relationship between fat and muscle mass.The mechanisms linking fat mass and muscle mass are poorly understood.The loss of muscle mass once weight loss exceeds 10% suggests that another checkpoint in energy balance may be energy needs.Myostatin has been conceptualized as having homeostatic effects on muscle mass.Our data suggests that myostatin may also work to convey energy needs.
Conventional VDR knockout mice have very low serum leptin and muscle speci c VDR knockout mice have excess production of myostatin mRNA 3,8 .We sought to examine the relationship between vitamin D signaling, fat mass, muscle mass, and muscle function.Further, we wanted to examine this relationship not only by comparing normal-to-low dietary vitamin D but also by comparing normal-to-high dietary vitamin D.
To evaluate the hypothesis that high-dose dietary vitamin D provides muscle function bene t beyond normal-dietary vitamin D, we sought to determine the effect of increasing vitamin D within the normal range (from 20-30 ng/dL to above 30 ng/dL) improved muscle function in adult wild-type mice.Normal dietary vitamin D increased strength over low-dose vitamin D without altering lean mass.High-dose dietary vitamin D signi cantly improved strength beyond normal vitamin D.Moreover, high-dose vitamin D also increased lean mass without affecting weight.That is, high-dose vitamin D effectively redistributed calories from fat to muscle.
Replenishing vitamin D from low-to-normal decreased serum myostatin and increased the amount of leptin produced per fat mass.High-dose vitamin D increased sensitivity to leptin without signi cantly affecting the amount of leptin produced per fat mass.This increased leptin sensitivity did not alter appetite but did increase energy expenditure.High-dose vitamin D also increased linear growth and lean mass proportion of weight in mice.Mendelian randomization revealed that vitamin D increases linear growth in humans, con rming the clinical importance of these ndings.We also found that high-dose vitamin D improved the length of early zebra sh, supporting the evolutionary signi cance of our ndings.
Thus, here we report for the rst time that high dose dietary vitamin D preferentially allocates excess calories to muscle and growth instead of storing them as fat by decreasing myostatin signaling and increasing leptin production and sensitivity.This result provides evidence to update the conventional model of energy sensing (Fig. 1a) to a new model of energy balance sensing (Fig. 1b) where integration of leptin and myostatin signaling allows control of calorie allocation.

High dose vitamin D signi cantly improves grip strength compared to normal vitamin D
To examine calciometabolic-independent effects of vitamin D on muscle, we used the vitamin D receptor knockout rescue diet (VDRKR diet) and varied vitamin D in wild-type (C57BL/6) male mice.We used three levels of added vitamin D in the diet: 0 IU/kg (no-D), 2000 IU/kg (normal-D) and 10,000 IU/kg (high-D).After 4 weeks, these diets allowed us to achieve target serum 25(OH)D of less than 5 ng/mL, between 20-30 ng/mL and above 30 ng/mL (experimental schematic in Fig. 2a).
To determine whether high-D improves muscle function in a dose-responsive fashion above the bottom of the normal range, we measured grip strength as previously described 17 .Normal-D signi cantly improved grip strength over no-D.High-D more dramatically increased grip strength over normal-D (Fig. 2b, ***p < 0.001 for each by Tukey's Post-hoc test).While the improvement in going from no-D to normal-D is expected, the more substantial improvement in going from normal-D to high-D is novel.

High dose vitamin D increases lean mass, but normal vitamin D does not
To examine whether the high-dose vitamin D stimulated increase in strength re ected changes in muscle mass, we measured mouse body composition via NMR as described previously 17 .Normal-D did not increase lean mass over low-D but high-D increased lean mass and decreased fat mass relative to normal-D without altering weight (Fig. 2c for lean mass, *: p < 0.05 by ANOVA with Tukey post-tests; Fig. 2d for fat mass, *: p < 0.05 by ANOVA with Tukey post-tests; Fig. 2e for weight, p > 0.05 by ANOVA).Thus, the high-dose vitamin D stimulated increase in strength is mediated in part by increased calorie allocation to build muscle mass instead of fat mass (physiologic schematic in Fig. 2f).

Increased vitamin D inhibits myostatin production
Recent work in muscle-speci c VDR knockout mice as well as in mouse models of vitamin D de ciency reveal that normal vitamin D concentrations facilitate VDR-dependent decreases in myostatin mRNA in muscle 11 .To examine the possibility that vitamin D dose alters circulating myostatin to regulate the proportion of muscle mass and fat mass, we measured serum myostatin in all three diet groups.Normal D decreased serum myostatin signi cantly relative to no-D (Fig. 3, no-D vs normal-D, p < 0.05, ANOVA with Tukey post-tests).High-D did not further alter serum myostatin relative to normal-D (Fig. 3, normal-D vs high-D, p > 0.05, ANOVA with Tukey post-tests).This rst decrease in myostatin (Fig. 3a) occurred without any change in fat free mass (Fig. 2c) suggesting that normal-D decreased average production of myostatin by muscle mass.The unchanged average myostatin in the high-D diet compared to normal-D occurred in the context of increased average lean mass (Fig. 2b), suggesting that high-D further decreased average myostatin production per muscle mass.Overall, these results are consistent with increasing doses of dietary vitamin D inhibiting myostatin production (Fig. 3b).

Normalizing vitamin D improves leptin generation per fat mass, but high-dose vitamin D increases leptin sensitivity
Vitamin D receptor de cient mice are hypoleptinemic and are largely de cient in white fat, the type of fat largely responsible for leptin secretion.To examine the possibility that vitamin D dose alters leptin signaling to regulate the proportion of muscle mass and fat mass, we measured serum leptin in all three diet groups of wild-type mice.Normal-D increased serum leptin signi cantly relative to no-D (Fig. 4a, normal-D vs no-D).High-D decreased leptin signi cantly relative to normal-D (Fig. 4a, high-D vs normal-D).To better understand how this complex pattern related to leptin production and sensitivity, we examined the relationship between serum leptin, fat mass, and vitamin D treatment group using linear regression.Within a vitamin D treatment group, total fat mass determined serum leptin (Fig. 4b, for no-D r = 0.98 (F < 0.01 for slope being signi cantly different than zero), for normal-D r = 0.96 (F < 0.01 for slope being signi cantly different than zero) and for no-D r = 0.85 (F < 0.05, for slope being signi cantly different than zero)).However the slope of this relationship was different between groups (* p < 0.05 for normal-D and high-D vs no-D by ANOVA with Tukey post-tests).This graph reveals how the relationship between leptin and fat mass shifted signi cantly and meaningfully between groups (Graph in Fig. 4b, schematic in Fig. 4c).The slopes in this graph (Fig. 4b) are representative of leptin produced per fat mass.Raising vitamin D from low to normal signi cantly increased the slope of the curve for leptin vs fat mass (red arrow in Fig. 4b, red lettering in Fig. 4c): that is, normalizing vitamin D increased the amount of leptin produced per fat mass (red arrow in Fig. 4b).Raising vitamin D from normal-D to high-D did not further change the slope of this relationship.However, increasing vitamin D from normal-D vs high-D shifted the distribution on this line (blue arrow in Fig. 4b, blue lettering in Fig. 4c).That is, further raising vitamin D from normal to high did not alter the slope of the curve for leptin vs fat mass but instead shifted the distribution of fat mass down the curve.This is a shift consistent with high dose vitamin D increasing sensitivity to leptin.

High dose dietary vitamin D signi cantly increases energy expenditure without altering activity level or intake
Previously, another group reported that acute paraventricular hypothalamic injection of 1,25D (the active form of vitamin D) decreases appetite in mice 1 .They interpreted this result to indicate that 1,25D increased leptin sensitivity 1 .While some actions of vitamin D may be acute, most actions of vitamin D are mediated by slower transcriptional changes.Thus, it is not clear that this acute response represents a physiologic effect of vitamin D. Furthermore, leptin has actions on both appetite and energy expenditure, but leptin action is generally thought to alter energy expenditure more than appetite.We wanted to better understand the apparent increase in leptin sensitivity described above (Fig. 4) due to high-dose dietary vitamin D. To further examine this relationship, we measured intake and energy expenditure.
High dose dietary vitamin D had no effect on mass-adjusted intake (Fig. 5a and Fig. 5b, unadjusted intake was also not signi cantly different between groups).High dose dietary vitamin D had no effect on activity level (data not shown).However, high dose dietary vitamin D signi cantly increased fat free mass adjusted energy expenditure (Fig. 5c and 5d, * p < 0.05 by t-test).Raw-energy expenditure (unadjusted for fat free mass) was also signi cantly different between groups (* p < 0.05 by t-test, data not shown)) without altering activity level.Thus high-dose dietary vitamin D signals to differentially allocate calories for use by muscle instead of storage as fat without altering weight by increasing leptin secretion and leptin sensitivity.

Dietary vitamin D rescues growth factor de cit in the clinic
We replenished dietary vitamin D for 2 months (50000 IU weekly for 4 weeks and 4000 IU/d subsequently), and we repeated these labs (Table 1).As we expected, repleting vitamin D normalized serum 25(OH)D and calcium.Surprisingly, repleting vitamin D improved IGF-1 to just above the middle of the normal range for age (z-score 0.05).The initially normal prealbumin and albumin indicated that this young adolescent had appropriate overall energy and protein nutrition and that her low IGF-1 at that time was not related to calorie or protein-calorie availability.In the context of our other data supporting the model that vitamin D conveys energy stores centrally to modulate calorie allocation (Fig. 5e), this result, novelly, suggests that vitamin D also conveys nutrient availability to enable growth.

High dose dietary vitamin D signi cantly increases linear growth in mice
Our results in mice to this point suggested to us a model where myostatin was not simply playing a homeostatic role at muscle alone, but was also conveying nutrient needs centrally (Fig. 1b for overall model).That is, myostatin conveys not how much energy is being used but what the energy needs are likely to be.If we use the metaphor of the muscles as an engine then myostatin conveys not just how much gas the engine is consuming minute to minute, but how large the engine is.This model would predict that the high-D mediated inhibition of myostatin and increase in leptin signaling would not only increase energy expenditure but would also facilitate increased growth.To examine this possibility, we measured length in anesthetized mice from our diet groups.As we hypothesized based on this model, high-D increased nose-to-tail and nose-to-rump length in mice (Fig. 6b-c; p < 0.05 for each by t-test).This result is consistent with vitamin D facilitating increased central energy sensing across modalities (e.g. for allocation to muscle as well as for use in linear growth).

A genetic predisposition to higher Vitamin D increases nal height in humans
Based on our results in mice, and the interesting result we observed in clinic in an adolescent with early rickets, we theorized that vitamin D might play a similar role in humans to facilitate energy sensing.To examine the possibility that vitamin D might modulate height by regulating calorie allocation in a clinically signi cant way we wanted to look whether vitamin D in uenced height in humans.There are a variety of recent studies that attempt to address this question 18,19 .Interpretation of this work is bedeviled by issues around selection of treatment group (age, vitamin D status, season, etc) as well as duration and dose of treatment.One approach that we have used in other contexts to avoid these issues is Mendelian randomization 20 .We used the most recent genome wide association study (GWAS) to generate instrumental variables for serum 25(OH)D 21 .We applied these results to the most recent and comprehensive GWAS of height 22 .Using this approach we found that our instrumental variables identi ed signi cant positive relationships between SNPs that are associated with increased vitamin D and height; (Fig. 6d: Manousakis beta = 0.19, P = 2.09E-3).This result supports the notion that 25(OH)D may in uence nal height via conveying energy status.
Raising vitamin D from normal to high-normal in zebra sh embryos increases length Based on our intriguing results in mice and in humans, we wondered if the role vitamin D plays to convey nutrient status re ects that calories are more plentiful in late summer and fall when vitamin D is generated and stored.This model would predict that the role of vitamin D to convey nutrient status would be evolutionarily conserved in non-mammalian species.To address this hypothesis, we measured growth in zebra sh over the rst ve days of life (schematic in Fig. 7a) when dosed with normal (25 ng/mL -four typical 5 day old larvae in Fig. 7b) compared with high-normal (50 ng/mL -four typical 5 day old zygotes in Fig. 7c) concentrations of 25(OH)D.We found that high-normal 25(OH)D signi cantly increased length in 5 day old zebra sh relative to normal (Fig. 7d: 50 ng/mL vs 25 ng/mL **** p < 0.0001 by unpaired t-test, n = 20 for each treatment).Other researchers have previously described that developmental vitamin D de ciency increases fat storage and decreases leanness and length relative to normal vitamin D in zebra sh at older time points.Similar to our results in mice, this result suggests that there are metabolic and growth bene ts to raising vitamin D to high-normal relative to low-normal.Overall, these results provide support for the model that 25(OH)D is signi cantly associated with growth via conveying energy status.

DISCUSSION
Replenishing vitamin D to normal decreased myostatin production, but further increases of vitamin D did not alter serum myostatin (Fig. 3).Measuring leptin concentrations across dietary groups revealed that replenishment of vitamin D to normal increased the amount of leptin produced per fat mass while highdose vitamin D increased sensitivity to leptin (Fig. 4).Consistent with the large majority of work on leptin actions, this increased leptin sensitivity did not alter appetite, but did increase fat free mass adjusted energy expenditure (Fig. 5).Thus, here we report for the rst time that high dose dietary vitamin D preferentially allocates excess calories to build muscle instead of be stored as fat by increasing leptin production and sensitivity and decreasing myostatin signaling.
In addition, we nd that the effect of high-dose vitamin D to increases linear growth (Fig. 6).Using mendelian randomzation we found that the genes that predispose to incrased serum vitamin D (25D) also increase nal height.This result con rms the clinical signi ciance of our work in humans, supporting the model that vitamin D conveys nutrient availability.
We also found that high dose vitamin D increases growth in early zebra sh.This effect con rms the evolutionary importance of our work and lends further backing to the model vitamin D conveys nutrient availability.
Strengths of this work include 1) a multidimensional assessment of strength and muscle function simultaneous with metabolism and fat mass, 2) an examination of the effects of low, normal and highnormal vitamin D muscle function and body composition in the context of healthy (e.g.lean) mice, 3) con rmation of the clinical signi cance of our ndings using mendelian randomization and nally, 4) con rmation of the evolutionary signi cance of our ndings using zebra sh.A weakness of this work is that we have not identi ed each of the mechanistic steps in how vitamin D decreases myostatin signaling and increases leptin production and sensitivity.
Our results summarized in Table 2 at left give rise to our proposed new models for vitamin D action (Fig. 1b) and for energy balance sensing (Fig. 8, as opposed to energy stores sensing (Fig. 1a)).In our proposed model raising vitamin D from low to normal increases leptin production by fat, and further raising serum vitamin D levels from normal to high-normal concentrations increases leptin sensitivity (Fig. 1b.i.).Concurrent with these effects, raising vitamin D decreases myostatin signaling (Fig. 1b.ii.).Overall, these changes increase allocation of excess calories to muscle mass (Fig. 1b.iv.) and to linear growth (Fig. 1b.iii.)rather the storage of excess calories as fat.
Notably, these ndings for vitamin D give rise to a new model for understanding energy balance (Fig. 8 a model of energy balance sensing rather than energy stores sensing).In this new model of Energy balance sensing there are two critical differences from the old model of energy stores sensing (Fig. 1a); rst, anticipated energy needs, as conveyed by myostatin, play a critical role in relaying the su ciency of energy stores; and, second, in addition to being used as energy expenditure or stored as fat, calorie intake will be allocated also to linear growth, fertility or to build muscle.
A variety of common pathologies including obesity, aging and diseases with chronic in ammation or wasting are associated with decreased muscle function and mass (e.g.sarcopenia).Furthermore, many of these pathologies are associated with low circulating vitamin D because of decreased conversion of calciferols (D2 and D3) to calcidiol 23,24 .Attempts to increase and bene t from an increase in serum Vitamin D have had mixed results in the context of aging, but, recent work in pediatric diseases with chronic in ammation including sickle cell and congenital-HIV identify signi cant improvements in muscle function with high-dose vitamin D 25,26 .In many diseases with chronic in ammation, weight gain is a proxy for increased health.However, careful assessment of how this weight gain is allocated often reveals that increases in lean mass as opposed to weight in and of itself improve functional measures of disease (e.g.measures of pulmonary function in cystic brosis) 27 .Thus, our work provides a physiologic framework for how high-dose vitamin D may be used in these contexts to increase allocation of excess calories to muscle instead of storage as fat.A great deal of work has examined vitamin D effects on weight and BMI with overall equivocal results.There is a small body of prior work suggesting that high dose vitamin D may improve body composition without altering weight or BMI [28][29][30][31] .
Several groups have studied leptin as a potential therapeutic for obesity.However, aside from the notable exceptional success in leptin-de cient lipodystrophies, leptin has been largely ineffective in combating obesity in clinical trials 32,33 .The failure of exogenous leptin to effectively treat obesity may re ect that leptin signaling in obesity is past the dynamic portion of the leptin response curve.Nonetheless, the exploration ways to increase leptin signaling or alter leptin sensitivity has been relatively scarce.Our work here provides evidence for the possibility that manipulation of leptin sensitivity can address obesity, and also introduces vitamin D as an initial target for this manipulation.Another possible reason for the failure of exogenous leptin alone as a therapy is that leptin signaling may be only one of several checkpoints in energy balance.The body's catabolism of muscle once weight loss exceeds 10% suggests that another checkpoint in energy balance may be energy needs.Previously, myostatin has been thought to have homeostatic effects on muscle mass.Our work suggests that myostatin may have an additional role in conveying energy needs centrally.Thus, our work further suggests that addressing energy need signaling by manipulating myostatin pathways can be used to preserve or increase muscle mass in the context of weight loss.
Aside from revealing the role of vitamin D in the interplay between fat and muscle mass, our results are deeply meaningful in at least two other respects.First, these data provide a context for understanding other vitamin D effects beyond its well-recognized function in calcium homeostasis.Viewed through the lens of metabolism, this model provides a chronic or sub-chronic signaling pathway to begin to understand metabolism.This model is a departure from energy stores signaling pathways such as insulin, ghrelin or leptin which convey nutrient status that changes on a day to day or week to week basis.High-dose dietary vitamin D alters the set points of the leptin and myostatin pathways to convey season-speci c nutrient availability and use expectations.With this long term signaling that we have identi ed, calories from a large feast in the winter (when vitamin D is at its nadir) will not be used to increase muscle mass or for a growth spurt but would instead be stored in fat as metabolic insurance against the possibility of scarcity for the remainder of the winter.By contrast, this model would predict that calories from a feast in the late summer or early fall (when vitamin D is peaking) are more likely to be used to improve muscle mass, strength and linear growth.Second, our work reveals how physiologic seasonal variation in vitamin D may be important in controlling season-speci c growth patterns.Humans have long been known to grow more in the summer and fall than the spring and winter.This pattern has been ascribed to historic nutrient availability.However, this pattern has persisted in the developed world where nutrient availability is stable throughout the year.
Another possible season speci c effect may be on fertility: vitamin D improves fertility in females with polycystic ovarian syndrome (PCOS).PCOS is a disease with abnormal or dysfunctional energy balance signaling.Vitamin D also improves fertility in males.In both of these contexts, vitamin D may also facilitate season-speci c fertility.
Overall our work identi es a novel role for vitamin D in nutrient sensing, nutrient needs sensing and calorie allocation (Fig. 1b).This role reveals novel physiology underlying the interplay between fat and muscle and gives rise to a new paradigm for understanding and exploiting control of body composition and calorie allocation (Fig. 8).

Statistics
Animals were randomized to treatment group by cage.Experimenters were blinded in all experiments to the treatment group of each mouse or zebra sh.All values are presented as Mean ± SEM.Differences were considered statistically signi cant if p < 0.05 (*) or F < 0.05 (*).For three treatment experiments (e.g.comparisons of low-D, normal-D and high-D) means were compared using a one-way ANOVA with Tukey's correction for post-tests.For two sample experiments (e.g.comparisons of normal-D vs high-D) a two tailed t-test was used.

Mouse diets and husbandry
All animal work was reviewed and approved by the Institutional Animal Care and Use Committee of the Children's Hospital of Philadelphia (Protocol # 0988).To examine calciometabolic independent effects of vitamin D on muscle we used the vitamin D receptor knockout rescue diet (VDRKR diet) and varied vitamin D. We used three levels of vitamin D in the context of de ned CHOW VDRKR diets: 0 IU/kg (low -ENVIGO #140078), 2000 IU/kd (normal -ENVIGO #140079) and 10,000 IU/kg (high -ENVIGO #140080).After 4 weeks these diets allowed us to achieve target serum 25(OH)D of less than 5 ng/mL, between 20-30 ng/mL and above 30 ng/mL.The serum calcium, phosphate and PTH were within the normal range in all three groups and were not different between groups.The 1,25(OH)D was undetectable in all three groups.Twelve-week-old male wild-type (WT) C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were housed (n = 5 per cage) under a 12:12-h light-dark cycle (light on at 0700) and an ambient temperature of 22°C, and allowed free access to water and diet.Food intake was measured weekly, and body composition was assessed 12 weeks later with nuclear magnetic resonance (NMR) (Echo Medical Systems, Houston, TX).

Grip strength
Mouse grip strength was measured at the University of Pennsylvania Penn Muscle Institute muscle core with a grip meter (TSE; Bad Hamburg, Germany) as described previously 17 .Brie y, mice were trained to grasp a horizontal metal bar while being pulled by their tail and the force was detected by a sensor.Ten measurements were determined for each mouse and averaged.
MN performed and conceptualized some experiments, acquired data, and performed initial analysis of data.
LD performed and conceptualized some experiments, acquired data, and performed initial analysis of data.
ML performed and conceptualized some experiments, acquired data, and performed initial analysis of data.
JM revised the manuscript and approved the nal manuscript as submitted.
JR conceptualized and designed the study, drafted the outline of the initial manuscript, and approved the nal manuscript as submitted.
Figures     ii) and calorie allocation to build muscle, for linear growth or for immediate use (Fig

Figure 1 Visual
Figure 1

Figure 2 High
Figure 2