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

Abstract

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.

Keywords: calorie allocation; energy sensing; growth; leptin; metabolism; myostatin; seasonal; vitamin D.

Figure 1. Visual Abstract: High dose dietary vitamin D preferentially allocates excess calories to muscle and growth instead of fat by increasing leptin production and sensitivity and decreasing myostatin production.

a) In the Conventional Model (left side) energy stores sensing is mediated by leptin and by default excess calories are stored as fat. b) in High dose Vitamin D (b, at bottom)). Our results below reveal a role for vitamin D in modulating energy balance sensing as well as calorie allocation. i)Vitamin D increases (+) leptin production and sensitivity (overall increasing leptin signaling (bold arrow)), and ii.) vitamin D decreases myostatin production (decreasing myostatin signaling (thin arrow)), leading to iii)increased energy expenditure, linear growth and improved fertility as well as iv) increased allocation of excess calories to muscle (bold arrow)

Figure 2. High dose vitamin D builds muscle without altering weight.

a. Wild-type mice were maintained on a defined vitamin D receptor knockout rescue (VDRKR) diet with each of different doses of vitamin D for 12 weeks and then the effects of each dose of vitamin D on strength, body composition and physiology were assessed. b. High-D improves grip strength (*** 0.001>p for each by Tukey post-tests) c. High-D improves lean mass (*= p<0.05 for each by Tukey post-tests). d. High-D decreases fat mass (*= p<0.05 for each by Tukey post-tests). e. High-D does not alter body weight. F. High-D shifts the curve of the relationship between weight and lean mass. In the linear relationship between lean mass and weight: weight gain decreases the proportion of lean mass and weight loss increases the proportion of lean mass. High-D shifts this curve vertically such that lean mass proportion increases for the same weight.

Figure 3. Increasing vitamin D inhibits myostatin production.

Wild-type mice were maintained on defined CHOW diets with different doses of vitamin D. At sacrifice serum was harvested and myostatin was measured by ELISA. a. Normal-D and high-D decrease myostatin concentrations vs no-D (*= p<0.05, for each ANOVA with Tukey post-tests). b.Normal-D and high-D inhibit myostatin production.

Figure 4. Increasing vitamin D increases leptin production and improves leptin sensitivity.

Wild-type mice were maintained on defined CHOW diets with different doses of vitamin D. At sacrifice leptin was measured by ELISA. a: Dietary vitamin D alters serum leptin concentration (*= p<0.05 for each ANOVA with Tukey post-tests). b: Dietary vitamin D alters serum leptin production per fat mass; the CHOW no-D line slope is different from both the reg-D and high-D lines (*= p<0.05 for each of Normal-D vs no-D, and High-D vs no-D by ANOVA with Tukey post-tests), all three correlations are significant: for no-D: r = 0.98 (F < 0.01), for normal-D: r = 0.96 (F < 0.01) and for high-D: r =0.85 (F < 0.05)). c. Normalizing vitamin D increased the amount of leptin produced per fat mass (red arrow in b, red text in c). Raising D from normal to high shifts the distribution of fat masses (blue arrow in b, blue text in c) consistent with increased leptin sensitivity.

Figure 5. Increasing vitamin D increases energy expenditure without altering intake.

Wild-type mice were maintained on defined CHOW diets with different doses of vitamin D. Metabolic cages were used to assay intake, energy expenditure and activity level over one week after acclimatization. a, b: High dose vitamin D did not alter weight adjusted intake over normal-dose vitamin D. c, d: High dose vitamin D significantly increased fat-free-mass adjusted 24-energy expenditure relative to normal-dose vitamin D. e: High dose vitamin D increases leptin production and sensitivity thus raising energy expenditure.

Figure 6. Increasing vitamin D within the normal range increases length.

c: Vitamin D deficiency caused a premature growth plateau in a single peri-menarchal female that was associated with low IGF-1 (Table 1). IGF-1 recovered with vitamin D supplementation. b, c: Wild-type mice were maintained on defined CHOW diets with different doses of vitamin D. Metabolic cages were used to assay intake, energy expenditure and activity level over one week after acclimatization. High dose vitamin D significantly increased length in wild type mice (a: nose to tail, b: nose to rump). d: Mendelian randomization reveals a significant relationship between genes that predispose to increased vitamin D and final height. e: Schema of how high-D allocates calories to growth.

Figure 7. Raising vitamin D to high-normal in zebrafish embryos increases length.

a: Zebrafish zygotes were injected with vitamin D at fertilization, matured for 5 days and measured, b: Typical 5 day embryos injected to achieve 25 ng/mL 25(OH)D, c: Typical 5 day embryos injected to achieve 50 ng/mL 25(OH)D, d: 50 ng/ml vitamin D significantly increased 5 day embryo length over 25 ng/ml by t-test (*** p < 0.001).

Figure 8. Energy balance sensing.

In the (Fig 1a above) energy stores are conveyed by leptin and by default excess calories are stored as fat. in this model, calorie intake can either be used (in energy expenditure) or is excess and thus stored as fat. Our results above (summarized in Fig 1b) reveal roles for vitamin D in modulating energy stores sensing via leptin (Fig 1b.i), energy needs sensing via myostatin (Fig 1b.ii) and calorie allocation to build muscle, for linear growth or for immediate use (Fig 1b.iii–iv). These novel roles give rise to this new model of Energy balance sensing. In this new model of there are two critical differences from the old model: first, anticipated energy needs (as conveyed by myostatin) play a critical role in relaying the sufficiency of energy stores, and, second, depending on the sufficiency of energy stores calorie intake will be allocated to linear growth, fertility or to build muscle.