01/27/2013 Brienna Larrick

Age-Related Changes in Skeletal Muscle Korhonen MT et al. J Appl Physiol (1985). 2006 Sep;101(3):906-17. Fiber composition of vastus lateralis in young (age 26) and old (age 71) runners. Progressive loss of muscle mass and strength with age, with an increase in skeletal muscle fat infiltration. Orange: Fat Infiltration Blue: High Density Muscle Green: Low Density Muscle The New York Academy of Sciences: Skeletal Muscle Power: a Critical Determinant of Physical Functioning in Older Adults

Vitamin D Deficiency is Associated with Changes in Skeletal Muscle Form and Function Muscle weakness, pain Decline in muscle function Changes in gait, inability to rise from a chair Increased occurrence of falls and fractures Thought to be due to: Loss of muscle fiber size; type II fibers Increased fat infiltration within muscle Origin of intramuscular adipocytes? Can be reversed with vitamin D supplementation  Effect of vitamin D supplementation unknown

Intramuscular Fat may Occur through Transdifferentiation of Myogenic Precursor Cells to Adipocytes Previous studies have demonstrated the ability of myogenic precursor cells to transdifferentiate towards the adipogenic lineage Does 1,25(OH)2D3 affect skeletal muscle transdifferentiation? Aim: To investigate the effect of a broad range of concentrations of 1,25(OH)2D3 on skeletal muscle differentiation or transdifferentiation. H0: Vitamin D deficiency (low 1,25(OH)2D3) stimulates skeletal muscle transdifferentiation towards an adipogenic lineage

C2C12 Myoblasts Model: Cultured for 6 Days 1,25(OH)2D3 0 (Vehicle) 0.0001 nM (Low Physiological) 0.01 nM (Low Physiological) 1 nM (High Physiological) 100 nM (Supraphysiological) 10,000 nM (Supraphysiological) Adipogenic Media Fetal Bovine Serum, Insulin, Isobutylmethylxanthine, T3, Dexamethasone, Rosiglitazone (PPARγ agonist) Myogenic Media Horse Serum 70-80% confluence 1,25(OH)2D3 0 (Vehicle) 0.0001 nM (Low Physiological) 0.01 nM (Low Physiological) 1 nM (High Physiological) 100 nM (Supraphysiological) 10,000 nM (Supraphysiological) Cultured for 6 Days

Effects of 1,25(OH)2D3 on Adipogenic Media-Stimulated C2C12 Lipid Droplet Formation Myogenic Media Adipogenic Media Fully differentiatied myotubes by day 6 Supraphysiological vitD inhibited myotube formation LD formed in adipogenic media Low vitD stimulated LD formation, while high physiological and supraphysiological inhibited it A/G = Vehicle; B-F/H-L = 1,25(OH)2D3 (0.0001-10,000 nM). Day 6 of differentiation.

Dose-Dependent 1,25(OH)2D3 Inhibition of C2C12 Lipid Droplet Formation in Adipogenic Media

Dose-Dependent Effects of 1,25(OH)2D3 on White Adipocyte Marker Genes (Adipocyte-specific)  (Detected in myogenic and adipogenic cell cultures; 1,25D effect in adipogenic media only) Data shown for C2C12 cells cultured in adipogenic media PPAR: peroxisome proliferator-activated receptor; FABP: fatty acid binding protein; AdipoQ: adiponectin

Dose-Dependent Effects of 1,25(OH)2D3 on Measure of Myogenic Differentiation -CK activity is reduced with adipogenic media, but activity is restored with 1,25(OH)2D3 treatment. -Supraphysiological 1,25(OH)2D3 treatment inhibited CK activity. -High protein and DNA in adipogenic media suggests increased proliferation Higher protein and DNA in adipogenic media indicative of increased proliferation, likely due to high FBS content. CK Activity: Quantitative measure of myogenic differentiation

Dose-Dependent Effects of 1,25(OH)2D3 on Markers of Myogenic Differentiation In myogenic media, high 1,25(OH)2D3 inhibited differentiation marker expression (data not shown). Differentiation marker expression was reduced in adipogenic media, but was increased by physiological 1,25(OH)2D3 treatment. Data shown for cells cultured in adipogenic media

No Effect of 1,25(OH)2D3 on Gene Markers of Brown Adipocytes Brown adipose markers Elovl3 and CIDEA were detected in cells cultured in adipogenic media on day 6. No effect of 1,25(OH)2D3 on brown adipocyte marker genes. Suggests 1,25D is not inducing conversion of myoblasts to brown adipocytes. However, longer treatment time might be needed. Data shown for cells cultured in adipogenic media. Expression of UCP1 and Prdm16 were undetectable.

Dose-Dependent Effects of 1,25(OH)2D3 on VDR, 1α-, 24-hydroxylase No effect of low or physiological 1,25(OH)2D3 on VDR, 24-hydroxylase, or 1α-hydroxylase expression. Supraphysiological 1,25(OH)2D3 induces VDR and 24-hydroxylase expression, possibly to minimize toxic effects of 1,25(OH)2D3. 1α-hydroxylase Data shown for cells cultured in adipogenic media

Conclusions 1,25(OH)2D3 modulates capacity of myogenic cells to transdifferentiate into adipocytes. In an adipogenic environment, low physiological 1,25(OH)2D3 concentrations (vitamin D-deficient state) induced adipogenesis, while high 1,25(OH)2D3 concentrations (physiological and supraphysiological) attenuated this effect.