Study of the adipose-muscle crosstalk in in vitro and ex vivo models

A growing body of evidence indicates that white adipose tissue (AT) can have detrimental effects on skeletal muscle integrity and function. Hypothesis: senescent and dysfunctional adipocytes impair skeletal muscle structure and regenerative capacity via paracrine signaling. Specific aims: (1) in vitro to test whether conditioned media (CM) from mature, aged, insulin-resistant or oxidative stress–exposed 3T3-L1 adipocytes alter C2C12 myotube morphology or/and differentiation, atrophy markers, protein synthesis and myostatin; (2) ex vivo to see whether adipocytes derived from elderly human SAT show impaired adipogenic features and a pro-inflammatory signature and whether their CM cause atrophic and anti-myogenic changes in human myotubes and finally to compare key histological features in muscle and adipose tissues from elderly versus control patients. In the present study, we first set up an in vitro model using conditioned culture medium obtained from cultures of mature, aged, insulin-resistant and oxidative stress-exposed 3T3-L1 murine adipocytes to treat C2C12 myocytes. Morphological analysis of C2C12 cells indicated a significant decrease in myotube diameter and fusion index in myotubes exposed to medium from aged or stressed adipocytes. Furthermore, we demonstrated a significant reduction in gene expression of myogenic marker MyHC-IIb and an increase in atrophy-related factor MuRF1 in myocytes treated with medium from aged or stressed adipocytes compared to controls. We also observed a significant decrease in protein synthesis and a significant increase in myostatin expression and protein levels in C2C12 cells treated with medium from aged or stressed adipocytes compared to controls. To explore whether similar phenomena occur under more physiological conditions, we extended our investigation to ex vivo human samples. Subcutaneous adipose tissues from elderly patients were isolated and differentiated into adipocytes before their conditioned medium was applied to human myotubes. Adipocytes derived from these samples showed a reduced expression of key adipogenic factors (PPARγ, C/EBP-α) and showed a trend toward higher pro-inflammatory mediators (TNF-α, IL-6) as well as variable leptin levels. Conditioned medium from these adipocytes was then used to treat primary human myotubes of a control patient. We observed increased Myostatin levels, decreased GLUT4 expression and lower Myf5 mRNA levels in myocytes exposed to conditioned medium obtained from elderly patients compared to those exposed to medium from control patient. These results are consistent with the induction of muscle atrophy and reduced myogenic potential. Furthermore, we observed a mild atrophy with a mixed atrophic pattern, moderate intracellular lipid accumulation and mild adipose infiltration in gluteus medius muscle biopsies from one of the two elderly patients from whom we previously cultured adipocytes , while biopsies from younger control subject, also the one from whom we cultured both adipocytes and myocytes didn’t show any sign of atrophy or significant adipose infiltration. These findings support the idea that senescent or dysfunctional adipocytes could negatively impact muscle structure and function in vivo. Taken together, these findings suggest that senescent or dysfunctional adipocytes can negatively affect muscle trophism and regenerative potential through paracrine signals. Further studies are needed to determine whether therapeutic targeting of aged AT by senolytic compounds or other interventions can improve age-related muscle deterioration and metabolic decline.