Conditioned media from AICAR-treated skeletal muscle cells increases neuronal differentiation of adult neural progenitor cells

Abstract

Exercise has profound benefits for brain function in animals and humans. In rodents, voluntary wheel running increases the production of new neurons and upregulates neurotrophin levels in the hippocampus, as well as improving synaptic plasticity, memory function and mood. The underlying cellular mechanisms, however, remain unresolved. Recent research indicates that peripheral organs such as skeletal muscle, liver and adipose tissue secrete factors during physical activity that may influence neuronal function. Here we used an in vitro cell assay and proteomic analysis to investigate the effects of proteins secreted from skeletal muscle cells on adult hippocampal neural progenitor cell (aNPC) differentiation. We also sought to identify the relevant molecules driving these effects. Specifically, we treated rat L6 skeletal muscle cells with the AMP-kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or vehicle (distilled water). We then collected the conditioned media (CM) and fractionated it using high-performance liquid chromatography (HPLC). Treatment of aNPCs with a specific fraction of the AICAR-CM upregulated expression of doublecortin (DCX) and Tuj1, markers of immature neurons. Proteomic analysis of this fraction identified proteins known to be involved in energy metabolism, cell migration, adhesion and neurogenesis. Culturing differentiating aNPCs in the presence of one of the factors, glycolytic enzyme glucose-6-phosphate isomerase (GPI), or AICAR-CM, increased the proportion of neuronal (Tuj1⁺) and astrocytic, glial fibrillary acidic protein (GFAP⁺) cells. Our study provides further evidence that proteins secreted from skeletal muscle cells may serve as a critical communication link to the brain through factors that enhance neural differentiation.

Keywords: AICAR; AMPK; Doublecortin; Exercise; HPLC; Muscle; Myokines; Neurogenesis.

Fig. 1.

Schematic model of experiments and HPLC chromatogram results (A) Flow chart shows collection and analysis of CM. (B,C) HPLC chromatograms show that samples from (B) vehicle-treated control and (C) AICAR-treated conditioned medium have distinct retention time of proteins in 215 nm UV wavelength.

Fig. 2.

Effect of AICAR- or vehicle-treated Conditioned Medium on aNPCs. (A) MTT analysis of the reconstituted fractions from vehicle (V3, V4) or AICAR (A9, A11) conditioned medium (10 μg/μl, respectively) on aNPCs for 24 h. Fetal Bovine Serum (FBS), was used as a positive control. Same volume of distilled water was used as a vehicle (Veh). The FBS (10%) treated samples were significantly different from all other groups (*P < 0.05). (B–D) Real-time PCR analysis of neuronal cell differentiation makers (Tuj1, DCX, NeuN), with administration of reconstituted fractions from vehicle or AICAR treated culture medium (10 μg/μl, respectively) or not treated (NT), on aNPCs for 24 h (B) Tuj1 levels in A11 treated samples were higher than in all other groups. (C) DCX was elevated in the cells treated with fraction A11 as compared to V3, V4, A9 and NT. (D) NeuN levels were enhanced by FBS (0.5%) as compared to all other conditions. Data is expressed as relative target gene expression levels compared with HSP90 expression and presented as the mean of at least three independent experiments, each conducted in triplicate (*P < 0.05).

Fig. 3.

Proteomic analysis of Conditioned Medium. (A) Number of differentially secreted proteins identified by LC/MS in CM fraction A11 from vehicle treated CM. (B) Table of unique proteins identified in AICAR-CM fraction A11. Proteins highlighted in green indicate peptide coverage greater than 5%. (C) Kegg pathway analysis describing the most common pathways represented by proteins secreted from AICAR-treated CM and relative number of proteins in each pathway.

Fig. 4.

Candidate factor glucose-6-phosphate isomerase (GPI) increases neural differentiation. (A) GPI (0.5 μg/ml and 1 μg/ml), AICAR CM (A-CM) and RA/FSK significantly increased the proportion of aNPCs differentiating into Tuj1-positive neurons as compared to Mock and Vehicle CM (V-CM) treatment conditions (*p < 0.05). (B) GPI (1 μg/ml) and AICAR CM also enhanced gliogenesis as evidenced by an increased number of GFAP-positive astrocytes as compared to Mock and Vehicle CM treatment (*p < 0.05). (C,E,G) Representative photomicrographs of (C) V-CM, (E) A-CM, and (G) GPI treated aNPCs expressing neuronal marker Tuj1 (red), nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), blue. (D,F,H) Representative photomicrographs of (D) V-CM, (F) A-CM, and (H) GPI treated cells expressing astroglial marker GFAP (red), nuclei were stained with DAPI (blue).