Research ArticleMetabolism

Muscle-generated BDNF is a sexually dimorphic myokine that controls metabolic flexibility

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Science Signaling  13 Aug 2019:
Vol. 12, Issue 594, eaau1468
DOI: 10.1126/scisignal.aau1468
  • Fig. 1 Fasting induces Bdnf expression in glycolytic muscle through CREB signaling.

    (A) Real-time PCR of Bdnf, Creb, and Ppargc1a (which encodes PGC-1α) expression in various mouse tissues after fasting (24 hours); n = 4 to 5 mice per group. *P < 0.05, ***P < 0.001 compared to fed; Student’s t test. (B) Circulating BDNF concentration in wild-type mice after fasting for various time intervals; n = 5 to 6 mice per group. ***P < 0.001 compared to 0 hours of the same sex; one-way ANOVA. (C) Bdnf expression in C2C12 myotubes after glucose depletion for various time intervals; n = 4 independent experiments. *P < 0.05, ***P < 0.001 compared to 0 hours; one-way ANOVA. (D) Real-time PCR of Bdnf expression in C2C12 myotubes stimulated with different concentrations of forskolin for 24 hours; n = 4 independent experiments. ***P < 0.001 compared to 0 μM; one-way ANOVA. (E) Real-time PCR (top) of Bdnf expression in C2C12 myotubes transfected with empty vector (control) or FLAG-tagged CREB (Flag-CREB) and cultured in glucose-free medium (24 hours). The abundance of FLAG-CREB and tubulin was determined by immunoblotting (IB) (middle and bottom); n = 3 independent experiments. **P < 0.01, ***P < 0.01; two-way ANOVA. Mw, molecular weight. (F) C2C12 myotubes were cultured in glucose-free medium for the indicated time points and immunoblotted for phosphorylated CREB and AMPK and total CREB, AMPKα, and tubulin. The immunoblots are representative of three independent experiments. Quantification is shown in the right panel. *P < 0.05 compared to 0 hours; one-way ANOVA. (G) Wild-type mice were fasted for 24 hours. Gastrocnemius muscles were collected, and the DNA binding activity of CREB was examined by ELISA; n = 4 mice per group. *P < 0.05; Student’s t test. (H) Genomic DNA and associated proteins were collected from C2C12 myotubes cultured in normal or glucose-free medium for 24 hours. ChIP assay was performed using control immunoglobulin G (Ctr IgG) or anti-CREB, and the associated Bdnf promoter was detected using real-time PCR; n = 4 independent experiments. **P < 0.01; Student’s t test. Rel, relative; IP, immunoprecipitation; p, phosphorylated; Ctr, control.

  • Fig. 2 BDNF increases mitochondrial content, cellular respiration, and lipid oxidation in muscle cells.

    (A) Palmitic (PA) oxidation in C2C12 myotubes treated with the indicated concentrations of BDNF for 24 hours was measured with an extracellular flux analyzer; n = 4 independent experiments. **P < 0.01 compared to 0 ng/ml; one-way ANOVA. (B) Immunoblotting of the mitochondrial protein content in C2C12 myotubes treated with BDNF (100 ng/ml) for the indicated time points. Immunoblots are representative of two independent experiments. (C) Real-time PCR of mitochondrial DNA in C2C12 myotubes treated with BDNF (100 ng/ml, 24 hours). Results were normalized to genomic DNA content; n = 3 independent experiments. *P < 0.05; Student’s t test. PBS, phosphate-buffered saline. (D) OCR traces for BDNF-stimulated (24 hours) C2C12 myotubes; n = 4 independent experiments. Oligo, oligomycin; Rot, rotenone. (E) Basal respiration, (F) maximal respiration, and (G) ATP production of C2C12 myotubes treated with different concentrations of BDNF (10 to 100 ng/ml) for 24 hours; n = 4 independent experiments. *P < 0.05, **P < 0.01 compared to 0 ng/ml; one-way ANOVA. (H) Analysis of various signaling pathways in C2C12 myotubes stimulated by BDNF (100 ng/ml) for the indicated time points. The phosphorylated and total abundance of the indicated proteins was determined by immunoblotting. Immunoblots are representative of three independent experiments. Quantification is shown in the right panel; n = 3 experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to 0 hours; one-way ANOVA.

  • Fig. 3 Deficiency in muscle-derived BDNF impairs systemic energy metabolism.

    (A) Growth curve of female Fl/Fl and MBKO mice fed a chow diet; n = 10 mice per group. ***P < 0.01; two-way repeated-measures ANOVA. (B) Total lean and fat mass of 28-week-old female Fl/Fl and MBKO mice; n = 6 mice per group. *P < 0.01; Student’s t test. (C) Adipocyte size in inguinal WAT collected from 28-week-old female Fl/Fl and MBKO mice; n = 5 to 6 mice per group. ***P < 0.01; Student’s t test. (D to H) Daily oxygen consumption (VO2) (D), daily CO2 production (VCO2) (E), daily energy expenditure (F), daily locomotion (G), and average RER (H) of 28-week-old female MBKO mice; n = 6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test. (I) RER of 28-week-old female Fl/Fl and MBKO mice after fasting (24 hours); n = 5 mice per group. **P < 0.01; Student’s t test. (J) FAO in gastrocnemius muscle isolated from 10-month-old female Fl/Fl and MBKO mice that had been fed or fasted for 16 hours; n = 4 mice per group. *P < 0.05, **P < 0.01; two-way ANOVA. (K) Metabolic signaling in female Fl/Fl and MBKO (28-week-old) gastrocnemius muscle after fasting (24 hours) as determined by immunoblotting. Each lane represents a different mouse. (L) Real-time PCR of FAO genes in the gastrocnemius muscle of fasted (24 hours) female MBKO (28-week-old) mice; n = 4 mice per group. *P < 0.05, **P < 0.01; Student’s t test. AcAA1a, encodes acetyl-CoA acyltransferase 1a; AcAA2, encodes acetyl-CoA acyltransferase 2; Acadl, encodes acyl-CoA dehydrogenase, long chain; Acadm, encodes acyl-CoA dehydrogenase, medium chain; Acads, encodes acyl-CoA dehydrogenase, short chain; Acadsb, encodes acyl-CoA dehydrogenase, short/branched chain; Acadvl, encodes acyl-CoA dehydrogenase, very long chain; Cpt1b, encodes carnitine palmitoyltransferase 1b; Cpt1b, encodes carnitine palmitoyltransferase 2; Echs1, encodes enoyl Co-A hydratase, short chain 1; Hadha, encodes enoyl-CoA hydratase; Lipe, encodes hormone-sensitive lipase; Lpl, encodes lipoprotein lipase.

  • Fig. 4 The skeletal muscle of MBKO mice has reduced energy content but has higher lipid metabolite accumulation.

    (A) Total triglyceride (TG), free fatty acid (FFA), and acylcarnitine content in the gastrocnemius and soleus muscles of female MBKO mice as determined by LC-MS; n = 4 mice per group. (B) Metabolites of glycogenolysis, glycolysis, and tricarboxylic acid (TCA) cycle in the gastrocnemius muscle of female Fl/Fl and MBKO mice after 24-hour fasting; n = 4 to 5 mice per group. Glc, glucose; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; 3PG, 3-phosphoglycerate; Pyr, pyruvate; Cit, citrate; Isoc, isocitrate; Suc, succinate; Fur, fumarate; Mal, malate; Lac, lactate. (C) Glycogen content in the gastrocnemius muscle of female Fl/Fl and MBKO mice after fasting; n = 6 to 7 mice per group. (D) Expression of glycogenolytic and glycolytic genes in the gastrocnemius muscle of female Fl/Fl and MBKO mice after 24-hour fasting as determined by RNA sequencing; n = 4 mice per group. Hk1, encodes hexokinase 1; Gpi1, encodes glucose phosphate isomerase 1; Pgam1, encodes phosphoglycerate mutase 1; Pgk1, encodes phosphoglycerate kinase 1; Pyg1, encodes glycogen phosphrylase 1; Pgm1, encodes phosphoglucomutase 1. (E) AA content in the gastrocnemius muscle from female Fl/Fl and MBKO mice fasted for 24 hours; n = 5 mice per group. (F) ATP content in the gastrocnemius muscle of female MBKO mice after 24-hour fasting; n = 5 mice per group. (G) Summary of the biochemical pathways of glucose and lipid metabolism affected by Bdnf ablation in the gastrocnemius during fasting. Up-regulated metabolites or genes are indicated in red, and the down-regulated metabolites or genes are indicated in blue. *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t test (A, B, and D to F) and two-way ANOVA (C).

  • Fig. 5 Diminished fasting-induced autophagy in MBKO muscle.

    (A) Changes in total lean and fat mass of female Fl/Fl and MBKO mice after 24-hour fasting; n = 10 to 12 mice per group. (B) Autophagy signaling in the gastrocnemius muscle of female Fl/Fl and MBKO mice after 24 hours of fasting as determined by immunoblotting. Each lane represents a different mouse. Quantification is also shown; n = 3 mice per group. (C) Representative LC3 immunofluorescent images of gastrocnemius muscle sections from fasted (24 hours) female Fl/Fl and MBKO mice (top). Scale bar, 50 μm. Magnified images of the selected regions (yellow boxes) are shown in the bottom. n = 3 mice per group. DAPI, 4′,6-diamidino-2-phenylindole. (D) Representative H&E staining of gastrocnemius muscle sections from fed or fasted female Fl/Fl and MBKO mice. Centronuclear myopathy (yellow asterisks), necrosis (black arrowheads), myositis (black arrows), and focal invasion of non-necrotic muscle fibers by inflammatory cells (yellow arrowhead) are indicated. Scale bar, 50 μm. n = 3 mice per group. (E) Total muscle strength of fed female Fl/Fl and MBKO mice (6 months old) as determined by the grip strength test; n = 6 mice per group. (F) The specific tetanic force, stiffness (SF0/SFt), specific twitch force, and fatigue rate of gastrocnemius muscles isolated from fed female Fl/Fl and MBKO mice (6 months old) as determined by ex vivo functional assessment; n = 6 mice per group. (G) Total time and distance run by fed female Fl/Fl and MBKO mice (6 months old on a treadmill; n = 6 mice per group. *P < 0.05, **P < 0.01; Student’s t test.

  • Fig. 6 MBKO mice display muscle-specific insulin resistance.

    (A) Glucose tolerance test (GTT) of 5-month-old female Fl/Fl and MBKO mice fasted for 16 hours; n = 6 mice per group. (B) Area under curve (AUC) for the GTT shown in Fig. 6A; n = 6 mice per group. (C) Glucose infusion rate for 16-hour fasted female Fl/Fl and MBKO (28-week-old) mice during hyperinsulinemic-euglycemic clamping; n = 6 to 8 mice per group. (D) Whole-body glucose turnover in female Fl/Fl and MBKO (28-week-old) mice during hyperinsulinemic-euglycemic clamping; n = 6 to 8 mice per group. (E) Insulin-stimulated glucose uptake by the gastrocnemius muscle of female Fl/Fl and MBKO (28-week-old) mice during hyperinsulinemic-euglycemic clamping; n = 6 mice per group. (F) Insulin-stimulated glucose uptake by WAT of female Fl/Fl and MBKO (28-week-old) mice during hyperinsulinemic-euglycemic clamping; n = 6 mice per group. (G) Insulin-stimulated signaling in gastrocnemius muscle isolated from 16-hour fasted female Fl/Fl and MBKO mice (6 months old). Each lane represents a different mouse. Quantification is shown in the right panels; n = 3 mice per group. (H) Differentiated C2C12 myotubes infected for 48 hours with control adenovirus (Ad-Ctr) or adenovirus expressing shBdnf (ad-shBdnf) were stimulated with human insulin (100 nM, 30 min) and immunoblotted as indicated. Each lane represents a different set of cells. (I) Hepatic glucose production (HGP) of Fl/Fl and MBKO (28-week-old) mice during hyperinsulinemic-euglycemic clamping; n = 6 mice per group. (J) Hepatic glycogen content in fed or 24-hour fasted female Fl/Fl and MBKO mice (6 months old); n = 4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001; two-way repeated-measures ANOVA (A), Student’s t test (B to E), and two-way ANOVA (G and J).

  • Table 1 Blood metabolite analysis of MBKO mice.

    Sera were collected from mice (6 months old; n = 4 to 8 mice per group) fed ad libitum or fasted for 24 hours. Results are expressed as the means ± SEM. CK, creatine kinase; FFA, free fatty acid; TG, triglyceride.

    FedFasted
    Fl/FlMBKOFl/FlMBKO
    Glucose (mg/dl)128.4 ± 8.2140.7 ± 6.892.1 ± 3.8b119.9 ± 10.2*
    Insulin (ng/ml)0.63 ± 0.080.88 ± 0.080.35 ± 0.06a0.74 ± 0.09*
    TG (mM)1.03 ± 0.081.51 ± 0.20*3.46 ± 0.54c5.90 ± 0.71*, c
    FFA (mM)0.70 ± 0.081.04 ± 0.161.09 ± 0.251.63 ± 0.14a
    BDNF (ng/ml)5.49 ± 2.833.61 ± 0.9342.46 ± 5.42c14.69 ± 5.64*
    Alanine (nM)
    CK activity (nmol/min per
    milliliter)
    0.33 ± 0.02
    39.36 ± 20.27
    0.30 ± 0.02
    53.19 ± 9.74
    0.67 ± 0.07b
    37.10 ± 18.32
    0.39 ± 0.01**,b
    98.47 ± 1.05**,b

    *P < 0.05 compared to Fl/Fl mice of the same treatment group, Student’s t test.

    **P < 0.01 compared to Fl/Fl mice of the same treatment group, Student’s t test.

    ***P < 0.001 compared to Fl/Fl mice of the same treatment group, Student’s t test.

    aP < 0.05 compared to the same genotype, Student’s t test.

    bP < 0.01 compared to the same genotype, Student’s t test.

    cP < 0.001 compared to the same genotype, Student’s t test.

    Supplementary Materials

    • stke.sciencemag.org/cgi/content/full/12/594/eaau1468/DC1

      Fig. S1. Regulation of Bdnf expression and secretion.

      Fig. S2. Effect of BDNF on cellular metabolism.

      Fig. S3. Generation of MBKO mice.

      Fig. S4. Metabolic characteristics of male MBKO mice.

      Fig. S5. Analysis of β-oxidation in gastrocnemius muscle from female MBKO mice.

      Fig. S6. Gene expression analysis in gastrocnemius muscle from female MBKO mice.

      Fig. S7. The ubiquitin-proteasome system is normal in the gastrocnemius muscle of female MBKO mice.

    • This PDF file includes:

      • Fig. S1. Regulation of Bdnf expression and secretion.
      • Fig. S2. Effect of BDNF on cellular metabolism.
      • Fig. S3. Generation of MBKO mice.
      • Fig. S4. Metabolic characteristics of male MBKO mice.
      • Fig. S5. Analysis of β-oxidation in gastrocnemius muscle from female MBKO mice.
      • Fig. S6. Gene expression analysis in gastrocnemius muscle from female MBKO mice.
      • Fig. S7. The ubiquitin-proteasome system is normal in the gastrocnemius muscle of female MBKO mice.

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