Supplementary Materials for · Wing Hoi Cheung, Jimmy Chun Yu Louie, Timothy M. Griffin, Wenyan Nong, Jerome Ho Lam Hui, Guan-hua Du, ... [atrogin and muscle RING-finger protein-1

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

Supplementary Materials for

Muscle-generated BDNF is a sexually dimorphic myokine that controls

metabolic flexibility

Xiuying Yang, Daniel Brobst, Wing Suen Chan, Margaret Chui Ling Tse, Oana Herlea-Pana, Palak Ahuja, Xinyi Bi, Aung Moe Zaw, Zara Sau Wa Kwong, Wei-hua Jia, Zhong-gou Zhang, Ning Zhang, Simon Kwoon Ho Chow,

Wing Hoi Cheung, Jimmy Chun Yu Louie, Timothy M. Griffin, Wenyan Nong, Jerome Ho Lam Hui, Guan-hua Du, Hye Lim Noh, Suchaorn Saengnipanthkul, Billy K. C. Chow, Jason K. Kim, Chi Wai Lee*, Chi Bun Chan*

*Corresponding author. Email: [email protected] (C.B.C.); [email protected] (C.W.L.)

Published 13 August 2019, Sci. Signal. 12, eaau1468 (2019)

DOI: 10.1126/scisignal.aau1468

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.

Fig. S1. Regulation of Bdnf expression and secretion. (A) Bdnf expression in the

gastrocnemius muscles of C57BL6/J mice (6-month-old; n = 6 mice per group). (B) Bdnf

expression in various muscles of C57BL6/J mice (6-month-old; n = 7–9 mice per group). (C)

BDNF content in the culture medium was measured by ELISA after culturing in glucose-free

(-Glc) medium for the indicated times; n = 3 independent experiments. (D) Immunoblotting

analysis of cellular BDNF content and secretion from C2C12 myotubes after glucose depletion

for the indicated times. The arrows indicate mature BDNF. Immunoblots are representative of

two independent experiments. (E) C2C12 myotubes were infected with control adenovirus or

shBdnf adenovirus for 48 h. BDNF content in the culture medium was assessed by ELISA (left

panel); n = 4 independent experiments. The efficiency of Bdnf knockdown was verified by

immunoblotting (right panel). Immunoblots are representative immunoblots of four independent

experiments. (F) C2C12 myotubes were cultured in medium containing the indicated

concentrations of cell permeable cAMP (sp-cAMP) for 24 h. Real-time PCR was performed to

determine Bdnf expression; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001;

Students t-test (A, B), two-way ANOVA (C, E), vs. 0 µM, one-way ANOVA (F).

Fig. S2. Effect of BDNF on cellular metabolism. (A) Non-mitochondrial respiration of C2C21

myotubes stimulated with the indicated concentrations of BDNF for 24 h as determined by

extracellular flux analyzer; n = 4 independent experiments. (B) ATP coupling efficiency of

BDNF-stimulated C2C12 myotubes as determined by extracellular flux analyzer; n = 4

independent experiments.

Fig. S3. Generation of MBKO mice. (A) Schematic representation of the mouse Bdnf gene

before and after targeted disruption. Exons are indicated by open rectangles and the coding

region is shaded. The locations of loxP sites are marked as solid triangles. (B) Expression of

Bdnf in various tissues of female Fl/Fl and MBKO mice (12-week-old) were determined by real-

time PCR. Results were normalized to endogenous β-actin in each tissue; n = 3 independent

experiments. (C) Phosphorylation and expression of TrkB in the gastrocnemius and cortex of

fasted female Fl/Fl and MBKO mice (12-week-old) were determined by immunoblotting. Each

lane represents a different mouse. Quantification of immunoblots is shown; n = 3 mice per

genotype. (D) Percentage of lean and fat mass of 28-week-old female Fl/Fl and MBKO mice; n =

6 mice per group. (E) Daily food intake of Fl/Fl and MBKO mice; n = 6 mice per genotype. *P <

0.05, ***P < 0.001; Students t-test.

Fig. S4. Metabolic characteristics of male MBKO mice. (A) Growth curve of male MBKO

mice fed a chow diet; n = 10 mice per genotype. (B) Body composition of male Fl/Fl and MBKO

(6-month-old; n = 5 mice per genotype). (C to H) Daily oxygen consumption (VO2) (C), daily

CO2 (VCO2) production (D), daily locomotion (E), daily energy expenditure (F), average RER

(G), and RER of 28-week-old male MBKO mice after fasting (24 h) (H). (I) Changes in total lean and fat mass of female Fl/Fl and MBKO mice after 24-h fasting; n = 5–6 mice per genotype. (J) Average cross-sectional area of myofibers from fed male Fl/Fl and MBKO mice (6-month-old; n = 5 mice per genotype). (K) Representative H&E staining of gastrocnemius muscle from fed or fasted male Fl/Fl and MBKO mice (6-month-old). Scale bar, 50 µm. n = 3 mice per genotype.

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

Gene expression analysis in (A) brown adipose tissues (BAT) and (B) white adipose tissues

(WAT) of fed female Fl/Fl and MBKO mice (6-month-old); n = 5 mice per genotype. (C) RER

of female Fl/Fl and MBKO mice after fasting as shown in Fig 3I. (D) Quantification of

immunoblots shown in Fig 3K; n = 3 mice per genotype. (E) Immunoblotting analysis of

gastrocnemius muscle from fed female Fl/Fl and MBKO mice (28-week-old). Each lane

represents a different mouse. Quantification is also shown; n = 3 mice per genotype. *P < 0.05,

**P < 0.01, ***P < 0.001; Student’s t-test.

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

Fgf21 expression in the gastrocnemius of fasted female Fl/Fl and MBKO mice; n = 9 mice per

genotype. (B) The expression of genes encoding fatty acid synthase (Fasn), the fatty acid

transporter CD36 (Cd36), adipocyte triglyceride lipase (Atgl), and monoglyceride lipase (Mgll)

were determined by real-time PCR in gastrocnemius muscles from fasted (24 h) female Fl/Fl and

MBKO mice (28-week-old). Results were normalized to β-actin; n = 4–5 mice per genotype. *P

< 0.05; Student’s t-test.

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

MBKO mice. (A) Total free AA content in 24-h fasted female Fl/Fl and MBKO gastrocnemius

muscle as determined by LC/MS; n = 5 mice per genotype. (B) Immunoblotting analysis of total

ubiquitination and atrophy-promoting ubiquitin E3 ligases [atrogin and muscle RING-finger

protein-1 (MuRF1)] in gastrocnemius muscle from female Fl/Fl and MBKO mice fasted for 24 h.

Tubulin was used as a loading control. Each lane represents a different mouse. (C)

Immunoblotting analysis of autophagy signaling in the extensor digitorum longus (EDL) and

tibialis anterior (TA) muscles from fasted (24 h) mice. Each lane represents a different mouse.

Quantification of immunoblots is shown; n=3 mice per genotype. *P < 0.05, **P < 0.01;

Student’s t-test.