We hypothesized that skeletal muscle contraction produces a cellular stress signal, triggering adipose tissue lipolysis to sustain fuel availability during exercise. The present study aimed at identifying exercise-regulated myokines, also known as exerkines, able to promote lipolysis. Human primary myotubes from lean healthy volunteers were submitted to electrical pulse stimulation (EPS) to mimic either acute intense or chronic moderate exercise. Conditioned media (CM) experiments with human adipocytes were performed. CM and human plasma samples were analyzed using unbiased proteomic screening and/or ELISA. Real-time qPCR was performed in cultured myotubes and muscle biopsy samples. CM from both acute intense and chronic moderate exercise increased basal lipolysis in human adipocytes. Growth and differentiation factor 15 (GDF15) gene expression and secretion increased rapidly upon skeletal muscle contraction. GDF15 protein was upregulated in CM from both acute and chronic exercise–stimulated myotubes. We further showed that physiological concentrations of recombinant GDF15 protein increased lipolysis in human adipose tissue, while blocking GDF15 with a neutralizing antibody abrogated EPS CM-mediated lipolysis. We herein provide the first evidence to our knowledge that GDF15 is a potentially novel exerkine produced by skeletal muscle contraction and able to target human adipose tissue to promote lipolysis.
Claire Laurens, Anisha Parmar, Enda Murphy, Deborah Carper, Benjamin Lair, Pauline Maes, Julie Vion, Nathalie Boulet, Coralie Fontaine, Marie Marquès, Dominique Larrouy, Isabelle Harant, Claire Thalamas, Emilie Montastier, Sylvie Caspar-Bauguil, Virginie Bourlier, Geneviève Tavernier, Jean-Louis Grolleau, Anne Bouloumié, Dominique Langin, Nathalie Viguerie, Fabrice Bertile, Stéphane Blanc, Isabelle de Glisezinski, Donal O’Gorman, Cedric Moro
Submitter: Logan Townsend | ltownsen@uoguelph.ca
Authors: Logan Townsend and David C. Wright
University of Guelph
Published March 27, 2020
A recent paper by Laurens et al. found that exercise and electrical stimulation promotes the secretion of GDF15 from skeletal muscle which can subsequently activate lipolysis in white adipose tissue (1). There are, however, some important issues that the authors did not discuss in sufficient detail that we believe should be highlighted.
First, using blood samples taken from the femoral artery and vein, it was shown that moderate-intensity exercise increases circulating GDF15, but that skeletal muscle is not a source of GDF15 (2). Another report using a similar EPS protocol in human primary myotubes showed that GDF15 is not responsive to contraction (3). Given these data, whether skeletal muscle secretes meaningful quantities of GDF15 in response to exercise is still in question. Indeed, GDF15 expression is much lower in muscle compared to other tissues (4).
Second, the conclusion that GDF15 stimulates adipose tissue lipolysis is difficult to reconcile with what is known regarding the expression of GFRAL, the receptor for GDF15 (5). Despite detectable gene expression, GFRAL protein content is undetectable in adipose tissue (6). Thus, it remains to be determined how GDF15 would signal to adipose tissue. One possibility, perhaps related to the supra-physiological concentrations of GDF15 used in their adipocyte cell culture model, is that GDF15 is engaging non-canonical signaling pathways. A consideration related to this is that recombinant sources of GDF15 have been reported to be contaminated with TGF beta (7).
Regardless, supra-physiological concentrations of recombinant GDF15 (1 or 100 ng/mL), elicited very modest (~5%) increases in lipolysis. Even if statistically significant, the physiological relevance of this is unclear as the concentration of GDF15 that was used was at least an order of magnitude higher than what is seen in the circulation following exercise.
In a key experiment, authors found that conditioned media from electrically stimulated skeletal muscle cells increased lipolysis and this was prevented with a GDF15 neutralizing antibody. It is surprising that in this case conditioned media increased glycerol release ~5-fold, a far greater response than even 100 ng/mL GDF15. This could suggest that another contraction-inducible myokine could be stimulating lipolysis, or the neutralizing antibody could be having non-specific effects on lipolysis.
Thus, what is not clear from the current data, is the degree to which skeletal muscle contributes to circulating GDF15 concentrations with exercise in vivo and to what extent this could contribute to exercise-induced increases in adipose tissue lipolysis.
1. Laurens C et al. Growth and Differentiation Factor 15 is secreted by skeletal muscle during exercise and promotes lipolysis in humans. JCI Insight [published online ahead of print: February 27, 2020]; doi:10.1172/jci.insight.131870
2. Kleinert M et al. Exercise increases circulating GDF15 in humans. Mol Metab 2018;9:187–191.
3. Raschke S, Eckardt K, Bjørklund Holven K, Jensen J, Eckel J. Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells. PLoS ONE 2013;8(4):e62008.
4. Campderrós L et al. Brown Adipocytes Secrete GDF15 in Response to Thermogenic Activation. Obesity (Silver Spring) 2019;27(10):1606–1616.
5. Mullican SE et al. GFRAL is the receptor for GDF15 and the ligand promotes weight loss in mice and nonhuman primates. Nature Medicine 2017;23(10):1150–1157.
6. Yang L et al. GFRAL is the receptor for GDF15 and is required for the anti-obesity effects of the ligand. Nature Medicine 2017;23(10):1158–1166.
7. Olsen OE, Skjærvik A, Størdal BF, Sundan A, Holien T. TGF-β contamination of purified recombinant GDF15. PLoS ONE 2017;12(11):e0187349.
Submitter: Cedric Moro | cedric.moro@inserm.fr
Authors: Claire Laurens, Fabrice Bertile, and Cedric Moro
Inserm, UMR1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse, France.
Published March 27, 2020
We appreciate the interest and criticisms of Townsend and Wright on our study (1). They raise some important points and limitations but also probably overlooked a number of experimental evidences pointing toward a potential systemic metabolic role of GDF15, particularly in adipose tissue (AT).
RNA-seq gene expression profile across 11 selected tissues of the Non-Human Primates Reference Transcriptome Resource indicates that skeletal muscle is the fifth organ mostly expressing GDF15 transcript in humans (2). In our study, GDF15 was detected by nanoLC-MS/MS through 12 different peptides and further quantified by ELISA in conditioned media. Raschke et al. (3) used a biased method of human cytokine array with single epitope detection and a different EPS protocol which may explain some of the discrepancies. Thus, GDF15 produced during exercise by skeletal muscle contraction could locally crosstalk with intramuscular adipose tissue to provide fuels.
First, subcutaneous AT is the highest site of GFRAL expression according to GTEx RNAseq public databases in humans, which contrasts with the absence of detectable levels of Gfral in mouse AT in agreement with several studies (1, 4, 5). There is currently no study reporting that GFRAL is a pseudogene. However, we cannot exclude that GDF15 binds to another hitherto unknown cognate receptor. This could explain some of the strong experimental evidences showing that GDF15 has profound systemic metabolic effects in mice independently of significant changes in energy intake as outlined in the studies below.
It was shown that mice ubiquitously expressing hGDF15 are protected from high fat diet-induced obesity and glucose intolerance by increasing thermogenesis and lipolysis in AT (6). Furthermore, Chung et al. recently reported that GDF15 behaves as a mitokine released upon mitochondrial stress and capable of enhancing systemic energy metabolism (7). They further show that GDF15 increases oxidative metabolism in white AT and stimulates lipolysis in 3T3L1 mouse adipocytes.
Finally, we used a different rhGDF15 protein (8146-GD, R&D Biosystems) than the one used by Olsen and colleagues (8), and, to the best of our knowledge, no study has ever reported a pro-lipolytic role of TGF-b itself in human adipocytes. In AT explants and isolated adipocytes studies, supraphysiological concentrations of lipolytic compounds are typically needed to achieve significant lipolytic responses (9).
Collectively, these studies argue for a potential biological activity of GDF15 on AT lipolysis. It is also physiologically meaningful that a hormone with appetite-suppressant effects may exhibit pro-catabolic actions in peripheral tissues, particularly AT.
1. Laurens C et al. Growth and Differentiation Factor 15 is secreted by skeletal muscle during exercise and promotes lipolysis in humans. JCI Insight [published online ahead of print: February 27, 2020]; doi:10.1172/jci.insight.131870
2. Available at : https://www.ncbi.nlm.nih.gov/ieb/research/acembly/av.cgi?db=human&term=GDF15&submit=Go
3. Raschke S, Eckardt K, Bjørklund Holven K, Jensen J, Eckel J. Identification and validation of novel contraction-regulated myokines released from primary human skeletal muscle cells. PLoS ONE 2013;8(4):e62008.
4. Yang L et al. GFRAL is the receptor for GDF15 and is required for the anti-obesity effects of the ligand. Nat Med 2017;23(10):1158–1166.
5. Emmerson PJ et al. The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL. Nat Med 2017;23(10):1215-1219.
6. Chrysovergis K et al. NAG-1/GDF-15 prevents obesity by increasing thermogenesis, lipolysis and oxidative metabolism. Int J Obes (Lond) 2014;38(12):1555-1564.
7. Chung HK et al. Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis. J Cell Biol 2017;216(1):149-165.
8. Olsen OE, Skjærvik A, Størdal BF, Sundan A, Holien T. TGF-β contamination of purified recombinant GDF15. PLoS ONE 2017;12(11):e0187349.
9. Arner P. Techniques for the measurement of white adipose tissue metabolism: a practical guide. Int J Obes Relat Metab Disord 1995;19(7):435-442.