# GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise

Axel Labour, Marlène Lac, Lucas Frassin, Benjamin Lair, Enda Murphy, Claire Maslo, Laurent Monbrun, Marie-Lou Calmy, Marie Marquès, Nathalie Viguerie, et al.

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## To cite this version:

Axel Labour, Marlène Lac, Lucas Frassin, Benjamin Lair, Enda Murphy, et al., GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise. Cell Reports, 2024, 43 (8), pp.114577. 10.1016/j CELREP.2024.114577 hal-04766608

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Distributed under a Creative Commons Attribution 4.0 International License
**Cell Reports**

# GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise

## Graphical abstract

<figure><img src="image_3.png" /></figure>

## Authors

Axel Labour, Marlène Lac,
Lucas Frassin, ..., Claire Laurens,
Alexandra Montagner, Cedric Moro

## Correspondence

cedric.moro@inserm.fr

## In brief

Labour et al. demonstrate that the exerkine growth and differentiation factor 15 (GDF15) produced during exercise is dispensable for the insulin-sensitizing effects of chronic exercise both in lean and obese mice and in humans with obesity.

## Highlights

* GDF15 deficiency has no effect on glucose homeostasis in mice irrespective of sex

* GDF15 is not required for the insulin-sensitizing effects of chronic exercise

* Plasma GDF15 negatively correlates with insulin sensitivity in humans with obesity

* Changes in plasma GDF15 and insulin sensitivity with chronic exercise are unrelated

<figure><img src="image_4.png" /></figure>
Article

# GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise

Axel Labour,<fnref n="1" />,<fnref n="5" /> Marlène Lac,<fnref n="1" />,<fnref n="5" /> Lucas Frassin,<fnref n="1" />,<fnref n="5" /> Benjamin Lair,<fnref n="1" /> Enda Murphy,<fnref n="2" /> Claire Maslo,<fnref n="1" /> Laurent Monbrun,<fnref n="1" /> Marie-Lou Calmy,<fnref n="1" /> Marie Marquès,<fnref n="1" /> Nathalie Viguerie,<fnref n="1" /> Geneviève Tavernier,<fnref n="1" /> Pierre Gourdy,<fnref n="1" />,<fnref n="3" /> Donal O’Gorman,<fnref n="2" /> Emilie Montastier,<fnref n="1" />,<fnref n="4" /> Claire Laurens,<fnref n="1" /> Alexandra Montagner,<fnref n="1" /> and Cedric Moro<fnref n="1" />,<fnref n="6" />,*

## SUMMARY

Growth and differentiation factor 15 (GDF15) has recently emerged as a weight loss and insulin-sensitizing factor. Growing evidence also supports a role for GDF15 as a physiological, exercise-induced stress signal. Here, we tested whether GDF15 is required for the insulin-sensitizing effects of exercise in mice and humans. At baseline, both under a standard nutritional state and high-fat feeding, GDF15 knockout (KO) mice display normal glucose tolerance, systemic insulin sensitivity, maximal speed, and endurance running capacity when compared to wild-type littermates independent of sex. When submitted to a 4-week exercise training program, both lean and obese wild-type and GDF15 KO mice similarly improve their endurance running capacity, glucose tolerance, systemic insulin sensitivity, and peripheral glucose uptake. Insulin-sensitizing effects of exercise training were also unrelated to changes in plasma GDF15 in humans. In summary, we here show that GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise.

## INTRODUCTION

Growth and differentiation factor 15 (GDF15) was initially identified as macrophage inhibitory cytokine 1 produced by macrophages.<fnref n="1" /> GDF15 recently emerged as a biomarker of cellular stress and the mitochondrial unfolded protein response released by a number of organs and tissues, such as the lungs, kidneys, adipose tissue, liver, and skeletal muscle.<fnref n="2" />⁻⁷ Recent studies demonstrate a key role of GDF15 in appetite suppression through non-canonical brain neuronal networks in the brain stem.⁸⁻¹¹ These effects appear to be largely mediated by food intake suppression through the GDF15 cognate receptor GFRAL (glial cell line-derived neurotrophic factor [GDNF] receptor alpha-like) which heterodimerizes with a co-receptor called RET (rearranged during transfection).⁹,¹¹ RET is a tyrosine kinase receptor for members of the GDNF family.⁹ GFRAL expression appears to be localized to the area postrema and the nucleus of the tractus solitarius in mice, while significant expression is found in adipose tissue in humans.<fnref n="5" />,¹¹ Pharmacological doses of GDF15 induce robust weight loss in obese mice fed a high-fat diet (HFD).¹⁰,¹²,¹³ Interestingly, a recent study reports that acute and chronic administration of recombinant GDF15 protein in rats and mice improves systemic insulin sensitivity in a GFRAL-dependent manner and independent of weight loss.¹² This effect seems to involve a brain-periphery crosstalk through

activation of the sympathetic nervous system and β-adrenergic signaling in adipose tissue and the liver.

GDF15 also emerged as a novel exerkine released during exercise in mice¹⁴ and humans.<fnref n="5" />,¹⁵,¹⁶ However, the exact physiological roles of GDF15 during exercise are currently unknown. Exercise is well known to improve systemic insulin sensitivity in mice¹⁷ and humans.¹⁸ Here, we tested whether GDF15 would be required for the insulin-sensitizing effects of exercise in lean and obese mice. Our results indicate that Gdf15-deficient mice display normal glucose and insulin tolerance both under standard and HFD irrespective of sex. However, both Gdf15 wild-type and knockout (KO) mice improve their glucose tolerance, systemic insulin sensitivity, and glucose uptake to a similar extent in response to exercise training, thus indicating that exercise-released GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise. In humans with obesity, changes in resting or exercise plasma GDF15 levels are not associated with the insulin-sensitizing effects of exercise training.

## RESULTS

### Normal glucose homeostasis and exercise capacity in lean GDF15 KO mice

Pharmacological doses of GDF15 induce appetite suppression in HFD-fed rodents, leading to a reduction in body mass and a

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<footnote n="1">¹Institute of Metabolic and Cardiovascular Diseases, INSERM/Paul Sabatier University, UMR1297, Toulouse, France</footnote>

<footnote n="2">²School of Health and Human Performance, Dublin City University, Dublin, Ireland</footnote>

<footnote n="3">³Department of Diabetology, Toulouse University Hospital, Toulouse, France</footnote>

<footnote n="4">⁴Department of Endocrinology, Metabolic Diseases and Nutrition, Toulouse University Hospital, Toulouse, France</footnote>

<footnote n="5">⁵These authors contributed equally</footnote>

<footnote n="6">⁶Lead contact</footnote>

<footnote>*Correspondence: cedric.moro@inserm.fr
https://doi.org/10.1016/j.celrep.2024.114577</footnote>
<figure><img src="image_6.png" /><figcaption>**Figure 1.** *Gdf15*-deficient mice display normal insulin sensitivity and exercise capacity irrespective of sex</figcaption></figure>

(A) Illustration of the study in chow diet-fed WT (n = 18, 9 female and 9 male) and *Gdf15* KO (n = 24; 11 female and 13 male) mice undergoing exercise and metabolic phenotyping in the sedentary pre-training state.

(B) Plasma GDF15 secretion in male (n = 5) and female (n = 7) WT mice, assessed during a stepwise incremental exercise test on a treadmill.

(C) Maximal running speed in male and female WT (n = 18; 9 female and 8 male) and *Gdf15* KO (n = 24; 11 female and 13 male) mice, assessed during a stepwise incremental exercise test on a treadmill.

(legend continued on next page)
concomitant improvement of glycemic control in obese animals.¹⁰ Thus, it is unclear whether GDF15 influences insulin sensitivity and body composition in lean mice, taking into account the potential influence of sex. To address this question, we explored the baseline metabolic and exercise phenotype in male and female wild-type (WT) and *Gdf15* KO mice (Figure 1A). We here confirm previous findings showing a significant increase of plasma GDF15 (+27%, *p* = 0.0003), irrespective of sex, during an incremental exercise test (Figure 1B). Our data indicate that genetic loss of GDF15 does not promote major changes in body weight (Figure S1A), lean mass (Figure S1B), and fat mass (Figure S1C) in female or male C57Bl6/J mice on a standard chow diet, most likely due to a lack of effect on daily food intake (Figure S1D). Since GDF15 has also been found to influence the motivation to exercise,¹⁶ we investigated both maximal speed and endurance running capacity of WT and *Gdf15* KO mice during forced exercise on a treadmill. WT and *Gdf15* KO male and female mice all exhibited a similar maximal running capacity (Figure 1C) and endurance running capacity (Figures 1E and 1F). Average time to exhaustion (Figure 1F) and running distance (Figure 1G) during the endurance test and during the maximal incremental test (Figures S1E and S1F) were similar between genotypes and irrespective of sex. Interestingly, we observed a stronger increase of exercise-induced plasma GDF15 levels (+56%, *p* < 0.05) with longer bouts of exercise during the endurance running test (Figure S1G).

Contrary to recent findings using pharmacological doses of GDF15 in mice and rats,¹² genetic loss of GDF15 had no effect on homeostasis model assessment of insulin resistance (HOMA-IR) (Figure 1H), fasting blood glucose (Figure S1H), fast-ing plasma insulin (Figure S1I), glucose tolerance (Figure 1I), and glucose-induced insulin secretion (Figure 1J) in female WT and *Gdf15* KO mice. Similar findings were observed in male mice (Figures 1K-1M). Consistent with this, insulin sensitivity measured during an insulin tolerance test remained strictly unaf-fected in both female (Figure 1N) and male (Figure 1O) WT and *Gdf15* KO mice. In summary, our results indicate that the lack of GDF15 does not significantly influence glucose homeostasis and exercise capacity in lean mice.

## Exercise training improves glucose homeostasis in lean mice independent of GDF15

A recent study indicates that GDF15 displays insulin-sensitizing effects independent of body weight loss in rodents.¹² We here

tested whether exercise-induced GDF15 would be required for the insulin-sensitizing effects of exercise. For this, we submitted adult (16-week-old) male and female WT and *Gdf15* KO mice to 4 weeks of endurance training on a treadmill. Exercise and meta-bolic phenotyping were performed before and at the end of the training program (Figure 2A). Body weight (Figures S2A and S2B), lean mass (Figure S2C), fat mass (Figure S2D), and average food intake (Figure S2E) remained comparable between WT and *Gdf15* KO mice irrespective of sex during the follow-up and in response to training. Training consisted of 30 min of endurance exercise per day, 4 times a week for 4 weeks. Although variable among individuals, training compliance reached ~80% and was similar between WT and *Gdf15* KO mice (Figure S2F). As expected, regular training for 4 weeks significantly improved the endurance running capacity of the whole cohort (Figure 2B) as well as in WT (Figure S2G) and *Gdf15* KO mice (Figure S2H).

We next assessed glucose homeostasis in response to training in the fasting state and in response to dynamic changes in blood glucose during oral glucose and insulin tolerance tests (Figure 2A). First, exercise training did not significantly influence HOMA-IR in WT mice (Figure 2C). However, exercise training greatly improved glucose tolerance (+40%, *p* = 0.001) (Figures 2D and 2E) as well as glucose-stimulated insulin secretion during the oral glucose tolerance test (oGTT) in WT mice (Figure 2F). Despite slightly lower plasma concentrations of GDF15 in female as compared to male WT mice (Figure S2I), strictly similar findings were observed in *Gdf15* KO male and female mice. HOMA-IR remained unchanged (Figure 2G), as did fasting blood glucose (Figure S2J) and insulin (Figure S2K), while glucose tolerance (+40%, *p* = 0.0005) (Figures 2H and 2I) and glucose-stimulated insulin secretion during the oGTT (Figure 2J) were significantly improved in the post-training period. In agreement with these findings, insulin sensitivity was also greatly improved post -training in both WT (+44%, *p* = 0.001) (Figures 2K and 2L) and *Gdf15* KO (+45%, *p* = 0.0003) mice (Figures 2M and 2N). In line with these observations, plasma GDF15 concentrations post training were unrelated to changes in incremental area under the curve (iAUC) measured during the oGTT (Figure S2L) and incremental area under the curve measured during insulin tolerance test (ITT) (Figure S2M) pre and post training in WT mice. Collectively, our data suggest that GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise in lean male and female mice.

(D and E) Percentage of (D) female (n = 9 and 10) and (E) male (n = 9 and 13) WT and *Gdf15* KO mice running as a function of time during the endurance running test in the sedentary pre-training state.

(F) Average time to exhaustion during the endurance running test at pre-training (female, n = 9 and 11; male, n = 9 and 13).

(G) Average running distance during the endurance running test at pre-training (female, n = 9 and 11; male, n = 9 and 13).

(H) HOMA-IR index at pre-training in female WT and *Gdf15* KO mice (n = 9 and 11).

(I) Blood glucose levels during an oGTT and iAUC of WT (n = 9) and *Gdf15* KO (n = 11) female mice.

(J) Plasma insulin at t = 15 min of the oGTT (peak) in WT (n = 9) and *Gdf15* KO (n = 11) male mice.

(K) HOMA-IR index at pre-training in male WT (n = 9) and *Gdf15* KO (n = 13) mice.

(L) Blood glucose levels during an oGTT and iAUC of the oGTT in WT (n = 9) and *Gdf15* KO (n = 13) male mice.

(M) Plasma insulin at t = 15 min of the oGTT (peak) in WT (n = 9) and *Gdf15* KO (n = 13) male mice.

(N and O) Blood glucose levels during an ITT and area above the curve of the ITT in (N) female (n = 9 and n = 11) and (O) male WT and *Gdf15* KO male mice (n = 9 and n = 13).

Data are presented as mean ± SEM and were analyzed by Student's t test (B, C, and F-O) or by Mantel-Cox test (D and E). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 versus the WT.
<figure><img src="image_7.png" /><figcaption>Figure 2. Exercise training improves insulin sensitivity independent of GDF15 in lean mice</figcaption></figure>

(A) Illustration of the study in chow diet-fed WT (n = 18) and Gdf15 KO (n = 24) mice undergoing exercise and metabolic phenotyping in response to a 4-week exercise training program on a treadmill.

(B) Percentage of mice running as a function of time during the endurance running test pre and post training (n = 42).

(C) HOMA-IR index in WT mice pre and post training (n = 18; 9 female and 9 male).

(D) Blood glucose levels during an oGTT in WT mice pre and post training (n = 18; 9 female and 9 male).

(E) iAUC of the oGTT in WT mice pre and post training (n = 18; 9 female and 9 male).

(F) Plasma insulin at t = 15 min of the oGTT (peak) in WT mice pre and post training (n = 18; 9 female and 9 male).

(G) HOMA-IR index in Gdf15 KO mice pre and post training (n = 24; 11 female and 13 male).

(H) Blood glucose levels during an oGTT in Gdf15 KO mice pre and post training (n = 24; 11 female and 13 male).

(I) iAUC of the oGTT in Gdf15 KO mice pre and post training (n = 24; 11 female and 13 male).

(J) Plasma insulin at t = 15 min of the oGTT (peak) in Gdf15 KO mice pre and post training (n = 24; 11 female and 13 male).

(K) Blood glucose levels during an ITT in WT mice pre and post training (n = 9; 4 female and 5 male).

(L) Area above the curve of the ITT in WT mice pre and post training (n = 9; 4 female and 5 male).

(legend continued on next page)
## Exercise training-induced molecular changes in insulin action and oxidative capacity are independent of GDF15

Glucose homeostasis and whole-body insulin sensitivity are determined by various insulin-sensitive tissues, such as skeletal muscle, adipose tissue, heart, and liver. We therefore studied whether the lack of GDF15 would influence tissue-specific glucose uptake in response to exercise training. Consistent with previous findings, blood glucose levels dropped by ~40%-45% (*p* < 0.0001) and to a similar extent in response to a supra-physiological insulin bolus in WT and *Gdf15* KO mice (Figure 3A). When examining tissue responses, insulin-stimulated 2-deoxy-glucose glucose uptake was similar in inguinal white adipose tissue (iWAT), epididymal WAT and interscapular brown adipose tissue (BAT), heart, liver, and the *soleus*, *gastrocnemius*, and *extensor digitorum longus* muscles between WT and *Gdf15* KO mice (Figure 3B). Skeletal muscle accounts for a large fraction of insulin-stimulated glucose uptake in lean mice.¹⁹ We next investigated insulin signaling in the *gastrocnemius* muscle. In agreement with whole-body and muscle-specific glucose uptake data, insulin-stimulated phosphorylation of Ser473-Akt (Figures 3C and 3D) and Thr308-Akt (Figures 3C and 3E) and GLUT4 protein content (Figures 3C and 3F) remained similar between WT and *Gdf15* KO mice. GLUT4 protein content remained also unchanged in iWAT of WT and *Gdf15* KO mice (Figures 3G and 3H).

Exercise is known as a powerful molecular transducer in various tissues.²⁰ We further studied canonical molecular adaptations of exercise training in skeletal muscle, BAT, and WAT. In line with similar improvement in endurance running capacity post training, we failed to observe differences in oxidative phosphorylation protein content (Figure S3A) and *Ppargc1a* gene expression (Figure S3B) in the *gastrocnemius* muscle of WT and *Gdf15* KO mice. No changes in expression of canonical genes of insulin-stimulated glucose metabolism, such as *Gsk3b* (Figure S3C) and *Glut4* (Figure S3D), in insulin-sensitive tissues (iWAT, BAT, and *gastrocnemius* muscle) were observed post training in both genotypes. Since exercise has been shown to induce thermogenic gene programs and WAT browning,²¹ here we assessed gene expression of thermogenic markers in BAT and iWAT. Gene expression of prototypical thermogenic markers such as *Ucp1*, *Ppargc1a*, and *Cidea* was comparable between WT and *Gdf15* KO mice in both BAT (Figure S3E) and iWAT (Figure S3F). Altogether, these results further confirm that exercise training-mediated improvement of insulin sensitivity and oxidative capacity is independent of GDF15.

### Normal glucose homeostasis and exercise capacity in obese GDF15 KO mice

It is still unclear whether glucose homeostasis is differently affected by obesity in WT and *Gdf15* KO mice according to sex. We here examined the metabolic and exercise phenotype of male and female WT and *Gdf15* KO mice after 4 weeks of

HFD (Figure 4A). Our data indicate that genetic loss of GDF15 does not alter body weight gain (Figures 4B and S4A) in female or male C57Bl6/J mice under HFD, most likely due to a lack of effect on daily food intake (Figure S4B). While resting plasma GDF15 levels are increased by HFD (Figure S4C), exercise increases plasma GDF15 levels (+35%) in HFD-fed WT mice similarly as in lean mice (Figure 4C). We then investigated both maximal speed and running capacity in HFD-fed WT and *Gdf15* KO mice during forced exercise on a treadmill. WT and *Gdf15* KO male and female mice all exhibited a similar maximal running capacity (Figure 4D) and endurance running capacity (Figures 4E and 4F).

As expected, HFD deteriorated glucose tolerance (Figure S4D) and increased fasting blood glucose (Figure S4E) and plasma insulin levels (Figures S4F-S4G) compared to a standard chow diet. However, genetic loss of GDF15 had no effect on HOMA-IR (Figure 4G), glucose tolerance (Figure 4H), and glucose-induced insulin secretion (Figure 4I) in female WT and *Gdf15* KO mice. Similar findings were observed in male mice (Figures 4J-4L). Consistent with this, insulin sensitivity measured during an insulin tolerance test remained strictly unaffected in both female (Figure 4M) and male (Figure 4N) WT and *Gdf15* KO mice. In summary, our results indicate that the lack of GDF15 does not significantly influence glucose homeostasis and exercise capacity in obese HFD-fed mice.

### Exercise training improves glucose homeostasis in obese mice independent of GDF15

Here, we tested whether exercise-induced GDF15 would be required for the insulin-sensitizing effects of chronic exercise in HFD-fed obese mice. For this, we submitted 4-week HFD-fed male and female WT and *Gdf15* KO mice to 4 weeks of endurance training on a treadmill while remaining under HFD. Exercise and metabolic phenotyping were performed before and at the end of the training program (Figure 5A). Body weight (Figures 5B and S4A) and average food intake (Figure S4H) remained comparable between WT and *Gdf15* KO mice irrespective of sex during the follow-up and in response to training. Exercise compliance during training was not significantly different in female or male WT and *Gdf15* KO mice (Figure S4I). As in lean mice, plasma GDF15 levels were increased during acute exercise (Figure 5C). After 4 weeks of endurance training, maximal running speed was similar between WT and *Gdf15* KO female or male mice (Figure 5D). As expected, endurance training significantly improved the endurance running capacity irrespective of sex and genotype (Figures 5E and 5F).

We next assessed glucose homeostasis in response to training in the fasted state and in response to dynamic changes in blood glucose during glucose and insulin tolerance tests (Figure 5A). Similarly to lean mice, exercise training did not significantly influence HOMA-IR in WT mice (Figure 5G). Exercise training improved glucose tolerance (Figure 5H) despite no

<footnote>(M) Blood glucose levels during an ITT in *Gdf15* KO mice pre and post training (n = 11; 5 female and 6 male).</footnote>

<footnote>(N) Area above the curve of the ITT in *Gdf15* KO mice pre and post training (n = 11; 5 female and 6 male).</footnote>

<footnote>Data are presented as mean ± SEM and were analyzed by Mantel-Cox test (B), paired t or Wilcoxon matched-pairs signed-rank test (C, E-G, I, L, and N), or by repeated measure analysis of variance (RM-ANOVA) (D, H, K, and M). \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 versus the WT. Female mice are indicated by pink symbols and male mice by blue symbols.</footnote>
<figure><img src="image_8.png" /><figcaption>Figure 3. Exercise training-induced molecular changes in insulin action and oxidative capacity are independent of GDF15</figcaption></figure>

(A) Blood glucose levels at t = 0 and t = 15 after intraperitoneal insulin bolus injection in WT (n = 9; 5 female and 4 male) and Gdf15 KO (n = 13; 6 female and 7 male) mice post training.

(B) Tissue-specific glucose uptake reflected by 2-deoxyglucose metabolic clearance in various tissues in WT (n = 9; 5 female and 4 male) and Gdf15 KO (n = 13; 6 female and 7 male) mice post training.

(C) Representative blots of Ser473-Akt and Thr308-Akt phosphorylation, total Akt, and GLUT4 expression in insulin-stimulated gastrocnemius muscle in WT and Gdf15 KO mice post training.

(D–F) Quantitative bar graphs of the relative phosphorylation level of (D) Ser473-Akt to total Akt, (E) Thr308-Akt to total Akt, and (F) GLUT4 protein content in insulin-stimulated gastrocnemius muscle in WT (n = 9; 5 female and 4 male) and Gdf15 KO (n = 13; 6 female and 7 male) mice post training.

(G) Representative blot of GLUT4 expression in iWAT of WT and Gdf15 KO mice post training.

(H) Quantitative bar graph of GLUT4 expression in iWAT of WT (n = 9; 4 female and 5 male) and Gdf15 KO (n = 11; 5 female and 6 male) mice post training.
Data are presented as mean ± SEM and were analyzed by two-way ANOVA with Tukey's post hoc test (A) or unpaired t test (B, D, and F). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.0001 versus the WT. Female mice are indicated by pink symbols and male mice by blue symbols.

change in the iAUC (Figure 5I). Glucose-stimulated insulin secretion during the oGTT was also improved in WT mice (Figures 5J and S4J). However, HOMA-IR (Figure 5K), glucose tolerance (Figures 5L and 5M), and glucose-induced insulin secretion (Figure 5N) were strictly similar in Gdf15 KO male and female mice versus the respective control in response to training. No change

in insulin sensitivity in response to training was observed in WT (Figures S4K and S4L) and Gdf15 KO (Figures S4M and S4N) mice despite a state of insulin resistance in HFD-fed versus normal chow diet (NCD)-fed mice (Figure S4O). Interestingly plasma GDF15 concentrations were correlated with fasting glucose levels at baseline in HFD-fed WT mice (Figure S4P).
<figure><img src="image_9.png" /><figcaption>**Figure 4.** *Gdf15*-deficient mice display normal insulin sensitivity and exercise capacity irrespective of sex under HFD</figcaption></figure>

(A) Illustration of the study in HFD-fed WT (n = 12; 6 female and 6 male) and Gdf15 KO (n = 12; 7 female and 5 male) undergoing exercise and metabolic phenotyping in the sedentary pre-training state.

(B) Body weight of male and female WT (n = 12; 6 female and 6 male) and Gdf15 KO (n = 12; 7 female and 5 male) mice, assessed after 4 weeks of HFD.

(C) Exercise-induced plasma GDF15 levels in male (n = 6) and female (n = 6) WT mice, assessed during an incremental exercise test on a treadmill.

(D) Maximal running speed in male and female WT (n = 12; 6 female and 6 male) and Gdf15 KO (n = 12; 7 female and 5 male) mice, assessed during an incremental exercise test on a treadmill.

*(legend continued on next page)*
However, this association was fairly lost in response training (Figure S4Q). Collectively, our data suggest that GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise in HFD-fed obese male and female mice.

## Associations between plasma GDF15 and insulin sensitivity in humans

Since GDF15 also behaves as a physiological exercise-induced stress signal in humans,¹⁶ we next investigated the relationship between plasma GDF15 at rest and during exercise with whole-body insulin sensitivity measured by euglycemic hyperinsulinemic clamp in 16 obese individuals before (pre) and at the end (post) of an 8-week exercise training program.²² As expected, we observed a significant influence of exercise training on systemic insulin sensitivity, as reflected by the increase in glucose disposal rate (GDR) (+8%, *p* = 0.033; Figure 6A). When normalizing to lean body mass, insulin sensitivity as determined by the M value remained significantly increased in response to exercise training (*p* = 0.024; Figure 6B). Plasma GDF15 levels measured at rest and after an overnight fast were unchanged in response to exercise training (Figure 6C). We observed a trend of negative correlation between resting plasma GDF15 levels and GDR in the pre-training state (Figure 6D). A similar correlation was observed with exercise plasma GDF15 levels (Figure S5A). Interestingly, changes in plasma GDF15 levels from rest to exercise were negatively associated with GDR at baseline prior to training (*r* = 0.60, *p* = 0.01) (Figure 6E). We further observed a tight negative relationship between resting plasma GDF15 levels and GDR after training (*r* = 0.61, *p* = 0.01) (Figure 6F). Again, this correlation remained significant with exercise plasma GDF15 levels post training (Figure S5B). However, no significant correlation between changes in plasma GDF15 levels from rest to exercise and GDR was observed after training (Figure 6G). Importantly, changes in GDR and resting plasma GDF15 levels in response to exercise training were unrelated (Figure 6H). Consistent with this, no association between changes in exercise plasma GDF15 levels and GDR was observed in response to exercise training (Figure S5C).

## DISCUSSION

GDF15 recently emerged as a major weight loss and insulin-sensitizing signal in rodents. GDF15 was also discovered to be a novel exerkine. Yet, its physiological role during exercise and in the control of systemic insulin sensitivity remains unknown. Here, we report that GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise in lean and obese mice. Consistent with this,

we observed that changes in GDF15 and systemic insulin sensitivity in response to exercise training are also unrelated in humans.

GDF15 can be secreted by numerous tissues and organs in response to various stressors,³,⁷ including exercise.⁵,¹⁴⁻¹⁶ Although GDF15 has been studied extensively in the context of metabolic diseases in recent years, little is currently known about its potential physiological effects during exercise. For instance, it is still unclear whether GDF15 released during exercise can mediate some of the beneficial effects of exercise on energy balance or insulin sensitivity. Since Sjoberg et al. suggested recently that GDF15 exerts insulin-sensitizing effects independent of weight loss in rodents,¹² here we tested whether GDF15 would be required for the insulin-sensitizing effects of exercise in mice and humans. At baseline in the pre-training state, Gdf15-deficient lean and obese mice displayed a similar metabolic phenotype and exercise capacity as WT littermates. This is in agreement with previous studies demonstrating a lack of apparent phenotype on insulin sensitivity²³ and exercise capacity¹⁴ under standard nutritional conditions. This is in contrast with several studies showing that Gdf15- and/or Gfral-ablated mice are more prone to diet-induced obesity and insulin resistance.⁸,¹⁰,¹¹,¹⁶,²³ In one of these studies, a sexual dimorphism in diet-induced obesity was observed, with male mice being more susceptible to diet-induced glucose intolerance.²³ Here, we did not observe any sexual dimorphism in glucose tolerance and whole-body insulin sensitivity. The discrepancy with other studies may be due to the short-term high-fat feeding (4 weeks) in the current study and the rather young age of the mice. Of note, GDF15 plasma concentrations in the fasted state were slightly lower in female mice. Although the reasons for this difference were not investigated, one can hypothesize that lower total and lean body mass in female mice triggers lower circulating levels of GDF15. In addition, although pharmacological doses of GDF15 and transgenic overexpression of Gdf15 at supra-physiological doses confer protection against obesity and improve glucose tolerance in diet-induced obese mice,⁶,¹²,²⁴ lack of GDF15 appears to be insufficient to significantly impact on energy balance, food intake, body composition, and systemic insulin sensitivity. This is in contrast with recent findings of Sjoberg et al. demonstrating a slightly reduced whole-body insulin sensitivity measured by euglycemic hyperinsulinemic clamps in Gfral KO mice under a standard chow diet.¹² The discrepancies between our studies could be due to different diet composition, genetic background, and/or the sanitary status of the animal care facility.

In agreement with other studies,¹⁶ we next confirmed that GDF15 behaves as an exerkine produced during acute endurance

<footnote>(E) Percentage of female (n = 6 and 7) and male (n = 6 and 5) WT and Gdf15 KO mice running as a function of time during the endurance running test in the sedentary pre-training state.</footnote>

<footnote>(F) Average running distance during the incremental exercise test at pre-training (female, n = 6 and 7; male, n = 6 and 5).</footnote>

<footnote>(G) HOMA-IR index at pre-training in female WT (n = 6) and Gdf15 KO (n = 7) mice.</footnote>

<footnote>(H) Blood glucose levels during an oGTT and iAUC of WT (n = 6) and Gdf15 KO (n = 7) female mice.</footnote>

<footnote>(I) Plasma insulin at t = 15 min of the oGTT (peak) in WT (n = 6) and Gdf15 KO (n = 7) female mice.</footnote>

<footnote>(J) HOMA-IR index at pre-training in male WT (n = 6) and Gdf15 KO (n = 5) mice.</footnote>

<footnote>(K) Blood glucose levels during an oGTT and iAUC of the oGTT in WT (n = 6) and Gdf15 KO (n = 5) male mice.</footnote>

<footnote>(L) Plasma insulin at t = 15 min of the oGTT (peak) in WT (n = 6) and Gdf15 KO (n = 5) male mice.</footnote>

<footnote>(M and N) Blood glucose levels during an ITT and area above curve of the ITT in (M) female (n = 6 and 7) and (N) male (n = 6 and 5) WT and Gdf15 KO mice.</footnote>

<footnote>Data are presented as mean ± SEM and were analyzed by paired t and Student's t tests (B-D and F-N) or by Mantel-Cox test (E). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 versus the WT.</footnote>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 5.** Exercise training improves endurance running capacity and glucose homeostasis independent of GDF15 in obese mice</figcaption></figure>

(A) Illustration of the study in HFD-fed WT (n = 12; 6 female and 6 male) and Gdf15 KO (n = 12; 7 female and 5 male) mice undergoing exercise and metabolic phenotyping in response to a 4-week exercise training program on a treadmill.

(B) Body weight of male and female WT (n = 12; 6 female and 6 male) and Gdf15 KO (n = 12; 7 female and 5 male) mice, assessed after 4 weeks of exercise training.

(C) Exercise-induced GDF15 secretion in male (n = 6) and female (n = 6) WT mice, assessed during an incremental exercise test on a treadmill.

(D) Maximal running speed in male and female WT (n = 12; 6 female and 6 male) and Gdf15 KO (n = 12; 7 female and 5 male) mice assessed during an incremental exercise test on treadmill.

(E) Percentage of female (n = 6 and 7) and male (n = 6 and 5) WT and Gdf15 KO mice running as a function of time during the endurance running test after pre- and post-training.

(F) Average running distance during the stepwise incremental exercise test at pre- and post-training (female, n = 6 and 7; male, n = 6 and 5).

(G) HOMA-IR index pre and post training in female (n = 6) and male (n = 6) WT mice.

(H) Blood glucose levels during an oGTT of female (n = 6) and male (n = 6) WT mice pre and post training.

(I) iAUC of the oGTT in WT female (n = 6) and male (n = 6) mice pre and post training.

(legend continued on next page)
exercise in mice. Plasma GDF15 was measured before and immediately after $V_{max}$ determination, where maximal running capacity was determined during an incremental exercise test until exhaustion. This test mimics a VO₂ peak test in humans and is usually short in duration (<25 min). Thus, plasma GDF15 increased by ~30% in lean and obese mice during the $V_{max}$ test and by ~50% during the endurance test in our study, which is much lower than the 3-fold increase observed in another study with long-duration exercise.¹⁶ Therefore, the exercise protocol (speed, duration, and treadmill incline) may be a significant determinant of plasma GDF15 levels. We further submitted WT and *Gdf15* KO mice to 4 week of exercise training to test whether GDF15 would be required for the insulin-sensitizing effects of exercise in lean and obese mice. Since GDF15 can influence exercise motivation,¹⁶ mice were submitted to forced exercise on treadmills. The exercise training program was prescribed according to individual maximal mouse capacity as determined by the $V_{max}$ test before training. The $V_{max}$ ranged between 0.35 and 0.60 m s⁻¹, which is quite typical for the C57BL/6J mouse strain.²⁵ Exercise training compliance was variable among individuals, reaching 80%-90% overall and being comparable between WT and *Gdf15* KO mice. Overall, 4 weeks of endurance training led to a significant improvement of endurance running capacity both in WT and *Gdf15* KO mice irrespective of sex. In this context, exercise training remarkably improved glucose tolerance (~40%) and whole-body insulin sensitivity (~45%) to a similar extent in both WT and *Gdf15* KO lean mice. However, short-term HFD (4 weeks) was sufficient to degrade glucose tolerance and systemic insulin sensitivity when comparing the glycemic response between NCD-fed mice and HFD-fed exercise-trained mice. Thus, the exercise training apparently protected HFD-fed mice from further degradation of systemic insulin sensitivity, thus explaining the lack of change between systemic insulin sensitivity in HFD-fed mice pre and post training. This systemic effect was also accompanied by a greater glucose-induced insulin secretion in response to chronic exercise in both lean and obese mice. This is consistent with a protective effect of exercise on β-cell mass and function, as observed recently in the context of type 1 and type 2 diabetes.²⁶ Consistent with the lack of effect of genetic loss of GDF15 on exercise capacity and insulin sensitivity, there was no difference in muscle glucose uptake, oxidative capacity, and insulin signaling in lean WT and *Gdf15* KO mice post training. In the same line, no change in GLUT4 protein content and thermogenic genes expression in iWAT was observed in lean mice.

Thus, the physiological role of GDF15 released during exercise remains unclear. Recent studies identified GDF15 as a novel exercise possibly produced by heart, liver, and skeletal muscle during exercise in humans.⁵,¹⁵,¹⁶ Here, we observed an unexpected negative correlation between plasma GDF15 concentrations measured at rest or during exercise with whole-body insulin

sensitivity both in lean healthy and obese individuals. Insulin sensitivity was measured using the gold-standard procedure of hyperinsulinemic euglycemic clamp, at a dose where most of the glucose disposal is accounted for by skeletal muscle.²²,²⁷ This negative association echoes a similar correlation between plasma GDF15 levels and physical fitness in humans.²⁸ Indeed, several studies previously reported a tight positive relationship between physical fitness and insulin sensitivity.²⁹ Thus, it might be speculated that GDF15 behaves as a biomarker of poor metabolic health. One can hypothesize that GDF15 is secreted in response to an energy crisis during exercise. Since chronic exercise improves physical fitness and insulin sensitivity, it may likely reduce this state of energy crisis. This hypothesis fits well with our observation that plasma GDF15 response to exercise correlates negatively with insulin sensitivity before training, but this correlation is lost after training both in mice and humans. This implies that improved metabolic health is accompanied by reduced release of GDF15 during exercise.

In summary, here we demonstrate that GDF15 is dispensable for the insulin-sensitizing effects of chronic exercise in lean and obese insulin-resistant mice. In addition, genetic loss of GDF15 has no effect on body composition, glucose homeostasis, and insulin sensitivity under standard nutritional conditions or high-fat feeding and irrespective of sex. We further corroborate our observations of a lack of association between changes in plasma GDF15 and changes in insulin sensitivity in response to exercise training in one human study on male individuals with obesity. Future studies are needed to uncover potential physiological roles of GDF15 in the context of exercise.

## Limitations of the study

Although we acknowledge that euglycemic hyperinsulinemic clamping is a gold standard procedure to assess whole-body insulin sensitivity in mouse models and may be more sensitive than other procedures, it is unlikely that mice presenting normal glucose tolerance, insulin response during the oGTT, insulin tolerance, and insulin-stimulated glucose uptake in various tissues during insulin bolus injection are insulin resistant systemically or in any tissue. Although we did not investigate the tissue source of circulating GDF15 during exercise, we observed that the liver may be a dominant source of circulating GDF15, in agreement with other studies.⁴,⁶

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* RESOURCE AVAILABILITY

* Lead contact

(J) Plasma insulin at t = 15 min of the oGTT (peak) in WT female (n = 6) and male (n = 6) mice pre and post training.

(K) HOMA-IR index in female (n = 7) and male (n = 5) *Gdf15* KO mice pre and post training.

(L) Blood glucose levels during an oGTT and iAUC of the oGTT in *Gdf15* KO female (n = 7) and male (n = 5) mice pre and post training.

(M) Incremental area under curve of the oGTT in *Gdf15* KO female (n = 7) and male (n = 5) mice pre and post training.

(N) Plasma insulin at t = 15 min of the oGTT (peak) in *Gdf15* KO female (n = 7) and male (n = 5) mice pre and post training.

Data are presented as mean ± SEM and were analyzed by paired t or Wilcoxon matched-pairs signed-rank test (B-D, F, G, I-K, M, and N), Mantel-Cox test (E), or RM-ANOVA (H and L). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001 versus the WT. Female mice are indicated by pink symbols and male mice by blue symbols.
A

<figure><img src="image_12.png" /></figure>

B

<figure><img src="image_13.png" /></figure>

C

<figure><img src="image_14.png" /></figure>

D

Pre-training

<figure><img src="image_15.png" /></figure>

E

<figure><img src="image_16.png" /></figure>

F

<figure><img src="image_17.png" /></figure>

G

Post-training

<figure><img src="image_18.png" /></figure>

H

Pre-/Post-training

<figure><img src="image_19.png" /><figcaption>Figure 6. Associations between resting plasma GDF15 levels and insulin sensitivity in humans</figcaption></figure>

(A) GDR pre and post training in obese individuals from study 1 (n = 16).

(B) M value pre and post training in obese individuals from study 1 (n = 16).

(C) Plasma GDF15 pre and post training in obese individuals from study 1 (n = 16).

(D) Correlation between GDR and resting plasma GDF15 pre-training in obese individuals from study 1 (n = 16).

(E) Correlation between GDR and changes in plasma GDF15 between exercise and rest (Ex-Rest) pre training in obese individuals from study 1 (n = 16).

(F) Correlation between GDR and resting plasma GDF15 post training in obese individuals from study 1 (n = 16).

(G) Correlation between GDR and changes in plasma GDF15 between exercise and rest (Ex-Rest) post training in obese individuals from study 1 (n = 16).

(H) Correlation between changes in GDR and changes in resting plasma GDF15 pre and post training in obese individuals from study 1 (n = 16).

Data are presented as mean ± SEM. Data were analyzed by Wilcoxon paired t test (A–C) and univariate linear regression models (E–H).
* Materials availability

* Data and code availability

* EXPERIMENTAL MODEL AND SUBJECT DETAILS

* Mouse study 1

* Mouse study 2

* Clinical studies and human subjects

* METHOD DETAILS

* Blood analyses

* Glucose and insulin tolerance tests

* Tissue specific 2-deoxyglucose uptake *in vivo*

* Acclimation to treadmill and maximal running capacity test

* Endurance exercise test

* Endurance training

* RNA extraction and real-time qPCR

* Western blot

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114577.

## ACKNOWLEDGMENTS

We are grateful to Morgane Rousseau and her staff (UMS006 CREFRE), Alexandre Lucas (We-Met), and Christelle Segura (Histology core facility) for excellent technical support. This work was supported by grants from INSERM, the Société Francophone du Diabète, the European Foundation for the Study of Diabetes (EFSD), the European Research Program on "New Targets for Type 2 Diabetes" supported by an educational grant from MSD and EFSD/Boehringer Ingelheim European Research Program on "Multi-System Challenges in Diabetes," Agence Nationale de la Recherche (ANR-21-CE14-0057-01), and the Fondation pour la Recherche Médicale (EQU202303016316) (to C.M.). L.F. is supported by a PhD fellowship from INSERM/Occitanie Region (ALDOCT-22009726).

## AUTHOR CONTRIBUTIONS

Conceptualization, A.L., M.L., L.F., and C.M.; methodology, A.L., M.L., L.F., and C.M.; investigation, A.L., M.L., L.F., B.L., E.M., C.M., L.M., M.-L.C., G.T., C.L., and C.M.; resources, N.V., E.M., P.G., D.O., A.M., and C.M.; writing – original draft, A.L. and C.M.; writing – review & editing, all co-authors; supervision, C.M.; funding acquisition, C.M.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: January 4, 2024

Revised: June 28, 2024

Accepted: July 18, 2024

Published: August 2, 2024

## REFERENCES

1. Fairlie, W.D., Moore, A.G., Bauskin, A.R., Russell, P.K., Zhang, H.P., and Breit, S.N. (1999). MIC-1 is a novel TGF-beta superfamily cytokine associated with macrophage activation. J. Leukoc. Biol. 65, 2–5. https://doi.org/10.1002/jlb.65.1.2.

2. Bottner, M., Suter-Crazzolara, C., Schober, A., and Unsicker, K. (1999). Expression of a novel member of the TGF-beta superfamily, growth/differentiation factor-15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues. Cell Tissue Res. 297, 103–110.

3. Patel, S., Alvarez-Guaita, A., Melvin, A., Rimmington, D., Dattilo, A., Miedzybrodzka, E.L., Cimino, I., Maurin, A.C., Roberts, G.P., Meek, C.L., et al. (2019). GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice

and Humans. Cell Metab. 29, 707–718.e8. https://doi.org/10.1016/j.cmet.2018.12.016.

4. Guillaume, M., Riant, E., Fabre, A., Raymond-Letron, I., Buscato, M., Davezac, M., Tramunt, B., Montagner, A., Smati, S., Zahreddine, R., et al. (2019). Selective Liver Estrogen Receptor alpha Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice. Hepatol. Commun. 3, 908–924. https://doi.org/10.1002/hep4.1363.

5. Laurens, C., Parmar, A., Murphy, E., Carper, D., Lair, B., Maes, P., Vion, J., Boulet, N., Fontaine, C., Marquès, M., et al. (2020). Growth and differentiation factor 15 is secreted by skeletal muscle during exercise and promotes lipolysis in humans. JCI Insight 5, e131870. https://doi.org/10.1172/jci.insight.131870.

6. Xie, B., Murali, A., Vandevender, A.M., Chen, J., Silva, A.G., Bello, F.M., Chuan, B., Bahudhanapati, H., Sipula, I., Dedousis, N., et al. (2022). Hepatocyte-derived GDF15 suppresses feeding and improves insulin sensitivity in obese mice. iScience 25, 105569. https://doi.org/10.1016/j.isci.2022.105569.

7. Lasaad, S., Walter, C., Rafael, C., Morla, L., Doucet, A., Picard, N., Blanchard, A., Fromes, Y., Matot, B., Crambert, G., and Cheval, L. (2023). GDF15 mediates renal cell plasticity in response to potassium depletion in mice. Acta Physiol. 239, e14046. https://doi.org/10.1111/apha.14046.

8. Hsu, J.Y., Crawley, S., Chen, M., Ayupova, D.A., Lindhout, D.A., Higbee, J., Kutach, A., Joo, W., Gao, Z., Fu, D., et al. (2017). Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15. Nature 550, 255–259. https://doi.org/10.1038/nature24042.

9. Chung, H.K., Ryu, D., Kim, K.S., Chang, J.Y., Kim, Y.K., Yi, H.S., Kang, S.G., Choi, M.J., Lee, S.E., Jung, S.B., et al. (2017). Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis. J. Cell Biol. 216, 149–165. https://doi.org/10.1083/jcb.201607110.

10. Emmerson, P.J., Wang, F., Du, Y., Liu, Q., Pickard, R.T., Gonciarz, M.D., Coskun, T., Hamang, M.J., Sindelar, D.K., Ballman, K.K., et al. (2017). The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL. Nat. Med. 23, 1215–1219. https://doi.org/10.1038/nm.4393.

11. Mullican, S.E., Lin-Schmidt, X., Chin, C.N., Chavez, J.A., Furman, J.L., Armstrong, A.A., Beck, S.C., South, V.J., Dinh, T.Q., Cash-Mason, T.D., et al. (2017). GFRAL is the receptor for GDF15 and the ligand promotes weight loss in mice and nonhuman primates. Nat. Med. 23, 1150–1157. https://doi.org/10.1038/nm.4392.

12. Sjoberg, K.A., Sigvardsen, C.M., Alvarado-Diaz, A., Andersen, N.R., Larance, M., Seeley, R.J., Schjerling, P., Knudsen, J.G., Katzilieris-Petras, G., Clemmensen, C., et al. (2023). GDF15 increases insulin action in the liver and adipose tissue via a beta-adrenergic receptor-mediated mechanism. Cell Metab. 35, 1327–1340.e5. https://doi.org/10.1016/j.cmet.2023.06.016.

13. Wang, D., Townsend, L.K., DesOrmeaux, G.J., Frangos, S.M., Batchuluun, B., Dumont, L., Kuhre, R.E., Ahmadi, E., Hu, S., Rebalka, I.A., et al. (2023). GDF15 promotes weight loss by enhancing energy expenditure in muscle. Nature 619, 143–150. https://doi.org/10.1038/s41586-023-06249-4.

14. Gil, C.I., Ost, M., Kasch, J., Schumann, S., Heider, S., and Klaus, S. (2019). Role of GDF15 in active lifestyle induced metabolic adaptations and acute exercise response in mice. Sci. Rep. 9, 20120. https://doi.org/10.1038/s41598-019-56922-w.

15. Kleinert, M., Clemmensen, C., Sjoberg, K.A., Carl, C.S., Jeppesen, J.F., Wojtaszewski, J.F.P., Kiens, B., and Richter, E.A. (2018). Exercise increases circulating GDF15 in humans. Mol. Metab. 9, 187–191. https://doi.org/10.1016/j.molmet.2017.12.016.

16. Klein, A.B., Nicolaisen, T.S., Ørtenblad, N., Gejl, K.D., Jensen, R., Fritzen, A.M., Larsen, E.L., Karstoft, K., Poulsen, H.E., Morville, T., et al. (2021). Pharmacological but not physiological GDF15 suppresses feeding and the motivation to exercise. Nat. Commun. 12, 1041. https://doi.org/10.1038/s41467-021-21309-x.
<figure><img src="image_20.png" /></figure>

17. Hoene, M., Kappler, L., Kollipara, L., Hu, C., Irmler, M., Bleher, D., Hoffmann, C., Beckers, J., Hrabě de Angelis, M., Häring, H.U., et al. (2021). Exercise prevents fatty liver by modifying the compensatory response of mitochondrial metabolism to excess substrate availability. Mol. Metab. 54, 101359. https://doi.org/10.1016/j.molmet.2021.101359.

18. Hingst, J.R., Onslev, J.D., Holm, S., Kjøbsted, R., Frøsig, C., Kido, K., Steenberg, D.E., Larsen, M.R., Kristensen, J.M., Carl, C.S., et al. (2022). Insulin Sensitization Following a Single Exercise Bout Is Uncoupled to Glycogen in Human Skeletal Muscle: A Meta-analysis of 13 Single-Center Human Studies. Diabetes 71, 2237-2250. https://doi.org/10.2337/db22-0015.

19. Ayala, J.E., Bracy, D.P., McGuinness, O.P., and Wasserman, D.H. (2006). Considerations in the design of hyperinsulinemic-euglycemic clamps in the conscious mouse. Diabetes 55, 390-397. https://doi.org/10.2337/diabetes.55.02.06.db05-0686.

20. Hawley, J.A., Hargreaves, M., Joyner, M.J., and Zierath, J.R. (2014). Integrative biology of exercise. Cell 159, 738-749. https://doi.org/10.1016/j.cell.2014.10.029.

21. Bostrom, P., Wu, J., Jedrychowski, M.P., Korde, A., Ye, L., Lo, J.C., Rasbach, K.A., Bostrom, E.A., Choi, J.H., Long, J.Z., et al. (2012). A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 481, 463-468. https://doi.org/10.1038/nature10777.

22. Vion, J., Sramkova, V., Montastier, E., Marquès, M.A., Caspar-Bauguil, S., Duparc, T., Martinez, L.O., Bourlier, V., Harant, I., Larrouy, D., et al. (2021). Metabolic and cardiovascular adaptations to an 8-wk lifestyle weight loss intervention in younger and older obese men. Am. J. Physiol. Endocrinol. Metab. 321, E325-E337. https://doi.org/10.1152/ajpendo.00109.2021.

23. Tran, T., Yang, J., Gardner, J., and Xiong, Y. (2018). GDF15 deficiency promotes high fat diet-induced obesity in mice. PLoS One 13, e0201584. https://doi.org/10.1371/journal.pone.0201584.

24. Chrysovergis, K., Wang, X., Kosak, J., Lee, S.H., Kim, J.S., Foley, J.F., Travlos, G., Singh, S., Baek, S.J., and Eling, T.E. (2014). NAG-1/GDF-15 prevents obesity by increasing thermogenesis, lipolysis and oxidative

metabolism. Int. J. Obes. 38, 1555-1564. https://doi.org/10.1038/ijo.2014.27.

25. Ericsson, M., Sjåland, C., Andersson, K.B., Sjaastad, I., Christensen, G., Sejersted, O.M., and Ellingsen, Ø. (2010). Exercise training before cardiac-specific Serca2 disruption attenuates the decline in cardiac function in mice. J. Appl. Physiol. 109, 1749-1755. https://doi.org/10.1152/japplphysiol.00282.2010.

26. Coomans de Brachene, A., Scoubeau, C., Musuaya, A.E., Costa-Junior, J.M., Castela, A., Carpentier, J., Faoro, V., Klass, M., Cnop, M., and Eizirik, D.L. (2023). Exercise as a non-pharmacological intervention to protect pancreatic beta cells in individuals with type 1 and type 2 diabetes. Diabetologia 66, 450-460. https://doi.org/10.1007/s00125-022-05837-9.

27. DeFronzo, R.A., Tobin, J.D., and Andres, R. (1979). Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am. J. Physiol. 237, E214-E223. https://doi.org/10.1152/ajpendo.1979.237.3.E214.

28. Conte, M., Martucci, M., Mosconi, G., Chiariello, A., Cappuccilli, M., Totti, V., Santoro, A., Franceschi, C., and Salvioli, S. (2020). GDF15 Plasma Level Is Inversely Associated With Level of Physical Activity and Correlates With Markers of Inflammation and Muscle Weakness. Front. Immunol. 11, 915. https://doi.org/10.3389/fimmu.2020.00915.

29. Gemmink, A., Schrauwen, P., and Hesselink, M.K.C. (2020). Exercising your fat (metabolism) into shape: a muscle-centred view. Diabetologia 63, 1453-1463. https://doi.org/10.1007/s00125-020-05170-z.

30. Strelau, J., Strzelczyk, A., Rusu, P., Bendner, G., Wiese, S., Diella, F., Altick, A.L., von Bartheld, C.S., Klein, R., Sendtner, M., and Unsicker, K. (2009). Progressive postnatal motoneuron loss in mice lacking GDF-15. J. Neurosci. 29, 13640-13648. https://doi.org/10.1523/JNEUROSCI.1133-09.2009.

31. Laurens, C., Bourlier, V., Mairal, A., Louche, K., Badin, P.M., Mouisel, E., Montagner, A., Marette, A., Tremblay, A., Weisnagel, J.S., et al. (2016). Perilipin 5 fine-tunes lipid oxidation to metabolic demand and protects against lipotoxicity in skeletal muscle. Sci. Rep. 6, 38310. https://doi.org/10.1038/srep38310.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td><strong>Antibodies</strong></td><td></td><td></td></tr><tr><td>Mouse monoclonal anti-OXPHOS</td><td>Abcam</td><td>Cat#ab110411</td></tr><tr><td>Rabbit monoclonal anti-Akt</td><td>Cell Signaling Technology</td><td>Cat# 4691L</td></tr><tr><td>Rabbit monoclonal anti-pS473 Akt</td><td>Cell Signaling Technology</td><td>Cat# 4060L</td></tr><tr><td>Rabbit monoclonal anti-pThr308 Akt</td><td>Cell Signaling Technology</td><td>Cat# 2965L</td></tr><tr><td>Mouse monoclonal anti-GLUT4</td><td>Cell Signaling Technology</td><td>Cat# 2213</td></tr><tr><td><strong>Biological samples</strong></td><td></td><td></td></tr><tr><td>Plasma samples from human study 1</td><td>Vion et al.<sup>22</sup></td><td>N/A</td></tr><tr><td><strong>Chemicals, peptides, and recombinant proteins</strong></td><td></td><td></td></tr><tr><td>Ripa buffer</td><td>Sigma-Aldrich</td><td>Cat#R0278</td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>Cat# P8340/P5726/P0044</td></tr><tr><td>Qiazol</td><td>Qiagen</td><td>Cat#79306</td></tr><tr><td>Fast SYBR green Master Mix</td><td>Life technology</td><td>Cat#4385614</td></tr><tr><td>Taqman Fast Master Mix</td><td>Life technology</td><td>Cat#4444558</td></tr><tr><td>2-[1,2-<sup>3</sup>H(N)]deoxy-D-glucose</td><td>PerkinElmer</td><td>Cat# NET328A250UC</td></tr><tr><td><strong>Critical commercial assays</strong></td><td></td><td></td></tr><tr><td>Mouse/Rat GDF-15 Quantikine ELISA Kit</td><td>R&amp;D Systems</td><td>Cat#MGD150</td></tr><tr><td>sMouse Ultrasensitive Insulin ELISA</td><td>ALPCO</td><td>Cat#80-INSMSU-E01</td></tr><tr><td>Qiagen RNeasy kit</td><td>Qiagen</td><td>Cat #74106</td></tr><tr><td>Kit High capacity cDNA Reverse Transcription kit</td><td>Applied Biosystem</td><td>Cat#4368814</td></tr><tr><td>BCA pierce kit</td><td>ThermoScientific</td><td>Cat # 23225</td></tr><tr><td><strong>Experimental models: Organisms/strains</strong></td><td></td><td></td></tr><tr><td>Gdf15 KO and WT C57BL6/J mice</td><td>N/A</td><td>Strelau et al.<sup>30</sup></td></tr><tr><td><strong>Oligonucleotides</strong></td><td></td><td></td></tr><tr><td>Tables S1 and S2</td><td>N/A</td><td>N/A</td></tr><tr><td><strong>Software and algorithms</strong></td><td></td><td></td></tr><tr><td>GraphPad Prism 10.0.2</td><td>GraphPad software</td><td>https://www.graphpad.com/</td></tr><tr><td>ImageLab 4.2 version software</td><td>Biorad</td><td>https://bio-rad.com/</td></tr><tr><td>BioRender</td><td>BioRender</td><td>https://app.biorender.com/</td></tr><tr><td><strong>Other</strong></td><td></td><td></td></tr><tr><td>Rodent chow diet</td><td>SAFE®</td><td>A04</td></tr><tr><td>Rodent high-fat diet</td><td>Ssniff</td><td>D12492</td></tr></tbody></table>

### RESOURCE AVAILABILITY

#### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Cedric Moro (cedric.moro@inserm.fr).

#### Materials availability

This study did not generate new unique reagents.

#### Data and code availability

* All data reported in this paper will be shared by the lead contact upon request.

* This paper does not report original code.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
<figure><img src="image_21.png" /></figure>

# EXPERIMENTAL MODEL AND SUBJECT DETAILS

## Mouse study 1

Sixteen weeks old male and female WT and Gdf15 KO mice under a C57BL/6J background were housed in a pathogen-free barrier facility (12h light/dark cycle) with ad libitum access to standard chow diet (SAFE A04) and water in standard animal care facility rooms at 21°C. Gdf15-deficient mice (Gdf15 KO) were generated as previously described and kindly provided by Prof. Klaus Unsicker (University of Freiburg, Freiburg, Germany).³⁰ Mice were weighted weekly and body composition was assessed by the use of a Mini-Spec (Minispec MQ-60, Bruker). At the end of the protocol, mice were killed and blood was collected into EDTA tubes containing protease inhibitors. Organs and tissues were rapidly excised and either snap frozen in liquid nitrogen before being stored at -80°C or processed for histology. All experimental procedures were approved by our institutional animal care and use committee CEEA-122 of UMS006 CREFRE (protocol APAFIS#7796-2016112509262218) and performed according to INSERM guidelines for the care and use of laboratory animals.

## Mouse study 2

Six weeks old male and female WT and Gdf15 KO mice under a C57BL/6J background were housed in 12h light/dark cycle with ad libitum access to high-fat diet (HFD; Ssniff D12492) and water in standard animal care facility rooms at 21°C. Gdf15-deficient mice (Gdf15 KO) were generated as previously described. Mice were weighted weekly and were subjected to exercise and metabolic phenotyping after 4 weeks under HFD. Mice were kept under HFD, acclimatized to and trained on treadmill according to the same protocol as described above. Exercise and metabolic phenotyping was assessed after 4 weeks of exercise training and overall 8 weeks of HFD. All experimental procedures were approved by our institutional animal care and use committee CEEA-122 of UMS006 CREFRE (protocol APAFIS#7796-2016112509262218) and performed according to INSERM guidelines for the care and use of laboratory animals.

## Clinical studies and human subjects

### Human study 1

Sixteen obese men (age 49.6 ± 3.6 years; BMI 33.7 ± 0.5 kg m⁻²; VO2max 24.9 ± 1.2 mL kg⁻¹.min⁻¹) participated in clinical trial NCT02161926. Core clinical data of this study have been published elsewhere.²² The protocol was approved by the Ethics Committee of Toulouse University Hospitals, and all subjects gave written informed consent. Subjected participated in an 8-week lifestyle intervention including an exercise training program. Maximal oxygen uptake (VO2max) was investigated on a bicycle ergometer by indirect calorimetry at baseline. Participants exercised 2 times per week under direct supervision and 3 times per week on their own following detailed instructions. All daily sessions consisted of 10 min warm-up at 40% VO2max on bicycle ergometer followed by endurance training with progressive increase in exercise intensity (at 60% VO2max) and duration (5-15 min), intense 1 min exercise bouts at 80-90% VO2max followed by 4 min at 40% VO2max (the number of which progressively increased during the intervention) and, muscle strengthening of the lower and upper limbs. Muscle stretching completed each session. Each subject performed an hyperinsulinemic euglycemic clamp before and at the end of the training program. Venous blood samples were drawn in the fasting state, before exercise, and immediately post-exercise.

### Hyperinsulinemic euglycemic clamp

The participants were asked to refrain from vigorous physical activity 48 h before presenting to the clinical investigation center, and they ate a weight-maintaining diet consisting of 35% fat, 15% protein, and 50% carbohydrates 2 days before the experiment. After an overnight fast, insulin (80 mU.m⁻².min⁻¹) and 20% glucose (to maintain plasma glucose at 90 mg/dL) were administered for 2h.²⁷ Glucose and insulin were measured in 3 independent blood plasma samples 10 min apart at baseline and again at steady-state during the last 30 min of the clamp. The glucose infusion rate was used to calculate the glucose disposal rate (GDR) which was further normalized to lean body mass (M value). Blood was collected on EDTA and immediately processed for plasma storage at -80°C.

### Blood analyses

GDF15 was measured in human plasma using the Quantikine ELISA Human GDF15 Immunoassay (ELISA, R&D systems, catalog no. DGD150). Insulin was measured with an ELISA kit (80-INSHU-E01.1, ALPCO Diagnostics, Salem, New Hampshire).

# METHOD DETAILS

## Blood analyses

Mouse plasma GDF15 was measured by ELISA (R&D Systems, Minneapolis, USA) following manufactory instructions. Plasma insulin was measured using an ultrasensitive ELISA kit (80-INSMSU-E01, ALPCO Diagnostics, Salem, New Hampshire).

## Glucose and insulin tolerance tests

After a 2h fast,⁵ a bolus of 1.5 g/kg of glucose (Sigma-Aldrich, Saint-Quentin Fallavier, France) was administered by oral gavage. For insulin tolerance test, mice were injected with 0.75 U/kg of insulin (NovoRapid 100 IU/mL) respectively. Blood glucose levels were monitored from the tip of the tail vein with a glucometer (Accuccheck; Roche, Meylan, France) at 0, 15, 30, 45, 60, 75 and 90 min iAUC (incremental area under the curve) and AAC (area above the curve) were calculated.
## Tissue specific 2-deoxyglucose uptake in vivo

To determine 2-DG uptake in muscle and adipose tissue, a bolus injection of 2-[1,2-³H(N)]deoxy-D-glucose (PerkinElmer, Boston, Massachusetts) (0.4 µCi/g body weight) and insulin (3 mU/g body weight) was injected intraperitoneally in mice as previously described.³¹ Then, 15 min after injection, blood glucose was measured, mice were euthanized and tissues were excised and quickly frozen at -80°C until further processing.

## Acclimation to treadmill and maximal running capacity test

Mice were acclimated to the treadmill (TSE Systems) four consecutive days by running for 2 min at a speed of 0.10 m/s, then 3 min at a speed of 0.10 ms/s and finally 7 min at a speed of 0.20 m/s at 0° incline during the week prior the maximal running capacity test. The maximal running capacity test to determine maximal speed was performed at 0.15 m/s and was increased by 0.05 m/s every 2 min until exhaustion.

## Endurance exercise test

For the endurance exercise test, after a warm up of 2 min at a speed of 0.10 m/s, mice ran 80 min at 60% of maximal capacity previously determined for each mouse. At exhaustion, mice were removed from the test and running time to exhaustion, distance covered and average running speed were measured.

## Endurance training

Then endurance training group ran 30 min per day, 4 days a week including two consecutive days' sessions for 4 weeks. Running speed was fixed at 60% of maximal capacity for each mouse.

## RNA extraction and real-time qPCR

Total RNA from tissue was isolated using Qiagen RNeasy kit (Qiagen, GmbH Hilden, Germany) following manufacturer's protocol. The quantity of the RNA was determined on a Nanodrop ND-1000 (Thermo Scientific, Rockford, IL, USA). Reverse-transcriptase PCR was performed using the Multiscribe Reverse Transcriptase method (Applied Biosystems, Foster City, CA). Quantitative Real-time PCR (qRT-PCR) was performed in duplicate using the ViiA 7 Real-time PCR system (Applied biosystems). All expression data were normalized by the 2^(-ΔCt) method using Tbp as housekeeping gene. Primer sequences are listed in Tables S1 and S2.

## Western blot

Proteins were extracted from tissues using RIPA buffer and protease inhibitor cocktail (Sigma-Aldrich). Tissues homogenates were centrifuged twice for 20 min at 12700 rpm and supernatants were quantified with BCA pierce kit (ThermoScientific). Equal amount of proteins was run on a 4-20% SDS-polyacrylamide gel electrophoresis (Biorad), transferred onto nitrocellulose membrane (Bio-Rad) and incubated overnight at 4°C with primary antibodies, Rabbit anti-pS473 AKT (1:1000, CST, #4060L), anti-pThr308 AKT (1:1000, CST, #2965L), anti-AKT (1:1000, CST, #4691L), and mouse anti-GLUT4 (1:1000, CST, #2213) and anti-OXPHOS (1:1000, Abcam, ab110411). Subsequently, immuno-reactive proteins were blotted with anti-rabbit or mouse horseradish peroxidase-labeled secondary antibodies for 1h at room temperature and revealed by enhanced chemiluminescence reagent (SuperSignal West Femto, Thermo Scientific), visualized using ChemiDoc MP Imaging System and data analyzed using the ImageLab 4.2 version software (Bio-Rad Laboratories, Hercules, USA).

## QUANTIFICATION AND STATISTICAL ANALYSIS

All statistical analyses were performed using GraphPad Prism 10.0.2 for Windows (GraphPad Software Inc., San Diego, CA). Normal distribution and homogeneity of variance of the data were tested using Shapiro-Wilk and F tests, respectively. Student's and Wilcoxon t-tests, Mann-Whitney test, Mantel-Cox test and two-way ANOVA followed by Bonferroni's post hoc tests were applied when appropriate. All values in Figures are presented as mean ± SEM. Statistical significance was set at *p* < 0.05.