Metformin-induced increases in GDF15 are important for suppressing appetite and promoting weight loss

Abstract

Metformin is the most commonly prescribed medication for type 2 diabetes, owing to its glucose-lowering effects, which are mediated through the suppression of hepatic glucose production (reviewed in refs. 1,2,3). However, in addition to its effects on the liver, metformin reduces appetite and in preclinical models exerts beneficial effects on ageing and a number of diverse diseases (for example, cognitive disorders, cancer, cardiovascular disease) through mechanisms that are not fully understood1,2,3. Given the high concentration of metformin in the liver and its many beneficial effects beyond glycemic control, we reasoned that metformin may increase the secretion of a hepatocyte-derived endocrine factor that communicates with the central nervous system4. Here we show, using unbiased transcriptomics of mouse hepatocytes and analysis of proteins in human serum, that metformin induces expression and secretion of growth differentiating factor 15 (GDF15). In primary mouse hepatocytes, metformin stimulates the secretion of GDF15 by increasing the expression of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP; also known as DDIT3). In wild-type mice fed a high-fat diet, oral administration of metformin increases serum GDF15 and reduces food intake, body mass, fasting insulin and glucose intolerance; these effects are eliminated in GDF15 null mice. An increase in serum GDF15 is also associated with weight loss in patients with type 2 diabetes who take metformin. Although further studies will be required to determine the tissue source(s) of GDF15 produced in response to metformin in vivo, our data indicate that the therapeutic benefits of metformin on appetite, body mass and serum insulin depend on GDF15.

Main

Metformin is one of the most widely used medications in the world. It is a strong base that exists in its protonated form at physiological pH and therefore does not pass through cellular membranes easily. In rodents, oral administration of metformin (250–300 mg kg–1 body weight) results in clinically relevant plasma concentrations of approximately 10–15 μM; however, concentrations in the liver are much higher (40–1,000 μM) than in other organs5,6. Similar tissue distributions7,8 and serum concentrations9 have been found in humans. This accumulation of metformin in the liver is important for the suppression of hepatic glucose production, which involves the inhibition of fructose-1-6-bisphosphatase10 and mitochondrial glycerol-3-phosphate6, and for the activation of AMP-activated protein kinase (AMPK), which improves insulin sensitivity through the phosphorylation and inhibition of acetyl-CoA carboxylase (ACC)11. Metformin may also lower blood glucose by acting in the gastrointestinal tract, where it alters the gut microbiome12,13 and stimulates glucagon-like peptide-1 (GLP-1) release14; however, increases in GLP-1 are not required for metformin-induced glucose lowering15.

In addition to lowering blood glucose, metformin consistently induces weight loss in people with or without type 2 diabetes16,17,18,19,20,21. This effect on weight loss is not due to increases in energy expenditure17,22, but instead involves the suppression of appetite18,23. Many preclinical studies have also observed beneficial effects of metformin for slowing ageing and treating a multitude of diseases, including cognitive disorders, several cancers, and cardiovascular disease. These findings have laid the foundation for the initiation of many clinical trials, but given the low concentrations of metformin outside of the gastrointestinal tract and the liver, the mechanisms by which metformin suppresses appetite and elicits multiple benefits remain unclear. Given the emerging role of hepatokines in regulating metabolism, we reasoned that metformin may increase the secretion of a hepatocyte-derived endocrine factor that communicates with the central nervous system to produce beneficial effects4.

We first examined transcriptional changes that occurred in response to acute metformin treatment in primary mouse hepatocytes from wild-type C57Bl6J mice and found significant changes in the expression of 1,403 transcripts (722 upregulated and 681 downregulated) (Extended Data Fig. 1a and Supplementary Table 1). To determine which of these transcripts could be secreted, we cross-referenced this list with the mouse secretome24 and found 51 (33 upregulated and 18 downregulated) secreted gene products whose expression was altered by metformin treatment (Supplementary Table 2). To determine which of these transcripts were of potential clinical relevance, we examined 900 proteins in the serum of 16 metformin-naive subjects in the Remission Evaluation of Metabolic Interventions in Type 2 Diabetes (REMIT) pilot trial25 who had been randomized to metformin (n = 10) or no metformin (continuing standard care, n = 6) treatment for 8 weeks. Subject characteristics are provided in Supplementary Table 3. Of the upregulated secreted gene products in mouse hepatocytes (Supplementary Table 2), the most significantly upregulated corresponding protein in the serum of human subjects who received metformin (upregulated 1.8-fold relative to subjects who received standard care) was growth differentiating factor 15 (GDF15) (Fig. 1a).

Fig. 1: Metformin increases serum GDF15 and is associated with reductions in body mass in subjects with type 2 diabetes.
figure1

a, Serum GDF15 measured at baseline and at 8-week follow-up after randomization to standard care (control, n = 6) or metformin (n = 10). Data are presented as mean ± s.e.m. *P < 0.05 for repeated measures two-way ANOVA with Sidak multiple comparison test. b, Change in body mass versus change in serum GDF15 in subjects receiving standard care (control, n = 6) or metformin (n = 10) for 8 weeks. A linear model was constructed with the change in protein level from baseline to 8 weeks as the dependent variable and metformin status as the independent variable. Two-sided P values are reported.

GDF15 is a member of the transforming growth factor beta (TGF-β) superfamily and is highly expressed in the liver26. Recent studies have indicated that recombinant GDF15 suppresses appetite and promotes weight loss through interactions with the GDNF family receptor α-like (GFRAL) receptor in the hindbrain27,28,29,30. Consistent with these observations, increases in serum GDF15 were associated with weight loss in patients with type 2 diabetes taking metformin and those receiving standard care (Fig. 1b). These data indicate that metformin increases GDF15 messenger RNA expression in mouse hepatocytes and that increases in serum GDF15 are associated with weight loss in people with type 2 diabetes; however, the primary tissue(s) contributing to this increase in GDF15 in vivo are unknown.

We conducted studies in primary mouse hepatocytes to examine potential mechanisms that might link metformin to elevated serum GDF15. Metformin increased GDF15 expression by 55% (Fig. 2a) and increased GDF15 release into the medium in a dose-dependent manner (Fig. 2b). The structurally similar biguanides phenformin and buformin also increased GDF15 release from hepatocytes (Extended Data Fig. 1b,c). It has been suggested that metformin regulates multiple pathways secondary to the inhibition of mitochondrial complex I (refs. 1,2,3). However, rotenone, a potent non-reversible complex I inhibitor, did not increase GDF15 release from hepatocytes (Extended Data Fig. 1d). Metformin increases AMPK and ACC phosphorylation11; however, GDF15 secretion was not altered in hepatocytes that genetically lacked the AMPK β1 subunit (which reduces AMPK activity in hepatocytes by approximately 90%) or AMPK phosphorylation sites on ACC (ACC DKI) (Extended Data Fig. 1e). These data suggest that the inhibition of complex I or the activation of AMPK are not required for metformin-stimulated GDF15 release from hepatocytes.

Fig. 2: Metformin increases GDF15 release from hepatocytes through an integrated stress response pathway.
figure2

a, Metformin treatment increases GDF15 mRNA levels (n = 4 per group). *P < 0.05 for t-test with 1,000 permutations of the data. b, Metformin increases GDF15 release from primary hepatocytes in a dose-dependent manner over 24 h of exposure at doses of 0 μM (n = 6), 100 μM (n = 3), 250 μM (n = 3), 500 μM (n = 6), 750 μM (n = 3) and 1,000 μM (n = 4). In each case, n is the number of biologically independent samples for which data were averaged from triplicate or quadruplicate measurements. **P < 0.01 and ***P < 0.001, one-way ANOVA with Sidak multiple comparison test. ce, Metformin increases protein expression of ATF4 (n = 5 for control and metformin) and CHOP (n = 4 for control and metformin) relative to β-actin in hepatocytes over 24 h. fh, siRNA knockdown of ATF4 blunts metformin-induced ATF4 expression and GDF15 release (n = 4 except for ATF4 siRNA without metformin, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA with Sidak multiple comparison test. i,j, Genetic deletion of CHOP prevents metformin-induced GDF15 secretion (n = 4 independent samples). ***P < 0.0001, two-way ANOVA with Sidak multiple comparison test. Data are presented as mean ± s.e.m.

Source data

In multiple cell types, a critical regulator of GDF15 transcription is the integrated stress response that culminates in interactions between CHOP and ATF4 (ref. 26). Consistent with previous reports that metformin acutely activates the integrated stress response31,32, metformin increased ATF4 and CHOP expression (Fig. 2c–e). To determine whether ATF4 and CHOP were essential for metformin-induced GDF15 secretion, we used ATF4 siRNA. This siRNA did not reduce basal levels of ATF4, possibly owing to low ATF4 turnover in hepatocytes; however, it did prevent the increase in ATF4 seen with metformin treatment (Fig. 2f,g). Importantly, ATF4 siRNA blunted the increase in GDF15 in response to metformin treatment (Fig. 2h). Hepatocytes generated from CHOP-null mice were also refractory to metformin (Fig. 2i,j). These data demonstrate that metformin increases the secretion of GDF15 from hepatocytes through ATF4 and CHOP.

To examine the mechanism and potential physiological importance of metformin-induced increases in GDF15, we generated GDF15-knockout (GDF15-KO) mice and performed experiments using a single oral gavage of metformin(250 mg kg–1 body weight). The dose of metformin was selected because it has been shown to elicit clinically equivalent serum concentrations of metformin in mice, and in agreement with previous studies5, we found serum metformin concentrations of approximately 150 μM (Extended Data Fig. 2a). On a control chow diet, treatment of wild-type mice with metformin increased serum GDF15 levels, but this effect was not observed in GDF15-KO mice (Fig. 3a). In separate experiments, mice were treated with a single oral gavage of saline or metformin (250 mg kg−1) while housed in metabolic cages to monitor food intake, physical activity levels, respiratory quotient and energy expenditure; metformin reduced food intake to a similar degree in both wild-type and GDF15-KO mice (Fig. 3b–d). Experiments were then repeated in mice fed a high-fat diet (HFD). Metformin increased serum GDF15 in wild-type but not GDF15-KO mice (Fig. 3e). However, in contrast to mice fed a chow diet, this was accompanied by reduced food intake in wild-type but not GDF15-KO mice (Fig. 3f–h). Differences in food intake between wild-type and GDF15-KO mice after metformin treatment were unlikely to have been due to GLP-1 levels, as these were comparable between genotypes (Extended Data Fig. 2b). Consistent with differences in food intake between genotypes, metformin also reduced the respiratory exchange ratio (RER) in wild-type but not GDF15-KO mice (Extended Data Fig. 2c,d). There were no differences in other metabolic parameters, including physical activity (beam breaks) and energy expenditure (Extended Data Fig. 2e,f). These data indicate that metformin acutely suppresses appetite through GDF15 in mice fed a HFD.

Fig. 3: Metformin reduces food intake through GDF15 in mice fed a HFD.
figure3

a, Serum GDF15 in mice fed a control diet 6 h after an oral gavage of metformin (250 mg kg–1) or an equal volume of saline (control). Wild-type control (n = 7), wild-type metformin (n = 7), GDF15-KO control (n = 6), GDF15-KO metformin (n = 6). bd, Food intake after gavage. Wild-type control (n = 7), wild-type metformin (n = 9), GDF15-KO control (n = 4), GDF15-KO metformin (n = 5). e, Serum GDF15 of mice fed a 45% HFD for 2–4 weeks, measured 6 h after a single oral gavage of metformin (250 mg kg–1) or an equal volume of saline (n = 6 per group). fh, Mice fed a 45% HFD treated with metformin (250 mg kg–1) or an equal volume of saline (control) by gavage 2 h before the onset of the dark period (indicated by arrow). Food intake was measured for 24 h after gavage. Wild-type control (n = 7), wild-type metformin (n = 8), GDF15-KO control (n = 6), GDF15-KO metformin (n = 6). Data are presented as mean ± s.e.m. *P < 0.05 for two-way ANOVA with Sidak multiple comparison test (a,d,e,h); § indicates overall effect of metformin (b,c,f). †P < 0.05 for overall effect of genotype (GDF15-KO, a,e).

We subsequently examined the potential chronic consequences of metformin exposure by treating wild-type and GDF15-KO mice fed a HFD with metformin in drinking water. In wild-type mice, chronic metformin treatment lowered food intake and reduced weight gain over time (Fig. 4a,b,d). By contrast, metformin did not suppress food intake or weight gain in GDF15-KO mice (Fig. 4a,c,d). Importantly, metformin lowered fasting insulin (Fig. 4e) and improved glucose tolerance in wild-type mice (Fig. 4f,h); effects that were eliminated in GDF15-KO mice (Fig. 4g,i). These data indicate that in mice fed a HFD, metformin suppresses appetite, induces weight loss, reduces serum insulin and improves glucose tolerance through increases in GDF15.

Fig. 4: Metformin reduces body mass and serum insulin, and improves glucose tolerance through GDF15.
figure4

Mice were fed a 45% HFD and provided ad libitum access to water (control) or water containing metformin. a, Food intake at week 7 of treatment in wild-type control (n = 11), wild-type metformin (n = 6), GDF15-KO control (n = 13) and GDF15-KO metformin (n = 8) mice. b,c, Body mass in wild-type control (n = 7), wild-type metformin (n = 9), GDF15-KO control (n = 8) and GDF15-KO metformin (n = 8) mice. *P < 0.05, repeated measures two-way ANOVA with Sidak multiple comparison test. d,e, Total weight gain over 10 weeks (d) and 12 h fasting serum insulin (e) in control (n = 4 for both genotypes) and metformin-treated (n = 5 for both genotypes) mice. *P < 0.05, t-test with Bonferroni–Sidak correction for multiple comparisons. fh, Glucose tolerance assessed after a 6 h fast and bolus of glucose (1.2 g kg−1 body weight). Wild-type control (n = 7), wild-type metformin (n = 9), GDF15-KO control (n = 7), GDF15-KO metformin (n = 7). AUC, area under curve. Data are presented as mean ± s.e.m. *P < 0.05 between control and metformin by 2-way ANOVA with Sidak multiple comparison test (a,d,e,h); § indicates overall effect of metformin using a repeated measures two-way ANOVA (b,f). †P < 0.05 for overall effect of genotype (GDF15-KO, e).

To further examine the mechanisms contributing to metformin-related weight loss, we assessed adiposity, physical activity, RER, energy expenditure and water consumption. Metformin tended to reduce adiposity in wild-type but not GDF15-KO mice without altering lean mass in either genotype (Extended Data Fig. 3a,b). There were no differences in physical activity, RER or energy expenditure (Extended Data Fig. 3c–e), even when these measurements were corrected for body mass or lean mass (Extended Data Fig. 3f–i). Reductions in feeding in wild-type mice treated with metformin were unlikely to be due to taste aversion, as water intake was unchanged with metformin supplementation and resulted in a daily metformin dose of approximately 250 mg kg–1 per day, as expected (Extended Data Fig. 4a,b). Consistent with previous literature5, this dose of metformin delivered through the drinking water resulted in serum metformin concentrations of approximately 5 μM that did not differ between groups (Extended Data Fig. 4c). Lastly, we examined whether the effects of metformin on serum GDF15 could be secondary to caloric restriction by matching the reduced food intake induced by metformin to mice that did not receive any metformin (Extended Data Fig. 4d). Only metformin-treated mice had increased serum GDF15 (Extended Data Fig. 4e). These data indicate that reductions in weight gain with metformin are due to GDF15 suppression of appetite.

Given the low systemic concentrations of metformin and its wide-ranging beneficial effects on whole-body parameters, including the suppression of appetite, we reasoned that metformin may induce the expression of a metformin-regulated endocrine factor or ‘metokine’4. We find that metformin induces the expression of GDF15 in hepatocytes through a mechanism requiring ATF4 and CHOP. Furthermore, clinically relevant dosing of metformin increases serum GDF15 in mice and humans. The finding that metformin acutely increases serum GDF15 is consistent with our previous research33 and that of others34, which show that serum GDF15 is correlated with metformin dose but not with the use of other glucose-lowering therapies in patients with insulin resistance or type 2 diabetes. Our data importantly establish that increases in serum GDF15 are not secondary to other actions of metformin, such as insulin sensitization, glucose-lowering and weight loss. Although our in vitro studies focused on the mechanisms by which metformin stimulates GDF15 release from hepatocytes, the primary tissue(s) that contribute to increases in serum GDF15 in vivo are not known. Given that metformin accumulates to high levels in both the liver and the gastrointestinal tract, future studies with tissue-selective GDF15-KO mice will be important to establish the relative contribution of these tissues to serum GDF15 levels and appetite regulation.

Our studies also establish the potential clinical significance of metformin-induced increases in GDF15 by demonstrating the importance of GDF15 for reductions in appetite and weight gain in mice fed a HFD. The GDF15-dependent effects of metformin to suppress appetite and weight gain in mice fed a HFD are consistent with findings that recombinant GDF15 induces weight loss through GFRAL inhibition of appetite without altering energy expenditure27,28,29,30. Interestingly, when mice were fed a control chow diet, metformin continued to suppress appetite in GDF15KO mice, thus suggesting that there are interactions between GDF15 and diet that require further investigation.

Our findings open a number of avenues of research. There are currently over 1,500 registered clinical trials to test the effects of metformin in different diseases, including cancers, cardiovascular disease and even ageing (ClinicalTrials.gov database, https://clinicaltrials.gov). Mice overexpressing GDF15 have enhanced lifespan and are protected from atherosclerotic cardiovascular disease35,36,37,38,39,40. These phenotypes are remarkably similar to those induced by metformin, which also reduces cardiovascular disease and potentially improves lifespan41,42. Therefore, the possibility that GDF15 has a causal role in multiple beneficial effects of metformin treatment warrants further investigation.

Methods

Cell culture

All animal experiments were approved by the McMaster University Animal Research Ethics Board. Mice were group-housed at conventional temperatures (22–23 °C) on a 12 h light–dark schedule with ad libitum access to food and water unless otherwise indicated. Primary hepatocytes were isolated from 10–20-week-old male AMPKβ1−/−(ref. 43), ACC DKI11, CHOP−/−(ref. 44) (purchased from JAX), and age-matched wild-type C57Bl6J control mice. After anaesthesia, livers were perfused with a solution containing 500 µM EGTA, followed by collagenase (320 U ml−1, Sigma, C5138), and cells were gently teased from the livers. Cells were plated in appropriate tissue culture dishes to approximately 85% confluency and allowed to adhere overnight in William’s Medium E supplemented to a final concentration of 10% FBS, 1% antibiotic-antimycotic solution and 1% l-glutamine (Gibco). The next morning, cells were washed with PBS and fresh 0.1% FBS medium was added. Metformin (500 μM, Sigma) or other treatments were added for 24 h at the concentrations indicated in the figure legends. For siRNA experiments, hepatocytes were isolated and plated. Ten hours later, 10 pmol of either scramble Negative Control siRNA (Qiagen, 1022076) or ATF4 siRNA (Qiagen, 1027417) was added with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) and Opti-MEM I reduced serum medium (Gibco) according to the manufacturer’s instructions. The next morning, medium and siRNA were replaced and metformin (1 mM) was added. Then 24 h after metformin addition, medium was collected and stored at −80 °C for further analysis.

Biochemical analysis

GDF15 levels in cell culture medium and serum were determined by enzyme-linked immunosorbent assay (ELISA, R&D Systems, DY6385). Serum insulin was assessed after a 12 h fast using the Millipore Sigma Rat/Mouse Insulin ELISA kit (EZRMI-13K) according to the manufacturer’s instructions. Serum GLP-1 was determined 10 min after oral gavage of metformin (250 mg kg–1) using the Millipore Sigma Multi Species GLP-1 Total ELISA kit (EZGLP1T-36K). Serum metformin was measured using liquid chromatography–tandem mass spectrometry, as described in ref. 45.

Immunoblotting

Cells were isolated in lysis buffer containing 50 mM HEPES, 150 mM NaCl, 100 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 250 mM sucrose, 1 mM dithiothreitol and 1 mM sodium orthovanadate, with 1% Triton X and one tablet of cOmplete Protease Inhibitor Cocktail (Roche, 11697498001) per 50 ml, then stored at −80 °C until analysis. Protein concentration was determined with the Pierce BCA Protein Assay kit (ThermoFisher). Lysates were then diluted with sample buffer and run on a polyacrylamide gel to separate proteins based on size. Next, samples were transferred to a polyvinylidene difluoride membrane and blocked in 5% BSA for 1 h at room temperature (20–25 °C). Membranes were incubated with primary antibody (1:1,000, except β-actin 1:1,500) overnight at 4 °C. For primary antibody catalogue numbers, please see the Nature Research Reporting Summary. Appropriate secondary antibodies were used at a concentration of 1:10,000. Bound antibodies were detected using Clarity Western ECL Substrate (BioRad).

Illumina sequencing

Primary hepatocytes (collected as described above) were treated in quadruplicate with or without 0.5 mM metformin for 24 h. Total RNA was extracted, and global gene expression patterns were determined using an Illumina MouseRef-8 BeadChip microarray according to the manufacturer’s instructions. Raw microarray probe data underwent background correction, log2-transformation, and normalization using the neqc function of the limma package (version 3.26.9) in R (version 3.2.3, 64-bit platform). Quality control metrics were assessed before and after these steps, and data were filtered to retain only those probes for which at least one sample exhibited detection above background (technical control, P < 0.05). The filtered and normalized dataset was imported into MeV (version 4.9.0)46, where the data were log2-transformed and median-centered prior to t-test analysis with α = 0.05 and 1,000 permutations of the data. To minimize false positives, rank product tests47 were performed on the significant probes as two-class unpaired analyses using α = 0.01, 5,000 permutations of the data, and a false discovery rate of ≤10%.

Human ethics and experiments

The REMIT pilot trial (ClinicalTrials.gov ID: NCT01181674) involved 83 participants with type 2 diabetes who were randomized to receive an 8-week metformin-containing intensive metabolic intervention, a 16-week metformin-containing intensive metabolic intervention, or standard care with or without metformin. Study methods have been described in detail previously25. In a subset of 19 participants who were not on metformin at baseline, serum was assessed at baseline and after 8 weeks using the SOMAscan 1.3k assay (SomaLogic). After manufacturer-recommended quality control, 900 proteins across 16 participants were measured and carried forward for subsequent analysis. Participants were stratified into two groups: intervention participants who were started on metformin (n = 10) or control participants who continued to receive standard care (n = 6). Only one control participant received metformin during follow-up. All protein data were ln-transformed. For each protein, a linear model was constructed with the change in protein level from baseline to 8 weeks as the dependent variable and metformin status as the independent variable. Statistical analyses were performed in R version 3.3.1.

Generation of GDF15-KO mice

The constitutive knockout allele of GDF15 was obtained through deletion of exons 1 and 2 (including the untranslated regions and approximately 2.3 kb of the proximal promoter) via CRISPR–Cas9-mediated gene editing. The proximal and distal guide RNAs were designed and checked for specificity and incidence of off-target binding using the 2012 GRCm38/mm10 Mus musculus genome assembly. The Cas9 and the proximal and distal gRNAs were injected into C57BL/6NJ zygotes. After recovery, 25–35 micro-injected one-cell-stage embryos were transferred to one of the oviducts of pseudopregnant female NMRI mice at 0.5 d post-conception. In total, 509 embryos were transferred in four independent sessions, and 84 pups were born. PCR screening and sequence validation confirmed that four females and three males were positive for the desired mutation. The founder 2112618 was used for expansion and production of study cohorts (hetero- and homozygotes). The genotypes were analysed and confirmed by PCR using genomic DNA extracted from tail biopsies with the following PCR primers: wild-type (WT)-FWD 5′-TTTGGGGGGTGATGATGC-3′, WT-REV 5′-GCGACTTTCTGGGGAAACC-3′, GDF15-KO-FWD 5′-TGCCCATGTGACCTGAGTACAC-3′.

Animal studies

For basal serum GDF15 measurements, samples were collected at 17:00 from 11–13-week-old male mice through heparinized capillary tubes via a small cut in the tail. For single oral gavage studies, male wild-type and GDF15-KO mice, aged 6–10 weeks, were maintained on either a standard chow diet or a 45% HFD for 2–4 weeks. For serum GDF15 measurements, mice were gavaged at the beginning of the light cycle, and blood was collected 6 h later through a small cut in the tail. For metformin measurements, mice were anaesthetized, and blood was collected retro-orbitally 90 min after gavage. For metabolic measurements, mice were placed into metabolic cages and allowed to acclimatize for approximately 24 h. At 17:00 (2 h before the onset of the dark cycle), mice were gavaged with either saline or metformin in saline (250 mg kg–1). Food and fluid intake, ambient locomotor activity, O2 consumption (VO2), CO2 output (VCO2), RER and energy expenditure were measured using a Comprehensive Laboratory Animal Monitoring System (Columbus Instruments) for 24 h after gavage. For chronic studies, 8–10-week-old male wild-type and GDF15-KO mice were switched from a conventional rodent chow diet to a 45% HFD diet for 4 weeks. After 4 weeks of HFD, mice were weight-matched and divided into groups to receive a control treatment (tap water) or 3 g l−1 metformin in H2O, a dose that has been shown to elicit clinically relevant serum metformin concentrations5. Weights were monitored weekly. Body composition measurments were obtained by time-domain nuclear magnetic resonance (Bruker). After 4 weeks of treatment, metabolic testing was initiated. At 7–8 weeks of treatment, food and fluid intake, ambient locomotor activity, VO2, VCO2, RER and energy expenditure were measured using a Comprehensive Laboratory Animal Monitoring System (Columbus Instruments). Glucose tolerance tests were performed after a 6 h fasting period, which began at 7:00. Blood glucose levels were monitored with an ACCU-CHEK Aviva handheld glucometer (Roche) following a small nick in the tail vein. An intraperitoneal injection of 1.2 g kg−1 (body weight) glucose was given at time zero, and blood glucose was measured at the time points indicated in the figure legends.

Statistical analyses

Unless otherwise noted, all statistical analyses were performed using GraphPad Prism (version 8). All data are expressed as mean ± s.e.m. In the case of multiple groups, a one- or two-way analysis of variance (ANOVA, performed as a repeated measures ANOVA for oral gavage experiments and analysis of body mass and glucose tolerance data) with a Tukey’s post-hoc test was used to determine the statistical significance of treatment and interaction effects. For analysis of energy expenditure data corrected for body mass or lean mass, linear regression analysis was used to examine whether slopes and intercepts differed between groups. When only two groups were compared, statistical significance was determined using Student’s t-test. P < 0.05 was considered statistically significant.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Gene array data are available at GEO with accession ID GSE138087. All other data that support the findings of this study are available from the corresponding author upon reasonable request. Source data for Fig. 2 are available online.

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Acknowledgements

The authors would like to thank A. Božović and V. Kulasingam for measuring serum metformin levels. E.A.D. was a recipient of an Ontario Graduate Scholarship (Queen Elizabeth II Graduate Scholarship in Science and Technology) and a Douglas C. Russell Memorial Scholarship. G.R.S. is a Canada Research Chair and the J. Bruce Duncan Chair in Metabolic Diseases. This study was supported by research grants from the Canadian Institutes of Health Research (201709FDN-CEBA-116200 to G.R.S.) and Diabetes Canada (DI-5-17-5302-GS). We thank Sanofi for providing heterozygous breeding pairs of GDF15-null mice.

Author information

E.A.D., R.J.F., B.K.S., N.M., S.H., G.P., H.C.G. and G.R.S. designed the experiments. E.A.D., R.J.F., B.K.S., P.M.S., M.R.M., R.L. and R.M.G. performed the experiments and/or analysed the data. A.R.R. and A.G.M. provided bioinformatics analysis and support. M.K. generated GDF15-KO mice. E.A.D., R.J.F. and G.R.S. wrote the manuscript. All authors edited the manuscript and provided comments.

Correspondence to Gregory R. Steinberg.

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Competing interests

S.H., G.P., H.C.G. and G.R.S. hold a patent entitled ‘Growth differentiation factor 15 as biomarker for metformin’ (WO/2017/108941). S.H. and M.K. are employees of Sanofi. All other authors have no competing interests.

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Peer review information Primary Handling Editor: Christoph Schmitt.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Metformin, phenformin and buformin increase GDF15 release independent of complex 1 inhibition or AMPK.

a, Volcano plot showing differentially regulated genes after 24 h of metformin treatment (metformin versus control, n = 4 per group). b,c, GDF15 release is stimulated in a dose-dependent manner by two biguanides that are structurally similar to metformin: (b) phenformin (0 μM n = 4, 10 μM n = 3, 30 μM n = 3 and 50 μM n = 4) and (c) buformin (0 μM n = 3, 10 μM n = 2, 30 μM n = 2 and 50 μM n = 3). d, The complex I inhibitor rotenone (0 μM, 0.1 μM, 1 μM, 5 μM n = 3) does not increase GDF15 release. e, Metformin increases GDF15 release in primary hepatocytes from WT (control n = 7, metformin n = 7), AMPK β1KO (control n = 4, metformin n = 3) and ACC DKI (control n = 3, metformin n = 3) mice. Data are presented as mean ± s.e.m. For bd, * indicates P < 0.05 and *** indicates P < 0.001 for one-way ANOVA with Sidak multiple comparison test. For e, * indicates P < 0.05 for two-way ANOVA with Sidak multiple comparison test.

Extended Data Fig. 2 Acute metformin treatment did not alter energy expenditure but reduced RER.

a, Serum metformin 1 h after acute saline (n = 4) or metformin gavage (n = 7). *P < 0.05, for unpaired two-sided t-test. b, Serum GLP-1 10 min after acute metformin gavage in wild-type (n = 8) and GDF15-KO (n = 6) mice fed a 45% HFD. cf, Wild-type (control n = 9, metformin n = 9) and GDF15-KO (control n = 6, metformin n = 6) mice were fed a 45% HFD, placed in metabolic cages, and allowed to acclimatize for approximately 24 h before a single oral gavage of metformin (250 mg kg–1) or appropriate volume of saline 2 h before the onset of the dark period. RER, beam breaks and energy expenditure were measured for 24 h after gavage, and data are presented as mean ± s.e.m. *P < 0.05 between control and metformin, two-way ANOVA with Sidak multiple comparison test.

Extended Data Fig. 3 Chronic metformin treatment does not alter lean mass, RER or energy expenditure.

Wild-type (control n = 8, metformin n = 9) and GDF15-KO (control n = 8, metformin n = 8) mice were fed a 45% HFD for 4 weeks prior to being switched to control (tap water) or metformin water (3 g l−1) for 10 weeks. a,b, Body composition was assessed at week 4 of treatment (wild-type control n = 8, wild-type metformin n = 9, GDF15-KO control n = 8 and GDF15-KO metformin n = 8). ci, Wild-type (control n = 7, metformin n = 8) and GDF15-KO (control n = 8, metformin n = 8) mice were placed in metabolic cages and allowed to acclimatize for approximately 24 h. Food intake, activity, RER and energy expenditure were measured over 48 h. Energy expenditure is shown (e) uncorrected, (f,g) corrected for body mass, and (h,i) corrected for lean mass, and data are presented as mean ± s.e.m.

Extended Data Fig. 4 Metformin in drinking water elicits clinically relevant serum metformin levels.

Wild-type and GDF15-KO mice were fed a 45% HFD for 4 weeks prior to being switched to control (tap water) or metformin water (3 g l−1) for 10 weeks. Wild-type (control n = 6, metformin n = 8) and GDF15-KO (control n = 6, metformin n = 7) mice were placed in metabolic cages and allowed to acclimatize for approximately 24 h. a, Water intake was measured over 48 h. b, Metformin dose was calculated based on water intake and body mass. c, Serum metformin was measured after mice were killed at the onset of the light period. d, Food intake of 45% HFD was monitored every 3–4 days in mice fed ad libitum (n = 9), mice fed ad libitum treated with metformin (n = 10), and mice pair-fed with metformin-treated animals (n = 10). e, Serum GDF15 was assessed at 3 weeks. Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.001 between control and metformin, two-way ANOVA with Sidak multiple comparison test (ac), one-way ANOVA with Sidak multiple comparison test (d,e).

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Day, E.A., Ford, R.J., Smith, B.K. et al. Metformin-induced increases in GDF15 are important for suppressing appetite and promoting weight loss. Nat Metab 1, 1202–1208 (2019). https://doi.org/10.1038/s42255-019-0146-4

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