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. 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. Full size image

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. 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. c–e, 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. f–h, 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 Full size image

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. 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). b–d, 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). f–h, 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). Full size image

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. 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. f–h, 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). Full size image

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.