Given the steady decline in metabolic function that typically ocurs with mammalian aging, we aimed to test if exogenous administration of rGDF11 to aged mice could improve their metabolic profile. Our experimental design is shown in Fig. 1A, and employs a dosing regimen that is based on our previous dose-response studies showing that daily administration of exogneous rGDF11 at a dose of 0.5 mg/kg to aged mice is sufficient to reduce age-related left ventricular cardiac hypertrophy in as little as 9 days7. Additionally, this experimental design allowed us to measure multiple metabolic parameters on the same cohort of mice before and after treatment by spacing the analyses over the course of 18 days (11 days of ligand administration and 7 days preceding ligand administration) and allowing the mice to recover between the various metabolic tests. Importantly, we also performed a head-to-head comparison between the phenotypic response observed after delivery of rGDF11 or delivery of rGDF8 in order to determine if a ligand-specific phenotypic outcome occurs.

Figure 1 rGDF11, but not rGDF8, improves glucose tolerance in aged mice. (A) Schematic representation of treatment schedule and analyses performed on young and aged mice fed a normal chow diet. (B,C) Fed body weights and relative weight loss (inset) in young (B, n = 6 mice/treatment) and aged (C, n = 6–8 mice/treatment) mice were measured prior to treatment (pre) and after (post) 9 days (D9) of treatment with saline (black), rGDF11 (orange) or rGDF8 (blue). Inset: the color of the p-value corresponds the to the group compared (black: saline; orange: rGDF11; blue: rGDF8). (D–G) Glucose tolerance tests (D,E; GTT) and insulin tolerance tests (F,G; ITT) were performed on young (D, n = 6 mice/treatment; F, n = 9 mice/treatment) and aged (E, n = 6–8 mice/treatment; G, 9–10 mice/treatment) mice. For GTT, mice were fasted for 18 hours overnight before a bolus of glucose was administered (2 g/kg). For ITT, mice were fasted for 5 hours before injected with insulin (young mice- 0.5 units/kg insulin; aged mice- 0.75 units/kg insulin). Data information: In (B–G), data are presented as mean ± standard deviation. For (B,C), 2-way ANOVA with either Sidak’s or Tukey’s post hoc test was used for comparison between pre vs. post treatment or comparison between groups, respectively. For the insets, an unpaired Student’s t-test was performed for comparison between pre vs. post treatment. For (D–G), 2-way ANOVA with Tukey’s post hoc test was used. For all graphs: * - saline vs. rGDF11; # - rGDF11 vs. rGDF8. Full size image

Administration of rGDF11, but not rGDF8, improved glucose tolerance in aged mice

We found that daily injection of rGDF11 or rGDF8 at equal doses prevented weight gain in wild-type C57BL/6 young male mice (8-week-old; Fig. 1B and Fig. S1), and caused a significant reduction in body weight in aged mice (24-month-old; Fig. 1C and Fig. S1), compared to saline controls. We next sought to determine the effect of each ligand on glucose metabolism in young or aged mice. In order to establish baseline metabolic parameters, we performed glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) on young and aged mice prior to administration of recombinant protein (Fig. S1A–D). Mice then received daily injections of either saline, rGDF11, or rGDF8 followed by repeated GTTs and ITTs following 7 total daily injections (Fig. 1A). Young and aged mice that received rGDF11 showed a small improvement in glucose tolerance (Fig. 1D,E) but showed little improvement in ITT performance (Fig. 1F,G). However, we observed significant differences in the absolute glucose levels during the course of ITT in both young and aged mice following rGDF11 administration, although these differences are likely due to significant differences in their baseline blood glucose levels following the 5 hour fast (Fig. S1G,H). The metabolic improvements observed in aged mice were also commensurate with a significant reduction in body weight which may explain the improved metabolic performance (Fig. 1C), although no weight loss was observed in young mice (Fig. 1B). In contrast, there was little to no effect on glucose tolerance or insulin sensitivity in young or aged mice that received rGDF8 (Fig. 1; Fig. S1G,H). Together, these data establish the impact of exogenous administration of rGDF11 in comparison to rGDF8 on glucose metabolism in the context of aging mice fed a normal chow diet.

Improved glucose tolerance in young and aged mice on a HFD following exogenous administration of rGDF11, but not rGDF8

Using a similar experimental approach as above, we next asked if exogenous delivery of rGDF11 would improve glucose metabolism in young or aged mice fed a long-term high fat diet (HFD; Fig. 2A). As expected, exposure to HFD resulted in an impaired metabolic phenotype in cohorts of young mice (Fig. S2A,B) and aged mice (Fig. S2C,D). These mice, which remained on HFD, were injected daily with either saline, rGDF11 (0.5 mg/kg), or rGDF8 (0.5 mg/kg) again for a total of 11 days. We found that the body weights of both young and aged mice that received rGDF11 were significantly lower at the conclusion of the experiment compared to body weights at the time that the ligand injections began (Fig. 2B,C). Aged mice that received rGDF8 showed a similar reduction in body weight; however, no difference in body weight was observed in the rGDF8-injected young cohort of mice (Fig. 2B,C). Both young and aged mice that received rGDF11 showed significant improvement in the GTT (Fig. 2D,E and Fig. S2E,F), but only young mice showed improved ITT compared to mice that received saline or rGDF8 (Fig. 2F,G and Fig. S2G,H). Despite the reduction in body weight, aged mice that received rGDF8 did not show an overall improvement in GTT or ITT performance (Fig. 2E,G; Fig. S2F,H). However, our GTT results indicate that aged mice injected with rGDF8 have significantly lower blood glucose levels at some timepoints suggesting that injection of rGDF8 may result in an intermediary effect on glucose homeostasis compared to the effects of rGDF11 (Fig. 2E), despite showing a similar reduction in body weight as aged mice that received rGDF11 (Fig. 2C).

Figure 2 Exogenous administration of rGDF11 and rGDF8 to mice fed a HFD results in reduced body weight and significantly improved GTT performance. (A) Schematic representation of treatment schedule and analyses performed on young and aged mice fed a high fat diet. (B,C) Fed body weights and relative weight loss (inset) in young (B, n = 7–8 mice/treatment) and aged (C, n = 6–8 mice/treatment) mice were measured prior to treatment (pre) and after (post) 9 days (D9) of treatment with saline (black), rGDF11 (orange) or rGDF8 (blue). Inset: the color of the p-value corresponds the to the group compared (black: saline; orange: rGDF11; blue: rGDF8). (D–G) Glucose tolerance tests (D,E; GTT) and insulin tolerance tests (F,G; ITT) were performed on young (D, n = 7–8 mice/treatment; F, n = 8–9 mice/treatment) and aged (E, n = 6–8 mice/treatment; G, n = 7–10 mice/treatment) mice. For GTT, mice were fasted for 18 hours overnight before a bolus of glucose was administered (2 g/kg). For ITT, mice were fasted for 5 hours before injected with 1.0 units/kg of insulin. Data information: In (B–G), data are presented as mean ± standard deviation. For (B, C), 2-way ANOVA with either Sidak’s or Tukey’s post hoc test was used for comparison between pre vs. post treatment or comparison between groups, respectively. For the insets, an unpaired Student’s t-test was performed for comparison between pre vs. post treatment. For (D–G), 2-way ANOVA with Tukey’s post hoc test was used. For all graphs: * - saline vs. rGDF11; # - rGDF11 vs. rGDF8; + - saline vs. rGDF8. Full size image

Exogeneous rGDF11 or rGDF8 does not increase insulin secretion, pancreatic β-cell replication, or promote cachexia in aged mice fed a HFD

Increased insulin secretion could account for the observed enhancements in glucose tolerance, and recent reports suggest that supplementation with rGDF11 or AAV-mediated GDF11 overexpression promotes higher plasma insulin and prevents loss of pancreatic β-cells in diabetic mice8,9. We therefore measured blood insulin levels during the GTTs and found no signifcant difference in fasting insulin levels in young or aged mice following injection of saline, rGDF11, or rGDF8 (Fig. 3A,D). Furthermore, neither rGDF11 nor rGDF8 affected glucose stimulated insulin secretion in aged mice fed a HFD (Fig. 3C,E). However, rGDF11 increased glucose stimulated insulin secretion in young mice fed a HFD (Fig. 3B,C). We also measured insulin c-peptide in these cohorts (Fig. S3). We found that young normal chow diet mice, which received rGDF11 or rGDF8 had sigificnatly reduced insulin c-peptide levels in their fed, but not fasted state (Fig. S3A,B). Further, young HFD mice that received rGDF11 showed significantly reduced insulin c-peptide in their fed, but not fasted state, compared to mice that received saline (Fig. S3A,B). We did not observe any differences in insulin c-peptide levels among different treatment groups in the aged chorts (Fig. S3C,D).

Figure 3 Exogenous rGDF11 or rGDF8 has little effect on fasting insulin levels or glucose stimulated insulin secretion (GSIS) in young or aged mice fed a HFD. (A,B) Fasting plasma insulin levels (A) and GSIS (B) in young mice fed a HFD (n = 7–8 mice/treatment). (C) Calculated stimulation index for young (n = 7–8 mice/treatment) and aged (n = 6–8 mice/treatment) mice fed a HFD. (D,E) Fasting plasma insulin levels (D) and GSIS (E) in aged mice fed a HFD (n = 6–8 mice/treatment). (F) Quantification of pancreatic β-cell replication in young (n = 4–6 mice/treatment) and aged (n = 3–6 mice/treatment) mice fed a HFD. (G,H) Gene expression analysis on the hearts (G) and skeletal muscle (H) from aged (n = 6–8 mice/treatment) mice fed a HFD. (I) Plasma GDF15 levels in young (n = 5–6 mice/treatment) and aged (n = 6–8 mice/treatment) mice fed a HFD. Individual values (open circles) for each mouse/treatment are superimposed on the bar graph. Data information: In (A–I), data are presented as mean ± standard deviation. For (A,D,F), 2-way ANOVA with either Sidak’s or Tukey’s post hoc test was used for comparison between pre vs. post treatment or comparison between groups, respectively. For (B,C,E,G–I), 1-way ANOVA with Tukey’s post hoc test was used. Full size image

It is well-described that TGFβ signaling, including GDF11-specific signaling, participates in pancreatic development and regulation of endocrine function26,27,28. Therefore, we next determined whether the improved glucose tolerance observed after 7 days of rGDF11 administration was associated with an increase in β-cell replication in young or aged mice. Pancreata were isolated from young or aged mice and processed for histological analysis. Replication rates were determined by Ki67, insulin, and Nkx6.1 immunofluorescence (Fig. 3F). Neither rGDF11 nor rGDF8 enhanced β-cell replication in young or aged mice (Fig. 3F). However, in young mice, rGDF8 lowered β-cell replication rates (Fig. 3F). Thus, rGDF11, and to a lesser extent rGDF8, enhanced glucose tolerance in aged mice on a HFD, without concomitant increases in insulin secretion or β-cell replication.

A recent report suggested that very high levels or supraphysiological overexpression of GDF11 promotes activin A mediated cachexia and directly invokes an increase in circulating levels of the TGFβ ligand, GDF1514, also known macrophage inhibitory cytokine-1 (MIC1). This study further suggested that elevated GDF15 acts as an appetite suppressant, resulting in weight loss14. As it is known that a reduction in body weight can have a positive influence on glucose homeostasis (reviewed in29), we asked if exogenous administration of either rGDF11 or rGDF8 resulted in upregulation of the TGFβ ligands Gdf15 and Inhba (activin A), or of markers of cachexia, Trim63 and Fbxo32, in the heart and skeletal muscle of aged mice on a long-term HFD (Fig. 3G,H). We found no differences in the mRNA levels of Gdf15, Inhba, Trim63, or Fbxo32 in the heart or skeletal muscle among aged mice that received saline, rGDF11, or rGDF8 (Fig. 3G,H). Furthermore, we evaluated the plasma of young and aged mice fed a long-term HFD following administration of either saline, rGDF11, or rGDF8 and found that there was no difference in the levels of GDF15 within the age groups (Fig. 3I). Though it is possible that exogenous rGDF11 affected GDF15 expression levels at earlier timepoints, our data indicate that GDF15 expression is unaffected following injection of rGDF11 for 11 days and that the exogenous dosing regimen of either rGDF11 or rGDF8 utilized in this study does not promote GDF15-mediated anorexia/weight loss or cachexia at this timepoint.The underlying mechanism responsbile for improved glucose metabolism following rGDF11 administration remains unknown.

Neither rGDF11 nor rGDF8 improves HFD- or age-induced hepatosteatosis

Hepatosteatosis is associated with aging and obesity, as well as with metabolic disorders such as diabetes, hypertension, and dyslipidemia. Defects in lipid metabolism and insulin resistance may contribute to hepatosteatosis (reviewed in30). Given that injection of rGDF11 prevented weight gain, as well as increased hepatic insulin action in aged mice concomitantly fed a HFD, we sought to determine whether rGDF11 could affect hepatic lipid accumulation. After 11 days, the livers from young and aged mice fed a normal chow diet were harvested and processed for histological analysis. We compared the liver histology from the young and aged mice injected with either saline, rGDF11, or rGDF8 from our normal chow diet and long-term HFD experiments. We did not see any differences within treatment groups in young or aged animals injected with saline, rGDF11, or rGDF8 (Fig. S4) suggesting that this duration of rGDF11 administration does not promote reversal of HFD- or age-induced hepatosteatosis.

Exogenous rGDF11, but not rGDF8, prevents weight gain in young and aged mice fed a short-term HFD

Our results provided evidence that exogeneous adminstration of rGDF11 to aged mice promotes an improvement in glucose metabolism. We next asked if supplementation with rGDF11 can provide a ‘protective’ benefit from HFD-induced metabolic stress. To address this question, we exogneously administered saline, rGDF11, or rGDF8 starting the same day as the mice were put on a HFD (Fig. 4A). Normal chow diet fed mice were weighed in order to assess their baseline weight and to match treatment groups with respect to body weights and glucose tolerance. Mice were subsequently fed a HFD and injected daily with either saline, rGDF11, or rGDF8. We measured the body weight of young and aged mice after 9 days of either saline, rGDF11, or rGDF8. As predicted, we found that saline-treated young and aged mice gained weight after 9 days on a HFD (Fig. 4A,B). We found that both rGDF11 and rGDF8 prevented HFD-induced weight gain in aged mice (Fig. 4C). In young mice under the same conditions, only rGDF11 was able to prevent HFD-induced weight gain (Fig. 4B). Cumulatively, these results suggest that while both rGDF11 and rGDF8 can prevent HFD-induced weight gain in aged mice, only rGDF11 treatment can prevent weight gain in young mice on a HFD at the dose and time point evaluated here.

Figure 4 Exogenous rGDF11, but not rGDF8, is protective of HFD-induced weight gain and HFD-induced glucose intolerance. (A) Schematic representation of treatment schedule and analyses performed on young and aged mice. Note that ligand delivery and initiation of HFD occur on the same day. (B,C) Fed body weights and relative weight loss (inset) in young (B, n = 11–12 mice/treatment) and aged (C, n = 11–12 mice/treatment) mice were measured prior to treatment (pre) and after 9 days (D9) of treatment with saline (black), rGDF11 (orange) or rGDF8 (blue). Inset: the color of the p-value corresponds the to the group compared (black: saline; orange: rGDF11; blue: rGDF8). (D,E) Glucose tolerance tests (GTT) were performed on young (D, n = 11–12 mice/treatment) and aged (E, n = 11–12 mice/treatment) mice. For GTT, mice were fasted for 18 hours overnight before a bolus of glucose was administered (2 g/kg). Data information: In (B–E), data are presented as mean ± standard deviation. For (B,C), 2-way ANOVA with either Sidak’s or Tukey’s post hoc test was used for comparison between pre vs. post treatment or comparison between groups, respectively. For the insets, an unpaired Student’s t-test was performed for comparison between pre vs. post treatment. For (D,E), 2-way ANOVA with Tukey’s post hoc test was used. For all graphs: * - saline vs. rGDF11; # - rGDF11 vs. rGDF8; + - saline vs. rGDF8. Full size image

Exogenous rGDF11, but not rGDF8, protects against HFD-induced glucose intolerance in young and aged mice

To determine if rGDF11 or rGDF8 is protective of HFD-induced metabolic stress, we performed GTTs on young and aged mice that received daily injections of either saline, rGDF11, or rGDF8 starting the same day that the mice were put on HFD (Fig. 4A). First, GTTs were performed on young and aged mice fed normal chow diet prior to injection of the recombinant protein and diet change in order to determine the baseline and to match the treatment groups with respect to glucose tolerance (Fig. 4A; Fig. S5A,B). Mice were subsequently fed a HFD and injected daily with either saline, rGDF11, or rGDF8. After 1 week, GTTs were repeated to assess the effects on glucose tolerance. Aged mice, and to a lesser extent, young mice that received rGDF11 showed significantly enhanced glucose clearance compared to saline and rGDF8 treated animals (Fig. 4D,E; Fig. S5C,D). Aged mice that received rGDF8 showed an intermediate phenotype with enhanced glucose tolerance compared to saline controls, but not to the same degree as compared to aged mice that received rGDF11 (Fig. 4E).

In addition to the effects on glucose tolerance, we found that 1 week of daily rGDF11 injections prevented HFD-induced fasting hyperglycemia in aged mice, but not in young mice (Fig. S6A,B). In contrast, fasting blood glucose levels rose in aged mice that received saline or rGDF8 after 1 week on a HFD (Fig. S6B). Furthermore, 9 days of daily injection of rGDF11, but not rGDF8, resulted in reduced fed blood glucose levels in both aging and young mice fed a HFD (Fig. S6C,D).

rGDF11, but not rGDF8, reduces food intake

Next, we aimed to determine the underlying mechanism behind the altered energy balance in rGDF11 supplemented mice. We performed metabolic cage analysis to measure food intake, energy expenditure, respiratory exchange ratio (RER), and physical activity in young and aged mice injected for 7 days with either saline, rGDF11, or rGDF8, starting the same day as the mice were put on HFD (Fig. 4A). Changes in body composition (whole body fat and lean mass) were assessed using 1H-MRS. While young mice injected with saline or rGDF8 gained weight on a HFD, young mice injected with rGDF11 and aged mice from all three cohorts failed to gain weight on the HFD in the CLAMS cages. Indeed, aged mice injected with rGDF11 lost weight during the experimental period (Fig. S7). Young mice, but not aged mice, which received saline or rGDF8 showed a significant increase in total fat mass and no change in total lean mass (Fig. 5A). This resulted in an overall significant increase body fat percentage in young mice treated with saline or rGDF8 (Fig. 5A). On the other hand, fat and lean mass did not change in young mice treated with rGDF11 (Fig. 5A). Interestingly, we did not observe any differences in actual fat or lean mass of aged mice treated with saline, rGDF11, or rGDF8, although aged mice treated with rGDF8 did show a significant reduction in lean mass percentage (Fig. 5B). Taken together, our data suggest that daily injection of rGDF11, but not rGDF8, helps to prevent HFD-induced fat accumulation in young, but not aged, mice and that rGDF11 treatment helps to prevent a loss of relative lean mass in aged mice.

Figure 5 Aged mice fed a short-term HFD with concomitant administration of exogenous rGDF11, but not rGDF8, show a transient reduction in food intake. (A,B) Body composition of young (A, n = 4–6 mice/treatment) and aged (B, n = 5–6 mice/treatment) mice before (pre) and 7 days after (post) concomittant HFD feeding and administration of saline (black), rGDF11 (orange), or rGDF8 (blue). Fat and lean mass values shown on the left and percent of body weight (BW) shown on the right. (C–F) Total daily food intake (C,D) and activity (E,F) were measured from young (C,E, n = 4–6 mice/treatment) and aged (D,F, n = 5–6 mice/treatment) mice before (pre) and 7 days after (post) concomittant HFD feeding and administration of saline (black), rGDF11 (orange), or rGDF8 (blue). Data information: In (A–F), data are presented as mean ± standard deviation. For (A–F), 2-way ANOVA with Tukey’s post hoc test was used for all comparisons. Full size image

Interestingly, we found that aged, but not young, mice injected with rGDF11, but not with rGDF8, showed a significant but transient decline in food intake as early as 3 days after initiation of recombinant protein injections (Fig. 5C,D). The decline in food intake persisted for 48 hours and the rGDF11 injected mice recovered a rate of food intake similar to that observed in the saline injected cohort by day 5 of treatment (Fig. 5D). This decline in food intake in rGDF11 treated mice was associated with a corresponding decline in respiratory exchange rate (RER; Fig. S8). Upon switching the mice from chow diet to HFD we observed a decline in RER in all three cohorts consistent with the diet change and the animals shifting from utilizing carbohydrates as fuel to utilizing fatty acids as fuel (Fig. S8A,B). However, rGDF11-injected mice exhibited reduced RER compared to saline-injected or rGDF8-injected mice at experimental day 3 (Fig. S8B) suggesting that supplementation with rGDF11 results in aged mice using their fat stores as fuel in response to a decline in food intake. Although analysis of energy expenditure showed a baseline difference between rGDF11- and saline-injected aged mice prior to treatment, the energy expenditures measured from rGDF11- or saline-injected mice showed little difference for the remainder of the experiment in both young and aged mice (Fig. S8C,D).

Reductions in food intake could indicate that rGDF11-injected mice were experiencing stress due to the experimental conditions despite our data showing that both young and aged mice injected with rGDF11 exhibited no change in total activity over the course of treatment (Fig. 5E,F). Based on the stability of activity patterns in rGDF11-injected mice, we believe it is unlikely that their transient behavioral change was due to illness, although we cannot rule out the possibility that administration of rGDF11 induced nausea leading to reduced food intake14. Taken together, the data presented here suggest that supplementation with rGDF11 is potentially protective from HFD-induced fat mass accumulation in part by transiently reducing food intake in aging mice leading to the mice utilizing their adipose stores as fuel. Overall, these measurements do not explain the primary tissue compartment or change in behavior responsible for the significant decrease in total body weight observed in aged mice that received rGDF11, suggesting that the weight loss may reflect a combination of changes occurring in multiple tissue depots. Interestingly, young mice injected with saline, rGDF11, or rGDF8 did not show differences in food intake, RER or total activity (Fig. 5E,F and Fig. S8), which may be expected since these mice were young and healthy when the experiment commenced.

rGDF11 increases hepatic insulin action in HFD-fed aged mice

In order to address whether GDF11 treatment increases insulin sensitivity, we performed a 2-hour hyperinsulinemic-euglycemic clamp (2.5 mU/kg/min insulin infusion rate) in young and aged mice injected daily for 7 days with either saline or rGDF11 (Fig. 6A). Steady-state glucose infusion rates required to maintain euglycemia during the clamp were significantly higher in aged mice that received rGDF11 compared to aged mice injected with saline (Fig. 6B), indicating that rGDF11 increased systemic insulin sensitivity in aged mice. This is consistent with improved glucose tolerance in rGDF11-injected mice (Fig. S6). Insulin-stimulated whole-body glucose turnover was significantly increased in aged mice that received rGDF11 as compared to saline-treated mice (Fig. 6C). Thus, these data demonstrate that supplementation with rGDF11 increases peripheral insulin sensitivity in HFD-fed aged mice. During the clamp, a bolus of 14C-labeled 2-deoxyglucose was injected to assess glucose uptake in individual organs31. Increased peripheral insulin sensitivity was largely due to a 2-fold increase in insulin-stimulated glucose uptake in white adipose tissue, while skeletal muscle glucose metabolism was not affected by rGDF11 treatment in aged mice (Fig. S9).

Figure 6 Aged mice fed a short-term HFD with concomitant administration of exogenous rGDF11 show increased insulin action. (A) Protocol design of the hyperinsulinemic-euglycemic clamp experiment following 7 days of exogenous administration of saline (black) or rGDF11 (orange) to young or aged mice. (B,C) Glucose infusion rates (B) and glucose turnover rates (C) for young (n = 6–7 mice/treatment) and aged (n = 7–10 mice/treatment) mice. (D) Calcuated hepatic insulin action for young (n = 6–7 mice/treatment) and aged (n = 7–10 mice/treatment) mice. (E,F) Plasma glucose levels of young (E, n = 6–7 mice/treatment) and aged (F, n = 7–10 mice/treatment) mice before (basal) and during the hyperinsulinemic-euglycemic clamp (clamp) experiment. (G,H) Hepatic glucose production for young (G, n = 6–7 mice/treatment) and aged (H, n = 7–10 mice/treatment) mice before (basal) and during the hyperinsulinemic-euglycemic clamp (clamp) experiment. Data information: In (B–H), data are presented as mean ± standard deviation. For (B,C,E–H), an unpaired Student’s t-test was performed. For (D), 2-way ANOVA with Sidaks’s post hoc test was used to compare each treatment (saline vs rGDF11). Full size image

Hepatic insulin resistance develops with aging and in obesity1,32,33,34. Our hyperinsulinemic-euglycemic clamp study showed that aging-associated hepatic insulin resistance was ameliorated after a 7-day dosing regimen of rGDF11. Plasma glucose levels were monitored before and during the clamp experiment in both young (Fig. 6E) and aged mice (Fig. 6F). Basal hepatic glucose production (HGP) was not affected by rGDF11 treatment in young mice (Fig. 6G,H). In contrast, clamp HGP was significantly reduced in rGDF11-injected aged mice as compared to saline-injected aged mice (Fig. 6H) which is consistent with our data showing that rGDF11 improved hepatic insulin action in aged mice (Fig. 6D). Furthermore, insulin sensitizing effects of rGDF11 were also observed in young mice, albeit less dramatic as glucose infusion rates tended to increase in young mice that received rGDF11 as compared to saline-injected young mice (Fig. 6B). Taken together, exogenous delivery of rGDF11 to aged mice resulted in an improved metabolic phenotype. However, our current approaches and data obtained to pinpoint a definitive mechanism of action suggest that there are likely multiple mechanisms responsible for the observed metabolic effects.