GDF11 Attenuates Development of Type 2 Diabetes Via Improvement of Islet B-Cell Function and Survival

1914 Diabetes Volume 66, July 2017

GDF11 Attenuates Development of Type 2 Diabetes via Improvement of Islet b-Cell Function and Survival

Huan Li,1 Yixiang Li,2 Lingwei Xiang,3 JiaJia Zhang,1 Biao Zhu,1 Lin Xiang,1 Jing Dong,1 Min Liu,1 and Guangda Xiang1

Diabetes 2017;66:1914–1927 | https://doi.org/10.2337/db17-0086

Growth differentiation factor 11 (GDF11) has been impli- reverses age-related phenotypes in multiple organs, includ- cated in the regulation of islet development and a variety ing the heart, skeletal muscle, and the cerebral vasculature of aging conditions, but little is known about the physio- (9–11). Strikingly, recently published data argued that logical functions of GDF11 in adult pancreatic islets. Here, GDF11 repletion inhibited muscle regeneration and failed we showed that systematic replenishment of GDF11 not to rejuvenate cardiac pathologies in mice (12,13). Very im- only preserved insulin secretion but also improved the portantly, Poggioli et al. (14) confirmed that administration b survival and morphology of -cells and improved glucose of recombinant GDF11 (rGDF11) reverses cardiac hypertro- metabolism in both nongenetic and genetic mouse mod- phy in both young and old mice. Furthermore, GDF11 pro- els of type 2 diabetes (T2D). Conversely, anti-GDF11 mono- motes the recovery of renal and cardiac function in old mice clonal antibody treatment caused b-cell failure and lethal (15,16). T2D. In vitro treatment of isolated murine islets and MIN6 Information regarding the role of GDF11 in pancreatic cells with recombinant GDF11 attenuated glucotoxicity- islets is scarce, however. Early studies showed that mice induced b-cell dysfunction and apoptosis. Mechanisti- lacking GDF11 exhibited markedly reduced b-cell numbers cally, the GDF11-mediated protective effects could be b attributed to the activation of transforming growth fac- and arrested -cell development (6). In addition, we pre- ISLET STUDIES tor-b/Smad2 and phosphatidylinositol-4,5-bisphosphate viously demonstrated that replenishment of GDF11 in mice 3-kinase–AKT–FoxO1 signaling. These findings suggest fed a high-fat diet (HFD) improves glucose tolerance (17). that GDF11 repletion may improve b-cell function and mass We therefore hypothesized that GDF11 may play an impor- and thus may lead to a new therapeutic approach for T2D. tant role in pancreatic b-cells under metabolic stress. In this study, we investigated the effects of GDF11 on adult islet biology using in vivo and in vitro experiments under di- Type 2 diabetes (T2D) is characterized by insulin resistance abetic conditions and investigated the possible mechanisms and insulinopenia caused by b-cell failure and decreases in involved. b-cell mass (1). As T2D develops, glycemic control gradually deteriorates over time, which is believed to be linked to the RESEARCH DESIGN AND METHODS progressive loss of b-cell function and mass (2–4). There- Animals and Treatments fore, approaches to preserve pancreatic b-cell function Mouse procedures were conducted in compliance with Na- and/or to arrest b-cell apoptosis would clearly be valuable tional Institutes of Health policies on the use of laboratory therapeutic modalities for diabetes. animals and were approved by the Animal Ethics Commit- Growth differentiation factor 11 (GDF11) plays a pleio- tee of the Wuhan General Hospital of Guangzhou Com- tropic role throughout mammalian development (5–8). Re- mand. All animals were housed at 21 6 2°C with a 12-h cently, GDF11 has attracted considerable attention for its light-dark cycle and free access to water and food. contradictory relationship with aging. Some studies have To determine the optimal dosage for the rGDF11 admin- shown that systemic restoration of GDF11 in old mice istration, we first performed a dose-response study, as shown

1Department of Endocrinology, Wuhan General Hospital of Guangzhou Command, This article contains Supplementary Data online at http://diabetes Wuhan, Hubei Province, China .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0086/-/DC1. 2 Radiation-Diagnostic/Oncology School of Medicine, Emory University, Atlanta, GA © 2017 by the American Diabetes Association. Readers may use this article as 3 Mathematics and Statistics Department, Georgia State University, Atlanta, GA long as the work is properly cited, the use is educational and not for profit, and the Corresponding author: Guangda Xiang, [email protected]. work is not altered. More information is available at http://www.diabetesjournals Received 19 January 2017 and accepted 18 April 2017. .org/content/license. diabetes.diabetesjournals.org Li and Associates 1915 in Supplementary Fig. 1A. Thirty male C57BL/6 mice aged sodium (60 mg/kg) and euthanized for blood tests, hormone 6 weeks were fed an HFD (60% of calories from fat; measurements, and histological assays. Research Diets, #D 12492) and randomized into five groups that received daily intraperitoneal (i.p.) injec- Glucose and Insulin Tolerance Tests and Biochemical tionsofrGDF11(0.1mg/kg,0.2mg/kg,0.3mg/kg,or Analyses 0.5 mg/kg) (PeproTech, Rocky Hill, NJ) or an equiva- Glucose tolerance tests (GTTs) and insulin tolerance lent volume of citrate buffer for 14 days before an i.p. tests (ITTs) were done at the beginning and end of the injection of 100 mg/kg streptozotocin (STZ; Sigma- studies based on a previous study (19). Blood samples Aldrich, St. Louis, MO). These mice were then maintained were obtained at the indicated times for the measure- on their initial protocols for an additional 14 days of ob- ment of plasma insulin levels during the GTT assays. servation (n =6pergroup). The area under the curve (AUC) was calculated by ap- Seventy-six male C57BL/6 mice aged 6 weeks (for the plying the trapezoidal rule. Measurements of GDF11/8, nongenetic mouse model) were fed an HFD (40 mice for the HbA1c, insulin, glucagon, total cholesterol, triglycerides, diabetic group) or normal chow (36 mice for the nondia- and free fatty acids were performed as previously described betic group). After 4 weeks, HFD mice were injected i.p. (17). with a single dose of STZ (HFD/STZ; 100 mg/kg). Mice fed Histological Analysis and Pancreatic Hormone Content normal chow received citrate buffer alone and were processed in parallel with the diabetic mice. All mice were Pancreatic tissues were weighed, divided longitudinally, and collected for histological examination or measure- maintained on their respective diets until the end of the ment of hormone content (20). For histological exami- study. nation, pancreatic tissues were fixedandprocessedas Blood samples were obtained by tail-tip bleeding, and previously reported (21). The antibodies used for immu- blood glucose levels were measured using a OneTouch Ultra nofluorescence staining were as follows: guinea pig anti- glucometer (LifeScan, Milpitas, CA) 14 days after the STZ insulin (1:100; Abcam), goat anti-guinea pig IgG/fluorescein or citrate buffer injection. The animals were considered isothiocyanate (FITC; 1:100; Abcam), rabbit anti-glucagon diabetic when nonfasting blood glucose levels exceeded 13.9 mmol/L for at least 2 consecutive days (18). A total of (1:400, Cell Signaling Technology), and Cy3-conjugated goat anti-rabbit antibody (1:100; Boster Bioengineering). 37 mice were considered diabetic from the HFD group The b-cell mass and a-cell mass were calculated as the (36 mice were used in the experiments). Diabetic and non- total insulin-positive/glucagon-positive area divided by diabetic mice were further randomized to receive vehicle or the total area and multiplied by the weight of the pan- rGDF11 (diabetic + vehicle, diabetic + rGDF11, nondiabetic creas. To calculate average b-cell size, the b-cell area was + vehicle, or nondiabetic + rGDF11; n = 10 per group) for divided by the number of b-cell nuclei in the covered area. 6 weeks, or isotype control antibody (isotype), or GDF11 For b-cell proliferation, pancreatic sections were immuno- antibody (Ab) (diabetic + isotype; diabetic + GDF11 Ab; nondiabetic + isotype; or nondiabetic + GDF11 Ab; n =8per stained for insulin and Ki67 (1:200; Abcam). To assess apoptotic b-cells, TUNEL was performed using the In Situ Cell group) for 6 weeks, as shown in Fig. 1A and Fig. 4A. Death Detection Kit (Roche, Mannheim, Germany). Nuclei To evaluate the effects of GDF11 in the genetic were counterstained with DAPI. The frequency of b-cell diabetic mouse model, 40 male 6-week-old db/db mice proliferation and apoptosis was calculated by dividing the and10age-matcheddb/m mice (The Jackson Laboratory, number of Ki67+ or TUNEL+ b-cells by the total b-cell Bar Harbor, ME) were used in the experiments. As shown in number. Fig. 2A, after acclimation for 2 weeks, db/m mice did not receive treatment and were used as nondiabetic controls. Islet Isolation and Culture The db/db mice were randomized into four groups: the Islets were isolated by collagenase V (Sigma-Aldrich) and vehicle, rGDF11, adenoassociated virus (AAV)-lacZ, and Ficoll gradient separation (22). Islets were then handpicked AAV-GDF11 treatment groups (n =10pergroup). under a stereomicroscope. For RNA or protein extraction, For rGDF11 treatment, mice were i.p. injected daily with freshly isolated islets were immediately frozen until ana- 0.3 mg/kg rGDF11 or an equivalent volume of vehicle lyzed. Islets for in vitro secretion assays were cultured in (citrate buffer) for 6 weeks. For GDF11 Ab treatment, mice RPMI containing 10% FBS for secretion studies. were injected intravenously (i.v.) with 100 mgGDF11Ab (clone #743833; R&D Systems, Minneapolis, MN) or Cell Culture and Cell Signaling Analyses 100 mg mouse IgG1 isotype Ab twice a week for 6 weeks. MIN6 cells were cultured as previously described (23). For For AAV treatment, mice were injected i.v. once with AAV- p-SMAD assays, MIN6 cells were fixedandprocessedas 12 GDF11 or AAV-lacZ at a dose of 1 3 10 viral genomes per previously described (17). mouse. The construction and efficiency of AAV-GDF11 have been verified as we previously reported (17). Secretion Studies In Vitro Body weight, food intake, and 6-h fasting glucose levels Secretion assays in MIN6 cells (5 3 105 cells per well) or were monitored weekly. At the end of the experiment, the size-matched islets (15 islets per tube) were performed as mice were anesthetized by i.p. administration of pentobarbital previously described (24). Results were presented as secreted 1916 GDF11 Preserves b-Cell Function and Mass Diabetes Volume 66, July 2017

Figure 1—GDF11 restoration improves glucose homeostasis and b-cell function in HFD/STZ mice. A: The experiment schedule of HFD-fed and STZ-induced mouse model studies. Male C57BL/6 mice aged 6 weeks were fed an HFD or normal chow. After 4 weeks, HFD mice were injected i.p. with a single dose of STZ (100 mg/kg). Normal chow mice received citrate buffer alone and were processed in parallel with the diabetic mice. Diabetic and nondiabetic mice were further randomized to receive vehicle or rGDF11 for 6 weeks: diabetic + vehicle, diabetic + rGDF11, nondiabetic + vehicle, or nondiabetic + rGDF11 (n = 10 per group). B: Blood glucose after a 6-h fast was monitored weekly (n = 10 per group). HbA1c levels (C) and serum insulin concentration (D) were determined at the termination of the study (n =6). E: GTT was performed at the termination of the study. Mice fasted overnight (12 h) were given a glucose challenge (2 g/kg glucose, i.p. injection), and blood glucose was monitored at the indicated times (n =10).F: The AUC for glucose tolerance was calculated (n =10). G: Plasma insulin levels were measured at the indicated times during the GTT assays (n =10).H: The mRNA expression of genes encoding NKX6.1, MafA, PDX-1, and Insulin2 in the mouse islets were determined by real-time PCR, with b-actin as an internal control (n =3).I:ITTwas performed by a single i.p. injection of 0.75 units/kg insulin in 6-h fasted mice at the end of the experiment (n = 10). J: The AUC for insulin tolerance was calculated (n = 10). Data are presented as mean 6 SEM. *P < 0.05 vs. nondiabetic + vehicle group. †P < 0.05, ††P < 0.01 vs. diabetic + vehicle group.

hormone concentrations that were normalized to the total Western Blot hormone content as analyzed by cold acid/ethanol extrac- Western blot was performed as previously reported (26). tion and ELISA (Millipore). The following antibodies were used: Smad2, phosphorylated (p)-Smad2, Smad3, p-Smad3, AKT, p-AKT (Ser473), ribo- MIN6 Proliferation and Apoptosis Assay somal protein S6 kinase (S6K), p-S6K (Thr389), FoxO1, MIN6 cell proliferation was measured by the Click-iT EdU p-FoxO1 (Ser256), Bax, Bcl-2, cleaved caspase-3 (all from Kit (Invitrogen, Carlsbad, CA), and apoptosis was deter- Cell Signaling Technology); anti-GDF11 (R&D Systems), mined using flow cytometry (BD, Franklin Lakes, NJ), after anti-myostatin (GDF8; Abcam), and b-actin (Boster double staining with Annexin V-FITC (BD Pharmingen) and Bioengineering). propidium iodide. Statistical Analysis RNA Extraction and Real-Time PCR All values are expressed as mean 6 SEM. Statistical signif- Total RNA samples were prepared and measured as pre- icance between groups was analyzed by unpaired Student t viously described (25). Primer sequences are listed in Sup- test or one-way ANOVA with a least significant difference test plementary Table 1. for multiple comparisons. Data were considered significant if diabetes.diabetesjournals.org Li and Associates 1917

Figure 2—GDF11 restoration ameliorates hyperglycemia and glucose intolerance as well as preserves b-cell function in db/db mice. A: Schematic representation of the treatment protocol for the genetic mouse model of T2D. Diabetic db/db mice (2 months old) were randomized to receive rGDF11, vehicle (citrate buffer), or the AAV vectors (AAV-GDF11 or AAV-lacZ) for 6 weeks. The db/m mice were used as normal controls. B: Blood glucose levels after a 6-h fast were measured weekly (n = 10 mice in each group). Analysis of serum HbA1c (C) and insulin levels (D) at the end of the study (n =6).E: After the 6-week intervention, GTT (1 g/kg glucose for db/db mice, 2 g/kg glucose for db/m mice, i.p. injection) was performed on mice after a 12-h fast (n =10).F: The AUC analysis of GTT (n = 10). G: Plasma insulin levels were assayed the indicated times during the GTT assays (n = 10). H: The real-time PCR analysis of key b-cell genes (Insulin2, PDX-1, MafA, and NKX6.1) in mouse islets (n =3).I: ITT was performed at the end of the study. Mice were fasted for 6 h and then injected i.p. with insulin (1 unit/kg for db/db mice, 0.75 units/kg for db/m mice) (n = 10). J: The AUC analysis of ITT (n = 10). Data are presented as mean 6 SEM. *P < 0.05, **P < 0.01 vs. vehicle group; †P < 0.05, ††P < 0.01 vs. AAV-lacZ group.

P was ,0.05. All statistical analyses were performed using Fig. 1A–E), and thus, we chose 0.3 mg/kg as the optimum SPSS 19.0 software (IBM Corp, Armonk, NY). dose in the following animal study (Fig. 1A). Second, the results from the nongenetic mouse model RESULTS study indicated that: GDF11 Restoration Improves Glucose Homeostasis and b-Cell Function in Diabetic Mice 1. Circulating GDF11/8 levels were markedly decreased To evaluate the potential effect of GDF11 on b-cell function in the HFD/STZ mice compared with nondiabetic in diabetic mice, we first treated the nongenetic diabetic mice at the end of the study. However, rGDF11 treat- mice with rGDF11. First, the dose-response study showed ment elevated GDF11/8 levels in both diabetic and that compared with vehicle-treated mice, rGDF11 treat- nondiabetic mice (Supplementary Table 2). ment decreased blood glucose levels and increased serum 2. The diabetic groups did not significantly differ in body insulin levels and b-cell mass in a dose-dependent manner. weight or food intake (Supplementary Table 2). Notably, no significant differences were observed between 3. Although vehicle-treated HFD/STZ mice devel- mice injected with 0.3 or 0.5 mg/kg rGDF11 (Supplementary oped severe hyperglycemia and higher HbA1c levels, 1918 GDF11 Preserves b-Cell Function and Mass Diabetes Volume 66, July 2017

rGDF11 treatment attenuated the progression of GDF11 Restoration Prevents the Loss of b-Cells in B hyperglycemia and reduced HbA1c levels (Fig. 1 Diabetic Mice and C). We next examined whether GDF11 replenishment 4. The rGDF11-treated HFD/STZ mice had higher fasting could prevent b-cell loss in the HFD/STZ mice. Histolog- serum insulin levels than vehicle-treated HFD/STZ ical examination showed that islets from rGDF11-treated mice (Fig. 1D). diabetic mice exhibited normal distribution, with a 5. Administration of rGDF11 to HFD/STZ mice miti- large insulin cell core surrounded by a mantle of a-cells, gated serum total cholesterol, free fatty acids, and which differed from the disorganized islet architecture triglyceride levels (Supplementary Table 2), indicating observed in vehicle-treated diabetic mice (Fig. 3A). Mea- that chronic treatment of rGDF11 alleviates hyperlip- surement of b-cell mass of vehicle-treated HFD/STZ pan- idemia beyond improved glycemic control. creatic sections revealed a 61% decrease compared with fi 6. Before these interventions, there was no signi cant vehicle-treated nondiabetic mice (Fig. 3B). However, difference in glucose and insulin tolerance between rGDF11 treatment elevated b-cell mass and pancreatic in- A–D the diabetic groups (Supplementary Fig. 2 ). sulin content in HFD/STZ mice (Fig. 3B and C). We did not observe differences in islet b-cell size, suggesting that the After the 6-week intervention, rGDF11-treated diabetic increase in b-cell mass observed in the rGDF11 group was mice displayed a remarkable improvement in glucose and b insulin tolerance (Fig. 1E and I). These were further corrob- unlikely to be caused by enhanced -cell size (Supplemen- B orated by AUC analyses (Fig. 1F and J). tary Fig. 4 ). Regarding proliferation, animals injected with + b Third, we assessed the insulin secretory response after rGDF11 tended to have a higher percentage of Ki67 -cells i.p. glucose administration. Indeed, fasting and glucose- than their diabetic littermates; however, this difference was induced insulin levels of rGDF11-treated HFD/STZ mice not statistically significant (Fig. 3D). Quantification of + were both augmented, indicating that GDF11 enhanced TUNEL b-cells, an index for cell apoptosis, indicated that b-cell secretory function in vivo (Fig. 1G). To further eluci- rGDF11 treatment decreased b-cell apoptosis (Fig. 3E). date how GDF11 repletion might promote insulin secretion, In line with the HFD/STZ model, both rGDF11 and we analyzed isolated islets for the expression of genes AAV-GDF11 treatment improved islet architecture as well encoding insulin transcription. As a result, the expression of as augmented b-cell mass and pancreatic insulin content PDX-1, NKX6.1, MafA, and insulin2 was reduced in diabetic in db/db mice (Fig. 3F and G and Supplementary Fig. 4A). islets, but the mRNA levels were significantly upregulated Islet histology showed no significant differences in the by rGDF11 treatment (Fig. 1H). Notably, injection of rGDF11 frequency of b-cell proliferation and b-cell size among in a group of nondiabetic mice did not affect any of the diabetic mice (Fig. 3H and Supplementary Fig. 4C). In parameters examined in the study, and these animals contrast, the number of TUNEL+ b-cells in the rGDF11 were indistinguishable from vehicle-treated nondiabetic and AAV-GDF11 groups was reduced by 33% and 51%, A–J mice (Fig. 1 ). respectively, compared with the vehicle or AAV-lacZ Next, we explored whether augmenting GDF11 levels groups (Fig. 3I). Overall, these data suggest that GDF11 b db db could improve -cell function in / mice, a genetic repletion may primarily protect b-cell mass via an anti- mouse model that recapitulates many of the features apoptotic mechanism rather than by an induction of b-cell of T2D in humans (Fig. 2A). GDF11/8 levels were dramat- proliferation. ically reduced in diabetic db/db mice, and injection of AAV- GDF11 and rGDF11 augmented the circulating GDF11/8 GDF11 Restoration Reduces Islet Glucagon Secretion levels by the end of the study (Supplementary Table 3). Both In Vivo and In Vitro As in the HFD/STZ models, we observed attenuated hyper- To investigate whether GDF11 has an effect on a-cells, we glycemia and HbA levels as well as improved plasma in- 1c assessed glucagon secretion and production as well as a-cell sulin levels and lipid profile in db/db mice after a 6-week mass. Critically, both rGDF11 and AAV-GDF11 treatment treatment with rGDF11 or AAV-GDF11, an effect that decreased circulating glucagon levels in the two different seemed to be independent of changes in body weight and mouse models of T2D (Supplementary Fig. 5A and D). Fur- food intake (Fig. 2B–D and Supplementary Table 3). Al- though glucose tolerance and insulin sensitivity were com- thermore, compared with vehicle-treated islets, rGDF11 2 P , parable among db/db mice at the beginning of the study treatment reduced glucagon secretion at 1 ( 22%, 2 P , (Supplementary Fig. 3A–D), the rGDF11 and AAV-GDF11 0.05), 6 ( 31%, 0.05), and 20 mmol/L glucose 2 P , G interventions both attenuated glucose intolerance and in- ( 42%, 0.05) in vitro (Supplementary Fig. 5 ). As db db sulinresistanceattheendofstudy(Fig.2E and I), which expected, HFD/STZ mice and / mice exhibited a re- the AUC data confirmed (Fig. 2F and J). In addition, im- markable increase in a-cell mass compared with nondiabetic proved glucose-stimulated insulin secretion (GSIS) and the + vehicle mice or db/m mice. However, a-cell mass and elevated expression of b-cell–specific genes were observed pancreatic glucagon content in diabetic mice were unaf- in rGDF11-treated and AAV-GDF11–treated db/db mice fected by rGDF11 or AAV-GDF11 treatment (Supplemen- (Fig. 2G and H). tary Fig. 5B, C, E,andF). diabetes.diabetesjournals.org Li and Associates 1919

Figure 3—Chronic treatment with GDF11 preserves b-cell mass in diabetic mice. At the end of the study, the mice were anesthetized by the i.p. administration of pentobarbital sodium (60 mg/kg) and euthanized for histological assays. A: Pancreata were isolated from mice fed a normal chow diet or HFD plus STZ injection (100 mg/kg). Representative images of pancreatic sections stained for hematoxylin-eosin (H&E) or double- stained for insulin/glucagon (left panel), insulin/Ki67 (middle panel), and insulin/TUNEL (right panel). The nuclei were stained with DAPI (blue).The white arrows indicate proliferating b-cells (middle panel) or apoptotic b-cells (right panel). Scale bar, 20 mm. B:Theb-cell mass was calculated as the total insulin-positive area divided by the total area and multiplied by the weight of the pancreas (n =4micepergroup).C: Pancreatic insulin content (n =4).D: Quantification of b-cell proliferation was assessed by Ki67 staining in insulin-stained sections (n =4).E: Apoptosis of b-cell was determined by quantification of TUNEL+-to-insulin+ ratio (n = 4). Data are presented as mean 6 SEM. **P < 0.01 vs. diabetic + vehicle group. F: Quantitative analysis of b-cell mass in db/db models (n =4).G: Pancreatic insulin content in db/db models (n =4).H: Proliferation of b-cells was determined by quantification of Ki67+-to-insulin+ (n =3).I: Percentage of b-cell apoptosis in db/db models (n =4).Dataarepresented as mean 6 SEM. *P < 0.05, **P < 0.01 vs. vehicle group; †P < 0.05, ††P < 0.01 vs. AAV-lacZ group.

Neutralization of GDF11 Promotes b-Cell Failure in were observed between the nondiabetic mice treated with HFD/STZ Mice GDF11 Ab or isotype Ab (Fig. 4B–H). However, GDF11 Ab Next, we sought to confirm the physiological role of GDF11 treatment further elevated 6-h fasting blood glucose and b in -cellfunctionandmass.Thus,nondiabeticorHFD/STZ HbA1c levels and reduced serum insulin levels in diabetic mice were treated with GDF11 Ab for 6 weeks (Fig. 4A). mice (Fig. 4B–D), without changing their body weight or Given that GDF11 is highly related to GDF8 and shares food intake (Supplementary Fig. 7A and B). Notably, glucose 90% amino acid sequence identity in their mature active intolerance was comparable between the diabetic groups at forms (27), we first verified the specificity of the antibody baseline (Supplementary Fig. 7C and D) but was worse in used in this study. Western blot analysis confirmed that the GDF11 Ab–treated diabetic mice after the 6-week in- this antibody specifically detects GDF11 without cross- tervention (Fig. 4E and F), which partially resulted from reaction with GDF8 (Supplementary Fig. 6A–C). Furthermore, perturbed acute insulin response to glucose (Fig. 4I). Con- compared with the vehicle-treated group, the stimulatory sistent with this, GDF11 Ab treatment reduced the expres- effects of GDF11 on GSIS and insulin content were dose- sion of essential b-cell transcription factors (Fig. 4J). dependently reversed by the GDF11 Ab treatment in vitro Insulin tolerance was also significantly decreased, induced (Supplementary Fig. 6D–G). by GDF11 Ab administration (Fig. 4G and H and Supple- We next assessed whether neutralization of GDF11 in the mentary Fig. 7E and F). Furthermore, the GDF11 Ab–treated blood impairs glucose homeostasis in mice. No differences diabetic mice displayed significantly reduced b-cell mass, 1920 GDF11 Preserves b-Cell Function and Mass Diabetes Volume 66, July 2017

Figure 4—Neutralization of GDF11 impairs b-cell function and mass in diabetic mice. A: Schematic representation of the treatment protocol for HFD-fed and STZ-induced (HFD/STZ) mouse model studies. Male C57BL/6 mice aged 6 weeks were fed an HFD or normal chow. After 4 weeks, HFD mice were injected i.p. with a single dose of STZ (100 mg/kg). Normal chow mice received citrate buffer alone and were processed in parallel with the diabetic mice. Diabetic and nondiabetic mice were further randomized to receive isotype control antibody (isotype) or GDF11 Ab for 6 weeks (diabetic + isotype, diabetic + GDF11 Ab, nondiabetic + isotype, or nondiabetic + GDF11 Ab; n = 8 per group). At the end of the experiment, the mice were anesthetized by the i.p. administration of pentobarbital sodium (60 mg/kg) and euthanized for blood tests and histological assays. B: Level of 6-h fasting blood glucose (n =8micepergroup).C: Analysis of serum HbA1c levels (n =5).D: Analysis of serum insulin levels (n =5).E: GTT (2 g/kg glucose, i.p. injection) (n =8).F: The AUC for GTT results (n =8). G: ITT (0.75 units/kg insulin, i.p. injection) (n =8).H: The AUC for ITT results (n =8).I:InvivoGSISduringtheGTTassays(n =8).J:Relative mRNA expression levels of Insulin2, PDX-1, MafA, and NKX6.1 in mouse islets (n =4).b-Cell mass (K) and pancreatic insulin content (L) (n =4).M: Percentage of b-cell apoptosis (n = 4). Data are presented as mean 6 SEM. *P < 0.05 vs. nondiabetic + isotype group; †P < 0.05 vs. diabetic + isotype Ab group.

which was partly caused by the increased b-cell apoptosis exposure of mouse islets to high glucose (25 mmol/L) for (Fig. 4K–M and Supplementary Fig. 8). 72 h impaired GSIS and insulin content, whereas preincubation with rGDF11 improved insulin secretion and GDF11 Attenuates Hyperglycemia-Induced b-Cell storage. Similar to mouse islets, rGDF11 partially re- DysfunctionInVitro stored GSIS and insulin content in MIN6 cells under high To further corroborate the direct effects of GDF11 on b-cell glucose conditions (Fig. 5G–L), whereas both SB431542 (an function, we explored the effects of rGDF11 on GSIS and inhibitor of transforming growth factor-b [TGF-b]typeI insulin content in MIN6 cells and in mouse islets. rGDF11 receptor [TbRI]) and LY294002 (the phosphatidylinositol- pretreatment stimulated a dose-dependent and time- 4,5-bisphosphate 3-kinase [PI3K] inhibitor), but not the dependent increase in insulin secretion and biosynthesis in MIN6 mechanistic target of rapamycin (mTOR), partially abol- cells (Fig. 5A–D). Accordingly, we chose 100 ng/mL rGDF11 ished the stimulatory effects of rGDF11 on insulin secre- and 24 h as the optimum concentration and time condi- tion and production in MIN6 cells (Fig. 5I–L). Furthermore, tions in the following study. As shown in Fig. 5E and F, rGDF11 pretreatment augmented insulin secretion at diabetes.diabetesjournals.org Li and Associates 1921

Figure 5—GDF11 improves hyperglycemia-induced b-cell dysfunction in vitro. A: For the analysis of insulin secretion, MIN6 cells were incubated in media with 2.8 mmol/L (basal) or 16.7 mmol/L (stimulated) glucose for 1 h at 37°C. The indicated concentrations of rGDF11 (0–300 ng/mL) were added during both the basal and stimulatory glucose incubations. Insulin secretion is presented as a percentage of insulin content. White bars = 2.8 mmol/L, black bars = 16.7 mmol/L. B: Insulin content in MIN6 cells exposed to the indicated concentrations of rGDF11 (0–300 ng/mL) for 24 h. C: MIN6 cells were pretreated with 100 ng/mL rGDF11 for 0, 4, 8, 24, and 48 h before exposure to 2.8 mmol/L (white bars) or 16.7 mmol/L (black bars) glucose for 1 h. D: Insulin content in MIN6 cells. E: Insulin secretion from mouse islets at 2.8 mmol/L (white bars) or 16.7 mmol/L (black bars) glucose after exposure to 5.5 mmol/L or 25 mmol/L glucose (high glucose [HG]) for 72 h in the absence or presence of 100ng/mLrGDF11pretreatmentfor24h.F: Insulin content in mouse islets. G: GSIS analysis in MIN6 cells treated with high glucose (25 mmol/L) for 72 h with or without the preincubation of rGDF11 (100 ng/mL) for 24 h. White bars = 2.8 mmol/L, black bars = 16.7 mmol/L. H: Insulin content in MIN6 cells. I and J: MIN6 cells were pretreated with 10 mmol/L SB431542 for 30 min before treatment with 100 ng/mL rGDF11 for 24 h, which was followed by a 72-h incubation with 5.5 or 25 mmol/L glucose. I: Insulin secretion from MIN6 cells at 2.8 mmol/L (white bars) or 16.7 mmol/L (black bars). J: Insulin content in MIN6 cells. K and L: MIN6 cells were preincubated with 50 mmol/L LY294002 or 100 nmol/L rapamycin for 30 min before treatment with 100 ng/mL rGDF11 for 24 h, which was followed by a 72-h incubation with 5.5 mmol/L or 25 mmol/L glucose. K: GSIS assay measurements were performed in MIN6 cells. White bars = 2.8 mmol/L, black bars = 16.7 mmol/L. L:InsulincontentinMIN6cells. M and N: Total RNA was isolated from mouse islets or MIN6 cells cultured in medium containing 5.5 (normal glucose [NG]) or 25 mmol/L glucose (HG) with or without 100 ng/mL rGDF11. M: Quantitative real-time PCR was used to measure PDX-1, MafA, NKX6.1, and Insulin2 mRNA levels in primary islets. N: Real-time PCR detection and quantification of key b-cell gene expression in MIN6 cells. Data are mean 6 SEM of five independent experiments. *P < 0.05; NS, not significant.

16.7 mmol/L glucose but did not affect basal (2.8 mmol/L) First, exposure of MIN6 cells to rGDF11 showed a tendency insulin release. In addition, mRNA levels of PDX-1, MafA, to promote b-cell proliferation (Supplementary Fig. 9A and NKX6.1, and Insulin2 were upregulated by rGDF11 pre- B). Next, to determine whether GDF11 has a direct anti- treatment in both primary islets and MIN6 cells (Fig. 5M apoptotic effect on b-cells, we preincubated MIN6 cells with and N). rGDF11. As expected, hyperglycemia robustly increased the percentage of apoptotic MIN6 cells compared with controls. GDF11 Partially Alleviates Glucotoxicity-Induced b-Cell However, the effect was partially blocked by rGDF11 pre- Apoptosis In Vitro treatment (Fig. 6A and B). Consistently, rGDF11 treatment We next investigated whether rGDF11 pretreatment enhanced the expression of the antiapoptotic protein Bcl-2 could affect b-cell proliferation and apoptosis in vitro. level but suppressed the expression of the proapoptotic 1922 GDF11 Preserves b-Cell Function and Mass Diabetes Volume 66, July 2017

Figure 5—Continued.

proteins Bax and cleaved-caspase3 (Fig. 6C and D). How- phosphorylation in islets from vehicle-treated diabetic ever, treatment of MIN6 cells with SB431542 or LY294002 mice compared with vehicle-treated nondiabetic mice. In- partially abrogated the antiapoptotic effects of GDF11 (Fig. triguingly, both rGDF11 and AAV-GDF11 treatment in- 6A–D). creased Smad2 phosphorylation but had no influence on Smad3 phosphorylation (Fig. 7A–D). In addition, immuno- GDF11 Activates TGF-b/Smad and PI3K/AKT/FoxO1 fluorescence analyses showed that preincubation of MIN6 Pathways cells with rGDF11 restored Smad2 phosphorylation but To explore the possible pathways whereby GDF11 exerts that this activation was partially blocked by SB431542 protective effects on b-cells in vivo and in vitro, Western (Fig. 7E and F). Indeed, rGDF11 pretreatment had no effect blot and immunofluorescence staining were performed. It is on p-Smad3 expression in vitro. commonly believed that GDF11 transmits its signals via We further explored the noncanonical signaling cascade dual serine/threonine kinase receptors and transcription of GDF11. Western blotting of islet lysates showed that factors called Smads (28). By Western blotting, we detected GDF11 enhanced AKT phosphorylation in HFD/STZ and decreased Smad2 phosphorylation and increased Smad3 db/db models (Fig. 8A–D). FoxO1 and S6K are evolutionarily diabetes.diabetesjournals.org Li and Associates 1923

Figure 6—GDF11 alleviates high glucose–related b-cell apoptosis in vitro. MIN6 cells were pretreated with LY294002 (50 mmol/L) or SB431542 (10 mmol/L) for 30 min and then incubated with rGDF11 (100 ng/mL) for 24 h, which was followed by a 72-h incubation with high glucose (HG; 25 mmol/L). A: After incubation, apoptosis of MIN6 cells was stained with Annexin V-FITC and propidium iodide and assessed by flow cytometry. B: Quantitative analysis of A. Representative immunoblots (C) and densitometric quantification (D) for the expression of the proteins Bcl-2, Bax, and cleaved-caspase3. Data are mean 6 SEM of five independent experiments. *P < 0.05.

conserved substrates of AKT (29). Compared with vehicle- data show for the first time that GDF11 restoration may treated diabetic mice, FoxO1 phosphorylation was increased curtail the progression of diabetes. in islet lysates from both the rGDF11- and AAV-GDF11– A prominent feature of T2D is blunted insulin response treated diabetic mice (Fig. 8A–D). Meanwhile, S6K phos- caused by b-cell dysfunction (1,2,30). We showed that phorylation was unchanged by rGDF11 treatment (Fig. GDF11 promoted insulin biosynthesis and secretion. In 8A–D). To further evaluate this noncanonical signaling path- particular, in vivo and in vitro GSIS experiments both in- wayinvitro,wetreatedMIN6cellswithrGDF11.High dicated that GDF11 promoted insulin release in a glucose- glucose reduced AKT and FoxO1 phosphorylation in MIN6 dependent manner, which is of vital importance for the cells, but preincubation with rGDF11 partially rescued this precise regulation of blood glucose levels. Recent studies defect. Notably, coincubation with LY294002, but not rapa- have also shown that b-cell transcription factors are critical mycin, exerted a suppressive effect on rGDF11-mediated for maintaining b-cell function (31,32). Moreover, PDX-1, AKT and FoxO1 phosphorylation (Supplementary Fig. 10A MafA, and NKX6.1 work synergistically to regulate insulin and B). As expected, no significant difference in the p-S6K transcription and exocytosis, and their expression is strik- expression was observed among the groups, suggesting that ingly decreased under diabetic conditions, which partially the protective mechanism of GDF11 may not be dependent accounts for the b-cell dysfunction observed in individuals on the mTOR signaling pathway (Supplementary Fig. 10C). with diabetes (23,33). Very importantly, MafA expression is diminished in the islets of GDF11-knockout mice (6,34). DISCUSSION Consistently, GDF11 treatment elevated the expression lev- The major findings of this study are the following: 1) els of these genes both in vivo and in vitro. In contrast, GDF11 improves b-cell function both in vivo and in vitro; neutralization of endogenous GDF11 impaired glycemic 2) GDF11 protects against b-cell apoptosis; 3) GDF11 in- control and GSIS as well as reduced the expression of key hibits inappropriate glucagon secretion; 4) neutralization of b-cell transcription factors. Together, these results provide GDF11 impairs GSIS and reduces b-cell mass in diabetic evidence that GDF11 improves b-cell function, in part by mice; and 5) the molecular mechanisms underlying these maintaining the expression of key b-cell genes. beneficial effects of GDF11 may involve the activation of That b-cell mass is reduced in T2D mainly as a result of the canonical TGF-b/Smad2 and the noncanonical PI3K- increased b-cell apoptosis has been established (4,35,36). AKT-FoxO1 pathways. To the best of our knowledge, our Hence, remission of b-cell apoptosis would be valuable in 1924 GDF11 Preserves b-Cell Function and Mass Diabetes Volume 66, July 2017

Figure 7—GDF11 activates TGF-b/Smad pathway both in vivo and in vitro. A–D: At the end of the study, mice were anesthetized by the i.p. administration of pentobarbital sodium (60 mg/kg) and euthanized for islet isolation. A: Islets were isolated from mice in the nondiabetic + vehicle, diabetic + vehicle, and diabetic + rGDF11 groups. Western blots of total islet lysates for p-Smad2, Smad2, p-Smad3, and Smad3. b-Actin was used as a loading control. B: The relative expression of the proteins p-Smad2 and p-Smad3 normalized to each total protein are shown (n =3per group). Data are presented as mean 6 SEM. **P < 0.01 vs. diabetic + vehicle group. C: After the 6-week intervention, Western blot of total islet lysates for p-Smad2, Smad2, p-Smad3, and Smad3 (db/db mouse models). b-Actin was used as a loading control. D: Quantitative analysis of C (n = 3). Data are presented as mean 6 SEM. **P < 0.01 vs. vehicle group. ††P < 0.01 vs. AAV-lacZ group. E: Representative images and quantiﬁcation of the density of p-SMAD2+ (upper panel), p-SMAD3+ cells (lower panel) in MIN6 cell cultures treated with rGDF11 (100 ng/mL) alone or pretreated with TbRI inhibitor SB431542 (10 mmol/L) under high glucose (HG) conditions (25 mmol/L). Scale bar, 20 mm. F: Quantitative analysis of E. Each experiment was repeated ﬁve times. Data are presented as mean 6 SEM. *P < 0.05.

the treatment of diabetes. In this study, GDF11 im- Extensive data support the notion that inappropriate proved the islet architecture and increased b-cell num- glucagon secretion is associated with excess hepatic glucose bers by protecting against b-cell apoptosis caused by output and hyperglycemia in diabetes (20,24,37). In- metabolic stress. Moreover, GDF11 attenuated glucose- deed, the inhibition of glucagon signaling has proven to induced b-cell apoptosis in vitro, which supports the direct be beneﬁcial in various animal models and in humans effects of GDF11 on b-cells. However, although GDF11 (38). In this study, we observed that GDF11 restoration promotes proliferation across other cell types (9,15–17), improved glucose-induced suppression of glucagon secre- here, we showed that b-cell proliferation, as measured by tion in mice and in primary islets via a direct effect on Ki67 immunostaining or 5-ethynyl-2’-deoxyuridine (EdU) a-cells. Of note, given that insulin secretion from b-cells incorporation, was unaffected by GDF11 repletion both directly inhibits glucagon secretion (39,40), the decreased in vitro and in vivo. The reason for these results may be glucagon levels in animals may be partially attributed to that the functions of GDF11 are dependent on the cell type. GDF11-induced insulin secretion. In addition, as reported However, neutralization of endogenous GDF11 promoted previously (20,41), we observed increased a-cell mass and b-cell apoptosis and reduced b-cell mass. Overall, these data pancreatic glucagon content in diabetic animals. However, indicate that GDF11 prevents b-cell loss under diabetic those remained unchanged after GDF11 repletion. These conditions. results suggest that the glucose-lowering effect of GDF11 diabetes.diabetesjournals.org Li and Associates 1925

Figure 8—Replenishment of GDF11 activates PI3k-AKT-FoxO1 signaling in vivo. At the end of the study, the mice were anesthetized by the i.p. administration of pentobarbital sodium (60 mg/kg) and euthanized for islet isolation. A: Western blot analyses for p-AKT (Ser473), AKT, p-FoxO1 (Ser256), FoxO1, p-S6K (Thr389), and S6K normalized to b-actin protein in the islets from mice in the nondiabetic + vehicle, diabetic + vehicle, and diabetic + rGDF11 groups. B: Bar graphs show averages of the ratios (phosphorylated protein signal vs. total protein signal) of the band intensities (n = 3 per group). Data are presented as mean 6 SEM. **P < 0.01 vs. diabetic + vehicle group. C: Western blot analysis of p-AKT, AKT, p-S6K, S6K, p-FoxO1, and FoxO1 in islets from db/db models. D: Quantitative analysis of C (n = 3). Data are presented as mean 6 SEM. **P < 0.01 vs. vehicle group. ††P < 0.01 vs. AAV-lacZ group.

may be dependent on the improvement of a-cell function significant decrease in the body weight of mice (14). How- but not a-cell expansion. ever, we found that injection of 0.3 mg/kg rGDF11 had no As observed previously (17), GDF11 replenishment im- effect on body weight in normal or diabetic mice. This dis- proved lipid metabolism and insulin sensitivity, which crepancy may be partly caused by the dose-dependent effect might be partially responsible for the improvement in glu- of GDF11. cose homeostasis. There are two possible mechanisms by Next, we explored the molecular signaling pathways that which GDF11 might attenuate insulin resistance. First, hy- may underlie the protective effect of GDF11 on islets. It perglycemia and hyperlipidemia are critical determinants of is well documented that GDF11 exerts its function by insulin sensitivity (42), and thus, improvements in glucose interacting with heterotetrameric membrane receptors and lipid metabolism might partially explain improvements composed of activin type II and activin type I receptors to in insulin sensitivity. Second, previous studies showed that activate the canonical Smad signaling pathway (43). Fur- GDF11 can promote skeletal muscle regeneration (9) and thermore, Smad2 is requisite for the maintenance of GSIS reduce adipose tissue (14), which may be partially responsible and b-cell mass (44,45). GDF11 may act through Smad2 to for the elevation in insulin sensitivity. In addition, the allevi- facilitate b-cell maturation during development, because ated hyperlipidemia may be a result of the beneficial effects of the phenotype of mice that are heterozygous for Smad2 GDF11 on glucose metabolism, insulin sensitivity, and adipose is strikingly reminiscent of that of GDF11-knockout mice tissue (14). Notably, a previous study showed that the ad- (6). Here, we showed that GDF11 treatment elevated the ministration of 0.5 or 1.0 mg/kg rGDF11 caused a p-Smad2 expression both in vivo and in vitro. The TbRI 1926 GDF11 Preserves b-Cell Function and Mass Diabetes Volume 66, July 2017 inhibitor partially blocked the ability of GDF11 to rescue 7. Kim J, Wu HH, Lander AD, Lyons KM, Matzuk MM, Calof AL. GDF11 controls b-cells from hyperglycemia-induced dysfunction and the timing of progenitor cell competence in developing retina. Science 2005;308: apoptosis. In addition, GDF11 can activate several non- 1927–1930 Smad signaling pathways in a context-dependent manner, 8. Liu JP. The function of growth/differentiation factor 11 (Gdf11) in rostrocaudal patterning of the developing spinal cord. Development 2006;133:2865–2874 including the PI3K pathway, which can cross talk with the 9. Sinha M, Jang YC, Oh J, et al. Restoring systemic GDF11 levels reverses age- Smad pathways (46). Moreover, AKT-mediated phosphory- related dysfunction in mouse skeletal muscle. Science 2014;344:649–652 lation of FoxO1 promotes its nuclear exclusion and medi- 10. Katsimpardi L, Litterman NK, Schein PA, et al. Vascular and neurogenic re- ates the increase of PDX-1, NKX6.1, and MafA expression juvenation of the aging mouse brain by young systemic factors. Science 2014;344: and b-cell survival (47). Here, our data support the notion 630–634 that GDF11 exerts its biological activity through the PI3K 11. Loffredo FS, Steinhauser ML, Jay SM, et al. Growth differentiation factor 11 is a pathway. GDF11 increased AKT and FoxO1 phosphoryla- circulating factor that reverses age-related cardiac hypertrophy. Cell 2013;153:828– tion both in diabetic mice and in MIN6 cells, and the PI3K 839 inhibitor partially abrogated the GDF11-mediated protec- 12. Smith SC, Zhang X, Zhang X, et al. GDF11 does not rescue aging-related tion against hyperglycemia-induced b-cell malfunction and pathological hypertrophy. Circ Res 2015;117:926–932 demise. Cumulatively, we conclude that the GDF11-mediated 13. Egerman MA, Cadena SM, Gilbert JA, et al. GDF11 increases with age and – beneficial effects may be dependent on the activation of the inhibits skeletal muscle regeneration. Cell Metab 2015;22:164 174 14. Poggioli T, Vujic A, Yang P, et al. Circulating growth differentiation factor 11/8 Smad2 and PI3K-AKT-FoxO1 cascades. levels decline with age. Circ Res 2016;118:29–37 This study has some limitations. First, GDF8 is a close 15. 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