MST1 is a key regulator of beta cell apoptosis and dysfunction in diabetes

Abstract

Apoptotic cell death is a hallmark of the loss of insulin-producing beta cells in all forms of diabetes mellitus. Current treatments fail to halt the decline in functional beta cell mass, and strategies to prevent beta cell apoptosis and dysfunction are urgently needed. Here, we identified mammalian sterile 20-like kinase-1 (MST1) as a critical regulator of apoptotic beta cell death and function. Under diabetogenic conditions, MST1 was strongly activated in beta cells in human and mouse islets and specifically induced the mitochondrial-dependent pathway of apoptosis through upregulation of the BCL-2 homology-3 (BH3)-only protein BIM. MST1 directly phosphorylated the beta cell transcription factor PDX1 at T11, resulting in the latter's ubiquitination and degradation and thus in impaired insulin secretion. MST1 deficiency completely restored normoglycemia, beta cell function and survival in vitro and in vivo. We show MST1 as a proapoptotic kinase and key mediator of apoptotic signaling and beta cell dysfunction and suggest that it may serve as target for the development of new therapies for diabetes.

Figure 1. MST1 is activated in diabetes

(a-c) Activated MST1 (cleaved and phosphorylated) in human (a) and mouse (b) islets and INS1-E cells (c) exposed to diabetogenic conditions (22.2-33.3 mM glucose or the mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3Palm) or IL-1β/IFNγ (IL/IF) for 72h. MST1, pMST1, pJNK, pH2B and caspase-3 cleavage were analyzed by western blotting, right panels show densitometry analysis from at least 3 independent experiments normalized to actin or tubulin. (d,e) Activated MST1 in diabetic islets. (d) Human isolated islets from non-diabetic controls (n=7) and subjects with T2D (n=4), all with documented fasting plasma glucose >150 mg/dl and (e) from 10-week old diabetic db/db (n=5) and their heterozygous db/+ littermates (n=5). MST1 and pMST1 were analyzed by western blotting, right panels show densitometry analysis normalized to actin. Right panels show double immunostaining for pMST1 in red and insulin in green in sections from human isolated islets from non-diabetic controls and subjects with T2D and from 6-week old diabetic db/db mice (representative analyses from 10 pancreases from subjects with T2D and >10 controls and from 7 db/db and 7 db/+ controls are shown, bar is 100 μM). (f) INS-1E cells transfected with GFP control or Myr-AKT1 expression-plasmids and exposed to 22.2 mM glucose for 72h. (g,h) PI3K/AKT was inhibited in INS-1E cells by exposure to (g) PI3K inhibitor, LY294002 (10 μM for 8h) or (h) AKT inhibitor Triciribine (10 μM for 6h). (i) INS-1E infected with Ad-GFP or Ad-MST1 or transfected with shMST1 or shScr control expression plasmids for 48h, serum-starved for 12h and then stimulated with insulin. Mst1, pMst1, pAkt, tAkt, pGsk3, pFoxo1 and caspase-3 cleavage were analyzed by western blotting. Right panels show densitometry analysis from at least 3 independent experiments normalized to actin. All western blots show representative results from at least 3 independent experiments from 3 different donors or mice. Tubulin/Actin was used as loading control. Results shown are means ±SE. *p<0.05 compared to untreated or nondiabetic control. #p<0.05 Myr-AKT1 compared to GFP at 22.2 mM glucose.

Figure 1. MST1 is activated in diabetes

(a-c) Activated MST1 (cleaved and phosphorylated) in human (a) and mouse (b) islets and INS1-E cells (c) exposed to diabetogenic conditions (22.2-33.3 mM glucose or the mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3Palm) or IL-1β/IFNγ (IL/IF) for 72h. MST1, pMST1, pJNK, pH2B and caspase-3 cleavage were analyzed by western blotting, right panels show densitometry analysis from at least 3 independent experiments normalized to actin or tubulin. (d,e) Activated MST1 in diabetic islets. (d) Human isolated islets from non-diabetic controls (n=7) and subjects with T2D (n=4), all with documented fasting plasma glucose >150 mg/dl and (e) from 10-week old diabetic db/db (n=5) and their heterozygous db/+ littermates (n=5). MST1 and pMST1 were analyzed by western blotting, right panels show densitometry analysis normalized to actin. Right panels show double immunostaining for pMST1 in red and insulin in green in sections from human isolated islets from non-diabetic controls and subjects with T2D and from 6-week old diabetic db/db mice (representative analyses from 10 pancreases from subjects with T2D and >10 controls and from 7 db/db and 7 db/+ controls are shown, bar is 100 μM). (f) INS-1E cells transfected with GFP control or Myr-AKT1 expression-plasmids and exposed to 22.2 mM glucose for 72h. (g,h) PI3K/AKT was inhibited in INS-1E cells by exposure to (g) PI3K inhibitor, LY294002 (10 μM for 8h) or (h) AKT inhibitor Triciribine (10 μM for 6h). (i) INS-1E infected with Ad-GFP or Ad-MST1 or transfected with shMST1 or shScr control expression plasmids for 48h, serum-starved for 12h and then stimulated with insulin. Mst1, pMst1, pAkt, tAkt, pGsk3, pFoxo1 and caspase-3 cleavage were analyzed by western blotting. Right panels show densitometry analysis from at least 3 independent experiments normalized to actin. All western blots show representative results from at least 3 independent experiments from 3 different donors or mice. Tubulin/Actin was used as loading control. Results shown are means ±SE. *p<0.05 compared to untreated or nondiabetic control. #p<0.05 Myr-AKT1 compared to GFP at 22.2 mM glucose.

Figure 2. MST1 induces beta-cell death

(a-d) MST1 overexpression in human islets (a,b) and INS-1E cells (c) for 48h. Beta-cell apoptosis was analyzed by triple staining for DAPI (blue), TUNEL (red) and insulin (green; a). An average number of 18501 insulin-positive beta-cells were counted in 3 independent experiments from 3 different donors. (b,c) Efficient adenovirus-mediated up-regulation of MST1 and profiling expression levels of proteins of the mitochondrial death pathway. BIM, BAX, caspase-9 cleavage, BCL-2 and BCL-xL together with JNK activation and caspase-3 and PARP cleavage were analyzed by western blotting. (d) Exposure of Ad-GFP- or Ad-MST1-infected human islets to Bax-inhibitory peptide V5 or negative control (NC) peptide for 36h. Caspase-3 cleavage was analyzed by western blotting. (e,f) Human islets transfected with BIM siRNA or control siScr were infected with Ad-GFP or Ad-MST1 for 48h. (e) Beta-cell apoptosis analyzed by double staining of TUNEL and insulin. An average number of 10378 insulin-positive beta-cells were counted in 3 independent experiments from 3 different donors. (f) MST1, BIM, caspase-3 and PARP cleavage were analyzed by western blotting. (g) Human islets transfected with GFP or dnJNK1 expressing-plasmids and infected with Ad-GFP or Ad-MST1 for 48h. PC-Jun, Bim and caspase-3 cleavage were analyzed by western blotting. (h) Human islets transfected with GFP or Myr-AKT1 expressing-plasmids and infected with Ad-GFP or Ad-MST1 for 48h. BIM and caspase-3 cleavage were analyzed by western blotting. Right (b,d,f,g,h)/ left (c) panels show densitometry analysis from at least 3 independent experiments normalized to actin. All western blots show representative results from at least 3 independent experiments from 3 different donors (human islets). Actin was used as loading control. Results shown are means ±SE. *p<0.05 MST-OE compared to GFP control, §p<0.05 V5-MST1 compared to MST1, **p<0.05 siBim-MST1 compared to siScr-MST1, #p<0.05 dnJNK-MST1 compared to MST1, +p<0.05 AKT1-MST1 compared to MST1.

Figure 3. MST1 impairs beta-cell function by destabilizing PDX1

(a,b) Adenovirus-mediated GFP or MST1 overexpression in human islets for 96h. (a) Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content and basal secretion at GFP control. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively and insulin content analyzed after GSIS and normalized to whole islet protein. (b) MST1 and PDX1 immunoreactivity were analyzed by Western blotting. Lower panel shows densitometry analysis from at least 3 independent experiments normalized to actin. Right panel shows PDX1 target genes including SLC2A2, GCK and Insulin analyzed by RT-PCR. (c-d) HEK293 cells were transfected with plasmids encoding Myc-MST1 and GFP-PDX1. (c) A kinase-dead MST1 (dn-MST1: K59R) was co-transfected with GFP-PDX1 (left panel). At 48 h after transfection, HEK293 cells were treated with cycloheximide (CHX) for 8h (middle panel). At 36h after transfection, HEK293 cells were treated with the proteasome inhibitor MG-132 for 6h (right panel). PDX1 and MST1 were analyzed by western blotting. (d) In vivo ubiquitination assay in HEK293 cells transfected with GFP-PDX1 and HA-ubiquitin, alone or together with Myc-MST1 or MST1-K59 expression plasmids for 48h (left) and human islets transfected with HA-ubiquitin and infected with Ad-GFP or Ad-MST1 for 48h (right; 2 different donors). MG-132 was added during the last 6h of the experiment. Cell lysates were immunoprecipitated with an anti-PDX1 antibody followed by immunoblotting with ubiquitin antibody to detect ubiquitinated PDX1. (e) HEK293 cells were transfected with GFP-PDX1 alone or together with Myc-MST1 for 48h. Reciprocal co-immunoprecipitations performed using anti-GFP and anti-Myc antibodies and western blot analysis performed with precipitates and input fraction using anti-Myc and anti-GFP antibodies, respectively. (f) In vitro kinase assay was performed by incubating recombinant MST1 and PDX1 proteins and analyzed by NuPAGE followed by western blotting using pan-phospho-threonine specific, PDX1 and MST1 antibodies. (g) Lysates of HEK293 cells transfected with PDX1-WT or PDX1-T11A expression-plasmids were immunoprecipitated with PDX1 antibody and subjected to an in vitro kinase assay using recombinant MST1. Phosphorylation reactions were analyzed by Western blotting using p-T11-PDX1 specific and pan-phospho threonine antibodies (left panel). HEK293 cells were transfected with PDX1-WT or PDX1-T11A alone or together with MST1 expression-plasmids for 48h. MST1 and PDX1 were analyzed by western blotting (middle panel). PDX-1-WT or PDX1-T11A co-transfected with MST1 in HEK293 cells for 36h and treated with CHX, western blot analysis for PDX1 and densitometry analysis of bands (right panel). (h) Human islets transfected with GFP, PDX1-WT or PDX1-T11A expression-plasmids and western blot analysis for PDX1. (i,j) human islets were infected with Ad-GFP or Ad-MST1 for 72h. (i) Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content and basal secretion at control. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively. (j) PDX1 target genes in human islets analyzed by RT-PCR and levels normalized to tubulin and shown as change from PDX1-WT transfected islets. All western blots show representative results from at least 3 independent experiments from 3 different donors (human islets). Tubulin/Actin was used as loading control. RT-PCR (b,j) and GSIS (a,i) show pooled results from 3 independent experiments from 3 different donors. Results shown are means ±SE. *p<0.05 MST-OE compared to control, **p<0.05 PDX-1T11A-MST1 compared to PDX-1WT-MST1.

Figure 4. MST1 deficiency improves beta-cell survival and function

(a-d) Human islets transfected with MST1 siRNA or control siScr and treated with the cytokines mixture IL/IF, 33.3 mM glucose or the mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3Pal) for 72h. (a) Beta-cell apoptosis analyzed by double staining of TUNEL and insulin. An average number of 11390 insulin-positive beta-cells were counted for each treatment condition in 3 independent experiments from 3 different donors. (b) Western blotting confirmed successful (~80%) MST1 depletion in human islets. MST1, pMST1, BIM, pH2B, caspase-9 and caspase-3 cleavage analyzes by western blotting. Right panel shows densitometry analysis from at least 3 independent experiments normalized to actin. (c) RT-PCR for BCL2L11 performed in human islets and levels normalized to tubulin shown as change from siScr control transfected islets. (d) Insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 1h-incubation with 2.8 mM glucose. (e,f) Islets were isolated from Mst1^−/− mice and their WT littermates and exposed to the cytokines mixture IL/IF or the mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3Pal) for 72 hours. (e) beta-cell apoptosis analyzed by double staining for TUNEL and insulin. An average number of 24180 insulin-positive beta-cells were counted for each treatment condition in 3 independent experiments. (f) Insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 1h-incubation with 2.8 mM glucose. (g-i) Stable INS-1E clones were generated by transfection of vectors for shMst1 and shScr control and treated with the cytokines mixture IL/IF or 22.2 or 33.3 mM glucose for 72h. (g) Mst1, Bim, Pdx1, caspase-3 and PARP cleavage were analyzed by western blotting. Right panel shows densitometry analysis from at least 3 independent experiments normalized to actin. (h) Insulin stimulatory index. (i) PDX1 target genes in shMst1 and shScr control INS-1E cells normalized to tubulin and shown as change from shScr control INS1-E clones. Western blots (b,g) show representative results from 3 independent experiments from 3 different donors (human islets). Actin was used as loading control. TUNEL data (a,e), GSIS (d,f,h) or RT-PCR (c,i) show pooled results from 3 independent experiments. Results shown are means ±SE. *p<0.05 compared to siScr (a,b,c,d), WT (e,f) or shScr untreated controls (g,h,i), **p<0.05 compared to siScr (a,b,c,d), WT (e,f) or shScr (g,h,i) at the same treatment conditions.

Figure 5. Mst1 deletion protects from diabetes in vivo

(a-g) Mst1^−/− mice (n=15) and their WT littermates (n=14) were injected with 40 mg/kg streptozotocin or citrate buffer for 5 consecutive days. (a) Random fed blood glucose measurements after last STZ injection (day 0) over 21 days and intraperitoneal glucose tolerance test (ipGTT) performed at day 17. (b) Insulin secretion during an ipGTT measured before (0 min) and 30 min after glucose injection and data are expressed as ratio of secreted insulin at 30 min/0 min (stimulatory index). (c) The ratio of secreted insulin and glucose is calculated at fed state. (d-g) Mice were sacrificed at day 22. (d) Beta-cell mass and quantitative analyses from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. The mean number of beta-cells scored was 23121 for each treatment condition. (e) The pancreatic area of alpha- (stained in red) and beta-cells (stained in green) are given as percentage of the whole pancreatic section from 10 sections spanning the width of the pancreas. (f,g) Representative double-staining for Bim (red, f) or Pdx1 (red, g) and insulin (green) is shown from STZ-treated Mst1^−/− mice and controls. White arrows indicate areas of cytosolic Pdx1 localization and its total absence in WT-STZ mice. (h-j) bMst1^−/− mice with specific deletion in the beta-cells using the Cre-Lox system (n=5) and their Rip-Cre (n=3) and fl/fl controls (n=3) were injected with 40 mg/kg STZ for 5 consecutive days. (h) Random fed blood glucose measurements after last STZ injection (day 0) over 32 days and ipGTT at day 32. (i) Insulin secretion during an ipGTT measured before (0 min) and 30 min after glucose injection and data are expressed as ratio of secreted insulin at 30 min/0 min (stimulatory index). The ratio of secreted insulin and glucose is calculated at fed state (right panel). (j) Mice were sacrificed at day 32. Beta-cell mass analysis and results from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. Data show means ± SE. *p<0.05 WT-STZ compared to WT saline injected mice, **p<0.05 MST1^−/−-STZ compared to WT-STZ mice. #p<0.05 bMST-STZ compared to fl/fl-STZ or Cre-STZ mice.

Figure 6. Beta-cell specific disruption of Mst1 prevents hyperglycemia and high-fat diet induced diabetes progression

(a-e) bMST1^−/− mice (fl/fl-Cre; n=12) and the Cre control mice (n=12) were fed a normal (ND) or high fat/ high sucrose diet (“Surwit”; HFD) for 20 weeks. (a) Random fed blood glucose measurements, (b) intraperitoneal glucose tolerance test (ipGTT) and (c) insulin secretion during an ipGTT measured before (0 min), 15 and 30 min after glucose injection. (d,e) Mice were sacrificed at week 21. (d) Islets were isolated from all 4 treatment groups, cultured overnight and subjected to an in vitro GSIS assay. Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively. (e) Beta-cell mass analysis and results from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. The mean number of beta-cells scored was 23121 for each treatment condition. *p<0.05 Cre HFD compared Cre ND mice. **p<0.05 bMst1^−/−-HFD compared to Cre HFD mice. (f) Our view on how diabetic stimuli lead to activation of MST1. Active MST1 triggers cytochrome c release and mitochondrial-dependent apoptosis by modulating Bim/Bax/Bcl2/Bcl-xL through JNK/AKT signaling. Active caspase-9 then triggers cleavage of caspase-3, which triggers the caspase-3-dependent cleavage of MST1 to its constitutively active fragment, which leads to further MST1 activation and processing of caspase-3 by a positive feedback mechanism, and acceleration of beta-cell death occurs. Cleaved MST1 translocates to the nucleus and directly phosphorylates PDX1 (we do not exclude the possibility that MST1 targets PDX1 also in cytoplasm) and histone H2B. PDX1 then shuttles to cytosol, where it marks for ubiquitination and subsequent degradation by proteasome machinery and beta-cell function is impaired. Histone H2B phosphorylation by MST1 also induces chromatin condensation, one of the characteristic features of apoptosis.