Checkpoint kinase 2 controls insulin secretion and glucose homeostasis

After the discovery of insulin, a century ago, extensive work has been done to unravel the molecular network regulating insulin secretion. Here we performed a chemical screen and identified AZD7762, a compound that potentiates glucose-stimulated insulin secretion (GSIS) of a human β cell line, healthy and type 2 diabetic (T2D) human islets and primary cynomolgus macaque islets. In vivo studies in diabetic mouse models and cynomolgus macaques demonstrated that AZD7762 enhances GSIS and improves glucose tolerance. Furthermore, genetic manipulation confirmed that ablation of CHEK2 in human β cells results in increased insulin secretion. Consistently, high-fat-diet-fed Chk2 − / − mice show elevated insulin secretion and improved glucose clearance. Finally, untargeted metabolic profiling demonstrated the key role of the CHEK2–PP2A–PLK1–G6PD–PPP pathway in insulin secretion. This study successfully identifies a previously unknown insulin secretion regulating pathway that is conserved across rodents, cynomolgus macaques and human β cells in both healthy and T2D conditions.


Article
https://doi.org/10.1038/s41589-023-01466-4 Among the 21 primary hits, AZD7762 (1), prostratin and tyrphostin AG1296 are the top three compounds that have the strongest effects on insulin secretion.Prostratin is known to activate protein kinase C, which has a well-documented role in regulating insulin secretion in β cells 11 .Tyrphostin AG1296 is an inhibitor of platelet-derived growth factor receptor (PDGFR).PDGFR belongs to receptor tyrosine kinases, whose activation has been shown to be involved in β-cell exocytosis 12,13 and proliferation 14 .AZD7762 (Fig. 1b) is a competitive dual CHEK1/CHEK2 inhibitor 15 .The role of CHEK2 in insulin secretion is largely unknown.Because AZD7762 has been used in human clinical trials 16 , this small molecule represents a useful tool for further elucidating the role of CHEK2 in insulin secretion in vitro and in vivo.AZD7762 increased luminescent signals from NLuc-MIN6 cells in a dose-dependent manner in the presence of 20 mM d-glucose, but not in the absence of glucose (Supplementary Fig. 1a).In addition, AZD7762 increased luminescent signals from human EndoC-βH1 carrying proinsulin-NanoLuc reporter (NLuc-EndoC-βH1 cells) through a dose-dependent manner (Supplementary Fig. 1b).We further confirmed that AZD7762 stimulated insulin and C-peptide secretion from MIN6 cells and human EndoC-βH1 cells using ELISA.Consistent with the dose curve, AZD7762 significantly increased both insulin (Extended Data Fig. 1b) and C-peptide (Extended Data Fig. 1c) secretion in MIN6 cells in the presence of 20 mM d-glucose, but not in the absence of glucose.In addition, AZD7762 treatment significantly increased insulin (Extended Data Fig. 1d) and C-peptide (Extended Data Fig. 1e) secretion of human EndoC-βH1 cells at both 0.5 mM and 20 mM d-glucose conditions.AZD7762 also increases insulin secretion of EndoC-βH1 cells through a glucose-dose-dependent manner at 2 mM, 5 mM and 11 mM d-glucose (Extended Data Fig. 1f).Consistent with the observed activity of 10 µM AZD7762, a lower dose of 1 µM AZD7762 also significantly increased insulin secretion from MIN6 and EndoC-βH1 cells during GSIS (Extended Data Fig. 1g,d).Moreover, EndoC-βH1 cells treated with AZD7762 for 24 h also exhibit elevated insulin secretion (Extended Data Fig. 1h).To determine whether AZD7762 functions through insulin processing, we measured the total cellular insulin and proinsulin levels in EndoC-βH1 cells treated with 1 µM and 10 µM AZD7762 and did not detect any difference (Supplementary Fig. 1c), indicating that AZD7762 does not promote insulin secretion through enhanced proinsulin to insulin processing.Notably, AZD7762 did not increase β-cell death as evidenced by unchanged percentages of propidium iodide (PI) + EndoC-βH1 cells after 1 h (Supplementary Fig. 1d) or 24 h (Supplementary Fig. 1e) of AZD7762 treatment.In addition, AZD treatment promotes insulin secretion in the absence of starvation (Extended Data Fig. 1i).To investigate whether AZD7762 increases GSIS by relieving cellular stress on β cells caused by starvation, we assessed stress markers, including phospho-eIF2α and phospho-CHEK2, by western blot and found that starvation in 0.5 mM glucose did not increase the phosphorylation of either CHEK2 or eIF2α (Supplementary Fig. 1f-i).Therefore, AZD7762 does not increase GSIS by relieving cellular stress caused by glucose deprivation.Together, these data show that AZD7762 treatment potentiates GSIS in both mouse and human β cell lines in the presence of glucose.

AZD7762 increases insulin secretion in human islets
We next tested if AZD7762 can improve insulin secretion from primary human islets.Consistent with the results using β cell lines, AZD7762-treated intact normal human islets (Supplementary Table 4) showed enhanced insulin secretion at both 2 mM and 20 mM d-glucose (Fig. 1c).We further validated the activity of AZD7762 on pseudoislets 17 , which showed a more robust and consistent response to glucose than intact islets.Each pseudoislet comprised approximately 2,000 dissociated human islet cells.Dynamic GSIS showed that AZD7762 treatment significantly increased secretion of insulin (Fig. 1d,e) and C-peptide (Fig. 1f,g), as well as the total area under the curve (AUC; Fig. 1e (insulin) and Fig. 1g (C-peptide)).To evaluate the activity of AZD7762 on pseudoislets that are similar to those in T2D condition, we cultured pseudoislets factors, such as paracrine hormones-glucagon-like peptide-1 (GLP-1) and somatostatin (SST) [1][2][3][4] .
Several major metabolic cycles that have been shown to regulate GSIS include pyruvate-malate cycle, pyruvate-citrate cycle, pyruvateisocitrate cycle 5 , phosphoenolpyruvate (PEP) cycle 6 , TCA cycle, mitochondrial oxidative phosphorylation and glycerolipid-free-fatty-acid (GL/FFA) cycle 1,2,4,7 .Potential crosstalk between metabolic cycles allows for a well-coordinated response of insulin secretion that ultimately converges to common downstream events that trigger exocytosis.Over the years, researchers have identified many metabolic mediator signals generated from these pathways that are critical for GSIS.These include ATP generated from the PEP cycle, TCA cycle and mitochondria oxidative phosphorylation; cytosolic NADPH from the pyruvatemalate cycle and pyruvate-isocitrate cycle; monoacylglycerols from GL/FFA, etc 1,2 .Evidence suggests that these intermediates influence the insulin secretion process at various steps and are responsible for tuning the overall strength of the cascade leading up to exocytosis.Because clinical studies suggested that both the triggering and amplifying phases are impaired in type 2 diabetes 8 , a detailed characterization and molecular understanding of insulin secretion cascades would help identify new target and strategy for therapy.
Chemical screens offer an unbiased approach to identify chemical tools to dissect biological processes related with human β cells.Most screens have focused on β cell identities and survival.Other screens focused on specific pathways and molecular targets that are known to regulate β-cell functions.Few studies 9 have focused on insulin secretion, partially due to the lack of a high-throughput approach to monitor insulin secretion.In 2015, a proinsulin-NanoLuc fusion reporter was developed to allow real-time monitoring of insulin secretion 9 .Using this type of reporter, chemical screens were performed to identify compounds that improve insulin secretion 9,10 .However, the in vivo activity and molecular mechanism of these hit compounds are largely unknown.
Here we performed a focused chemical screen and identified AZD7762, which significantly increased insulin secretion from mouse and human β cells stimulated with high glucose.Using pharmacological and genetic approaches, we systematically confirmed the role of CHEK2 function in insulin secretion of EndoC-βH1 cells, healthy and T2D human islets, cynomolgus macaque islets, as well as in chowfed, high-fat-diet (HFD)-fed and genetically obese leptin-deficient Lep ob/ob (ob/ob) mice.By combining chemical screening, pharmacological, genetic and metabolomics approaches, we discovered a previously unknown role of the CHEK2-PP2A-PLK1-glucose-6phosphate dehydrogenase (G6PD)-pentose phosphate pathway (PPP) in insulin secretion.

Small molecule AZD7762 enhances insulin secretion
To identify small molecules that acutely enhance insulin secretion from β cells, we performed a focused chemical screen using the mouse insulinoma β cell line (MIN6) carrying a proinsulin-NanoLuc reporter (NLuc-MIN6) 9 with chemicals from an in-house chemical library containing 223 compounds targeting different signaling pathways (Supplementary Tables 1 and 2).After 1 h of treatment, cells were used for GSIS using luminescence signals as a surrogate for insulin levels (Fig. 1a).The compounds that enhanced luciferase signals under high glucose by more than 1.5-fold from the mean were chosen as primary hits.We further confirmed the activities of the hit compounds with a subsequent GSIS experiment and observed that 62% of the primary hits elevated high glucose-stimulated insulin secretion (Extended Data Fig. 1a).Among the 21 primary hits, 19 compounds were found to target signaling pathways that have been previously implicated to be involved in regulating insulin secretion, insulin sensitivity, β-cell proliferation, β-cell apoptosis or glucose metabolism (Supplementary Table 3).This finding validates the effectiveness of our screening platform.

Article
https://doi.org/10.1038/s41589-023-01466-4generated from healthy islets in 0.4 mM sodium oleate for 2 d to induce lipotoxicity and then assessed the short-term effect of AZD7762 on GSIS.One-hour treatment with AZD7762 significantly increased GSIS from pseudoislets cultured in lipotoxic condition (Extended Data Fig. 2a,b).The effect of AZD7762 was also confirmed using intact islets from T2D donors.Consistent with the impact on human EndoC-βH1 cells and healthy human islets, AZD7762 significantly improved GSIS (Fig. 1h) and glucose-stimulated C-peptide secretion (GSCS; Fig. 1i) in static condition.The perfusion experiments further validated that AZD7762 increased C-peptide secretion in response to 20 mM d-glucose (Fig. 1j).AUC was significantly increased in AZD7762-treated T2D islets (Fig. 1k).Then, we investigated if AZD7762 altered the secretion of other islet hormones and found that AZD7762 treatment did not alter secretion of SST from intact human islets (Extended Data Fig. 2c).AZD7762 treatment significantly reduced glucagon (GCG) secretion from intact normal human islets, possibly due to the inhibited effects of insulin on glucagon secretion, an observation reported by several groups 18,19 (Extended Data Fig. 2d).Furthermore, immunostaining experiments confirmed that AZD7762 treatment does not change the percentages of insulin (INS)-, GCG-, SST-and Ki67-positive cells in human islets (Extended Data Fig. 2e-l).These results suggest that AZD7762 stimulates insulin secretion in primary human islets without changing the islet cellular composition.

AZD7762 improves insulin secretion in mouse models
Because AZD7762 effectively increased insulin secretion from β cells in vitro, we further evaluated its impact on glucose metabolism in vivo using both healthy and T2D mouse models.In the glucose tolerance test (GTT), overnight-fasted chow-fed CD-1/ICR mice were first intraperitoneally (IP) treated with 25 mg kg −1 of AZD7762.One hour later, mice were injected IP with glucose for GTT and GSIS experiments.AZD7762 significantly improved glucose tolerance in chow-fed CD-1/ICR mice (Fig. 2a,b).Consistently, AZD7762-treated mice show significantly increased insulin secretion compared to vehicle-treated mice at 0-and 15-min postglucose injection (Fig. 2c).Because 25 mg kg −1 AZD7762 led to a slight reduction in glucose levels in mice before glucose administration, we evaluated the effect of lower dose of AZD7762 on glucose tolerance and GSIS.At a dose of 12 mg kg −1 , AZD7762 treatment did not cause hypoglycemia at 0 min, while still significantly improving glucose tolerance and insulin secretion at both 0-and 15-min postglucose  injection (Extended Data Fig. 3a,b).Notably, even when no glucose was administered for a total period of 3 h, treatment with 12 mg kg −1 AZD7762 did not result in hypoglycemia (Extended Data Fig. 3c).The effect of AZD7762 on glucose tolerance is short-term as there was no change in glucose tolerance in mice 24 h after AZD7762 treatment (Extended Data Fig. 3d).This result is consistent with the half-life of AZD7762 reported to be only 1-2 h in mice 20 .Next, two T2D mouse models, HFD-fed C57BL/6J mice and genetically obese leptin-deficient Lep ob/ob (hereafter ob/ob mice) mice were used to evaluate the effect of AZD7762.Similar to the effect on chow-fed mice, 1-h pretreatment with AZD7762 significantly improved glucose tolerance during intraperitoneal glucose tolerance test (IPGTT) in C57BL/6J mice fed with HFD for 4 months (Supplementary Fig. 2a,b).Insulin secretion was also higher in AZD7762-treated mice, but the increase in insulin secretion only reached significance at 30 min postglucose administration (Supplementary Fig. 2c).Additionally, 16-week-old ob/ob mice treated with AZD7762 showed improved glucose tolerance (Fig. 2d,e) and enhanced insulin secretion at 15 min postglucose administration compared with vehicle-treated ob/ob mice (Fig. 2f).We calculated the homeostasis model assessment of insulin resistance (HOMA-IR) in AZD-treated and control chow-fed CD-1/ICR, HFD-fed C57BL/6J and ob/ob mice, and found no significant differences between the vehicle and AZD7762 treatment groups (Extended Data Fig. 3e and Supplementary Fig. 2d).
To determine the possible effect of AZD7762 on incretin hormones, we measured active GLP-1 and total GIP levels during in vivo GSIS of CD-1 mice at 0-and 15-min postglucose injection.We did not detect a difference in GLP-1 levels between AZD7762-and vehicle-treated mice (Extended Data Fig. 3f).We observed a reduction in GIP levels at 0-and 15-min postglucose injection in AZD7762 mice treated with AZD7762 (Extended Data Fig. 3g), which is consistent with increased insulin levels as it was reported that insulin can inhibit GIP secretion in vivo [21][22][23][24] .Furthermore, we investigated if AZD7762 increases secretion of other islet hormones from primary mouse islets.Similar to primary human islets (Extended Data Fig. 2c,d), mouse islets treated with AZD7762 did not show any change in SST secretion but revealed insignificant reductions in glucagon secretion (Extended Data Fig. 3h,i).Furthermore, AZD7762-treated mice did not show an improvement in insulin sensitivity during an insulin tolerance test (Extended Data Fig. 3j).Together, the in vivo data strongly suggest that AZD7762 improves glucose homeostasis by stimulating insulin secretion in β cells without altering insulin sensitivity.

AZD7762 enhances GSIS in cynomolgus macaques
We further evaluated AZD7762's activity using cynomolgus macaques, a nonhuman primate model.AZD7762-treated cynomolgus macaque islets showed a significant increase in insulin and C-peptide secretion when stimulated with 20 mM d-glucose (Fig. 3a,b).To determine the in vivo activity of AZD7762 on cynomolgus macaques, we performed an intravenous glucose tolerance test (IVGTT).The animals were injected with 1.6 mg kg −1 of AZD7762 1 h before the experiment (Fig. 3c).Pretreatment with AZD7762 significantly increased glucose tolerance (Fig. 3d,e) with approximately 20% reduction in AUC.The improvement in glucose tolerance was accompanied by a significant increase in insulin secretion (Fig. 3f) and C-peptide secretion (Fig. 3g) at 0-, 1, 3-, 5-and 15-min post-IV-glucose injection.These results show that AZD7762 does not only improve GSIS in primary human islets in vitro but also improves GSIS and glucose homeostasis in cynomolgus macaques in vivo, supporting a possible physiological role of CHEK2 in modulating insulin secretion in response to glucose in primates.

Reduced CHEK2 in human β cells improves insulin secretion
Because both AZD7762 (a competitive CHEK1/CHEK2 inhibitor) 15 and CCT241533 (2, a selective CHEK2 inhibitor) 25 (Supplementary Table 3) were identified to potentiate GSIS in the primary screen, we decided to apply genetic approaches to confirm and determine the role of CHEK2 in β-cell function.We generated CHEK2-deficient EndoC-βH1 (hereafter known as sgCHEK2 EndoC-βH1) cells by using a CRISPR-Cas9-mediated knockout approach.In brief, EndoC-βH1 cells were infected with a lentivirus carrying single guide RNA (sgRNA) against exon 2 of CHEK2 (sgCHEK2) or a scrambled control sgRNA (Supplementary Table 5).After 1 week of puromycin selection, western blotting confirmed a significant reduction in CHEK2 protein levels (Fig. 4a,b).No detectable difference in total insulin was observed between EndoC-βH1 cells carrying control scrambled sgRNA or sgCHEK2 (Extended Data Fig. 4a), suggesting that CHEK2 is not involved in insulin synthesis.Static GSIS confirmed that EndoC-βH1 cells carrying sgCHEK2 showed an increase in insulin (Fig. 4c) and C-peptide (Fig. 4d) secretion when stimulated with 20 mM d-glucose, suggesting that the reduction of CHEK2 led to an increased response to glucose stimulation.EndoC-βH1 cells carrying scrambled sgRNA or sgCHEK2 were then aggregated to form pseudoislets and used for dynamic GSIS.Consistent with the static GSIS results, the pseudoislets containing EndoC-βH1 cells carrying sgCHEK2 showed enhanced insulin response (Fig. 4e) and AUC (Fig. 4f), as well as C-peptide secretion (Fig. 4g,h).
To further examine the impact of the loss of CHEK2 on β-cell function in T2D condition, NLuc-EndoC-βH1 cells carrying control scrambled sgRNA or sgCHEK2 were cultured in the presence of 2 mM sodium oleate for 72 h and then assessed for their insulin secretory response to glucose.Similar to other reports on β-cell dysfunction induced by lipotoxicity, NLuc-EndoC-βH1 cells carrying scrambled sgRNA cultured in sodium oleate showed diminished insulin secretion in response to glucose stimulation (Fig. 4i), while NLuc-EndoC-βH1 cells carrying sgCHEK2 showed higher insulin response to glucose stimulation than the control (Fig. 4i,j).These data suggest that the pathway that mediates CHEK2's effect on GSIS is at least partially preserved in lipotoxic T2D-like condition.
Considering that AZD7762 is known to target other kinases, we conducted a shRNA knockdown experiment to evaluate the effect of AZD7762's off-targets on GSIS.We first performed a kinase screening assay using AZD7762 and observed that 36 of the 37 reported kinase targets of AZD7762 (ref.15), including CHEK2, were inhibited by more than 40% (Supplementary Tables 6 and 7).Subsequently, we designed two shRNAs to knockdown each of the off-target genes in EndoC-βH1 cells (Supplementary Table 8) and evaluated the effects of these gene knockdowns on GSIS (Extended Data Fig. 4b).Notably, knockdown of any of the off-target genes of AZD7762, including CHEK1, did not result in an increase in GSIS (Extended Data Fig. 4c), further confirming that AZD7762 primarily exerts its effects through CHEK2.We also evaluated the impact of AZD7762 on shCHEK2 EndoC-βH1 cells and Chk2 −/− mouse islets and found that the GSIS response to AZD7762 treatment was significantly reduced in CHEK2-deficient β cells (Extended Data Fig. 4d,e).Consistent with the impact of CHEK2 deficiency on GSIS, overexpression of CHEK2 suppressed insulin secretion from shCHEK2 EndoC-βH1 cells (Extended Data Fig. 4f,g).Overall, these results confirm that the effect of AZD7762 on GSIS is mediated via suppressing CHEK2 functions, and suppressing CHEK2 functions can improve insulin secretion in both normal and related conditions in vitro.
Our previous data showed that the reduction of CHEK2 improved human β-cell function in T2D conditions in vitro (Fig. 4i,j).We therefore evaluated the glucose metabolism in Chk2 −/− mice fed with 60% HFD for 6 months.Male Chk2 −/− mice exhibited decreased fed glucose levels (Fig. 5c) and increased fed insulin levels (Fig. 5d).Moreover, HFD-fed male Chk2 −/− mice displayed significant improvement in oral glucose tolerance test (OGTT; Fig. 5e), accompanied by an increase in insulin secretory response after glucose administration (Fig. 5f).Similar to AZD7762-treated mice (Extended Data Fig. 3f), active GLP-1 levels were not significantly different between wild-type and Chk2 −/− mice, suggesting that the increased insulin secretion during OGTT was not due to changes in active GLP-1 levels (Fig. 5g).Similar to the male Chk2 −/− mice, HFD-fed female Chk2 −/− mice also showed improved glucose tolerance (Fig. 5h) and increased insulin secretion (Fig. 5i) during OGTT.We further isolated islets from HFD-fed Chk2 −/− mice to confirm the increase in insulin secretion in vitro.HFD Chk2 −/− mouse islets showed enhanced insulin secretion in response to 20 mM d-glucose when compared to HFD-fed wild-type mouse islets (Extended Data Fig. 5a).Together, these results show that the loss of CHEK2 improves GSIS in normal and T2D conditions and Chk2 −/− mice exhibit increased resistance to HFD-induced β-cell dysfunction.
To further elucidate the effect of Chk2 loss-of-function in vivo in islets, we performed immunostaining of islet cell markers on pancreatic sections from HFD-fed wild-type and Chk2 −/− mice to determine if Chk2 loss-of-function led to any changes in the cellular composition of the pancreatic islet (Extended Data Fig. 5b).Immunostaining of INS, GCG, SST and PP did not uncover any changes in percentage islet area or islet cell markers in HFD-fed Chk2 −/− mice (Extended Data Fig. 5c-h).Consistent with unchanged percentage of INS-positive cells in HFD-fed Chk2 −/− mouse islets, the percentage of cells expressing neurogenin-3 (NGN3), a dedifferentiation marker of β cell 29 , in pancreatic islets was also not changed in HFD-fed Chk2 −/− mice (Extended Data Fig. 5i,j).Hematoxylin and eosin (H&E) staining of pancreatic section also did not reveal any changes in HFD-fed Chk2 −/− mouse islets (Supplementary Fig. 3e).Together, these data suggest that loss of CHEK2 does not change the islet structure or cellular composition.

Metabolomics reveal CHEK2-PP2A-PLK1-G6PD-PPP pathway
To understand how reduced CHEK2 function potentiates insulin secretion, we performed an untargeted metabolomics profiling using MIN6 cells treated with control or AZD7762 in the absence or presence of 20 mM d-glucose.Partial least square-discriminant analysis (PLS-DA) scores plot confirmed that four different treatment groups clustered separately (Extended Data Fig. 6a).The heatmap highlighted a group of metabolites that were increased in AZD7762-treated cells only in the presence of 20 mM d-glucose (Fig. 6a).Metabolites reported to be associated with insulin secretion such as fructose, sedoheptulose and ribose-5-phosphate 30 were only increased by AZD7762 treatment at 20 mM d-glucose, consistent with our observation that AZD7762 augmented insulin secretion at 20 mM d-glucose, but not at 0 mM glucose (Extended Data Fig. 1i).QIAGEN Ingenuity Pathway Analysis (IPA) analysis of these metabolites identified that PPP was significantly increased in AZD7762-treated cells upon 20 mM d-glucose stimulation (Fig. 6b).
To confirm the changes in PPP in AZD7762-treated cells upon 20 mM d-glucose stimulation, we assessed the activity of G6PD, the rate-limiting enzyme of PPP 31 .AZD7762 significantly increased G6PD activities (Extended Data Fig. 6b).Consistent with the changes in PPP and G6PD activity, cytosolic NADPH/NADP ratio was also significantly elevated in the presence of AZD7762 (Extended Data Fig. 6c).These data showed that acute pharmacological inhibition of CHEK2 activated PPP and increased G6PD activities, and cytosolic NADPH/NADP ratio.Consistently, AZD7762 significantly stimulated G6PD activities in EndoC-βH1 cells (Extended Data Fig. 6d).We then confirmed the change of PPP in genetically modified EndoC-βH1 cells.Similar to the results of pharmacological inhibition of CHEK2 with AZD7762, sgCHEK2 EndoC-βH1 cells also showed higher G6PD activity when compared to control EndoC-βH1 cells stimulated with 20 mM glucose (Fig. 6c).Together, our data show that potentiation of GSIS by CHEK2 inhibition requires activation of PPP and G6PD activities.
Given the absence of previous reports on a direct interaction between CHEK2 and G6PD, we hypothesize that other effectors might be involved in mediating the effect of CHEK2 on G6PD activity.Studies have demonstrated that CHEK2 binds and activates PP2A 32 , which in turn can deactivate PLK1 through dephosphorylation 33 .PLK1 has been shown to directly activate G6PD 34 , the rate-limiting enzyme for the PPP.We then used a shRNA knockdown strategy to explore the relationship between CHEK2, PP2A, PLK1 and G6PD, as well as to evaluate their contribution to regulating insulin secretion in β cells.
First, we demonstrated that sgCHEK2 EndoC-βH1 cells exhibit a reduction in PP2A activity (Fig. 6d), which aligns with a previous report that CHEK2 phosphorylated and increased catalytic activity of PP2A (ref.32).Subsequently, we evaluated the effect of PP2A loss-of-function in β cells by knocking down PP2A expression in EndoC-βH1 cells using a shRNA strategy (Supplementary Fig. 4a).We refer to these cells as shPP2A EndoC-βH1 cells.Consistent with the previous report 33 , activation phosphorylation (T210) status of PLK1 was increased in shPP2A EndoC-βH1 cells (Fig. 6e,f).shPP2A EndoC-βH1 cells exhibited a reduction in AZD7762-potentiated GSIS (Fig. 6g,h).This is consistent with the role of PP2A in mediating CHEK2's effect on insulin secretion.Next, we evaluated the function of PLK1 in β cells by infecting EndoC-βH1 cells with lentivirus carrying shRNA against PLK1 (hereafter known as shPLK1 EndoC-βH1 cells; Supplementary Fig. 4b).Reduction of PLK1 resulted in a decrease of G6PD activity (Fig. 6i), which is consistent with the reported effect of PLK1 on activating G6PD activity 34 .Aligned with PLK1's role in regulating GSIS, shPLK1 EndoC-βH1 cells exhibited reduced GSIS (Fig. 6j), as well as response to AZD7762 treatment (Fig. 6k).We further evaluated the role of G6PD in EndoC-βH1 cells by generating shRNA knockdown G6PD-deficient EndoC-βH1 cells (hereafter known as shG6PD EndoC-βH1 cells; Supplementary Fig. 4c).Similar to the decrease in PLK1, the decrease in G6PD in EndoC-βH1 cells resulted in a reduction of GSIS (Fig. 6l) and GSIS potentiation by AZD7762 (Fig. 6m).In summary, these data demonstrate that reduced CHEK2 activity leads to a decrease in PP2A activity, resulting in an increase in PLK1 activity, followed by an elevation in G6PD activity (Extended Data Fig. 6e).
Interestingly, the effect of AZD7762 was glucose-dependent and CHEK2 phosphorylation was reduced in a glucose-dose-dependent manner (Supplementary Fig. 4d,e).Tolbutamide, a sulfonylurea drug that inhibits flux through the K ATP channel and mimics glucose's response, however, did not reduce CHEK2 phosphorylation (Supplementary Fig. 4f,g), confirming that inhibition of CHEK2 phosphorylation is not an event secondary to the closure of K ATP channel and depolarization of the cell membrane.Consistent with other studies [35][36][37][38][39] , we observed an increase in G6PD activities and 6-phosphogluconate levels in MIN6 cells exposed to 20 mM glucose (Supplementary Fig. 4h,i).Bulk RNA sequencing (RNA-seq) on AZD7762-treated EndoC-βH1 cells showed that gene expression patterns between control and AZD7762-treated conditions are highly similar (Extended Data Fig. 6f).Furthermore, the volcano plot revealed that only a limited number of genes were significantly changed (Extended Data Fig. 6g).These evidence supporting the conclusion that AZD7762 primarily exerts its effects through the kinase pathway CHEK2-PP2A-PLK1-G6PD-PPP to acutely regulate GSIS response in β cells 40 .Nevertheless, we cannot exclude the possibility that other downstream targets of CHEK2 could be involved in regulating insulin secretion.

Discussion
CHEK2 is classically known to be involved in DNA repair mechanisms.CHEK2 variations have been associated with an increased risk of type 2 diabetes in multiple populations 41 .Checkpoint activation is also associated with low exocytosis in T2D β cells 42 .However, there are currently no reports of a direct link between CHEK2 and β-cell function.We identified CHEK2 inhibitors that effectively potentiated insulin secretion upon glucose stimulation using chemical screening.Improvements in glucose tolerance and in vivo insulin secretion were detected only in CHEK2 −/− mice that were fed with HFD.One possibility is that the loss of function of a protein from birth can be compensated developmentally [43][44][45] , which might mask the phenotype caused by the loss of gene function.Through untargeted metabolomics profiling, we discovered that inhibition of CHEK2 activity leads to activation of PPP that can potentially regulate GSIS 31,46 .Several studies have reported rapid increase in PPP metabolites in β cells after glucose exposure 1,30,36,37,[47][48][49][50] .Moreover, extracellular glucose stimulates an increase in ribose-5-phosphate in β cells, indicating that β cells possess an active and responsive PPP 7,50,51 .Pathway perturbation study by removing function of the rate-limiting enzymes of PPP, G6PD, also resulted in impaired GSIS 50,52 .Furthermore, cholecystokinin-8, a gut hormone that potentiates GSIS, has been shown to increase PPP activity in pancreatic islets 53,54 ; in clinical settings, G6PD deficiency is associated with an increase in the prevalence of T2D (ref.55) and G6PD activities are reduced in T2D (ref.56); patients with G6PD deficiency also have reduced insulin response to IVGTT 57 and impaired fasting glucose 40,51,58,59 .
In summary, we discovered a previously unreported role of CHEK2 in modulating insulin secretion in both healthy and T2D conditions.While our study provides compelling evidence supporting the role of CHEK2 in AZD7762-potentiated insulin secretion in β cells, we recognize potential contribution of non-CHEK2 targets in this process, which will require further investigation.Due to the small chemical library used for screening in this study, only a limited number of new Article https://doi.org/10.1038/s41589-023-01466-4pathways were identified.Future research, using larger and diverse chemical libraries along with advanced screening techniques, holds promise for uncovering a broader array of therapeutic targets.Our findings strongly support the role of the PPP metabolic pathway in augmenting GSIS by inhibition of CHEK2.We used metabolomics and genetic manipulation strategies to discover a pathway that involves CHEK2, PP2A, PLK1 and G6PD in regulating insulin secretion.Manipulation of this axis could rescue the function of glucose-unresponsive β cells in T2D and enrich our comprehension of the molecular mechanism of GSIS regulation.
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Methods
All mouse studies have been approved by the Institutional Animal Care and Use Committee (IACUC) at Weill Cornell Medicine (2011-0024).All cynomolgus macaque work has been approved by IACUC at the University of Pennsylvania under protocol 806688.The pancreatic organs were obtained from the local organ procurement organization under the United Network for Organ Sharing (UNOS).The informed consent was obtained for research purposes.
For knocking down AZD7762 target genes in EndoC-βH1 cells, we designed two different shRNAs to target each using the Broad Institute GPP Web Portal (https://portals.broadinstitute.org/gpp/public/).The shRNA target sequences are listed in Supplementary Table 8.Each sgRNA was then cloned into pLKO.1-blast(Addgene, 26655) and packaged along with psPAX2 and pMD2.G.The transfection and lentivirus infection protocol used was similar to that used for shRNA knockdown experiment, as described above.Cells infected by lentivirus were then cultured with 5 µg ml −1 blasticidin (Invitrogen) for 1 week.

Bulk RNA-seq
Total RNA was extracted in TRIzol (Invitrogen) and treated with DNase I using the Directzol RNA Miniprep kit (Zymo Research).RNA-seq libraries of polyadenylated RNA were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) or TruSeq Stranded mRNA Library Prep Kit (Illumina).cDNA libraries were sequenced with pair-end 51 bps using an Illumina NovaSeq6000 platform.The resulting reads were checked for quality using FastQC (v0.10.1, https://www.bioinformatics.babraham.ac.uk/projects/fastqc) and were trimmed for adaptor sequences and low-quality bases using cutadapt (v1.18).To measure gene expression, the trimmed reads were aligned to the human refe rence genome (GRCh37).Raw gene counts were quantified using HTSeq-count (v0.11.2).The counts data were subjected to a regularized logarithm transformation using the rlog function within the DESeq2 package (v1.36.0).The transformed data were used to perform a principal component analysis (PCA) using the plotPCA function within the DESeq2 package.Additionally, the counts data were converted into fragment counts normalized per kilobase of feature-length per million mapped fragments (FPKM) using the fpkm function within the DESeq2 package, and an unsupervised hierarchical clustering on samples was conducted using the Pearson correlation coefficient metric.The R heatmap package (v1.0.12) was used to visualize the clustering result.

Quantitative RT-PCR
To validate shRNA knockdown efficiency, we measured gene expression levels in control and knock-downed cells using quantitative RT-PCR.We isolated total RNA from EndoC-βH1 cells using the RNeasy Plus Universal Kit (Qiagen) and synthesized cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).We used a Light-Cycler 480 SYBR Green I Master System (Roche) in the quantitative RT-PCR experiments.We normalized the expression of target genes against Β actin.Primers used for quantitative RT-PCR experiments can be found in Supplementary Table 9.

Insulin tolerance test
Before the insulin tolerance test, mice were fasted for 6 h.During the experiment, mice were injected IP with 1 IU kg −1 body weight of insulin and blood glucose levels were monitored at 0, 15, 30, 45, 60 and 120 min after insulin injection.

PP2A activity
sgCHEK2 and control EndoC-βH1 cells were starved in 0.5 mM glucose for 1 h and then stimulated with 20 mM glucose for 1 h.Cells were then lysed to assess PP2A activity with the PP2A Immunoprecipitation Phosphatase Assay Kit (MilliporeSigma).

6-Phosphogluconate levels
MIN6 cells were starved in 0 mM glucose for 1 h and then in 0 mM glucose for an additional 1 h.Then they were stimulated with either 0 mM or 20 mM glucose for 30 min.AZD7762 or control was included starting from the second starvation and throughout the rest of the experiments.Cells were lysed with Dounce homogenizer, and 6-phosphogluconate levels were assessed by 6-phosphogluconic acid Assay Kit (BioVision).

H&E staining protocol
Slides were processed as follows for H&E staining: first, they were washed in PBS for 1 min and dipped in water once, then immersed in hematoxylin for 1 min and rinsed twice in water.After that, the slides were dipped in lithium carbonate once, washed again in water and then dipped in eosin.Next, the slides were dipped in 95% EtOH twice, followed by 100% EtOH twice, and then dipped two times in histoclear.Finally, the tissue sections on the slides were mounted using Cytoseal (Thermo Fisher Scientific).

Focused chemical screen
A total of 2 × 10 5 of MIN6 cells were cultured for 4 d and were starved in 0 mM glucose Krebs-Ringer Bicarbonate (KRBH) buffer for 1 h and then followed by an additional hour of starvation including the compounds.The source and purity of chemicals used in the screen are included in Supplementary Table 2.After the initial starvation, cells were treated with 20 mM glucose KRBH buffer for 30 min before 10 µl of supernatant was collected for assessing the luminescence levels using the Promega Nano-Glo Luciferase Assay System (N1120).The 96-well assay plate was then read by the Biotek Synergy H1 microplate reader.

Pancreatic islets isolation
The islets were isolated in the Human Islet Core at the University of Pennsylvania following the guidelines of the Clinical Islet Transplantation Consortium protocol.Gift of Life as well as any other organ procurement organization who recovers the organs obtain consent from the deceased donor's family.The collected organs could be used for research.There is no compensation for participants.All procedures are in compliance with the University of Pennsylvania IRB, Gift of Life leadership team and UNOS.Our research complies with all regulations and standards by the University of Pennsylvania Institutional Review Board, which is responsible for approval of the protocol.Cynomolgus macaque islet isolation was performed based on a modified protocol using Liberase enzyme (Roche).The islets were purified from the digested pancreas using a three-layer, discontinuous Euro-Ficoll gradient (densities 1.108, 1.096 and 1.037) and a COBE blood cell processor (COBE Laboratories).Samples were collected from different layers after islet purification to assess the purity of cell isolation.Final samples were stained with dithizone, counted manually and sized using a formula to calculate islet number and islet equivalents based on a 150-mm diameter.Islet preparations with purity >85% were used for this study.The isolated islets were cultured overnight in CRML 1066 (Mediatech) containing 10% heat-inactivated FBS at 25-28 °C in 95% O 2 and 5% CO 2 .Mouse islets were isolated following a previously published protocol 60 .
For in vivo studies with AZD7762, AZD7762 is dissolved in 11.3% (2-hydroxypropyl)-β-cyclodextrin.All animals were fasted overnight and treated with AZD7762 IP 1 h before the experiments.For mouse GTT and GSIS, 12 or 25 mg kg −1 AZD7762 was given by IP injection, and then 0.5-2 g kg −1 glucose was given either by IP injection or orally.Glucose levels were measured at −60, 0, 15, 30, 60, 90 and 120 min postglucose.For GSIS experiments, blood was drawn from the tail vein at −60, 0, 15 and 30 min postglucose.For HOMA-IR calculation for vehicle-and AZD7762-treated CD-1/ICR, HFD-fed C57BL/6J mice and ob/ob mice, we used the method as described in ref. 61.

Cynomolgus macaque models
Cynomolgus macaque experiments are approved under the animal Protocol title-Cellular approaches for the modulation of alloresponses in nonhuman primates (protocol 806688).Adult Mauritius-origin male cynomolgus macaques (Macaca fascicularis) were provided by Alpha Genesis.After overnight fasting, the animal was sedated with ketamine (3-4 mg kg −1 ) mixed with dexmedetomidine (0.15 mg kg −1 intramuscular).Baseline blood samples were collected before the IV infusion of vehicle or 1.55 mg kg −1 AZD.Blood glucose was monitored at −20 and −40 min after infusion.At time 0 min, glucose (0.5 g kg −1 body weight) was infused IV via the IV catheter.Blood glucose was analyzed using a bedside glucometer (whole blood), and serum was tested for insulin/C-peptide levels.A small blood drop (~0.3 µl) was produced by a pinprick (using either a lancet or a needle) at 1, 3, 5, 7, 10, 15, 20, 30 and 60 min after administration of glucose.Additional blood samples (0.5 ml) were collected at 0, 1, 3, 5, 10, 15 and 30 min to measure insulin/C-peptide levels.

Insulin secretion assays
For human, mouse and cynomolgus macaque islets experiment, islets were starved in 2 mM glucose KRBH buffer for 2 h at 37 °C, and then stimulated in 2 mM glucose KRBH buffer for 1 h and subsequently with 20 mM glucose KRBH buffer for 1 h.To measure the total level of insulin in samples, cells were lysed in 0.1% Triton X-100.MIN6 cells were starved in 0 mM glucose for 1 h and then in 0 mM glucose for an additional 1 h.Then the cells were stimulated with either 0 mM or 20 mM glucose for 30 min.EndoC-βH1 cells were starved in 0.5 mM glucose for 1 h and then stimulated with either 0.5 mM or 20 mM glucose for 1 h.AZD7762 was included during the starvation and glucose stimulation steps.For MIN6 experiments, AZD7762 was included starting from the second starvation and throughout the rest of the experiments.For pseudoislet experiments, 4,000 EndoC-βH1 cells or 2,000 dissociated human islet cells were aggregated in v-bottom plate in culture media for 4 d before GSIS.A total of 50 mM KCl was used for KCl-stimulated insulin secretion experiments.At the end of each stimulation, 100 µl buffer was collected to assess insulin or C-peptide levels with ELISA kits (Alpco and Novus Biologicals).The islet perifusion experiment was carried out in a BioRep Perifusion System.During perifusion dynamic GSIS experiments, islets or pseudoislets were perfused at 100 µl min −1 of KRBH buffer containing 2 mM glucose for 30 min, then 45 min with 20 mM glucose and 15 min with 2 mM glucose again, and finally with 50 mM KCl KRBH buffer for 15 min.Samples were collected for assessment of insulin and C-peptide levels every 90 s.Data were normalized to baseline insulin secretion at 2 mM glucose for human islets and 0.5 mM for EndoC-βH1 cells.

Fig. 1 |
Fig. 1 | A focused chemical screen identified AZD7762 that increases glucosestimulated insulin secretion of mouse and human islets.a, Schematic diagram of the chemical screen.b, Chemical structure of AZD7762.c, Static GSIS of intact human islets in the presence of control or 1 µM AZD7762.Low glucose (LG), 2 mM glucose (P = 0.0001); High glucose (HG), 20 mM glucose (P = 0.013).n = 11 (control) and n = 22 (AZD7762) biological replicates.d,e, Dynamic GSIS (d)and AUC (e) of human pseudoislets in the presence of control or 1 µM AZD776 (P = 0.0008).n = 3 biological replicates for each group.The data were normalized to baseline.f,g, Dynamic GSCS (f) and AUC (g, P = 0.0007) of human pseudoislets in the presence of control or 1 µM AZD7762.n = 3 biological replicates.The data