Decreased islet amyloid polypeptide staining in the islets of insulinoma patients.

Islet amyloid polypeptide (IAPP) is a factor that regulates food intake and is secreted from both pancreatic islets and insulinoma cells. Here, we aimed to evaluate IAPP immunohistochemically in islets or insulinoma cells in association with clinical characteristics. We recruited six insulinoma patients and six body mass index-matched control patients with pancreatic diseases other than insulinoma whose glucose tolerance was confirmed to be normal preoperatively. IAPP and IAPP-insulin double staining were performed on pancreatic surgical specimens. We observed that the IAPP staining level and percentage of IAPP-positive beta cells tended to be lower (p = 0.1699) in the islets of insulinoma patients than in those of control patients, which might represent a novel IAPP expression pattern under persistent hyperinsulinemia and hypoglycemia.

Serotonin Influences Insulin Secretion in Rat Insulinoma INS-1E Cells.

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine that plays a critical role in insulin secretion, energy metabolism, and mitochondrial biogenesis. However, the action of serotonin in insulin production and secretion by pancreatic ß cells has not yet been elucidated. Here, we investigated how exogenous nanomolar serotonin concentrations regulate insulin synthesis and secretion in rat insulinoma INS-1E cells. Nanomolar serotonin concentrations (10 and 50 nM) significantly increased insulin protein expression above the constant levels in untreated control cells and decreased insulin protein levels in the media. The reductions in insulin protein levels in the media may be associated with ubiquitin-mediated protein degradation. The levels of membrane vesicle trafficking-related proteins including Rab5, Rab3A, syntaxin6, clathrin, and EEA1 proteins were significantly decreased by serotonin treatment compared to the untreated control cells, whereas the expressions of Rab27A, GOPC, and p-caveolin-1 proteins were significantly reduced by serotonin treatment. In this condition, serotonin receptors, Gαq-coupled 5-HT2b receptor (Htr2b), and ligand-gated ion channel receptor Htr3a were significantly decreased by serotonin treatment. To confirm the serotonylation of Rab3A and Rab27A during insulin secretion, we investigated the protein levels of Rab3A and Rab27A, in which transglutaminase 2 (TGase2) serotonylated Rab3A but not Rab27A. The increases in ERK phosphorylation levels were consistent with increases in the expression of p-Akt. Also, the expression level of the Bcl-2 protein was significantly increased by 50 and 100 nM serotonin treatment compared to the untreated control cells, whereas the levels of Cu/Zn-SOD and Mn-SOD proteins decreased. These results indicate that nanomolar serotonin treatment regulates the insulin protein level but decreases this level in media through membrane vesicle trafficking-related proteins (Rab5, Rab3A, syntaxin6, clathrin, and EEA1), the Akt/ERK pathway, and Htr2b/Htr3a in INS-1E cells.

Implications of noncoding regulatory functions in the development of insulinomas.

Insulinomas are rare neuroendocrine tumors arising from pancreatic ß cells, characterized by aberrant proliferation and altered insulin secretion, leading to glucose homeostasis failure. With the aim of uncovering the role of noncoding regulatory regions and their aberrations in the development of these tumors, we coupled epigenetic and transcriptome profiling with whole-genome sequencing. As a result, we unraveled somatic mutations associated with changes in regulatory functions. Critically, these regions impact insulin secretion, tumor development, and epigenetic modifying genes, including polycomb complex components. Chromatin remodeling is apparent in insulinoma-selective domains shared across patients, containing a specific set of regulatory sequences dominated by the SOX17 binding motif. Moreover, many of these regions are H3K27me3 repressed in ß cells, suggesting that tumoral transition involves derepression of polycomb-targeted domains. Our work provides a compendium of aberrant cis-regulatory elements affecting the function and fate of ß cells in their progression to insulinomas and a framework to identify coding and noncoding driver mutations.

Bisphenol a accelerates the glucolipotoxicity-induced dysfunction of rat insulinoma cell lines: An implication for a potential risk of environmental bisphenol a exposure for individuals susceptible to type 2 diabetes.

Epidemiological studies have suggested a correlation between bisphenol A (BPA) and type 2 diabetes (T2DM). The effects of BPA on ß-cell dysfunction may reveal the risks from an in vitro perspective. We used the rat insulinoma (INS-1) cell lines (a type of ß-cells) to set up normal or damaged models (DM), which were exposed to various concentrations of BPA (0.001, 0.01, 0.1, 1, 10 and 100 µM). An increase in reactive oxygen species (ROS) and apoptosis, and a decrease in cell viability were observed in INS-1 cells exposed to high doses of BPA for 48 h. Interestingly, exposure to lower doses of BPA for 24 h resulted in increased ROS levels and apoptosis rates in INS-1 in the DM group, along with decreased cell viability, suggesting that BPA exerts toxicity to INS-1 cells, particularly to the DM group. Insulin levels and Glut2 expression, glucose consumption, intracellular Ca2+ and insulin secretion were increased in INS-1 cells after 48 h exposure to high dose of BPA. Stronger effects were observed in the DM group, even those exposed to low doses of BPA for 24 h. Moreover, BPA inhibited high glucose-stimulated insulin secretion in these cells. Our research suggests that low doses of BPA exacerbate the dysfunction caused by glucolipotoxicity, implying environmental BPA exposure poses a risk for individuals with prediabetes or T2DM.

A rare case report of reversible glucose counterregulation in an insulinoma patient with type 2 diabetes.

CONTEXT: Insulinoma is a neuroendocrine tumor derived from pancreatic ß -cells whose clinical manifestation is recurrent hypoglycemia. Insulinoma in a patient with preexisting diabetes is extraordinarily rare, and the unmasking of type 2 diabetes (T2DM) after insulinoma surgery is even rarer. CASE REPORT: This article reports a 49-year-old male patient with insulinoma that masked the diagnosis of T2DM. The patient was admitted to the hospital with symptoms of hypoglycemia, such as repeated sweating, palpitations, and asthenia for over 4 years. The patient was diagnosed with insulinoma after completing relevant examinations. The emergence of hyperglycemia after the removal of insulinoma is attributable to the coexistence of T2DM. Surprisingly, a reversible decrease in cortisol levels was observed during the diagnostic process. We searched the previously published reports of this type of case from PubMed to determine why type 2 diabetes was covered by insulinoma and why glucocorticoids decreased. CONCLUSIONS: The diagnosis of T2DM in the patient after surgery may be related to increased food intake and insulin resistance induced by hyperinsulinemia caused by long-term hypoglycemia. The reversible decrease in cortisol levels, not adrenocortical insufficiency during the diagnostic process, may be caused by a transient abnormality in glucose counterregulation.

TMEM219 regulates the transcription factor expression and proliferation of beta cells.

Pancreatic beta cells replenishment is considered the next therapeutic option for type 1 diabetes; while stimulating endogenous beta cells proliferation is the "holy grail" for those patients with exhausted beta cell mass. Here we are demonstrating that the pro-apoptotic receptor TMEM219 is expressed in fetal pancreas, in beta cell precursors and in in vitro embryonic-derived endocrine progenitors. TMEM219 signaling negatively regulates beta cells at early stages and induces Caspase 8-mediated cell death. Pharmacological blockade of TMEM219 further rescued beta cell precursor and proliferation markers, and decreased cell death, both in islets and in in vitro-derived endocrine progenitors, allowing for beta cell preservation. While addressing the upstream controlling TMEM219 expression, we determined the TMEM219 miRNet; indeed, one of those miRNAs, miR-129-2, is highly expressed in human islets, particularly in patients at risk or with established type 1 diabetes. miR-129-2 mimic downregulated TMEM219 expression in islets, in in vitro embryonic-derived endocrine progenitors and in highly proliferating insulinoma-derived cells. Moreover, miR-129-2 inhibitor induced a TMEM219 overexpression in insulinoma-derived cells, which restored cell proliferation and functional markers, thus acting as endogenous regulator of TMEM219 expression. The TMEM219 upstream regulator miR129-2 controls the fate of beta cell precursors and may unleash their regenerative potentials to replenish beta cells in type 1 diabetes.

Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging.

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.

Exploring dual effects of dinutuximab beta on cell death and proliferation of insulinoma.

Insulinoma INS-1 cells are pancreatic beta cell tumors. Dinutuximab beta (DB) is a monoclonal antibody used in the treatment of neuroblastoma. The aim of this study is to investigate the effects of DB on pancreatic beta cell tumors at the molecular level. DB (Qarziba®) was available from EUSA Pharma. Streptozotocin (STZ) was used induce to cell cytotoxicity. DB was applied to the cells before or after the STZ application. KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were analyzed by q-RT-PCR, and protein levels were analyzed by Western blotting. Analysis of glucose-stimulated insulin secretion was performed. Ca+2 and CA19-9 levels were determined by the ELISA kit. PERK, CHOP, HSP90, p-c-Jun, p-Atf2, and p-Elk1 protein levels were analyzed by simple WES. Decreased KCND3, KCNK1, and PTHrP protein levels and increased KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were observed with DB applied after STZ application. Cell dysfunction was detected with DB applied before and after STZ application. Ca19-9 and Ca+2 levels were increased with DB applied after STZ application. PERK, CHOP, and p-Elk1 levels decreased, while HSP90 levels increased with DB applied after STZ application. CHOP, p-Akt-2, and p-c-Jun levels increased in the DB group. As a result, INS-1 cells go to cell death via the ERK signaling pathway without ER stress and release insulin with the decrease of K+ channels and an increase in Ca+2 levels with DB applied after STZ application. Moreover, the cells proliferate via JNK signaling with DB application. DB holds promise for the treatment of insulinoma. The study should be supported by in vivo studies.

Immunohistochemical Glucagon-like Peptide-1 Receptor Expression in Human Insulinomas.

Insulinomas are rare functional pancreatic neuroendocrine tumours, which metastasize in 10% of cases. As predicting the prognosis can be challenging, there is a need for the determination of clinicopathological factors associated with metastatic potential. The aim of this study is to evaluate the glucagon-like peptide-1 receptor (GLP-1R) expression in insulinomas and to analyse its association with clinicopathological features and patient outcome. This retrospective study involves pancreatic tumour tissue samples from fifty-two insulinoma patients. After histological re-evaluation, formalin-fixed paraffin-embedded tissue samples were processed into tissue microarrays and stained immunohistochemically with a monoclonal GLP-1R antibody. Forty-eight of the forty-nine (98%) non-metastatic tumours expressed GLP-1R, while one non-metastatic, multiple endocrine neoplasia type 1 (MEN1)-related tumour and all three of the metastatic tumours lacked GLP-1R expression. The lack of GLP-1R expression was associated with impaired overall survival, larger tumour diameter, higher Ki-67 PI and weaker insulin staining. Somatostatin receptor 1-5 expression did not differ between GLP-1R-positive and GLP-1R-negative insulinomas. In conclusion, the lack of GLP-1R expression is associated with metastatic disease and impaired survival in insulinoma patients. Thus, GLP-1R expression could be a useful biomarker in estimating the metastatic potential of the tumour and the prognosis of surgically treated patients.

Rab26 restricts insulin secretion via sequestering Synaptotagmin-1.

Rab26 is known to regulate multiple membrane trafficking events, but its role in insulin secretion in pancreatic ß cells remains unclear despite it was first identified in the pancreas. In this study, we generated Rab26-/- mice through CRISPR/Cas9 technique. Surprisingly, insulin levels in the blood of the Rab26-/- mice do not decrease upon glucose stimulation but conversely increase. Deficiency of Rab26 promotes insulin secretion, which was independently verified by Rab26 knockdown in pancreatic insulinoma cells. Conversely, overexpression of Rab26 suppresses insulin secretion in both insulinoma cell lines and isolated mouse islets. Islets overexpressing Rab26, upon transplantation, also failed to restore glucose homeostasis in type 1 diabetic mice. Immunofluorescence microscopy revealed that overexpression of Rab26 results in clustering of insulin granules. GST-pulldown experiments reveal that Rab26 interacts with synaptotagmin-1 (Syt1) through directly binding to its C2A domain, which interfering with the interaction between Syt1 and SNAP25, and consequently inhibiting the exocytosis of newcomer insulin granules revealed by TIRF microscopy. Our results suggest that Rab26 serves as a negative regulator of insulin secretion, via suppressing insulin granule fusion with plasma membrane through sequestering Syt1.

ATP6AP2 is robustly expressed in pancreatic ß cells and neuroendocrine tumors, and plays a role in maintaining cellular viability.

ATP6AP2, also known as (pro)renin receptor, has been shown to be expressed in several tissues including pancreatic ß cells. Whereas ATP6AP2 plays an important role in regulating insulin secretion in mouse pancreatic ß cells, the expression profiles and roles of ATP6AP2 in human pancreatic endocrine cells and neuroendocrine tumor cells remain unclear. Here in this study, we investigated the expression profiles of ATP6AP2 in pancreatic endocrine cells, and found that ATP6AP2 is robustly expressed in pancreatic insulinoma cells as well as in normal ß cells. Although ATP6AP2 was also expressed in low-grade neuroendocrine tumors, it was not or faintly detected in intermediate- and high-grade neuroendocrine tumors. Knockdown experiments of the Atp6ap2 gene in rat insulinoma-derived INS-1 cells demonstrated decreased cell viability accompanied by a significant increase in apoptotic cells. Taken together, these findings suggest that ATP6AP2 plays a role in maintaining cellular homeostasis in insulinoma cells, which could lead to possible therapeutic approaches for endocrine tumors.

5-Iodotubercidin Inhibits the Growth of Insulinoma Cells by Inducing Apoptosis.

INTRODUCTION: 5-Iodotubercidin, a type of purine derivative, has attracted increasing attention in tumor chemotherapy because of its potential as an antitumor agent in recent years. In this study, we confirmed the effects on apoptosis in insulinoma cell lines induced by 5-iodotubercidin and tried to illuminate the underlying mechanisms. METHODS: We used 5-iodotubercidin in the treatment of insulinoma cells and the cell proliferation was examined using CCK-8 assay, colony-forming assays, and insulinoma animal models. Cell apoptosis was examined using TUNEL assays and Western blotting. Cellular DNA damage was shown by comet assay and immunofluorescence. The expression of apoptosis-regulating proteins and DNA damage biomarker was investigated by Western blotting. Subcutaneous inoculation of the insulinoma cells into nude mice was to measure blood glucose, insulin levels, and tumor growth. ATM siRNA and p53 siRNA were used as loss-of-function targets to evaluate 5-iodotubercidin treatment. RESULTS: 5-Iodotubercidin inhibited the proliferation of insulinoma cells and induced DNA damage and cell apoptosis. Moreover, 5-iodotubercidin induced ATM and p53 activated. In vivo, 5-iodotubercidin inhibited the growth of Ins-1 and Min-6 cells xenografts in nude mice. CONCLUSION: 5-Iodotubercidin induces DNA damage leading to insulinoma cells apoptosis by activating ATM/p53 pathway. Therefore, this is a potential strategy for treating insulinoma.

Immunohistochemical somatostatin receptor expression in insulinomas.

Insulinomas are rare pancreatic neuroendocrine tumours. Most patients can be cured with surgery, but patients with a metastatic disease show impaired survival. The aim of this study was to evaluate somatostatin receptor (SSTR) 1-5 expression in insulinomas and to correlate the expression profile with clinicopathological variables and with patient outcome. This retrospective study involved 52 insulinoma patients. After histological re-evaluation, formalin-fixed paraffin-embedded tissue samples were processed into tissue microarrays and stained immunohistochemically with monoclonal SSTR1-5 antibodies. All the 52 tumours (49 non-metastatic, 3 metastatic) expressed at least one SSTR subtype. SSTR2 was expressed most frequently (71%), followed by SSTR3 (33%), SSTR1 (27%), SSTR5 (6%) and SSTR4 (0%). SSTR3 expression was associated with a larger tumour size (median diameter 19 mm vs. 13 mm, p = 0.043), and SSTR3 and SSTR5 expression were associated with impaired overall survival [HR 3.532 (95% CI 1.106-11,277), p = 0.033, and HR 6.805 (95% CI 1.364-33.955), p = 0.019 respectively]. Most insulinomas express SSTR2, which may be utilized in diagnostic imaging, and in planning individualized treatment strategies for insulinoma patients. Further studies are needed to clarify the association between SSTR profile and overall survival.

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39).

PURPOSE: Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. METHODS: We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. RESULTS: Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). CONCLUSION: We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

In Vivo Imaging of Naked and Microencapsulated Islet Cell Transplantation.

We describe step-by-step methods to label human pancreatic islet cells and murine insulinoma cells and their subsequent transplantation into type I diabetic mouse models with a focus on in vivo imaging using clinically applicable scanners. We also cover islets that are microencapsulated within alginate hydrogels loaded with imaging agents. By following these methods, it is possible to image cell grafts using T1-weighted and T2/T2*-weighted 1H magnetic resonance imaging (MRI), 19F MRI, computed tomography, ultrasound imaging, and bioluminescence imaging in vivo. Considering a myriad of factors that may affect the outcome of proper in vivo detection, we discuss potential issues that may be encountered during and after the process of labeling. The ultimate goal is to use these in vivo imaging approaches to determine and optimize naked and encapsulated islet cell survival, therapeutic function, and engraftment procedures.

Dibutyl phthalate affects insulin synthesis and secretion by regulating the mitochondrial apoptotic pathway and oxidative stress in rat insulinoma cells.

Dibutyl phthalate (DBP) is a typical phthalate (PAEs). The environmental health risks of DBP have gradually attracted attention due to the common use in the production of plastics, cosmetics and skin care products. DBP was associated with diabetes, but its mechanism is not clear. In this study, an in vitro culture system of rat insulinoma (INS-1) cells was established to explore the effect of DBP on insulin synthesis and secretion and the potential mechanisms. INS-1 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and treated with 15, 30, 60 and 120 µmol/L of DBP and dimethyl sulfoxide (vehicle, < 0.1%) for 24 h. The contents of insulin in the intracellular fluid and the extracellular fluid of the cells were measured. The results showed that insulin synthesis and secretion in INS-1 cells were significantly decreased in 120 µmol/L DBP group. The apoptosis rate and mitochondrial membrane potential of INS-1 cells were measured by flow cytometry with annexin V-FITC conjugate and PI, and JC-1, respectively. The results showed that DBP caused an increase in the apoptosis rate and a significant decrease in the mitochondrial membrane potential in INS-1 cells in 60 µmol/L and 120 µmol/L DBP group. The results of western blot showed that the expression of Bax/Bcl-2, caspase-3, caspase-9 and Cyt-C were significantly increased. Meanwhile, the level of oxidative stress in INS-1 cells was detected by fluorescent probes DCFH-DA and western blot. With the increase of DBP exposure, the oxidative stress levels (MDA, GSH/GSSG) were increased; and the antioxidant index (SOD) levels were decreased. Our experimental results provide reliable evidence that DBP induced apoptosis and functional impairment in INS-1 cells through the mitochondrial apoptotic pathway and oxidative stress. Therefore, we hypothesized that interference with these two pathways could be considered in the development of preventive protection measures.

Identification and Validation of a New Peptide Targeting Pancreatic Beta Cells.

Noninvasive targeted visualization of pancreatic beta cells or islets is becoming the focus of molecular imaging application in diabetes and islet transplantation studies. In this study, we aimed to produce the beta-cell-targeted peptide for molecular imaging of islet. We used phage display libraries to screen a beta-cell-targeted peptide, LNTPLKS, which was tagged with fluorescein isothiocyanate (FITC). This peptide was validated for targeting beta-cell with in vitro and in vivo studies. Immunocytochemistry (ICC) and fluorescence-activated cell sorting (FACS) analysis were used to validate the target specificity of the peptide. FITC-LNTPLKS displayed much higher fluorescence in beta cells vs. control cells in ICC. This discrimination was consistently observed using primary rodent islet. FACS analysis showed right shift of peak point in beta cells compared to control cells. The specific bind to in situ islet was verified by in vitro experiments using rodent and human pancreatic slices. The peptide also showed high affinity of islet grafts under the renal capsule. In the insulinoma animal model, we could find FITC-LNTPLKS accumulated specifically to the tumor, thus indicating a potential clinical application of molecular imaging of insulinoma. In conclusion, LNTPLKS showed a specific probe for beta-cells, which might be further utilized in targeted imaging/monitoring beta cells and theragnosis for beta-cells-related disease (diabetes, insulinoma, etc.).

Characterization of the Secretome, Transcriptome, and Proteome of Human ß Cell Line EndoC-ßH1.

Early diabetes research is hampered by limited availability, variable quality, and instability of human pancreatic islets in culture. Little is known about the human ß cell secretome, and recent studies question translatability of rodent ß cell secretory profiles. Here, we verify representativeness of EndoC-ßH1, one of the most widely used human ß cell lines, as a translational human ß cell model based on omics and characterize the EndoC-ßH1 secretome. We profiled EndoC-ßH1 cells using RNA-seq, data-independent acquisition, and tandem mass tag proteomics of cell lysate. Omics profiles of EndoC-ßH1 cells were compared to human ß cells and insulinomas. Secretome composition was assessed by data-independent acquisition proteomics. Agreement between EndoC-ßH1 cells and primary adult human ß cells was ∼90% for global omics profiles as well as for ß cell markers, transcription factors, and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-ßH1 cells compared to adult ß cells. Consistently, similarity was slightly higher with benign nonmetastatic insulinomas. EndoC-ßH1 secreted 783 proteins in untreated baseline state and 3135 proteins when stressed with nontargeting control siRNA, including known ß cell hormones INS, IAPP, and IGF2. Further, EndoC-ßH1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. EndoC-ßH1 secretome contained an unexpectedly high proportion of predicted extracellular vesicle proteins. We believe that secretion of extracellular vesicles and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.

EZH2 suppresses insulinoma development by epigenetically reducing KIF4A expression via H3K27me3 modification.

Kinesin family member 4A (KIF4A), located in the human chromosome band Xq13.1, is aberrantly overexpressed in various cancers. Our study intended to assess the expression of KIF4A in insulinoma and to gain new insights into the molecular mechanisms of this rare disease. First, KIF4A was significantly recruited in pancreatic endocrine cells relative to other cell types. A significant correlation existed between the overexpression of KIF4A and the poor survival of pancreatic adenocarcinoma patients. As revealed by CCK-8, TUNEL assay, flow cytometry, wound healing, Matrigel-transwell, senescence-associated ß-galactosidase staining, ELISA, and subcutaneous tumor formation analysis in nude mice, knocking down KIF4A significantly inhibited the growth and metastasis of insulinoma cells in vivo and in vitro. Mechanistically, we observed that KIF4A promoter sequences had reduced H3K27me3 modifications, and decline in enhancer of zeste homolog-2 (EZH2) expression promoted KIF4A expression by reducing the modification, thus leading to insulinoma. Moreover, EZH2 knockdown-induced insulinoma cell proliferation was dependent on KIF4A overexpression since KIF4A knockdown eradicated shEZH2-induced proliferation of insulinoma cells. In summary, KIF4A was identified as a possible therapeutic target for insulinoma.

Divergent effects of HIV reverse transcriptase inhibitors on pancreatic beta-cell function and survival: Potential role of oxidative stress and mitochondrial dysfunction.

Antiretroviral therapy (ART), a life-saving treatment strategy in HIV/AIDS, has been implicated in increasing the risk of type 2 diabetes mellitus (T2DM). Direct damaging effects on beta-cell function and survival by either non-nucleoside reverse transcriptase inhibitors (NNRTIs) or nucleoside/tide reverse transcriptase inhibitors (NRTIs) may predispose individuals to developing T2DM or if already type 2 diabetic, to insulin dependency. The aim of this study was to investigate the effects of the NNRTIs efavirenz, rilpivirine and doravirine, and the NRTIs tenofovir disoproxil fumarate and emtricitabine, on beta-cell function and survival while suggesting potential cellular and molecular mechanism(s). Our results show contrasting effects within the NNRTI class as doravirine did not cause damaging effects in the rat insulinoma INS-1E cells while efavirenz and rilpivirine reduced insulin release and cell viability, and induced apoptosis in INS-1E cells. Additionally, efavirenz and rilpivirine increased ROS generation, disrupted Δψm and upregulated the mRNA and protein expression of CHOP and GRP78, key markers of endoplasmic reticulum stress. In silico docking studies predict a possible inhibition of the mitochondrial ATP synthase by rilpivirine. On the contrary, both the NRTIs tenofovir disoproxil fumarate and emtricitabine did not affect GSIS, cell viability and apoptosis/necrosis levels in INS-1E cells. The deleterious effects observed in beta-cells exposed to efavirenz or rilpivirine may be, at least partially, mediated by oxidative stress and mitochondrial toxicity. These findings provide potential mechanism(s) by which efavirenz and rilpivirine may contribute to the pathogenesis of T2DM and the progression of T2DM to insulin dependency in HIV-infected type 2 diabetics.