Insulinoma Cell Line Research Articles

The Influence of BMP6 on Serotonin and Glucose Metabolism.

Previous studies have suggested a potential role of bone morphogenetic protein 6 (BMP6) in glucose metabolism, which also seems to be regulated by serotonin (5-hydroxytryptamine, 5HT), a biogenic amine with multiple roles in the organism. In this study, we explored possible interactions between BMP6, serotonin, and glucose metabolism regulation. The effect of BMP6 or 5HT on pancreatic β-cells has been studied in vitro using the INS-1 832/13 rat insulinoma cell line. Studies in vivo have been performed on mice with the global deletion of the Bmp6 gene (BMP6-/-) and included glucose and insulin tolerance tests, gene expression studies using RT-PCR, immunohistochemistry, and ELISA analyses. We have shown that BMP6 and 5HT treatments have the opposite effect on insulin secretion from INS-1 cells. The effect of BMP6 on the 5HT system in vivo depends on the tissue studied, with no observable systemic effect on peripheral 5HT metabolism. BMP6 deficiency does not cause diabetic changes, although a mild difference in insulin tolerance test between BMP6-/- and WT mice was observed. In conclusion, BMP6 does not directly influence glucose metabolism, but there is a possibility that its deletion causes slowly developing changes in glucose and serotonin metabolism, which would become more expressed with ageing.

Chronic Epinephrine-Induced Endoplasmic Reticulum and Oxidative Stress Impairs Pancreatic β-Cells Function and Fate.

Epinephrine influences the function of pancreatic β-cells, primarily through the α2A-adrenergic receptor (α2A-AR) on their plasma membrane. Previous studies indicate that epinephrine transiently suppresses insulin secretion, whereas prolonged exposure induces its compensatory secretion. Nonetheless, the impact of epinephrine-induced α2A-AR signaling on the survival and function of pancreatic β-cells, particularly the impact of reprogramming after their removal from sustained epinephrine stimulation, remains elusive. In the present study, we applied MIN6, a murine insulinoma cell line, with 3 days of high concentration epinephrine incubation and 2 days of standard incubation, explored cell function and activity, and analyzed relevant regulatory pathways. The results showed that chronic epinephrine incubation led to the desensitization of α2A-AR and enhanced insulin secretion. An increased number of docked insulin granules and impaired Syntaxin-2 was found after chronic epinephrine exposure. Growth curve and cell cycle analyses showed the inhibition of cell proliferation. Transcriptome analysis showed the occurrence of endoplasmic reticulum stress (ER stress) and oxidative stress, such as the presence of BiP, CHOP, IRE1, ATF4, and XBP, affecting cellular endoplasmic reticulum function and survival, along with UCP2, OPA1, PINK, and PRKN, associated with mitochondrial dysfunction. Consequently, we conclude that chronic exposure to epinephrine induces α2A-AR desensitization and leads to ER and oxidative stress, impairing protein processing and mitochondrial function, leading to modified pancreatic β-cell secretory function and cell fate.

Muscle-derived Stem Cell Exosomes Enhance Autophagy through the Regulation of the mTOR Signaling Pathway to Attenuate Glucolipotoxicity-induced Pancreatic Β-cell Injury

Background: Diabetes mellitus (DM) refers to a series of metabolic disorders, including elevated blood glucose level diseases due to insufficient insulin secretion or insulin resistance. Objective: To investigate the effect and protective mechanism of muscle-derived stem cell exosomes (MDSC-Exo) on glucolipotoxicity-induced pancreatic β cell injury. Methods: Primary rat muscle-derived stem cells (MDSCs) were isolated and cultured. After the completion of the third-generation culture for MDSCs, MDSC-Exo was isolated. Then, the morphology and diameter of exosomes were observed by means of electron microscopy and nanoparticle tracking analysis (NTA) instrument. The expression of exosome-related proteins CD63, TSG101 and Calnexin was detected by western blot. After stimulation of rat insulinoma cell line INS- 1 with high glucose/palmitic acid (HG/PA) and/or MDSC-Exo, cell viability and apoptosis were measured through MTT and flow cytometry (FCT), respectively. Biochemical reagents were utilized for the examination of the levels of superoxide dismutase (SOD) and malondialdehyde (MDA); enzyme-linked immunosorbent assay (ELISA) for the levels of cellular insulin secretion, and the western blot for the expression level of LC3, p62, AKT, p-AKT, mTOR and p-mTOR. Results: MDSC-Exo was successfully isolated and identified, and it was found that MDSC-Exo could reduce HG/PA-induced apoptosis as well as MDA levels in INS-1 cells. Also, MDSC-Exo could significantly increase cell viability, insulin secretion ability within 24 hours and SOD level. Besides, MDSC-Exo was able to significantly increase the LC3-II/I ratio, decrease the expression level of p62, and promote autophagy in the cells. Aside from what has been mentioned, MDSC- Exo showed a significant reduction effect on p-Akt and p-mTOR level as well as p-Akt/Akt and p-mTOR/mTOR ratios. Conclusion: MDSC-Exo can alleviate oxidative stress and enhance autophagy by inhibiting Akt/ mTOR signaling pathway activation. Then, the inhibition of apoptosis and the promotion of insulin secretion can be achieved to alleviate glucolipotoxicity-induced pancreatic β cell injury.

Mitochondrial ALDH2 improves ß-cell survival and function against doxorubicin-induced apoptosis by targeting CK2 signaling.

The aim of this study was to better understand how the chemotherapy drug doxorubicin contributes to the development of β-cell dysfunction and to explore its relationship with mitochondrial aldehyde dehydrogenase-2 (ALDH2). In order to investigate this hypothesis, doxorubicin was administered to INS-1 cells, a rat insulinoma cell line, either with or without several target protein activators and inhibitors. ALDH2 activity was detected with a commercial kit and protein levels were determined with western blot. Mitochondrial ROS, membrane potential, and lipid ROS were determined by commercial fluorescent probes. The cell viability was measured by CCK-assay. Exposure of INS-1 cells to doxorubicin decreased active insulin signaling resulting in elevated ALDH2 degradation, compared with control cells by the induction of acid sphingomyelinase mediated ceramide induction. Further, ceramide induction potentiated doxorubicin induced mitochondrial dysfunction. Treatment with the ALDH2 agonist, ALDA1, blocked doxorubicin-induced acid sphingomyelinase activation which significantly blocked ceramide induction and mitochondrial dysfunction mediated cell death. Treatment with the ALDH2 agonist, ALDA1, stimulated casein kinase-2 (CK2) mediated insulin signaling activation. CK2 silencing neutralized the function of ALDH2 in the doxorubicin treated INS-1 cells. Mitochondrial ALDH2 activation could inhibit the progression of doxorubicin induced pancreatic β-cell dysfunction by inhibiting the acid sphingomyelinase induction of ceramide, by regulating the activation of CK2 signaling. Our research lays the foundation of ALDH2 activation as a therapeutic target for the precise treatment of chemotherapy drug induced β-cell dysfunction.

X Ray-Induced Insulinoma Cell Line Rin-5F Has a Novel Mutation Site, C.A1459G (P.T487A), in Death Domain Associated Protein (DAXX) Gene

A popular toxicological and pharmacological research cell line is the insulin-secreting pancreatic cell line Rin-5F. The cell line originates from insulinomas induced by X-ray exposure. The author of this report looked at the mutation status of the DAXX gene in the Rin-5F cell line clone.<em> </em>The complete DNA and RNA were extracted from the cultivated cells as well. Double-stranded cDNA was then synthesized using the RNA template. Sequencing was done using a 3730xl DNA Analyzer.<em> </em>In the present study, c.A1459G (p.T487A) in Exon 5 in the DAXX gene was detected in Rin-5F cell lines, one of the X-ray-induced insulinomas. An NCBI homology search reveals that the 487 amino acid site in rats is the 497 amino acid of humans, based on the genomic cDNA homology between rats and humans. In humans, the COSMIC database suggests that mutations involving 497 amino acids have not been detected in all human cancers. However, the mutation of 496 amino acids was detected in human stomach and colon cancers. This is the first account of the DAXX gene's state in a cell line created by exposure to X-rays. This may point to the need for additional data and research on unique gene alterations involved in the development of X-ray-induced insulinoma tumors.

Comparison of the effects of monounsaturated fatty acids and polyunsaturated fatty acids on the lipotoxicity of islets.

Monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) have been reported to combat saturated fatty acid (SFA)-induced cellular damage, however, their clinical effects on patients with metabolic diseases such as diabetes and hyperlipidemia are still controversial. Since comparative studies of the effects of these two types of unsaturated fatty acids (UFAs) are still limited. In this study, we aimed to compare the protective effects of various UFAs on pancreatic islets under the stress of SFA-induced metabolic disorder and lipotoxicity. Rat insulinoma cell line INS-1E were treated with palmitic acid (PA) with or without UFAs including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (AA), and oleic acid (OA) to determine cell viability, apoptosis, endoplasmic reticulum (ER) stress, and inflammatory. In vivo, male C57BL/6 mice were fed a 60% high-fat diet (HFD) for 12w. Then the lard in HFD was partially replaced with fish oil (FO) and olive oil (OO) at low or high proportions of energy (5% or 20%) to observe the ameliorative effects of the UFA supplement. All UFAs significantly improved PA-induced cell viability impairment in INS-1E cells, and their alleviation on PA induced apoptosis, ER stress and inflammation were confirmed. Particularly, OA had better effects than EPA, DHA, and AA on attenuating cellular ER stress. In vivo, the diets with a low proportion of UFAs (5% of energy) had limited effects on HFD induced metabolic disorder, except for a slight improved intraperitoneal glucose tolerance in obese mice. However, when fed diets containing a high proportion of UFAs (20% of energy), both the FO and OO groups exhibited substantially improved glucose and lipid metabolism, such as decrease in total cholesterol (TC), low-density lipoprotein (LDL), fasting blood glucose (FBG), and fasting blood insulin (FBI)) and improvement of insulin sensitivity evidenced by intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT). Unexpectedly, FO resulted in abnormal elevation of the liver function index aspartate aminotransferase (AST) in serum. Pathologically, OO attenuated HFD-induced compensatory hyperplasia of pancreatic islets, while this effect was not obvious in the FO group. Both MUFAs and PUFAs can effectively protect islet β cells from SFA-induced cellular lipotoxicity. In particular, both OA in vitro and OO in vivo showed superior activities on protecting islets function and enhance insulin sensitivity, suggesting that MUFAs might have greater potential for nutritional intervention on diabetes.

Evaluating the Antioxidant and Antidiabetic Properties of Medicago sativa and Solidago virgaurea Polyphenolic-Rich Extracts.

The present study evaluated the antioxidant and antidiabetic properties of Medicago sativa and Solidago virgaurea extracts enriched in polyphenolic compounds. The extracts were obtained by accelerated solvent extraction (ASE) and laser irradiation. Then, microfiltration was used for purification, followed by nanofiltration used to concentrate the two extracts. The obtained extracts were analyzed to determine their antioxidant activity using DPPH radical scavenging and reducing power methods. The antidiabetic properties have been investigated in vitro on a murine insulinoma cell line (β-TC-6) by the inhibition of α-amylase and α-glucosidase. M. sativa obtained by laser irradiation and concentrated by nanofiltration showed the highest DPPH• scavenging (EC50 = 105.2 ± 1.1 µg/mL) and reducing power activities (EC50 = 40.98 ± 0.2 µg/mL). M. sativa extracts had higher inhibition on α-amylase (IC50 = 23.9 ± 1.2 µg/mL for concentrated extract obtained after ASE, and 26.8 ± 1.1), while S. virgaurea had the highest α-glucosidase inhibition (9.3 ± 0.9 µg/mL for concentrated extract obtained after ASE, and 8.6 ± 0.7 µg/mL for concentrated extract obtained after laser extraction). The obtained results after evaluating in vitro the antidiabetic activity showed that the treatment with M. sativa and S. virgaurea polyphenolic-rich extracts stimulated the insulin secretion of β-TC-6 cells, both under normal conditions and under hyperglycemic conditions as well. This paper argues that M. sativa and S. virgaurea polyphenolic-rich extracts could be excellent natural sources with promising antidiabetic potential.

Metabolomic identification of biochemical changes induced by fluoxetine in an insulinoma cell line (MIN6).

The use of fluoxetine raises the risk of pancreatic beta-cell dysfunction. However, the specific mechanism behind its mechanism of action in beta cells is unknown. This study investigated the cellular response of MIN6 cells to fluoxetine using untargeted cell-based metabolomics. Metabolic profiling of MIN6 cells was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis on samples prepared under optimized conditions, followed by principal component analysis, partial least squares-discriminant analysis, and pair-wise orthogonal projections to latent structures discriminant analyses. Sixty-six metabolites that had been differentially expressed between the control and fluoxetine-treated groups demonstrated that the citric acid cycle is mainly perturbed by fluoxetine treatment. The current study provides insights into the molecular mechanisms of fluoxetine effects in MIN6 cells.

Imatinib prevents dexamethasone-induced pancreatic β-cell apoptosis via decreased TRAIL and DR5.

Prolonged administration of dexamethasone, a potent anti-inflammatory drug, can lead to steroid-induced diabetes. Imatinib, a medication commonly prescribed for chronic myeloid leukemia (CML), has been shown to improve diabetes in CML patients. Our recent study demonstrated that dexamethasone induces pancreatic β-cell apoptosis by upregulating the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 5 (DR5). We hypothesized that imatinib may protect against dexamethasone-induced pancreatic β-cell apoptosis by reducing the expression of TRAIL and DR5, thereby favorably modulating downstream effectors in apoptotic pathways. We test this hypothesis by assessing the effects of imatinib on dexamethasone-induced apoptosis in rat insulinoma cell line cells. As anticipated, dexamethasone treatment led to increased TRAIL and DR5 expression, as well as an elevation in superoxide production. Conversely, expression of the TRAIL decoy receptor (DcR1) was decreased. Moreover, key effectors in the extrinsic and intrinsic apoptosis pathways, such as B-cell lymphoma 2 (BCL-2) associated X (BAX), nuclear factor kappa B (NF-κb), P73, caspase 8, and caspase 9, were upregulated, while the antiapoptotic protein BCL-2 was downregulated. Interestingly and importantly, imatinib at a concentration of 10 µM reversed the effect of dexamethasone on TRAIL, DR5, DcR1, superoxide production, BAX, BCL-2, NF-κB, P73, caspase 3, caspase 8, and caspase 9. Similar effects of imatinib on dexamethasone-induced TRAIL and DR5 expression were also observed in isolated mouse islets. Taken together, our findings suggest that imatinib protects against dexamethasone-induced pancreatic β-cell apoptosis by reducing TRAIL and DR5 expression and modulating downstream effectors in the extrinsic and intrinsic apoptosis pathways.

288-LB: CHIP Haplodeficiency Exacerbates Experimental Type 1 Diabetes via the Regulation of DNA Damage Response and Apoptosis

Type 1 diabetes is a disease characterized by hyperglycemia resulting from defects in insulin secretion. Streptozotocin (STZ)-induced pancreatic β-cell damage is commonly used as causing hyperglycemia of type 1 diabetes model. It has been established that the carboxyl terminus of heat shock protein 70-interacting protein (CHIP) contributes to the pathogeneses of diabetic complications. However, the mechanisms linking type 1 diabetes and CHIP-dependent antidiabetic signaling pathway remain to be addressed. Here we utilized STZ-induced murine model of type 1 diabetes to examine DNA damage responses (DDR) during pancreatic β-cell apoptosis. This study was undertaken to explore the mechanism whereby CHIP instigates pancreatic β-cell apoptosis via DDR-dependent signaling pathway. Wild type and CHIP haplodeficient mice were i.p. injected with 50 or 100 mg/kg STZ in order to induce experimental mouse model of type I diabetes. STZ-induced hyperglycemia, glucose intolerance, and inflammatory responses were exacerbated in CHIP haplodeficient mice compared to Wild type. In addition, immunoblotting data revealed that depletion of CHIP with siRNA against rat stub1 enhanced STZ-induced apoptosis and DDR pathway in INS-1 cells, a rat insulinoma cell line. Notably, CHIP inhibition deteriorates STZ-induced cell cycle arrest and oxidative stress in INS-1 cells. In addition to in vitro study, immunohistochemical and immunoblotting study showed that CHIP haplodeficiency exacerbates DDR in pancreas from an experimental mouse model of type I diabetes. These results suggest that CHIP protects against STZ-induced pancreatic β-cell apoptosis and hyperglycemia by interrupting the DDR-mediated apoptotic pathway. Key words: diabetes, CHIP, DNA damage response, apoptosis, β-cell Disclosure A. Hwang: None.

GHRH agonist MR-409 protects β-cells from streptozotocin-induced diabetes

Patients with type 1 diabetes (T1D) suffer from insufficient functional β-cell mass, which results from infiltration of inflammatory cells and cytokine-mediated β-cell death. Previous studies demonstrated the beneficial effects of agonists of growth hormone-releasing hormone receptor (GHRH-R), such as MR-409 on preconditioning of islets in a transplantation model. However, the therapeutic potential and protective mechanisms of GHRH-R agonists on models of T1D diabetes have not been explored. Using in vitro and invivo models of T1D, we assessed the protective propertie of the GHRH agonist, MR409 on β-cells. The treatment of insulinoma cell lines and rodent and human islets with MR-409 induces Akt signaling by induction of insulin receptor substrate 2 (IRS2), a master regulator of survival and growth in β-cells, in a PKA-dependent manner. The increase in cAMP/PKA/CREB/IRS2 axis by MR409 was associated with decrease in β-cell death and improved insulin secretory function in mouse and human islets exposed to proinflammatory cytokines. The assessment of the effects of GHRH agonist MR-409 in a model of T1D induced by low-dose streptozotocin showed that mice treated with MR-409 exhibited better glucose homeostasis, higher insulin levels, and preservation of β-cell mass. Increased IRS2 expression in β-cells in the group treated with MR-409 corroborated the invitro data and provided evidence for the underlying mechanism responsible for beneficial effects of MR-409 invivo. Collectively, our data show that MR-409 is a novel therapeutic agent for the prevention and treatment of β-cells death in T1D.

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 β 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.

RyR2 regulates store-operated Ca2+ entry, phospholipase C activity, and electrical excitability in the insulinoma cell line INS-1.

The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 μM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates β-cell electrical activity by controlling Cav current density and SK channel activation.

VNN1 overexpression in pancreatic cancer cells inhibits paraneoplastic islet function by increasing oxidative stress and inducing β‑cell dedifferentiation.

Vanin‑1 (VNN1) may be a potential biomarker for the early screening of pancreatic cancer (PC)‑associated diabetes (PCAD). A previous study by the authors reported that cysteamine secreted by VNN1‑overexpressing PC cells induced the dysfunction of paraneoplastic insulinoma cell lines by increasing oxidative stress. In the present study, it was observed that both cysteamine and exosomes (Exos) secreted by VNN1‑overexpressing PC cells aggravated the dysfunction of mouse primary islets. PC‑derived VNN1 could be transported into islets through PC cell‑derived Exos (PC‑Exos). However, β‑cell dedifferentiation, and not cysteamine‑mediated oxidative stress, was responsible for the islet dysfunction induced by VNN1‑containing Exos. VNN1 inhibited the phosphorylation of AMPK and GAPDH, and prevented Sirt1 activation and FoxO1 deacetylation in islets, which may be responsible for the induction of β‑cell dedifferentiation induced by VNN1‑overexpressing PC‑Exos. Furthermore, it was demonstrated that VNN1‑overexpressing PC cells further impaired the functions of paraneoplastic islets invivo using diabetic mice with islets transplanted under the kidney capsule. On the whole, the present study demonstrates that PC cells overexpressing VNN1 exacerbate the dysfunction of paraneoplastic islets by inducing oxidative stress and β‑cell dedifferentiation.

Construction of PLGA Porous Microsphere-Based Artificial Pancreatic Islets Assisted by the Cell Centrifugation Perfusion Technique.

Pancreatic islet transplantation is a promising treatment that could potentially reverse diabetes, but its clinical applicability is severely limited by a shortage of organ donors. Various cell loading approaches using polymeric porous microspheres (PMs) have been developed for tissue regeneration; however, PM-based multicellular artificial pancreatic islets' construction has been scarcely reported. In this study, MIN6 (a mouse insulinoma cell line) and MS1 (a mouse pancreatic islet endothelial cell line) cells were seeded into poly(lactic-co-glycolic acid) (PLGA) PMs via an upgraded centrifugation-based cell perfusion seeding technique invented and patented by our group. Cell morphology, distribution, viability, migration, and proliferation were all evaluated. Results from glucose-stimulated insulin secretion (GSIS) assay and RNA-seq analysis suggested that MIN6 and MS1-loaded PLGA PMs exhibited better glucose responsiveness, which is partly attributable to vascular formation during PM-dependent islet construction. The present study suggests that the PLGA PM-based artificial pancreatic islets may provide an alternative strategy for the potential treatment of diabetes in the future.

The antidiabetic effect of methanolic extract of Holarrhena pubescens seeds is mediated through multiple mechanisms of action

Holarrhena pubescens is widely used in Indian and Chinese medicine in the treatment of diabetes. The current work determined the oral hypoglycemic and antidiabetic effects of seed extract in rats. The probable mechanism of action was evaluated in-vitro by α - glucosidase inhibition, glucose metabolism in insulinoma (INS-1) cells to reflect secretion of insulin, and protein glycation inhibition. Its potential for herb-drug interaction was evaluated in the cytochrome P450 3A4 (CYP3A4) inhibition assay. The seed extract increased serum insulin levels and reduced serum blood glucose levels in the oral glucose tolerance test. It also reduced the serum glucose levels in streptozocin-induced diabetes. The extract also inhibited α –glucosidase enzyme activity and demonstrated that it can increase the secretion of insulin from INS to 1-rat insulinoma cell line cells in-vitro in a concentration-dependent manner. However, it had a very weak inhibitory effect on protein glycation and it did not affect the activity of CYP3A4. The results of the study showed that H. pubescens seed extract increases insulin secretion and inhibits glucose absorption both in-vivo and in-vitro with a weak protein glycation inhibitory effect. The herb is devoid of CYP3A4 inhibitory effect indicating that it may not have pharmacokinetic interaction with the drug metabolized by this enzyme.

Nuclear factor-Y mediates pancreatic β-cell compensation by repressing reactive oxygen species-induced apoptosis under metabolic stress.

Pancreatic β-cells elevate insulin production and secretion through a compensatory mechanism to override insulin resistance under metabolic stress conditions. Deficits in β-cell compensatory capacity result in hyperglycemia and type 2 diabetes (T2D). However, the mechanism in the regulation of β-cell compensative capacity remains elusive. Nuclear factor-Y (NF-Y) is critical for pancreatic islets' homeostasis under physiological conditions, but its role in β-cell compensatory response to insulin resistance in obesity is unclear. In this study, using obese ( ob/ob ) mice with an absence of NF-Y subunit A (NF-YA) in β-cells ( ob , Nf-ya βKO) as well as rat insulinoma cell line (INS1)-based models, we determined whether NF-Y-mediated apoptosis makes an essential contribution to β-cell compensation upon metabolic stress. Obese animals had markedly augmented NF-Y expression in pancreatic islets. Deletion of β-cell Nf-ya in obese mice worsened glucose intolerance and resulted in β-cell dysfunction, which was attributable to augmented β-cell apoptosis and reactive oxygen species (ROS). Furthermore, primary pancreatic islets from Nf-ya βKO mice were sensitive to palmitate-induced β-cell apoptosis due to mitochondrial impairment and the attenuated antioxidant response, which resulted in the aggravation of phosphorylated c-Jun N-terminal kinase (JNK) and cleaved caspase-3. These detrimental effects were completely relieved by ROS scavenger. Ultimately, forced overexpression of NF-Y in INS1 β-cell line could rescue palmitate-induced β-cell apoptosis, dysfunction, and mitochondrial impairment. Pancreatic NF-Y might be an essential regulator of β-cell compensation under metabolic stress.

Expression Silencing of Mitogen-Activated Protein Kinase 8 Interacting Protein-1 Conferred Its Role in Pancreatic β-Cell Physiology and Insulin Secretion.

Mitogen-activated protein kinase 8 interacting protein-1 (MAPK8IP1) gene has been recognized as a susceptibility gene for diabetes. However, its action in the physiology of pancreatic β-cells is not fully understood. Herein, bioinformatics and genetic analyses on the publicly available database were performed to map the expression of the MAPK8IP1 gene in human pancreatic islets and to explore whether this gene contains any genetic variants associated with type 2 diabetes (T2D). Moreover, a series of functional experiments were executed in a rat insulinoma cell line (INS-1 832/13) to investigate the role of the Mapk8ip1 gene in β-cell function. Metabolic engineering using RNA-sequencing (RNA-seq) data confirmed higher expression levels of MAPK8IP1 in human islets compared to other metabolic tissues. Additionally, comparable expression of MAPK8IP1 expression was detected in sorted human endocrine cells. However, β-cells exhibited higher expression of MAPK8IP1 than ductal and PSC cells. Notably, MAPK8IP1 expression was reduced in diabetic islets, and the expression was positively correlated with insulin and the β-cell transcription factor PDX1 and MAFA. Using the TIGER portal, we found that one genetic variant, "rs7115753," in the proximity of MAPK8IP1, passes the genome-wide significance for the association with T2D. Expression silencing of Mapk8ip1 by small interfering RNA (siRNA) in INS-1 cells reduced insulin secretion, glucose uptake rate, and reactive oxygen species (ROS) production. In contrast, insulin content, cell viability, and apoptosis without cytokines were unaffected. However, silencing of Mapk8ip1 reduced cytokines-induced apoptosis and downregulated the expression of several pancreatic β-cell functional markers including, Ins1, Ins2, Pdx1, MafA, Glut2, Gck, Insr, Vamp2, Syt5, and Cacna1a at mRNA and/or protein levels. Finally, we reported that siRNA silencing of Pdx1 resulted in the downregulation of MAPK8IP1 expression in INS-1 cells. In conclusion, our findings confirmed that MAPK8IP1 is an important component of pancreatic β-cell physiology and insulin secretion.

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.

OVEREXPRESSION OF PTEN GENE INCREASES INS2 GENE MRNA EXPRESSION, NOT INS1 GENE MRNA EXPRESSION, IN INSULINOMA CELL LINE RIN-5F.

One functional neuroendocrine tumor that causes hypoglycemia due to inappropriately high insulin production is an insulinoma. In rats, two genes coding for insulin, insulin 1 (Ins1) and insulin 2 (Ins2) are found on chromosome 1. Ins1 was produced from an Ins2 transcript, and it was inserted into the genome via an RNA-mediated duplication-transposition event, according to some structural feature analyses. In this study, the author has looked at how overexpression of the PTEN gene in the insulinoma cell line Rin-5F affects the expression of the insulin genes, Ins 1 and Ins 2. In the insulinoma cell line, overexpression of the PTEN gene boosts Ins2 gene mRNA expression but not Ins1 gene mRNA expression. It has been reported that PTEN upregulates insulin signaling by increasing insulin receptor substrate (IRS)-2 mRNA levels. Also, PTEN has been reported to be secreted in exosomes and thereafter, into extracellular space. The present study suggested that overexpression of PTEN might induce the increasing Ins 2 gene expression, one of the phosphorylated genes against the IRS-2 through the insulin/IGF-1 receptor. Our knowledge of the molecular pathways of PTEN relating the synthesis of insulin has been increased by the present study.