Insulin Secretion In MIN6 Cells Research Articles

Structural characterization of squash polysaccharide and its effect on STZ-induced diabetes mellitus model in MIN6 cells

A novel natural water-soluble acidic polysaccharide (PWESP-3) was isolated from squash with a molecular mass of 140.519 kDa, which was composed of arabinose (Ara, 35.30 mol%), galactose (Gal, 61.20 mol%), glucose (Glc, 1.80 mol%), and Mannuronic acid (ManA, 1.70 mol%) and contained Araf-(1→, →3)-Araf-(1→, →5)-Araf-(1→, Glcp-(1→, Galp-(1→, →3,5)-Araf-(1→, →2)-Glcp-(1→, →2)-Manp-(1→, →3)-Glcp-(1→, →4)-Galp-(1→, →3)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp-(1→, →4,6)-Galp-(1→ residues in the backbone. Moreover, the structure of PWESP-3 was identified by NMR spectra. The branch chain was connected to the main chain by the O-3 and O-4 atom of Gal. In addition, the effect of PWESP-3 on STZ-induced type I diabetes mellitus model in MIN6 cells was investigated. The results showed that PWESP-3 can increase the viability and insulin secretion of MIN6 cells and reduce the oxidative stress caused by ROS and NO. Meanwhile, PWESP-3 can also reduce the content of ATP, Ca2+, mitochondrial membrane potential and Caspase-3 activity in MIN6 cells. Furthermore, treatment with PWESP-3 can prevent single or double stranded DNA breaking to form DNA fragments and improve DNA damage in MIN6 cells, thereby avoiding apoptosis. Therefore, the above data highlight that PWESP-3 can improve the function of insulin secretion in STZ-induced MIN6 cells in vitro and can be used as an alternative food supplement to diabetes drugs.

From the Clinical to the Bench: Exploring the Insulin Modulation Effects of Tacrolimus and Belatacept.

Calcineurin inhibitors (CNIs) are critical in preventing rejection posttransplantation but pose an increased risk of post-transplant diabetes (PTD). Recent studies show that late conversion from CNIs to belatacept, a costimulation blocker, improves HbA1c in kidney transplant recipients with PTD or de novo diabetes. This study investigates whether the observed effects on PTD stem solely from CNI withdrawal or if belatacept influences PTD independently. The study assessed the impact of tacrolimus and belatacept on insulin secretion in MIN6 cells (a beta cell line) and rat islets. Tacrolimus and belatacept were administered to the cells and islets, followed by assessments of cell viability and insulin secretion. Tacrolimus impaired insulin secretion without affecting cell viability, while belatacept showed no detrimental effects on either parameter. These findings support clinical observations of improved HbA1c upon switching from tacrolimus to belatacept. Belatacept holds promise in islet or pancreas transplantation, particularly in patients with unstable diabetes. Successful cases of islet transplantation treated with belatacept without severe hypoglycemia highlight its potential in managing PTD. Further research is needed to fully understand the metabolic changes accompanying the transition from CNIs to belatacept. Preserving insulin secretion emerges as a promising avenue for investigation in this context.

Pharmacological and mechanistic study of PS1, a Pdia4 inhibitor, in β-cell pathogenesis and diabetes in db/db mice

Pdia4 has been characterized as a key protein that positively regulates β-cell failure and diabetes via ROS regulation. Here, we investigated the function and mechanism of PS1, a Pdia4 inhibitor, in β-cells and diabetes. We found that PS1 had an IC50 of 4 μM for Pdia4. Furthermore, PS1 alone and in combination with metformin significantly reversed diabetes in db/db mice, 6 to 7 mice per group, as evidenced by blood glucose, glycosylated hemoglobin A1c (HbA1c), glucose tolerance test, diabetic incidence, survival and longevity (P < 0.05 or less). Accordingly, PS1 reduced cell death and dysfunction in the pancreatic β-islets of db/db mice as exemplified by serum insulin, serum c-peptide, reactive oxygen species (ROS), islet atrophy, and homeostatic model assessment (HOMA) indices (P < 0.05 or less). Moreover, PS1 decreased cell death in the β-islets of db/db mice. Mechanistic studies showed that PS1 significantly increased cell survival and insulin secretion in Min6 cells in response to high glucose (P < 0.05 or less). This increase could be attributed to a reduction in ROS production and the activity of electron transport chain complex 1 (ETC C1) and Nox in Min6 cells by PS1. Further, we found that PS1 inhibited the enzymatic activity of Pdia4 and mitigated the interaction between Pdia4 and Ndufs3 or p22 in Min6 cells (P < 0.01 or less). Taken together, this work demonstrates that PS1 negatively regulated β-cell pathogenesis and diabetes via reduction of ROS production involving the Pdia4/Ndufs3 and Pdia4/p22 cascades.

Dapagliflozin improves pancreatic islet function by attenuating microvascular endothelial dysfunction in type 2 diabetes.

Sodium-glucose co-transporter 2 inhibitors, including dapagliflozin, improve ß cell function in type 2 diabetic individuals. Whether dapagliflozin can protect islet microvascular endothelial cells (IMECs) and thus contribute to the improvement of ß cell function remains unknown. The db/db mice were treated with dapagliflozin or vehicle for 6weeks. ß cell function, islet capillaries and the levels of inflammatory chemokines in IMECs were detected. The mouse IMEC cell line MS-1 cells were incubated with palmitate and/or dapagliflozin for 24h. Angiogenesis and inflammatory chemokine levels were evaluated, and the involved signalling pathways were analysed. The mouse ß cell line MIN6 cells, in the presence or absence of co-culture with MS-1 cells, were treated with palmitate and/or dapagliflozin for 24h. The expression of ß cell specific markers and insulin secretion in MIN6 cells were determined. Dapagliflozin significantly improved ß cell function, increased islet capillaries and decreased the levels of inflammatory chemokines of IMECs in db/db mice. In the palmitate-treated MS-1 cells, angiogenesis was enhanced and the levels of inflammatory chemokines were downregulated by dapagliflozin. Either a PI3K inhibitor or mTOR inhibitor eliminated the dapagliflozin-mediated effects. Importantly, dapagliflozin attenuated the palmitate-induced downregulation of ß cell function-related gene expression and insulin secretion in MIN6 cells co-cultured with MS-1 cells but not in those on mono-culture. Dapagliflozin restores islet vascularisation and attenuates the inflammation of IMECs in type 2 diabetic mice. The dapagliflozin-induced improvement of ß cell function is at least partially accounted for by its beneficial effects on IMECs in a PI3K/Akt-mTOR-dependent manner.

214-OR: Effects of Polyphenols on Cholesterol Efflux and Insulin Secretion in MIN6 Cells

Background: Diabetic dyslipidemia is characterized by elevated levels of triglyceride-rich lipoproteins and reduced levels of HDL-cholesterol (HDL-C) . Low levels of HDL-C are associated with an increased risk of atherosclerotic cardiovascular disease, because HDL protects against atherosclerosis through multiple mechanisms include removal of excess cholesterol from macrophages. HDL can also influence cholesterol homeostasis in pancreatic β-cell. It is noteworthy that absence of ATP-binding cassette transporter A1 (ABCA1) , which is a cellular cholesterol transporter and regulates islet cholesterol efflux, results in islet cholesterol overload and impaired insulin release in mice. We examined the associations of cholesterol efflux and insulin secretion in MIN6 cells with polyphenols that is reported to increase cholesterol efflux. Methods: Murine MIN6 cells were labeled with 3H-cholesterol and incubated with ApoA1 (μg/ml) or HDL (25 μg/ml) for 24 hours. The percentage cholesterol efflux was calculated by dividing the media-derived radioactivity by the sum of the radioactivity in the media and the cells. The effect of polyphenols on the cholesterol efflux was studied, adding that into the medium before the analysis of the cholesterol efflux. Insulin secretion was assessed by ELISA as the concentration of insulin in the culture medium. The mRNA expression of ABCA1 and ABCG1 were determined by real-time Quantitative RT-PCR. Results: Under the existence of polyphenols (isorhamnetin) , the ApoA1 or HDL-mediated cholesterol efflux had significantly increased by 1.4% or 1.8%, respectively (n=4, p&amp;lt;0.01) . Isorhamnetin had increased insulin secretion by 882 ng/mg protein/h (n=3, p&amp;lt;0.05) and significantly decreased ABCA1 and ABCG1 expressions. Conclusion: These data suggest that isorhamnetin increases cholesterol efflux and insulin secretion in MIN6 cells. Isorhamnetin could be the interesting candidate to investigate the new treatment for patients with diabetes mellitus. Disclosure K.Matsuki: None. Y.Kimura: None. K.Matsumura: None. J.Tanabe: None. H.Murakami: None. M.Daimon: None. Funding JSPS KAKENHI Grant Number 20K11549

1518-PUB: Role of PPARd in Regulating Autophagy Pathway in Improving Islet ß-Cell Function with GLP-1 Receptor Agonist

Aim: To investigate the effect and mechanism of glucagon-like peptide-1 receptor agonist (GLP-1 RA) on the function of islet β cells. Methods: Type 2 diabetes C57BL/6J mice were successfully induced by high fat diet (HFD) feeding for 12 weeks then were randomly divided into HFD+NaCl group (n=5) , HFD+Exenatide group (n=5, 24 nmol•kg-1­•d‑1) and NC group (n=5) . Tolerance tests and insulin measurement were performed after 8 weeks of drug therapy. Weight was measured weekly and fasting blood glucose was measured every 4 weeks. The pancreas were stained with LC3B Immunofluorescence at the end of the experiment. The PA induced primary islet β cells were treated with exendin-4 and PA induced Min6 cells were treated with exendin-4, GW501516 for 24h, which BSA as the control group, then detected the protein levels of PPARδ, LC3B-II and P62. At the same time, PA induced MIN6 cells were treated with GSIS. What's more, we silenced PPARδ transfected with siRNA to verify the role of PPARδ in GLP-1 receptor agonist activation of β-cell autophagy. Results: Compared with the normal control group, the body weight and fasting blood glucose increase and significant peripheral insulin resistance in the high fat group, while the above indexes are significantly improved in the high-fat fed mice after GLP-1 RA treatment. Immunofluorescence biopsies of pancreatic tissue show that LC3B expression decrease after high fat diet, while GLP-1 RA intervention significantly increase. In addition, GLP-1 RA not only can promote the expression of PPARδ and LC3B-II protein and inhibit the expression of P62 protein in primary islet β cells and MIN6 cells, but also significantly increase glucose stimulation insulin secretion in MIN6 cells. More importantly, GW501516 is found to be consistent with GLP-1 RA in MIN6 cells, while GLP-1 RA promote autophagy disappeare after PPARδ expression is silenced. Conclusion: GLP-1 RA can improve the function of islet β cells by activating the autophagy pathway through PPARδ. Disclosure Y.Su: None. B.Lin: None. Y.Chen: None. Z.Liu: None. L.Gan: None. W.Xu: None. Funding Science &amp; Technology Project of Guangzhou（202102010175）

1468-P: Effect of Kcnq1ot1 on Influencing the Function of Pancreatic ß-Cells Targeting miR-92a-1-5p

Objective: To study the role and mechanism of Kcnq1ot1 by targeting to regulate miR-92a-1-5p to effect insulin secretion in pancreatic β cells. Methods: The qRT-PCR was used to detect the expression of Kcnq1ot1 in different tissues of C57BL/6J mice, Min6 with PA intervention, islets of T2D model mice and the serum of T2D patients. The biological information website was used to predict that Kcnq1ot1 had complementary binding sites with miR-92a-1-5p, which was verified by the luciferase reporting gene system. The si-Kcnq1ot1, miR-92a-1-5p mimics, miR-92a-1-5p inhibitors and their respective controls were respectively transfected into Min6, and the effect of Ins1, Ins2, Insr, Nkx6.1, Irs1 and Ngn3 were detected by qRT-PCR, the Western blot method was used to detect the expression of insulin and Insr, and the GSIS experiment was used to detect its effect on insulin secretion. Results: The expression of Kcnq1ot1 in pancreas was significantly higher than that in other tissues. Compared with the control group, the expression of Kcnq1ot1 in T2D patients' serum, Min6 after PA treatment and the islets of T2D model mice were lower. And the expression of Kcnq1ot1 in serum is negatively correlated with FBG or HbA1c. Low expression of Kcnq1ot1 led to remarkable reduction of insulin secretion in Min6 cells. Down-regulation of Kcnq1ot1 or overexpression of miR-92a-1-5p reduced the mRNA expression levels of Ins1, Ins2, Insr, Nkx6.1, Irs1 and Ngn3, and the protein levels of insulin and Insr. We screened miR-92a-1-5p as targets of Kcnq1ot1, and their interaction was validated with dual luciferase reporter gene experiment. After knocking down Kcnq1ot1, the expression of miR-92a-1-5p was found to increase. Further rescue experiments showed that down-regulation of miR-92a-1-5p could reverse the effect of low Kcnq1ot1 expression on the function of pancreatic β-cells. Conclusion: Kcnq1ot1 may regulate the function of pancreatic β cells by targeting to negatively regulate miR-92a-1-5p. Disclosure Y.Chen: None. Y.Li: None. W.Xu: None. B.Lin: None. X.Mai: None. Y.Peng: None. D.Chen: None. Y.Su: None. W.Li: n/a. Funding Science &amp; Technology Project of Guangzhou（202102010175）

Growth of MIN-6 Cells on Salmon Fibrinogen Scaffold Improves Insulin Secretion

The incidence of type I diabetes has been increasing worldwide at an annual rate of approximately 3%. One of the strategies to treat type I diabetes is islet transplantation, in which damaged β-cells are replaced with new islets. To improve β-cells’ expansion and pseudoislet formation, studies are focusing on using extracellular-matrix-resembling substrates. We evaluated the potential of salmon fibrinogen and chitosan electrospun scaffold as cell substrate for cultivating MIN-6 cells. The morphology of cells, insulin secretion and gene expression was evaluated and compared with other substrates (nanofibrous scaffold, microporous scaffold and tissue culture polystyrene). We found that all tested 3D conditions favored the pseudoislet formation of MIN-6 cells. The insulin secretion of MIN-6 cells after stimulation with high-glucose media shows approximately a 9-fold increase compared to the control group when a fibrinogen/chitosan-based electrospun scaffold was used for cultivation. The differences in insulin secretion were corroborated by differences in gene expression. The differences in insulin secretion could probably be attributed to the differences in the mechanical and/or chemical nature of the tested substrates.

Cadmium perturbed metabolomic signature in pancreatic beta cells correlates with disturbed metabolite profile in human urine.

Cd exposure has been demonstrated to induce a variety of metabolic disorders accompanied with imbalance of glucose and lipid homeostasis. The metabolic toxicity of Cd exposure at metabolome-wide level remains elusive. In our study, we demonstrated that Cd exposure via drinking water increased blood glucose levels, decreased serum insulin levels, led to glucose intolerance and suppressed insulin expression in the pancreas of C57/6J mice. Cd exposure significantly inhibited cell viability and suppressed insulin secretion in MIN6 cells in vitro. Since pancreatic β-cells are the only source of insulin production in the body and play a pivotal role in modulating glucose and lipid metabolisms, we further delineated the metabolomic signatures of Cd exposure in insulin-secreting MIN6 cells by using non-target metabolomics. PCA and OPLS-DA analysis clearly suggested that Cd exposure led to a marked metabolic alteration in MIN6 cells. 76 perturbed metabolites were identified after Cd exposure. Classification of metabolites suggested that Cd perturbed metabolites belong to nucleosides, nucleotides and analogues, organic acids and derivatives, and lipids and lipid-like molecules. 28 perturbed metabolites existed in mitochondrion, suggesting mitochondrion as the major target organelle in metabolic toxicity of Cd exposure. KEGG pathway analysis revealed that 20 metabolic pathways were disturbed by Cd exposure. Mitochondrial TCA cycle and glycerophospholipid metabolism were remarkably disturbed. The mRNA expressions of genes in mitochondrial TCA cycle and fatty acid oxidation in pancreas and MIN6 cells were significantly dysregulated by Cd exposure. Disturbances in mitochondrial TCA cycle and glycerophospholipid metabolism result in producing perturbed metabolites in pancreatic β-cells. Moreover, 14 perturbed metabolites identified in MIN6 cells co-existed in the urine of Cd exposed workers. 11 biomarkers of diabetes mellitus were also found to be significantly altered in the urine of Cd exposed workers. In conclusion, findings of this study greatly extend our understanding of metabolic toxicity of Cd exposure in pancreatic β-cells at metabolome-wide level and offer some new clues for linking Cd exposure to development of diabetes mellitus. Results of this study also support the notion that Cd induced metabolic toxicity could be monitored by examining perturbed urinary metabolites in humans and highlight the significance of reducing Cd exposure via drinking water at population level.

Membrane tethering of CreER decreases uninduced cell labeling and cytotoxicity while maintaining recombination efficiency

Genetic lineage tracing is indispensable to unraveling the origin, fate, and plasticity of cells. However, the intrinsic leakiness in the CreER-loxP system raises concerns on data interpretation. Here, we reported the generation of a novel dual inducible CreER-loxP system with superior labeling characteristics. This two-component system consists of membrane localized CreER (mCreER: CD8α-FRB-CS-CreER) and TEV protease (mTEVp: CD8α-FKBP-TEVp), which are fusion proteins incorporated with the chemically induced dimerization machinery. Rapamycin and tamoxifen induce sequential dimerization of FKBP and FRB, cleavage of CreER from the membrane, and translocation into the nucleus. The labeling leakiness in Ad293 cells reduced dramatically from more than 70% to less than 5%. This tight labeling feature depends largely on the association of mCreER with HSP90, which conceals the TEV protease cutting site between FRB and CreER and thus preventing uninduced cleavage of the membrane-tethering CreER. Membrane-bound CreER also diminished significantly cytotoxicity. Our studies showed mCreER under the control of the rat insulin promoter increased labeling specificity in MIN6 islet beta-cells. Viability and insulin secretion of MIN6 cells remained intact. Our results demonstrate that this novel system can provide more stringent temporal and spatial control of gene expression and will be useful in cell fate probing.

Anti-Hyperglycemic Effects of Refined Fractions from Cyclocarya paliurus Leaves on Streptozotocin-Induced Diabetic Mice.

To identify the chemical components responsible for the anti-hyperglycemic effect of Cyclocarya paliurus (Batal.) Iljinsk (Juglandaceae) leaves, an ethanol extract (CPE) and a water extract (CPW) of C. paliurus leaves, as well as their total flavonoids (CPF), triterpenoids (CPT) and crude polysaccharides (CPP), were prepared and assessed on streptozotocin (STZ)-induced diabetic mice. After being orally administrated once a day for 24 days, CPF (300 mg/kg), CPP (180 mg/kg), or CPF+CPP (300 mg/kg CPF + 180 mg/kg CPP) treatment reversed STZ-induced body weight and muscle mass losses. The glucose tolerance tests and insulin tolerance tests suggested that CPF, CPP, and CPF+CPP showed anti-hyperglycemic effect in STZ-induced diabetic mice. Furthermore, CPF enhances glucose-stimulated insulin secretion in MIN6 cells and insulin-stimulated glucose uptake in C2C12 myotubes. CPF and CPP suppressed inflammatory cytokine levels in STZ-induced diabetic mice. Additionally, CPF and CPP improved STZ-induced diabetic nephropathy assessed by H&E staining, blood urea nitrogen content, and urine creatinine level. The molecular networking and Emperor analysis results indicated that CPF showed potential anti-hyperglycemic effects, and HPLC–MS/MS analysis indicated that CPF contains 3 phenolic acids and 9 flavonoids. In contrast, CPT (650 mg/kg) and CPC (300 mg/kg CPF + 180 mg/kg CPP + 650 mg/kg CPT) did not show anti-hyperglycemic effect. Taken together, polysaccharides and flavonoids are responsible for the anti-hyperglycemic effect of C. paliurus leaves, and the clinical application of C. paliurus need to be refined.

Berberine Potentiates Insulin Secretion and Prevents β-cell Dysfunction Through the miR-204/SIRT1 Signaling Pathway.

Pancreatic β-cell dysfunction is a key link during the progression of type 2 diabetes (T2DM), and SIRT1 participates in the regulation of various physiological activities of islet β-cells. However, as a key link in signal transduction, it is not clear how SIRT1 is regulated. By TargetScan prediction, we found that miR-204, which is enriched in islets, has highly complementary binding sites with SIRT1. Therefore, we speculate that miR-204 may be the upstream regulatory target of SIRT1 in islets and thus participate in the occurrence of β-cell dysfunction. In this study, we explored the association between miR-204 and β-cell dysfunction, the therapeutic effects of berberine (BBR) on β-cell function and the possible mechanisms. We found that miR-204 increased and SIRT1 mRNA and protein levels decreased significantly in islets both in vivo and in vitro. MIN6 cells induced by palmitic acid exhibited increased apoptosis, and the accumulation of insulin and ATP in the supernatant decreased. Importantly, palmitic acid treatment combined with miR-204 silencing showed opposite changes. MiR-204 overexpression in MIN6 cells increased apoptosis and decreased insulin and ATP production and SIRT1 expression. SIRT1 overexpression reversed the damage to β-cells caused by miR-204. The BBR treatment effectively improved insulin synthesis, reduced miR-204 levels, and increased SIRT1 expression in islet tissue in diabetic mice. Overexpression of miR-204 reversed the protective effect of BBR on apoptosis and insulin secretion in MIN6 cells. Our study identifies a novel correlation between miR-204 and β-cell dysfunction in T2DM and shows that administration of BBR leads to remission of β-cell dysfunction by regulating the miR-204/SIRT1 pathway.

Analysis of miRNA and mRNA expression in the dysregulation of insulin secretion in MIN6 cells exposed to microcystin-leucine-arginine

Microcystin -leucine-arginine (MC-LR), produced by freshwater cyanobacteria, is a potential pancreatic β-cell toxin. In this study, the function of the mouse pancreatic β-cell line, MIN6, was evaluated after MC-LR exposure, and the underlying molecular mechanisms were explored. Exposure to MC-LR for 24 h was found to inhibit cell viability and impair insulin secretion. Such findings indicate that β-cell function would be impaired following MC-LR treatment. The microarray results revealed altered miRNA and mRNA expression profiles that might be responsible for the abnormal function of MIN6 cells. Further, miRNA-gene network analysis demonstrated that miR-29b-3p, miR-6967–5p, miR-3473, miR-7061–5p, Xkr4, Tmem178b, Scp2, Ypel2, and Kcnj11 are key miRNAs and genes in the MC-LR-induced MIN6-cell toxicity. The altered expression levels of several miRNAs (e.g., miR-320–5p, miR-770–5p, miR-99a-3p, and miR-375–5p) and genes (e.g., Pklr and Gpd2) involved in insulin secretion or the onset of diabetes were also identified in MIN6 cells after treatment with MC-LR. Collectively, these findings provide evidence of the toxic effects of MC-LR on β-cells and the underlying molecular mechanisms of its glycometabolism toxicity. MCs may thus possibly play an important role in the development of diabetes mellitus in humans.

Tacrolimus inhibits insulin release and promotes apoptosis of Min6 cells through the inhibition of the PI3K/Akt/mTOR pathway.

As a calcineurin inhibitor, tacrolimus is commonly used as a first‑line immunosuppressant in organ transplant recipients. Post‑transplantation diabetes mellitus (PTDM) is a common complication following kidney transplantation and is associated with immunosuppressant drugs, such as tacrolimus. PTDM caused by tacrolimus may be related to its influence on insulin secretion and insulin resistance. However, the specific mechanism has not been fully elucidated. The aim of the present study was to investigate whether the PI3K/Akt/mTOR signaling pathway served an important role in the pathogenesis of PTDM induced by tacrolimus. In the present study, the Cell Counting Kit‑8 assay was used to measure the effect of tacrolimus on the viability of Min6 mouse insulinoma cells. The effects of tacrolimus on the insulin secretion and the activity of caspase‑3 of Min6 cells stimulated by glucose exposure were measured by ELISA. Superoxide dismutase (SOD) and malondialdehyde(MDA) levels were measured using WST‑8 and thiobarbituric acid assays, respectively. The effects of tacrolimus on the mRNA expression levels of PI3K, Akt and mTOR were detected by reverse transcription‑quantitative PCR (RT‑qPCR), whereas the protein expression levels of PI3K, Akt, mTOR, phosphorylated (p)‑AKT and p‑mTOR in Min6 cells were assessed using western blotting. The present data indicated that, compared with the control group, 5, 25and50ng/ml tacrolimus treatment could inhibit the insulin secretion of Min6 cells stimulated by glucose solution, and 50ng/ml tacrolimus could notably decrease the stimulation index (P<0.05). Moreover, 50ng/ml tacrolimus markedly increased the activity of caspase‑3 by 175.1% (P<0.05), it also decreased the SOD activity (P<0.01) and increased MDA levels (P<0.05). The RT‑qPCR results demonstrated that the mRNA expression levels of PI3K, Akt and mTOR were downregulated by 25and50ng/ml tacrolimus (P<0.01). Furthermore, the western blotting results suggested that tacrolimus had no significant effects on the expression levels of total PI3K, Akt and mTOR proteins (P>0.05), but 25and50ng/ml tacrolimus could significantly inhibit the expression levels of p‑Akt and p‑mTOR (P<0.01). In conclusion, tacrolimus decreased the activity and insulin secretion of pancreatic β cells and induced the apoptosis of islet β cells by inhibiting the mRNA expression levels of PI3K, Akt and mTOR and reducing the phosphorylation of Akt and mTOR proteins in the PI3K/Akt/mTOR signaling pathway, which may ultimately lead to the occurrence of diabetes mellitus, and may be considered as one of the specific mechanisms of PTDM caused by tacrolimus.

MiRNA-770-5p expression is upregulated in patients with type 2 diabetes and miRNA-770-5p knockdown protects pancreatic β-cell function via targeting BAG5 expression.

MicroRNA (miR)-770-5p expression is increased in patients with type 2 diabetes mellitus (T2DM) compared with healthy controls; however, the roles and molecular mechanism underlying miR-770-5p in T2DM are not completely understood. In the present study, the reverse transcription-quantitative PCR (RT-qPCR) results indicated that miR-770-5p expression was significantly increased and Bcl-2 associated athanogene 5 (BAG5) expression was significantly decreased in the serum of patients with T2DM compared with healthy volunteers. TargetScan and a dual luciferase reporter gene system were used to predict and verify BAG5 as a target gene of miR-770-5p. Additionally, the RT-qPCR results demonstrated that miR-770-5p expression was significantly increased and BAG5 expression was significantly decreased in uric acid (UA)-treated Min6 cells compared with control cells. Min6 cells were transfected with miR-770-5p inhibitor and BAG5-small interfering (si)RNA to alter expression levels. The results indicated that miR-770-5p negatively regulated BAG5. The effect of miR-770-5p knockdown on UA-induced pancreatic β-cell damage and dysfunction was subsequently assessed. Min6 cells were transfected with miR-770-5p inhibitor or miR-770-5p inhibitor + BAG5-siRNA for 48 h, followed by treatment with or without 5 mg/dl UA for 24 h. Cell viability, apoptosis, apoptosis-related factor expression levels and insulin secretion were assessed. The results demonstrated that UA treatment significantly reduced cell viability, increased cell apoptosis and reduced insulin secretion in Min6 cells compared with the control group. miR-770-5p inhibitor significantly attenuated UA-induced injury and dysfunction of Min6 cells, whereas BAG5 knockdown abolished the protective effects of miR-770-5p inhibitor on UA-damaged Min6 cells. In conclusion, miR-770-5p was highly expressed in the serum of patients with T2DM compared with healthy volunteers. In UA-treated pancreatic β-cells, compared with the inhibitor control group, miR-770-5p knockdown regulated the expression of apoptosis-related genes, increased cell viability, inhibited cell apoptosis and increased insulin secretion by targeting BAG5. Therefore, the present study suggested that miR-770-5p inhibitor may serve a protective role in T2DM.

Vascular endothelial growth factor B inhibits insulin secretion in MIN6 cells and reduces Ca2+ and cyclic adenosine monophosphate levels through PI3K/AKT pathway

BACKGROUNDType 2 diabetes (T2D) is characterized by insufficient insulin secretion caused by defective pancreatic β-cell function or insulin resistance, resulting in an increase in blood glucose. However, the mechanism involved in this lack of insulin secretion is unclear. The level of vascular endothelial growth factor B (VEGF-B) is significantly increased in T2D patients. The inactivation of VEGF-B could restore insulin sensitivity in db/db mice by reducing fatty acid accumulation. It is speculated that VEGF-B is related to pancreatic β-cell dysfunction and is an important factor affecting β-cell secretion of insulin. As an in vitro model of normal pancreatic β-cells, the MIN6 cell line can be used to analyze the mechanism of insulin secretion and related biological effects.AIMTo study the role of VEGF-B in the insulin secretion signaling pathway in MIN6 cells and explore the effect of VEGF-B on blood glucose regulation.METHODSThe MIN6 mouse pancreatic islet β-cell line was used as the model system. By administering exogenous VEGF-B protein or knocking down VEGF-B expression in MIN6 cells, we examined the effects of VEGF-B on insulin secretion, Ca2+ and cyclic adenosine monophosphate (cAMP) levels, and the insulin secretion signaling pathway.RESULTSExogenous VEGF-B inhibited the secretion of insulin and simultaneously reduced the levels of Ca2+ and cAMP in MIN6 cells. Exogenous VEGF-B also reduced the expression of phospholipase C gamma 1 (PLCγ1), phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (AKT), and other proteins in the insulin secretion pathway. Upon knockdown of VEGF-B, MIN6 cells exhibited increased insulin secretion and Ca2+ and cAMP levels and upregulated expression of PLCγ1, PI3K, AKT, and other proteins.CONCLUSIONVEGF-B can regulate insulin secretion by modulating the levels of Ca2+ and cAMP. VEGF-B involvement in insulin secretion is related to the expression of PLCγ1, PI3K, AKT, and other signaling proteins. These results provide theoretical support and an experimental basis for the study of VEGF-B in the pathogenesis of T2D.

MiR-720 Regulates Insulin Secretion by Targeting Rab35.

miRNAs pose a good prospect in the diagnosis and treatment of type 2 diabetes (T2D). This study is aimed at investigating whether miR-720 targets Rab35 to regulate insulin secretion in MIN6 cells and its molecular mechanism and the clinical value of miR-720 as a specific biomarker of T2D. Fifty-five samples of new diagnosis T2D patients and normal control were collected. Levels of miR-720, fasting blood glucose, insulin, and other indicators of glucose and lipid metabolism were determined. We increased and decreased the miR-720 expression using miR-720 mimic and inhibitor to identify the effect of miR-720 on insulin secretion in MIN6 cells, respectively. Then, we used miR-720 mimic, miR-720 inhibitor, and dual luciferase reporter gene assays to prove miR-720 which regulates insulin secretion by targeting Rab35 in MIN6 cells. In addition, we overexpressed and silenced the Rab35 gene to detect the expression of PI3K, Akt, and mTOR in MIN6 cells by RT-PCR and western blot. In this study, circulating miR-720 was significantly higher in the T2D group than the control group, and miR-270 was positive correlated with FBG, while negatively correlated with FINS. The overexpression of miR-720 inhibited insulin secretion, and miR-720 downregulation promoted insulin secretion. miR-720 regulated insulin secretion by targeting Rab35 in MIN6 cells. Compared with the control group, the expression of PI3K, Akt, and mTOR was significantly decreased by the overexpression of the Rab35 gene, while the silencing Rab35 gene could induce the expression of PI3K, Akt, and mTOR. Furthermore, miR-720 mimic could activate the PI3K pathway. We conclude that miR-720 may be a potential biomarker for the diagnosis of T2D. Increase of miR-720 reduced the Rab35 expression then activate the PI3K/Akt/mTOR signal pathway, thus inhibiting insulin secretion.

Protective effect of fermented Diospyros lotus L. extracts against the high glucose-induced apoptosis of MIN6 cells.

Date plum persimmon (Diospyros lotus L.) is a fruit crop from the Ebenaceae family. Its microorganism-fermented extract (DPEML) was shown to exhibit a hypoglycemic effect in our previous work. Here, we investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF, and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. In summary, our study suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus. PRACTICAL APPLICATIONS: We investigated the effects of DPEML fermented by Microbacterium flavum YM18-098 and Lactobacillus plantarum B7 on the high glucose-induced apoptosis of MIN6 cells and explored its potential cell protective mechanisms. DPEML ameliorated the apoptosis of MIN6 cells cultured under high glucose conditions, thereby improving cell viability. DPEML upregulated the Bcl-2/Bax mRNA ratio to obstruct an intrinsic apoptotic pathway and concomitantly downregulated the expression of the apoptosis-linked proteins, AIF and Cyt-C, in high glucose-induced MIN6 cells. Furthermore, DPEML promoted the insulin secretion of MIN6 cells grown under chronically high-glucose conditions by upregulating Ins mRNA expression. We suggested that DPEML is a promising functional food for the development of therapeutics for the treatment of Type 2 diabetes mellitus.

Glucose-induced cAMP elevation in β-cells involves amplification of constitutive and glucagon-activated GLP-1 receptor signalling.

AimcAMP typically signals downstream of Gs‐coupled receptors and regulates numerous cell functions. In β‐cells, cAMP amplifies Ca2+‐triggered exocytosis of insulin granules. Glucose‐induced insulin secretion is associated with Ca2+‐ and metabolism‐dependent increases of the sub‐plasma‐membrane cAMP concentration ([cAMP]pm) in β‐cells, but potential links to canonical receptor signalling are unclear. The aim of this study was to clarify the role of glucagon‐like peptide‐1 receptors (GLP1Rs) for glucose‐induced cAMP signalling in β‐cells.MethodsTotal internal reflection microscopy and fluorescent reporters were used to monitor changes in cAMP, Ca2+ and ATP concentrations as well as insulin secretion in MIN6 cells and mouse and human β‐cells. Insulin release from mouse and human islets was also measured with ELISA.ResultsThe GLP1R antagonist exendin‐(9‐39) (ex‐9) prevented both GLP1‐ and glucagon‐induced elevations of [cAMP]pm, consistent with GLP1Rs being involved in the action of glucagon. This conclusion was supported by lack of unspecific effects of the antagonist in a reporter cell‐line. Ex‐9 also suppressed IBMX‐ and glucose‐induced [cAMP]pm elevations. Depolarization with K+ triggered Ca2+‐dependent [cAMP]pm elevation, an effect that was amplified by high glucose. Ex‐9 inhibited both the Ca2+ and glucose‐metabolism‐dependent actions on [cAMP]pm. The drug remained effective after minimizing paracrine signalling by dispersing the islets and it reduced basal [cAMP]pm in a cell‐line heterologously expressing GLP1Rs, indicating that there is constitutive GLP1R signalling. The ex‐9‐induced reduction of [cAMP]pm in glucose‐stimulated β‐cells was paralleled by suppression of insulin secretion.ConclusionAgonist‐independent and glucagon‐stimulated GLP1R signalling in β‐cells contributes to basal and glucose‐induced cAMP production and insulin secretion.

Controlled 3D co-culture of beta cells and endothelial cells in a micropatterned collagen sheet for reproducible construction of an improved pancreatic pseudo-tissue.

The co-culture of beta cells and endothelial cells in constructing a pancreatic pseudo-tissue can provide a functional advancement for in vitro diabetic-related drug testing and biological studies or in vivo transplantation. In order to mimic the pancreatic tissue more similar to in vivo, it is necessary to control the microenvironment, including cell–cell and cell–extracellular matrix interactions. In this study, we report a geometrically controlled three-dimensional (3D) pancreatic model where MIN6 and MS1 cells are co-cultured within a micropatterned collagen sheet. In 4–10 days, depending on the cell seeding concentration, the MIN6 cells formed islet-like clusters surrounded by an endothelial MS1 cell monolayer. The MS1 cells also formed monolayers at the edge of the micropatterns connecting between the clusters, resulting in a blood vessel-like structure in which no cells were found. It was confirmed that the 3D co-culture structure was not formed in a non-patterned sheet and the structure also helped insulin secretion of MIN6 cells. By simply embedding the cell mixture and the hexagonal micropattern into the collagen sheet, we were also able to achieve the highly reproducible fabrication of a 3D pancreatic pseudo-tissue construct for in vivo and in vitro applications.