Insulin Secretion In INS-1 Cells Research Articles

Investigating the Impact of IL6 on Insulin Secretion: Evidence from INS-1 Cells, Human Pancreatic Islets, and Serum Analysis

Interleukin-6 (IL6) is a pleiotropic cytokine implicated in metabolic disorders and inflammation, yet its precise influence on insulin secretion and glucose metabolism remains uncertain. This study examined IL6 expression in pancreatic islets from individuals with/without diabetes, alongside a series of functional experiments, including siRNA silencing; IL6 treatment; and assessments of glucose uptake, cell viability, apoptosis, and expression of key β-cell genes, which were conducted in both INS-1 cells and human islets to elucidate the effect of IL6 on insulin secretion. Serum levels of IL6 from Emirati patients with type 2 diabetes (T2D) were measured, and the effect of antidiabetic drugs on IL6 levels was studied. The results revealed that IL6 mRNA expression was higher in islets from diabetic and older donors compared to healthy or young donors. IL6 expression correlated negatively with PDX1, MAFB, and NEUROD1 and positively with SOX4, HES1, and FOXA1. Silencing IL6 in INS-1 cells reduced insulin secretion and glucose uptake independently of apoptosis or oxidative stress. Reduced expression of IL6 was associated with the downregulation of Ins, Pdx1, Neurod1, and Glut2 in INS-1 cells. In contrast, IL6 treatment enhanced insulin secretion in INS-1 cells and human islets and upregulated insulin expression. Serum IL6 levels were elevated in patients with T2D and associated with higher glucose, HbA1c, and triglycerides, regardless of glucose-lowering medications. This study provides a new understanding of the role of IL6 in β-cell function and the pathophysiology of T2D. Our data highlight differences in the response to IL6 between INS-1 cells and human islets, suggesting the presence of species-specific variations across different experimental models. Further research is warranted to unravel the precise mechanisms underlying the observed effects of IL-6 on insulin secretion.

Simultaneous TIRF imaging of subplasmalemmal Ca2+ dynamics and granule fusions in insulin-secreting INS-1 cells reveals coexistent synchronized and asynchronous release

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca2+-dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3′,5′-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca2+]i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca2+]i microdomains around single or clustered Ca2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca2+ spikes or incidentally overlap with subplasmalemmal Ca2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca2+ microdomains around voltage-gated Ca2+ channels.

Endoplasmic reticulum stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.

Dense core vesicles (DCVs) and synaptic vesicles are specialised secretory vesicles in neurons and neuroendocrine cells, and abnormal release of their cargo is associated with various pathophysiologies. Endoplasmic reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using thapsigargin. DCV exocytosis was severely compromised in ER-stressed PC12 cells and was reversed to varying magnitudes by ER stress attenuators. Experiments with tunicamycin, an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in insulin-secreting INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB-binding site. The effects of loss of function of ATF4 in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the eIF2α (EIF2A)-ATF4 axis.

The role of peripheral β-amyloid in insulin resistance, insulin secretion, and prediabetes: in vitro and population-based studies.

Previous experimental studies have shown that mice overexpressing amyloid precursor protein, in which β-amyloid (Aβ) is overproduced, exhibit peripheral insulin resistance, pancreatic impairment, and hyperglycemia. We aimed to explore the effects of Aβ on insulin action and insulin secretion in vitro and the association of plasma Aβ with prediabetes in human. We examined the effects of Aβ40 and Aβ42 on insulin-inhibited glucose production in HepG2 cells, insulin-promoted glucose uptake in C2C12 myotubes, and insulin secretion in INS-1 cells. Furthermore, we conducted a case-control study (N = 1142) and a nested case-control study (N = 300) within the prospective Tongji-Ezhou cohort. Odds ratios (ORs) and 95% confidence intervals (CIs) for prediabetes were estimated by using conditional logistic regression analyses. In the in vitro studies, Aβ40 and Aβ42 dose-dependently attenuated insulin-inhibited glucose production in HepG2 cells, insulin-promoted glucose uptake in C2C12 myotubes, and basal and glucose-stimulated insulin secretion in INS-1 cells. In the case-control study, plasma Aβ40 (adjusted OR: 2.00; 95% CI: 1.34, 3.01) and Aβ42 (adjusted OR: 1.94; 95% CI: 1.33, 2.83) were positively associated with prediabetes risk when comparing the extreme quartiles. In the nested case-control study, compared to the lowest quartile, the highest quartile of plasma Aβ40 and Aβ42 were associated with 3.51-fold (95% CI: 1.61, 7.62) and 2.75-fold (95% CI: 1.21, 6.22) greater odds of prediabetes, respectively. Elevated plasma Aβ40 and Aβ42 levels were associated with increased risk of prediabetes in human subjects, which may be through impairing insulin sensitivity in hepatocytes and myotubes and insulin secretion in pancreatic β-cells.

Disrupting of family with sequence similarity 105, member A (Fam105a) deteriorates pancreatic β-cell physiology and insulin secretion in INS-1 cells

The role of “Family with sequence similarity 105, member A” (FAM105A) in pancreatic β-cell function in relation to type 2 diabetes mellitus (T2D) is not fully understood. To address this issue, various molecular and functional experiments were conducted on primary human islets and INS-1 cells. RNA-seq expression analysis showed that FAM105A is highly expressed in human islets and its expression is reduced in diabetic islets compared to healthy islets. FAM105A expression correlated negatively with HbA1c levels and body mass index (BMI). Co-expression analysis showed a significant correlation between FAM105A with PDX1, GCK, GLUT1 and INSR, but not the INS gene. Silencing of Fam105a impaired insulin release, content, glucose uptake, and mitochondria ATP content but did not affect cell viability, reactive oxygen species (ROS) or apoptosis levels. Silencing of Fam105a was associated with reduced Pdx1 and Glut2 expression at mRNA and protein levels. RNA-seq analysis of dysregulated genes in Fam105a-silenced cells showed an overall downregulation of gene expression in β-cells and insulin secretion pathway. Disrupting Pdx1 did not affect Fam105a expression in INS-1 cells. Overall, the results suggest that FAM105A plays an important role in pancreatic β-cells biology and may be involved in the development of T2D.

Vachellia farnesiana Pods or a Polyphenolic Extract Derived from Them Exert Immunomodulatory, Metabolic, Renoprotective, and Prebiotic Effects in Mice Fed a High-Fat Diet.

Obesity causes systemic inflammation, hepatic and renal damage, as well as gut microbiota dysbiosis. Alternative vegetable sources rich in polyphenols are known to prevent or delay the progression of metabolic abnormalities during obesity. Vachellia farnesiana (VF) is a potent source of polyphenols with antioxidant and anti-inflammatory activities with potential anti-obesity effects. We performed an in vivo preventive or an interventional experimental study in mice and in vitro experiments with different cell types. In the preventive study, male C57BL/6 mice were fed with a Control diet, a high-fat diet, or a high-fat diet containing either 0.1% methyl gallate, 10% powdered VFP, or 0.5%, 1%, or 2% of a polyphenolic extract (PE) derived from VFP (Vachellia farnesiana pods) for 14 weeks. In the intervention study, two groups of mice were fed for 14 weeks with a high-fat diet and then one switched to a high-fat diet with 10% powdered VFP for ten additional weeks. In the in vitro studies, we evaluated the effect of a VFPE (Vachellia farnesiana polyphenolic extract) on glucose-stimulated insulin secretion in INS-1E cells or of naringenin or methyl gallate on mitochondrial activity in primary hepatocytes and C2C12 myotubes. VFP or a VFPE increased whole-body energy expenditure and mitochondrial activity in skeletal muscle; prevented insulin resistance, hepatic steatosis, and kidney damage; exerted immunomodulatory effects; and reshaped fecal gut microbiota composition in mice fed a high-fat diet. VFPE decreased insulin secretion in INS-1E cells, and its isolated compounds naringenin and methyl gallate increased mitochondrial activity in primary hepatocytes and C2C12 myotubes. In conclusion VFP or a VFPE prevented systemic inflammation, insulin resistance, and hepatic and renal damage in mice fed a high-fat diet associated with increased energy expenditure, improved mitochondrial function, and reduction in insulin secretion.

The cardioprotective role of sirtuins is mediated in part by regulating KATP channel surface expression.

Sirtuins are NAD+-dependent deacetylases with beneficial roles in conditions relevant to human health, including metabolic disease, type II diabetes, obesity, cancer, aging, neurodegenerative diseases, and cardiac ischemia. Since ATP-sensitive K+ (KATP) channels have cardioprotective roles, we investigated whether they are regulated by sirtuins. Nicotinamide mononucleotide (NMN) was used to increase cytosolic NAD+ levels and to activate sirtuins in cell lines, isolated rat and mouse cardiomyocytes or insulin-secreting INS-1 cells. KATP channels were studied with patch clamping, biochemistry techniques, and antibody uptake experiments. NMN led to an increase in intracellular NAD+ levels and an increase in the KATP channel current, without significant changes in the unitary current amplitude or open probability. An increased surface expression was confirmed using surface biotinylation approaches. The rate of KATP channel internalization was diminished by NMN, which may be a partial explanation for the increased surface expression. We show that NMN acts via sirtuins since the increased KATP channel surface expression was prevented by blockers of SIRT1 and SIRT2 (Ex527 and AGK2) and mimicked by SIRT1 activation (SRT1720). The pathophysiological relevance of this finding was studied using a cardioprotection assay with isolated ventricular myocytes, in which NMN protected against simulated ischemia or hypoxia in a KATP channel-dependent manner. Overall, our data draw a link between intracellular NAD+, sirtuin activation, KATP channel surface expression, and cardiac protection against ischemic damage.

2,3-Dihydrosorbicillin and chrysopanol stimulate insulin secretion in INS-1 cells

Pancreatic β-cell function and insulin secretion are important in antidiabetic drug development. In an effort to discover small molecules to regulate insulin secretion, an endophytic fungus, Penicillium sp. SSP-1CLG, was selected for chemical investigation. Large scale cultures of the strain followed by extraction and chromatographic analysis led to the isolation of 10 anthraquinone and alkaloid-type compounds. The isolated compounds were identified by comprehensive analysis of NMR, MS, and ECD data. The effect of compounds 1–10 on insulin secretion in INS-1 cells was investigated. 2,3-Dihydrosorbicillin (1), chrysophanol (2), and glandicolin B (10) at non-cytotoxic concentrations resulted in an increase of glucose-stimulated insulin secretion (GSIS) in rat INS-1 pancreatic β-cells. Furthermore, we investigated the signaling pathway involved in 2,3-dihydrosorbicillin (1) and chrysophanol (2) action in the activation of peroxisome proliferator-activated receptor γ (PPARγ), pancreatic and duodenal homeobox-1 (PDX-1), insulin receptor substrate-2 (IRS-2), phosphatidylinositol 3-kinase (PI3K), and Akt. Treatment of INS-1 cells with 2,3-dihydrosorbicillin (1) and chrysophanol (2) increased the expression of these proteins. Our findings indicate that 2,3-dihydrosorbicillin and chrysophanol may play roles in the regulation of insulin secretion in pancreatic β-cells, at least in part, by targeting PPARγ and PDX-1 via the IRS-2/PI3K/Akt signaling pathway.

GDF15 plays a critical role in insulin secretion in INS-1 cells and human pancreatic islets.

Mounting evidence points to a link between growth differentiation factor-15 (GDF15) expression and the onset and progression of diabetes mellitus. However, the exact role of GDF15 in pancreatic β-cell function is unclear. To examine the role of GDF15 in β-cell function, bioinformatics analysis and functional experiments involving GDF15 silencing and overexpression were performed in INS-1 cells and human islets. Public microarray and RNA-seq expression data showed that islets obtained from diabetic donors express high levels of GDF15 compared to islets obtained from normal donors. Moreover, analysis of RNA-seq expression data revealed that GDF15 expression correlates positively with that of insulin (INS), KCNJ11, GLUT1, MAFA, INSR and negatively with that of Glucokinase (GCK) and Alpha-Ketoglutarate Dependent Dioxygenase (FTO). No T2D-associated genetic variants in the GDF15 were found to pass genome-wide significance in the TIGER portal. Expression silencing of Gdf15 in INS-1 cells reduced insulin release, glucose uptake levels, increased reactive oxygen species (ROS) production and apoptosis levels. While Gdf15-silenced cells downregulated mRNA expression of Ins, Pdx1, Mafa, and Glut2 genes, its overexpression human islets was associated with increased insulin secretion and upregulated expression of MAFA and GLUT1 but not INS or GCK. Silencing of Pdx1 or Mafa in INS-1 cells did not affect the expression of GDF15. These findings suggest that GDF15 plays a significant role in pancreatic β-cell function.

Deficiency of transcription factor Nkx6.1 does not prevent insulin secretion in INS-1E cells

Pancreatic-β-cell-specifying transcription factor Nkx6.1, indispensable for embryonic development of the pancreatic epithelium and commitment to β-cell lineage, directly controls the expression of a glucose transporter (Glut2), pyruvate carboxylase (Pcx), and genes for insulin processing (endoplasmic reticulum oxidoreductase-1β, Ero1lb; zinc transporter-8, Slc30a8). The Nkx6.1 decline in aging diabetic Goto-Kakizaki rats contributes to β-cell trans-differentiation into δ-cells. Elucidating further Nkx6.1 roles, we studied Nkx6.1 ablation in rat INS-1E cells, prepared by CRISPR/Cas9 gene editing from single colonies. INS-1ENkx6.1–/– cells exhibited unchanged glucose-stimulated insulin secretion (GSIS), moderately decreased phosphorylating/non-phosphorylating respiration ratios at high glucose; unchanged but delayed ATP-elevation responses to glucose; delayed uptake of fluorescent glucose analog, but slightly improved cytosolic Ca2+-oscillations, induced by glucose; despite approximately halved Glut2, Pcx, Ero1lb, and Slc30a8 expression, and reduced nuclear receptors Nr4a1 and Nr4a3. Thus, ATP synthesis was time-compensated, despite the delayed GLUT2-mediated glucose uptake and crippled pyruvate-malate redox shuttle (owing to the PCX-deficiency) in INS-1ENkx6.1–/– cells. Nkx6.1 thus controls the expression of genes that are not essential for acute insulin secretion, the function of which can be compensated for. Considerations that Nkx6.1 deficiency is an ultimate determinant of β-cell pathology beyond cell trans-(de-)differentiation or β-cell identity are not supported by our results.

Luminal H2 O2 promotes ER Ca2+ dysregulation and toxicity of palmitate in insulin-secreting INS-1E cells.

The endoplasmic reticulum (ER) lumen is not only the major site for the assembly and folding of newly synthesized proteins but also the main intracellular Ca2+ store. Ca2+ ions are involved in versatile biochemical processes, including posttranslational processing and folding of nascent proteins. Disruption of ER Ca2+ homeostasis is usually accompanied by an ER stress response that can ultimately lead to apoptosis if unresolved. Abnormal ER Ca2+ depletion has been linked to pancreatic β-cell dysfunction and death under lipotoxic conditions. However, the underlying mechanisms how the β-cell toxic saturated free fatty acid palmitate perturbs ER Ca2+ homeostasis and its interplay with other organelles are not fully understood. In the present study, we demonstrate that treatment of insulin-secreting INS-1E cells with palmitate diminished ER Ca2+ levels, elevated cytosolic/mitochondrial Ca2+ content, lowered the mitochondrial membrane potential, and ATP content. In addition, palmitate-pretreated β-cells contained significantly less luminal Ca2+ , revealed a severely impaired ER Ca2+ reuptake rate, and substantially lower insulin content. Importantly, detoxification of luminal H2 O2 by expression of the ER-resident glutathione peroxidase 8 (GPx8) abrogated the lipotoxic effects of palmitate. Moreover, GPx8 supported oxidative protein folding and preserved insulin content under lipotoxic conditions. A direct involvement of luminal H2 O2 in palmitate-mediated ER Ca2+ depletion could be corroborated by the ectopic expression of an ER-luminal active catalase. Our data point to the critical role of luminal H2 O2 in palmitate-mediated depletion of ER Ca2+ through redox-dependent impairment of Ca2+ ATPase pump activity upstream of mitochondrial dysfunction in insulin-secreting INS-1E cells.

Responses of INS-1 cells to glucose stimulation patterns

Diabetes has become a major public health problem in the world for many years, and it is driving us to probe into its complex mechanism of insulin secretion in pancreatic β cells. The nanoscale resolution characterization of pancreatic β cells in response to glucose led to insights into diverse mechanical and functional processes at the single cell level. Recent advances allowed the direct observations of cytoskeleton dynamics which were quantitatively determined. Here, we firstly performed the glucose stimulation with multiple physiologically relevant glucose patterns. Atomic force microscopy (AFM) produced high spatial resolution mechanical images together with the insulin secretions linking the physical interactions to the biochemical process of INS-1 cells. Altered material properties of the INS-1 cells revealed the regulation of multiple glucose stimulation patterns. Rapidly responded to high glucose (HG), INS-1 cells presented the unique meshing networks of elasticities. The decreases of Young's modulus (YM) and insulin secretion suggested that mechanical changes affected the insulin release. Furthermore, the frequency and gradient of glucose patterns induced nanomechanical and secreting changes of the INS-1 cells and gained the knowledge on the potential controllability of glucose. The relationships between the cellular mechanics and insulin secretion of INS-1 cells could contribute to establish a mechanical cell model for the study of β cells in diabetes. The results also indicated the cell mechanics as promising mechanical biomarkers for β cells, and promoted the understanding of specific mechanical mechanism of glucose regulation, which lighted on the further application of functional glucose regulation in therapy.

Beneficial role of Boehmeria nivea in health and phytochemical constituents.

The leaf and stem extracts of Boehmeria nivea (BN) collected from three different regions in Korea were screened for their antioxidant, neuroprotective, estrogenic, insulin secretion, and α-glucosidase inhibitory activity. We also examined whether BN extracts regulate cancer cell growth, inflammatory-related gene expression, and lipid accumulation in cellular system. Leaf extracts possessed greater antioxidant, anti-proliferative in cancer cells, neuroprotective, estrogenic activity, and inhibitory effect on pro-inflammatory gene expression than stem extracts. Leaf and stem extracts inhibited lipid accumulation in three T3-L1 adipocytes but did not affect glucose-stimulated insulin secretion in INS-1 cells. We isolated and identified the phytochemical constituents in the n-butanol and ethyl acetate fractions of BN leaves by combining silica gel column chromatography with mass spectrometry and 1 H- and 13 C-NMR analysis. The active compounds (caffeic acid, isoquercitrin, p-coumaric acid, and rutin) exhibited ABTS and DPPH radical scavenging activity, which may contribute to the biological activities of BN leaf extract. An analytical method was developed to quantify marker compounds for the discrimination of BN collected from different regions. Our results support the use of this analysis method for accurate identification and quantification of marker compounds in BN for the development of functional foods. PRACTICAL APPLICATIONS: Boehmeria nivea (BN) has been used as a raw material for the textile industry or traditional herbal medicine. The current study established the biological activities and active components of BN. Our results showed that BN leaf and stem extracts exhibit antioxidant, neuroprotective, and estrogenic activity. BN leaf extract also inhibited cancer cell growth, inflammatory mediators and cytokines production, and lipid accumulation in vitro. Moreover, the bioactive compounds, such as caffeic acid, isoquercitrin, p-coumaric acid, and rutin, exert ABTS and DPPH radical scavenging activities. Therefore, BN could potentially be a promising source of bioactive phytochemicals for the development of functional foods or drugs.

Palmitic acid impairs INS-1 cells and alters the global gene expression profile

Chronic elevated free fatty acids (FFAs) impair pancreatic β cells, but the mechanisms remain elusive. In this study, palmitic acid (PA) impaired viability and glucose-stimulated insulin secretion of INS-1 cells. Microarray analysis showed that PA markedly altered the expression of 277 probe sets of genes with 232 upregulated and 45 downregulated (fold change ≥ 2.0 or ≤ -2.0; P < 0.05). Gene Ontology analysis displayed a series of the biological process of the differentially expressed genes, such as intrinsic apoptotic signaling pathway in response to endoplasmic reticulum (ER) stress and oxidative stress, inflammatory response, positive regulation of macroautophagy, regulation of insulin secretion, cell proliferation and cycle, fatty acid metabolic process, glucose metabolic process and so on. Kyoto Encyclopedia of Genes and Genomes analysis demonstrated molecular pathways with which the differentially expressed genes associated, including NOD-like receptor, NF-κB and PI3K-Akt signaling pathways, apoptosis, adipocytokine signaling pathway, ferroptosis, protein processing in ER, fatty acid biosynthesis and cell cycle. Moreover, PA promoted protein expression of CHOP, cleaved caspase-3, microtubule-associated proteins light chain 3 (LC3)-II, NOD-like receptor pyrin domain containing 3 (NLRP3), cleaved IL-1β and Lcn2, increased reactive oxygen species, apoptosis and the ratio of LC3-II/I, and reduced p62 protein expression, intracellular glutathione peroxidase and catalase levels, suggesting activation of ER stress, oxidative stress, autophagy and NLRP3 inflammasome. The results indicate the impaired role of PA and the global gene expression profile of INS-1 cells following PA intervention, providing new insights into the mechanisms involving the damage of pancreatic β cells by FFAs.

A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples.

Targeted and nontargeted metabolomics has the potential to evaluate and detect global metabolite changes in biological systems. Direct infusion mass spectrometric analysis enables detection of all ionizable small molecules, thus simultaneously providing information on both metabolites and lipids in chemically complex samples. However, to unravel the heterogeneity of the metabolic status of cells in culture and tissue a low number of cells per sample should be analyzed with high sensitivity, which requires low sample volumes. Here, we present the design and characterization of the direct infusion probe, DIP. The DIP is simple to build and position directly in front of a mass spectrometer for rapid metabolomics of chemically complex biological samples using pneumatically assisted electrospray ionization at 1 μL/min flow rate. The resulting data is acquired in a square wave profile with minimal carryover between samples that enhances throughput and enables several minutes of uniform MS signal from 5 μL sample volumes. The DIP was applied to study the intracellular metabolism of insulin secreting INS-1 cells and the results show that exposure to 20 mM glucose for 15 min significantly alters the abundance of several small metabolites, amino acids, and lipids.

Loss of Nexmif results in the expression of phenotypic variability and loss of genomic integrity

We identified two NEXMIF variants in two unrelated individuals with non-autoimmune diabetes and autistic traits, and investigated the expression of Nexmif in mouse and human pancreas and its function in pancreatic beta cells in vitro and in vivo. In insulin-secreting INS-1E cells, Nexmif expression increased strongly in response to oxidative stress. CRISPR Cas9-generated Nexmif knockout mice exhibited a reduced number of proliferating beta cells in pancreatic islets. RNA sequencing of pancreatic islets showed that the downregulated genes in Nexmif mutant islets are involved in stress response and the deposition of epigenetic marks. They include H3f3b, encoding histone H3.3, which is associated with the regulation of beta-cell proliferation and maintains genomic integrity by silencing transposable elements, particularly LINE1 elements. LINE1 activity has been associated with autism and neurodevelopmental disorders in which patients share characteristics with NEXMIF patients, and can cause genomic instability and genetic variation through retrotransposition. Nexmif knockout mice exhibited various other phenotypes. Mortality and phenotypic abnormalities increased in each generation in both Nexmif mutant and non-mutant littermates. In Nexmif mutant mice, LINE1 element expression was upregulated in the pancreas, brain, and testis, possibly inducing genomic instability in Nexmif mutant mice and causing phenotypic variability in their progeny.

Anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol against H_2O_2-induced oxidative damage in pancreatic β cells (INS-1 cells)

The present study investigated the anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol on the H_2O_2-induced pancreatic β-cells(INS-1 cells).The oxidative damage model of INS-1 cells was induced and optimized by the stimulation of H_2O_2 of different concentrations for different time.CCK-8 assay was used to detect cell viability after catalpol intervention(1, 5, 10, 20, 40, 80, and 160 μmol·L~(-1)) for 24 h.Intracellular reactive oxygen species(ROS), superoxide dismutase(SOD), and lipid peroxide malondialdehyde(MDA) were measured by DCFH-DA fluorescent probe, WST-1, and TBA respectively.Moreover, the apo-ptotic effect was detected by AO-EB and Annexin V-FITC/PI staining.In addition, the protein expression levels were detected by Wes-tern blot, and intracellular insulin concentration was measured by ELISA.The results showed that the oxidative damage model of INS-1 cells was stably induced by 50 μmol·L~(-1) H_2O_2 treatment for 2 h, and catalpol at 1-80 μmol·L~(-1) did not affect cell viability of INS-1 cells.Compared with the conditions in the model group, 1, 5, and 10 μmol·L~(-1) catalpol intervention for 2 h could protect INS-1 cells from oxidative damage(P&lt;0.001), reduce ROS and MDA, increase SOD, and inhibit excessive cell apoptosis.Moreover, 1, 5, and 10 μmol·L~(-1) catalpol could also up-regulate the phosphorylation of nuclear transcription factor NF-E2 related factors, negatively regulate Kelch-like ECH-associated protein 1(Keap1), phosphorylation of extracellular signal-regulated kinase(ERK), and heme oxyge-nase 1(HO-1), and promote the protein expression of pancreatic-duodenal homeobox factor-1(PDX-1) and glucose transporter 2(GLUT2).In addition, 1, 5, and 10 μmol·L~(-1) catalpol increased insulin secretion of INS-1 cells under oxidative damage in the high-glucose culture medium, indicating function recovery of pancreatic β cells.PDX-1 is a key nuclear transcription factor of pancreatic β cell function that directly regulates GLUT2 and insulin synthesis, and affects glucose homeostasis.In conclusion, catalpol can reduce the oxidative damage and apoptosis of INS-1 cells, activate antioxidant pathway, protect the function of pancreatic β cells, and improve insulin synthesis and secretion.

Targeting ferroptosis with miR-144-3p to attenuate pancreatic β cells dysfunction via regulating USP22/SIRT1 in type 2 diabetes

BackgroundRecently, ferroptosis has been implicated in the pathologic process of several diseases including type 2 diabetes mellitus (T2DM). However, molecular mechanisms underlying ferroptosis in T2DM remain obscure.MethodsTwenty four mice were included in this study. T2DM model mice were established by a high-fat diet combined with streptozotocin injection. INS-1 cells were stimulated with high glucose (HG). Cell viability was detected by CCK-8 kit. The levels of GSH, MDA, iron, and lipid ROS, and SOD activity, were detected by the corresponding kits. The interaction between miR-144-3p and USP22 was validated by dual-luciferase reporter assay. The relationship between USP22 and its substrate was verified using Co-IP and ubiquitination assays. The mRNA and protein expressions were examined by RT-qPCR and western blot, respectively. The functions of β cells in vitro and in vivo were evaluated glucose-stimulated insulin secretion test and HOMA-β, respectively.ResultsFerroptosis occurred in the pancreas of T2DM mice and HG-induced INS-1 cells. Silencing miR-144-3p blocked the effect of HG on the cell viability and accumulation of lipid peroxides, thereby improving the insulin secretion in INS-1 cells. Mechanistically, USP22 is a direct target of miR-144-3p, which could stabilize SIRT1 expression, thereby suppressing ferroptosis. Overexpressing USP22 attenuated deleterious roles of HG in INS-1 cells; but its roles were reversed by up-regulating miR-144-3p. In vivo study demonstrated that miR-144-3p antagomir exerted an anti-hyperglycemic effect and regulated the ferroptosis-related proteins in the pancreas.ConclusionThe up-regulation of miR-144-3p suppressed USP22/SIRT1 to induce ferroptosis, which causes pancreatic β cells dysfunction, thereby promoting T2DM development.

Antioxidant Effect of Tyr-Ala Extracted from Zein on INS-1 Cells and Type 2 Diabetes High-Fat-Diet-Induced Mice.

Type 2 diabetes mellitus (T2DM) is associated with an oxidative milieu that often leads to adverse health problems. Bioactive peptides of zein possess outstanding antioxidant activity; however, their effects on hyperglycemia-related oxidative stress remain elusive. In the present study, the dipeptide Tyr-Ala (YA), a functional peptide with typical health benefits, was applied to alleviate oxidative stress in pancreatic islets under hyperglycemic conditions. By detecting viability, antioxidant ability, and insulin secretion in INS-1 cells, YA showed excellent protection of INS-1 cells from H2O2 oxidative stress, erasing reactive oxygen species (ROS) and promoting insulin secretion. Moreover, by Western blotting, we found that YA can regulate the PI3K/Akt signaling pathway associated with glycometabolism. After establishing a T2DM mice model, we treated mice with YA and measured glucose, insulin, hemoglobin A1C (HbA1c), total cholesterol (TC), triglyceride (TG), and malonaldehyde (MDA) levels and activities of superoxide dismutase (SOD) and glutathione (GSH) from blood samples. We observed that YA could reduce the production of glucose, insulin, HbA1c, TC, TG, and MDA, in addition to enhancing the activities of SOD and GSH. YA could also repair the function of the kidneys and pancreas of T2DM mice. Along with the decline in fasting blood glucose, the oxidative stress in islets was alleviated in T2DM mice after YA administration. This may improve the health situation of diabetic patients in the future.

IGF-1 ameliorates streptozotocin-induced pancreatic β cell dysfunction and apoptosis via activating IRS1/PI3K/Akt/FOXO1 pathway.

Type 2 diabetes mellitus (T2DM) is an endocrine disorder with pancreatic β cell dysfunction and/or reduced insulin sensitivity. IGF-1 is critically involved in pancreatic β cell growth, differentiation, and insulin secretion. Insulin-mediated IRS1/PI3K/Akt/FOXO1 signaling has been proved to be closely associated with pancreatic β cell function, hepatic glucose metabolism, and the development of T2DM. This present work was designed to demonstrate the protective role of IGF-1 against pancreatic β cell dysfunction and to probe into the underlying mechanisms. Herein, cell viability, cell apoptosis, insulin secretion, oxidative stress, and glycolysis in STZ-treated INS-1 cells were measured, so as to determine the biological function of IGF-1 against pancreatic β cell dysfunction in T2DM. Additionally, whether IGF-1 could activate IRS1/PI3K/Akt/FOXO1 signaling pathway to manipulate the progression of T2DM was also investigated. It was discovered that IGF-1 treatment enhanced the viability and suppressed the apoptosis of STZ-treated INS-1 cells. Besides, IGF-1 treatment augmented insulin secretion of INS-1 cells in response to STZ. Moreover, IGF-1 exerted protective role against oxidative damage and displayed inhibitory effect on glycolysis in STZ-treated INS-1 cells. Mechanistically, IGF-1 treatment markedly boosted the activation of IRS1/PI3K/Akt/FOXO1 pathway. Furthermore, treatment with AG1024 (an inhibitor of IGF-1R) partially abolished the actions of IGF-1 on cell viability, cell apoptosis, insulin secretion, oxidative stress, and glycolysis in STZ-treated INS-1 cells. To conclude, IGF-1 could improve the viability and inhibit the apoptosis of STZ-treated pancreatic β cells, induce insulin secretion, alleviate oxidative damage, as well as arrest glycolysis by activating IRS1/PI3K/Akt/FOXO1 pathway.