Β-cell Line MIN6 Research Articles

Artemisinin and dihydroartemisinin promote β-cell apoptosis induced by palmitate via enhancing ER stress.

Artemisinin (ART) and dihydroartemisinin (DHA) are first-line antimalarial drugs and have been reported to have anti-obesity effects. Hyperlipidemia is associated with β-cell damage in obese subjects, which could contribute to the pathogenesis of type 2 diabetes. In addition to their anti-obesity effects, ART and DHA also have protective roles in some diseases. Thus, we investigated the effects of ART and DHA in palmitate-induced β-cell apoptosis and the underlying mechanism. In this study, the rat pancreatic β-cell line INS-1 and mouse pancreatic β-cell line MIN6 were cultured with palmitate (PA) (0.1mM) to induce cell apoptosis in the presence or absence of ART or DHA. Cell apoptosis was investigated by using flow cytometry, and the expression of ER stress markers, including CHOP, GRP78 and PDI, was detected by Western blotting and/or qRT-PCR. The results showed that ART and DHA significantly increased the apoptosis of β-cells induced by PA and exacerbated the ER stress caused by PA. An inhibitor of ER stress, 4-phenylbutyric acid (4-PBA), significantly ameliorated cell apoptosis caused by ART and DHA in PA-treated β-cells, consistent with the inhibition of ER stress. Together, the findings from the current study suggested that ART and DHA may promote lipid disorder-associated β-cell injury via enhancing ER stress when they were used to treat obesity.

Internalization of AMPA-type Glutamate Receptor in the MIN6 Pancreatic β-cell Line.

The activity of AMPA-type glutamate receptor is involved in insulin release from pancreatic β-cells. However, the mechanism and dynamics that underlie AMPA receptor-mediated insulin release in β-cells is largely unknown. Here, we show that AMPA induces internalization of glutamate receptor 2/3 (GluR2/3), AMPA receptor subtype, in the mouse β-cell line MIN6. Immunofluorescence experiments showed that GluR2/3 appeared as fine dots that were distributed throughout MIN6 cells. Intracellular GluR2/3 co-localized with AP2 and clathrin, markers for clathrin-coated pits and vesicles. Immunoelectron microscopy revealed that GluR2/3 was also localized at plasma membrane. Surface biotinylation and immunofluorescence measurements showed that addition of AMPA caused an approximate 1.8-fold increase in GluR2/3 internalization under low-glucose conditions. Furthermore, internalized GluR2 largely co-localized with EEA1, an early endosome marker. In addition, GluR2/3 co-immunoprecipitated with cortactin, a F-actin binding protein. Depletion of cortactin by RNAi in MIN6 cells altered the intracellular distribution of GluR2/3, suggesting that cortactin is involved in internalization of GluR2/3 in MIN6 cells. Taken together, our results suggest that pancreatic β-cells adjust the amount of AMPA-type GluR2/3 on the cell surface to regulate the receptive capability of the cell for glutamate.Key words: endocytosis, GluR2, AMPA, cortactin, MIN6.

The beneficial effects of a muscarinic agonist on pancreatic \u03b2-cells

The brain and nervous system play an important role in pancreatic β-cell function. This study investigated the role of muscarinic agonists or acetylcholine, which is the major neurotransmitter in the vagal nerve, in regulating pancreatic β-cell mass and glucose homeostasis. Administration of the muscarinic agonist bethanechol increased insulin secretion and improved glucose tolerance in insulin-receptor substrate 2 (IRS2)-knockout (IRS-2−/−) mice and diet-induced obesity mice. Oral administration of bethanechol increased β-cell mass and proliferation in wild-type mice, but not IRS-2−/− mice. The muscarinic agonist also increased the incorporation of 5-bromo-2′-deoxyuridine (BrdU) into islets isolated from wild-type mice and pancreatic β-cell line MIN6. The phosphorylation of protein kinase B (Akt) induced by oral administration of bethanechol was observed in wild-type mice, but not IRS-2−/− mice. The secretion of glucagon-like peptide-1 (GLP-1) was also stimulated by bethanechol in wild-type mice, and a GLP-1 antagonist partially inhibited the bethanechol-induced increase in β-cell mass. These results suggest that the muscarinic agonist exerted direct and indirect effects on β-cell proliferation that were dependent on the IRS-2/Akt pathway. The bethanechol-stimulated release of GLP-1 may be indirectly associated with β-cell proliferation.

Genome-Wide Association Study on the Early-Phase Insulin Response to a Liquid Mixed Meal: Results From the NEO Study.

Early-phase insulin secretion is a determinant of postprandial glucose homeostasis. In this study, we aimed to identify novel genetic variants associated with the early-phase insulin response to a liquid mixed meal by a genome-wide association study using a discovery and replication design embedded in the Netherlands Epidemiology of Obesity (NEO) study. The early-phase insulin response was defined as the difference between the natural logarithm-transformed insulin concentrations of the postprandial state at 30 min after a meal challenge and the fasting state (Δinsulin). After Bonferroni correction, rs505922 (β: -6.5% [minor allele frequency (MAF) 0.32, P = 3.3 × 10-8]) located in the ABO gene reached genome-wide significant level (P < 5 × 10-8) and was also replicated successfully (β: -7.8% [MAF 0.32, P = 7.2 × 10-5]). The function of the ABO gene was assessed using in vitro shRNA-mediated knockdown of gene expression in the murine pancreatic β-cell line MIN6. Knocking down the ABO gene led to decreased insulin secretion in the murine pancreatic β-cell line. These data indicate that the previously identified elevated risk of type 2 diabetes for carriers of the ABO rs505922:C allele may be caused by decreased early-phase insulin secretion.

Oxygen-releasing polycaprolactone/calcium peroxide composite microspheres.

Oxygen-releasing polycaprolactone/calcium peroxide (PCL/CaO2 ) composite microspheres were fabricated via homogenization, electrospray with a single nozzle, and electrospray with a co-axial nozzle, resulting in homogenized, single-walled, and double-walled microspheres, respectively. Scanning electron microscopy revealed that homogenized microspheres had pores, while electrosprayed microspheres did not. Alizarin Red S staining showed a core-shell structure for double-walled microspheres. In a hypoxia incubator, single-walled, double-walled, and homogenized microspheres could maintain oxygen tension in PBS at or above 10% for approximately 5, 4, and 3 days, respectively. All the PCL/CaO2 microspheres could support viability of pancreatic β-cell line MIN6 cells in 2D cultures in a hypoxia incubator for 1 week, with the cells supported by double-walled and homogenized microspheres exhibiting the highest and the lowest metabolic activity, respectively. For 3D MIN6 cell cultures in a hypoxia incubator, single-walled and homogenized PCL/CaO2 microspheres led to the highest and the lowest live cell densities, respectively. Double-walled and single-walled microspheres provided the best support for 2D and 3D cultures, respectively, suggesting that they are suitable for different applications.

Mitochondrial unfolded protein response gene CLPP changes mitochondrial dynamics and affects mitochondrial function.

Mitochondrial dynamics is associated with mitochondrial function, which is associated with diabetes. Although an important indicator of the mitochondrial unfolded protein response, to the best of our knowledge, CLPP and its effects on mitochondrial dynamics in islet cells have not been studied to date. We analyzed the effects of CLPP on mitochondrial dynamics and mitochondrial function in the mice islet β-cell line Min6 under high glucose and high fat conditions. Min6 cells were assigned to: Normal, HG, HG+NC, HG+siCLPP, HF, HF+NC and HF+ siCLPP groups. High glucose and high fat can promote the mRNA and protein expression of CLPP in mitochondria. The increase of mitochondrial fission, the decrese of mitochondrial fusion, and the damage of mintocondrial ultrastructure were significant in the siCLPP cell groups as compared to no-siCLPP treated groups. Meanwhile, mitochondrial functions of MIN6 cells treated with siCLPP were impaired, such as ATP decreased, ROS increased, mitochondrial membrane potential decreased. In addition, cell insulin secretion decreased and cell apoptosis rate increased in siCLPP groups. These results revealed that mitochondrial unfolded protein response geneCLPP alleviated high glucose and high fat-induced mitochondrial dynamics imbalance and mitochondrial dysfunction.

Islet β-cell-produced NUCB2/nesfatin-1 maintains insulin secretion and glycemia along with suppressing UCP-2 in β-cells.

Nesfatin-1 is a hypothalamic anorexigenic peptide processed from nucleobindin 2 (NUCB2). Central and peripheral administration of NUCB2/nesfatin-1 enhances glucose metabolism and insulin release. NUCB2/nesfatin-1 is also localized in pancreatic islets, while its function remains unknown. To explore the role of pancreatic β-cell-produced NUCB2/nesfatin-1, we developed pancreatic β-cell-specific NUCB2 knockout (βNUCB2 KO) mice and NUCB2 gene knockdown (shNUCB2) MIN6 β-cell line. In βNUCB2 KO mice, casual blood glucose was elevated from 12weeks of age. In a glucose tolerance test at 12weeks, insulin secretion at 15min was reduced and blood glucose at 2h increased in βNUCB2 KO mice fasted 8h. In islets isolated from βNUCB2 KO mice, high glucose-stimulated insulin secretion (GSIS) was impaired. In shNUCB2 MIN6 cells, GSIS was reduced and UCP-2 mRNA expression was elevated. These results show impaired GSIS possibly associated with UCP-2 overexpression in NUCB2-silenced β-cells, suggesting that β-cell-produced NUCB2/nesfatin-1 maintains GSIS and thereby glycemia.

MicroRNA-18 promotes apoptosis of islet β-cells via targeting NAV1.

The detailed pathogenesis of diabetes mellitus (DM) remains to be fully elucidated. The purpose of the present study was to explore the role of microRNA (miR)-18 in DM and its underlying mechanisms, providing novel ideas for the treatment of the disease. After inflammatory factor-mediated induction, miR-18 expression in the islet β-cell line MIN6 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). miR-18 mimics and miR-18 inhibitor were then constructed and transfected into MIN6 cells. The mRNA levels of pro-insulin in MIN6 cells were also detected by RT-qPCR. Released insulin levels and insulin secretion function of MIN6 cells were accessed by ELISA and glucose-stimulated insulin secretion assay, respectively. Apoptosis of MIN6 cells was detected by a terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end labeling assay and western blot analysis of apoptotic proteins. The binding interaction of miR-18 and neuron navigator 1(NAV1), a constituent of the phosphoinositide 3-kinase (PI3K)/AKT pathway, was assessed using a dual-luciferase reporter gene assay. Finally, the regulatory effect of miR-18 on the PI3K/AKT pathway was determined by western blot analysis. After induction of inflammatory factors in MIN6 cells, miR-18 expression was markedly upregulated. Transfection with miR-18 mimics inhibited pro-insulin levels, as well as insulin production and secretion capacity. miR-18 knockdown partially abrogated the inhibited insulin secretion capacity induced by interleukin-1β (IL-1β) treatment. In addition, apoptosis of MIN6 cells was increased by miR-18 mimics. The dual-luciferase reporter gene assay confirmed the direct binding of miR-18 to NAV1. Western blot analysis suggested that miR-18 markedly inhibited the PI3K/AKT pathway in MIN6 cells. In conclusion, miR-18 expression is upregulated by IL-1β induction in islet β-cells. It was demonstrated that miR-18 promotes apoptosis of islet β-cells at least partially by inhibiting NAV1 expression and insulin production via suppression of the PI3K/AKT pathway.

Culture in 10% O2 enhances the production of active hormones in neuro-endocrine cells by up-regulating the expression of processing enzymes

To closely mimic physiological conditions, low oxygen cultures have been employed in stem cell and cancer research. Although in vivo oxygen concentrations in tissues are often much lower than ambient 21% O2 (ranging from 3.6 to 12.8% O2), most cell cultures are maintained at 21% O2 To clarify the effects of the O2 culture concentration on the regulated secretion of peptide hormones in neuro-endocrine cells, we examined the changes in the storage and release of peptide hormones in neuro-endocrine cell lines and endocrine tissues cultured in a relatively lower O2 concentration. In both AtT-20 cells derived from the mouse anterior pituitary and freshly prepared mouse pituitaries cultured in 10% O2 for 24 h, the storage and regulated secretion of the mature peptide hormone adrenocorticotropic hormone were significantly increased compared with those in cells and pituitaries cultured in ambient 21% O2, whereas its precursor proopiomelanocortin was not increased in the cells and tissues after being cultured in 10% O2 Simultaneously, the prohormone-processing enzymes PC1/3 and carboxypeptidase E were up-regulated in cells cultured in 10% O2, thus facilitating the conversion of prohormones to their active form. Similarly, culturing the mouse β-cell line MIN6 and islet tissue in 10% O2 also significantly increased the conversion of proinsulin into mature insulin, which was secreted in a regulated manner. These results suggest that culture under 10% O2 is more optimal for endocrine tissues/cells to efficiently generate and secrete active peptide hormones than ambient 21% O2.

TRPV2 channels mediate insulin secretion induced by cell swelling in mouse pancreatic β-cells.

β-Cell swelling induces membrane depolarization, which has been suggested to be caused at least partly by the activation of cation channels. Here, we show the identification of the cation channels. In isolated mouse pancreatic β-cells, the exposure to 30% hypotonic solution elicited an increase in cytosolic Ca2+ concentration ([Ca2+]c). The [Ca2+]c elevation was partially inhibited by ruthenium red, a blocker of several Ca2+-permeable channels including transient receptor potential vanilloid receptors [transient receptor potential cation channel subfamily V (TRPV)], and by nicardipine, but not by the depletion of intracellular Ca2+ stores with thapsigargin and caffeine. The hypotonic stimulation also increased insulin secretion from isolated mouse islets, which was significantly suppressed by ruthenium red. Expression of TRPV2 and TRPV4 was confirmed in mouse pancreatic islets and the MIN6 β-cell line by RT-PCR, Western blot, and immunohistochemical analyses. However, neither 4α-phorbol 12,13-didecanoate nor GSK1016790A, TRPV4 activators, showed any apparent effect on [Ca2+]c in isolated mouse β-cells or in MIN6 cells. In contrast, probenecid, a TRPV2 activator, induced an increase in [Ca2+]c in MIN6 cells, which was attenuated by ruthenium red. Moreover, the [Ca2+]c elevation induced by 30% hypotonic stimulation was significantly reduced by knockdown of TRPV2 with siRNA and by tranilast, a TRPV2 inhibitor. The knockdown of TRPV2 also decreased insulin secretion induced by the hypotonic stimulation. In addition, glucose-stimulated insulin secretion was also significantly reduced in the TRPV2-knockdown MIN6 cells. These results suggest that osmotic cell swelling activates TRPV2 in mouse β-cells, thereby causing membrane depolarization and subsequent activation of voltage-dependent Ca2+ channels and insulin secretion.

Design, synthesis, and biological evaluations of phenylpropiolic acid derivatives as novel GPR40 agonists

GPR40, also known as free fatty acid receptor 1 (FFAR1), is a member of G protein-coupled receptors (GPCR) family and has emerged as an attractive target for the treatment of type 2 diabetes mellitus. So far, most of the synthetic GPR40 agonists, including several drug candidates discontinued in clinical trials, were derived from the phenylpropionic acid scaffold. For discovering novel GPR40 agonists with diverse chemical structures, a series of phenylpropiolic acid derivatives were designed, synthesized, and evaluated under a battery of bioassays. Compound 9, the most potent compound in this series, exhibited submicromolar agonist activity and similar agonistic efficacy compared to that of TAK-875. In addition, compound 9 was able to dose-dependently amplify glucose-stimulated insulin secretion (GSIS) in pancreatic β-cell line MIN6, which could be reversed by a selective GPR40 antagonist GW1100. In addition, compound 9 was found to have potent glucose-lowering effects during an oral glucose tolerance test in normal C57BL/6 mice.

Baicalin prevents tumor necrosis factor-α-induced apoptosis and dysfunction of pancreatic β-cell line Min6 via upregulation of miR-205.

Baicalin (BAI), one major flavonoid from Scutellaria baicalensis, possesses anticancer and anti-inflammatory properties. However, the effect of BAI on diabetes mellitus has not been investigated. This study explored the antidiabetic effect of BAI on pancreatic β-cell line Min6. Min6 cells were treated with tumor necrosis factor-α (TNF-α) to mimic β-cell destruction in type 1 diabetes mellitus. The effects of BAI on viability and apoptosis of Min6 cells were analyzed by the cell counting kit-8 assay and Annexin V-fluoresceine isothiocyanate/propidium iodide staining method. The insulin secretion of Min6 cells was determined using radioimmunoassay. Expression of apoptosis-associated proteins and inducible nitric oxide synthase (iNOS), and activation of phosphatidylinositol 3'-kinase/protein kinase B (PI3K/AKT) and nuclear factor ΚB (NF-κB) pathways were analyzed by Western blot analysis. Relative microRNA-205 (miR-205) expression was determined by quantitative real time polymerase chain reaction. TNF-α treatment inhibited cell growth and insulin secretion, but promoted iNOS expression. All of these effects were reversed by BAI treatment. BAI promoted viability; suppressed apoptosis; regulated caspase-3, B-cell lymphoma 2 and Bcl-2-associated X protein; decreased iNOS level; and increased insulin production. BAI protected Min6 cells by upregulating miR-205. Besides, the Min6 cell-protective effect of BAI was PI3K/AKT pathway and NF-κB pathway dependent. BAI activated the PI3K/AKT pathway and inhibited the NF-κB pathway by regulating miR-205. In conclusion, BAI protected Min6 cells from TNF-α-induced injury by upregulating miR-205, which acts, at least in part, via activation of the PI3K/AKT pathway and inactivation of the NF-κB pathway.

SAD-A Promotes Pancreatic ß-Cell Proliferation by Regulating ß-Cell Cycle as a Downstream Target of mTORC1 Signaling

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and β-cell dysfunction, which leads to the depletion of β-cell mass. Understanding the molecular mechanisms driving islet β-cell dysfunction is paramount for developing therapies to increase β-cell function. The mammalian target of rapamycin complex 1 (mTORC1) plays a key role in regulating β-cell function by integrating growth factors and mitogens to direct cell cycle and survival; yet, mechanistically this process is not fully understood. Synapses of amphids defective protein kinase A (SAD-A) is a member of AMP-activated protein kinase (AMPK)-related family kinases, with high expression in the pancreas. My earlier work demonstrated that SAD-A is a β-cell specific mediator of insulin secretion and islet β-cell size. Furthermore, glucose dramatically stimulated SAD-A protein translation in islets that was potently inhibited by rapamycin, an inhibitor of mTORC1. My recent preliminary studies showed that 1) SAD-A overexpression increases β-cell proliferation in the MIN6 β-cell line and islets; 2) Ablation of SAD-A in pancreas of mice (pSADKO) promotes β-cell apoptosis and attenuates compensatory islet growth in obese mice; 3) SAD-A depletion in the pancreas increases mRNA expression of cyclin D2 in islets of pSADKO mice. Based on these observations, I hypothesize that SAD-A promotes pancreatic β-cell proliferation by regulating β-cell cycle as a downstream target of mTORC1 signaling. Disclosure J. Nie: None. N. Musi: None. Y. Shi: None.

Distinct roles of systemic and local actions of insulin on pancreatic β-cells

ObjectivePancreatic β-cell mass and function are critical in glucose homeostasis. Their regulatory mechanisms have been studied principally under experimental conditions of reduced β-cell numbers, such as β-cell ablation and partial pancreatectomy. In the present study, we generated an opposite mouse model with an excessive amount of ectopic β-cells, and analyzed its consequence on β-cell mass and survival. MethodsMice underwent sub-renal transplantation (SRT) of pseudo-islets generated from a pancreatic β-cell line MIN6 or intra-pancreatic transplantation (IPT) of MIN6 cells, and morphological and functional changes of their endocrine pancreata were analyzed. Cellular fate of pancreatic β-cells after transplantation was traced using RipCre:Rosa26-tdTomato mice. By using MIN6 cells, we evaluated the roles of extracellular glucose, membrane potential, and insulin signaling on β-cell survival. ResultsSRT mice developed severe, progressive hypoglycemia associated with marked reduction in insulin-positive (Ins+) cell mass and apparent increase in apoptotic Ins+ cells. In in vitro experiments of MIN6 cells, insulin signaling blockade potently induced cell death, suggesting that local insulin action is required for β-cell survival. In fact, IPT (i.e. transplantation close to endogenous β-cells) resulted in fewer apoptotic Ins+ cells compared with those induced by SRT. On the other hand, β-cell mass was decreased in proportion to the decrease in blood glucose levels in both SRT and IPT mice, suggesting a contribution of hypoglycemia induced by systemic hyperinsulinemia. ConclusionInsulin plays distinct roles in β-cell survival and β-cell mass regulation through its local and systemic actions on β-cells, respectively.

Identifying Signalling Pathways Regulated by GPRC5B in β-Cells by CRISPR-Cas9-Mediated Genome Editing

Background/Aims: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 β-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. Methods: The optimal transfection reagent for use in MIN6 β-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 β-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 β-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. Results: JetPRIME® was the best candidate for MIN6 β-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 β-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-β and IFNγ. Conclusion: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 β-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to β-cell proliferation and apoptosis.

56 - β-cells accumulate the age-related protein aggregate lipofuscin - Impact on functionality

The formation of cross-linked protein aggregates such as lipofuscin is an accepted hallmark of cellular aging and is inversely related to longevity. Lipofuscin is an intralysosomal aggregate, composed mainly of cross-linked proteins and formed due to oxidative processes. It is a prominent source of oxidants and able to inhibit proteolytic systems such as the 20S proteasome and the autophagy-lysosomal pathway. Because it is undegradable and cannot be removed via exocytosis, lipofuscin accumulation in postmitotic and senescent cells, such as neurons and skeletal muscle cells is inevitable, whereas proliferative cells dilute it during division. Adult pancreatic β-cells are also considered to be long-living cells with a very low proliferation rate. In 2010 it was shown by Cnop et al. [1], that these cells are also characterized by an age-dependent increase in lipofuscin, but little is known about the process of accumulating protein aggregates and their impact on β-cell functionality. We used the pancreatic β-cell line MIN6 and artificial lipofuscin to investigate different parameters of β-cell functionality and the impact of lipofuscin on the insulin-degrading enzyme (IDE). We were able to show a significantly higher insulin amount and secretion in the aggregate-fed MIN6 cells and evidence that both, gene expression and degradation are impaired. Furthermore our results show that insulin functionality seems to be affected.

Oxygen-Releasing Coatings for Improved Tissue Preservation.

Current organ transplantation protocols require the rapid transport of freshly isolated donor tissue to the recipient patient at the site where the procedure is to be conducted. During transport, the tissue graft can quickly deteriorate as a result of oxygen starvation. In this study, we report the fabrication of oxygen-releasing coatings for improved tissue preservation. The coatings were prepared via the encapsulation of calcium peroxide or urea peroxide microparticles between layers of octadiene plasma polymer films. By varying the thickness of the plasma polymer coating and type of peroxide, formulations were obtained that generate oxygen upon contact with aqueous solutions, while at the same time limiting the amount of toxic reactive oxygen species produced. The optimized coatings were tested under hypoxic conditions using the MIN6 β-cell line, which resulted in a 3-fold increase in the viability of cultured cells. These thin oxygen-releasing coatings can be deposited on a wide range of surfaces, creating a platform for oxygen delivery with the potential to extend the viability of transported tissues and increase the time frame available for graft transport.

Long noncoding RNAs are dynamically regulated during β-cell mass expansion in mouse pregnancy and control β-cell proliferation in vitro.

Pregnancy is associated with increased β-cell proliferation driven by prolactin. Long noncoding RNAs (lncRNA) are the most abundant RNA species in the mammalian genome, yet, their functional importance is mainly elusive. Aims/hypothesis: This study tests the hypothesis that lncRNAs regulate β-cell proliferation in response to prolactin in the context of β-cell mass compensation in pregnancy. Methods: The expression profile of lncRNAs in mouse islets at day 14.5 of pregnancy was explored by a bioinformatics approach, further confirmed by quantitative PCR at different days of pregnancy, and islet specificity was evaluated by comparing expression in islets versus other tissues. In order to establish the role of the candidate lncRNAs we studied cell proliferation in mouse islets and the MIN6 β-cell line by EdU incorporation and cell count. Results: We found that a group of lncRNAs is differentially regulated in mouse islets at 14.5 days of pregnancy. At different stages of pregnancy, these lncRNAs are dynamically expressed, and expression is prolactin dependent in mouse islets and MIN6 cells. One of those lncRNAs, Gm16308 (Lnc03), is dynamically regulated during pregnancy, prolactin-dependent and islet-enriched. Silencing Lnc03 in primary β-cells and MIN6 cells inhibits, whereas over-expression stimulates, proliferation even in the absence of prolactin, demonstrating that Lnc03 regulates β-cell growth. Conclusions/interpretation: During pregnancy mouse islet proliferation is correlated with dynamic changes of lncRNA expression. In particular, Lnc03 regulates mouse β-cell proliferation and may be a crucial component of β-cell proliferation in β-cell mass adaptation in both health and disease.

P 162 - Impact of the age-related protein aggregate lipofuscin on β-cell functionality

The formation of cross-linked protein aggregates such as lipofuscin is a typical hallmark of cellular aging and especially affects long-living and postmitotic cells such as neurons and skeletal muscle cells. Lipofuscin can neither be degraded nor exocytosed, it is a prominent source of oxidants and able to inhibit proteolytic systems such as the 20 S proteasome and the autophagy-lysosomal pathway. Adult pancreatic β-cells are also considered to be long-living cells which may last for years or even a life-time. It was shown, that these cells are also characterized by an age-dependent increase in lipofuscin content, but little is known about the process of accumulating protein aggregates and their impact on β-cell functionality. We used the pancreatic β-cell line MIN6 with a well-established artificial lipofuscin to investigate different parameters of β-cell functionality and the impact of lipofuscin on the insulin-degrading enzyme (IDE). Interestingly, we observed both, a significantly higher insulin amount and secretion in the aggregate-fed MIN6 cells. While the expression of insulin does not seem to be affected by lipofuscin treatment, the presence of lipofuscin is able to decrease the activity of IDE by an unknown mechanism.

168 - Impact of Artificial Lipofuscin on β-Cell Functionality

Aging is a multi-causal irreversible process characterized by a progressive degeneration affecting all higher organisms. The formation of insoluble protein aggregates such as lipofuscin is one typical hallmark of aging. The accumulation of lipofuscin has already been shown in many different tissues, but especially long-living and postmitotic cells such as neurons and skeletal muscle cells are affected by the intralysosomal accumulation of this protein aggregate. Lipofuscin can neither be degraded by lysosomal enzymes nor exocytosed, furthermore postmitotic cells are not able to protect themselves from lipofuscin accumulation by cell division. Lipofuscin is a source of reactive oxygen species, able to inhibit proteolytic systems leading to a decline in proteasomal activity. Therefore, lipofuscin is an important contributor limiting the lifespan of especially postmitotic cells. Pancreatic β-cells are also considered to be long-living cells in adults and it was shown, that these cells are characterized by an age-dependent increase in lipofuscin content. However, even though β-cell replication is a rare event in aged humans and mice, little is known about the accumulation of protein aggregates and the intracellular effects of lipofuscin in β-cells. Therefore the pancreatic β-cell line MIN6 was treated with artificial lipofuscin to investigate the impact of this aggregate on the insulin-degrading enzyme (IDE) and different parameters of β-cell functionality. Interestingly, we observed a significant rise in insulin secretion and insulin content in the aggregate-fed MIN6 cells. While the expression of insulin does not seem to be affected by lipofuscin treatment, the prescence of lipofuscin is able to decrease the activity of IDE by an unknown mechanism.