Primary Mouse Beta Cells Research Articles

Synchrotron fluorescence imaging of individual mouse beta-cells reveals changes in zinc, calcium, and iron in a model of low-grade inflammation.

Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158± 57.7 femtograms (fg) to 65.7± 29.7 fg; calcium decreased from 216± 67.4 to 154.3± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4± 12.2 to 47.2± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

Glucose but not KCl diminishes submembrane granule turnover in mouse beta-cells.

KCl depolarization is widely used to mimic the depolarization during glucose-stimulated insulin secretion. Consequently, the insulin secretion elicited by KCl is often regarded as the equivalent of the first phase of glucose-induced insulin secretion. Here, the effects of both stimuli were compared by measuring the secretion of perifused mouse islets, the cytosolic Ca2+ concentration of single beta-cells and the mobility of submembrane insulin granules by TIRF microscopy of primary mouse beta-cells. Two cargo-directed granule labels were used namely insulin-EGFP and C-peptide-emGFP. The granule behaviour common to both was used to compare the effect of sequential stimulation with 40 mM KCl and 30 mM glucose and sequential stimulation with the same stimuli in reversed order. At the level of the cell secretory response, the sequential pulse protocol showed marked differences depending on the order of the two stimuli. KCl produced higher maximal secretion rates and diminished the response to the subsequent glucose stimulus, whereas glucose enhanced the response to the subsequent KCl stimulus. At the level of granule behaviour, a difference developed during the first stimulation phase in that the total number of granules, the short-term resident granules and the arriving granules, which are all parameters of granule turnover, were significantly smaller for glucose than for KCl. These differences at both the level of the cell secretory response and granule behaviour in the submembrane space are incompatible with identical initial response mechanisms to KCl and glucose stimulation.

Precise expression of Fis1 is important for glucose responsiveness of beta cells.

Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucose-stimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein.

Insulin granule mobility, cytosolic calcium concentration, and stimulated insulin secretion in MIN6 cells and beta cells: differences and similarities

Background and aims: The conventional view that insulin granules are sequentially recruited to release insulin has been superseded by newer, more complex models, resulting from TIRF imaging. Here, we compared insulin secretion, cytosolic Ca2+ concentration ([Ca2+]i) and granule mobility in MIN6 cells and in primary mouse beta cells.

The Talk-1 C-Terminus is a Charge-Sensitive Module Regulating Channel Activity

Human beta-cell insulin secretion is dependent on Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs). K+ channels modulate the beta-cell membrane potential, which influences VDCC activity. The most abundant K+ channel of the human islet is the two-pore domain K+ (K2P) channel TALK-1; however, its function is unknown. We characterized TALK-1 currents in primary mouse beta-cells and find that K2P currents are significantly reduced after genetic ablation of TALK-1. To identify mechanisms that regulate TALK-1 channel activity, we used a Tl+-sensitive fluorescent screen to assess if TALK-1 is regulated by kinases. Cells expressing TALK-1 channels were monitored for changes in Tl+ flux in response to co-expression with a constitutively active kinase (examining a total of 192 kinases), revealing 9 kinases that modify Tl+ flux through TALK-1. Interestingly, three phosphatidylinositol phosphate kinases (PIKs) including PIK3CG, PI4K2B, and PIKFYVE, increased TALK-1 channel activity. To determine if activation of TALK-1 channels by PIKs is due to build-up of PIPs, we depleted membrane PIP2 with a rapamycin-recruitable phosphatase and found that TALK-1 channel activity was reduced. As charged residues of other K+ channels mediate PIP2 sensitivity, we assessed if PIP2 influences TALK-1 channel activity through a charged residue enriched domain in the C-terminus. We found that neutralization of these charged residues significantly reduced PIP2 activation of TALK-1 channels. Importantly, PIP2 introduced via patch pipette into primary mouse beta-cells also significantly enhanced TALK-1 currents. As glucose induces Ca2+-dependent oscillations in beta-cell PIP2, we then assessed how TALK-1 regulates islet Ca2+ dynamics. We find that Ca2+ oscillation frequency is significantly increased in islets lacking TALK-1, which results in enhanced glucose-stimulated insulin secretion (GSIS). These data provide compelling evidence that TALK-1 influences GSIS by limiting excitability, and suggests a novel mechanism for phospholipid-dependent regulation of beta-cell Ca2+ entry.

Real-time analysis of intracellular glucose and calcium in pancreatic beta cells by fluorescence microscopy

Glucose is the physiological stimulus for insulin secretion in pancreatic beta cells. The uptake and phosphorylation of glucose initiate and control downstream pathways, resulting in insulin secretion. However, the temporal coordination of these events in beta cells is not fully understood. The recent development of the FLII12Pglu-700μ-δ6 glucose nanosensor facilitates real-time analysis of intracellular glucose within a broad concentration range. Using this fluorescence-based technique, we show the shift in intracellular glucose concentration upon external supply and removal in primary mouse beta cells with high resolution. Glucose influx, efflux, and metabolism rates were calculated from the time-dependent plots. Comparison of insulin-producing cells with different expression levels of glucose transporters and phosphorylating enzymes showed that a high glucose influx rate correlated with GLUT2 expression, but was largely also sustainable by high GLUT1 expression. In contrast, in cells not expressing the glucose sensor enzyme glucokinase glucose metabolism was slow. We found no evidence of oscillations of the intracellular glucose concentration in beta cells. Concomitant real-time analysis of glucose and calcium dynamics using FLII12Pglu-700μ-δ6 and fura-2-acetoxymethyl-ester determined a glucose threshold of 4mM for the [Ca2+]i increase in beta cells. Indeed, a glucose concentration of 7mM had to be reached to evoke large amplitude [Ca2+]i oscillations. The KATP channel closing agent glibenclamide was not able to induce large amplitude [Ca2+]i oscillations in the absence of glucose. Our findings suggest that glucose has to reach a threshold to evoke the [Ca2+]i increase and subsequently initiate [Ca2+]i oscillations in a KATP channel independent manner.

Endocannabinoid Inhibition of Ion Channels of Pancreatic Beta Cells

The secretion of insulin from pancreatic beta cells is regulated by many factors, chiefly the plasma glucose concentration. Glucose initiates a chain of events that raises the ATP:ADP ratio within the cell. When the K(ATP) channel is inhibited by this increased ratio, the cell depolarizes, calcium action potentials ensue, and the raised cytosolic calcium concentration leads to exocytosis of insulin secretory granules. Preliminary evidence from our lab indicates that endocannabinoids are negative regulators of insulin secretion, and we previously demonstrated that the endocannabinoid 2-arachidonylglycerol (2-AG) inhibits sodium, delayed rectifier potassium, and high voltage-activated (mostly L-type) calcium channels at low micromolar concentrations in the insulinoma cell line R7T1.In the R7T1 cell line, inside-out patches (+60 mV) displayed unitary currents of about 4.2 pA and various multiples, which were identified as K(ATP) channels by being nearly completely inhibited by 1 mM ATP. 2-AG inhibited the K(ATP) channel with an IC50 of 1 uM. This block was irreversible, even in the presence of lipid free bovine serum albumin. To test whether the block by 2-AG were mediated by the CB1 receptor, the CB1 antagonist AM-251 (10 uM) was added to the pipette solution. Though a >95% antagonism was predicted, no effect of the AM-251 was observed, suggesting that the CB1 receptor did not mediate the 2-AG blockade. This block was evident in current clamped (perforated patch) primary mouse beta cells, which showed a small (2.5 mV median) but consistent depolarization in the presence of 10 uM 2-AG. Moreover, the depolarization induced by 10 mM glucose was blunted by 10 uM 2-AG, evidence that the ion channel blockade seen in the insulinoma cells can apply to primary beta cells.

Glucose-Induced Cyclic AMP Oscillations Regulate Pulsatile Insulin Secretion

Cyclic AMP (cAMP) and Ca(2+) are key regulators of exocytosis in many cells, including insulin-secreting beta cells. Glucose-stimulated insulin secretion from beta cells is pulsatile and involves oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse beta cells. These oscillations were preceded and enhanced by elevations of [Ca(2+)](i). However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca(2+)](i) rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.

Lactogens Promote Beta Cell Survival through JAK2/STAT5 Activation and Bcl-XL Upregulation

One of the goals in the treatment for diabetes is to enhance pancreatic beta cell function, proliferation, and survival. This study explores the role of lactogenic hormones, prolactin (PRL) and placental lactogen (PL), in beta cell survival. We have previously shown that transgenic mice expressing mouse placental lactogen-1 (mPL1) in beta cells under the rat insulin II promoter (RIP) are resistant to the diabetogenic and cytotoxic effects of streptozotocin (STZ) in vivo. The current study demonstrates that lactogens protect rat insulinoma (INS-1) cells and primary mouse beta cells against two distinct beta cell death inducers, STZ and dexamethasone (DEX), in vitro. Further, we identify the mechanism through which lactogens protect beta cells against DEX-induced death. The signaling pathway mediating this protective effect is the janus-activated-kinase-2/signal transducer and activator of transcription-5 (JAK2/STAT5) pathway. This is demonstrated in INS-1 cells and primary mouse beta cells using three separate approaches, pharmacological inhibitors, JAK2-specific siRNAs and a dominant-negative STAT5 mutant. Furthermore, lactogens specifically and significantly increase the anti-apoptotic protein Bcl-XL in insulinoma cells and mouse islets. Bcl-XL-specific siRNA significantly inhibits lactogen-mediated protection against DEX-induced beta cell death. We believe this is the first direct demonstration of lactogens mediating their protective effect through the JAK2/STAT5 pathway in the beta cell and through Bcl-XL in any cell type.

Increased KATP channel current in pancreatic beta cells over expressing fatty acyl CoA synthetase I

Closure of KATP channels links glucose metabolism to enhanced electrical activity and insulin secretion in the pancreatic beta cell. Long chain acyl CoAs can potently activate KATP when perfused directly across excised membrane patches, which could account for impaired glucose-stimulated insulin secretion in obese and type II diabetic individuals via reduced beta cell excitability. However, the effects of chronic elevation of acyl CoAs within beta cells have not been documented. We have enhanced endogenous acyl CoA levels within intact primary mouse beta cells using virally delivered over expression of fatty acyl CoA synthetase-1 (FACS-1), and assessed the effects of this manipulation on beta cell excitability using the perforated whole-cell patch-clamp technique. Infection with AdFACS-1 produced a large increase in FACS-1 protein expression compared to controls (AdGFP-treated), as confirmed by Western blot. Patch-clamp data indicated a significant enhancement (p<0.05, n=7) of basal KATP current measured at low glucose in AdFACS-1 infected cells compared to controls. Furthermore, membrane potential recordings showed a decreased spiking frequency in response to high glucose in AdFACS-1 infected cells. We provide direct evidence for reduced beta cell excitability by a FACS-1 driven cytosolic accumulation of acyl CoAs. These observations suggest a strong and direct link between intracellular fatty esters and KATP activity and may have major implications for our current understanding of the involvement of diet and obesity in the development of type II diabetes. Work is sponsored by CIHR and AHFMR.

Dynamic imaging of endoplasmic reticulum Ca2+ concentration in insulin-secreting MIN6 Cells using recombinant targeted cameleons: roles of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2 and ryanodine receptors.

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca(2+) concentrations ([Ca(2+)](cyt)) and hence in insulin secretion from pancreatic beta-cells. However, the molecular mechanisms involved in both the uptake and release of Ca(2+) from the ER are only partially defined in these cells, and the presence and regulation of ER ryanodine receptors are a matter of particular controversy. To monitor Ca(2+) fluxes across the ER membrane in single live MIN6 beta-cells, we have imaged changes in the ER intralumenal free Ca(2+) concentration ([Ca(2+)](ER)) using ER-targeted cameleons. Resting [Ca(2+)](ER) (approximately 250 micromol/l) was markedly reduced after suppression (by approximately 40%) of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca(2+)-ATPase in this cell type. Nutrient secretagogues that elevated [Ca(2+)](cyt) also increased [Ca(2+)](ER), an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca(2+)](ER). Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca(2+) and marked increases in [Ca(2+)](cyt). Furthermore, photolysis of caged cyclic ADP ribose increased [Ca(2+)](cyt), and this effect was largely abolished by emptying ER/Golgi stores with thapsigargin. Expression of RyR protein in living MIN6, INS-1, and primary mouse beta-cells was also confirmed by the specific binding of cell-permeate BODIPY TR-X ryanodine. RyR channels are likely to play an important part in the regulation of intracellular free Ca(2+) changes in the beta-cell and thus in the regulation of insulin secretion.