scholarly journals Electrophysiological Characterization of Pancreatic Islet Cells in the Mouse Insulin Promoter-Green Fluorescent Protein Mouse

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4766-4775 ◽  
Author(s):  
Yuk M. Leung ◽  
Ishtiaq Ahmed ◽  
Laura Sheu ◽  
Robert G. Tsushima ◽  
Nicholas E. Diamant ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Katp Channels ◽  
Β Cells ◽  
Β Cell ◽  
Mouse Islet ◽  
Channel Density ◽  
Na Currents ◽  
Insulin Promoter ◽  
Green Fluorescent ◽  
Ca2 Channels

We recently reported a transgenic [mouse insulin promoter (MIP)-green fluorescent protein (GFP)] mouse in which GFP expression is targeted to the pancreatic islet β-cells to enable convenient identification of β-cells as green cells. The GFP-expressing β-cells of the MIP-GFP mouse were functionally indistinguishable from β-cells of normal mice. Here we characterized the ionic channel properties and exocytosis of MIP-GFP mouse islet β- and α-cells. β-Cells displayed delayed rectifying K+ and high-voltage-activated Ca2+ channels and exhibited Na+ currents only at hyperpolarized holding potential. α-Cells were nongreen and had both A-type and delayed rectifier K+ channels, both low-voltage-activated and high-voltage-activated Ca2+ channels, and displayed Na+ currents readily at −70 mV holding potential. α-Cells had ATP-sensitive K+ channel (KATP) channel density as high as that in β-cells, and, surprisingly, α-cell KATP channels were more sensitive to ATP inhibition (IC50 = 0.16 ± 0.03 mm) than β-cell KATP channels (IC50 = 0.86 ± 0.10 mm). Whereas α-cells were rather uniform in size [2–4.5 picofarad (pF)], β-cells varied vastly in size (2–12 pF). Of note, small β-cells (<4.5 pF) showed little exocytosis, whereas medium β-cells (5–8 pF) exhibited vigorous exocytosis, but large β-cells (>8 pF) had weaker exocytosis. We found no correlation between β-cell size and their Ca2+ channel density, suggesting that Ca2+ influx may not be the cause of the heterogeneity in exocytotic responses. The MIP-GFP mouse therefore offers potential to further explore the functional heterogeneity in β-cells of different sizes. The MIP-GFP mouse islet is therefore a reliable model to efficiently examine α-cell and β-cell physiology and should greatly facilitate examination of their pathophysiology when the MIP-GFP mice are crossed with diabetic models.

scholarly journals Insulin Regulates Islet α-Cell Function by Reducing KATP Channel Sensitivity to Adenosine 5′-Triphosphate Inhibition

Endocrinology ◽  
2006 ◽  
Vol 147 (5) ◽  
pp. 2155-2162 ◽  
Author(s):  
Yuk M. Leung ◽  
Ishtiaq Ahmed ◽  
Laura Sheu ◽  
Xiaodong Gao ◽  
Manami Hara ◽  
...  
Keyword(s):  
Katp Channel ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Glucagon Secretion ◽  
Katp Channels ◽  
Β Cell ◽  
Major Mechanism ◽  
Insulin Promoter ◽  
Atp Inhibition ◽  
Green Fluorescent

Glucose regulates pancreatic islet α-cell glucagon secretion directly by its metabolism to generate ATP in α-cells, and indirectly via stimulation of paracrine release of β-cell secretory products, particularly insulin. How the cellular substrates of these pathways converge in the α-cell is not well known. We recently reported the use of the MIP-GFP (mouse insulin promoter-green fluorescent protein) mouse to reliably identify islet α- (non-green cells) and β-cells (green cells), and characterized their ATP-sensitive K+ (KATP) channel properties, showing that α-cell KATP channels exhibited a 5-fold higher sensitivity to ATP inhibition than β-cell KATP channels. Here, we show that insulin exerted paracrine regulation of α-cells by markedly reducing the sensitivity of α-cell KATP channels to ATP (IC50 = 0.18 and 0.50 mm in absence and presence of insulin, respectively). Insulin also desensitized β-cell KATP channels to ATP inhibition (IC50 = 0.84 and 1.23 mm in absence and presence of insulin, respectively). Insulin effects on both islet cell KATP channels were blocked by wortmannin, indicating that insulin acted on the insulin receptor-phosphatidylinositol 3-kinase signaling pathway. Insulin did not affect α-cell A-type K+ currents. Glutamate, known to also inhibit α-cell glucagon secretion, did not activate α-cell KATP channel opening. We conclude that a major mechanism by which insulin exerts paracrine control on α-cells is by modulating its KATP channel sensitivity to ATP block. This may be an underlying basis for the proposed sequential glucose-insulin regulation of α-cell glucagon secretion, which becomes distorted in diabetes, leading to dysregulated glucagon secretion.

scholarly journals Protective Role of Autophagy in Palmitate-Induced INS-1 β-Cell Death

Endocrinology ◽  
2008 ◽  
Vol 150 (1) ◽  
pp. 126-134 ◽  
Author(s):  
Sung-E Choi ◽  
Sung-Mi Lee ◽  
Youn-Jung Lee ◽  
Ling-Ji Li ◽  
Soo-Jin Lee ◽  
...  
Keyword(s):  
Cell Death ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Mammalian Target Of Rapamycin ◽  
Protective Role ◽  
Target Of Rapamycin ◽  
Β Cells ◽  
Β Cell ◽  
Green Fluorescent

Autophagy, a vacuolar degradative pathway, constitutes a stress adaptation that avoids cell death or elicits the alternative cell-death pathway. This study was undertaken to determine whether autophagy is activated in palmitate (PA)-treated β-cells and, if activated, what the role of autophagy is in the PA-induced β-cell death. The enhanced formation of autophagosomes and autolysosomes was observed by exposure of INS-1 β-cells to 400 μm PA in the presence of 25 mm glucose for 12 h. The formation of green fluorescent protein-LC3-labeled structures (green fluorescent protein-LC3 dots), with the conversion from LC3-I to LC3-II, was also distinct in the PA-treated cells. The phospho-mammalian target of rapamycin level, a typical signal pathway that inhibits activation of autophagy, was gradually decreased by PA treatment. Blockage of the mammalian target of rapamycin signaling pathway by treatment with rapamycin augmented the formation of autophagosomes but reduced PA-induced INS-1 cell death. In contrast, reduction of autophagosome formation by knocking down the ATG5, inhibition of fusion between autophagosome and lysosome by treatment with bafilomycin A1, or inhibition of proteolytic degradation by treatment with E64d/pepstatin A, significantly augmented PA-induced INS-1 cell death. These findings showed that the autophagy system could be activated in PA-treated INS-1 β-cells, and suggested that the induction of autophagy might play an adaptive and protective role in PA-induced cell death. Autophagy is activated in palmitate-treated insulinoma-1 beta cells, and the induction of autophagy plays a protective role in palmitate-induced beta cell death.

Transgenic mice with green fluorescent protein-labeled pancreatic β-cells

2003 ◽  
Vol 284 (1) ◽  
pp. E177-E183 ◽  
Author(s):  
Manami Hara ◽  
Xiaoyu Wang ◽  
Toshihiko Kawamura ◽  
Vytas P. Bindokas ◽  
Restituto F. Dizon ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Cell Biology ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Gene Promoter ◽  
Β Cells ◽  
Β Cell ◽  
Β Cell Function ◽  
Green Fluorescent

We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with coexpression of insulin and GFP in the β-cells of all islets. We observed GFP expression in islets from embryonic day E13.5 through adulthood. Studies of β-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP-transgenic mice and sorted the β-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4 ± 0.2% ( n = 6) of the cells in the pancreas of newborn (P1) and 0.9 ± 0.1% ( n = 5) of 8-wk-old mice were β-cells. The MIP-GFP-transgenic mouse may be a useful tool for studying β-cell biology in normal and diabetic animals.

scholarly journals Reprogramming Mouse Cells With a Pancreatic Duct Phenotype to Insulin-Producing β-Like Cells

Endocrinology ◽  
2015 ◽  
Vol 156 (6) ◽  
pp. 2029-2038 ◽  
Author(s):  
Takatsugu Yamada ◽  
Claudia Cavelti-Weder ◽  
Francisco Caballero ◽  
Philippe A. Lysy ◽  
Lili Guo ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Pancreatic Duct ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Β Cells ◽  
Β Cell ◽  
Cell Markers ◽  
Adenoviral Delivery ◽  
Glucagon Like Peptide 1 ◽  
Green Fluorescent

Abstract Reprogramming technology has opened the possibility of converting one cell type into another by forced expression of transgenes. Transduction of adenoviral vectors encoding 3 pancreatic transcription factors, Pdx1, Ngn3, and MafA, into mouse pancreas results in direct reprogramming of exocrine cells to insulin-producing β-like cells. We hypothesized that cultured adult pancreatic duct cells could be reprogrammed to become insulin-producing β-cells by adenoviral-mediated expression of this same combination of factors. Exocrine were isolated from adult mouse insulin 1 promoter (MIP)-green fluorescent protein (GFP) transgenic mice to allow new insulin-expressing cells to be detected by GFP fluorescence. Cultured cells were transduced by an adenoviral vector carrying a polycistronic construct Ngn3/Pdx1/MafA/mCherry (Ad-M3C) or mCherry sequence alone as a control vector. In addition, the effects of glucagon-like peptide-1 (GLP-1) receptor agonist, exendin-4 (Ex-4) on the reprogramming process were examined. GFP+ cells appeared 2 days after Ad-M3C transduction; the reprogramming efficiency was 8.6 ± 2.6% by day 4 after transduction. Ad-M3C also resulted in increased expression of β-cell markers insulin 1 and 2, with enhancement by Ex-4. Expression of other β-cell markers, neuroD and GLP-1 receptor, were also significantly up-regulated. The amount of insulin release into the media and insulin content of the cells were significantly higher in the Ad-M3C-transduced cells; this too was enhanced by Ex-4. The transduced cells did not secrete insulin in response to increased glucose, indicating incomplete differentiation to β-cells. Thus, cultured murine adult pancreatic cells with a duct phenotype can be directly reprogrammed to insulin-producing β-like cells by adenoviral delivery of 3 pancreatic transcription factors.

Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA

2001 ◽  
Vol 280 (1) ◽  
pp. C175-C182 ◽  
Author(s):  
Michihiro Tateyama ◽  
Shuqin Zong ◽  
Tsutomu Tanabe ◽  
Rikuo Ochi
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Foreign Gene ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent ◽  
Ca2 Channels

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.

scholarly journals Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic β-cells

2005 ◽  
Vol 185 (1) ◽  
pp. 57-67 ◽  
Author(s):  
L B Hays ◽  
B Wicksteed ◽  
Y Wang ◽  
J F McCuaig ◽  
L H Philipson ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Fluorescent Protein ◽  
Zymogen Granule ◽  
Β Cells ◽  
Rat Islets ◽  
Intracellular Ca ◽  
Specific Localization ◽  
Glucagon Like Peptide 1 ◽  
Green Fluorescent ◽  
Insulin Exocytosis

Several proteins play a role in the mechanism of insulin exocytosis. However, these ‘exocytotic proteins’ have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet β-cells as experimental tools. Syncollin is not normally expressed in β-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of β-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5μM). Consistent with specific localization of syncollin to β-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in β-cells has highlighted the importance of intralumenal β-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a ‘fluorescent tag’ to track β-granule transport and exocytosis in real time.

Effects of palmitate on ER and cytosolic Ca2+ homeostasis in β-cells

2009 ◽  
Vol 296 (4) ◽  
pp. E690-E701 ◽  
Author(s):  
Kamila S. Gwiazda ◽  
Ting-Lin B. Yang ◽  
Yalin Lin ◽  
James D. Johnson
Keyword(s):  
Type 2 Diabetes ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acids ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Β Cells ◽  
Β Cell ◽  
Β Cell Function

There are strong links between obesity, elevated free fatty acids, and type 2 diabetes. Specifically, the saturated fatty acid palmitate has pleiotropic effects on β-cell function and survival. In the present study, we sought to determine the mechanism by which palmitate affects intracellular Ca2+, and in particular the role of the endoplasmic reticulum (ER). In human β-cells and MIN6 cells, palmitate rapidly increased cytosolic Ca2+ through a combination of Ca2+ store release and extracellular Ca2+ influx. Palmitate caused a reversible lowering of ER Ca2+, measured directly with the fluorescent protein-based ER Ca2+ sensor D1ER. Using another genetically encoded indicator, we observed long-lasting oscillations of cytosolic Ca2+ in palmitate-treated cells. In keeping with this observed ER Ca2+ depletion, palmitate induced rapid phosphorylation of the ER Ca2+ sensor protein kinase R-like ER kinase (PERK) and subsequently ER stress and β-cell death. We detected little palmitate-induced insulin secretion, suggesting that these Ca2+ signals are poorly coupled to exocytosis. In summary, we have characterized Ca2+-dependent mechanisms involved in altered β-cell function and survival induced by the free fatty acid palmitate. We present the first direct evidence that free fatty acids reduce ER Ca2+ and shed light on pathways involved in lipotoxicity and the pathogenesis of type 2 diabetes.

scholarly journals Diabetes induced by gain-of-function mutations in the Kir6.1 subunit of the KATP channel

2016 ◽  
Vol 149 (1) ◽  
pp. 75-84 ◽  
Author(s):  
Maria S. Remedi ◽  
Jonathan B. Friedman ◽  
Colin G. Nichols
Keyword(s):  
Insulin Secretion ◽  
Katp Channel ◽  
Vascular System ◽  
Wild Type Mouse ◽  
Neonatal Diabetes ◽  
Katp Channels ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Gain Of Function ◽  
Insulin Promoter

Gain-of-function (GOF) mutations in the pore-forming (Kir6.2) and regulatory (SUR1) subunits of KATP channels have been identified as the most common cause of human neonatal diabetes mellitus. The critical effect of these mutations is confirmed in mice expressing Kir6.2-GOF mutations in pancreatic β cells. A second KATP channel pore-forming subunit, Kir6.1, was originally cloned from the pancreas. Although the prominence of this subunit in the vascular system is well documented, a potential role in pancreatic β cells has not been considered. Here, we show that mice expressing Kir6.1-GOF mutations (Kir6.1[G343D] or Kir6.1[G343D,Q53R]) in pancreatic β cells (under rat-insulin-promoter [Rip] control) develop glucose intolerance and diabetes caused by reduced insulin secretion. We also generated transgenic mice in which a bacterial artificial chromosome (BAC) containing Kir6.1[G343D] is incorporated such that the transgene is only expressed in tissues where Kir6.1 is normally present. Strikingly, BAC-Kir6.1[G343D] mice also show impaired glucose tolerance, as well as reduced glucose- and sulfonylurea-dependent insulin secretion. However, the response to K+ depolarization is intact in Kir6.1-GOF mice compared with control islets. The presence of native Kir6.1 transcripts was demonstrated in both human and wild-type mouse islets using quantitative real-time PCR. Together, these results implicate the incorporation of native Kir6.1 subunits into pancreatic KATP channels and a contributory role for these subunits in the control of insulin secretion.

scholarly journals MafA Is a Glucose-regulated and Pancreatic β-Cell-specific Transcriptional Activator for the Insulin Gene

2002 ◽  
Vol 277 (51) ◽  
pp. 49903-49910 ◽  
Author(s):  
Kohsuke Kataoka ◽  
Song-iee Han ◽  
Setsuko Shioda ◽  
Momoki Hirai ◽  
Makoto Nishizawa ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Regulatory Element ◽  
Transcriptional Activator ◽  
Dominant Negative ◽  
Insulin Gene ◽  
Pcr Analysis ◽  
Β Cells ◽  
Β Cell ◽  
Basic Leucine Zipper ◽  
Insulin Promoter

The insulin gene is specifically expressed in β-cells of the Langerhans islets of the pancreas, and its transcription is regulated by the circulating glucose level. Previous reports have shown that an unidentified β-cell-specific nuclear factor binds to a conservedcis-regulatory element called RIPE3b and is critical for its glucose-regulated expression. Based on the sequence similarity of the RIPE3b element and the consensus binding sequence of the Maf family of basic leucine zipper transcription factors, we here identified mammalianhomologueof avian MafA/L-Maf, an eye-specific member of the Maf family, as the RIPE3b-binding transcriptional activator. Reverse transcription-PCR analysis showed thatmafAmRNA is detected only in the eyes and in pancreatic β-cells and not in α-cells. MafA protein as well as its mRNA is up-regulated by glucose, consistent with the glucose-regulated binding of MafA to the RIPE3b element in β-cell nuclear extracts. In transient luciferase assays, we also showed that expression of MafA greatly enhanced insulin promoter activity and that a dominant-negative form of MafA inhibited it. Therefore, MafA is a β-cell-specific and glucose-regulated transcriptional activator for insulin gene expression and thus may be involved in the function and development of β-cells as well as in the pathogenesis of diabetes.

scholarly journals A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

2016 ◽  
Vol 36 (23) ◽  
pp. 2918-2930 ◽  
Author(s):  
Heather L. Hayes ◽  
Lu Zhang ◽  
Thomas C. Becker ◽  
Jonathan M. Haldeman ◽  
Samuel B. Stephens ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Islet Cell ◽  
Transforming Growth Factor ◽  
Cell Function ◽  
Transforming Growth Factor Β ◽  
Human Islet ◽  
Soluble Factor ◽  
Β Cells ◽  
Β Cell ◽  
Insulin Promoter

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation.

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