Michael W. Roe

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function.

Cell physiology of cAMP sensor Epac.

Epac is an acronym for the exchange proteins activated directly by cyclic AMP, a family of cAMP‐regulated guanine nucleotide exchange factors (cAMPGEFs) that mediate protein kinase A (PKA)‐independent signal transduction properties of the second messenger cAMP. Two variants of Epac exist (Epac1 and Epac2), both of which couple cAMP production to the activation of Rap, a small molecular weight GTPase of the Ras family. By activating Rap in an Epac‐mediated manner, cAMP influences diverse cellular processes that include integrin‐mediated cell adhesion, vascular endothelial cell barrier formation, and cardiac myocyte gap junction formation. Recently, the identification of previously unrecognized physiological processes regulated by Epac has been made possible by the development of Epac‐selective cyclic AMP analogues (ESCAs). These cell‐permeant analogues of cAMP activate both Epac1 and Epac2, whereas they fail to activate PKA when used at low concentrations. ESCAs such as 8‐pCPT‐2′‐O‐Me‐cAMP and 8‐pMeOPT‐2′‐O‐Me‐cAMP are reported to alter Na+, K+, Ca2+ and Cl− channel function, intracellular [Ca2+], and Na+–H+ transporter activity in multiple cell types. Moreover, new studies examining the actions of ESCAs on neurons, pancreatic beta cells, pituitary cells and sperm demonstrate a major role for Epac in the stimulation of exocytosis by cAMP. This topical review provides an update concerning novel PKA‐independent features of cAMP signal transduction that are likely to be Epac‐mediated. Emphasized is the emerging role of Epac in the cAMP‐dependent regulation of ion channel function, intracellular Ca2+ signalling, ion transporter activity and exocytosis.

Conditional gene targeting in mouse pancreatic β-cells: Analysis of ectopic Cre transgene expression in the brain

OBJECTIVE Conditional gene targeting has been extensively used for in vivo analysis of gene function in β-cell biology. The objective of this study was to examine whether mouse transgenic Cre lines, used to mediate β-cell– or pancreas-specific recombination, also drive Cre expression in the brain. RESEARCH DESIGN AND METHODS Transgenic Cre lines driven by Ins1, Ins2, and Pdx1 promoters were bred to R26R reporter strains. Cre activity was assessed by β-galactosidase or yellow fluorescent protein expression in the pancreas and the brain. Endogenous Pdx1 gene expression was monitored using Pdx1tm1Cvw lacZ knock-in mice. Cre expression in β-cells and co-localization of Cre activity with orexin-expressing and leptin-responsive neurons within the brain was assessed by immunohistochemistry. RESULTS All transgenic Cre lines examined that used the Ins2 promoter to drive Cre expression showed widespread Cre activity in the brain, whereas Cre lines that used Pdx1 promoter fragments showed more restricted Cre activity primarily within the hypothalamus. Immunohistochemical analysis of the hypothalamus from Tg(Pdx1-cre)89.1Dam mice revealed Cre activity in neurons expressing orexin and in neurons activated by leptin. Tg(Ins1-Cre/ERT)1Lphi mice were the only line that lacked Cre activity in the brain. CONCLUSIONS Cre-mediated gene manipulation using transgenic lines that express Cre under the control of the Ins2 and Pdx1 promoters are likely to alter gene expression in nutrient-sensing neurons. Therefore, data arising from the use of these transgenic Cre lines must be interpreted carefully to assess whether the resultant phenotype is solely attributable to alterations in the islet β-cells.

Defective Pancreatic β-Cell Glycolytic Signaling in Hepatocyte Nuclear Factor-1α-deficient Mice

Mutations in the hepatocyte nuclear factor-1α (HNF-1α) gene cause maturity onset diabetes of the young type 3, a form of type 2 diabetes mellitus. In mice lacking the HNF-1α gene, insulin secretion and intracellular calcium ([Ca2+] i ) responses were impaired following stimulation with nutrient secretagogues such as glucose and glyceraldehyde but normal with non-nutrient stimuli such as potassium chloride. Patch clamp recordings revealed ATP-sensitive K+currents (KATP) in β-cells that were insensitive to suppression by glucose but normally sensitive to ATP. Exposure to mitochondrial substrates suppressed KATP, elevated [Ca2+] i , and corrected the insulin secretion defect. NAD(P)H responses to glucose were substantially reduced, and inhibitors of glycolytic NADH generation reproduced the mutant phenotype in normal islets. Flux of glucose through glycolysis in islets from mutant mice was reduced, as a result of which ATP generation in response to glucose was impaired. We conclude that hepatocyte nuclear factor-1α diabetes results from defective β-cell glycolytic signaling, which is potentially correctable using substrates that bypass the defect.

Increases in Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Calcium Responses in Pulmonary Artery Smooth Muscle Cells

Mitochondria have been implicated as a potential site of O2 sensing underlying hypoxic pulmonary vasoconstriction (HPV), but 2 disparate models have been proposed to explain their reaction to hypoxia. One model proposes that hypoxia-induced increases in mitochondrial reactive oxygen species (ROS) generation activate HPV through an oxidant-signaling pathway, whereas the other proposes that HPV is a result of decreased oxidant signaling. In an attempt to resolve this debate, we use a novel, ratiometric, redox-sensitive fluorescence resonance energy transfer (HSP-FRET) probe, in concert with measurements of reduced/oxidized glutathione (GSH/GSSG), to assess cytosolic redox responses in cultured pulmonary artery smooth muscle cells (PASMCs). Superfusion of PASMCs with hypoxic media increases the HSP-FRET ratio and decreases GSH/GSSG, indicating an increase in oxidant stress. The antioxidants pyrrolidinedithiocarbamate and N-acetyl-l-cysteine attenuated this response, as well as the hypoxia-induced increases in cytosolic calcium ([Ca2+]i), assessed by the Ca2+-sensitive FRET sensor YC2.3. Adenoviral overexpression of glutathione peroxidase or cytosolic or mitochondrial catalase attenuated the hypoxia-induced increase in ROS signaling and [Ca2+]i. Adenoviral overexpression of cytosolic Cu, Zn-superoxide dismutase (SOD-I) had no effect on the hypoxia-induced increase in ROS signaling and [Ca2+]i, whereas mitochondrial matrix–targeted Mn-SOD (SOD-II) augmented [Ca2+]i. The mitochondrial inhibitor myxothiazol attenuated the hypoxia-induced changes in the ROS signaling and [Ca2+]i, whereas cyanide augmented the increase in [Ca2+]i. Finally, simultaneous measurement of ROS and Ca2+ signaling in the same cell revealed that the initial increase in these 2 signals could not be distinguished temporally. These results demonstrate that hypoxia triggers increases in PASMC [Ca2+]i by augmenting ROS signaling from the mitochondria.

Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism.

This study investigated the role of intracellular free Ca2+ concentration ([Ca2+]i) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), caused a time- and concentration-dependent decrease in the viability of MIN6 cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic of apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+]i by other mechanisms did not induce apoptosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellular Ca2+ stores were completely depleted in cells treated by SERCA inhibitors, but not by other agents that increase [Ca2+]i. The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ influx or by clamping [Ca2+]i with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+]i per se. However, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic acid and nordihydroguaiaretic acid partially prevented MIN6 cell apoptosis, while exposure of cells to the product of lipoxygenase, 12-hydroxy-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, inhibition of cyclooxygenase with indomethacin did not abolish thapsigargin-induced apoptosis in MIN6 cells. Our findings indicate that thapsigargin causes apoptosis in MIN6 cells by depleting intracellular Ca2+ stores and leading to release of intermediate metabolites of arachidonic acid metabolism.

EXPRESSION AND FUNCTION OF PANCREATIC BETA -CELL DELAYED RECTIFIER K+ CHANNELS : ROLE IN STIMULUS-SECRETION COUPLING

Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (βTC3-neo) and β-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in βTC3-neo insulinoma cells and purified rodent pancreatic β-cells was detected and confirmed by immunoblotting in the insulinoma cells. βTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on βTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized β-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.

A cAMP and Ca2+ coincidence detector in support of Ca2+-induced Ca2+ release in mouse pancreatic β cells

The blood glucose‐lowering hormone glucagon‐like peptide‐1 (GLP‐1) stimulates cAMP production, promotes Ca2+ influx, and mobilizes an intracellular source of Ca2+ in pancreatic β cells. Here we provide evidence that these actions of GLP‐1 are functionally related: they reflect a process of Ca2+‐induced Ca2+ release (CICR) that requires activation of protein kinase A (PKA) and the Epac family of cAMP‐regulated guanine nucleotide exchange factors (cAMPGEFs). In rat insulin‐secreting INS‐1 cells or mouse β cells loaded with caged Ca2+ (NP‐EGTA), a GLP‐1 receptor agonist (exendin‐4) is demonstrated to sensitize intracellular Ca2+ release channels to stimulatory effects of cytosolic Ca2+, thereby allowing CICR to be generated by the uncaging of Ca2+ (UV flash photolysis). This sensitizing action of exendin‐4 is diminished by an inhibitor of PKA (H‐89) or by overexpression of dominant negative Epac. It is reproduced by cell‐permeant cAMP analogues that activate PKA (6‐Bnz‐cAMP) or Epac (8‐pCPT‐2′‐O‐Me‐cAMP) selectively. Depletion of Ca2+ stores with thapsigargin abolishes CICR, while inhibitors of Ca2+ release channels (ryanodine and heparin) attenuate CICR in an additive manner. Because the uncaging of Ca2+ fails to stimulate CICR in the absence of cAMP‐elevating agents, it is concluded that there exists in β cells a process of second messenger coincidence detection, whereby intracellular Ca2+ release channels (ryanodine receptors, inositol 1,4,5‐trisphosphate (IP3) receptors) monitor a simultaneous increase of cAMP and Ca2+ concentrations. We propose that second messenger coincidence detection of this type may explain how GLP‐1 interacts with β cell glucose metabolism to stimulate insulin secretion.

Store-Operated Ca2+ Entry Activates the CREB Transcription Factor in Vascular Smooth Muscle

Ca2+-regulated gene transcription is a critical component of arterial responses to injury, hypertension, and tumor-stimulated angiogenesis. The Ca2+/cAMP response element binding protein (CREB), a transcription factor that regulates expression of many genes, is activated by Ca2+-induced phosphorylation. Multiple Ca2+ entry pathways may contribute to CREB activation in vascular smooth muscle including voltage-dependent Ca2+ channels and store-operated Ca2+ entry (SOCE). To investigate a role for SOCE in CREB activation, we measured CREB phosphorylation using immunofluorescence, intracellular Ca2+ levels using a fluorescence resonance energy transfer (FRET)–based Cameleon indicator, and c-fos transcription using RT-PCR. In this study, we report that SOCE activates CREB in both cultured smooth muscle cells and intact arteries. Depletion of intracellular Ca2+ stores with thapsigargin increased nuclear phospho-CREB levels, intracellular Ca2+ concentration, and transcription of c-fos. These effects were abolished by inhibiting SOCE through lowering extracellular Ca2+ concentration or by application of 2-aminoethoxydiphenylborate and Ni2+. Inhibition of Ca2+ influx through voltage-dependent Ca2+ channels using nimodipine partially blocked intact artery responses, but was without effect in cultured smooth muscle cells. Our findings indicate that Ca2+ entry through store-operated Ca2+ channels leads to CREB activation, suggesting that SOCE contributes to the regulation of gene expression in vascular smooth muscle.

Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans

Glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2’-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2’-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2’-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of β-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.