George G. Holz

Leptin Suppression of Insulin Secretion by the Activation of ATP-Sensitive K+ Channels in Pancreatic β-Cells

In the genetic mutant mouse models ob/ob or db/db, leptin deficiency or resistance, respectively, results in severe obesity and the development of a syndrome resembling NIDDM. One of the earliest manifestations in these mutant mice is hyperinsulinemia, suggesting that leptin may normally directly suppress the secretion of insulin. Here, we show that pancreatic islets express a long (signal-transducing) form of leptin-receptor mRNA and that β-cells bind a fluorescent derivative of leptin (Cy3-leptin). The expression of leptin receptors on insulin-secreting β-cells was also visualized utilizing antisera generated against an extracellular epitope of the receptor. A functional role for the β-cell leptin receptor is indicated by our observation that leptin (100 ng/ml) suppressed the secretion of insulin from islets isolated from ob/ob mice. Furthermore, leptin produced a marked lowering of ]Ca2+]i in ob/ob β-cells, which was accompanied by cellular hyperpolarization and increased membrane conductance. Cell-attached patch measurements of ob/ob β-Cells demonstrated that leptin activated ATP-sensitive potassium channels (KATP) by increasing the open channel probability, while exerting no effect on mean open time. These effects were reversed by the sulfonylurea tolbutamide, a specific inhibitor of KATP. Taken together, these observations indicate an important physiological role for leptin as an inhibitor of insulin secretion and lead us to propose that the failure of leptin to inhibit insulin secretion from the β-Cells of ob/ob and db/db mice may explain, in part, the development of hyperinsulinemia, insulin resistance, and the progression to NIDDM.

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.

cAMP-dependent Mobilization of Intracellular Ca2+ Stores by Activation of Ryanodine Receptors in Pancreatic β-Cells A Ca2+ SIGNALING SYSTEM STIMULATED BY THE INSULINOTROPIC HORMONE GLUCAGON-LIKE PEPTIDE-1-(7–37)

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes mellitus. In vitro studies of pancreatic islets of Langerhans demonstrated that GLP-1 interacts with specific β-cell G protein-coupled receptors, thereby facilitating insulin exocytosis by raising intracellular levels of cAMP and Ca2+. Here we report that the stimulatory influence of GLP-1 on Ca2+ signaling results, in part, from cAMP-dependent mobilization of ryanodine-sensitive Ca2+ stores. Studies of human, rat, and mouse β-cells demonstrate that the binding of a fluorescent derivative of ryanodine (BODIPY FL-X ryanodine) to its receptors is specific, reversible, and of high affinity. Rat islets and BTC3 insulinoma cells are shown by reverse transcriptase polymerase chain reaction analyses to express mRNA corresponding to the type 2 isoform of ryanodine receptor-intracellular Ca2+ release channel (RYR2). Single-cell measurements of [Ca2+] i using primary cultures of rat and human β-cells indicate that GLP-1 facilitates Ca2+-induced Ca2+ release (CICR), whereby mobilization of Ca2+ stores is triggered by influx of Ca2+ through l-type Ca2+ channels. In these cells, GLP-1 is shown to interact with metabolism ofd-glucose to produce a fast transient increase of [Ca2+] i . This effect is reproduced by 8-Br-cAMP, but is blocked by a GLP-1 receptor antagonist (exendin-(9–39)), a cAMP antagonist ((Rp)-cAMPS), anl-type Ca2+ channel antagonist (nimodipine), an antagonist of the sarco(endo)plasmic reticulum Ca2+ ATPase (thapsigargin), or by ryanodine. Characterization of the CICR mechanism by voltage clamp analysis also demonstrates a stimulation of Ca2+ release by caffeine. These findings provide new support for a model of β-cell signal transduction whereby GLP-1 promotes CICR by sensitizing intracellular Ca2+ release channels to the stimulatory influence of cytosolic Ca2+.

Dihydropyridine inhibition of neuronal calcium current and substance P release

Dihydropyridine (DHP) calcium channel antagonists, which inhibit the slowly inactivating or L-type cardiac calcium (Ca) current, have been shown to be ineffective in blocking45Ca influx and Ca-dependent secretion in a number of neuronal preparations. In the studies reported here, however, the antagonist DHP nifedipine inhibited both the L-type Ca current and potassium-evoked substance P (SP) release from embryonic chick dorsal root ganglion (DRG) neurons. These results suggest that, in DRG neurons. Ca entry through L-type channels is critical to the control of secretion. The inhibition of Ca current by nifedipine was both voltage and time-dependent, significant effects being observed only on currents evoked from relatively positive holding potentials maintained for several seconds. As expected from these results, nifedipine failed to inhibit L-type Ca current underlying the brief plateau phase of the action potential generated from the cells normal resting potential; likewise, no significant effect of the drug was observed on action potential-stimulated SP release evoked by electrical field stimulation. The results of this work are discussed in terms of an assessment of the role of L-type Ca channels in neurosecretion.

cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic β-cells

1 The signal transduction pathway responsible for cAMP‐dependent Ca2+‐induced Ca2+ release (CICR) from endoplasmic reticulum Ca2+ stores was assessed in the insulin‐secreting cell line INS‐1. 2 CICR was triggered by the GLP‐1 receptor agonist exendin‐4, an effect mimicked by caffeine, Sp‐cAMPS or forskolin. CICR required influx of Ca2+ through L‐type voltage‐dependent Ca2+ channels, and was blocked by treatment with nimodipine, thapsigargin, or ryanodine, but not by the IP3 receptor antagonist xestospongin C. 3 Treatment with the cAMP antagonist 8‐Br‐Rp‐cAMPS blocked CICR in response to exendin‐4, whereas the PKA inhibitor H‐89 was ineffective when tested at a concentration demonstrated to inhibit PKA‐dependent gene expression. 4 RT‐PCR of INS‐1 cells demonstrated expression of mRNA coding for the type‐II isoform of cAMP‐regulated guanine nucleotide exchange factor (cAMP‐GEF‐II, Epac2). 5 CICR in response to forskolin was blocked by transient transfection and expression of a dominant negative mutant isoform of cAMP‐GEF‐II in which inactivating mutations were introduced into the exchange factors two cAMP‐binding domains. 6 It is concluded that CICR in INS‐1 cells results from GLP‐1 receptor‐mediated sensitization of the intracellular Ca2+ release mechanism, a signal transduction pathway independent of PKA, but which requires cAMP‐GEF‐II.

G proteins as regulators of ion channel function

Virtually unknown just a decade ago, GTP-binding proteins (G proteins) have become a major focus of current research. This family of closely related proteins transduce extracellular signals (such as hormones, neurotransmitters and sensory stimuli) into effector responses (1,2). It is now evident that ion channel permeability is one such effector response. In fact, the striking increase in the frequency of reports that demonstrate G protein-regulated ion channel function suggests that channels whose permeability mechanism can be altered by a G protein-mediated process may be more the rule than the exception. It is well-known that the cAMP-dependent modulation of ion channels is under the control of G proteins that regulate adenylate cyclase activity(3,4). However recent studies demonstrate that G proteins also transduce agonist-induced changes in channel activity that do not involve adenylate cyclase. It is on this aspect of G protein signal transduction that this review will focus.

Glucagon-like peptide-1 synthetic analogs: new therapeutic agents for use in the treatment of diabetes mellitus.

Glucagon-like peptide-1-(7-36)-amide (GLP-1) is a potent blood glucose-lowering hormone now under investigation for use as a therapeutic agent in the treatment of type 2 (adult onset) diabetes mellitus. GLP-1 binds with high affinity to G protein-coupled receptors (GPCRs) located on pancreatic beta-cells, and it exerts insulinotropic actions that include the stimulation of insulin gene transcription, insulin biosynthesis, and insulin secretion. The beneficial therapeutic action of GLP-1 also includes its ability to act as a growth factor, stimulating formation of new pancreatic islets (neogenesis) while slowing beta-cell death (apoptosis). GLP-1 belongs to a large family of structurally-related hormones and neuropeptides that include glucagon, secretin, GIP, PACAP, and VIP. Biosynthesis of GLP-1 occurs in the enteroendocrine L-cells of the distal intestine, and the release of GLP-1 into the systemic circulation accompanies ingestion of a meal. Although GLP-1 is inactivated rapidly by dipeptidyl peptidase IV (DDP-IV), synthetic analogs of GLP-1 exist, and efforts have been directed at engineering these peptides so that they are resistant to enzymatic hydrolysis. Additional modifications of GLP-1 incorporate fatty acylation and drug affinity complex (DAC) technology to improve serum albumin binding, thereby slowing renal clearance of the peptides. NN2211, LY315902, LY307161, and CJC-1131 are GLP-1 synthetic analogs that reproduce many of the biological actions of GLP-1, but with a prolonged duration of action. AC2993 (Exendin-4) is a naturally occurring peptide isolated from the lizard Heloderma, and it acts as a high affinity agonist at the GLP-1 receptor. This review summarizes structural features and signal transduction properties of GLP-1 and its cognate beta-cell GPCR. The usefulness of synthetic GLP-1 analogs as blood glucose-lowering agents is discussed, and the applicability of GLP-1 as a therapeutic agent for treatment of type 2 diabetes is highlighted.

cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic β cells and rat INS-1 cells

The Epac family of cAMP‐regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic β cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide‐binding fold‐1 (NBF‐1) of the β‐cell sulphonylurea receptor‐1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit β‐cell ATP‐sensitive K+ channels (KATP channels; a hetero‐octomer of SUR1 and Kir6.2). If so, Epac‐mediated inhibition of KATP channels might explain prior reports that cAMP‐elevating agents promote β‐cell depolarization, Ca2+ influx and insulin secretion. Here we report that Epac‐selective cAMP analogues (2′‐O‐Me‐cAMP; 8‐pCPT‐2′‐O‐Me‐cAMP; 8‐pMeOPT‐2′‐O‐Me‐cAMP), but not a cGMP analogue (2′‐O‐Me‐cGMP), inhibit the function of KATP channels in human β cells and rat INS‐1 insulin‐secreting cells. Inhibition of KATP channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6‐Bnz‐cAMP, a cAMP analogue that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac‐selective cAMP analogues at KATP channels are mimicked by a cAMP agonist (8‐Bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Sp‐isomer, Sp‐8‐Br‐cAMPS), but not a cAMP antagonist (8‐Bromoadenosine‐3′, 5′‐cyclic monophosphorothioate, Rp‐isomer, Rp‐8‐Br‐cAMPS), and are abrogated following transfection of INS‐1 cells with a dominant‐negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 coimmunoprecipitate with full‐length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of KATP channel activity in pancreatic β cells.

Signal transduction crosstalk in the endocrine system: pancreatic β-cells and the glucose competence concept

Crosstalk between intracellular signalling systems is recognized as the principal means by which a cell orchestrates coordinate responses to stimulation by neurotransmitters, hormones or growth factors. The functional consequences of crosstalk are evident at multiple levels within a given signalling cascade, including the regulation of receptor-ligand interactions, guanine nucleotide-binding proteins, enzyme activities, ion channel function and gene expression. Here we focus on the pancreatic beta-cells of the islets of Langerhans to illustrate the important role crosstalk plays in the regulation of glucose-induced insulin secretion. Recent studies indicating a synergistic interaction in beta-cells between the glucose-regulated ATP-dependent signalling system and the hormonally regulated cAMP-dependent signalling system are emphasized. This interaction gives beta-cells the ability to match the ambient concentration of glucose to an appropriate insulin secretory response, a process we refer to as the induction of glucose competence. The glucose competence concept may provide new insights into the etiology and treatment of non-insulin-dependent diabetes mellitus (Type II diabetes).

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.