Evaluation of cerebrospinal fluid heparan sulfate as a biomarker of neuropathology in a murine model of mucopolysaccharidosis type II using high-sensitivity LC/MS/MS.

Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes glycosaminoglycans (GAGs) including heparan sulfate (HS) and dermatan sulfate (DS). GAG accumulation leads to severe neurological and somatic impairments. At present, the most common treatment for MPS II is intravenous enzyme replacement therapy; however, the inability of recombinant IDS to cross the blood-brain barrier (BBB) restricts therapeutic efficacy for neurological manifestations. We recently developed a BBB-penetrating IDS fusion protein, JR-141, and demonstrated its ability to reduce GAG accumulation in the brain of human transferrin receptor knock-in and Ids knock-out mice (TFRC-KI/Ids-KO), an animal model of MPS II, following intravenous administration. Given the impossibility of measuring GAG accumulation in the brains of human patients with MPS II, we hypothesized that GAG content in the cerebrospinal fluid (CSF) might serve as an indicator of brain GAG burden. To test this hypothesis, we optimized a high-sensitivity method for quantifying HS and DS in low-volume samples by combining acidic methanolysis and liquid chromatography-tandem mass spectrometry (LC/MS/MS). We employed this method to quantify HS and DS in samples from TFRC-KI/Ids-KO mice and revealed that HS but not DS accumulated in the central nerve system (CNS). Moreover, concentrations of HS in CSF correlated with those in brain. Finally, intravenous treatment with JR-141 reduced levels of HS in the CSF and brain in TFRC-KI/Ids-KO mice. These results suggest that CSF HS content may be a useful biomarker for evaluating the brain GAG accumulation and the therapeutic efficacy of drugs in patients with MPS II.

Structure and functional roles of Epac2 (Rapgef4).

Epac (exchange protein activated by cyclic-AMP) 2 is a direct target of 3'-5'-cyclic adenosine monophosphate (cAMP) and is involved in cAMP-mediated signal transduction through activation of the Ras-like small GTPase Rap. Crystallographic analyses revealed that activation of Epac2 by cAMP is accompanied by dynamic structural changes. Epac2 is expressed mainly in brain, neuroendocrine and endocrine tissues, and is involved in diverse cellular functions in the tissues. In this review, we summarize the structure and function of Epac2. We also discuss the physiological and pathophysiological roles of Epac2, and the possibility of Epac2 as a therapeutic target.

Role of Epac2A/Rap1 signaling in interplay between incretin and sulfonylurea in insulin secretion.

Incretin-related drugs and sulfonylureas are currently used worldwide for the treatment of type 2 diabetes. We recently found that Epac2A, a cAMP binding protein having guanine nucleotide exchange activity toward Rap, is a target of both incretin and sulfonylurea. This suggests the possibility of interplay between incretin and sulfonylurea through Epac2A/Rap1 signaling in insulin secretion. In this study, we examined the combinatorial effects of incretin and various sulfonylureas on insulin secretion and activation of Epac2A/Rap1 signaling. A strong augmentation of insulin secretion by combination of GLP-1 and glibenclamide or glimepiride, which was found in Epac2A(+/+) mice, was markedly reduced in Epac2A(-/-) mice. In contrast, the combinatorial effect of GLP-1 and gliclazide was rather mild, and the effect was not altered by Epac2A ablation. Activation of Rap1 was enhanced by the combination of an Epac-selective cAMP analog with glibenclamide or glimepiride but not gliclazide. In diet-induced obese mice, ablation of Epac2A reduced the insulin secretory response to coadministration of the GLP-1 receptor agonist liraglutide and glimepiride. These findings clarify the critical role of Epac2A/Rap1 signaling in the augmenting effect of incretin and sulfonylurea on insulin secretion and provide the basis for the effects of combination therapies of incretin-related drugs and sulfonylureas.

Glutamate acts as a key signal linking glucose metabolism to incretin/cAMP action to amplify insulin secretion.

Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting ß cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.

Antidiabetic sulfonylureas and cAMP cooperatively activate Epac2A.

Sulfonylureas are widely used drugs for treating insulin deficiency in patients with type 2 diabetes. Sulfonylureas bind to the regulatory subunit of the pancreatic ß cell potassium channel that controls insulin secretion. Sulfonylureas also bind to and activate Epac2A, a member of the Epac family of cyclic adenosine monophosphate (cAMP)-binding proteins that promote insulin secretion through activation of the Ras-like guanosine triphosphatase Rap1. Using molecular docking simulation, we identified amino acid residues in one of two cyclic nucleotide-binding domains, cNBD-A, in Epac2A predicted to mediate the interaction with sulfonylureas. We confirmed the importance of the identified residues by site-directed mutagenesis and analysis of the response of the mutants to sulfonylureas using two assays: changes in fluorescence resonance energy transfer (FRET) of an Epac2A-FRET biosensor and direct sulfonylurea-binding experiments. These residues were also required for the sulfonylurea-dependent Rap1 activation by Epac2A. Binding of sulfonylureas to Epac2A depended on the concentration of cAMP and the structures of the drugs. Sulfonylureas and cAMP cooperatively activated Epac2A through binding to cNBD-A and cNBD-B, respectively. Our data suggest that sulfonylureas stabilize Epac2A in its open, active state and provide insight for the development of drugs that target Epac2A.

Actin dynamics regulated by the balance of neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin activities determines the biphasic response of glucose-induced insulin secretion.

Actin dynamics in pancreatic ß-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic ß-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic ß-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic ß-cells (MIN6-K8 ß-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 ß-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 ß-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.

GLP-1 receptor activation and Epac2 link atrial natriuretic peptide secretion to control of blood pressure.

Glucagon-like peptide-1 receptor (GLP-1R) agonists exert antihypertensive actions through incompletely understood mechanisms. Here we demonstrate that cardiac Glp1r expression is localized to cardiac atria and that GLP-1R activation promotes the secretion of atrial natriuretic peptide (ANP) and a reduction of blood pressure. Consistent with an indirect ANP-dependent mechanism for the antihypertensive effects of GLP-1R activation, the GLP-1R agonist liraglutide did not directly increase the amount of cyclic GMP (cGMP) or relax preconstricted aortic rings; however, conditioned medium from liraglutide-treated hearts relaxed aortic rings in an endothelium-independent, GLP-1R-dependent manner. Liraglutide did not induce ANP secretion, vasorelaxation or lower blood pressure in Glp1r(-/-) or Nppa(-/-) mice. Cardiomyocyte GLP-1R activation promoted the translocation of the Rap guanine nucleotide exchange factor Epac2 (also known as Rapgef4) to the membrane, whereas Epac2 deficiency eliminated GLP-1R-dependent stimulation of ANP secretion. Plasma ANP concentrations were increased after refeeding in wild-type but not Glp1r(-/-) mice, and liraglutide increased urine sodium excretion in wild-type but not Nppa(-/-) mice. These findings define a gut-heart GLP-1R-dependent and ANP-dependent axis that regulates blood pressure.

Dynamics of insulin secretion and the clinical implications for obesity and diabetes.

Insulin secretion is a highly dynamic process regulated by various factors including nutrients, hormones, and neuronal inputs. The dynamics of insulin secretion can be studied at different levels: the single ß cell, pancreatic islet, whole pancreas, and the intact organism. Studies have begun to analyze cellular and molecular mechanisms underlying dynamics of insulin secretion. This review focuses on our current understanding of the dynamics of insulin secretion in vitro and in vivo and discusses their clinical relevance.

[Regulatory mechanism of incretin secretion].

Incretin hormones GIP(glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1) improve glycemic control by potentiating glucose-induced insulin secretion in pancreatic beta-cells and also have beneficial effects on appetite control and body weight. In response to food ingestion, GIP and GLP-1 are secreted from enteroendocrine K- and L-cells, respectively. In these cells, it is shown that a variety of molecular sensors are involved in the detection of carbohydrates, lipids, and proteins. In view of development of new incretin-related drugs, these sensors are attractive targets to enhance the endogenous pools of incretins.

Rim2alpha determines docking and priming states in insulin granule exocytosis.

Insulin secretion is essential for maintenance of glucose homeostasis, but the mechanism of insulin granule exocytosis, the final step of insulin secretion, is largely unknown. Here, we investigated the role of Rim2alpha in insulin granule exocytosis, including the docking, priming, and fusion steps. We found that interaction of Rim2alpha and Rab3A is required for docking, which is considered a brake on fusion events, and that docking is necessary for K(+)-induced exocytosis, but not for glucose-induced exocytosis. Furthermore, we found that dissociation of the Rim2alpha/Munc13-1 complex by glucose stimulation activates Syntaxin1 by Munc13-1, indicating that Rim2alpha primes insulin granules for fusion. Thus, Rim2alpha determines docking and priming states in insulin granule exocytosis depending on its interacting partner, Rab3A or Munc13-1, respectively. Because Rim2alpha(-/-) mice exhibit impaired secretion of various hormones stored as dense-core granules, including glucose-dependent insulinotropic polypeptide, growth hormone, and epinephrine, Rim2alpha plays a critical role in exocytosis of these dense-core granules.

Pancreatic beta-cell signaling: toward better understanding of diabetes and its treatment.

Pancreatic beta-cells play a central role in the maintenance glucose homeostasis by secreting insulin, a key hormone that regulates blood glucose levels. Dysfunction of the beta-cells and/or a decrease in the beta-cell mass are associated closely with the pathogenesis and pathophysiology of diabetes mellitus, a major metabolic disease that is rapidly increasing worldwide. Clarification of the mechanisms of insulin secretion and beta-cell fate provides a basis for the understanding of diabetes and its better treatment. In this review, we discuss cell signaling critical for the insulin secretory function based on our recent studies.

GLP-1 inhibits and adrenaline stimulates glucagon release by differential modulation of N- and L-type Ca2+ channel-dependent exocytosis.

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).

Sulfonylurea action re-revisited.

Sulfonylureas (SU), commonly used in the treatment of type 2 diabetes mellitus (T2DM), stimulate insulin secretion by inhibiting adenosine triphosphate (ATP)-sensitive K(+) (KATP) channels in pancreatic ß-cells. SU are now known to also activate cyclic adenosine monophosphate (cAMP) sensor Epac2 (cAMP-GEFII) to Rap1 signaling, which promotes insulin secretion. The different effects of various SU on Epac2/Rap1 signaling, as well as KATP channels in different tissues, underlie the diverse pancreatic and extra-pancreatic actions of SU. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00014.x, 2010).

Establishment of new clonal pancreatic ß-cell lines (MIN6-K) useful for study of incretin/cyclic adenosine monophosphate signaling.

Incretin/cyclic adenosine monophosphate (cAMP) signaling is critical for potentiation of insulin secretion. Although several cell lines of pancreatic ß-cells are currently available, there are no cell lines suitable for investigation of incretin/cAMP signaling. In the present study, we have newly established pancreatic ß-cell lines (named MIN6-K) from the IT6 mouse, which develops insulinoma. MIN6-K8 cells respond to both glucose and incretins, such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), as is the case in pancreatic islets, whereas MIN6-K20 cells respond to glucose, but not to incretins. Despite the difference in incretin-potentiated insulin secretion between these two cell lines, the accumulation of cAMP after stimulation of GLP-1 is comparable in these cells. Interestingly, we also found that incretin responsiveness is drastically induced by the formation of pseudoislets from MIN6-K20 cells to a level comparable to that of pancreatic islets. Thus, these cell lines are useful for studying incretin/cAMP signaling in ß-cells. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00026.x, 2010).

The cAMP sensor Epac2 is a direct target of antidiabetic sulfonylurea drugs.

Epac2, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rap1, is activated by adenosine 3',5'-monophosphate. Fluorescence resonance energy transfer and binding experiments revealed that sulfonylureas, widely used antidiabetic drugs, interact directly with Epac2. Sulfonylureas activated Rap1 specifically through Epac2. Sulfonylurea-stimulated insulin secretion was reduced both in vitro and in vivo in mice lacking Epac2, and the glucose-lowering effect of the sulfonylurea tolbutamide was decreased in these mice. Epac2 thus contributes to the effect of sulfonylureas to promote insulin secretion. Because Epac2 is also required for the action of incretins, gut hormones crucial for potentiating insulin secretion, it may be a promising target for antidiabetic drug development.

Rab11 and its effector Rip11 participate in regulation of insulin granule exocytosis.

Rab GTPases and their effectors play important roles in membrane trafficking between cellular compartments in eukaryotic cells. In the present study, we examined the roles of Rab11B and its effectors in insulin secretion in pancreatic beta-cells. In the mouse insulin-secreting cell line MIN6, Rab11 was co-localized with insulin-containing granules, and over-expression of the GTP- or the GDP-bound form of Rab11B significantly inhibited regulated secretion, indicating involvement of Rab11B in regulated insulin secretion. To determine the downstream signal of Rab11-mediated insulin secretion, we examined the effects of various Rab11-interacting proteins on insulin secretion, and found that Rip11 is involved in cAMP-potentiated insulin secretion but not in glucose-induced insulin secretion. Analyses by immunocytochemistry and subcellular fractionation revealed Rip11 to be co-localized with insulin granules. The inhibitory effect of the Rip11 mutant was not altered in MIN6 cells lacking Epac2, which mediates protein kinase A (PKA)-independent potentiation of insulin secretion, compared with wild-type MIN6 cells. In addition, Rip11 was found to be phosphorylated by PKA in MIN6 cells. The present study shows that both Rab11 and its effector Rip11 participate in insulin granule exocytosis and that Rip11, as a substrate of PKA, regulates the potentiation of exocytosis by cAMP in pancreatic beta-cells.

Critical role of the N-terminal cyclic AMP-binding domain of Epac2 in its subcellular localization and function.

cAMP is a well-known regulator of exocytosis, and cAMP-GEFII (Epac2) is involved in the potentiation of cAMP-dependent, PKA-independent regulated exocytosis in secretory cells. However, the mechanisms of its action are not fully understood. In the course of our study of Epac2 knockout mice, we identified a novel splicing variant of Epac2, which we designate Epac2B, while renaming the previously identified Epac2 Epac2A. Epac2B, which lacks the first cAMP-binding domain A in the N-terminus but has the second cAMP-binding domain B of Epac2A, possesses GEF activity towards Rap1, as was found for Epac2A. Immunocytochemical analysis revealed that exogenously introduced Epac2A into insulin-secreting MIN6 cells was localized near the plasma membrane, while Epac2B was found primarily in the cytoplasm. Interestingly, cAMP-binding domain A alone introduced into MIN6 cells was also localized near the plasma membrane. In MIN6 cells, Epac2A was involved in triggering hormone secretion by stimulation with 5.6 mM glucose plus 1 mM 8-Bromo-cAMP, but Epac2B was not. The addition of a membrane-targeting signal to the N-terminus of Epac2B was able to mimic the effect of Epac2A on hormone secretion. Thus, the present study indicates that the N-terminal cAMP-binding domain A of Epac2A plays a critical role in determining its subcellular localization and potentiating insulin secretion by cAMP. J. Cell. Physiol. 219: 652-658, 2009. (c) 2009 Wiley-Liss, Inc.

Involvement of Exoc3l, a protein structurally related to the exocyst subunit Sec6, in insulin secretion.

The exocyst is an octameric complex involved in docking or tethering of secretory vesicles to fusion sites of the plasma membrane. Sec6 is the core subunit of the exocyst complex. Here we identify an isoform of Sec6, deposited as Exocyst complex component 3-like (Exoc3l) in the database, by in silico screening using rat Sec6 as a probe. The amino acid sequence of Exoc3l has 31% identity and 53% similarity with that of Sec6. RT-PCR analysis reveals that Exoc3l is expressed in insulin-secreting MIN6 cells as well as in various tissues including pancreatic islets and brain. In co-immunoprecipitation experiments, Exoc3l was found to interact with Sec5, Sec8, and Sec10, all of which are binding partners of Sec6 in the exocyst complex. Furthermore, overexpression of a deletion mutant of Exoc3l in MIN6 cells suppressed glucose-stimulated secretion. These results suggest that Exoc3l is involved in regulated exocytosis of insulin granules.

Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP.

cAMP is well known to regulate exocytosis in various secretory cells, but the precise mechanism of its action remains unknown. Here, we examine the role of cAMP signaling in the exocytotic process of insulin granules in pancreatic beta cells. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucose-induced fusion events. Interestingly, all granules responsible for this potentiation are newly recruited and immediately fused to the plasma membrane without docking (restless newcomer). Importantly, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in mice lacking the cAMP-binding protein Epac2 (Epac2(ko/ko)). In addition, the small GTPase Rap1, which is activated by cAMP specifically through Epac2 in pancreatic beta cells, mediates cAMP-induced insulin secretion in a protein kinase A-independent manner. We also have developed a simulation model of insulin granule movement in which potentiation of the first phase is associated with an increase in the insulin granule density near the plasma membrane. Taken together, these data indicate that Epac2/Rap1 signaling is essential in regulation of insulin granule dynamics by cAMP, most likely by controlling granule density near the plasma membrane.