Robert G. Tsushima

Nesfatin-1 exerts a direct, glucose-dependent insulinotropic action on mouse islet β- and MIN6 cells

Nesfatin-1 is a recently discovered multifunctional metabolic hormone abundantly expressed in the pancreatic islets. The main objective of this study is to characterize the direct effects of nesfatin-1 on insulin secretion in vitro using MIN6 cells and islets isolated from C57BL/6 mice. We also examined the expression of the nesfatin-1 precursor protein, nucleobindin 2 (NUCB2) mRNA, and nesfatin-1 immunoreactivity (ir) in the islets of normal mice and in the islets from mice with streptozotocin-induced type 1 diabetes and diet-induced obese (DIO) mice with type 2 diabetes. Nesfatin-1 stimulated glucose-induced insulin release in vitro from mouse islets and MIN6 cells in a dose-dependent manner. No such stimulation in insulin secretion was found when MIN6 cells/islets were incubated with nesfatin-1 in low glucose. In addition, a fourfold increase in nesfatin-1 release from MIN6 cells was observed following incubation in high glucose (16.7  mM) compared to low glucose (2  mM). Furthermore, we observed a significant reduction in both NUCB2 mRNA expression and nesfatin-1-ir in the pancreatic islets of mice with type 1 diabetes, while a significant increase was observed in the islets of DIO mice. Together, our findings indicate that nesfatin-1 is a novel insulinotropic peptide and that the endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity.

Nutrient Responsive Nesfatin-1 Regulates Energy Balance and Induces Glucose-Stimulated Insulin Secretion in Rats

Nesfatin-1 is a recently discovered anorexigen, and we first reported nesfatin-like immunoreactivity in the pancreatic β-cells. The aim of this study was to characterize the effects of nesfatin-1 on whole-body energy homeostasis, insulin secretion, and glycemia. The in vivo effects of continuous peripheral delivery of nesfatin-1 using osmotic minipumps on food intake and substrate partitioning were examined in ad libitum-fed male Fischer 344 rats. The effects of nesfatin-1 on glucose-stimulated insulin secretion (GSIS) were examined in isolated pancreatic islets. L6 skeletal muscle cells and isolated rat adipocytes were used to assess the effects of nesfatin-1 on basal and insulin-mediated glucose uptake as well as on major steps of insulin signaling in these cells. Nesfatin-1 reduced cumulative food intake and increased spontaneous physical activity, whole-body fat oxidation, and carnitine palmitoyltransferase I mRNA expression in brown adipose tissue but did not affect uncoupling protein 1 mRNA in the brown adipose tissue. Nesfatin-1 significantly enhanced GSIS in vivo during an oral glucose tolerance test and improved insulin sensitivity. Although insulin-stimulated glucose uptake in L6 muscle cells was inhibited by nesfatin-1 pretreatment, basal and insulin-induced glucose uptake in adipocytes from nesfatin-1-treated rats was significantly increased. In agreement with our in vivo results, nesfatin-1 enhanced GSIS from isolated pancreatic islets at both normal (5.6 mM) and high (16.7 mM), but not at low (2 mM), glucose concentrations. Furthermore, nesfatin-1/nucleobindin 2 release from rat pancreatic islets was stimulated by glucose. Collectively, our data indicate that glucose-responsive nesfatin-1 regulates insulin secretion, glucose homeostasis, and whole-body energy balance in rats.

Phosphatidylinositol 3-Kinase γ Is a Critical Mediator of Myocardial Ischemic and Adenosine-Mediated Preconditioning

Ischemic preconditioning (IPC) is a potent cellular protective mechanism whereby brief periods of sublethal ischemia protect the myocardium from prolonged ischemia-induced injury. We demonstrate the selective role of phosphatidylinositol 3-kinase (PI3K) isoforms in IPC. Hearts from PI3K&ggr; knockout mice (PI3K&ggr;−/−) displayed poorer functional recovery and greater tissue injury following IPC compared to wild-type and PI3K&ggr;+/− hearts. Examination of the cell-signaling pathways revealed restored phosphorylation levels of Akt and glycogen synthase kinase (GSK)3&bgr; in wild-type hearts, which were abolished in PI3K&ggr;−/− hearts subjected to IPC. Inhibition of GSK3&bgr; by LiCl reversed the loss in protection in PI3K&ggr;−/− hearts. In contrast, mice expressing a cardiac-specific kinase-deleted PI3K&agr; (PI3K&agr;DN) were resistant to injury induced by 30 minutes of ischemia followed by 40 minutes of reperfusion. Furthermore, the resistance of PI3K&agr;DN hearts to ischemia/reperfusion correlated with the persistent expression of p110&ggr; and was blocked by the PI3K inhibitor wortmannin, suggesting the possible enhanced cell signaling through the PI3K&ggr; pathway. These results demonstrate the importance of the PI3K&ggr;-Akt-GSK3&bgr; signaling pathway in IPC. Selective activation of myocardial PI3K&ggr; may be an attractive target for the treatment of ischemic heart disease.

Role of phosphoinositide 3-kinase {alpha}, protein kinase C, and L-type Ca2+ channels in mediating the complex actions of angiotensin II on mouse cardiac contractility.

Although angiotensin II (Ang II) plays an important role in heart disease associated with pump dysfunction, its direct effects on cardiac pump function remain controversial. We found that after Ang II infusion, the developed pressure and +dP/dtmax in isolated Langendorff-perfused mouse hearts showed a complex temporal response, with a rapid transient decrease followed by an increase above baseline. Similar time-dependent changes in cell shortening and L-type Ca2+ currents were observed in isolated ventricular myocytes. Previous studies have established that Ang II signaling involves phosphoinositide 3-kinases (PI3K). Dominant-negative inhibition of PI3K&agr; in the myocardium selectively eliminated the rapid negative inotropic action of Ang II (inhibited by ≈90%), whereas the loss of PI3K&ggr; had no effect on the response to Ang II. Consistent with a link between PI3K&agr; and protein kinase C (PKC), PKC inhibition (with GF 109203X) reduced the negative inotropic effects of Ang II by ≈50%. Although PI3K&agr; and PKC activities are associated with glycogen synthase kinase-3&bgr; and NADPH oxidase, genetic ablation of either glycogen synthase kinase-3&bgr; or p47phox (an essential subunit of NOX2-NADPH oxidase) had no effect on the inotropic actions of Ang II. Our results establish that Ang II has complex temporal effects on contractility and L-type Ca2+ channels in normal mouse myocardium, with the negative inotropic effects requiring PI3K&agr; and PKC activities.

Cross-talk between glycogen synthase kinase 3β (GSK3β) and p38MAPK regulates myocyte enhancer factor 2 (MEF2) activity in skeletal and cardiac muscle

Characterizing the signaling network that controls MEF2 transcription factors is crucial for understanding skeletal and cardiac muscle gene expression. Glycogen synthase kinase 3β (GSK3β) regulates MEF2 activity indirectly through reciprocal regulation of p38MAPK. Cross-talk between GSK3β and p38MAPK regulates MEF2 activity in skeletal and cardiac muscle. Understanding cross-talk in the signaling network converging at MEF2 control has therapeutic implications in cardiac and skeletal muscle pathology. Glycogen synthase kinase 3β (GSK3β) is a known regulator of striated muscle gene expression suppressing both myogenesis and cardiomyocyte hypertrophy. Since myocyte enhancer factor 2 (MEF2) proteins are key transcriptional regulators in both systems, we assessed whether MEF2 is a target for GSK3β. Pharmacological inhibition of GSK3β resulted in enhanced MEF2A/D expression and transcriptional activity in skeletal myoblasts and cardiac myocytes. Even though in silico analysis revealed GSK3β consensus (S/T)XXX(S/T) sites on MEF2A, a subsequent in vitro kinase assay revealed that MEF2A is only a weak substrate. However, we did observe a posttranslational modification in MEF2A in skeletal myoblasts treated with a GSK3β inhibitor which coincided with increased p38MAPK phosphorylation, a potent MEF2A activator, indicating that GSK3β inhibition may de-repress p38MAPK. Heart specific excision of GSK3β in mice also resulted in up-regulation of p38MAPK activity. Interestingly, upon pharmacological p38MAPK inhibition (SB203580), GSK3β inhibition loses its effect on MEF2 transcriptional activity suggesting potent cross-talk between the two pathways. Thus we have documented that cross-talk between p38MAPK and GSK3β signaling converges on MEF2 activity having potential consequences for therapeutic modulation of cardiac and skeletal muscle gene expression.

Exogenous Glucocorticoids and a High-Fat Diet Cause Severe Hyperglycemia and Hyperinsulinemia and Limit Islet Glucose Responsiveness in Young Male Sprague-Dawley Rats

Corticosterone (CORT) and other glucocorticoids cause peripheral insulin resistance and compensatory increases in β-cell mass. A prolonged high-fat diet (HFD) induces insulin resistance and impairs β-cell insulin secretion. This study examined islet adaptive capacity in rats treated with CORT and a HFD. Male Sprague-Dawley rats (age ∼6 weeks) were given exogenous CORT (400 mg/rat) or wax (placebo) implants and placed on a HFD (60% calories from fat) or standard diet (SD) for 2 weeks (N = 10 per group). CORT-HFD rats developed fasting hyperglycemia (>11 mM) and hyperinsulinemia (∼5-fold higher than controls) and were 15-fold more insulin resistant than placebo-SD rats by the end of ∼2 weeks (Homeostatic Model Assessment for Insulin Resistance [HOMA-IR] levels, 15.08 ± 1.64 vs 1.0 ± 0.12, P < .05). Pancreatic β-cell function, as measured by HOMA-β, was lower in the CORT-HFD group as compared to the CORT-SD group (1.64 ± 0.22 vs 3.72 ± 0.64, P < .001) as well as acute insulin response (0.25 ± 0.22 vs 1.68 ± 0.41, P < .05). Moreover, β- and α-cell mass were 2.6- and 1.6-fold higher, respectively, in CORT-HFD animals compared to controls (both P < .05). CORT treatment increased p-protein kinase C-α content in SD but not HFD-fed rats, suggesting that a HFD may lower insulin secretory capacity via impaired glucose sensing. Isolated islets from CORT-HFD animals secreted more insulin in both low and high glucose conditions; however, total insulin content was relatively depleted after glucose challenge. Thus, CORT and HFD, synergistically not independently, act to promote severe insulin resistance, which overwhelms islet adaptive capacity, thereby resulting in overt hyperglycemia.

Functional characterization of hyperpolarization-activated cyclic nucleotide-gated channels in rat pancreatic β cells

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate pacemaker activity in some cardiac cells and neurons. In the present study, we have identified the presence of HCN channels in pancreatic beta-cells. We then examined the functional characterization of these channels in beta-cells via modulating HCN channel activity genetically and pharmacologically. Voltage-clamp experiments showed that over-expression of HCN2 in rat beta-cells significantly increased HCN current (I(h)), whereas expression of dominant-negative HCN2 (HCN2-AYA) completely suppressed endogenous I(h). Compared to control beta-cells, over-expression of I(h) increased insulin secretion at 2.8 mmol/l glucose. However, suppression of I(h) did not affect insulin secretion at both 2.8 and 11.1 mmol/l glucose. Current-clamp measurements revealed that HCN2 over-expression significantly reduced beta-cell membrane input resistance (R(in)), and resulted in a less-hyperpolarizing membrane response to the currents injected into the cell. Conversely, dominant negative HCN2-AYA expression led to a substantial increase of R(in), which was associated with a more hyperpolarizing membrane response to the currents injected. Remarkably, under low extracellular potassium conditions (2.5 mmol/l K(+)), suppression of I(h) resulted in increased membrane hyperpolarization and decreased insulin secretion. We conclude that I(h) in beta-cells possess the potential to modulate beta-cell membrane potential and insulin secretion under hypokalemic conditions.

ATP Modulates Interaction of Syntaxin-1A with Sulfonylurea Receptor 1 to Regulate Pancreatic β-Cell KATP Channels

ATP-sensitive potassium (KATP) channels are regulated by a variety of cytosolic factors (adenine nucleotides, Mg2+, phospholipids, and pH). We previously reported that KATP channels are also regulated by endogenous membrane-bound SNARE protein syntaxin-1A (Syn-1A), which binds both nucleotide-binding folds of sulfonylurea receptor (SUR)1 and 2A, causing inhibition of KATP channel activity in pancreatic islet β-cells and cardiac myocytes, respectively. In this study, we show that ATP dose-dependently inhibits Syn-1A binding to SUR1 at physiological concentrations, with the addition of Mg2+ causing a decrease in the ATP-induced inhibitory effect. This ATP disruption of Syn-1A binding to SUR1 was confirmed by FRET analysis in living HEK293 cells. Electrophysiological studies in pancreatic β-cells demonstrated that reduced ATP concentrations increased KATP channel sensitivity to Syn-1A inhibition. Depletion of endogenous Syn-1A in insulinoma cells by botulinum neurotoxin C1 proteolysis followed by rescue with exogenous Syn-1A showed that Syn-1A modulates KATP channel sensitivity to ATP. Thus, our data indicate that although both ATP and Syn-1A independently inhibit β-cell KATP channel gating, they could also influence the sensitivity of KATP channels to each other. These findings provide new insight into an alternate mechanism by which ATP regulates pancreatic β-cell KATP channel activity, not only by its direct actions on Kir6.2 pore subunit, but also via ATP modulation of Syn-1A binding to SUR1.

Trafficking defect and proteasomal degradation contribute to the phenotype of a novel KCNH2 long QT syndrome mutation.

The Kv11.1 (hERG) K+ channel plays a fundamental role in cardiac repolarization. Missense mutations in KCNH2, the gene encoding Kv11.1, cause long QT syndrome (LQTS) and frequently cause channel trafficking-deficiencies. This study characterized the properties of a novel KCNH2 mutation discovered in a LQT2 patient resuscitated from a ventricular fibrillation arrest. Proband genotyping was performed by SSCP and DNA sequencing. The electrophysiological and biochemical properties of the mutant channel were investigated after expression in HEK293 cells. The proband manifested a QTc of 554 ms prior to electrolyte normalization. Mutation analysis revealed an autosomal dominant frameshift mutation at proline 1086 (P1086fs+32X; 3256InsG). Co-immunoprecipitation demonstrated that wild-type Kv11.1 and mutant channels coassemble. Western blot showed that the mutation did not produce mature complex-glycosylated Kv11.1 channels and coexpression resulted in reduced channel maturation. Electrophysiological recordings revealed mutant channel peak currents to be similar to untransfected cells. Co-expression of channels in a 1∶1 ratio demonstrated dominant negative suppression of peak Kv11.1 currents. Immunocytochemistry confirmed that mutant channels were not present at the plasma membrane. Mutant channel trafficking rescue was attempted by incubation at reduced temperature or with the pharmacological agents E-4031. These treatments did not significantly increase peak mutant currents or induce the formation of mature complex-glycosylated channels. The proteasomal inhibitor lactacystin increased the protein levels of the mutant channels demonstrating proteasomal degradation, but failed to induce mutant Kv11.1 protein trafficking. Our study demonstrates a novel dominant-negative Kv11.1 mutation, which results in degraded non-functional channels leading to a LQT2 phenotype.

Syntaxin-1A Actions on Sulfonylurea Receptor 2A Can Block Acidic pH-induced Cardiac K ATP Channel Activation *

During cardiac ischemia, ATP stores are depleted, and cardiomyocyte intracellular pH lowers to <7.0. The acidic pH acts on the Kir6.2 subunit of KATP channels to reduce its sensitivity to ATP, causing channel opening. We recently reported that syntaxin-1A (Syn-1A) binds nucleotide binding folds (NBF)-1 and NBF2 of sulfonylurea receptor 2A (SUR2A) to inhibit channel activity (Kang, Y., Leung, Y. M., Manning-Fox, J. E., Xia, F., Xie, H., Sheu, L., Tsushima, R. G., Light, P. E., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 47125–47131). Here, we examined Syn-1A actions on SUR2A to influence the pH regulation of cardiac KATP channels. KATP channel currents from inside-out patches excised from Kir6.2/SUR2A expressing HEK293 cells and freshly isolated cardiac myocytes were increased by reducing intracellular pH from 7.4 to 6.8, which could be blocked by increasing concentrations of Syn-1A added to the cytoplasmic surface. Syn-1A had no effect on C-terminal truncated Kir6.2 (Kir6.2-ΔC26) channels expressed in TSA cells without the SUR subunit. In vitro binding and co-immunoprecipitation studies show that Syn-1A binding to SUR2A or its NBF-1 and NBF-2 domain proteins increased progressively as pH was reduced from 7.4 to 6.0. The enhancement of Syn-1A binding to SUR2A by acidic pH was further regulated by Mg2+ and ATP. Therefore, pH regulates Kir.6.2/SUR2A channels not only by its direct actions on the Kir6.2 subunit but also by modulation of Syn-1A binding to SUR2A. The increased Syn-1A binding to the SUR2A at acidic pH would assert some inhibition of the KATP channels, which may serve as a “brake” to temper the fluctuation of low pH-induced KATP channel opening that could induce fatal reentrant arrhythmias.