Interactive pediatric emergency checklists to the palm of your hand - How the Pedi Crisis App traveled around the world.

BACKGROUND: Cognitive aids help clinicians manage critical events and have been shown to improve outcomes by providing critical information at the point of care. Critical event guidelines, such as the Society of Pediatric Anesthesia's Critical Events Checklists described in this article, can be distributed globally via interactive smartphone apps. From October 1, 2013 to January 1, 2014, we performed an observational study to determine the global distribution and utilization patterns of the Pedi Crisis cognitive aid app that the Society for Pediatric Anesthesia developed. We analyzed distribution and utilization metrics of individuals using Pedi Crisis on iOS (Apple Inc., Cupertino, CA) devices worldwide. We used Google Analytics software (Google Inc., Mountain View, CA) to monitor users' app activity (eg, screen views, user sessions). METHODS: The primary outcome measurement was the number of user-sessions and geographic locations of Pedi Crisis user sessions. Each user was defined by the use of a unique Apple ID on an iOS device. RESULTS: Google Analytics correlates session activity with geographic location based on local Internet service provider logs. Pedi Crisis had 1 252 active users (both new and returning) and 4 140 sessions across 108 countries during the 3-month study period. Returning users used the app longer and viewed significantly more screens that new users (mean screen views: new users 1.3 [standard deviation +/-1.09, 95% confidence interval 1.22-1.55]; returning users 7.6 [standard deviation +/-4.19, 95% confidence interval 6.73-8.39]P<.01) CONCLUSIONS: Pedi Crisis was used worldwide within days of its release and sustained utilization beyond initial publication. The proliferation of handheld electronic devices provides a unique opportunity for professional societies to improve the worldwide dissemination of guidelines and evidence-based cognitive aids.

PEDSnet: a National Pediatric Learning Health System.

A learning health system (LHS) integrates research done in routine care settings, structured data capture during every encounter, and quality improvement processes to rapidly implement advances in new knowledge, all with active and meaningful patient participation. While disease-specific pediatric LHSs have shown tremendous impact on improved clinical outcomes, a national digital architecture to rapidly implement LHSs across multiple pediatric conditions does not exist. PEDSnet is a clinical data research network that provides the infrastructure to support a national pediatric LHS. A consortium consisting of PEDSnet, which includes eight academic medical centers, two existing disease-specific pediatric networks, and two national data partners form the initial partners in the National Pediatric Learning Health System (NPLHS). PEDSnet is implementing a flexible dual data architecture that incorporates two widely used data models and national terminology standards to support multi-institutional data integration, cohort discovery, and advanced analytics that enable rapid learning.

Demonstrated brain insulin resistance in Alzheimer's disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline.

While a potential causal factor in Alzheimer's disease (AD), brain insulin resistance has not been demonstrated directly in that disorder. We provide such a demonstration here by showing that the hippocampal formation (HF) and, to a lesser degree, the cerebellar cortex in AD cases without diabetes exhibit markedly reduced responses to insulin signaling in the IR→IRS-1→PI3K signaling pathway with greatly reduced responses to IGF-1 in the IGF-1R→IRS-2→PI3K signaling pathway. Reduced insulin responses were maximal at the level of IRS-1 and were consistently associated with basal elevations in IRS-1 phosphorylated at serine 616 (IRS-1 pS6¹6) and IRS-1 pS6³6/6³9. In the HF, these candidate biomarkers of brain insulin resistance increased commonly and progressively from normal cases to mild cognitively impaired cases to AD cases regardless of diabetes or APOE ε4 status. Levels of IRS-1 pS6¹6 and IRS-1 pS6³6/6³9 and their activated kinases correlated positively with those of oligomeric Aß plaques and were negatively associated with episodic and working memory, even after adjusting for Aß plaques, neurofibrillary tangles, and APOE ε4. Brain insulin resistance thus appears to be an early and common feature of AD, a phenomenon accompanied by IGF-1 resistance and closely associated with IRS-1 dysfunction potentially triggered by Aß oligomers and yet promoting cognitive decline independent of classic AD pathology.

Liver-specific overexpression of pancreatic-derived factor (PANDER) induces fasting hyperglycemia in mice.

The pancreas-derived hormones, insulin and glucagon, are the two main regulators of glucose homeostasis. However, their actions can be modulated by the presence of other circulating factors including cytokines. Pancreatic-derived factor (PANDER) is a novel cytokine-like molecule secreted from the endocrine pancreas, but its biological function is currently unknown. To address this, we employed adenoviral gene delivery to develop a novel murine model of PANDER overexpression, which we used to study PANDER's effect on glucose homeostasis. Although serum metabolites in fed mice were unaffected by PANDER overexpression, fasting glucose, insulin, and corticosterone levels were significantly elevated. Additionally, PANDER-overexpressing mice displayed elevated glucose and insulin levels during a glucose tolerance test, indicating that glucose tolerance was impaired. However, there were no defects in glucose-stimulated insulin secretion or peripheral insulin sensitivity. Elevated transcription of hepatic gluconeogenic genes, PEPCK and G6Pase accompanied the fasting hyperglycemia observed in PANDER-overexpressing animals. Similarly, treatment of primary hepatocytes with PANDER-expressing adenovirus or PANDER-enriched conditioned medium elevated gluconeogenic gene expression and glucose output. PANDER treatment also resulted in higher levels of Ser133-phosphorylated cAMP-response element-binding protein in hepatocytes stimulated with 8-bromo-cAMP and dexamethasone and higher levels of intracellular cAMP upon stimulation with forskolin. In summary, we provide the first report that identifies PANDER as a regulator of hepatic glucose metabolism, where it serves as a novel factor that amplifies hepatic cAMP and cAMP-response element-binding protein signaling to induce gluconeogenic gene expression and glucose output.

Characterization of the expression, localization, and secretion of PANDER in alpha-cells.

The novel islet-specific protein PANcreatic DERived Factor (PANDER; FAM3B) has been extensively characterized with respect to the beta-cell, and these studies suggest a potential function for PANDER in the regulation of glucose homeostasis. Little is known regarding PANDER in pancreatic -cells, which are critically involved in maintaining euglycemia. Here we present the first report elucidating the expression and regulation of PANDER within the alpha-cell. Pander mRNA and protein are detected in alpha-cells, with primary localization to a glucagon-negative granular cytosolic compartment. PANDER secretion from alpha-cells is nutritionally and hormonally regulated by l-arginine and insulin, demonstrating similarities and differences with glucagon. Signaling via the insulin receptor (IR) through the PI3K and Akt/PKB node is required for insulin-stimulated PANDER release. The separate localization of PANDER and glucagon is consistent with their differential regulation, and the effect of insulin suggests a paracrine/endocrine effect on PANDER release. This provides further insight into the potential glucose-regulatory role of PANDER.

Targeted disruption of pancreatic-derived factor (PANDER, FAM3B) impairs pancreatic beta-cell function.

OBJECTIVE: Pancreatic-derived factor (PANDER, FAM3B) is a pancreatic islet-specific cytokine-like protein that is secreted from beta-cells upon glucose stimulation. The biological function of PANDER is unknown, and to address this we generated and characterized a PANDER knockout mouse. RESEARCH DESIGN AND METHODS: To generate the PANDER knockout mouse, the PANDER gene was disrupted and its expression was inhibited by homologous recombination via replacement of the first two exons, secretion signal peptide and transcriptional start site, with the neomycin gene. PANDER(-/-) mice were then phenotyped by a number of in vitro and in vivo tests to evaluate potential effects on glucose regulation, insulin sensitivity, and beta-cell morphology and function. RESULTS: Glucose tolerance tests demonstrated significantly higher blood glucose levels in PANDER(-/-) versus wild-type male mice. To identify the mechanism of the glucose intolerance, insulin sensitivity and pancreatic beta-cell function were examined. Hyperinsulinemic-euglycemic clamps and insulin tolerance testing showed similar insulin sensitivity for both the PANDER(-/-) and wild-type mice. The in vivo insulin response following intraperitoneal glucose injection surprisingly produced significantly higher insulin levels in the PANDER(-/-) mice, whereas insulin release was blunted with arginine administration. Islet perifusion and calcium imaging studies showed abnormal responses of the PANDER(-/-) islets to glucose stimulation. In contrast, neither islet architecture nor insulin content was impacted by the loss of PANDER. Interestingly, the elevated insulin levels identified in vivo were attributed to decreased hepatic insulin clearance in the PANDER(-/-) islets. Taken together, these results demonstrated decreased pancreatic beta-cell function in the PANDER(-/-) mouse. CONCLUSIONS: These results support a potential role of PANDER in the pancreatic beta-cell for regulation or facilitation of insulin secretion.

Leucine metabolism in regulation of insulin secretion from pancreatic beta cells.

Leucine, a branched-chain amino acid that must be supplied in the daily diet, plays an important role in controlling protein synthesis and regulating cell metabolism in various cell types. In pancreatic beta cells, leucine acutely stimulates insulin secretion by serving as both metabolic fuel and allosteric activator of glutamate dehydrogenase to enhance glutaminolysis. Leucine has also been shown to regulate gene transcription and protein synthesis in pancreatic islet beta cells via both mTOR-dependent and -independent pathways at physiological concentrations. Long-term treatment with leucine has been shown to improve insulin secretory dysfunction of human diabetic islets via upregulation of certain key metabolic genes. In vivo, leucine administration improves glycemic control in humans and rodents with type 2 diabetes. This review summarizes and discusses the recent findings regarding the effects of leucine metabolism on pancreatic beta-cell function.

PANDER binds to the liver cell membrane and inhibits insulin signaling in HepG2 cells.

PANDER is a cytokine co-secreted with insulin from islet beta-cells. To date, the physiological function of PANDER remains largely unknown. Here we show that PANDER binds to the liver membrane by (125)I-PANDER saturation and competitive binding assays. In HepG2 cells, pre-treatment with PANDER ranging from 4 pM to 4 nM for 8h resulted in a maximal inhibition of insulin-stimulated activation of insulin receptor and insulin receptor substrate 1 by 52% and 63%, respectively. Moreover, PANDER treatment also reduced insulin-stimulated PI3K and pAkt levels by 55% and 48%, respectively. In summary, we have identified the liver as a novel target for PANDER, and PANDER may be involved in the progression of diabetes by regulating hepatic insulin signaling pathways.

PDX-1 interaction and regulation of the Pancreatic Derived Factor (PANDER, FAM3B) promoter.

Pancreatic Derived Factor (PANDER) is a novel cytokine-like protein dominantly expressed within the endocrine pancreas. Our previous study demonstrated that the PANDER promoter was both tissue-specific and glucose-responsive. Surrounding the PANDER transcriptional start site are several putative A- and E-Box elements that may bind to the various pancreatic transcriptional factors of MafA, BETA2/NeuroD, and Pancreatic Duodenal Homeobox-1 (PDX-1). To characterize the transcriptional regulatory factors involved in PANDER gene expression, we performed co-transfection reporter gene analysis and demonstrated upregulation by all three transcription factors, with the greatest individual increase stemming from PDX-1. Potential binding of PDX-1 to A box (TAAT) regions of the PANDER promoter was demonstrated by chromatin immunoprecipitation (ChIP) and further corroborated by electrophoretic mobility shift assay (EMSA). Binding of PDX-1 to the A box regions was inhibited by mutagenized (TAGT) oligonucleotides. Site-directed mutagenesis of the three PDX-1 A box binding motifs revealed that A box sites 2 and 3 in combination were critical for maximal gene expression and deletion resulted in a 82% reduction in promoter activity. Furthermore, deletion of A box sites 2 and 3 completely diminished the glucose-responsiveness of the PANDER promoter. Our findings demonstrate that PANDER is a potential PDX-1 target gene and the A box sites within the promoter region are critical for basal and glucose-stimulated PANDER expression.

Over-expression of CLN3P, the Batten disease protein, inhibits PANDER-induced apoptosis in neuroblastoma cells: further evidence that CLN3P has anti-apoptotic properties.

Juvenile neuronal ceroid-lipofuscinosis (JNCL) or Batten/Spielmeyer-Vogt-Sjogren disease (OMIM #204200) is one of a group of nine clinically related inherited neurodegenerative disorders (CLN1-9). JNCL results from mutations in CLN3 on chromosome 16p12.1. The neuronal loss in Batten disease has been shown to be due to a combination of apoptosis and autophagy suggesting that CLN3P, the defective protein, may have an anti-neuronal death function. PANDER (PANcreatic-DERived factor) is a novel cytokine that was recently cloned from pancreatic islet cells. PANDER is specifically expressed in the pancreatic islets, small intestine, testis, prostate, and neurons of the central nervous system, and has been demonstrated to induce apoptosis. In this study, we over-expressed CLN3P in SH-SY5Y neuroblastoma cells and monitored the effects on PANDER-induced apoptosis. CLN3P significantly increased the survival rate of the SH-SY5Y cells in this system. This study provides additional evidence that the function of CLN3P is related to preventing neuronal apoptosis.

PANDER-induced cell-death genetic networks in islets reveal central role for caspase-3 and cyclin-dependent kinase inhibitor 1A (p21).

PANcreatic DERived factor is an islet-specific cytokine that promotes apoptosis in primary islets and islet cell lines. To elucidate the genetic mechanisms of PANDER-induced cell death we performed expression profiling using the mouse PancChip version 5.0 in conjunction with Ingenuity Pathway Analysis. Murine islets were treated with PANDER and differentially expressed genes were identified at 48 and 72 h post-treatment. 64 genes were differentially expressed in response to PANDER treatment. 22 genes are associated with cell death. In addition, the genes with the highest fold change were linked with cell death or apoptosis. The most significantly affected gene at 48 h was the downregulated cyclin-dependent kinase inhibitor 1A (CDKN1A or p21). Approximately half of the genes impacted at 72 h were linked to cell death. Cell death differentially expressed genes were confirmed by quantitative RT-PCR. Further analysis identified cell death genetic networks at both time points with 21 of the 22 cell death genes related in various biological pathways. Caspase-3 (CASP3) was biologically linked to CDKN1A in several genetic networks and these two genes were further examined. Elevated cleaved CASP3 levels in PANDER-treated beta-TC3 insulinoma cells were found to abrogate CDKN1A expression. Levels of CDKN1A were not affected in the absence of cleaved CASP3. PANDER-induced downregulation of CDKN1A expression coupled with induced CASP3-activation may serve a central role in islet cell death and offers further insight into the mechanisms of cytokine-induced beta-cell apoptosis.

Leucine regulation of glucokinase and ATP synthase sensitizes glucose-induced insulin secretion in pancreatic beta-cells.

We have recently shown that leucine culture upregulates ATP synthase beta-subunit (ATPSbeta) and increases ATP level, cytosolic Ca(2+), and glucose-induced insulin secretion in rat islets. The aim is to test whether glucokinase expression is also affected in rat islets and its role in glucose sensitization during leucine culture. Leucine culture increased glucose-induced NAD(P)H level at 1 and 2 days but not at 1 week. The half-maximal effective concentration of the glucose response curve for NAD(P)H was left-shifted from 5-7 to 2-3 mmol/l. The effect was dose dependent and rapamycin insensitive. Leucine culture did not affect glyceraldehyde effects on NAD(P)H. Leucine pretreatment for 30 min had no effects on NAD(P)H levels. Leucine culture for 2 days also increased glucose-induced cytosolic Ca(2+) elevation, ATP level, and insulin secretion. Leucine increase of glucokinase mRNA levels occurred as early as day 1 and lasted through 1 week. That of ATPSbeta did not occur until day 2 and lasted through 1 week. Leucine effects on both mRNAs were dose dependent. The upregulation of both genes was confirmed by Western blotting. Leucine culture also increased glucose-induced insulin secretion, ATP level, glucokinase, and ATPSbeta levels of type 2 diabetic human islets. In conclusion, leucine culture upregulates glucokinase, which increases NAD(P)H level, and ATPSbeta, which increases oxidation of NADH and production of ATP. The combined upregulation of both genes increases glucose-induced cytosolic Ca(2+) and insulin secretion.

Mechanisms of glucose-induced secretion of pancreatic-derived factor (PANDER or FAM3B) in pancreatic beta-cells.

Pancreatic-derived factor (PANDER) is an islet-specific cytokine present in both pancreatic alpha- and beta-cells, which, in vitro, induces beta-cell apoptosis of primary islet and cell lines. In this study, we investigated whether PANDER is secreted by pancreatic alpha- and beta-cells and whether PANDER secretion is regulated by glucose and other insulin secretagogues. In mouse-derived insulin-secreting beta-TC3 cells, PANDER secretion in the presence of stimulatory concentrations of glucose was 2.8 +/- 0.4-fold higher (P < 0.05) than without glucose. Insulin secretion was similarly increased by glucose in the same cells. The total concentration of secreted PANDER in the medium was approximately 6-10 ng/ml (0.3-0.5 nmol/l) after a 24-h culture with glucose. L-Glucose failed to stimulate PANDER secretion in beta-TC3 cells. KCl stimulated PANDER secretion 2.1 +/- 0.1-fold compared with control without glucose. An L-type Ca2+ channel inhibitor, nifedipine, completely blocked both glucose- or KCl-induced insulin and PANDER secretion. In rat-derived INS-1 cells, glucose (20 mmol/l) stimulated PANDER secretion 4.4 +/- 0.9-fold, while leucine plus glutamine stimulated 4.4 +/- 0.7-fold compared with control without glucose. In mouse islets overexpressing PANDER, glucose (20 mmol/l) stimulated PANDER secretion 3.2 +/- 0.5-fold (P < 0.05) compared with basal (3 mmol/l glucose). PANDER was also secreted by alpha-TC3 cells but was not stimulated by glucose. Mutations of cysteine 229 or of cysteines 91 and 229 to serine, which may form one disulfide bond, and truncation of the COOH-terminus or NH2-terminus of PANDER all resulted in failure of PANDER secretion, even though these mutant or truncated PANDERs were highly expressed within the cells. In conclusion, we found that 1) PANDER is secreted from both pancreatic alpha- and beta-cells, 2) glucose stimulates PANDER secretion dose dependently in beta-cell lines and primary islets but not in alpha-cells, 3) PANDER is likely cosecreted with insulin via the same regulatory mechanisms, and 4) structure and conformation is vital for PANDER secretion.

Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells.

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.

Tissue-specific and glucose-responsive expression of the pancreatic derived factor (PANDER) promoter.

Pancreatic derived factor (PANDER) is a recently identified cytokine-like protein that is dominantly expressed in the islets of Langerhans of the pancreas. To investigate the mechanism of tissue-specific regulation of PANDER, we identified and characterized the promoter region. The transcriptional start site was identified 520 bp upstream of the translational start codon by 5'-RLM-RACE. Computer algorithms identified several islet-associated and glucose-responsive binding motifs that included A and E boxes, hepatocyte nuclear factors 1 and 4, Oct-1, and signal transducer and activator of transcription 3, and 5. Reporter gene analysis revealed cell type-specific PANDER promoter expression in islet and liver-derived cell lines. Levels of PANDER mRNA were directly concordant to the observed cell type-specific PANDER promoter gene expression. The minimal element was mapped to the 5'-UTR and located between +200 and +491 relative to the transcriptional start site and imparted maximal gene expression. In addition, several putative glucose-responsive binding sites were further functionally characterized to reveal critical regulatory elements of PANDER. The PANDER promoter was demonstrated to be glucose-responsive in a dose-dependent manner in murine insulinoma beta-TC3 cells and primary murine islets, but unresponsive in glucagon-secreting alpha-TC3 cells. Our findings revealed that the 5'-UTR of PANDER contains the minimal element for gene expression and imparts both tissue-specificity and glucose-responsiveness. The regulation of PANDER gene expression mimics that of insulin and suggests a potential biological function of PANDER involved in metabolic homeostasis.

Interferon-gamma-induced regulation of the pancreatic derived cytokine FAM3B in islets and insulin-secreting betaTC3 cells.

The pancreatic-derived factor (PANDER, FAM3B) is a novel protein that is beta-cell specific and induces beta-cell death. PANDER is localized to insulin-containing granules-based on confocal microscopy and immunogold electron microscopy. PANDER protein was detected in the conditioned medium of betaTC3 cells. Using real-time reverse transcription-polymerase chain reaction, treatment of betaTC3 cells with IL-1beta + TNFalpha + IFNgamma induced a significant seven-fold increase in PANDER mRNA expression (n = 3; p < 0.01 at 24 h, p < 0.05 at 48 h), while IFNgamma alone caused a 3.2-fold increase (n = 3; p < 0.01 at 24 h) compared to unstimulated and time-matched vehicle controls. IL-1beta or TNFalpha alone had no effect. Under those conditions, a similar up-regulation was also observed in mouse islet cells, with increases in PANDER mRNA of 5.9-fold and 5.0-fold after treatment with IL-1beta + TNFalpha + IFNgamma or IFNgamma alone. Because PANDER mRNA expression is up-regulated by IFNgamma, a cytokine implicated in the pathogenesis of type 1 diabetes, PANDER may contribute to the pathogenesis of beta-cell death.

Effects of overexpression of pancreatic derived factor (FAM3B) in isolated mouse islets and insulin-secreting betaTC3 cells.

PANcreatic DERived factor (PANDER, FAM3B) is a recently discovered islet-specific cytokine. We have previously shown that, in vitro, truncated recombinant PANDER isoforms (20 and 21 kDa) are cytotoxic to beta-cell lines but the effects of full-length PANDER on islet biology remain unclear. In this study, we used adenovirus (Ad-PANDER) to overexpress full-length cDNA of PANDER in islets and betaTC3 cells. BetaTC3 cells were infected with Ad-PANDER or control vector. After 48 h, cell viability was significantly decreased as evaluated by MTT assay. The number of dead cells was significantly increased as indicated by the fluorescent intensity of the propidium iodide-stained cells (160 +/- 13 vs. control 100 +/- 7%, P = 0.001). Flow cytometric Tunel assay showed that overexpressing PANDER induced a significant fourfold increase in beta-cell apoptosis (19.4 +/- 6.3 vs. control 4.1 +/- 0.8%, P < 0.05). There was a significant increase in the number of annexin V-positive (apoptotic) cells and propidium iodide-positive (dead) cells in mouse islets infected with Ad-PANDER compared with control cells infected with Ad-LacZ. Addition of 4 nM recombinant PANDER protein to betaTC3 cells or infection of Ad-PANDER did not affect Akt and STAT1 phosphorylation, Bcl-2, Fas, and NF-kappaB protein levels. However, activation of caspase-3 was observed in betaTC3 and islets infected with Ad-PANDER. Overexpression of PANDER in mouse islets or addition of recombinant PANDER decreased insulin secretion induced by carbachol plus glucose or high potassium but not that by glucose alone. Culture with recombinant PANDER did not affect glucose-induced NAD(P)H elevation in mouse islets. In conclusion, Ad-PANDER infection is as effective as truncated recombinant PANDER to induce betaTC3 cell and mouse islet apoptosis.

Leucine culture reveals that ATP synthase functions as a fuel sensor in pancreatic beta-cells.

Our goal was to investigate whether leucine culture affects beta-cell glucose sensing. One-day culture of rat islets with 10 mM leucine had no effect on glucose-induced insulin secretion. One-week leucine culture decreased the threshold for glucose-induced insulin secretion and increased maximal insulin secretion at 30 mM glucose. Glucose-induced cytosolic free Ca(2+) was increased at 1 week but not at 1 day of leucine culture. Without glucose, ATP content was not different with or without leucine culture for 1 week. With 20 mM glucose, ATP content was higher by 1.5-fold in islets cultured for 1 week with leucine than those without leucine. Microarray experiments indicated that culture of RINm5F cells with leucine increased expression of ATP synthase beta subunit 3.2-fold, which was confirmed by real time reverse transcription-PCR analysis (3.0- +/- 0.4-fold) in rat islets at 1 week but not after 1 day with leucine culture. Down-regulation of ATP synthase beta subunit by siRNA decreased INS1 cell ATP content and insulin secretion with 20 mM glucose. Overexpression of ATP synthase beta subunit in INS1 cell increased insulin secretion in the presence of 5 and 20 mM glucose. In conclusion, one-week leucine culture of rat islets up-regulated ATP synthase and increased ATP content, which resulted in elevated [Ca(2+)] levels and more insulin exocytosis by glucose. Depletion of ATP synthase beta subunit with siRNA produced opposite effects. These data reveal the fuel-sensing role of mitochondrial ATP synthase in the control of ATP production from glucose and the control of glucose-induced insulin secretion.

Interleukin-1beta upregulates functional expression of neurokinin-1 receptor (NK-1R) via NF-kappaB in astrocytes.

Cytokines and neuropeptides are modulators of neuroimmunoregulation in the central nervous system (CNS). The interaction of these modulators may have important implications in CNS diseases. We investigated whether interleukin-1beta (IL-1beta) modulates the expression of neurokinin-1 receptor (NK-1R), the primary receptor for substance P (SP), a potent neuropeptide in the CNS. IL-1beta upregulated NK-1R expression in human astroglioma cells (U87 MG) and primary rat astrocytes at both mRNA and protein levels. IL-1beta treatment of U87 MG cells and primary rat astrocytes led to an increase in cytosolic Ca(2+) in response to SP stimulation, indicating that IL-1beta-induced NK-1R is functional. CP-96,345, a specific non-peptide NK-1R antagonist, inhibited SP-induced rise of [Ca(2+)](i) in the astroglioma cells. Investigation of the mechanism responsible for IL-1beta action revealed that IL-1beta has the ability of activating nuclear factor-kappab (NF-kappaB). Caffeic acid phenethyl ester (CAPE), a specific inhibitor of NF-kappaB activation, not only abrogated IL-1beta-induced NF-kappaB promoter activation, but also blocked IL-1beta-mediated induction of NK-1R gene expression. These findings provide additional evidence that there is a biological interaction between IL-1beta and the neuropeptide SP in the CNS, which may have important implications in the inflammatory diseases in the CNS.

Rapamycin has a deleterious effect on MIN-6 cells and rat and human islets.

Rapamycin (sirolimus) is a macrolide fungicide with immunosuppressant properties that is used in human islet transplantation. Little is known about the effects of rapamycin on MIN-6 cells and islets. Rapamycin had a dose-dependent, time-dependent, and glucose-independent deleterious effect on MIN-6 cell viability. At day 1, using the MTT method, 0.01 nmol/l rapamycin reduced cell viability to 83 +/- 6% of control (P < 0.05). Using the calcein AM method, at day 2, 10 nmol/l rapamycin caused a reduction in cell viability to 73 +/- 5% of control (P < 0.001). Furthermore, 10 and 100 nmol/l rapamycin caused apoptosis in MIN-6 cells as assessed by the transferase-mediated dUTP nick-end labeling assay. Compared with control, there was a 3.1 +/- 0.6-fold increase (P < 0.01) in apoptosis in MIN-6 cells treated with 10 nmol/l rapamycin. A supra-therapeutic rapamycin concentration of 100 nmol/l significantly impaired glucose- and carbachol-stimulated insulin secretion in rat islets and had a deleterious effect on the viability of rat and human islets, causing apoptosis of both alpha- and beta-cells.