The association of a frailty index derived from laboratory tests and vital signs with clinical outcomes in critical care patients with septic shock: a retrospective study based on the MIMIC-IV database.

PURPOSE: Frailty is a vulnerable state to stressors due to the loss of physiological reserve as a result of multisystem dysfunction. The physiological and laboratory-based frailty index (FI-Lab), depending on laboratory values and vital signs, is a powerful tool to capture frailty status. The aim of this study was to assess the relationship between FI-Lab and in-hospital mortality in patients with septic shock. METHODS: Baseline data for patients with sepsis in the intensive care unit were retrieved from the Critical Care Medicine Database (MIMIC-IV, v2.2). The primary outcome was mortality during hospitalization. The propensity score matching (PSM) method was used to analyze the basic conditions during hospitalization between groups.The FI-Lab was analysed for its relationship with in-hospital mortality using logistic regression according to continuous and categorical variables, respectively, and described using the restricted cubic spline (RCS). Survival was compared between groups using Kaplan-Meier (KM) curves. Subgroup analyses were used to improve the stability of the results. RESULTS: A total of 9219 patients were included. A cohort score of 1803 matched patients was generated after PSM. The analyses showed that non-surviving patients with septic shock in the ICU had a high FI-Lab index (P<0.001). FI-Lab, whether used as a continuous or categorical variable, increased with increasing FI-Lab and increased in-hospital mortality (P<0.001).Subgroup analyses showed similar results. RCS depicts this non-linear relationship. KM analysis shows the cumulative survival time during hospitalisation was significantly lower as FI-Lab increased (log-rank test, P<0.001). CONCLUSION: Elevated FI-Lab is associated with increased in-hospital mortality in patients with septic shock.

Groundwater hydrochemistry, source identification and health assessment based on self-organizing map in an intensive mining area in Shanxi, China.

The Changzhi Basin in Shanxi is renowned for its extensive mining activities. It's crucial to comprehend the spatial distribution and geochemical factors influencing its water quality to uphold water security and safeguard the ecosystem. However, the complexity inherent in hydrogeochemical data presents challenges for linear data analysis methods. This study utilizes a combined approach of self-organizing maps (SOM) and K-means clustering to investigate the hydrogeochemical sources of shallow groundwater in the Changzhi Basin and the associated human health risks. The results showed that the groundwater chemical characteristics were categorized into 48 neurons grouped into six clusters (C1-C6) representing different groundwater types with different contamination characteristics. C1, C3, and C5 represent uncontaminated or minimally contaminated groundwater (Ca-HCO3 type), while C2 signifies mixed-contaminated groundwater (HCO3-Ca type, Mixed Cl-Mg-Ca type, and CaSO4 type). C4 samples exhibit impacts from agricultural activities (Mixed Cl-Mg-Ca), and C6 reflects high Ca and NO3- groundwater. Anthropogenic activities, especially agriculture, have resulted in elevated NO3- levels in shallow groundwater. Notably, heightened non-carcinogenic risks linked to NO3-, Pb, F-, and Mn exposure through drinking water, particularly impacting children, warrant significant attention. This research contributes valuable insights into sustainable groundwater resource development, pollution mitigation strategies, and effective ecosystem protection within intensive mining regions like the Changzhi Basin. It serves as a vital reference for similar areas worldwide, offering guidance for groundwater management, pollution prevention, and control.

Influence of Geometric Characteristics on Water Flow and Solute Transport at Fracture Intersections.

Laboratory experiments and numerical simulations were performed to explore the influence of intersection geometry on fluid flow and solute transport in fractures. Fractures were engraved and sealed into an acrylic plate and two orthogonal intersections with different geometry were constructed. The effects of curvature and relative shear displacement at intersections on preferential flow and solute transport were investigated. By solving the Navier-Stokes (NS) equation, the fluid mixing and solute distribution were predicted. The results showed that the geometric characteristics at the intersection have a significant effect on the preferential flow and solute distribution. The results agreed well with the experimental results, in terms of flow direction, preferential flow rate, and heterogeneous solute distribution. With an increase in curvature, the flow difference between the two outlets increases gradually. Increasing curvature can reduce the preferential flow and weaken the inhomogeneity of solute distribution. An increase of relative shear displacement decreases the pressure gradient and flow rate at the entrance of the two branch fractures, and thereby increases preferential flow and inhomogeneity of solute distribution. The results provide a basis and reference for further exploring the relationship between the geometric characteristics of fracture intersections and flow behaviors.

Use of Hydrogeochemistry and Isotopes for Evaluation of Groundwater in Qilian Coal Base of China.

The Jiangcang Basin is an important mining area of the former Qilian Mountain large coal base in Qinghai Province, and understanding the groundwater circulation mechanism is the basis for studying the hydrological effects of permafrost degradation in alpine regions. In this study, hydrogeochemical and multiple isotope tracer analysis methods are used to understand the chemical evolution and circulation mechanisms of the groundwater in the typical alpine region of the Jiangcang Basin. The diversity of the groundwater hydrochemistry in the study area reflects the complexity of the hydrogeochemical environment in which it is located. The suprapermafrost water and intrapermafrost water are recharged by modern meteoric water. The groundwater is closely hydraulically connected to the surface water with weak evaporation overall. The high δ34 S value of deep groundwater is due to SO4 reduction, and SO4 2- -rich snow recharge with lixiviated sulfate minerals are the main controlling factor for the high SO4 2- concentration in groundwater. According to the multivariate water conversion relationships, it reveals that the river receives more groundwater recharge, suprapermafrost water is recharged by the proportion of meteoric water, which is closely related to the mountainous area at the edge of the basin, while intrapermafrost water is mainly recharged by the shallow groundwater. This study provides a data-driven approach to understanding groundwater recharge and evolution in alpine regions, in addition to having significant implications for water resource management and ecological environmental protection in coal bases of the Tibetan Plateau.

Application of Levosimendan in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection Patients with Myocardial Injury.

Objective: This study aims to investigate the effectiveness of levosimendan in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection complicated by cardiac insufficiency and myocardial injury. Methods: A total of 22 patients with SARS-CoV-2 infection and myocardial injury, admitted to the Cardiology Department of our hospital between December 2022 and February 2023, are randomly divided into two groups: a dobutamine general treatment group and a levosimendan observation treatment group. The treatment outcomes of the two groups are compared and analyzed. Results: The overall improvement rate in the general treatment group is 80%, while the levosimendan treatment group shows a 100% improvement rate. There is a statistically significant difference between the two groups (P < .05). Post-treatment, the left ventricular ejection fraction for the general treatment group and the levosimendan treatment group are (48 ± 7)% and (54 ± 6)%, respectively. Additionally, the left ventricular end-diastolic diameter is (55.0 ± 3.0) mm in the general treatment group and (51 ± 5.0) mm in the levosimendan group, with a statistically significant difference (P < .05). After active treatment, the plasma levels of B-type natriuretic peptide (Brain Natriuretic Peptide, NT-proBNP) are significantly lower in the levosimendan treatment group than in the general treatment group (P < .05). Moreover, the plasma levels of interleukin-6 (IL-6) and C-reactive protein (CRP) in the levosimendan group decrease slightly faster than those in the general treatment group, with a statistically significant difference (P < .05). The length of hospital stay in the levosimendan group is (12 ± 3) days, significantly lower than the general treatment group (16 ± 5) days, with a statistically different result (P < .05). Conclusions: Levosimendan demonstrates significant efficacy in patients with novel coronavirus infection complicated by myocardial injury, resulting in improved clinical symptoms, enhanced cardiac function, shorter hospital stays, early discharge, and cost savings.

Chloroplast genome characterization of Rubus arcticus L.

Rubus arcticus Linnaeus (1753) is a medicinal and edible plant in the Rosaceae with wide distribution in northeast China. The total length of the genome was 156,668 bp with a GC content of 37.1%, including a large single-copy (LSC, 85,958 bp) region, a small single-copy region (SSC, 18,756 bp), and inverted repeat (IR, 51,954 bp) regions. A total of 129 genes were identified. The numbers of protein genes tRNAs and rRNAs were 85, 36, and 8, respectively. Phylogenetic analysis indicated that R. arcticus belongs to the Rubus genus. Published R. arcticus chloroplast genomes have yielded insights into the closely related species identification, phylogenetic position and Rubus evolution.

Identification of origin and runoff of karst groundwater in the glacial lake area of the Jinsha River fault zone, China.

Karst groundwater plays important roles as a water supply and in sustaining the biodiversity and ecosystems of the eastern Qinghai-Xizang Plateau. Owing to the stratigraphic structure, high tectonic activity, and changeable climate of the region, the recharge source, runoff path, and dynamic characteristics of karst groundwater are highly complex, which poses challenges with regard to the protection of water resources and ecology. This study identified the origin and flow processes of karst groundwater in the glacial lake area of the Jinsha River fault zone using satellite remote sensing, hydrochemical and isotope analyses, and flow measurements. Results showed that active faults control the distribution of glacial lakes and the recharge, runoff, and discharge of karst groundwater. Glacial lake water is an important source of karst groundwater in the Jinsha River fault zone area. Specifically, glacial lake water continuously recharges the karst system via faults, fractures, and karst conduits, thereby maintaining the relative stability of karst spring flows. Through hierarchical cluster analysis, two main runoff conduits of karst water were distinguished: one along the Dingqu Fault and the other along the Eastern Zhairulong Fault, which together account for 59% of the total regional karst groundwater flow. The elevation difference between the recharge and discharge areas of the main karst springs is > 1000 m. Groundwater runoff is fast and residence time in the aquifer is short. The dissolution of calcite and dolomite mainly occurs during transit through the groundwater system, and cation exchange is weak. Therefore, the regional karst springs are predominantly HCO3-Ca·Mg type. To protect regional karst water resources and ecology, the monitoring and protection of glacial lakes should be strengthened.

The complete chloroplast genome sequence of Dictamnus albus L.

Dictamnus albus L. refers to a perennial herb with both ornamental and medicinal value. In the present study, we obtained the complete chloroplast genome sequence of D. albus through high-throughput sequencing. The length of the chloroplast genome was 157,139 bp, while the large single-copy and small single-copy regions were 84,478 bp and 18,587 bp, respectively. The pair of inverted repeat sequences was 27,037 bp, and the GC content was 38.5%. A total of 132 genes were annotated, including 87 protein-coding genes (PCGs), eight ribosomal RNA (rRNA) genes, and 37 transfer RNA (tRNA) genes. The chloroplast genomes of D. albus and eight species of Rutaceae were subjected to maximum-likelihood phylogenetic tree analysis. D. albus was found to be most closely related to Orixa japonica.

STAT1 determines aggressiveness of glioblastoma both in vivo and in vitro through wnt/ß-catenin signalling pathway.

Glioblastoma is one of the most malignant tumors and causes the high mortality in cancer patients. Currently, there is no highly efficient therapy against glioblastoma. Therefore, searching for a new molecular target to anti-glioblastoma therapy is urgent and necessary. In this study, we elucidated the role of Signal transducer and activator of transcription 1 (STAT1) in proliferation, migration and apoptosis of glioblastoma cells. We found that STAT1 downregulation could weaken the aggressiveness of glioblastoma cells. Besides, the glioblastoma growth in vivo was also inhibited with the STAT1 downregulation by shRNA as well as by pharmacological stimulation withSTAT1inhibitors. This negative regulation of tumor growth was accompanied by the inhibition in epithelial-mesenchymal transition (EMT), whereas the STAT1 overexpression promoted EMT. Furthermore, the involvement of wnt/ß-catenin was observed in STAT1 downregulation mediated weakness in glioblastoma aggressiveness since application of activator wnt agonist 1 could counteract the inhibitory effect induced by STAT1 downregulation. Collectively, this work provided the evidence to support the conclusion that STAT1 can regulate the glioblastoma growth and migration, potentially serving as a therapeutic target against glioblastoma. SIGNIFICANCE OF THE STUDY: Glioblastoma is one of the most malignant tumors with very high mortality. Until now, there is no efficient therapy against glioblastoma. In this study, we found downregulation of Signal transducer and activator of transcription 1 (STAT1) could weaken the aggressiveness of glioblastoma cells through inhibition in epithelial-mesenchymal transition, mediated through wnt/ß-catenin signalling pathway. Thus, this work supported the regulatory role of STAT1 in glioblastoma growth and migration. This potentially serves as a new therapeutic target against glioblastoma.

Evaluation of an Autologous Bone Mesenchymal Stem Cell-Derived Extracellular Matrix Scaffold in a Rabbit and Minipig Model of Cartilage Repair.

BACKGROUND This study aimed to evaluate an autologous bone mesenchymal stem cell (MSC)-derived extracellular matrix (ECM) scaffold in two animal models of cartilage repair. MATERIAL AND METHODS A rabbit model (n=16) and a minipig model (n=8) of cartilage repair were created with cartilage defects of the knee joints treated with bone marrow stimulation (BMS). In the ECM group, autologous bone MSC-derived ECM scaffolds were implanted into the cartilage defects after bone marrow stimulation. In the BMS group, the cartilage defects were treated by bone marrow stimulation only. The renewal capacity of bone MSCs was measured with a colony-forming unit fibroblast (CFU-F) in vitro assay. The extent of cartilage repair was as-sessed at 6 months after surgery. RESULTS In the rabbit model, the macroscopic appearance of the exudate of the healing wounds in the ECM group showed less fibrosis, and the histology showed more evenly distributed chondrocytes compared with the BMS group. The CFU-F assay showed that the number of bone MSCs in the ECM group was approximately was twice that of the BMS group. In the minipig model, the macroscopic appearance and magnetic resonance imaging (MRI) findings of the ECM group were improved when compared with the BMS group. The repaired tissue in ECM group had similar histological characteristics and biochemical content to normal hyaline cartilage. CONCLUSIONS In two animal models of knee joint cartilage repair, the use of an ECM scaffold increased the number of bone MSCs and improved the extent of cartilage repair.

An improved wavelet transform and multi-block forecast engine based on a novel training mechanism.

In this paper, a novel prediction technique is proposed to predict wind-power generation. Because of the growth of wind-generated electricity as a component of power grids, various wind-power prediction methods have been proposed recently by researchers. To achieve accurate prediction, a novel approach using a dual-tree complex wavelet transform, a new feature selection procedure, and a combinatorial prediction engine has been implemented to forecast wind-power generation. To improve feature selection to reduce diagnostic efficiency degradation caused by outliers in data-driven diagnostics, an outlier-insensitive combinatorial feature selection procedure has been used to determine candidate subgroup characteristics. Furthermore, a multi-stage forecast engine equipped with a new training mechanism for optimizing free parameters and based on the Elman neural network (ENN) is presented in this work. This training mechanism was developed using an efficient stochastic search method to attain the high learning capabilities of the proposed ENN-based forecast engine. The proposed model has been applied to real-world engineering data from Alberta, Canada, and Oklahoma, United States. The outcomes achieved by the different forecasting methods are compared, proving the effectiveness of the proposed procedure.

lncRNA H19/miR-675 axis regulates cardiomyocyte apoptosis by targeting VDAC1 in diabetic cardiomyopathy.

We previously established a rat model of diabetic cardiomyopathy (DCM) and found that the expression of lncRNA H19 was significantly downregulated. The present study was designed to investigate the pathogenic role of H19 in the development of DCM. Overexpression of H19 in diabetic rats attenuated oxidative stress, inflammation and apoptosis, and consequently improved left ventricular function. High glucose was associated with reduced H19 expression and increased cardiomyocyte apoptosis. To explore the molecular mechanisms involved, we performed in vitro experiments using cultured neonatal rat cardiomyocytes. Our results showed that miR-675 expression was decreased in cardiomyocytes transfected with H19 siRNA. The 3'UTR of VDAC1 was cloned downstream of a luciferase reporter construct and cotransfected into HEK293 cells with miR-675 mimic. The results of luciferase assay indicated that VDAC1 might be a direct target of miR-675. The expression of VDAC1 was upregulated in cardiomyocytes transfected with miR-675 antagomir, which consequently promotes cellular apoptosis. Moreover, enforced expression of H19 was found to reduce VDAC1 expression and inhibit apoptosis in cardiomyocytes exposed to high glucose. In conclusion, our study demonstrates that H19/miR-675 axis is involved in the regulation of high glucose-induced apoptosis by targeting VDAC1, which may provide a novel therapeutic strategy for the treatment of DCM.

[Current therapy status and research progress of cartilage defects of knees].

To demonstrate the current strategies for treating cartilage defects of knees and the related research. Published papers about cartilage defects were searched and reviewed. The current strategies for the treatment were summarized. Based on the research of our study and others, the conclusion how to treat cartilage defects was made. The current ways for treating cartilage defects include micro-fractures, chondrocytes transplantation, mosaicplasty and tissue engineering; Research on functional magnetic resonance imaging in the early diagnosis of cartilage defects, cartilage degeneration is gradually increasing. There is still no effective treatment of cartilage defects and tissue engineering has brought new hopes for the treatment of cartilage defects , functional magnetic resonance imaging has some significance in early diagnosis of cartilage defects, cartilage degeneration.

Alterations in lipid signaling underlie lipodystrophy secondary to AGPAT2 mutations.

Congenital generalized lipodystrophy (CGL), secondary to AGPAT2 mutation is characterized by the absence of adipocytes and development of severe insulin resistance. In the current study, we investigated the adipogenic defect associated with AGPAT2 mutations. Adipogenesis was studied in muscle-derived multipotent cells (MDMCs) isolated from vastus lateralis biopsies obtained from controls and subjects harboring AGPAT2 mutations and in 3T3-L1 preadipocytes after knockdown or overexpression of AGPAT2. We demonstrate an adipogenic defect using MDMCs from control and CGL human subjects with mutated AGPAT2. This defect was rescued in CGL MDMCs with a retrovirus expressing AGPAT2. Both CGL-derived MDMCs and 3T3-L1 cells with knockdown of AGPAT2 demonstrated an increase in cell death after induction of adipogenesis. Lack of AGPAT2 activity reduces Akt activation, and overexpression of constitutively active Akt can partially restore lipogenesis. AGPAT2 modulated the levels of phosphatidic acid, lysophosphatidic acid, phosphatidylinositol species, as well as the peroxisome proliferator-activated receptor γ (PPARγ) inhibitor cyclic phosphatidic acid. The PPARγ agonist pioglitazone partially rescued the adipogenic defect in CGL cells. We conclude that AGPAT2 regulates adipogenesis through the modulation of the lipome, altering normal activation of phosphatidylinositol 3-kinase (PI3K)/Akt and PPARγ pathways in the early stages of adipogenesis.

Bcl10 links saturated fat overnutrition with hepatocellular NF-kB activation and insulin resistance.

Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. With high-fat feeding, FFAs activate NF-kB in target tissues, initiating negative crosstalk with insulin signaling. However, the mechanisms underlying FFA-dependent NF-kB activation remain unclear. Here, we demonstrate that the saturated FA, palmitate, requires Bcl10 for NF-kB activation in hepatocytes. Uptake of palmitate, metabolism to diacylglycerol, and subsequent activation of protein kinase C (PKC) appear to mechanistically link palmitate with Bcl10, known as a central component of a signaling complex that, along with CARMA3 and MALT1, activates NF-kB downstream of selected cell surface receptors. Consequently, Bcl10-deficient mice are protected from hepatic NF-kB activation and insulin resistance following brief high-fat diet, suggesting that Bcl10 plays a major role in the metabolic consequences of acute overnutrition. Surprisingly, while CARMA3 also participates in the palmitate response, MALT1 is completely dispensable, thereby revealing an apparent nonclassical role for Bcl10 in NF-kB signaling.

DNA methylation and microRNAs in cancer.

DNA methylation is a type of epigenetic modification in the human genome, which means that gene expression is regulated without altering the DNA sequence. Methylation and the relationship between methylation and cancer have been the focus of molecular biology researches. Methylation represses gene expression and can influence embryogenesis and tumorigenesis. In different tissues and at different stages of life, the level of methylation of DNA varies, implying a fundamental but distinct role for methylation. When genes are repressed by abnormal methylation, the resulting effects can include instability of that gene and inactivation of a tumor suppressor gene. MicroRNAs have some aspects in common with this regulation of gene expression. Here we reviewed the influence of gene methylation on cancer and analyzed the methods used to profile methylation. We also assessed the correlation between methylation and other epigenetic modifications and microRNAs. About 55,845 research papers have been published about methylation, and one-fifth of these are about the appearance of methylation in cancer. We conclude that methylation does play a role in some cancer types.

PKA-dependent potentiation of glucose-stimulated insulin secretion by Epac activator 8-pCPT-2'-O-Me-cAMP-AM in human islets of Langerhans.

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2'-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM was associated with depolarization and an increase of [Ca(2+)](i) that reflected both Ca(2+) influx and intracellular Ca(2+) mobilization in islet beta-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca(2+)-dependent exocytosis.

Conditional ablation and recovery of forebrain neurogenesis in the mouse.

Forebrain neurogenesis persists throughout life in the rodent subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Several strategies have been employed to eliminate adult neurogenesis and thereby determine whether depleting adult-born neurons disrupts specific brain functions, but some approaches do not specifically target neural progenitors. We have developed a transgenic mouse line to reversibly ablate adult neural stem cells and suppress neurogenesis. The nestin-tk mouse expresses herpes simplex virus thymidine kinase (tk) under the control of the nestin 2nd intronic enhancer, which drives expression in neural progenitors. Administration of ganciclovir (GCV) kills actively dividing cells expressing this transgene. We found that peripheral GCV administration suppressed SVZ-olfactory bulb and DG neurogenesis within 2 weeks but caused systemic toxicity. Intracerebroventricular GCV infusion for 28 days nearly completely depleted proliferating cells and immature neurons in both the SVZ and DG without systemic toxicity. Reversibility of the effects after prolonged GCV infusion was slow and partial. Neurogenesis did not recover 2 weeks after cessation of GCV administration, but showed limited recovery 6 weeks after GCV that differed between the SVZ and DG. Suppression of neurogenesis did not inhibit antidepressant responsiveness of mice in the tail suspension test. These findings indicate that SVZ and DG neural stem cells differ in their capacity for repopulation, and that adult-born neurons are not required for antidepressant responses in a common behavioral test of antidepressant efficacy. The nestin-tk mouse should be useful for studying how reversible depletion of adult neurogenesis influences neurophysiology, other behaviors, and neural progenitor dynamics.

Enhanced Rap1 activation and insulin secretagogue properties of an acetoxymethyl ester of an Epac-selective cyclic AMP analog in rat INS-1 cells: studies with 8-pCPT-2'-O-Me-cAMP-AM.

To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live cell imaging and high throughput screening of a fluorescence resonance energy transfer-based cAMP sensor (Epac1-camps) to validate the selectivity with which acetoxymethyl esters (AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or Bt(2)cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Because 2.4-fold more insulin was secreted in response to a saturating concentration (10 microm) of Bt(2)cAMP-AM as compared with 8-pCPT-2'-O-Me-cAMP-AM, and because the action of Bt(2)cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 microm of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 microm of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 microm of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM-ester did not. Thus, the membrane permeability of 8-pCPT-2'-O-Me-cAMP in insulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog.

Glucose-dependent potentiation of mouse islet insulin secretion by Epac activator 8-pCPT-2'-O-Me-cAMP-AM.

Epac2 is a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) that is proposed to mediate stimulatory actions of the second messenger cAMP on mouse islet insulin secretion. Here we have used methods of islet perifusion to demonstrate that the acetoxymethyl ester (AM-ester) of an Epac-selective cAMP analog (ESCA) penetrates into mouse islets and is capable of potentiating both first and second phases of glucose-stimulated insulin secretion (GSIS). When used at low concentrations (1-10 µM), 8-pCPT-2'-O-Me-cAMP-AM activates Rap1 GTPase but exhibits little or no ability to activate protein kinase A (PKA), as validated in assays of in vitro PKA activity (phosphorylation of Kemptide), Ser (133) CREB phosphorylation status, RIP1-CRE-Luc reporter gene activity, and PKA-dependent AKAR3 biosensor activation. Since quantitative PCR demonstrates Epac2 mRNA to be expressed at levels ca. 5.3-fold greater than that of Epac1, available evidence indicates that Epac2 does in fact mediate stimulatory actions of cAMP on mouse islet GSIS.