Phosphatidylinositol 3-kinase P85 Research Articles

Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling

Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as cysteine (Cys) in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed phosphatidylinositol-3 kinase (PI3K)–Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor (EGF) receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K–Akt signaling by MVK reversed EGF-induced negative regulation of autophagy, and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K–Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K–Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.

Altered Placental Insulin Signaling in the Absence of Systemic Insulin Resistance in Hypertensive Disorders of Pregnancy among South Indian Tamil Population

A cross sectional study was conducted to explore the possible derangements in placental insulin signaling and also to investigate the presence or absence of maternal systemic insulin resistance in different types of hypertensive disorders of pregnancy (HDP). Study Design: Healthy pregnant women (n=32) and pregnant women with gestational hypertension (GH) (n=18), late onset Pre-eclampsia (LP) (n=18) and early onset Pre-eclampsia (EP) (n=19) were recruited for the study. Maternal venous blood and placental samples were collected from the study subjects at 35±7 weeks of gestation. Insulin resistance parameters such as fasting glucose, fasting insulin, homeostatic model assessment of insulin resistance, beta cell function and insulin sensitivity and glycated hemoglobin were estimated in the maternal venous blood samples. Placental protein expressions of insulin signaling molecules were analyzed by western blotting method. Results: Maternal venous blood insulin resistance parameters were not significantly different between the study groups. However, placental content of phospho insulin receptor beta was significantly decreased in GH, LP and EP groups in comparison to controls. We also found a significant decrease in placental protein expression of phosphatidyl inositol 3-kinase p85 alpha in LP group when compared to other groups.

Hydrophobic Bile Salts Induce Pro-Fibrogenic Proliferation of Hepatic Stellate Cells through PI3K p110 Alpha Signaling.

Bile salts accumulating during cholestatic liver disease are believed to promote liver fibrosis. We have recently shown that chenodeoxycholate (CDC) induces expansion of hepatic stellate cells (HSCs) in vivo, thereby promoting liver fibrosis. Mechanisms underlying bile salt-induced fibrogenesis remain elusive. We aimed to characterize the effects of different bile salts on HSC biology and investigated underlying signaling pathways. Murine HSCs (mHSCs) were stimulated with hydrophilic and hydrophobic bile salts. Proliferation, cell mass, collagen deposition, and activation of signaling pathways were determined. Activation of the human HSC cell line LX 2 was assessed by quantification of α-smooth muscle actin (αSMA) expression. Phosphatidyl-inositol-3-kinase (PI3K)-dependent signaling was inhibited both pharmacologically and by siRNA. CDC, the most abundant bile salt accumulating in human cholestasis, but no other bile salt tested, induced Protein kinase B (PKB) phosphorylation and promoted HSC proliferation and subsequent collagen deposition. Pharmacological inhibition of the upstream target PI3K-inhibited activation of PKB and pro-fibrogenic proliferation of HSCs. The PI3K p110α-specific inhibitor Alpelisib and siRNA-mediated knockdown of p110α ameliorated pro-fibrogenic activation of mHSC and LX 2 cells, respectively. In summary, pro-fibrogenic signaling in mHSCs is selectively induced by CDC. PI3K p110α may be a potential therapeutic target for the inhibition of bile salt-induced fibrogenesis in cholestasis.

Circ_0000523 regulates miR-1184/COL1A1/PI3K/Akt pathway to promote nasopharyngeal carcinoma progression.

The present study is to investigate the biological functions and mechanisms of circular RNA_0000523 (circ_0000523) in nasopharyngeal carcinoma (NPC). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to examine the expression levels of circ_0000523 and microRNA-1184 (miR-1184) in NPC tissues and cells. Collagen type 1 alpha 1 chain (COL1A1) expression was assessed by qRT-PCR and immunohistochemistry (IHC) assay. Cell proliferation, cell cycle progression, migration and invasion were examined by cell counting kit-8 (CCK-8), 5-bromo-2'-deoxyuridine (BrdU), flow cytometry and Transwell assays. Xenograft nude mouse models were used to investigate the metastatic potential of NPC cells in vivo. The binding relationships between circ_0000523 and miR-1184, and between miR-1184 and COL1A1 were detected by dual-luciferase reporter gene assay. The protein expressions of COL1A1, phosphatidylinositol 3-kinase (p85), phosphorylated (p)-p85, protein kinase B (Akt) and p-Akt were detected through Western blot. The DAVID database was used for the enrichment analysis of the potential targets of miR-1184. Circ_0000523 and COL1A1 mRNA expressions were significantly increased in NPC tissues and cell lines. Circ_0000523 overexpression promoted NPC cell proliferation and accelerated cell cycle progression, whereas miR-1184 overexpression reversed these effects; circ_0000523 knockdown suppressed NPC cell proliferation and induced cell cycle arrest, while miR-1184 inhibition counteracted these effects. MiR-1184 was the downstream target of circ_0000523, and COL1A1 was the target gene of miR-1184 and could be positively modulated by circ_0000523. COL1A1 overexpression increased the expression levels of p-p85 and p-Akt, whereas knocking down COL1A1 repressed their expressions. Circ_0000523 facilitates NPC progression through regulating the miR-1184/COL1A1 axis.

Synergism between the phosphatidylinositol 3-kinase p110\u03b2 isoform inhibitor AZD6482 and the mixed lineage kinase 3 inhibitor URMC-099 on the blockade of glioblastoma cell motility and focal adhesion formation

BackgroundGlioblastoma multiforme, the most aggressive and malignant primary brain tumor, is characterized by rapid growth and extensive infiltration to neighboring normal brain parenchyma. Our previous studies delineated a crosstalk between PI3K/Akt and JNK signaling pathways, and a moderate anti-glioblastoma synergism caused by the combined inhibition of PI3K p110β (PI3Kβ) isoform and JNK. However, this combination strategy is not potent enough. MLK3, an upstream regulator of ERK and JNK, may replace JNK to exert stronger synergism with PI3Kβ.MethodsTo develop a new combination strategy with stronger synergism, the expression pattern and roles of MLK3 in glioblastoma patient’s specimens and cell lines were firstly investigated. Then glioblastoma cells and xenografts in nude mice were treated with the PI3Kβ inhibitor AZD6482 and the MLK3 inhibitor URMC-099 alone or in combination to evaluate their combination effects on tumor cell growth and motility. The combination effects on cytoskeletal structures such as lamellipodia and focal adhesions were also evaluated.ResultsMLK3 protein was overexpressed in both newly diagnosed and relapsing glioblastoma patients’ specimens. Silencing of MLK3 using siRNA duplexes significantly suppressed migration and invasion, but promoted attachment of glioblastoma cells. Combined inhibition of PI3Kβ and MLK3 exhibited synergistic inhibitory effects on glioblastoma cell proliferation, migration and invasion, as well as the formation of lamellipodia and focal adhesions. Furthermore, combination of AZD6482 and URMC-099 effectively decreased glioblastoma xenograft growth in nude mice. Glioblastoma cells treated with this drug combination showed reduced phosphorylation of Akt and ERK, and decreased protein expression of ROCK2 and Zyxin.ConclusionTaken together, combination of AZD6482 and URMC-099 showed strong synergistic anti-tumor effects on glioblastoma in vitro and in vivo. Our findings suggest that combined inhibition of PI3Kβ and MLK3 may serve as an attractive therapeutic approach for glioblastoma multiforme.

KLHL18 inhibits the proliferation, migration, and invasion of non-small cell lung cancer by inhibiting PI3K/PD-L1 axis activity

BackgroundThe expression of Kelch-like protein 18 (KLHL18) in non-small cell lung cancer (NSCLC) is lower than that in normal lung tissue according to the Gene Expression Profiling Interactive Analysis database. KLHL18 is a BTB domain protein and binds cullin 3 (CUL3). However, whether this complex participates in ubiquitination-mediated protein degradation in NSCLC is unclear. Therefore, we aimed to investigate the role of KLHL18 in human NSCLC cells.ResultsWe found that KLHL18 is downregulated in cancer cells and is associated with poor prognosis. Further, its expression was significantly associated with tumor node metastasis (TNM) stage, lymph node metastasis, and tumor size. In vitro analysis of NSCLC cells showed that overexpressing KLHL18 inhibited cell proliferation, migration, and invasion. We found that the tumor-inhibitory effect of the KLHL18 protein was achieved by promoting the ubiquitination and degradation of phosphatidylinositol 3-kinase (PI3K) p85α and inhibiting the expression of PD-L1 protein, ultimately preventing tumor cell immune escape.ConclusionsOur results identified the tumor-suppressive mechanism of KLHL18 and suggested that it is closely related to NSCLC occurrence and development. Further investigation of the underlying mechanism may provide new targets for NSCLC treatment.

Involvement of intracellular caveolin-1 distribution in the suppression of antigen-induced mast cell activation by cationic liposomes.

Cationic liposomes are commonly used as vectors to effectively introduce foreign genes into target cells. In another function, we recently showed that cationic liposomes bound to the mast cell surface suppress the degranulation induced by the cross-linking of high-affinity immunoglobulin E receptor in a time- and dose-dependent manner. This suppression is mediated by the impairment of the sustained level of intracellular Ca2+ concentration ([Ca2+ ]i ) via the inhibition of store-operated Ca2+ entry. Further, we revealed that the mechanism underlying an impaired [Ca2+ ]i increase is the inhibition of the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Yet, how cationic liposomes inhibit the PI3K-Akt pathway is still unclear. Here, we focused on caveolin-1, a major component of caveolae, which is reported to be involved in the activation of the PI3K-Akt pathway in various cell lines. In this study, we showed that caveolin-1 translocated from the cytoplasm to the plasma membrane after the activation of mast cells and colocalized with the p85 subunit of PI3K, which seemed to be essential for PI3K activity. Meanwhile, cationic liposomes suppressed the translocation of caveolin-1 to the plasma membrane and the colocalization of caveolin-1 with PI3K p85 also at the plasma membrane. This finding provides new information for the development of therapies using cationic liposomes against allergies.

Dietary Leucine Supplementation Restores Serum Glucose Levels, and Modifying Hepatic Gene Expression Related to the Insulin Signal Pathway in IUGR Piglets.

Simple SummaryIntrauterine malnutrition may compromise the size and structure of fetal organs and tissues, which leads to lower birth weight and a slower rate of growth after weaning. Intrauterine growth restriction/retardation (IUGR) impairs pancreas function, resulting in the decreased glucose levels in serum. Leucine, one of branched chain amino acids, is an essential amino acid and the substrate of protein synthesis. Leucine also acts as a major regulator of hormone signal transduction, like insulin. Dietary branched chain amino acids or leucine have beneficial effects on the glucose metabolism and glycogen synthesis of muscle. Leucine supplementation improves the insulin sensitivity in liver and muscle and then influences the systemic glucose homeostasis. However, it is still unclear whether leucine supplementation would alter insulin sensitivity in IUGR neonatal piglets. Our results showed that dietary leucine supplementation restored serum glucose concentrations, increased insulin and creatinine concentrations, and enhanced protein kinase adenosine monophosphate-activated γ 3-subunit and glucose transporter type 2 expression. These findings suggest that leucine might play a positive role in hepatic lipid metabolism and glucose metabolism in IUGR.This study aimed to investigate the effects of leucine with different levels on the insulin resistance in intrauterine growth restriction/retardation (IUGR) piglets. Thirty-two weaned piglets were arranged in a 2 × 2 factorial design and four treatments (n = 8) were as follow: (1) normal weaned piglets fed a basal diet (CONT), (2) IUGR weaned piglets fed a basal diet (IUGR), (3) normal weaned piglets fed a basal diet with the addition of 0.35% l-leucine (C-LEU), and (4) IUGR fed a basal diet with the addition of 0.35% l-leucine (I-LEU) for a 21-days trial. The results showed that compared to the IUGR group, the I-LEU group had higher final body weight and body weight gain, higher serum glucose concentrations, and higher serum insulin concentrations (p < 0.05). The gene expression of phosphatidylinositol 3-kinase p110 gamma, protein kinase adenosine monophosphate-activated γ 3-subunit, glycogen synthase kinase-3 alpha, and glucose transporter type 2 were increased in the I-LEU group as compared to the IUGR group (p < 0.05). It was concluded that dietary leucine supplementation restored serum glucose concentrations, increased insulin and creatinine concentrations, and enhanced protein kinase adenosine monophosphate-activated γ 3-subunit and glucose transporter type 2 expression, suggesting that leucine might play a positive role in hepatic lipid metabolism and glucose metabolism in IUGR.

Torin2 Exploits Replication and Checkpoint Vulnerabilities to Cause Death of PI3K-Activated Triple-Negative Breast Cancer Cells.

Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

305-LB: BRD7 Deficiency Leads to the Development of Obesity

Elevated endoplasmic reticulum (ER) stress is tightly associated with the development of obesity and insulin resistance. Previous reports showed that haploinsufficiency of X-box binding protein 1 (XBP1), one of the master regulators of ER protein folding function, results in obesity and type 2 diabetes. In our previous work, we showed that bromodomain-containing protein 7 (BRD7) regulates the nuclear translocation of XBP1 through its interaction with the regulatory subunits of phosphatidylinositol 3 kinase (PI3K), p85s. We reported that the expression levels of BRD7 are significantly reduced in the liver of obese mice and acute upregulation of hepatic BRD7 in obese and type 2 diabetic mice improves glucose homeostasis. In our current unpublished work, we show that nuclear translocation of BRD7 is independent of PI3K activity and p110, the catalytic subunit of PI3K, is not involved in the degradation of BRD7. We show that both whole-body reduction and liver-specific deletion of BRD7 lead to increased weight gain. We also show that sustained restoration of BRD7 in the liver starting at an early age protects mice from developing obesity, glucose intolerance, and insulin resistance even when challenged with a high-fat diet. Disclosure J.M. Lee: None. Y. Kim: None. M.A. Salazar Hernandez: None. S. Park: None. Funding American Diabetes Association (1-17-IBS-104 to S.P.); National Institutes of Health

Laminin promotes differentiation of rat embryonic stem cells into cardiomyocytes by activating the integrin/FAK/PI3K p85 pathway

The generation of germline competent rat embryonic stem cells (rESCs) allows the study of their lineage commitment. Here, we developed a highly efficient system for rESC‐derived cardiomyocytes, and even the formation of three‐dimensional (3D)‐like cell clusters with cTNT and α‐Actinin. We have validated that laminin can interact with membrane integrin to promote the phosphorylation of both phosphatidylinositol 3‐kinase (PI3K) p85 and the focal adhesion kinase (FAK). In parallel, GATA4 was up‐regulated. Upon inhibiting the integrin, laminin loses the effect on cardiomyocyte differentiation, accompanied with a down‐regulation of phosphorylation level of PI3K p85 and FAK. Meanwhile, the expression of Gata4 was inhibited as well. Taken together, laminin is a crucial component in the differentiation of rESCs into cardiomyocytes through increasing their proliferation via interacting with integrin pathway. These results provide new insights into the pathways mediated by extracellular laminin involved in the fate of rESC‐derived cardiomyocytes.

MiR-409 Inhibits Human Non-Small-Cell Lung Cancer Progression by Directly Targeting SPIN1

Lung cancers, the leading cause of cancer mortality worldwide, are characterized by a high metastatic potential. Growing evidence reveals that Spindlin 1 (SPIN1) is involved in tumor progression and carcinogenesis. However, the role of SPIN1 in non-small-cell lung cancer (NSCLC) and the molecular mechanisms underlying SPIN1 in human NSCLC remain undetermined. Here we examined the function of SPIN1 in human NSCLC and found that the expression of SPIN1 was closely correlated with the overall survival and poor prognosis of NSCLC patients. Aberrant regulation of microRNAs (miRNAs) has an important role in cancer progression. We revealed that miR-409 inhibits the expression of SPIN1 by binding directly to the 3′ UTR of SPIN1 using dual-luciferase reporter assays. Overexpression of miR-409 significantly suppressed cell migration, growth, and proliferation by inhibiting SPIN1 in vitro and in vivo. SPIN1 overexpression in miR-409-transfected NSCLC cells effectively rescued the suppression of cell migration, growth, and proliferation regulated by miR-409. miR-409 regulates the PI3K/AKT (protein kinase B) pathway in NSCLC. Moreover, clinical data showed that NSCLC patients with high levels of miR-409 experienced significantly better survival. miR-409 expression was also negatively associated with SPIN1 expression. Taken together, these findings highlight that the miR-409/SPIN1 axis is a useful pleiotropic regulatory network and could predict the metastatic potential in NSCLC patients early, indicating the possibility that miR-409 and SPIN1 might be attractive prognostic markers for treating NSCLC patients.

PIK3R1 gene polymorphisms are associated with type 2 diabetes and related features in the Turkish population.

The phosphatidylinositol 3-kinase p85 alpha regulatory subunit 1 gene (PIK3R1) encodes the PIK3R1 protein, which plays a direct role in insulin signaling. PIK3R1 (p85 regulatory subunit) connects firmly with the p110 catalytic subunit, and together these proteins form the phosphatidylinositol 3-kinase (PI3K) protein. PI3K is a key protein in the Akt signaling pathway, which regulates cell survival, growth, differentiation, glucose trafficking, and utilization. Defects in the insulin signaling cascade play an important role in the development of insulin resistance, which shares a common genetic basis for metabolic diseases such as type 2 diabetes (T2D), obesity and cardiovascular diseases. In our study, we investigated the effect of single nucleotide polymorphisms (SNPs) rs3756668 in 3'UTR region, rs706713 and rs3730089 in exons 1 and 6, respectively, rs7713645 and rs7709243 in intron 1, and rs1550805 in intron 6 of PIK3R1 gene on T2D. This study enrolled a total of 840 individuals, including 427 diabetic individuals (206 obese and 221 non-obese) and 413 nondiabetic individuals (138 obese and 275 non-obese). The target SNPs were analyzed using real-time polymerase chain reaction (RT-PCR). Statistical analysis was performed using SPSS18.0 (IBM Corp., Armonk, USA). The p-values ≤0.05 were considered statistically significant. The SNPs rs706713 (Tyr73Tyr) and rs3730089 (Met326Ile) located in exons, and rs7713645, rs7709243 and rs1550805 located in introns were determined to be significantly associated with T2D and phenotypic features such as obesity, insulin resistance and the lipid parameters. The association with SNP rs3756668, which is located in the 3'UTR, was not significant. Our study supports the role of PIK3R1, an important candidate gene due to its critical role in insulin signal transduction, in T2D development.

IGF1 receptor signaling pathways.

Insulin-like growth factors (IGFs) bind specifically to the IGF1 receptor on the cell surface of targeted tissues. Ligand binding to the α subunit of the receptor leads to a conformational change in the β subunit, resulting in the activation of receptor tyrosine kinase activity. Activated receptor phosphorylates several substrates, including insulin receptor substrates (IRSs) and Src homology collagen (SHC). Phosphotyrosine residues in these substrates are recognized by certain Src homology 2 (SH2) domain-containing signaling molecules. These include, for example, an 85 kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase), growth factor receptor-bound 2 (GRB2) and SH2-containing protein tyrosine phosphatase 2 (SHP2/Syp). These bindings lead to the activation of downstream signaling pathways, PI 3-kinase pathway and Ras-mitogen-activated protein kinase (MAP kinase) pathway. Activation of these signaling pathways is known to be required for the induction of various bioactivities of IGFs, including cell proliferation, cell differentiation and cell survival. In this review, the well-established IGF1 receptor signaling pathways required for the induction of various bioactivities of IGFs are introduced. In addition, we will discuss how IGF signals are modulated by the other extracellular stimuli or by themselves based on our studies.

Protective effects of PI3KCG gene on acute myocardial infarction.

To study the protective effects of recombinant phosphatidylinositol 3-kinase p110 gamma (rPLV-PI3KCG) lentiviral vector in Sprague-Dawley (SD) rats with acute myocardial infarction (AMI). The AMI rat models were established by ligaturing left anterior descending coronary artery. The rPLV-PI3KCG or empty lentiviral vectors were injected at the edge of the infarct zone. The experiment was divided randomly into four groups (n=8): (I) Sham group; (II) AMI group; (III) AMI + empty vector injection group (AMI + E group); and (IV) AMI + PLV-PI3KCG injection group (AMI + PLV-PI3KCG group). The ultrasonic cardiogram (UCG) was used to compare the structural or functional changes among the four groups after operation for 10 days. Meanwhile, the rats were sacrificed and HE staining was used to compare the myocardial tissue changes among the four groups. The immunofluorescence and western blots were performed to compare the angiogenesis in the infarct region and explore the mechanism of the protective effects of PI3KCG gene on AMI rats. Compared with AMI group and AMI + E group, in the AMI + PLV-PI3KCG group, left ventricular end diastolic diameter (LVEDd) was decreased, left ventricular ejection fraction (LVEF%) was significantly increased, and vascular endothelial growth factor (VEGF) expression was significantly increased in the infarct region (P<0.05); PI3KCG, pAkt/Akt, HIF-1a, and Bcl-2/Bax protein expressions were significantly increased (P<0.05). The rPLV-PI3KCG injection could improve the cardiac function, relieve the cardiac injury after the AMI operation. PI3KCG gene could play the protection role in the AMI process possibly by activating PI3K/Akt signal pathway, inhibiting apoptosis and promoting angiogenesis.

Jia-Wei-Jiao-Tai-Wan ameliorates type 2 diabetes by improving \u03b2 cell function and reducing insulin resistance in diabetic rats

BackgroundJia-Wei-Jiao-Tai-Wan (JWJTW), composed of Jiao-Tai-Wan (Cinnamomum cassia and Rhizoma coptidis) and other antidiabetic herbs, including Astragalus membranaceus, Herba Gynostemmatis, Radix Puerariae Lobatae, Folium Mori and Semen Trigonellae, is widely used to treat diabetes and has demonstrated a curative effect in the clinic, but the potential mechanism is unknown. This study aimed to explore the effects of JWJTW on diabetic rats and to clarify the underlying mechanism.MethodsJWJTW was prepared, and the main components contained in the formula were identified by high-performance liquid chromatography (HPLC) fingerprint analysis. Diabetic rats induced by streptozotocin (STZ) and a high-sucrose-high-fat diet were treated with two concentrations of JWJTW (1.025 and 2.05 g/kg/d) for 100 days. The oral glucose tolerance test (OGTT), insulin release test (IRT) and insulin tolerance test (ITT) were performed to measure the glycometabolism of the diabetic rats at the end of the treatment period. Blood was collected to determine the serum lipid levels of the diabetic rats. Nitric oxide (NO), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were detected in pancreas homogenates to analyze the oxidative stress in the pancreata of diabetic rats, and the expression levels of pancreatic and duodenal homeobox 1 (PDX-1) and insulin in the pancreas were tested by Western blot to measure pancreatic islet function. In addition, Western blots were used to measure the expression of proteins related to the insulin signaling pathway in skeletal muscle of the diabetic rats.ResultsThe results showed that the administration of JWJTW could ameliorate impairments in glucose tolerance, insulin release function and insulin tolerance in diabetic rats. JWJTW could also dose-dependently reduce serum lipid levels in diabetic rats. JWJTW restrained oxidative stress by decreasing the expression of NO and MDA and increasing the expression of SOD and GSH-px. JWJTW improved the function of pancreatic β cells by increasing PDX-1 and insulin expression. In addition, JWJTW restored the impaired insulin signaling; upregulated phospho-insulin receptor (pInsR) expression, insulin receptor substrate (IRS) tyrosine phosphorylation, phosphatidylinositol 3-kinase (PI3K) (p85), and glucose transporter 4 (GLUT4) expression; and downregulated the serine phosphorylation of IRS.ConclusionsThis study suggests that JWJTW can ameliorate type 2 diabetes by improving β cell function and reducing insulin resistance in diabetic rats.

Phosphatidylinositol 3-kinase inhibition potentiates glucocorticoid response in B-cell acute lymphoblastic leukemia.

Despite remarkable progress in polychemotherapy protocols, pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains fatal in around 20% of cases. Hence, novel targeted therapies are needed for patients with poor prognosis. Glucocorticoids (GCs) are drugs commonly administrated for B-ALL treatment. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway is frequently observed in B-ALL and contributes to GC-resistance. Here, we analyzed for the first time to our knowledge, the therapeutic potential of pan and isoform-selective PI3K p110 inhibitors, alone or combined with dexamethasone (DEX), in B-ALL leukemia cell lines and patient samples. We found that a pan PI3K p110 inhibitor displayed the most powerful cytotoxic effects in B-ALL cells, by inducing cell cycle arrest and apoptosis. Both a pan PI3K p110 inhibitor and a dual γ/δ PI3K p110 inhibitor sensitized B-ALL cells to DEX by restoring nuclear translocation of the GC receptor and counteracted stroma-induced DEX-resistance. Finally, gene expression analysis documented that, on one hand the combination consisting of a pan PI3K p110 inhibitor and DEX strengthened the DEX-induced up- or down-regulation of several genes involved in apoptosis, while on the other, it rescued the effects of genes that might be involved in GC-resistance. Overall, our findings strongly suggest that PI3K p110 inhibition could be a promising strategy for treating B-ALL patients by improving GC therapeutic effects and/or overcoming GC-resistance.

Interaction between CD133 and phosphatidylinositol 3-kinase p85 promote the chemoresistance capacity of gastric cancer cells

Objective By building stable CD133 knock-down and overexpression cell lines, we will study its effects on the ability of multiple chemoresistance and the possible relevant mechanism. Methods Firstly, we established KATOⅢ-shCD133 and MKN45-CD133 cell lines, and then tested the interference and overexpression efficiency of CD133 by Western blotting. Secondly, we detected the cells sensibility to 5-fluorouracil (5-Fu) and cisplatin (DDP) by cell counting kit-8 (CCK-8), and measured the protein level of chemoresistance related factors: P-gp, B cell lymphoma/leukemia-2 (bcl-2), B cell lymphoma/leukemia-2 associated X protein (bax) and the activation level of protein kinase B (Akt) by Western blotting. Finally, we examined the interaction of CD133 and p85 by immunoprecipitation. Results By Western blotting, we found the protein level of CD133 of the knock-down or overexpression cell lines were significantly lower or higher than the control group. Under the same drug concentrations, 5-Fu and DDP showed higher inhibition rate in the CD133 konck-down group than that of the vector group with the half maximal inhibitory concentration (IC50) value [(54.030 0±0.647 9) μmol/L vs. (133.300 0±4.918 0) μmol/L, P=0.005] and [(2.312 0±0.223 6) μmol/L vs. (24.590 0±2.159 0) μmol/L, P=0.001], while the overexpression group took the opposite sides with the IC50 value [(8.102 0±0.433 1) μmol/L vs. (2.217 0±0.132 4) μmol/L, P=0.010] and [(24.487 0±2.132 0) μmol/L vs. (11.620 0±1.534 0) μmol/L, P=0.030]. After stablyknock-down the expression of CD133, the protein levels of chemoresistance related factor: P-gp, bcl-2 and p-Akt were significantly lower, and the promoting apoptosis factor bax was significantly increased; the overexpression group was still the opposite. Conclusion CD133 could obviously increase the chemoresistance capacity of gastric cancer cells, which might be realized through the activation of phosphoinositide 3-kinase (PI3K)/Akt pathway by CD133-p85 interaction. Key words: Gastric cancer; CD133; Chemoresistance; Phosphatidylinositol 3-kinase p85

Wu-Mei-Wan Reduces Insulin Resistance via Inhibition of NLRP3 Inflammasome Activation in HepG2 Cells

Wu-Mei-Wan (WMW) is a Chinese herbal formula used to treat type 2 diabetes. In this study, we aimed to explore the effects and mechanisms of WMW on insulin resistance in HepG2 cells. HepG2 cells were pretreated with palmitate (0.25 mM) to impair the insulin signaling pathway. Then, they were treated with different doses of WMW-containing medicated serum and stimulated with 100 nM insulin. Results showed that palmitate could reduce the glucose consumption rate in HepG2 cells and impair insulin signaling related to phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), thereby regulating the downstream signaling pathways. However, medicated serum of WMW restored impaired insulin signaling, upregulated the expression of phospho-IR (pIR), phosphatidylinositol 3-kinase p85 subunit, phosphoprotein kinase B, and glucose transporter 4, and decreased IRS serine phosphorylation. In addition, it decreased the expression of interleukin-1β and tumor necrosis factor-α, which are the key proinflammatory cytokines involved in insulin resistance; besides, it reduced the expression of NLRP3 inflammasome. These results suggested that WMW could alleviate palmitate-induced insulin resistance in HepG2 cells via inhibition of NLRP3 inflammasome and reduction of proinflammatory cytokine production.

Effects of electroacupuncture on insulin resistance and hypothalamic insulin signal molecule in rats with diet-induced obesity

To observe the effects of electroacupuncture (EA) on body weight, insulin resistance (IR) and hypothalamic insulin signal molecule in rats with diet-induced obesity (DIO), and to explore the action mechanism of EA on DIO. Fifty SD male rats were randomly divided into a low fat diet (LFD) group (10 rats) and a high fat diet (HFD) group (40 rats). Rats were fed with LFD and HFD, respectively, and the DIO model was established in the HFD group. After the model was established, the rats were randomly divided intoa model group, an EA group and a medication group, ten rats in each one. The rats in the EA group were treated with EA at "Housanli" (ST 36) and "Quchi" (LI 11) for 20 min, once a day for totally 28 days. The rats in the medication group were treated with intragastric administration of orlistat, once a day for 28 days. The rats in the LFD group and model group received no treatment. After treatment, HE staining method was applied to observe the morphological changes of liver; the biochemistry technique and radioimmunoassay method were applied to detect the fasting plasma glucose (FPG) and fasting insulin (FINS); Western blot method was applied to measure the expression of phosphatidylinositol-3 kinase p85 subunit (PI3K-p85) and insulin receptor substrate 2 (IRS2). Under light microscope, compared with the model group, the fatty degenerative cells were below 1/2 in the EA group and the medication group, accompanied with decreased lipid droplet, mild edema and none inflammatory infiltration. The body weight, FPG, FINS, homeostasis model assessment-insulin resistance index (HOMA-IR) and PI3K-p85 in the EA group were significantly lower than those in the model group (P<0.01, P<0.05), but the expression of IRS2 was not significantly different from the model group (P>0.05). The body weight, HOMA-IR and PI3K-p85 in the medication group were lower than those in the model group (P<0.01, P<0.05), but the expression of IRS2 was higher than that in the model group (P<0.05). The differences of each index were not significant between the EA group and the medication group (all P>0.05). In addition, rats in the medication group showed watery defecation, decreased activity, fatigue mentality and yellow hairs, while rats in the EA group showed normal defecation and vivid hair. EA can decrease the expression of PI3K-p85 to prompt the IR of DIO rats, inhibit the weight body and improve hepatic steatosis, which is probably one of the action mechanisms of EA on DIO. Besides, the adverse effects as the medication group can be avoided.