Global Phosphoproteomic Analysis of Insulin/Akt/mTORC1/S6K Signaling in Rat Hepatocytes

Insulin resistance is a hallmark of type 2 diabetes. Although multiple genetic and physiological factors interact to cause insulin resistance, deregulated signaling by phosphorylation is a common underlying mechanism. In particular, the specific phosphorylation-dependent regulatory mechanisms and signaling outputs of insulin are poorly understood in hepatocytes, which represents one of the most important insulin-responsive cell types. Using primary rat hepatocytes as a model system, we performed reductive dimethylation (ReDi)-based quantitative mass spectrometric analysis and characterized the phosphoproteome that is regulated by insulin as well as its key downstream kinases including Akt, mTORC1, and S6K. We identified a total of 12 294 unique, confidently localized phosphorylation sites and 3805 phosphorylated proteins in this single cell type. Detailed bioinformatic analysis on each individual data set identified both known and previously unrecognized targets of this key insulin downstream effector pathway. Furthermore, integrated analysis of the hepatic Akt/mTORC1/S6K signaling axis allowed the delineation of the substrate specificity of several close-related kinases within the insulin signaling pathway. We expect that the data sets will serve as an invaluable resource, providing the foundation for future hypothesis-driven research that helps delineate the molecular mechanisms that underlie the pathogenesis of type 2 diabetes and related metabolic syndrome

Inhibition of Glycogen Synthase Kinase-3β Prevents Remifentanil-Induced Hyperalgesia via Regulating the Expression and Function of Spinal N-Methyl-D-Aspartate Receptors In Vivo and Vitro

<div><p>A large number of experimental and clinical studies have confirmed that brief remifentanil exposure can enhance pain sensitivity presenting as opioid-induced hyperalgesia (OIH). N-methyl-D-aspartate (NMDA) receptor antagonists have been reported to inhibit morphine analgesic tolerance in many studies. Recently, we found that glycogen synthase kinase-3β (GSK-3β) modulated NMDA receptor trafficking in a rat model of remifentanil-induced postoperative hyperalgesia. In the current study, it was demonstrated that GSK-3β inhibition prevented remifentanil-induced hyperalgesia via regulating the expression and function of spinal NMDA receptors in vivo and in vitro. We firstly investigated the effects of TDZD-8, a selective GSK-3β inhibitor, on thermal and mechanical hyperalgesia using a rat model of remifentanil-induced hyperalgesia. GSK-3β activity as well as NMDA receptor subunits (NR1, NR2A and NR2B) expression and trafficking in spinal cord L₄-L₅ segments were measured by Western blot analysis. Furthermore, the effects of GSK-3β inhibition on NMDA-induced current amplitude and frequency were studied in spinal cord slices by whole-cell patch-clamp recording. We found that remifentanil infusion at 1 μg·kg^-1·min^-1 and 2 μg·kg^-1·min^-1 caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate of the spinal cord dorsal horn and increased GSK-3β activity in spinal cord dorsal horn. GSK-3β inhibitor TDZD-8 significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in dorsal horn neurons, which was prevented with the application of TDZD-8. These results suggest that inhibition of GSK-3β can significantly ameliorate remifentanil-induced hyperalgesia via modulating the expression and function of NMDA receptors, which present useful insights into the mechanistic action of GSK-3β inhibitor as potential anti-hyperalgesic agents for treating OIH.</p> </div

Quantitative Secretomic Analysis Identifies Extracellular Protein Factors That Modulate the Metastatic Phenotype of Non-Small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related deaths for men and women in the United States, with non-small cell lung cancer (NSCLC) representing 85% of all diagnoses. Late stage detection, metastatic disease and lack of actionable biomarkers contribute to the high mortality rate. Proteins in the extracellular space are known to be critically involved in regulating every stage of the pathogenesis of lung cancer. To investigate the mechanism by which secreted proteins contribute to the pathogenesis of NSCLC, we performed quantitative secretomic analysis of two isogenic NSCLC cell lines (NCI-H1993 and NCI-H2073) and an immortalized human bronchial epithelial cell line (HBEC3-KT) as control. H1993 was derived from a chemo-naïve metastatic tumor, while H2073 was derived from the primary tumor after etoposide/cisplatin therapy. From the conditioned media of these three cell lines, we identified and quantified 2713 proteins, including a series of proteins involved in regulating inflammatory response, programmed cell death and cell motion. Gene Ontology (GO) analysis indicates that a number of proteins overexpressed in H1993 media are involved in biological processes related to cancer metastasis, including cell motion, cell–cell adhesion and cell migration. RNA interference (RNAi)-mediated knock down of a number of these proteins, including SULT2B1, CEACAM5, SPRR3, AGR2, S100P, and S100A14, leads to dramatically reduced migration of these cells. In addition, meta-analysis of survival data indicates NSCLC patients whose tumors express higher levels of several of these secreted proteins, including SULT2B1, CEACAM5, SPRR3, S100P, and S100A14, have a worse prognosis. Collectively, our results provide a potential molecular link between deregulated secretome and NSCLC cell migration/metastasis. In addition, the identification of these aberrantly secreted proteins might facilitate the development of biomarkers for early detection of this devastating disease

Additional file 1 of Hydrogen alleviated cognitive impairment and blood‒brain barrier damage in sepsis-associated encephalopathy by regulating ABC efflux transporters in a PPARα-dependent manner

Supplementary Material

GSK-3β inhibition prevents the enhancement effect of remifentanil on NMDA receptor-mediated mEPSCs in dorsal horn neurons

<p>NMDA receptor-mediated mEPSCs in dorsal horn neurons were recorded at the holding potential of -70mV in the presence of TTX (10 μM), GABA receptor antagonist bicuculline (BIM, 20 μM) and AMPA receptor antagonist CNQX (20 μM). Representative traces of mEPSCs under control conditions (C group) and Remifentanil (R group) and Remifentainil+TDZD-8 treatment (RT group) were showed in A. <i>Scale </i><i>bar</i>, 100 pA, 30 s. Cumulative probability plots of mEPSCs amplitude distribution showed significant shift after remifentanil treatment in spinal dorsal horn neurons (Kolmogorov-Smirnov test, <i>P</i> < 0.01), but no shift after Remifentainil+TDZD-8 treatment (Kolmogorov-Smirnov test, <i>P</i> = 0.881), (B). The distribution of cumulative interevent intervals (IEIs) for mEPSCs showed significant shift after remifentanil treatment in spinal dorsal horn neurons (Kolmogorov-Smirnov test, <i>P</i> < 0.01), however there is no shift after Remifentainil+TDZD-8 treatment (Kolmogorov-Smirnov test, <i>P</i> = 0.932), (C). Summary bar graph of mEPSCs amplitude in dorsal horn neurons treated with different treatments (D). n = 8 for each group, vs C group, **<i>P</i> < 0.01, ANOVA. Bar graph of mEPSCs IEIs in dorsal horn neurons treated with different treatments (E). n = 8 for each group. vs C group, **<i>P</i> < 0.01, ANOVA.</p

Remifentanil infusion increases the GSK-3β activity in spinal dorsal horn.

<p>The total GSK-3β and phosphorylated GSK-3β in spinal dorsal horn were tested by Western blot. β-actin was used as the internal standard (a). The band intensity of C group was assigned a value of 1. Remifentanil resulted in significant decreases of pGSK-3β (ser9) and p-GSK-3β (ser9)/GSK-3β ratio, but had no effect on the total protein level of GSK-3β (b). GSK-3β inhibitor TDZD-8 prevented the changes of pGSK-3β (ser9) and pGSK-3β (ser9)/GSK-3β ratio. n = 5 for each group. Compaired with C group, * <i>P</i> < 0.05, ANOVA.</p

GSK-3β regulates the expression of NMDA receptors in spinal dorsal horn.

<p>Western blot for membrane NR1, NR2A and NR2B subunit was performed on rat spinal cord dorsal horn neuron (A). Epidermal growth factor receptor (EGFR) was used as the loading control. Pooled densitometric results for NR1, NR2A and NR2B, with the band intensity of C group assigned the value of 1. Remifentanil induced significant increases of both membrane NR1 and NR2B, but had no effect on membrane protein level of NR2A. GSK-3β inhibitor TDZD-8 prevented the changes of membrane NR1 and NR2B. n = 5 for each group, compared with C group, * <i>P</i> < 0.05, ANOVA (B). The expression of total NR1, NR2A and NR2B protein in spinal dorsal horn was tested by Western blot (C). β-actin was used as the loading control. Densitometry measurements from 5 groups were pooled and the band intensity of C group was assigned a value of 1. Remifentanil increased the total protein level of NR1 and NR2B, but had no effect on total protein level of NR2A. TDZD-8 prevented the changes of total protein expression of NR1 and NR2B (D). n = 5 for each group, compared with C group, * <i>P</i> < 0.05, ANOVA.</p

Outcome of hypothermia and normothermia.

<p>Outcome of hypothermia and normothermia.</p

Risk factors associated with intraoperative hypothermia.

<p>Risk factors associated with intraoperative hypothermia.</p

Incidence of intraoperative hypothermia, patient warming and clinical outcome (N = 3132).

<p>Incidence of intraoperative hypothermia, patient warming and clinical outcome (N = 3132).</p