Lipid-correlated alterations in the transcriptome are enriched in several specific pathways in the postmortem prefrontal cortex of Japanese patients with schizophrenia.

AIMS: Schizophrenia is a chronic relapsing psychiatric disorder that is characterized by many symptoms and has a high heritability. There were studies showing that the phospholipid abnormalities in subjects with schizophrenia (Front Biosci, S3, 2011, 153; Schizophr Bull, 48, 2022, 1125; Sci Rep, 7, 2017, 6; Anal Bioanal Chem, 400, 2011, 1933). Disturbances in prefrontal cortex phospholipid and fatty acid composition have been reported in subjects with schizophrenia (Sci Rep, 7, 2017, 6; Anal Bioanal Chem, 400, 2011, 1933; Schizophr Res, 215, 2020, 493; J Psychiatr Res, 47, 2013, 636; Int J Mol Sci, 22, 2021). For exploring the signaling pathways contributing to the lipid changes in previous study (Sci Rep, 7, 2017, 6), we performed two types of transcriptome analyses in subjects with schizophrenia: an unbiased transcriptome analysis solely based on RNA-seq data and a correlation analysis between levels of gene expression and lipids. METHODS: RNA-Seq analysis was performed in the postmortem prefrontal cortex from 10 subjects with schizophrenia and 5 controls. Correlation analysis between the transcriptome and lipidome from 9 subjects, which are the same samples in the previous lipidomics study (Sci Rep, 7, 2017, 6). RESULTS: Extraction of differentially expressed genes (DEGs) and further sequence and functional group analysis revealed changes in gene expression levels in phosphoinositide 3-kinase (PI3K)-Akt signaling and the complement system. In addition, a correlation analysis clarified alterations in ether lipid metabolism pathway, which is not found as DEGs in transcriptome analysis alone. CONCLUSIONS: This study provided results of the integrated analysis of the schizophrenia-associated transcriptome and lipidome within the PFC and revealed that lipid-correlated alterations in the transcriptome are enriched in specific pathways including ether lipid metabolism pathway.

Oroxylum indicum Stem Bark Extract Reduces Tumor Progression by Inhibiting the EGFR-PI3K-AKT Pathway in an In Vivo 4NQO-Induced Oral Cancer Model.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is the predominant type of oral cancer. Its incidence is high in certain geographic regions, and it is correlated with chewing tobacco. Epidermal growth factor receptor (EGFR), induced by tobacco carcinogens, is overexpressed in OSCC, leading to poor prognosis. Thus, EGFR inhibitors are promising agents against OSCC. High cost and toxicity of existing EGFR inhibitors necessitate alternative EGFR-targeted therapy. Here, we tested the antitumor potential of ethyl acetate fraction of an ethnomedicinal tree, Oroxylum indicum stem bark extract (OIEA) in a 4-nitroquinoline-1-oxide (4NQO)-induced oral carcinogenesis model. METHODS: OIEA was prepared by solvent extraction method, and subsequently its in vitro radical scavenging activities were measured. High-performance liquid chromatography (HPLC) analysis of OIEA was done to identify the constituent active compounds. Hemolytic, trypan blue exclusion, and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assays were performed in normal and cancer cells to select an optimum dose of OIEA for antitumor activity study in 4NQO-induced oral cancer in F344 rats. Measurement of tumor volume, weight, and cell count was followed by tumor cell cycle analysis and comet and annexin V/Propidium Iodide (PI) assay. Pro-apoptotic markers were detected by western blot testing. Molecular docking was done to predict the interaction between OIEA active component and EGFR or phosphatidylinositol-3-kinase (PI3K), which was further validated biologically. Finally, hepatic and renal function testing and histopathology were performed. RESULTS: OIEA reduced tumor burden and increased survivability of the tumor-bearing rats significantly as compared to untreated tumor bearers. HPLC revealed oroxylin A as the predominant bioactive component in OIEA. Molecular docking predicted significant binding between oroxylin A and EGFR as well as PI3K, which was confirmed by western blot analysis of in vivo samples. OIEA also ameliorated hepato-, renal- and myelotoxicity induced by 4NQO. CONCLUSION: OIEA reduces 4NQO-induced OSCC by modulating the EGFR/PI3K/AKT signaling cascade and also ameliorated toxicity in tumor bearers.

Proteomic profile of extracellular matrix from native and decellularized chorionic canine placenta.

Placental plasticity, employing rapid growth and remodeling to supply the growing fetus, is majorly related to its extracellular matrix (ECM) components. Thus, we studied the proteome profiled of canine native and decellularized placenta to characterize the proteome related to maintenance of a microenvironment and structure suitable for tissue engineering applications. Protein was profiled from native (n=3) and decellularized (n=3) 35-days old canine placenta using the mass spectrometer Orbitrap Fusion Lumos. A total of 52 proteins were filtered and revealed ontologies connected to skeleton structuration, collagen processing, germ layers formation, cell adhesion, response to amino acids, and others. Also, the major enriched pathways were ECM-receptor interaction, focal adhesion, PI3K-Akt signaling, protein digestion and absorption. Aside, proteins related to structure (collagens), cell adhesion (laminin and fibronectin), ECM remodeling (MMP2 and TIMP3) and vascularization (VEGF and RLN) were present in decellularized condition. Our findings support the requirement of a proteomic profile to visualize the maintenance of essential protein groups for ECM structuring and physiology, that should support functions related to cell adhesion, vasculogenesis and as a reservoir of soluble molecules. Altogether, the 35-days old decellularized canine placenta can provide an adequate microenvironment for cell anchoring for further regenerative medicine application.

The effectiveness of monotherapy with PI3K/AKT/mTOR pathway inhibitors in ovarian cancer: A meta-analysis.

OBJECTIVE: To determine the clinical benefit of monotherapy with PI3K/AKT/mTOR inhibitors in patients diagnosed with advanced or recurrent ovarian cancer and to investigate the predictive value of current PI3K/AKT/mTOR biomarkers on therapy response. METHODS: A systematic search was conducted in PubMed, Embase and the Cochrane Library for articles reporting on treatment with PI3K/AKT/mTOR inhibitors in ovarian cancer. The primary endpoint was defined as the clinical benefit rate (CBR), including the proportion of patients with complete (CR) and partial response (PR) and stable disease (SD). Secondary endpoints included the overall response rate (ORR, including CR and PR) and drug-related grade 3 and 4 adverse events. RESULTS: We included 233 patients from 19 studies and observed a pooled CBR of 32% (95% CI 20-44%) and ORR of 3% (95% CI 0-6%) in advanced or recurrent ovarian cancer patients treated with PI3K/AKT/mTOR inhibitors. Subgroup analysis tended to favor the studies who selected patients based on current PI3K/AKT/mTOR biomarker criteria (e.g. genomic alterations or loss of PTEN protein expression), but the difference in CBR was not statistically significant from studies with unselected populations (respectively, CBR of 42% (95% CI 23-62%) and 27% (95% CI 14-42%), P = 0.217). To better reflect true patient benefit, we excluded SD <6 months as a beneficial outcome which resulted in a pooled CBR of 7% (95% CI 2-13%). The overall proportion of patients with drug-related grade 3 and 4 adverse events was 36%. CONCLUSIONS: The efficacy of monotherapy with PI3K/AKT/mTOR inhibitors in advanced recurrent ovarian cancer patients is limited to a small subgroup and selection of patients with the use of current biomarkers did not improved the CBR significantly. Given the toxicity profile, we suggest that current treatment with PI3K/AKT/mTOR inhibitors should not be initiated unless in clinical trials. Furthermore, improved biomarkers to measure functional PI3K/AKT/mTOR pathway activity are needed to optimize patient selection.

Simultaneous Detection of Phosphoinositide Lipids by Radioactive Metabolic Labeling.

Phosphoinositide (PPI) lipids are a crucial class of low-abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-3H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.

Label-Free Quantification of Phosphoinositides in Drosophila by Mass Spectrometry.

Phosphoinositides (PIs), the seven phosphorylated derivatives of phosphatidylinositol, are recognized as key molecules in the control of multiple molecular events in eukaryotic cells. Within cells, PIs are low-abundance lipids making their detection and quantification challenging. While many methods that allow radiolabeling and quantification of PIs in the context of cultured cells are available, these are not useful in the context of in vivo animal models where cell and developmental processes are best studied. In this chapter, we describe radionuclide-free, mass spectrometry-based methods for the detection and quantification of PIs from Drosophila tissues in vivo. The use of these methods should facilitate the discovery of novel modes by which PIs regulate cellular and developmental processes in complex metazoans.

Profiling of Phosphoinositide Molecular Species in Resting or Activated Human or Mouse Platelets by a LC-MS Method.

Our knowledge of the role and biology of the different phosphoinositides has greatly expanded over recent years. Reversible phosphorylation by specific kinases and phosphatases of positions 3, 4, and 5 on the inositol ring is a highly dynamic process playing a critical role in the regulation of the spatiotemporal recruitment and binding of effector proteins. The specific phosphoinositide kinases and phosphatases are key players in the control of many cellular functions, including proliferation, survival, intracellular trafficking, or cytoskeleton reorganization. Several of these enzymes are mutated in human diseases. The impact of the fatty acid composition of phosphoinositides in their function is much less understood. There is an important molecular diversity in the fatty acid side chains of PI. While stearic and arachidonic fatty acids are the major acyl species in PIP, PIP2, and PIP3, other fatty acid combinations are also found. The role of these different molecular species is still unknown, but it is important to quantify these different molecules and their potential changes during cell stimulation to better characterize this emerging field. Here, we describe a sensitive high-performance liquid chromatography-mass spectrometry method that we used for the first time to profile the changes in phosphoinositide molecular species (summed fatty acyl chain profiles) in human and mouse platelets under resting conditions and following stimulation. This method can be applied to other hematopoietic primary cells isolated from human or experimental animal models.

Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways in vivo.

PURPOSE: To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. METHODS: Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. RESULTS: Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. CONCLUSIONS: The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

11C-Labeled Pictilisib (GDC-0941) as a Molecular Tracer Targeting Phosphatidylinositol 3-Kinase (PI3K) for Breast Cancer Imaging.

Pictilisib (GDC-0941) is an inhibitor of phosphatidylinositol 3-kinase (PI3K), part of a signaling cascade involved in breast cancer development. The purpose of this study was to evaluate the pharmacokinetics of pictilisib noninvasively by radiolabeling it with 11C and to assess the usability of the resulting [11C]-pictilisib as a positron-emission tomography (PET) tracer to screen for pictilisib-sensitive tumors. In this study, pictilisib was radiolabeled with [11C]-methyl iodide to obtain 11C-methylated pictilisib ([11C]-pictilisib) using an automated synthesis module with a high radiolabeling yield. Considerably higher uptake ratios were observed in MCF-7 (PIK3CA mutation, pictilisib-sensitive) cells than those in MDA-MB-231 (PIK3CA wild-type, pictilisib-insensitive) cells at all evaluated time points, indicating good in vitro binding of [11C]-pictilisib. Dynamic micro-PET scans in mice and biodistribution results showed that [11C]-pictilisib was mainly excreted via the hepatobiliary tract into the intestines. MCF-7 xenografts could be clearly visualized on the static micro-PET scans, while MDA-MB-231 tumors could not. Biodistribution results of two xenograft models showed significantly higher uptake and tumor-to-muscle ratios in the MCF-7 xenografts than those in MDA-MB-231 xenografts, exhibiting high in vivo targeting specificity. In conclusion, [11C]-pictilisib was first successfully prepared, and it exhibited good potential to identify pictilisib-sensitive tumors noninvasively, which may have a great impact in the treatment of cancers with an overactive PI3K/Akt/mTOR signal pathway. However, the high activity in hepatobiliary system and intestines needs to be addressed.

MiR-647 promotes proliferation and migration of ox-LDL-treated vascular smooth muscle cells through regulating PTEN/PI3K/AKT pathway.

OBJECTIVE: Aberrant microRNAs (miRNAs) play vital roles in various human diseases, including atherosclerosis (AS). MiR-647 expression was highly elevated in AS samples. Therefore, this study aimed at exploring the role and mechanism of miR-647 on AS progression. PATIENTS AND METHODS: Human aorta vascular smooth muscle cells (HA-VSMCs) were treated with oxidized modified low-density lipoprotein (ox-LDL) to establish the AS model in vitro. The qRT-PCR assay was used to detect the expression of miR-647 and PTEN mRNA. The levels of PTEN protein, PI3K, AKT, p-PI3K, and p-AKT were measured using Western blot. Cell proliferation and migration were determined by Cell Counting Kit-8 (CCK-8) assay and transwell assay, respectively. The target of miR-647 was verified using the dual-luciferase reporter assay. RESULTS: Our data supported that miR-647 was upregulated and PTEN was downregulated in the serum of AS patients and ox-LDL-treated HA-VSMCs. The proliferation and migration of ox-LDL-treated HA-VSMCs were promoted by miR-647 overexpression or PTEN knockdown, while they were suppressed following miR-647 depletion or high PTEN expression. Moreover, PTEN was a direct target of miR-647. PTEN antagonized miR-647-mediated regulatory effects on cell proliferation and migration. Additionally, the PI3K/AKT signaling pathway was involved in miR-647/PTEN-mediated regulation in ox-LDL-treated HA-VSMCs. CONCLUSIONS: MiR-647 promoted the proliferation and migration of ox-LDL-treated HA-VSMCs at least partly by targeting the PTEN/PI3K/AKT pathway. Targeting miR-647 may be a promising method for AS treatment.

A kinome-wide shRNA screen uncovers vaccinia-related kinase 3 (VRK3) as an essential gene for diffuse intrinsic pontine glioma survival.

Diffuse intrinsic pontine glioma (or DIPG) are pediatric high-grade gliomas associated with a dismal prognosis. They harbor specific substitution in histone H3 at position K27 that induces major epigenetic dysregulations. Most clinical trials failed so far to increase survival, and radiotherapy remains the most efficient treatment, despite only transient tumor control. We conducted the first lentiviral shRNA dropout screen in newly diagnosed DIPG to generate a cancer-lethal signature as a basis for the development of specific treatments with increased efficacy and reduced side effects compared to existing anticancer therapies. The analysis uncovered 41 DIPG essential genes among the 672 genes of human kinases tested, for which several distinct interfering RNAs impaired cell expansion of three different DIPG stem-cell cultures without deleterious effect on two control neural stem cells. Among them, PLK1, AURKB, CHEK1, EGFR, and GSK3A were previously identified by similar approach in adult GBM indicating common dependencies of these cancer cells and pediatric gliomas. As expected, we observed an enrichment of genes involved in proliferation and cell death processes with a significant number of candidates belonging to PTEN/PI3K/AKT and EGFR pathways already under scrutiny in clinical trials in this disease. We highlighted VRK3, a gene involved especially in cell cycle regulation, DNA repair, and neuronal differentiation, as a non-oncogenic addiction in DIPG. Its repression totally blocked DIPG cell growth in the four cellular models evaluated, and induced cell death in H3.3-K27M cells specifically but not in H3.1-K27M cells, supporting VRK3 as an interesting and promising target in DIPG.

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells.

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

Role of Thymoquinone in Cardiac Damage Caused by Sepsis from BALB/c Mice.

Sepsis is a major health complication causing patient mortality and increased healthcare costs. Cardiac dysfunction, an important consequence of sepsis, affects mortality. We previously reported that thymoquinone (TQ) protected against hyperlipidemia and doxorubicin-induced cardiac damage. This study investigated the possible protective effects of TQ against cardiac damage in septic BALB/c mice. Eight-week-old male BALB/c mice were divided into four groups: control, TQ, cecal ligation and puncture (CLP), and TQ + CLP. CLP was performed after 2-week TQ gavage. After 48 h, we measured the histopathological alterations of the cardiac tissue and the plasma levels of troponin-T (cTnT) and ATP. We evaluated autophagy (p62 and beclin 1), pyroptosis (NLRP3, caspase-1, interleukin [IL]-1ß, and IL-18) at the gene and protein levels and IL-6 and tumor necrosis factor-α (TNF-α) at the gene level. Our results demonstrated that TQ administration significantly reduced intestinal histological alterations. TQ inhibited plasma cTnT levels; improved ATP; significantly inhibited p62, NLRP3, caspase-1, IL-1ß, IL-18, IL-6, TNF-α, and MCP-1expressions; and increased beclin 1 and IL-10 level. The phosphatidylinositide 3-kinase level was significantly decreased in the TQ + CLP group versus the CLP group. These results suggest that TQ effectively modulates autophagy, pyroptosis, and pro-inflammatory, making it important in the treatment of sepsis-induced cardiac damage.

Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways in vivo

Abstract Purpose To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. Methods Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. Results Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. Conclusions The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

PIK3R3 Promotes Metastasis of Pancreatic Cancer via ZEB1 Induced Epithelial-Mesenchymal Transition.

BACKGROUND/AIMS: PIK3R3 is a regulatory subunit of phosphatidylinositol 3-kinase (PI3K) which plays an essential role in the metastasis of several types of cancer. However, whether PIK3R3 can promote the metastasis of pancreatic cancer (PC) is still unclear. In this study, we characterized the role of PIK3R3 in metastasis of PC and underlying potential mechanisms. METHODS: RT-PCR, western blot, immunofluorescence (IF) and immunohistochemistry (IHC) were applied to investigate the expression of genes and proteins in different cell lines and tissues. To assess the function of PIK3R3 and related mechanisms, the cells with RNAi-mediated knockdown or overexpression were used to perform a series of in vitro and in vivo assays. RESULTS: PIK3R3 was significantly overexpressed in pancreatic cancer tissues, especially in metastatic cancer tissues, as well as in pancreatic cancer cells. Functional assays suggested that overexpression or knockdown of PIK3R3 could respectively promote or suppress the migration and invasion of PC cells in vitro and in vivo. Further mechanism related studies demonstrated that ERK1/2-ZEB1 pathway-triggered epithelial-mesenchymal transition (EMT) might be responsible for the PIK3R3-induced PC cell migration and invasion. CONCLUSION: PIK3R3 could promote the metastasis of PC by facilitating ZEB1 induced EMT, and could act as a potential therapeutic target to limit PC metastasis.

Comparison of Rejection-Specific Genes in Peripheral Blood and Allograft Biopsy From Kidney Transplant.

BACKGROUND: Although improved understanding and assessment of organ rejection significantly contribute to long-term allograft survival after kidney transplantation, reliable and predictive biomarkers that enable diagnoses of rejection state are lacking. Patient rejection of a kidney graft displays a specific blood and biopsy transcriptional pattern, raising the question of whether transcript biomarkers in blood could reflect events within the allograft. METHODS: Differential expression genes were screened on large-scale transcriptomic data from blood and allograft biopsies, which included recipients undergoing rejection and recipients with stable renal function. RESULTS: We found that the number of rejection-related genes in biopsy samples was much greater than in blood. We observed only one overlapping gene, HIST1H4A, consistently expressed in biopsy samples and blood. Functional association of the identified genes in biopsies implicated a strong involvement of inflammatory-immune pathways. Rejection-related genes in the mammalian target of rapamycin-signaling pathway were down-regulated, and genes related to allograft rejection and graft-versus-host disease were up-regulated in allograft biopsy samples. We also recognized the core signaling elements (PIK3R2 and EGFR) in inflammatory-immune pathways based on biopsy samples. CONCLUSIONS: We have expanded our understanding of rejection-specific gene expression pattern in allograft biopsy and peripheral blood, and provided a candidate set of overlapping genes for screening of rejection in kidney transplant recipients.

Effects of Quercetin, Kaempferol, and Exogenous Glutathione on Phospho- and Total-AKT in 3T3-L1 Preadipocytes.

Obesity has been reported to be a risk factor for some types of cancer, such as prostate and lung. The AKT or PI3K-AKT is a signal transduction pathway that promotes survival and growth in response to extracellular signals. The aim of this study was to investigate the effects of two flavonoids, quercetin and kaempferol, and exogenous glutathione (GSH) on the expressions of phospho- and total-AKT levels in 3T3-L1 preadipocytes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels were measured in the treated samples and used as the internal standard. 3T3-L1 preadipocytes were exposed to each flavonoid and GSH at concentrations of 0, 5, 10, 15, 20, and 25 µM, and the levels of phospho- and total-Akt were measured by the MILLIPLEX MAP mates protocol, based on the Luminex xMAP technology (Millipore Corp., St. Charles, MI, USA). GAPDH levels in the preadipocytes were not significantly different at the doses tested for the flavonoids and exogenous GSH. However, significant (p <.05) decreases in phospho-AKT levels in cells treated with quercetin, kaempferol, and GSH at certain doses were observed compared to their respective controls. Total-AKT levels showed the same profile for all the tested compounds. Significant (p <.01) differences were observed for kaempferol (15-25 µM), quercetin at 10 and 20 µM, and GSH at 10 µM compared to their respective controls. Findings suggest that exposure of 3T3-L1 preadipocytes to quercetin, kaempferol, and GSH may block the activation of AKT, suggesting the role such compounds play in cell differentiation in 3T3-L1 cells.

Regulatory Efficacy of Brown Seaweed Lessonia nigrescens Extract on the Gene Expression Profile and Intestinal Microflora in Type 2 Diabetic Mice.

SCOPE: In this study, the antidiabetic activity of Lessonia nigrescens ethanolic extract (LNE) is investigated in streptozotocin (SZT)-induced type 2 diabetic mice fed with a high-sucrose/high-fat diet. METHODS AND RESULTS: Ultra high performance liquid chromatography coupled with photo-DAD and electospray ionization-mass spectrometry (ESI-MS) is employed to analyze the major compounds in LNE. The components of the intestinal microflora in type 2 diabetic mice are analyzed by high-throughput next-generation 16S rRNA gene sequencing. Fasting blood glucose levels in diabetic mice are significantly decreased after LNE administration. The histology reveals that LNE could protect the cellular architecture of liver and kidney. LNE treatment significantly increases Bacteroidetes and decreases Firmicutes populations in intestinal microflora. Specifically, It could selectively enrich the amounts of beneficial bacteria, Barnesiella, as well as reduce the abundances of Clostridium and Alistipes. The increased gene and protein expression levels of phosphatidylinositol 3-kinase (PI3K) in the liver are observed in LNE treatment groups, while the expressions of c-Jun N-terminal kinase (JNK) are significantly downregulated. CONCLUSION: The above findings suggest that LNE could be considered as a functional food for reducing blood glucose and regulating intestinal microflora.

Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells.

Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox. Across various conditions, including glycolytic or mitochondrial inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including both stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations that indicate a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells.

Ginsenoside Rg1 protects human renal tubular epithelial cells from lipopolysaccharide-induced apoptosis and inflammation damage.

Ginsenoside Rg1, one of the most notable active components of Panax ginseng, has been widely reported to exert anti-inflammatory actions. This study aimed to reveal whether ginsenoside Rg1 also exhibits beneficial roles against lipopolysaccharide (LPS)-induced apoptosis and inflammation in human renal tubular epithelial cells, and to evaluate the potential role of the component on tubulointerstitial nephritis treatment. HK-2 cells were treated with various doses of ginsenoside Rg1 (0, 50, 100, 150, and 200 µM) in the absence or presence of 5 µg/mL LPS. Thereafter, CCK-8 assay, flow cytometry, western blot, migration assay, reactive oxygen species (ROS) assay, and ELISA were carried out to respectively assess cell viability, apoptosis, migration, ROS activity, and the release of inflammatory cytokines. As a result, ginsenoside Rg1 protected HK-2 cells from LPS-induced injury, as cell viability was increased, cell apoptosis was decreased, and the release of MCP-1, IL-1ß, IL-6, and TNF-α was reduced. Ginsenoside Rg1 functioned to HK-2 cells in a dose-dependent manner, and the 150 µM dose exhibited the most protective functions. Ginsenoside Rg1 had no significant impact on cell migration and ROS activity, while it alleviated LPS-induced ROS release and migration impairment. Furthermore, the down-regulations of p-PI3K, p-AKT, and up-regulations of PTEN, p-IκBα, p-p65, Bcl-3 induced by LPS were recovered to some extent after ginsenoside Rg1 treatment. In conclusion, ginsenoside Rg1 protects HK-2 cells against LPS-induced inflammation and apoptosis via activation of the PI3K/AKT pathway and suppression of NF-κB pathway.