Chemical inhibition of mitochondrial fission improves insulin signaling and subdues hyperglycemia induced stress in placental trophoblast cells.

BACKGROUND: Gestational diabetes mellitus (GDM) is a metabolic complication that affects millions of pregnant women in the world. Placental tissue function is endangered by hyperglycemia during GDM, which is correlated to increased incidences of pregnancy complications. Recently we showed that due to a significant decrease in mitochondrial fusion, mitochondrial dynamics equilibrium is altered in placental tissues from GDM patients. Evidence for the role of reduced mitochondrial fusion in the disruption of mitochondrial function in placental cells is limited. METHODS AND RESULTS: Here we show that chemical inhibition of mitochondrial fission in cultured placental trophoblast cells leads to an increase in mitochondrial fusion and improves the physiological state of these cells and hence, their capacity to cope in a hyperglycemic environment. Specifically, mitochondrial fission inhibition led to a reduction in reactive oxygen species (ROS) generation, mitochondrial unfolded protein marker expressions, and mitochondrial depolarization. It supported the increase in mitochondrial antioxidant enzyme expressions as well. Mitochondrial fission inhibition also increases the placental cell insulin sensitivity during hyperglycemia. CONCLUSION: Our results suggest that mitochondrial fusion/fission equilibrium is critical for placental cell function and signify the therapeutic potential of small molecule inhibitors of fission during GDM.

Transforming growth factor ß1 upregulates 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-4 expression in A549 and MCF-10A cells.

Transforming growth factor ß1 (TGFß1) induces a cellular process known as epithelial-mesenchymal transition (EMT) associated with metabolic reprogramming, including enhanced glycolysis. Given the involvement of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFKFB) enzymes in glycolysis, we aimed to investigate whether TGFß1 regulates expressions of PFKFB genes and if PFKFBs are required for TGFß1-driven phenotypes. A549 and MCF-10A cell lines were used as TGFß1-driven EMT models. Messenger RNA expressions of PFKFB and EMT genes were determined by real-time quantitative polymerase chain reaction. A small interfering RNA approach was used to deplete PFKFB4 expression. A Matrigel invasion assay was conducted to assess the effect of PFKFB4 silencing on the TGFß1-enhanced invasion of A549 cells. F2,6BP levels were analyzed using an enzyme-coupled assay. Glucose and lactate concentrations were determined using colorimetric assays. TGFß1 robustly induced expression of the fourth isoform of PFKFBs, PFKFB4, in both cell lines. PFKFB4 depletion partially inhibits mesenchymal transdifferentiation caused by TGFß1 in A549 cells, as assessed by microscopy. Inductions of Snail in MCF-10A cells and Fibronectin in A549 cells and repressions of E-cadherin in both cell lines by TGFß1 are attenuated by PFKFB4 silencing. PFKFB4 silencing reduces F2,6BP and glycolytic activity, although TGFß1 alone does not affect these parameters. Finally, PFKFB4 depletion suppresses the TGFß1-driven invasion of A549 cells through Matrigel. Presented data suggest that TGFß1 induces the expression of PFKFB4 in A549 and MCF-10 cells, and PFKFB4 may be required for TGFß1-driven phenotypes such as EMT and invasion in these models.

Expression of dual-specificity phosphatases in TGFß1-induced EMT in SKOV3 cells.

BACKGROUND: The study aims to profile the dual-specificity phosphatases (DUSP) expression in response to Transforming growth factor ß1 (TGFß1)-induced epithelial- mesenchymal transition (EMT) in ovarian adenocarcinoma cells. METHODS: The ovarian adenocarcinoma cell line SKOV3 was used as a TGFß1-induced EMT model. Cells were incubated with 5 ng/mL TGFß1 to induce EMT. EMT was confirmed with real-time qPCR, western blot, and immunofluorescence analyses of various EMT markers. Western blot was used to analyze phospho- and total MAPK protein levels. Typical and atypical DUSPs mRNA expression profile was determined by real-time qPCR. RESULTS: The epithelial marker E-cadherin expressions were decreased and mesenchymal EMT markers Snail and Slug expression levelswere increased after TGFß1 induction. Phosphorylation of ERK1/2 and p38 MAPK were enhanced in response to TGFß1 treatment. The expression of DUSP2, DUSP6, DUSP8, DUSP10, and DUSP13 were decreased while DUSP7, DUSP16, DUSP18, DUSP21, and DUSP27 were increased by TGFß1. DISCUSSION: TGFß1 induced EMT which was accompanied by increased activity of MAPKs, and led to marked changes in expressions of several DUSPs in SKOV3 cells.

Expression of Pfkfb isoenzymes during in vitro differentiation of mouse embryonic stem cells into insulin-producing cells.

Background/aim: Type 1 diabetes mellitus (T1DM) is caused by the autoimmune-mediated destruction of insulin-producing cells (IPCs) and still has no effective cure. Better understanding of the molecular mechanisms involved in the differentiation of embryonic stem cells (ESCs) into IPCs may help us improve the therapeutic strategies for treating T1DM. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (Pfkfb1-4) are key regulators of glucose metabolism. Although Pfkfb3 has been shown to be required for the growth of early differentiated mouse ESCs (mESCs), more studies are needed to further assess the roles of Pfkfb isoenzymes in embryonic development and differentiation, particularly into specific cell types. In this study, we aimed to elucidate the changes in the expression of Pfkfb isoenzymes on the differentiation of mESCs into IPCs. Materials and methods: A 3-step protocol was used to differentiate R1 and J1 mESCs into IPCs. The changes in the gene expression of MafA, MafB, Ins2, and Nkx6.1 (IPC specific markers) and Pfkfb1-4 were analyzed using real-time quantitative polymerase chain reaction (qPCR). Insulin expression and secretion were determined by immunofluorescence (IF) staining and the enzyme linked immunosorbent assay (ELISA), respectively. Results: Upon differentiation, the IPC specific markers in differentiated cells were upregulated. Continued differentiation was confirmed by the development of insulin-positive islet-like clusters that secreted insulin in response to glucose uptake. Expressions of the Pfkfb2 and Pfkfb3 isoenzymes were markedly increased in various stages of differentiation. Conclusion: These findings suggest that Pfkfb2 and Pfkfb3 may impact the differentiation of mESCs into IPCs and the regulation of the insulin response to glucose levels. This study also lays a foundation for researchers to further probe the roles of Pfkfb isoenzymes on the differentiation of mESCs into IPCs and may open new avenues for regenerative medicine.

Overexpression of dual-specificity phosphatases 4 and 13 attenuates transforming growth factor ß1-induced migration and drug resistance in A549 cells in vitro.

Transforming growth factor-beta (TGFß) proteins induce an epithelial-mesenchymal transition (EMT) programme that is associated with increased invasive and drug-resistant phenotype of carcinoma cells. In addition to the canonical pathway involving SMAD proteins, the mitogen-activated kinase (MAPK) pathway via extracellular signal-regulated kinases ½ (ERK1/2) is also involved in promoting and maintaining a mesenchymal phenotype by tumor cells following TGFß signal activation. As dual-specificity phosphatases (DUSPs) regulate ERK1/2 activity by dephosphorylation, we aimed to examine DUSPs' expression upon TGFß stimulation and whether DUSPs play a role in the EMT and related phenotypes promoted by TGFß1 in A549 cells. We found that TGFß1 stimulation led to marked changes in several DUSP proteins, including significant decreases in DUSP4 and DUSP13 expressions. We then showed that the ectopic co-expression of DUSP4/13 suppresses TGFß1-induced ERK1/2 phosphorylation and protein levels of the EMT transcription factors Snail and Slug proteins. We then demonstrated that DUSP4/13 co-expression partially inhibited TGFß1-promoted migration, invasion, and chemoresistance in A549 cells. Collectively, this report provides data for the involvement of DUSP4/13 in malignant phenotypes regulated by TGFß1 in A549 cells.

Upregulation of dual-specificity phosphatase-26 is required for transforming growth factor ß1(TGFß1)-induced Epithelial-mesenchymal transition in A549 and PANC1 cells.

BACKGROUND: Transforming Growth Factor ß (TGFß) proteins are potent inducers of the epithelial-mesenchymal transition (EMT) in tumor cells. Although mitogen-activated protein kinase (MAPK) family has been shown to be involved in TGFß-induced EMT, role of Dual Specificity Phosphatases (DUSP), key regulators of MAPK activity, in TGFß-induced EMT is largely unkonwn. METHODS AND RESULTS: Real-time qPCR analyses were performed to determine the effect of TGFß1 on expression of EMT genes and DUSP proteins in the non-small cell lung cancer model A549 and pancreatic adenocarcinoma model PANC1 cells. Western blot analyses were conducted to study the changes in protein levels of EMT proteins and select DUSP proteins, as well as phosphorylations of MAPK proteins upon TGFß1 stimulation. Small interfering RNA (siRNA) was utilized to reduce expressions of DUSP genes. We observed that the EMT phenotype coincided with increases in phosphorylations of the MAPK proteins ERK1/2, p38MAPK, and JNK upon TGFß1 stimulation. Real-time qPCR analysis showed increases in DUSP15 and DUSP26 mRNA levels and Western blot analysis confirmed the increase in DUSP26 protein levels in both A549 and PANC1 cells treated with TGFß1 relative to control. Silencing of DUSP26 expression by siRNA markedly suppressed the effect of TGFß1 on E-cadherin and mesenchymal genes in the cells. CONCLUSIONS: Data provided suggest that TGFß1 modulates the expression of DUSP genes and that upregulation of DUSP26 may be required for TGFß1-promoted EMT in A549 and PANC1 cells. Further studies should be carried out to elucidate the requirement of individual DUSPs in TGFß1-associated EMT in tumor cells.

The expression of glutamate metabolism modulators in the intracranial tumors and glioblastoma cell line.

BACKGROUND: The accumulation of excess glutamate in the synapse leads to excitotoxicity, which is the underlying reason of neuronal death in intracranial tumors. METHODS AND RESULTS: We identified the expression levels of glutamate dehydrogenase, glutamine synthetase and sirtuin 4 in U87 cell line and various intracranial tumors. mRNA expressions of glutamate dehydrogenase (GDH), glutamine synthetase (GS) and sirtuin 4 (SIRT4) were analyzed in various intracranial tumors using qPCR. GDH, GS and SIRT4 protein expressions were analyzed in glioblastoma (U87) and glial (IHA-immortalized human astrocytes) cell lines via western blotting. The protein expressions of SIRT4 and GS were shown to be elevated and GDH protein expression was reduced in U87 cells in comparison to IHA cells. All types of intracranial tumors displayed lower GS mRNA expressions compared to controls. SIRT4 mRNA expressions were also shown to be lower in all the tumors and grades, although not significantly. GDH mRNA expression was found to be similar in all groups. CONCLUSION: The molecular mechanisms of glutamate metabolism and excitotoxicity should be discovered to develop therapies against intracranial tumors.

Simultaneous inhibition of PFKFB3 and GLS1 selectively kills KRAS-transformed pancreatic cells.

Activating mutations of the oncogenic KRAS in pancreatic ductal adenocarcinoma (PDAC) are associated with an aberrant metabolic phenotype that may be therapeutically exploited. Increased glutamine utilization via glutaminase-1 (GLS1) is one such feature of the activated KRAS signaling that is essential to cell survival and proliferation; however, metabolic plasticity of PDAC cells allow them to adapt to GLS1 inhibition via various mechanisms including activation of glycolysis, suggesting a requirement for combinatorial anti-metabolic approaches to combat PDAC. We investigated whether targeting the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) in combination with GLS1 can selectively prevent the growth of KRAS-transformed cells. We show that KRAS-transformation of pancreatic duct cells robustly sensitizes them to the dual targeting of GLS1 and PFKFB3. We also report that this sensitivity is preserved in the PDAC cell line PANC-1 which harbors an activating KRAS mutation. We then demonstrate that GLS1 inhibition reduced fructose-2,6-bisphosphate levels, the product of PFKFB3, whereas PFKFB3 inhibition increased glutamine consumption, and these effects were augmented by the co-inhibition of GLS1 and PFKFB3, suggesting a reciprocal regulation between PFKFB3 and GLS1. In conclusion, this study identifies a novel mutant KRAS-induced metabolic vulnerability that may be targeted via combinatorial inhibition of GLS1 and PFKFB3 to suppress PDAC cell growth.

PFKFB2 regulates glycolysis and proliferation in pancreatic cancer cells.

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.

6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 is required for transforming growth factor ß1-enhanced invasion of Panc1 cells in vitro.

Transforming growth factor ß1 (TGFß1) is a well-established inducer of the epithelial-mesenchymal transition (EMT) that is essential for the acquisition of malignant properties, such as invasion, in tumor cells. Although recent studies suggest that the EMT in tumor cells is associated with reprogramming of energy metabolism and TGFß1 has been shown to stimulate glycolysis in multiple primary cell lines, little is known about TGFß1's effect on glycolysis and glycolytic regulators in transformed cells. Given the known regulatory role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 (PFKFB3) in glycolysis and association of glycolytic activity with malignant features such as invasion, we sought to investigate whether TGFß1 regulates PFKFB3 expression and if PFKFB3 is involved in the TGFß1-mediated increase in the invasive ability of the Panc1 cell cline-a well-established model of TGFß1-initiated EMT. Herein we demonstrate that TGFß1 induces PFKFB3 expression and stimulates glycolysis in Panc1 cells. We also show that siRNA silencing of PFKFB3 prevents the stimulation of glycolysis and in vitro invasive ability of Panc1 cells by TGFß1. Furthermore, PFKFB3 silencing suppresses the TGFß1-mediated induction of the Snail protein, suggesting that PFKFB3 is required for the regulation of Snail expression by TGFß1. Taken together, our study identifies PFKFB3 as a key TGFß1 effector protein that mediates TGFß1's effect on Snail expression, invasion, and glycolysis.

Functional RNA interference (RNAi) screen identifies system A neutral amino acid transporter 2 (SNAT2) as a mediator of arsenic-induced endoplasmic reticulum stress.

Exposure to the toxic metalloid arsenic is associated with diabetes and cancer and causes proteotoxicity and endoplasmic reticulum (ER) stress at the cellular level. Adaptive responses to ER stress are implicated in cancer and diabetes; thus, understanding mechanisms of arsenic-induced ER stress may offer insights into pathogenesis. Here, we identify genes required for arsenite-induced ER stress response in a genome-wide RNAi screen. Using an shRNA library targeting ∼20,000 human genes, together with an ER stress cell model, we performed flow cytometry-based cell sorting to isolate cells with defective response to arsenite. Our screen discovered several genes modulating arsenite-induced ER stress, including sodium-dependent neutral amino acid transporter, SNAT2. SNAT2 expression and activity are up-regulated by arsenite, in a manner dependent on activating transcription factor 4 (ATF4), an important mediator of the integrated stress response. Inhibition of SNAT2 expression or activity or deprivation of its primary substrate, glutamine, specifically suppressed ER stress induced by arsenite but not tunicamycin. Induction of SNAT2 is coincident with the activation of the nutrient-sensing mammalian target of rapamycin (mTOR) pathway, which is at least partially required for arsenite-induced ER stress. Importantly, inhibition of the SNAT2 or the System L transporter, LAT1, suppressed mTOR activation by arsenite, supporting a role for these transporters in modulating amino acid signaling. These findings reveal SNAT2 as an important and specific mediator of arsenic-induced ER stress, and suggest a role for aberrant mTOR activation in arsenic-related human diseases. Furthermore, this study demonstrates the utility of RNAi screens in elucidating cellular mechanisms of environmental toxins.

Increased expression of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 is required for growth of mouse embryonic stem cells that are undergoing differentiation.

The unlimited proliferation capacity of embryonic stem cells (ESCs) coupled with their capability to differentiate into several cell types makes them an attractive candidate for studying the molecular mechanisms regulating self-renewal and transition from pluripotent state. Although the roles of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family (PFKFB1-4) in cell survival, proliferation, and differentiation in tumor cells have been studied, their role in mouse ESC (mESC) biology is currently unkown. In the current study, Pfkfb isoenzyme expressions were analyzed in R1 and J1 mESCs that were cultured in the presence and absence of leukemia inhibitory factor (LIF). We report that expression of the Pfkfb3 isoenzyme was markedly increased when mESCs were promoted to differentiate upon LIF removal. We then demonstrated that Pfkfb3 silencing induced the differentiation marker Brachyury suggesting that Pfkfb3 may be required for the regulation of mesodermal differentiation of mESCs. Furthermore, we show that the increase in Pfkfb3 expression is required for the growth of early differentiated mESCs. Although these results provide important insights into the early differentiation of mESCs with regard to Pfkfb expressions, further mechanistic studies will be needed for understanding the pathways and mechanisms involved in regulation of proliferation and early differentiation of mESCs through Pfkfb3.

Kainic Acid-Induced Excitotoxicity Leads to the Activation of Heat Shock Response.

Heat shock response (HSR) which is regulated by heat shock factor 1 (HSF1) is the most important mechanism and the major regulator that prevents protein aggregation in neurodegenerative diseases. Excitotoxicity, which is the accumulation of excess glutamate in synaptic cleft, is observed in age-dependent neurodegenerative diseases and also in stroke, epilepsy, and brain trauma. Only a few studies in the literature show the link between excitotoxicity and HSR. In this study, we aimed to show the molecular mechanism underlying this link. We applied heat shock (HS) treatment and induced excitotoxicity with kainic acid (KA) in neuroblastoma (SHSY-5Y) and glia (immortalized human astrocytes (IHA)) cells. We observed that, only in SHSY-5Y cells, heat shock preconditioning increases cell survival after KA treatment. GLT-1 mRNA expression is increased as a result of KA treatment and HS due to the elevation of HSF1 binding to GLT-1 promoter which was induced by HSF1 phosphorylation and sumolation in SHSY-5Y cells. Additionally, glutamine synthetase and glutaminase expressions are increased after HS preconditioning in SHSY-5Y cells indicating that HS activates glutamate metabolism modulators and accelerates the glutamate cycle. In glia cells, we did not observe the effect of HS preconditioning. In summary, heat shock preconditioning might be protective against excitotoxicity-related cell death and degeneration.

The role of protein kinase R in placental inflammation, mtUPR and apoptosis.

INTRODUCTION: Placental inflammation is implicated in the pathophysiology of many pregnancy complications, including fetal growth restriction, preeclampsia, gestational diabetes, and choriocarcinoma. Mitochondrial dysfunction, one of the outcomes of placental inflammation, is characterized by loss of membrane potential, accumulation of oxygen radicals, mitochondrial protein folding defects, and disturbances in mitochondrial dynamics. Protein kinase R (PKR) is stimulated by double-stranded RNA and bacterial endotoxins in the presence of pathogens and is a critical immune response enzyme. PKR is also correlated with the cell death response during endoplasmic reticulum stress. In this study, we aim to investigate the effects of PKR activity stimulated by lipopolysaccharide (LPS), and double-stranded RNA analog (Poly I:C) on mitochondrial unfolded protein response (mtUPR), mitochondrial membrane potential, apoptosis, and oxidative stress in placental trophoblasts. METHODS: We applied LPS and Poly I:C to BeWo cells to induce PKR activation. In addition, cells were treated with 2-aminopurine (2-AP) to inhibit the kinase activity of PKR. Protein levels of ATP-dependent Clp protease proteolytic subunit (CLPP) and heat shock protein 60 (HSP60) were determined after treatments. Apoptotic markers were detected by real-time PCR and flow cytometry. PKR-induced reactive oxygen radicals (ROS) accumulation and mitochondrial membrane potential change were assessed by flow cytometry. RESULTS: It was determined that PKR activation-induced apoptosis in BeWo cells by reducing the levels of mtUPR proteins (CLPP and HSP60) and caused a decrease in mitochondrial membrane potential. PKR inhibition was sufficient for decreases in apoptotic markers and caused a reduction in the ratio of depolarized and ROS (+) cells. DISCUSSION: Our results showed that LPS and Poly I:C administration stimulated PKR in BeWo cells in vitro. Furthermore, PKR activation is correlated with the levels of proteins involved in mitochondrial homeostasis and apoptosis. Our findings will contribute to understanding the role of PKR activation in placental inflammation and related diseases.

Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth.

BACKGROUND: Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). RESULTS: We found that the introduction of activated H-Ras(V12) into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. CONCLUSION: Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.

A diagnostic unified classification model for classifying multi-sized and multi-modal brain graphs using graph alignment.

BACKGROUND: Presence of multimodal brain graphs derived from different neuroimaging modalities is inarguably one of the most critical challenges in building unified classification models that can be trained and tested on any brain graph regardless of its size and the modality it was derived from. EXISTING METHODS: One solution is to learn a model for each modality independently, which is cumbersome and becomes more time-consuming as the number of modalities increases. Another traditional solution is to build a model inputting multimodal brain graphs for the target prediction task; however, this is only applicable to datasets where all samples have joint neuro-modalities. NEW METHOD: In this paper, we propose to build a unified brain graph classification model trained on unpaired multimodal brain graphs, which can classify any brain graph of any size. This is enabled by incorporating a graph alignment step where all multi-modal graphs of different sizes and heterogeneous distributions are mapped to a common template graph. Next, we design a graph alignment strategy to the target fixed-size template and further apply linear discriminant analysis (LDA) to the aligned graphs as a supervised dimensionality reduction technique for the target classification task. RESULTS: We tested our method on unpaired autistic and healthy brain connectomes derived from functional and morphological MRI datasets (two modalities). CONCLUSION: Our results showed that our unified model method not only has great promise in solving such a challenging problem but achieves comparable performance to models trained on each modality independently.

The effect of gestational diabetes on the expression of mitochondrial fusion proteins in placental tissue.

INTRODUCTION: Gestational diabetes mellitus (GDM) poses a risk factor for fetal mortality and morbidity by directly affecting the placenta and fetus. Mitochondria are dynamic organelles that play a key role in energy production and conversion in placental tissue. Mitochondrial fusion and fission proteins are important in terms of providing mitochondrial dynamics, the adaptation of the cell to different conditions, and maintaining the metabolic stability of the cells. Although GDM shares many features with Type 2 diabetes mellitus (T2DM), different effects of these conditions on the mother and the child suggest that GDM may have specific pathological effects on placental cells. The aim of this study is to investigate the expression of mitochondrial dynamics, and mitochondrial protein folding markers in placentas from GDM patients and women with pre-existing diabetes mellitus. METHODS: Placentas were properly collected from women, who had pre-existing diabetes (Pre-DM), from women with gestational diabetes mellitus (GDM) and from healthy (non-diabetic) pregnant women. Levels of mitochondrial fusion markers were determined in these placentas by real time quantitative PCR and Western blot experiments. RESULTS: mRNA expressions and protein levels of mitochondrial fusion markers, mitofusin 1, mitofusin 2 (MFN1 and MFN2) and optical atrophy 1 (OPA1) proteins were found to be significantly lower in both Pre-DM placentas and those with GDM compared to healthy (non-diabetic) control group. Likewise, proteins involved in mitochondrial protein folding were also found to be significantly reduced compared to control group. DISCUSSION: Diabetes during pregnancy leads to processes that correlate with mitochondria dysfunction in placenta. Our results showed that mitochondrial fusion markers significantly decrease in placental tissue of women with GDM, compared to the healthy non-diabetic women. The decrease in mitochondrial fusion markers was more severe during GDM compared to the Pre-DM. Our results suggest that there may be differences in the pathophysiology of these conditions.

Intercellular Transmission of Hepatic ER Stress in Obesity Disrupts Systemic Metabolism.

Endoplasmic reticulum stress (ERS) has a pathophysiological role in obesity-associated insulin resistance. Yet, the coordinated tissue response to ERS remains unclear. Increased connexin 43 (Cx43)-mediated intercellular communication has been implicated in tissue-adaptive and -maladaptive response to various chronic stresses. Here, we demonstrate that in hepatocytes, ERS results in increased Cx43 expression and cell-cell coupling. Co-culture of ER-stressed "donor" cells resulted in intercellular transmission of ERS and dysfunction to ERS-naive "recipient" cells ("bystander response"), which could be prevented by genetic or pharmacologic suppression of Cx43. Hepatocytes from obese mice were able to transmit ERS to hepatocytes from lean mice, and mice lacking liver Cx43 were protected from diet-induced ERS, insulin resistance, and hepatosteatosis. Taken together, our results indicate that in obesity, the increased Cx43-mediated cell-cell coupling allows intercellular propagation of ERS. This novel maladaptive response to over-nutrition exacerbates the tissue ERS burden, promoting hepatosteatosis and impairing whole-body glucose metabolism.

PKR inhibitors suppress endoplasmic reticulum stress and subdue glucolipotoxicity-mediated impairment of insulin secretion in pancreatic beta cells.

Type 2 diabetes mellitus is characterized by insulin resistance and hypersecretion of insulin from the pancreas to compensate for decreased insulin sensitivity in the peripheral tissues. In later stages of the disease insulin-secreting beta cell degeneration commences and patients require insulin replacement therapy in order to accomplish proper regulation of their blood glucose. Endoplasmic reticulum (ER) stress in the beta cells is one of the factors contributing to this detrimental effect. Protein kinase R (PKR) is a cellular stress kinase activated by ER stress and contributing to degeneration of pancreatic islets. In order to determine whether inhibition of PKR activation by specific small molecule inhibitors of PKR ameliorates pancreatic insulin secretion capacity, we treated beta cells with two imidazole/oxindole-derived inhibitors of PKR kinase, imoxin (C16) and 2-aminopurine (2-AP), in the presence of ER stress. Our results demonstrate that PKR inhibition suppresses tunicamycin-mediated ER stress without altering the insulin production capacity of the cells. Palmitic acid-mediated suppression of insulin secretion, however, was subdued significantly by PKR inhibitor treatment through an ER stress-related mechanism. We suggest that PKR inhibitor treatment may be used to increase the insulin secretion capacity of the pancreas in later stages of diabetes.

Glutamate Transporter 1 expression in human glioblastomas.

PURPOSE: The purpose of our study was to investigate the mRNA expression profile of glutamate transporter 1 (GLT-1) in different types and grades of brain tumors, such as glioblastoma multiforme, astrocytomas (pilocytic, diffuse, anaplastic), oligodendrogliomas, ependydomas, medulloblastomas, and meningiomas using Real Time Quantitative PCR technique (qRT-PCR). METHODS: A total of 66 surgically removed primary brain tumors were collected retrospectively and the total RNA was isolated from each tumor sample. cDNA was generated and GLT-1 mRNA expression was evaluated with quantitative qRT-PCR. RESULTS: The mRNA expression of GLT-1 was significantly lower in primary brain tumors when compared to control brain tissues. GLT-1 expression was inversely correlated with the tumor grade, implicating its potential role in tumor progression. GLT-1 mRNA expression was lowest in grade 4 tumors, such as glioblastoma multiforme and medulloblastomas. The tumors with grade 3 and 4 combined displayed lower expression compared to tumors with grades 1 and 2. In grade 4 tumors, female patients displayed lower GLT-1 expression compared to male patients. In addition, glioblastoma multiforme patients older than 65 years of age showed lower GLT-1 expression when compared to the patients younger than 65. CONCLUSION: qRT-PCR was found to be a sensitive method in detecting GLT-1 expression in brain tumors. This study may lay the foundation for the future research about the excitotoxicity and brain tumors and GLT-1 might be a potential biomarker. Targeted therapies based on excitotoxic molecular pathways against gliomas should be designed to effectively combat these diseases.