The Complex of p-Tyr42 RhoA and p-p65/RelA in Response to LPS Regulates the Expression of Phosphoglycerate Kinase 1.

Inflammation plays a crucial role in tumorigenesis, primarily mediated by NF-κB. RhoA GTPases are instrumental in regulating the activation of NF-κB. Specifically, the phosphorylation of Tyrosine 42 on RhoA ensures the activation of NF-κB by directly activating the IKKß associated with IKKγ (NEMO). This study aimed to uncover the molecular mechanism through which p-Tyrosine 42 RhoA, in conjunction with NF-κB, promotes tumorigenesis. Notably, we observed that p-Tyrosine 42 RhoA co-immunoprecipitated with the p-Ser 536 p65/RelA subunit in NF-κB in response to LPS. Moreover, both p-Tyrosine 42 RhoA and p-p65/RelA translocated to the nucleus, where they formed a protein complex associated with the promoter of phosphoglycerate kinase 1 (PGK1) and regulated the expression of PGK1. In addition, p-p65/RelA and p-Tyr42 RhoA co-immunoprecipitated with p300 histone acetyltransferase. Intriguingly, PGK1 exhibited an interaction with ß-catenin, PKM1 and PKM2. Of particular interest, si-PGK1 led to a reduction in the levels of ß-catenin and phosphorylated pyruvate dehydrogenase A1 (p-PDHA1). We also found that PGK1 phosphorylated ß-catenin at the Thr551 and Ser552 residues. These findings discovered that PGK1 may play a role in transcriptional regulation, alongside other transcription factors.

Association of Phosphorylated Pyruvate Dehydrogenase with Pyruvate Kinase M2 Promotes PKM2 Stability in Response to Insulin.

Insulin is a crucial signalling molecule that primarily functions to reduce blood glucose levels through cellular uptake of glucose. In addition to its role in glucose homeostasis, insulin has been shown to regulate cell proliferation. Specifically, insulin enhances the phosphorylation of pyruvate dehydrogenase E1α (PDHA1) at the Ser293 residue and promotes the proliferation of HepG2 hepatocellular carcinoma cells. Furthermore, we previously observed that p-Ser293 PDHA1 bound with pyruvate kinase M2 (PKM2) as confirmed by coimmunoprecipitation. In this study, we used an in silico analysis to predict the structural conformation of the two binding proteins. However, the function of the protein complex remained unclear. To investigate further, we treated cells with si-PDHA1 and si-PKM2, which led to a reduction in PKM2 and p-Ser293 PDHA1 levels, respectively. Additionally, we found that the PDHA S293A dephospho-mimic reduced PKM2 levels and its associated enzyme activity. Treatment with MG132 and leupeptin impeded the PDHA1 S293A-mediated PKM2 reduction. These results suggest that the association between p-PDHA1 and PKM2 promotes their stability and protects them from protein degradation. Of interest, we observed that p-PDHA1 and PKM2 were localized in the nucleus in liver cancer patients. Under insulin stimulation, the knockdown of both PDHA1 and PKM2 led to a reduction in the expression of common genes, including KDMB1. These findings suggest that p-PDHA1 and PKM2 play a regulatory role in these proteins' expression and induce tumorigenesis in response to insulin.

Extracellular pyruvate kinase M2 induces cell migration through p-Tyr42 RhoA-mediated superoxide generation and epithelial-mesenchymal transition.

In the tumor microenvironment (TME), communication between cancer cells and tumor-associated macrophages (TAMs) through secreted extracellular proteins promotes cancer progression. Here, we observed that co-culturing cancer cells (4T1) and macrophage cells (Raw264.7) significantly enhanced superoxide production in both cell types. Using MALDI-TOF, we identified PKM2 as a highly secreted protein by Raw264.7 cells and bone marrow-derived monocytes. The extracellular recombinant PKM2 protein not only enhanced cancer cell migration and invasion but also increased superoxide production. Additionally, PKM2 was found to associate with the cell surface, and its binding to integrin α5/ß1 receptor was inhibited by antibodies specifically targeting it. Furthermore, we investigated downstream signaling pathways involved in PKM2-induced superoxide production. We found that knock-down of RhoA and p47phox using siRNAs effectively abolished superoxide generation in response to extracellular PKM2. Notably, extracellular PKM2 triggered the phosphorylation of p47phox at Ser345 residue and RhoA at Tyr42 residue (p-Tyr42 RhoA). Moreover, extracellular PKM2 exerted regulatory control over the expression of key epithelial-mesenchymal transition (EMT) markers, including ZEB1, Snail1, vimentin, and E-cadherin. Interestingly, p-Tyr42 RhoA translocated to the nucleus, where it bound to the ZEB1 promoter region. In light of these findings, we propose that extracellular PKM2 within the TME plays a critical role in tumorigenesis by promoting cancer cell migration and invasion through RhoA/p47phox signaling pathway.

A p-Tyr42 RhoA Inhibitor Promotes the Regeneration of Human Corneal Endothelial Cells by Ameliorating Cellular Senescence.

The development of treatment strategies for human corneal endothelial cells (hCECs) disease is necessary because hCECs do not regenerate in vivo due to the properties that are similar to senescence. This study is performed to investigate the role of a p-Tyr42 RhoA inhibitor (MH4, ELMED Inc., Chuncheon) in transforming growth factor-beta (TGF-ß)- or H2O2-induced cellular senescence of hCECs. Cultured hCECs were treated with MH4. The cell shape, proliferation rate, and cell cycle phases were analyzed. Moreover, cell adhesion assays and immunofluorescence staining for F-actin, Ki-67, and E-cadherin were performed. Additionally, the cells were treated with TGF-ß or H2O2 to induce senescence, and mitochondrial oxidative reactive oxygen species (ROS) levels, mitochondrial membrane potential, and NF-κB translocation were evaluated. LC3II/LC3I levels were determined using Western blotting to analyze autophagy. MH4 promotes hCEC proliferation, shifts the cell cycle, attenuates actin distribution, and increases E-cadherin expression. TGF-ß and H2O2 induce senescence by increasing mitochondrial ROS levels and NF-κB translocation into the nucleus; however, this effect is attenuated by MH4. Moreover, TGF-ß and H2O2 decrease the mitochondrial membrane potential and induce autophagy, while MH4 reverses these effects. In conclusion, MH4, a p-Tyr42 RhoA inhibitor, promotes the regeneration of hCECs and protects hCECs against TGF-ß- and H2O2-induced senescence via the ROS/NF-κB/mitochondrial pathway.

Effects of Pyruvate Kinase M2 (PKM2) Gene Deletion on Astrocyte-Specific Glycolysis and Global Cerebral Ischemia-Induced Neuronal Death.

Ischemic stroke is caused by insufficient blood flow to the brain. Astrocytes have a role in bidirectionally converting pyruvate, generated via glycolysis, into lactate and then supplying it to neurons through astrocyte-neuron lactate shuttle (ANLS). Pyruvate kinase M2 (PKM2) is an enzyme that dephosphorylates phosphoenolpyruvate to pyruvate during glycolysis in astrocytes. We hypothesized that a reduction in lactate supply in astrocyte PKM2 gene deletion exacerbates neuronal death. Mice harboring a PKM2 gene deletion were established by administering tamoxifen to Aldh1l1-CreERT2; PKM2f/f mice. Upon development of global cerebral ischemia, mice were immediately injected with sodium l-lactate (250 mg/kg, i.p.). To verify our hypothesis, we compared oxidative damage, microtubule disruption, ANLS disruption, and neuronal death between the gene deletion and control subjects. We observed that PKM2 gene deletion increases the degree of neuronal damage and impairment of lactate metabolism in the hippocampal region after GCI. The lactate administration groups showed significantly reduced neuronal death and increases in neuron survival and cognitive function. We found that lactate supply via the ANLS in astrocytes plays a crucial role in maintaining energy metabolism in neurons. Lactate administration may have potential as a therapeutic tool to prevent neuronal damage following ischemic stroke.

Lipopolysaccharide Stimulates A549 Cell Migration through p-Tyr 42 RhoA and Phospholipase D1 Activity.

Cell migration is a crucial contributor to metastasis, a critical process associated with the mortality of cancer patients. The initiation of metastasis is triggered by epithelial-mesenchymal transition (EMT), along with the changes in the expression of EMT marker proteins. Inflammation plays a significant role in carcinogenesis and metastasis. Lipopolysaccharide (LPS), a typical inflammatory agent, promoted the generation of superoxide through the activation of p-Tyr42 RhoA, Rho-dependent kinase 2 (ROCK2), and the phosphorylation of p47phox. In addition, p-Tyr42 RhoA activated phospholipase D1 (PLD1), with PLD1 and phosphatidic acid (PA) being involved in superoxide production. PA also regulated the expression of EMT proteins. Consequently, we have identified MHY9 (Myosin IIA, NMIIA) as a PA-binding protein in response to LPS. MYH9 also contributed to cell migration and the alteration in the expression of EMT marker proteins. Co-immunoprecipitation revealed the formation of a complex involving p-Tyr42 RhoA, PLD1, and MYH9. These proteins were found to be distributed in both the cytosol and nucleus. In addition, we have found that p-Tyr42 RhoA PLD1 and MYH9 associate with the ZEB1 promoter. The suppression of ZEB1 mRNA levels was achieved through the knockdown of RhoA, PLD1, and MYH9 using si-RNAs. Taken together, we propose that p-Tyr42 RhoA and PLD1, responsible for producing PA, and PA-bound MYH9 are involved in the regulation of ZEB1 expression, thereby promoting cell migration.

Pyruvate Dehydrogenase A1 Phosphorylated by Insulin Associates with Pyruvate Kinase M2 and Induces LINC00273 through Histone Acetylation.

Insulin potently promotes cell proliferation and anabolic metabolism along with a reduction in blood glucose levels. Pyruvate dehydrogenase (PDH) plays a pivotal role in glucose metabolism. Insulin increase PDH activity by attenuating phosphorylated Ser293 PDH E1α (p-PDHA1) in normal liver tissue. In contrast to normal hepatocytes, insulin enhanced p-PDHA1 level and induced proliferation of hepatocellular carcinoma HepG2 cells. Here, we attempted to find a novel function of p-PDHA1 in tumorigenesis upon insulin stimulation. We found that p-Ser293 E1α, but not the E2 or E3 subunit of pyruvate dehydrogenase complex (PDC), co-immunoprecipitated with pyruvate kinase M2 (PKM2) upon insulin. Of note, the p-PDHA1 along with PKM2 translocated to the nucleus. The p-PDHA1/PKM2 complex was associated with the promoter of long intergenic non-protein coding (LINC) 00273 gene (LINC00273) and recruited p300 histone acetyl transferase (HAT) and ATP citrate lyase (ACL), leading to histone acetylation. Consequently, the level of transcription factor ZEB1, an epithelial-mesenchymal transition (EMT) marker, was promoted through increased levels of LINC00273, resulting in cell migration upon insulin. p-PDHA1, along with PKM2, may be crucial for transcriptional regulation of specific genes through epigenetic regulation upon insulin in hepatocarcinoma cells.

Cross-Talk between Wnt Signaling and Src Tyrosine Kinase.

Src, a non-receptor tyrosine kinase, was first discovered as a prototype oncogene and has been shown to critical for cancer progression for a variety of tissues. Src activity is regulated by a number of post-translational modifications in response to various stimuli. Phosphorylations of Src Tyr419 (human; 416 in chicken) and Src Tyr530 (human; 527 in chicken) have been known to be critical for activation and inactivation of Src, respectively. Wnt signaling regulates a variety of cellular functions including for development and cell proliferation, and has a role in certain diseases such as cancer. Wnt signaling is carried out through two pathways: ß-catenin-dependent canonical and ß-catenin-independent non-canonical pathways as Wnt ligands bind to their receptors, Frizzled, LRP5/6, and ROR1/2. In addition, many signaling components including Axin, APC, Damm, Dishevelled, JNK kinase and Rho GTPases contribute to these canonical and non-canonical Wnt pathways. However, the communication between Wnt signaling and Src tyrosine kinase has not been well reviewed as Src regulates Wnt signaling through LRP6 tyrosine phosphorylation. GSK-3ß phosphorylated by Wnt also regulates Src activity. As Wnt signaling and Src mutually regulate each other, it is noted that aberrant regulation of these components give rise to various diseases including typically cancer, and as such, merit a closer look.

Vehicle Load Estimation Using the Reaction Force of a Vertical Displacement Sensor Based on Fiber Bragg Grating.

Accurately calculating the vehicle load acting on a bridge at any one time is crucial to determining the integrity and safety of the bridge. To ensure this integrity and safety, information on the types, characteristics, and load of vehicles that regularly cross the bridge is very important in terms of its structural adequacy and maintenance. In this study, the vehicle load that a bridge will be subjected to was estimated using the reaction force response at the support. To estimate this response to the reaction force, a vertical displacement sensor, developed based on Fiber Bragg Grating (FBG), was applied to the Eradi Quake System (EQS), a commercially available bridge bearing. This vertical displacement sensor can measure the vertical load and has the advantage of being easy to attach and detach. To verify the performance and accuracy of this sensor, this study conducted numerical analysis and vehicle loading tests. It found that the vehicle load can be estimated from the reaction force response, as measured by the vertical displacement sensor on the bridge.

Multiple functions of pyruvate kinase M2 in various cell types.

Glucose metabolism is a mechanism by which energy is produced in form of adenosine triphosphate (ATP) by mitochondria and precursor metabolites are supplied to enable the ultimate enrichment of mature metabolites in the cell. Recently, glycolytic enzymes have been shown to have unconventional but important functions. Among these enzymes, pyruvate kinase M2 (PKM2) plays several roles including having conventional metabolic enzyme activity, and also being a transcriptional regulator and a protein kinase. Compared with the closely related PKM1, PKM2 is highly expressed in cancer cells and embryos, whereas PKM1 is dominant in mature, differentiated cells. Posttranslational modifications such as phosphorylation and acetylation of PKM2 change its cellular functions. In particular, PKM2 can translocate to the nucleus, where it regulates the transcription of many target genes. It is notable that PKM2 also acts as a protein kinase to phosphorylate several substrate proteins. Besides cancer cells and embryonic cells, astrocytes also highly express PKM2, which is crucial for lactate production via expression of lactate dehydrogenase A (LDHA), while mature neurons predominantly express PKM1. The lactate produced in cancer cells promotes tumor progress and that in astrocytes can be supplied to neurons and may act as a major source for neuronal ATP energy production. Thereby, we propose that PKM2 along with its different posttranslational modifications has specific purposes for a variety of cell types, performing unique functions.

Influence of the Depth of the Convolutional Neural Networks on an Artificial Intelligence Model for Diagnosis of Orthognathic Surgery.

The aim of this study was to investigate the relationship between image patterns in cephalometric radiographs and the diagnosis of orthognathic surgery and propose a method to improve the accuracy of predictive models according to the depth of the neural networks. The study included 640 and 320 patients requiring non-surgical and surgical orthodontic treatments, respectively. The data of 150 patients were exclusively classified as a test set. The data of the remaining 810 patients were split into five groups and a five-fold cross-validation was performed. The convolutional neural network models used were ResNet-18, 34, 50, and 101. The number in the model name represents the difference in the depth of the blocks that constitute the model. The accuracy, sensitivity, and specificity of each model were estimated and compared. The average success rate in the test set for the ResNet-18, 34, 50, and 101 was 93.80%, 93.60%, 91.13%, and 91.33%, respectively. In screening, ResNet-18 had the best performance with an area under the curve of 0.979, followed by ResNets-34, 50, and 101 at 0.974, 0.945, and 0.944, respectively. This study suggests the required characteristics of the structure of an artificial intelligence model for decision-making based on medical images.

RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to ß-catenin and contributes transcriptional regulation of vimentin upon Wnt3A.

In the Wnt canonical pathway, Wnt3A has been known to stabilize ß-catenin. In the non-canonical Wnt signaling pathway, Wnt is known to activate Rho GTPases. The correlation between canonical and non-canonical pathways by Wnt signaling, however, has not been well elucidated. Here, we identified that Wnt3A promoted superoxide generation, leading to Tyr42 phosphorylation of RhoA through activations of c-Src and Rho-dependent coiled coil kinase 2 (ROCK2) and phosphorylation of p47phox, a component of NADPH oxidase. Wnt3A also induced accumulation of ß-catenin along with activations of RhoA and ROCK1. Concurrently, ROCK1 was able to phosphorylate GSK-3ß at Ser9, which phosphorylated Src at Ser51 and Ser492 residues, leading to Src inactivation through dephosphorylation of Tyr416 during the late period of Wnt3A treatment. Meanwhile, p-Tyr42 RhoA bound to ß-catenin via the N-terminal domain of ß-catenin, thereby leading to the nuclear translocation of p-Tyr42 RhoA/ß-catenin complex. Notably, p-Tyr42 RhoA as well as ß-catenin was associated with the promoter of Vim, leading to increased expression of vimentin. In addition, stomach cancer patients harboring higher expressed p-Tyr42 Rho levels revealed the much poorer survival probability. Therefore, we propose that p-Tyr42 RhoA is crucial for transcriptional regulation of specific target genes in the nucleus by binding to their promoters and involved in tumorigenesis.

Distinct dual roles of p-Tyr42 RhoA GTPase in tau phosphorylation and ATP citrate lyase activation upon different Aß concentrations.

Both the accumulation of Amyloid-ß (Aß) in plaques and phosphorylation of Tau protein (p-Tau) in neurofibrillary tangles have been identified as two major symptomatic features of Alzheimer's disease (AD). Despite of critical role of Aß and p-Tau in AD progress, the interconnection of signalling pathways that Aß induces p-Tau remains elusive. Herein, we observed that a popular AD model mouse (APP/PS1) and Aß-injected mouse showed an increase in p-Tyr42 Rho in hippocampus of brain. Low concentrations of Aß (1 µM) induced RhoA-mediated Ser422 phosphorylation of Tau protein (p-Ser422 Tau), but reduced the expression of ATP citrate lyase (ACL) in the HT22 hippocampal neuronal cell line. In contrast, high concentrations of Aß (10 µM) along with high levels of superoxide production remarkably attenuated accumulation of p-Ser422 Tau, but augmented ACL expression and activated sterol regulatory element-binding protein 1 (SREBP1), leading to cellular senescence. Notably, a high concentration of Aß (10 µM) induced nuclear localization of p-Tyr42 Rho, which positively regulated NAD kinase (NADK) expression by binding to the NADK promoter. Furthermore, severe AD patient brain showed high p-Tyr42 Rho levels. Collectively, our findings indicate that both high and low concentrations of Aß are detrimental to neurons via distinct two p-Tyr42 RhoA-mediated signalling pathways in Ser422 phosphorylation of Tau and ACL expression.

P-Tyr42 RhoA GTPase amplifies superoxide formation through p47phox, phosphorylated by ROCK.

Optimal levels of reactive oxygen species (ROS) play a critical role in cellular physiological function. For production of intracellular superoxide, NADPH oxidase is one of the sources. Rac1/2 and RhoA GTPases are involved in regulation of NADPH oxidase activity and Tyr42 phosphorylation of RhoA (p-Tyr42 RhoA) seems significant in this regard as it was recently shown that hydrogen peroxide was able to increase p-Tyr42 RhoA levels. Phorbol myristate acetate (PMA), a tumor promoter, also induces production of superoxides; PMA activates Src, a tyrosine kinase, and increases p-Tyr42 RhoA levels. In exploring the mechanism of PMA effects, we reduced RhoA levels in test cells with si-RhoA and then restoration of various versions of RhoA for effect in response of the cells to PMA and producing superoxides. Restoration of RhoA Y42F (a dephospho-mimic form) still had reduced superoxide formation in response to PMA, compared with WT and Y42E RhoA. This was similarly seen with assays for cell migration and proliferation with cells responding to PMA. Y27632, a ROCK (Rho associated coiled coil kinase) inhibitor, also inhibited superoxide production, and also reduced p-Y416 Src and p-p47phox levels. A ROCK active fragment was also able to phosphorylate p47phox at Ser345 residue (p-Ser345 p47phox), a component of NADPH oxidase. Overall, we demonstrate that p-Tyr42 RhoA levels increase following PMA treatment and this is through production of superoxide and activation of Src. These in turn amplify superoxide production through ROCK phophorylation of p47phox and maintain a positive feedback loop for superoxide generation, and contribute to tumor progression.

Regulation of RhoA GTPase and novel target proteins for ROCK.

Rho GTPases play significant roles in cellular function and their activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), providing activation and inactivation of these GTPases, respectively. Active GTP-bound form of RhoA activates its effector proteins while the inactive GDP-bound form of RhoA exists in a RhoA-RhoGDI (guanine nucleotide dissociation inhibitor) complex in the cytosol. In particular, IκB kinase γ IKKγ/NF-κB essential modulator (NEMO) plays a role as a GDI displacement factor (GDF) for RhoA activation through binding to RhoA-RhoGDI complex. Meanwhile, prion protein inactivates RhoA despite RhoA/RhoGDI association. Novel target proteins for Rho-associated kinase (ROCK) such as glycogen synthase kinase (GSK)-3ß and IKKß are recently discovered. Here, we elaborate on a post-translationally modified version of RhoA, phosphorylated at Tyr42 and oxidized at Cys16/20. This form of RhoA dissociates from RhoA-RhoGDI complex and activates IKKß on IKKγ/NEMO, thus providing possibly a critical role for tumourigenesis.

Aß modulates actin cytoskeleton via SHIP2-mediated phosphoinositide metabolism.

Emerging evidences suggest that phospholipid metabolism is altered in Alzheimer's disease (AD), but molecular mechanisms on how this affects neurodegeneration in AD is poorly understood. SHIP2 is a phosphoinositide-metabolizing enzyme, which dephosphorylates PI(3,4,5)P3 resulting to PI(3,4)P2, and it has been recently shown that Aß directly increases the activity of SHIP2. Here we monitored, utilizing fluorescent SHIP2 biosensor, real-time increase of PI(3,4)P2-containing vesicles in HT22 cells treated with Aß. Interestingly, PI(3,4)P2 is accumulated at late endosomes and lysosomal vesicles. We further discovered that ARAP3 can be attracted to PI(3,4)P2-positive mature endosomes via its PH domain and this facilitates the degradation of ARAP3. The reduced level of ARAP3 then causes RhoA hyperactivation and filamentous actin, which are critical for neurodegeneration in AD. These results provide a novel molecular link between Aß and actin disruption through dysregulated phosphoinositide metabolism, and the SHIP2-PI(3,4)P2-ARAP3-RhoA signaling pathway can be considered as new therapeutic targets for synaptic dysfunctions in Alzheimer's disease.

Rap1 regulates hepatic stellate cell migration through the modulation of RhoA activity in response to TGF­ß1.

Although the migration of hepatic stellate cells (HSCs) is important for hepatic fibrosis, the regulation of this migration is poorly understood. Notably, transforming growth factor (TGF)­ß1 induces monocyte migration to sites of injury or inflammation during the early phase, but inhibits cell migration during the late phase. In the present study, the role of transforming protein RhoA signaling in TGF­ß1­induced HSC migration was investigated. TGF­ß1 was found to increase the protein and mRNA levels of smooth muscle actin and collagen type I in HSC­T6 cells. The level of RhoA­GTP in TGF­ß1­stimulated cells was significantly higher than that in control cells. Furthermore, the phosphorylation of cofilin and formation of filamentous actin (F­actin) were more marked in TGF­ß1­stimulated cells than in control cells. Additionally, TGF­ß1 induced the activation of nuclear factor­κB, and the expression of extracellular matrix proteins and several cytokines in HSC­T6 cells. The active form of Rap1 (Rap1 V12) suppressed RhoA­GTP levels, whereas the dominant­negative form of Rap1 (Rap1 N17) augmented RhoA­GTP levels. Therefore, the data confirmed that Rap1 regulated the activation of RhoA in TGF­ß1­stimulated HSC­T6 cells. These findings suggest that TGF­ß1 regulates Rap1, resulting in the suppression of RhoA, activation of and formation of F­actin during the migration of HSCs.

The Effects of Sodium Dichloroacetate on Mitochondrial Dysfunction and Neuronal Death Following Hypoglycemia-Induced Injury.

Our previous studies demonstrated that some degree of neuronal death is caused by hypoglycemia, but a subsequent and more severe wave of neuronal cell death occurs due to glucose reperfusion, which results from the rapid restoration of low blood glucose levels. Mitochondrial dysfunction caused by hypoglycemia leads to increased levels of pyruvate dehydrogenase kinase (PDK) and suppresses the formation of ATP by inhibiting pyruvate dehydrogenase (PDH) activation, which can convert pyruvate into acetyl-coenzyme A (acetyl-CoA). Sodium dichloroacetate (DCA) is a PDK inhibitor and activates PDH, the gatekeeper of glucose oxidation. However, no studies about the effect of DCA on hypoglycemia have been published. In the present study, we hypothesized that DCA treatment could reduce neuronal death through improvement of glycolysis and prevention of reactive oxygen species production after hypoglycemia. To test this, we used an animal model of insulin-induced hypoglycemia and injected DCA (100 mg/kg, i.v., two days) following hypoglycemic insult. Histological evaluation was performed one week after hypoglycemia. DCA treatment reduced hypoglycemia-induced oxidative stress, microglial activation, blood-brain barrier disruption, and neuronal death compared to the vehicle-treated hypoglycemia group. Therefore, our findings suggest that DCA may have the therapeutic potential to reduce hippocampal neuronal death after hypoglycemia.

Alcohol dependence treating agent, acamprosate, prevents traumatic brain injury-induced neuron death through vesicular zinc depletion.

Acamprosate, also known as N-acetyl homotaurine, is an N-methyl-d-aspartate receptor antagonist that is used for treating alcohol dependence. Although the exact mechanism of acamprosate has not been clearly established, it appears to work by promoting a balance between the excitatory and inhibitory neurotransmitters, glutamate, and gamma-aminobutyric acid, respectively. Several studies have demonstrated that acamprosate provides neuroprotection against ischemia-induced brain injury. However, no studies have been performed evaluating the effect of acamprosate on traumatic brain injury (TBI). In the present study, we sought to evaluate the therapeutic potential of acamprosate to protect against neuronal death following TBI. Rats were given oral acamprosate (200 mg/kg/d for 2weeks) and then subjected to a controlled cortical impact injury localized over the parietal cortex. Histologic analysis was performed at 3hours, 24hours, and 7days after TBI. We found that acamprosate treatment reduced the concentration of vesicular glutamate and zinc in the hippocampus. Consequently, this reduced vesicular glutamate and zinc level resulted in a reduction of reactive oxygen species production after TBI. When evaluated 24hours after TBI, acamprosate administration reduced the number of degenerating neurons, zinc accumulation, blood-brain barrier disruption, neutrophil infiltration, and dendritic loss. Acamprosate also reduced glial activation and neuronal loss at 7days after TBI. In addition, acamprosate rescued TBI-induced neurologic and cognitive dysfunction. The present study demonstrates that acamprosate attenuates TBI-induced brain damage by depletion of vesicular glutamate and zinc levels. Therefore, this study suggests that acamprosate may have high therapeutic potential for prevention of TBI-induced neuronal death.

Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.

Insulin is a critical signaling molecule in reducing blood glucose levels, and pyruvate dehydrogenase (PDH) is an essential enzyme in regulating glucose metabolism. However, the insulin effect on PDH function has not been well established. We observed that insulin attenuated the phosphorylation (p) of Ser264 (p-Ser264) in the PDH E1α subunit (PDHA1) in normal rat hepatocyte. In contrast, insulin induced an increase of p-Ser264 PDHA1 levels in hepatocellular carcinoma HepG2 and Huh7 cells. Insulin activated RhoA and Rho-dependent coiled coil kinase, an effector protein of active RhoA, which regulated p-Ser264 PDHA1 levels, along with both p-Ser9 and p-Tyr216 forms of glycogen synthase kinase-3ß (GSK-3ß) in HepG2 cells. Only p-Tyr216 GSK-3ß, the active form was involved in an increase of p-Ser264 PDHA1. Akt was also engaged in p-Ser9 of GSK-3ß, but neither in p-Tyr216 of GSK-3ß nor p-Ser264 of PDHA1 upon insulin. Reconstituted dephospho-mimic forms PDHA1 S264A and GSK-3ß Y216F impaired, but wild-types PDHA1 and GSK-3ß and phospho-mimic forms PDHA1 S264D and GSK-3ß Y216E increased cell proliferation upon insulin through expression of c-Myc and cyclin D1. Therefore, we propose that insulin-mediated p-PDHA1 is involved in the regulation of HepG2 cell proliferation through RhoA signaling pathway.-Islam, R., Kim, J.-G., Park, Y., Cho, J.-Y., Cap, K.-C., Kho, A.-R., Chung, W.-S., Suh, S.-W., Park, J.-B. Insulin induces phosphorylation of pyruvate dehydrogenase through RhoA activation pathway in HepG2 cells.