Sensing plasma membrane pore formation induces chemokine production in survivors of regulated necrosis.

Although overwhelming plasma membrane integrity loss leads to cell lysis and necrosis, cells can tolerate a limited level of plasma membrane damage, undergo ESCRT-III-mediated repair, and survive. Here, we find that cells which undergo limited plasma membrane damage from the pore-forming actions of MLKL, GSDMD, perforin, or detergents experience local activation of PKCs through Ca2+ influx at the damage sites. S660-phosphorylated PKCs subsequently activate the TAK1/IKKs axis and RelA/Cux1 complex to trigger chemokine expressions. We observe that in late-stage cancers, cells with active MLKL show expression of CXCL8. Similar expression induction is also found in ischemia-injured kidneys. Chemokines generated in this manner are also indispensable for recruiting immune cells to the dead and dying cells. This plasma membrane integrity-sensing pathway is similar to the well-established yeast cell wall integrity signaling pathway at molecular level, and this suggests an evolutionary conserved mechanism to respond to the cellular barrier damage.

Involvement of the Parathyroid Hormone-Related Protein on Changes in the CYP3A Expression in Cancer Cachexia.

Parathyroid hormone-related protein (PTHrP), which is secreted from a tumor, contributes to the progression of cachexia, a condition that is observed in half of all cancer patients. Although drug clearance was reported to decrease in patients with cancer cachexia, the details have not been clarified. The present study reports on an investigation of whether PTHrP is involved in the alternation of drug metabolism in cases of cancer cachexia. Cancer cachexia model rats with elevated serum PTHrP levels showed a significant decrease in hepatic and intestinal CYP3A2 protein expression. When midazolam, a CYP3A substrate drug, was administered intravenously or orally to the cancer cachexia rats, its area under the curve (AUC) was increased by about 2 and 5 times, as compared to the control group. Accordingly, the bioavailability of midazolam was increased by about 3 times, thus enhancing its pharmacological effect. In vitro experiments using HepG2 cells and Caco-2 cells showed that the addition of serum from cancer cachexia rats or active PTHrP (1-34) to each cell resulted in a significant decrease in the expression of CYP3A4 mRNA. Treatment with a cell-permeable cAMP analog also resulted in a decreased CYP3A4 expression. Pretreatment with protein kinase A (PKA), protein kinase C (PKC), and nuclear factor-kappa B (NF-κB) inhibitors recovered the decrease in CYP3A4 expression that was induced by PTHrP (1-34). These results suggest that PTHrP suppresses CYP3A expression via the cAMP/PKA/PKC/NF-κB pathway. Therefore, it is likely that PTHrP would be involved in the changes in drug metabolism observed in cancer cachexia.

Protein kinase Cι and SRC signaling define reciprocally related subgroups of glioblastoma with distinct therapeutic vulnerabilities.

We report that atypical protein kinase Cι (PKCι) is an oncogenic driver of glioblastoma (GBM). Deletion or inhibition of PKCι significantly impairs tumor growth and prolongs survival in murine GBM models. GBM cells expressing elevated PKCι signaling are sensitive to PKCι inhibitors, whereas those expressing low PKCι signaling exhibit active SRC signaling and sensitivity to SRC inhibitors. Resistance to the PKCι inhibitor auranofin is associated with activated SRC signaling and response to a SRC inhibitor, whereas resistance to a SRC inhibitor is associated with activated PKCι signaling and sensitivity to auranofin. Interestingly, PKCι- and SRC-dependent cells often co-exist in individual GBM tumors, and treatment of GBM-bearing mice with combined auranofin and SRC inhibitor prolongs survival beyond either drug alone. Thus, we identify PKCι and SRC signaling as distinct therapeutic vulnerabilities that are directly translatable into an improved treatment for GBM.

Photoactivation of mitochondrial reactive oxygen species-mediated Src and protein kinase C pathway enhances MHC class II-restricted T cell immunity to tumours.

High fluence low-level laser (HF-LLL), a mitochondria-targeted tumour phototherapy, results in oxidative damage and apoptosis of tumour cells, as well as damage to normal tissue. To circumvent this, the therapeutic effect of low fluence LLL (LFL), a non-invasive and drug-free therapeutic strategy, was identified for tumours and the underlying molecular mechanisms were investigated. We observed that LFL enhanced antigen-specific immune response of macrophages and dendritic cells by upregulating MHC class II, which was induced by mitochondrial reactive oxygen species (ROS)-activated signalling, suppressing tumour growth in both CD11c-DTR and C57BL/6 mice. Mechanistically, LFL upregulated MHC class II in an MHC class II transactivator (CIITA)-dependent manner. LFL-activated protein kinase C (PKC) promoted the nuclear translocation of CIITA, as inhibition of PKC attenuated the DNA-binding efficiency of CIITA to MHC class II promoter. CIITA mRNA and protein expression also improved after LFL treatment, characterised by direct binding of Src and STAT1, and subsequent activation of STAT1. Notably, scavenging of ROS downregulated LFL-induced Src and PKC activation and antagonised the effects of LFL treatment. Thus, LFL treatment altered the adaptive immune response via the mitochondrial ROS-activated signalling pathway to control the progress of neoplastic disease.

The Role of PKC in Regulating NMDARs in Aluminum-Induced Learning and Memory Impairment in Rats.

Aluminum is a widespread environmental neurotoxicant that can induce Alzheimer's disease (AD)-like damage, such as neuronal injury and impairment of learning and memory. Several studies have shown that aluminum could reduce the synaptic plasticity, but its molecular mechanism remains unclear. In this study, rats were treated with aluminum maltol (Al(mal)3) to establish a toxic animal model and PMA was used to interfere with the expression of PKC. The Morris water maze and open field test were used to investigate the behavioral changes of the rats. Western blotting and RT-PCR were used to detect the expression levels of NMDAR subunits, PKC and CaMKII. The results showed that Al(mal)3 damaged learning and memory function and reduced anxiety in rats. During this process, the expression of PKC was downregulated and it inhibited the expression of NMDARs through the phosphorylation of CaMKII.

Brain-derived neurotrophic factor produced long-term synaptic enhancement in the anterior cingulate cortex of adult mice.

Previous studies have demonstrated that brain-derived neurotrophic factor (BDNF) is one of the diffusible messengers for enhancing synaptic transmission in the hippocampus. Less information is available about the possible roles of BDNF in the anterior cingulate cortex (ACC). In the present study, we used 64-electrode array field recording system to investigate the effect of BDNF on ACC excitatory transmission. We found that BDNF enhanced synaptic responses in a dose-dependent manner in the ACC in C57/BL6 mice. The enhancement was long-lasting, and persisted for at least 3 h. In addition to the enhancement, BDNF also recruited inactive synaptic responses in the ACC. Bath application of the tropomyosin receptor kinase B (TrkB) receptor antagonist K252a blocked BDNF-induced enhancement. L-type voltage-gated calcium channels (L-VGCC), metabotropic glutamate receptors (mGluRs), but not NMDA receptors were required for BDNF-produced enhancement. Moreover, calcium-stimulated adenylyl cyclase subtype 1 (AC1) but not AC8 was essential for the enhancement. A selective AC1 inhibitor NB001 completely blocked the enhancement. Furthermore, BDNF-produced enhancement occluded theta burst stimulation (TBS) induced long-term potentiation (LTP), suggesting that they may share similar signaling mechanisms. Finally, the expression of BDNF-induced enhancement depends on postsynaptic incorporation of calcium-permeable AMPA receptors (CP-AMPARs) and protein kinase Mζ (PKMζ). Our results demonstrate that cortical BDNF may contribute to synaptic potentiation in the ACC.

Development of an arteriolar niche and self-renewal of breast cancer stem cells by lysophosphatidic acid/protein kinase D signaling.

Breast cancer stem cells (BCSCs) are essential for cancer growth, metastasis and recurrence. The regulatory mechanisms of BCSC interactions with the vascular niche within the tumor microenvironment (TME) and their self-renewal are currently under extensive investigation. We have demonstrated the existence of an arteriolar niche in the TME of human BC tissues. Intriguingly, BCSCs tend to be enriched within the arteriolar niche in human estrogen receptor positive (ER+) BC and bi-directionally interact with arteriolar endothelial cells (ECs). Mechanistically, this interaction is driven by the lysophosphatidic acid (LPA)/protein kinase D (PKD-1) signaling pathway, which promotes both arteriolar differentiation of ECs and self-renewal of CSCs likely via differential regulation of CD36 transcription. This study indicates that CSCs may enjoy blood perfusion to maintain their stemness features. Targeting the LPA/PKD-1 -CD36 signaling pathway may have therapeutic potential to curb tumor progression by disrupting the arteriolar niche and effectively eliminating CSCs.

HDAC6 inhibitors sensitize non-mesenchymal triple-negative breast cancer cells to cysteine deprivation.

Triple-negative breast cancer (TNBC) is a highly malignant type of breast cancer and lacks effective therapy. Targeting cysteine-dependence is an emerging strategy to treat the mesenchymal TNBC. However, many TNBC cells are non-mesenchymal and unresponsive to cysteine deprivation. To overcome such resistance, three selective HDAC6 inhibitors (Tubacin, CAY10603, and Tubastatin A), identified by epigenetic compound library screening, can synergize with cysteine deprivation to induce cell death in the non-mesenchymal TNBC. Despite the efficacy of HDAC6 inhibitor, knockout of HDAC6 did not mimic the synthetic lethality induced by its inhibitors, indicating that HDAC6 is not the actual target of HDAC6 inhibitor in this context. Instead, transcriptomic profiling showed that tubacin triggers an extensive gene transcriptional program in combination with erastin, a cysteine transport blocker. Notably, the zinc-related gene response along with an increase of labile zinc was induced in cells by the combination treatment. The disturbance of zinc homeostasis was driven by PKCγ activation, which revealed that the PKCγ signaling pathway is required for HDAC6 inhibitor-mediated synthetic lethality. Overall, our study identifies a novel function of HDAC6 inhibitors that function as potent sensitizers of cysteine deprivation and are capable of abolishing cysteine-independence in non-mesenchymal TNBC.

Atypical protein kinase C is essential for embryonic vascular development in mice.

The atypical PKC (aPKC) subfamily constitutes PKCζ and PKCλ in mice, and both aPKC isoforms have been proposed to be involved in regulating various endothelial cell (EC) functions. However, the physiological function of aPKC in ECs during embryonic development has not been well understood. To address this question, we utilized Tie2-Cre to delete PKCλ alone (PKCλ-SKO) or both PKCλ and PKCζ (DKO) in ECs, and found that all DKO mice died at around the embryonic day 11.5 (E11.5), whereas a small proportion of PKCλ-SKO mice survived till birth. PKCλ-SKO embryos also exhibited less phenotypic severity than DKO embryos at E10.5 and E11.5, suggesting a potential compensatory role of PKCζ for PKCλ in embryonic ECs. We then focused on DKO embryos and investigated the effects of aPKC deficiency on embryonic vascular development. At E9.5, deletion of both aPKC isoforms reduced the diameters of vitelline artery and vein, and decreased branching from both vitelline vessels in yolk sac. Ablation of both aPKC isoforms also disrupted embryonic angiogenesis in head and trunk at the same stage, increasing apoptosis of both ECs and non-ECs. Taken together, our results demonstrated that aPKC in ECs plays an essential role in regulating cell apoptosis, angiogenesis, and embryonic survival.

The protein kinase D1-mediated inflammatory pathway is involved in olanzapine-induced impairment of skeletal muscle insulin signaling in rats.

AIMS: Skeletal muscle insulin resistance (SMIR) contributes to the metabolic syndrome. Mounting evidence has demonstrated that the second generation antipsychotic olanzapine causes SMIR. The present study sought to investigate the molecular mechanisms underlying olanzapine-induced SMIR. MAIN METHODS: Male rats were given olanzapine (5 mg/kg, by a gavage method) for consecutive eight weeks. Plasma glucose and insulin concentrations were determined enzymatically or by ELISA. Gene/protein expression was analyzed by Real-Time PCR, Western blot and/or immunohistochemistry. KEY FINDINGS: Olanzapine increased fasting plasma insulin concentration, and decreased glucose clearance during insulin tolerance test in rats. In skeletal muscle, it decreased protein expression of membrane glucose transporter (GLUT) 4, the ratio of membrane to total GLUT4, and total insulin receptor substrate 1 (IRS1). However, it increased protein phosphorylation of Ser307 in IRS1, Y607 in phosphoinositide 3-kinase p85α and Ser307 in AKT. These results indicate olanzapine-induced impairment of skeletal muscle insulin signaling. Mechanistically, olanzapine upregulated mRNA expression of TNFα, IL6 and IL1ß, and protein phosphorylation of both IκB kinase (IKK)α/ß and nuclear factor (NF)κB p65. Furthermore, it increased protein phosphorylation of Ser485/491 in AMPKα2, whereas it decreased AMPKα2 activity. More importantly, both Western blot and immunohistochemical analyses revealed that olanzapine increased protein phosphorylation of Ser744/748 in protein kinase D1 (PKD1). SIGNIFICANCE: The present results suggest that the PKD1-mediated inflammatory pathway is involved in olanzapine-induced impairment of skeletal muscle insulin signaling in rats. Our findings may go new insight into the mechanisms underlying olanzapine-induced SMIR.

The role of protein kinase D (PKD) in intracellular nutrient sensing and regulation of adaptive responses to the obese environment.

Obesity is associated with ectopic accumulation of lipids, which is implicated in the development of insulin resistance, type 2 diabetes mellitus and cardiovascular disease. As the global prevalence of obesity continues to rise, it is becoming increasingly important to understand the underlying cellular mechanisms of this disease. Protein kinase D (PKD) is an intracellular signalling kinase with well characterized roles in intracellular vesicle transport and secretion, cancer cell proliferation and cardiac hypertrophy. However, emerging evidence also highlights PKD as a novel nutrient sensor. PKD activation is mediated by the accumulation of the lipid intermediate diacylglycerol, and PKD activity in the liver, heart and adipose tissue increases upon feeding. In obesity, PKD signalling is linked to reduced insulin signalling and dysfunction in adipose tissue, liver and heart, whilst in the pancreas, PKD is essential for the compensatory increase in glucose-stimulated insulin secretion from ß-cells during obesity. Collectively, these studies reveal aspects of PKD signalling that are involved in the tissue-specific responses to obesity. This review summarizes the emerging evidence suggesting that PKD plays an important role in regulating the adaptive response to the obese environment.

Functional characterization of uveal melanoma oncogenes.

Uveal melanoma (UM) is a currently untreatable form of melanoma with a 50% mortality rate. Characterization of the essential signaling pathways driving this cancer is critical to develop target therapies. Activating mutations in the Gαq signaling pathway at the level of GNAQ, GNA11, or rarely CYSLTR2 or PLCß4 are considered alterations driving proliferation in UM and several other neoplastic disorders. Here, we systematically examined the oncogenic signaling output of various mutations recurrently identified in human tumors. We demonstrate that CYSLTR2 → GNAQ/11 → PLCß act in a linear signaling cascade that, via protein kinase C (PKC), activates in parallel the MAP-kinase and FAK/Yes-associated protein pathways. Using genetic ablation and pharmacological inhibition, we show that the PKC/RasGRP3/MAPK signaling branch is the essential component that drives the proliferation of UM. Only inhibition of the MAPK branch but not the FAK branch synergizes with inhibition of the proximal cascade, providing a blueprint for combination therapy. All oncogenic signaling could be extinguished by the novel GNAQ/11 inhibitor YM-254890, in all UM cells with driver mutation in the Gαq subunit or the upstream receptor. Our findings highlight the GNAQ/11 → PLCß â†’ PKC → MAPK pathway as the central signaling axis to be suppressed pharmacologically to treat for neoplastic disorders with Gαq pathway mutations.

Equivocal, explicit and emergent actions of PKC isoforms in cancer.

The maturing mutational landscape of cancer genomes, the development and application of clinical interventions and evolving insights into tumour-associated functions reveal unexpected features of the protein kinase C (PKC) family of serine/threonine protein kinases. These advances include recent work showing gain or loss-of-function mutations relating to driver or bystander roles, how conformational constraints and plasticity impact this class of proteins and how emergent cancer-associated properties may offer opportunities for intervention. The profound impact of the tumour microenvironment, reflected in the efficacy of immune checkpoint interventions, further prompts to incorporate PKC family actions and interventions in this ecosystem, informed by insights into the control of stromal and immune cell functions. Drugging PKC isoforms has offered much promise, but when and how is not obvious.

Using rat operant delayed match-to-sample task to identify neural substrates recruited with increased working memory load.

The delayed match-to-sample task (DMS) is used to probe working memory (WM) across species. While the involvement of the PFC in this task has been established, limited information exists regarding the recruitment of broader circuitry, especially under the low- versus high-WM load. We sought to address this question by using a variable-delay operant DMS task. Male Sprague-Dawley rats were trained and tested to determine their baseline WM performance across all (0- to 24-sec) delays. Next, rats were tested in a single DMS test with either 0- or 24-sec fixed delay, to assess low-/high-load WM performance. c-Fos mRNA expression was quantified within cortical and subcortical regions and correlated with WM performance. High WM load up-regulated overall c-Fos mRNA expression within the PrL, as well as within a subset of mGlu5+ cells, with load-dependent, local activation of protein kinase C (PKC) as the proposed underlying molecular mechanism. The PrL activity negatively correlated with choice accuracy during high load WM performance. A broader circuitry, including several subcortical regions, was found to be activated under low and/or high load conditions. These findings highlight the role of mGlu5- and/or PKC-dependent signaling within the PrL, and corresponding recruitment of subcortical regions during high-load WM performance.

Bisindolylpyrrole triggers transient mitochondrial permeability transitions to cause apoptosis in a VDAC1/2 and cyclophilin D-dependent manner via the ANT-associated pore.

Bisindolylpyrrole at 0.1 µM is cytoprotective in 2% FBS that is counteracted by cyclosporin-A (CsA), an inhibitor of cyclophilin-D (CypD). We hypothesized that the cytoprotective effect might be due to transient mitochondrial permeability transition (tPT). This study tested the hypothesis that bisindolylpyrrole can trigger tPT extensively, thereby leading to cell death under certain conditions. Indeed, CsA-sensitive tPT-mediated apoptosis could be induced by bisindolylpyrrole at > 5 µM in HeLa cells cultured in 0.1% FBS, depending on CypD and VDAC1/2, as shown by siRNA knockdown experiments. Rat liver mitochondria also underwent swelling in response to bisindolylpyrrole, which proceeded at a slower rate than Ca2+-induced swelling, and which was blocked by the VDAC inhibitor tubulin and the ANT inhibitor bongkrekate, indicating the involvement of the ANT-associated, smaller pore. We examined why 0.1% FBS is a prerequisite for apoptosis and found that apoptosis is blocked by PKC activation, which is counteracted by the overexpressed defective PKCε. In mitochondrial suspensions, bisindolylpyrrole triggered CsA-sensitive swelling, which was suppressed selectively by pretreatment with PKCε, but not in the co-presence of tubulin. These data suggest that upon PKC inactivation the cytoprotective compound bisindolylpyrrole can induce prolonged tPT causing apoptosis in a CypD-dependent manner through the VDAC1/2-regulated ANT-associated pore.

Critical Signaling Events in the Mechanoactivation of Human Mast Cells through p.C492Y-ADGRE2.

A role for the adhesion G-protein coupled receptor ADGRE2 or EMR2 in mechanosensing was revealed by the finding of a missense substitution (p.C492Y) associated with familial vibratory urticaria. In these patients, friction of the skin induces mast cell hyper-degranulation through p.C492Y-ADGRE2, causing localized hives, flushing, and hypotension. We have now characterized the responses and intracellular signals elicited by mechanical activation in human mast cells expressing p.C492Y-ADGRE2 and attached to dermatan sulfate, a ligand for ADGRE2. The presence of p.C492Y-ADGRE2 reduced the threshold to activation and increased the extent of degranulation along with the percentage of mast cells responding. Vibration caused phospholipase C activation, transient increases in cytosolic calcium, and downstream activation of phosphoinositide 3-kinase and extracellular signal-regulated kinases 1 and 2 by Gßγ, Gαq/11, and Gαi/o-independent mechanisms. Degranulation induced by vibration was dependent on phospholipase C pathways, including calcium, protein kinase C, and phosphoinositide 3-kinase but not extracellular signal-regulated kinases 1/2 pathways, along with pertussis toxin-sensitive signals. In addition, mechanoactivation of mast cells stimulated the synthesis and release of prostaglandin D2, to our knowledge a previously unreported mediator in vibratory urticaria, and extracellular signal-regulated kinases 1/2 activation was required for this response together with calcium, protein kinase C, and to some extent, phosphoinositide 3-kinase. Our studies thus identified critical molecular events initiated by mechanical forces and potential therapeutic targets for patients with vibratory urticaria.

Transcriptomic Analysis of Cellular Pathways in Healing Flexor Tendons of Plasminogen Activator Inhibitor 1 (PAI-1/Serpine1) Null Mice.

Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Plasminogen activator inhibitor 1 (PAI-1/SERPINE1), a master suppressor of fibrinolysis and protease activity, is associated with adhesions. Here, we used next-generation RNA sequencing (RNA-Seq) to assess genome-wide differences in messenger RNA expression due to PAI-1 deficiency after zone II flexor tendon injury. We used the ingenuity pathway analysis to characterize molecular pathways and biological drivers associated with differentially expressed genes (DEG). Analysis of hundreds of overlapping and DEG in PAI-1 knockout (KO) and wild-type mice (C57Bl/6J) during tendon healing revealed common and distinct biological processes. Pathway analysis identified cell proliferation, survival, and senescence, as well as chronic inflammation as potential drivers of fibrotic healing and adhesions in injured tendons. Importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1-associated biological processes as unique transcriptional signatures of the healing tendon in the PAI-1/Serpine1 KO mice. Further, transcriptomic differences due to the genetic deletion of PAI-1 were mechanistically linked to PI3K/Akt/mTOR, PKC, and MAPK signaling cascades. These transcriptional observations provide novel insights into the biological roles of PAI-1 in tendon healing and could identify therapeutic targets to achieve scar-free regenerative healing of tendons. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:43-58, 2020.

aPKCi triggers basal extrusion of luminal mammary epithelial cells by tuning contractility and vinculin localization at cell junctions.

Metastasis is the main cause of cancer-related deaths. How a single oncogenic cell evolves within highly organized epithelium is still unknown. Here, we found that the overexpression of the protein kinase atypical protein kinase C ι (aPKCi), an oncogene, triggers basally oriented epithelial cell extrusion in vivo as a potential mechanism for early breast tumor cell invasion. We found that cell segregation is the first step required for basal extrusion of luminal cells and identify aPKCi and vinculin as regulators of cell segregation. We propose that asymmetric vinculin levels at the junction between normal and aPKCi+ cells trigger an increase in tension at these cell junctions. Moreover, we show that aPKCi+ cells acquire promigratory features, including increased vinculin levels and vinculin dynamics at the cell-substratum contacts. Overall, this study shows that a balance between cell contractility and cell-cell adhesion is crucial for promoting basally oriented cell extrusion, a mechanism for early breast cancer cell invasion.

Participation of PKG and PKA-related pathways in the IFN-γ induced modulation of the BKCa channel activity in human cardiac fibroblasts.

This study was devoted to elucidating the interferon (IFN)-γ-induced signaling pathway and the interaction between protein kinase G (PKG) and protein kinase A (PKA) through large-conductance Ca(2+)-activated K(+) channels in human cardiac fibroblasts. The IK currents were recorded using a whole-cell patch clamp method. A large depolarization (+50 mV) and a high Ca2+ concentration (pCa 6.0) were used in the internal pipette solution to activate only the KCa channels. Iberiotoxin (Ibtx), which selectively inhibits BKCa channels at a concentration of 100 nmol/l, caused a significant reduction of basal IK. Adding IFN-γ in the presence of Ibtx had no effect on IK. Application of the IFN-γ caused a significant reduction in total K+ current amplitude, recorded with a 500 ms depolarizing pulse duration, to +50 mV from a holding potential of -80 mV. We tested the involvement of the sGC/cGMP/PKG signaling pathway by using specific PKG inhibitor KT 5823, potent sGC inhibitor NS 2028, and specific sGC agonist BAY 41-8543. The obtained data confirmed that only sGC participated in the IFN-γ-mediated BKCa channel modulation, which was mediated further by PKA. This study represents first evidence about the participation of the IFN-γ in the mechanisms responsible for BKCa modulation in HCFs. We also believe that this process occurs via negative crosstalk between the PKG- and PKA-associated pathways.

Protein Kinase C Attenuates Insulin Signalling Cascade in Insulin-Sensitive and Insulin-Resistant Neuro-2a Cells.

Protein kinase C (PKC) family of enzymes is known to be a feedback regulator of insulin signalling pathway in peripheral insulin-responsive tissues. Insulin signalling is reported to be required for maintaining cognitive abilities in brain. PKCs are involved in innumerable neuronal processes including differentiation, apoptosis, survival, maintaining synaptic plasticity, long-term potentiation and memory formation. In the present study, we made an attempt to elucidate the role of PKC, if any, in regulating insulin signalling and insulin resistance in Neuro-2a (N2a) cells in vitro. We show that phorbol 12-myristate 13-acetate (PMA) -activated PKC inhibited Akt activation in neuronal cell, N2a. In the process of inhibiting Akt, PMA-activated PKC decreased downstream insulin signalling proteins like Akt substrate 160 kDa (AS160) and glycogen synthase kinase (GSK3ß), followed by a decrease of glucose uptake in N2a cells. PKC activation caused insulin resistance in N2a cells and worsened the resistant state of already insulin-resistant cells. Hence, our study demonstrated that the activation of PKC attenuates insulin signalling cascade and make N2a cells insulin-resistant.