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Background The noble gas helium induces pre- and postconditioning in animals and humans. Volatile anesthetics induce cardioprotection in humans undergoing coronary artery bypass graft (CABG) surgery. We hypothesized that helium induces pre- and postconditioning in CABG-patients, affecting signaling molecules protein kinase C-epsilon (PKC-ε), p38 mitogen activated protein kinase (p38 MAPK), extracellular signal-regulated kinase 1/2 (ERK-1/2) and heat shock protein 27 (HSP-27) within cardiac tissue, and reducing postoperative troponin levels. Methods After ethical approval and informed consent, 125 elective patients undergoing CABG surgery were randomised into this prospective, placebo controlled, investigator blinded, parallel arm single-centre study. Helium preconditioning (3 × 5 min of 70 % helium and 30 % oxygen) was applied before aortic cross clamping; postconditioning (15 min of helium) was applied before release of the aortic cross clamp. Signaling molecules were measured in right atrial appendix specimens. Troponin-T was measured at 4, 12, 24 and 48 h postoperatively. Results Baseline characteristics of all groups were similar. Helium preconditioning did not significantly alter the primary outcome (molecular levels of kinases PKC-ε and HSP-27, ratio of activated p38 MAPK or ERK ½). Postoperative troponin T was 11 arbitrary units [5, 31; area-under-the-curve (interquartile range)] for controls, and no statistically significant changes were observed after helium preconditioning [He-pre: 11 (6, 18)], helium postconditioning [He-post: 11 (8, 15)], helium pre- and postconditioning [He-PP: 14 (6, 20)] and after sevoflurane preconditioning [APC: 12 (8, 24), p = 0.13]. No adverse effects related to study treatment were observed in this study. Conclusions No effect was observed of helium preconditioning, postconditioning or the combination thereof on activation of p38 MAPK, ERK 1/2 or levels of HSP27 and PKC-ε in the human heart. Helium pre- and postconditioning did not affect postoperative troponin release in patients undergoing CABG surgery. Clinical trial number Dutch trial register ( http://www.trialregister.nl/ ) number NTR1226

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet-induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance.

Transient receptor potential vanillic acid 1 (TRPV1) is an ion channel activated by heat and inflammatory factors involved in the development of various types of pain. The P2X7 receptor is in the P2X family and is associated with pain mediated by satellite glial cells. There might be some connection between the P2X7 receptor and TRPV1 in neuropathic pain in diabetic rats. A type 2 diabetic neuropathic pain rat model was induced using high glucose and high-fat diet for 4 weeks and low-dose streptozocin (35 mg/kg) intraperitoneal injection to destroy islet B cells. Male Sprague Dawley rats were administrated by intrathecal injection of P2X7 shRNA and p38 inhibitor, and we recorded abnormal mechanical and thermal pain and nociceptive hyperalgesia. One week later, the dorsal root ganglia from the L4-L6 segment of the spinal cord were harvested for subsequent experiments. We measured pro-inflammatory cytokines, examined the relationship between TRPV1 on neurons and P2X7 receptor on satellite glial cells by measuring protein and transcription levels of P2X7 receptor and TRPV1, and measured protein expression in the PKCε/P38 MAPK/NF-κB signaling pathway after intrathecal injection. P2X7 shRNA and p38 inhibitor relieved hyperalgesia in diabetic neuropathic pain rats and modulated inflammatory factors in vivo. P2X7 shRNA and P38 inhibitors significantly reduced TRPV1 expression by downregulating the PKCε/P38 MAPK/NF-κB signaling pathway and inflammatory factors in dorsal root ganglia. Intrathecal injection of P2X7 shRNA alleviates nociceptive reactions in rats with diabetic neuropathic pain involving TRPV1 via PKCε/P38 MAPK/NF-κB signaling pathway.

The heart’s response to varying demands of the body is regulated by signaling pathways that activate protein kinases which phosphorylate sarcomeric proteins. Although phosphorylation of cardiac myosin binding protein-C (cMyBP-C) has been recognized as a key regulator of myocardial contractility, little is known about its mechanism of action. Here, we used protein kinase A (PKA) and Cε (PKCε), as well as ribosomal S6 kinase II (RSK2), which have different specificities for cMyBP-C’s multiple phosphorylation sites, to show that individual sites are not independent, and that phosphorylation of cMyBP-C is controlled by positive and negative regulatory coupling between those sites. PKA phosphorylation of cMyBP-C’s N terminus on 3 conserved serine residues is hierarchical and antagonizes phosphorylation by PKCε, and vice versa. In contrast, RSK2 phosphorylation of cMyBP-C accelerates PKA phosphorylation. We used cMyBP-C’s regulatory N-terminal domains in defined phosphorylation states for protein–protein interaction studies with isolated cardiac native thin filaments and the S2 domain of cardiac myosin to show that site-specific phosphorylation of this region of cMyBP-C controls its interaction with both the actin-containing thin and myosin-containing thick filaments. We also used fluorescence probes on the myosin-associated regulatory light chain in the thick filaments and on troponin C in the thin filaments to monitor structural changes in the myofilaments of intact heart muscle cells associated with activation of myocardial contraction by the N-terminal region of cMyBP-C in its different phosphorylation states. Our results suggest that cMyBP-C acts as a sarcomeric integrator of multiple signaling pathways that determines downstream physiological function.

Insulin resistance drives the development of type 2 diabetes (T2D). In liver, diacylglycerol (DAG) is a key mediator of lipid-induced insulin resistance. DAG activates protein kinase C ε (PKCε), which phosphorylates and inhibits the insulin receptor. In rats, a 3-day high-fat diet produces hepatic insulin resistance through this mechanism, and knockdown of hepatic PKCε protects against high-fat diet-induced hepatic insulin resistance. Here, we employed a systems-level approach to uncover additional signaling pathways involved in high-fat diet-induced hepatic insulin resistance. We used quantitative phosphoproteomics to map global in vivo changes in hepatic protein phosphorylation in chow-fed, high-fat–fed, and high-fat–fed with PKCε knockdown rats to distinguish the impact of lipid- and PKCε-induced protein phosphorylation. This was followed by a functional siRNA-based screen to determine which dynamically regulated phosphoproteins may be involved in canonical insulin signaling. Direct PKCε substrates were identified by motif analysis of phosphoproteomics data and validated using a large-scale in vitro kinase assay. These substrates included the p70S6K substrates RPS6 and IRS1, which suggested cross talk between PKCε and p70S6K in high-fat diet-induced hepatic insulin resistance. These results identify an expanded set of proteins through which PKCε may drive high-fat diet-induced hepatic insulin resistance that may direct new therapeutic approaches for T2D.

G-protein-gated inwardly rectifying potassium (GIRK) channel activity is regulated by the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PI 4,5P₂). Constitutive activity of cardiac GIRK channels in atrial myocytes, that is implicated in atrial fibrillation (AF), is mediated via a protein kinase C-ε (PKCε)-dependent mechanism. The novel PKC isoform, PKCε, is reported to enhance the activity of cardiac GIRK channels. Here, we report that PKCε stimulation leads to activation of GIRK channels in mouse atria and in human stem cell-derived atrial cardiomyocytes (iPSCs). We identified residue GIRK4(S418) which when mutated to Ala abolished, or to Glu, mimicked the effects of PKCε on GIRK currents. PKCε strengthened the interactions of the cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP₂, an effect that was reversed in the GIRK4(S418A) mutant. This mechanistic insight into the PKCε-mediated increase in channel activity because of GIRK4(S418) phosphorylation, provides a precise druggable target to reverse AF-related pathologies due to GIRK overactivity.

Insulin resistance is associated with nonalcoholic fatty liver disease (NAFLD) and is a major factor in the pathogenesis of type 2 diabetes. The development of hepatic insulin resistance has been ascribed to multiple causes, including inflammation, endoplasmic reticulum (ER) stress, and accumulation of hepatocellular lipids in animal models of NAFLD. However, it is unknown whether these same cellular mechanisms link insulin resistance to hepatic steatosis in humans. To examine the cellular mechanisms that link hepatic steatosis to insulin resistance, we comprehensively assessed each of these pathways by using flash-frozen liver biopsies obtained from 37 obese, nondiabetic individuals and correlating key hepatic and plasma markers of inflammation, ER stress, and lipids with the homeostatic model assessment of insulin resistance index. We found that hepatic diacylglycerol (DAG) content in cytoplasmic lipid droplets was the best predictor of insulin resistance (R = 0.80, P < 0.001), and it was responsible for 64% of the variability in insulin sensitivity. Hepatic DAG content was also strongly correlated with activation of hepatic PKCε (R = 0.67, P < 0.001), which impairs insulin signaling. In contrast, there was no significant association between insulin resistance and other putative lipid metabolites or plasma or hepatic markers of inflammation. ER stress markers were only partly correlated with insulin resistance. In conclusion, these data show that hepatic DAG content in lipid droplets is the best predictor of insulin resistance in humans, and they support the hypothesis that NAFLD-associated hepatic insulin resistance is caused by an increase in hepatic DAG content, which results in activation of PKCε.

Recent studies have revealed the critical role of microRNAs (miRNAs) in regulating cardiac injury. Among them, the cardiac enriched microRNA-1(miR-1) has been extensively investigated and proven to be detrimental to cardiac myocytes. However, solid in vivo evidence for the role of miR-1 in cardiac injury is still missing and the potential therapeutic advantages of systemic knockdown of miR-1 expression remained unexplored. In this study, miR-1 transgenic (miR-1 Tg) mice and locked nucleic acid modified oligonucleotide against miR-1 (LNA-antimiR-1) were used to explore the effects of miR-1 on cardiac ischemia/reperfusion injury (30 min ischemia followed by 24 h reperfusion). The cardiac miR-1 level was significantly increased in miR-1 Tg mice, and suppressed in LNA-antimiR-1 treated mice. When subjected to ischemia/reperfusion injury, miR-1 overexpression exacerbated cardiac injury, manifested by increased LDH, CK levels, caspase-3 expression, apoptosis and cardiac infarct area. On the contrary, LNA-antimiR-1 treatment significantly attenuated cardiac ischemia/reperfusion injury. The expression of PKCε and HSP60 was significantly repressed by miR-1 and enhanced by miR-1 knockdown, which may be a molecular mechanism for the role miR-1 in cardiac injury. Moreover, luciferase assay confirmed the direct regulation of miR-1 on protein kinase C epsilon (PKCε) and heat shock protein 60 (HSP60). In summary, this study demonstrated that miR-1 is a causal factor for cardiac injury and systemic LNA-antimiR-1 therapy is effective in ameliorating the problem.

Background The breast cancer recurrence and chemoresistance has increased over the years. A novel PKC, PKCε, may promote chemoresistance by causing hypoxia and cancer metabolic rewiring. A natural flavonoid, Zapotin, in colon cancer cells may modulate PKCε expression. Therefore, this study aimed to explore Zapotin impact on PKCε expression and the metabolic profile of breast cancer cells. Methods Pharmacophore analysis of Zapotin was performed and molecular dynamics (MD) simulations were employed to study PKCε and Zapotin interaction stability. The effect of Zapotin treatment on PKCε expression and various aspects of cancer cell viability and metabolism was studied in MCF-7 and MDA-MB-231 breast cancer cell lines using real-time PCR, growth and death assays, and Gas Chromatography-Mass Spectrometry. Results In silico analyses revealed good solubility and absorption of Zapotin with lower toxicity. Zapotin showed cancer cell-specific cytotoxicity ( P  < 0.0001). It’s treatment also reduced breast cancer cell viability, colony formation, and migratory potential by targeting PKCε and associated HIF-1ɑ and VEGF signaling ( P  < 0.01). Zapotin also impacted PKCε-mediated metabolic signaling by targeting glycolytic pathways. Conclusion This study demonstrated the role of PKCε mediated HIF-1ɑ, VEGF, and glycolytic pathways in promoting breast carcinogenicity and demonstrated Zapotin as a potential treatment option for different types of breast tumors.

Aims/hypothesis Athletes exhibit increased muscle insulin sensitivity, despite increased intramuscular triacylglycerol content. This phenomenon has been coined the ‘athlete’s paradox’ and is poorly understood. Recent findings suggest that the subcellular distribution of sn -1,2-diacylglycerols (DAGs) in the plasma membrane leading to activation of novel protein kinase Cs (PKCs) is a crucial pathway to inducing insulin resistance. Here, we hypothesised that regular aerobic exercise would preserve muscle insulin sensitivity by preventing increases in plasma membrane sn -1,2-DAGs and activation of PKCε and PKCθ despite promoting increases in muscle triacylglycerol content. Methods C57BL/6J mice were allocated to three groups (regular chow feeding [RC]; high-fat diet feeding [HFD]; RC feeding and running wheel exercise [RC-EXE]). We used a novel LC-MS/MS/cellular fractionation method to assess DAG stereoisomers in five subcellular compartments (plasma membrane [PM], endoplasmic reticulum, mitochondria, lipid droplets and cytosol) in the skeletal muscle. Results We found that the HFD group had a greater content of sn -DAGs and ceramides in multiple subcellular compartments compared with the RC mice, which was associated with an increase in PKCε and PKCθ translocation. However, the RC-EXE mice showed, of particular note, a reduction in PM sn -1,2-DAG and ceramide content when compared with HFD mice. Consistent with the PM sn -1,2-DAG–novel PKC hypothesis, we observed an increase in phosphorylation of threonine 1150 on the insulin receptor kinase (IRK T1150 ), and reductions in insulin-stimulated IRK Y1162 phosphorylation and IRS-1-associated phosphoinositide 3-kinase activity in HFD compared with RC and RC-EXE mice, which are sites of PKCε and PKCθ action, respectively. Conclusions/interpretation These results demonstrate that lower PKCθ/PKCε activity and sn -1,2-DAG content, especially in the PM compartment, can explain the preserved muscle insulin sensitivity in RC-EXE mice. Graphical abstract