Protein Kinase B Phosphorylation Research Articles

PHA-665752's Antigrowth and Proapoptotic Effects on HSC-3 Human Oral Cancer Cells.

c-Met is a tyrosine-kinase receptor, and its aberrant activation plays critical roles in tumorigenesis, invasion, and metastatic spread in many human tumors. PHA-665752 (PHA) is an inhibitor of c-Met and has antitumor effects on many hematological malignancies and solid cancers. However, the activation and expression of c-Met and its role and the antitumor effect of PHA on human oral squamous cell carcinoma (OSCC) cells remain unclear. Here, we investigated the activation and expression of c-Met and the effects of PHA on the growth of a highly tumorigenic HSC-3 human OSCC cell line with high c-Met phosphorylation and expression. Of note, c-Met was highly expressed and phosphorylated on Y1234/1235 in HSC-3 cells, and PHA treatment significantly suppressed the growth and induced apoptosis of these cells. Moreover, PHA that inhibited the phosphorylation (activation) of c-Met further caused the reduced phosphorylation and expression levels of Src, protein kinase B (PKB), mammalian target of rapamycin (mTtor), and myeloid cell leukemia-1 (Mcl-1) in HSC-3 cells. In addition, the antiangiogenic property of PHA in HSC-3 cells was shown, as evidenced by the drug's suppressive effect on the expression of hypoxia-inducible factor-1α (HIF-1α), a critical tumor angiogenic transcription factor. Importantly, genetic ablation of c-Met caused the reduced growth of HSC-3 cells and decreased Src phosphorylation and HIF-1α expression. Together, these results demonstrate that c-Met is highly activated in HSC-3 human oral cancer cells, and PHA exhibits strong antigrowth, proapoptotic, and antiangiogenic effects on these cells, which are mediated through regulation of the phosphorylation and expression of multiple targets, including c-Met, Src, PKB, mTOR, Mcl-1, and HIF-1α.

Oncogene-mediated nuclear accumulation of lactate promotes epigenetic alterations to induce cancer cell proliferation.

Homeobox gene families are associated with embryonic development and organogenesis. Pieces of evidence suggest that these Homeobox genes are also crucial in facilitating oncogenesis when mutated or overexpressed. Paired homeodomain transcription factor-2 (PITX2), one of the members of this family, is involved in oncogenic regulation apart from its different development regulatory functions. PITX2 has been earlier shown to induce ovarian cancer cell proliferation through the activation of different signaling cascades. Increased cancer cell proliferation requires a constant supply of nutrients for both adenosine triphosphate and biomass synthesis, which is facilitated by altered cancer cell metabolism that includes enhanced glucose uptake and increased glycolytic rate. This present study highlights the involvement of PITX2 in enhancing the cellular glycolysis pathway in ovarian cancer cells through protein kinase B-phosphorylation (phospho-AKT). PITX2 expression correlates positively with that of the glycolytic rate-determining enzyme, lactate dehydrogenase-A (LDHA), in both high-grade serous ovarian cancer tissues and common ovarian cancer cell lines. Interestingly, transient localization of enzymatically active LDHA in the nucleus was observed in PITX2-overexpressed ovarian cancer cells. This nuclear LDHA produces higher concentrations of the glycolytic end product, lactate, which accumulates in the nuclear compartment resulting in decreased histone deacetylase (HDAC1/2) expression and increased histone acetylation at H3/H4. However, the mechanistic details of lactate-HDAC interaction are still elusive in the earlier reports. Our in silico studies elaborated on the interaction dynamics of lactate in the HDAC catalytic core through ligand-binding studies and molecular dynamics simulation approaches. Blocking lactate production by silencing LDHA reduced cancer cell proliferation. Thus, PITX2-induced epigenetic changes can lead to high cellular proliferation and increase the size of tumors in syngeneic mice as well. Taken together, this is the first report of its kind to show that the developmental regulatory homeobox gene PITX2 could enhance oncogenesis through enhanced glycolysis of tumor cells followed by epigenetic modifications.

Molecular Insights into the Regulation of 3-Phosphoinositide-Dependent Protein Kinase 1: Modeling the Interaction between the Kinase and the Pleckstrin Homology Domains

The 3-phosphoinositide-dependent protein kinase 1 (PDK1) K465E mutant kinase can still activate protein kinase B (PKB) at the membrane in a phosphatidylinositol-3,4,5-trisphosphate (PIP3, PtdIns(3,4,5)P3) independent manner. To understand this new PDK1 regulatory mechanism, docking and molecular dynamics calculations were performed for the first time to simulate the wild-type kinase domain–pleckstrin homology (PH) domain complex with PH-in and PH-out conformations. These simulations were then compared to the PH-in model of the KD–PH(mutant K465E) PDK1 complex. Additionally, three KD–PH complexes were simulated, including a substrate analogue bound to a hydrophobic pocket (denominated the PIF-pocket) substrate-docking site. We find that only the PH-out conformation, with the PH domain well-oriented to interact with the cellular membrane, is active for wild-type PDK1. In contrast, the active conformation of the PDK1 K465E mutant is PH-in, being ATP-stable at the active site while the PIF-pocket is more accessible to the peptide substrate. We corroborate that both the docking-site binding and the catalytic activity are in fact enhanced in knock-in mouse samples expressing the PDK1 K465E protein, enabling the phosphorylation of PKB in the absence of PIP3 binding.

Insulin regulates Nedd4-2 via a PKB-dependent mechanism in HEI-OC1 auditory cells-crosstalks with sphingolipid and cAMP signaling

Background The mechanisms of association between diabetes and inner ear dysfunction are unknown, although endolymphatic hydrops may be involved. We have previously shown that insulin signaling components are expressed in human saccule and that insulin signaling takes place in HEI-OC1 auditory cells. Aim To explore Nedd4-2 as a target for insulin signaling. Materials and methods Effects of insulin were analyzed using western blot and confocal microscopy in HEI-OC1 auditory cells. Results Insulin induced phosphorylation of Nedd4-2 and increased the amount of ENaC at the plasma membrane. Also, protein kinase B (PKB) and NDRG1, a substrate for SGK1 (serum and glucocorticoid stimulated kinase), were phosphorylated in response to insulin. The SGK1 inhibitor GSK650394 prevented insulin-induced phosphorylation of NRDG1, but not of PKB and Nedd4-2, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin and the PKB inhibitor MK2206 inhibited phosphorylation of all components. Ceramides prevented insulin-induced phosphorylation of PKB and NDRG1, but not of Nedd4-2. The ceramide metabolite sphingosine 1-phosphate induced phosphorylation of Nedd4-2. Conclusions Insulin induces phosphorylation of Nedd4-2, most likely involving PI3K/PKB signaling. Sphingosine 1-phosphate might protect Nedd4-2 against ceramide-induced insulin resistance. Significance Insulin-mediated regulation of Nedd4-2 might impact on inner ear sodium homeostasis with implications for diabetes-induced inner ear damage.

Full Activation of Kinase Protein Kinase B by Phosphoinositide-Dependent Protein Kinase-1 and Mammalian Target of Rapamycin Complex 2 Is Required for Early Natural Killer Cell Development and Survival.

The role of PI3K-mTOR pathway in regulating NK cell development has been widely reported. However, it remains unclear whether NK cell development depends on the protein kinase B (PKB), which links PI3K and mTOR, perhaps due to the potential redundancy of PKB. PKB has two phosphorylation sites, threonine 308 (T308) and serine 473 (S473), which can be phosphorylated by phosphoinositide-dependent protein kinase-1 (PDK1) and mTORC2, respectively. In this study, we established a mouse model in which PKB was inactivated through the deletion of PDK1 and Rictor, a key component of mTORC2, respectively. We found that the single deletion of PDK1 or Rictor could lead to a significant defect in NK cell development, while combined deletion of PDK1 and Rictor severely hindered NK cell development at the early stage. Notably, ectopic expression of myristoylated PKB significantly rescued this defect. In terms of mechanism, in PDK1/Rictor-deficient NK cells, E4BP4, a transcription factor for NK cell development, was less expressed, and the exogenous supply of E4BP4 could alleviate the developmental defect of NK cell in these mice. Besides, overexpression of Bcl-2 also helped the survival of PDK1/Rictor-deficient NK cells, suggesting an anti-apoptotic role of PKB in NK cells. In summary, complete phosphorylation of PKB at T308 and S473 by PDK1 and mTORC2 is necessary for optimal NK cell development, and PKB regulates NK cell development by promoting E4BP4 expression and preventing cell apoptosis.

5946The immune protein MD-2 is associated with early death in dilated cardiomyopathy and increases M1 macrophage polarization and recruitment in vitro

Abstract Objective Dilated cardiomyopathy (DCM) is characterized by systolic dysfunction and dilatation of ventricles. Myocardial inflammation and leukocyte activation and recruitment play a major role in the development and progression of disease. Myeloid differentiation factor-2 (MD-2) is the TLR (toll like receptor)-4 co-receptor and has been shown to be an important risk predictor for mortality of DCM patients. It is expressed in various cell types and mediates TLR-4 dependent inflammation/activation processes. Purpose We examined the impact of MD-2 on mortality of DCM patients and on polarization and recruitment of monocytes in vitro. Methods In 77 DCM patients, divided by median time point of death after first hospital admission into early and late death and alive group, MD-2 was quantified by means of ELISA. In THP-1 monocytes, cytokine secretion was quantified by ELISA after 72h treatment with MD-2 (5μg/mL). Bone marrow derived macrophages (BMDM) were generated from MD-2 KO and WT mice. NFkB phosphorylation (10min) and changes in gene expression (4h) of different adhesion molecules was quantified after treatment with 1 or 10ng/mL LPS. In human umbilical vein endothelial cells (HUVEC), protein kinase B (PKB) phosphorylation was quantified after 15min of treatment with 10ng/mL LPS or 5μg/mL MD-2. CCL2 gene expression in lysed cells (4h) and CCL2 secretion in supernatants (48h) were quantified to. Adhesion of monocytes on treated HUVEC was determined by FACS (Fig.1a). Initial HUVEC treatment with MD-2 (5μg/mL) or LPS (10 or 100ng/mL) took place for 48h. Results We found significant increased MD-2 in early (591.3ng/mL; N=18) vs late death (p=0.015) (369.2ng/mL; N=17) and alive (p≤0.0001) (303.2ng/mL; N=42) patients. Treatment of THP-1 cells (N=5) with MD-2 lead to a significantly increased secretion of inflammatory cytokines IL-8 (p=0.012), IP-10 (p=0.029), and MCP-1 (p=0.032) but not of anti-inflammatory cytokines IL-4, IL-10 and IL-13. Treatment of BMDM obtained from MD-2 KO and WT mice with 10ng/mL LPS lead to a increased phosphorylation of NFkB (N=4; p=0.022) and increased gene expression (N=6) of adhesion molecules VLA-4 (p=0.006) and ICAM-1 (p=0.049) in WT mice but not in KO mice. In HUVEC, LPS (p=0.008) and MD-2 induced a comparable increased phosphorylation of PKB (p=0.008) as well as an increase of CCL2 gene expression (p=0.029) and protein amount (p=0.039). Furthermore, treatment of HUVEC with both MD-2 (p=0.015) and LPS (p=0.0001) lead to a significant increase in monocyte adhesion (Fig.1). Conclusion The impact of MD-2 on cardiac inflammation and macrophage recruitment has not been described yet. In this study, we showed that, in DCM, elevated levels of sMD-2 are associated with early death. Furthermore, we could demonstrate that MD-2 enhances the process of HUVEC based monocyte recruitment. Finally, we could show that MD-2 induces inflammatory monocyte activity and triggers polarization of macrophages towards an inflammatory phenotype.

Delayed treatment of propofol inhibits lipopolysaccharide-induced inflammation in microglia through the PI3K/PKB pathway.

Propofol is a short-acting intravenous anesthetic agent with potential neuroprotective effect. In this study, we aim to investigate whether delayed propofol treatment is protective against lipopolysaccharide (LPS)-stimulated inflammatory responses in microglial cells. Cultured BV2 microglial cells were exposed to propofol at various time points after initiation of LPS stimulation. Nitrite production and cell viability were assessed after stimulation with LPS for 24 h. The effect of propofol on mRNA levels of cyclooxygenase-2 (Cox-2), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) was analyzed using reverse transcription PCR (RT-PCR) 6 h after LPS stimulation. The production of TNF-α and reactive oxygen species was also studied. Propofol applied 0–4 h after the initiation of LPS dose-dependently inhibits nitric oxide production. Propofol application also decreased LPS-induced Cox-2, IL-6, iNOS, TNF-α, and IL-1β mRNA expression and induced significant protein kinase B (PKB) phosphorylation in BV2 cells. Treatment with phosphoinositide 3-kinase (PI3K)/PKB inhibitor wortmannin decreased PKB phosphorylation induced by propofol, and abolished the inhibitory effect of propofol on LPS-stimulated NO, reactive oxygen species and TNF-α production. Our results suggest that delayed propofol treatment can reduce LPS-induced activation of microglial cells. These effects may be mediated by activation of the PI3K/PKB pathway.

Silencing of ASPP2 promotes the proliferation, migration and invasion of triple-negative breast cancer cells via the PI3K/AKT pathway.

Apoptosis-stimulating p53 protein 2 (ASPP2) is an apoptosis inducer that acts via binding with p53 and then enhancing the transcriptional activities toward pro‑apoptosis genes. ASPP2 has recently been reported to serve a major role in p53‑independent pathways. Triple‑negative breast cancer(TNBC) is a type of breast cancer that is more aggressive and highly lethal when p53 is mutated. In the present study, the mRNA level of ASPP2 was found to be suppressed in breast tumors compared with that in adjacent normal breast tissues, and the expression of ASPP2 was also decreased in a series of breast cancer cell lines compared with that in MCF‑10A normal breast cells. Downregulation of ASPP2 by specific small interfering RNA(siRNA) transfection was able to promote cell growth, reduce cell apoptosis, and contribute to cell migration and invasion. Furthermore, downregulation of ASPP2 promoted cell epithelial‑mesenchymal transition(EMT) in MDA‑MB‑231 and HCC‑1937 TNBC cells. Furthermore, it was found that when ASPP2 siRNA was transfected into MDA‑MB‑231 and HCC‑1937 cells, the expression of phosphoinositide‑3‑kinase regulatory subunit1(p85α) decreased and phosphorylation of protein kinaseB(AKT) increased, which are key molecular regulators in the phosphatidylinositol3-kinase (PI3K)/AKT pathway. In conclusion, the present data indicated that ASPP2 had a crucial influence on the proliferation and metastasis in TNBC, and that the functional mechanism may be p53‑independent to a great extent. ASPP2 and its link with the PI3K/AKT pathway deserve further investigation and may provide novel insights into therapeutic targets for TNBC.

Amyloid formation reduces protein kinase B phosphorylation in primary islet β-cells which is improved by blocking IL-1β signaling.

Amyloid formation in the pancreatic islets due to aggregation of human islet amyloid polypeptide (hIAPP) contributes to reduced β-cell mass and function in type 2 diabetes (T2D) and islet transplantation. Protein kinase B (PKB) signaling plays a key role in the regulation of β-cell survival, function and proliferation. In this study, we used human and hIAPP-expressing transgenic mouse islets in culture as two ex vivo models of human islet amyloid formation to: 1. Investigate the effects of amyloid formation on PKB phosphorylation in primary islet β-cells; 2. Test if inhibition of amyloid formation and/or interleukin-1β (IL-1β) signaling in islets can restore the changes in β-cell phospho-PKB levels mediated by amyloid formation. Human and hIAPP-expressing mouse islets were cultured in elevated glucose with an amyloid inhibitor (Congo red) or embedded within collagen matrix to prevent amyloid formation. To block the IL-1β signaling, human islets were treated with an IL-1 receptor antagonist (anakinra) or a glucagon-like peptide-1 agonist (exenatide). β-cell phospho-PKB levels, proliferation, apoptosis, islet IL-1β levels and amyloid formation were assessed. Amyloid formation in both cultured human and hIAPP-expressing mouse islets reduced β-cell phospho-PKB levels and increased islet IL-1β levels, both of which were restored by prevention of amyloid formation either by the amyloid inhibitor or embedding islets in collagen matrix, resulting in improved β-cell survival. Furthermore, inhibition of IL-1β signaling by treatment with anakinra or exenatide increased β-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis in amyloid forming human islets during 7-day culture. These data suggest that amyloid formation leads to reduced PKB phosphorylation in β-cells which is associated with elevated islet IL-1β levels. Inhibitors of amyloid or amyloid-induced IL-1β production may provide a new approach to restore phospho-PKB levels thereby enhance β-cell survival and proliferation in conditions associated with islet amyloid formation such as T2D and clinical islet transplantation.

Signal-Regulated Protein Kinases/Protein Kinase B-p53-BH3-Interacting Domain Death Agonist Pathway Regulates Gingipain-Induced Apoptosis in Osteoblasts.

Gingipains are cysteine proteases produced by Porphyromonas gingivalis, the predominant pathogen in chronic periodontitis. The present study aims to examine the role of gingipains in promoting apoptosis in osteoblasts. Human calvarial osteoblasts and osteoblast MC3T3-E1 cells were treated with 8.348 U/L gingipains. Flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining were used to detect cell apoptosis. Protein expression was examined by Western blotting, and gene expression was detected by real-time polymerase chain reaction. Small interfering (si)RNA transfection was used to knock down BH3-interacting domain death agonist (Bid) expression. Treatment with 8.348 U/L gingipains from 4 to 72 hours increased apoptosis, accompanied by elevated cleaved caspase-3 levels. Notably, gingipain-induced apoptosis was associated with increase of Bid and its truncated form, tBid, as well as p53. Transfection with Bid siRNA resulted in suppression of gingipain-induced apoptosis. The p53 inhibitor, Pifithrin-α, blocked the gingipain-induced Bid. The ability of gingipains to stimulate p53 and Bid expression was mimicked by PD-0325901 and MK-2206, the specific extracellular signal-regulated protein kinases (ERK) and protein kinase B (PKB) inhibitors, respectively. Furthermore, treatment with gingipains reduced phospho-ERK and phospho-PKB levels, an effect correlated to gingipain-induced increase in p53 and tBid expression. The present findings suggest that Bid plays an essential role in gingipain-induced osteoblast apoptosis, which is dependent on inhibition of ERK and PKB phosphorylation, followed by the activation of p53.

Leptolide Improves Insulin Resistance in Diet-Induced Obese Mice.

Type 2 diabetes (T2DM) is a complex disease linked to pancreatic beta-cell failure and insulin resistance. Current antidiabetic treatment regimens for T2DM include insulin sensitizers and insulin secretagogues. We have previously demonstrated that leptolide, a member of the furanocembranolides family, promotes pancreatic beta-cell proliferation in mice. Considering the beneficial effects of leptolide in diabetic mice, in this study, we aimed to address the capability of leptolide to improve insulin resistance associated with the pathology of obesity. To this end, we tested the hypothesis that leptolide should protect against fatty acid-induced insulin resistance in hepatocytes. In a time-dependent manner, leptolide (0.1 µM) augmented insulin-stimulated phosphorylation of protein kinase B (PKB) by two-fold above vehicle-treated HepG2 cells. In addition, leptolide (0.1 µM) counteracted palmitate-induced insulin resistance by augmenting by four-fold insulin-stimulated phosphorylation of PKB in HepG2 cells. In vivo, acute intraperitoneal administration of leptolide (0.1 mg/kg and 1 mg/kg) improved glucose tolerance and insulin sensitivity in lean mice. Likewise, prolonged leptolide treatment (0.1 mg/kg) in diet-induced obese mice improved insulin sensitivity. These effects were paralleled with an ~50% increased of insulin-stimulated phosphorylation of PKB in liver and skeletal muscle and reduced circulating pro-inflammatory cytokines in obese mice. We concluded that leptolide significantly improves insulin sensitivity in vitro and in obese mice, suggesting that leptolide may be another potential treatment for T2DM.

Anti-metastatic effect of rhodomyrtone from Rhodomyrtus tomentosa on human skin cancer cells.

This study focused on the inhibitory effect of rhodomyrtone, a bioactive compound isolated from the leaves of Rhodomyrtus tomentosa (Aiton) Hassk., on cancer metastasis in epidermoid carcinoma A431 cells and on the verification of the underlying related molecular mechanisms of this event. We demonstrated that rhodomyrtone at the subcytotoxic concentration (0.5 and 1.5µg/ml) exhibited pronounced inhibition of cancer metastasis by reducing cell migration, cell adhesive ability and cell invasion of A431 cells in a dose-dependent manner. Data demonstrated that rhodomyrtone could inhibit the focal adhesion kinase (FAK) and phosphorylation of protein kinaseB(AKT), c-Raf, extracellular signal-regulated kinase1/2 (ERK1/2) and p38MAPK involved in the downregulation the enzyme activities and protein expression of matrix metalloproteinase-2(MMP-2) and MMP-9. Moreover, we found that rhodomyrtone increased the expression of TIMP-1 and TIMP-2, which are inhibitors of MMP-9 and MMP-2, respectively. Rhodomyrtone also inhibited the expression of NF-κB and phosphorylation of NF-κB in a dose-dependent manner. These results suggested that rhodomyrtone inhibited A431 cell metastasis by reducing MMP-2/9 activities and expression through inhibiting ERK1/2, p38 and FAK/Akt signaling pathways via NF-κB activities. This finding suggested that rhodomyrtone may be a novel antimetastasis agent for treatment of skin cancer cells.

The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitate-induced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1

Dietary administration of the histone deacetylase (HDAC) inhibitor butyric acid – a short chain fatty acid present in milk products and also bacterially produced in the intestine – has been shown to increase energy expenditure and favour insulin sensitivity in mice through induction of PGC1α (peroxisome proliferator-activated receptor gamma co-activator 1α) and AMPK (AMP-activated protein kinase) in skeletal muscle, and a consequential increase of mitochondrial fatty acid oxidation. Here, we investigate whether such physiological improvements are associated to epigenetic effects dependent on increased histone acetylation and whether butyrate exerts a direct action on skeletal muscle insulin signalling. We show that sodium butyrate (NaBut) ameliorates the insulin-resistant phenotype, induced in L6 myotubes by prolonged exposure to palmitate, by i) increasing the insulin-induced phosphorylation of both PKB (protein kinase B) and MAPK (mitogen activated protein kinase), the two branches of insulin signalling and ii) increasing histone H3 acetylation – even in the presence of palmitate - on chromatin in proximity of the Irs1 (insulin receptor substrate 1) transcriptional start site. Consequently, NaBut induced Irs1 mRNA and protein overexpression, which in turn relayed higher insulin-stimulated IRS1 tyrosine phosphorylation and PI 3-kinase (phosphoinositide 3-kinase) association, suggesting that the increased IRS1 expression may mediate the insulin-sensitizing effects of NaBut. Furthermore, downstream of PKB, NaBut induced GSK3β gene upregulation. Our observations indicate that NaBut – through its action as HDAC inhibitor – can promote insulin responsiveness in L6 myotubes under conditions of lipid-induced insulin resistance.

Paxillin and Focal Adhesion Kinase (FAK) Regulate Cardiac Contractility in the Zebrafish Heart.

An orchestrated interplay of adaptor and signaling proteins at mechano-sensitive sites is essential to maintain cardiac contractility and when defective leads to heart failure. We recently showed that Integrin-linked Kinase (ILK), ß-Parvin and PINCH form the IPP-complex to grant tuned Protein Kinase B (PKB) signaling in the heart. Loss of one of the IPP-complex components results in destabilization of the whole complex, defective PKB signaling and finally heart failure. Two components of IPP, ILK and ß-Parvin directly bind to Paxillin; however, the impact of this direct interaction on the maintenance of heart function is not known yet. Here, we show that targeted gene inactivation of Paxillin results in progressive decrease of cardiac contractility and heart failure in zebrafish without affecting IPP-complex stability and PKB phosphorylation. However, we found that Paxillin deficiency leads to the destabilization of its known binding partner Focal Adhesion Kinase (FAK) and vice versa resulting in degradation of Vinculin and thereby heart failure. Our findings highlight an essential role of Paxillin and FAK in controlling cardiac contractility via the recruitment of Vinculin to mechano-sensitive sites in cardiomyocytes.

Mitochondrial Inhibitory Factor Protein 1 Functions as an Endogenous Inhibitor for Coupling Factor 6.

Coupling factor 6 (CF6) forces a counter-clockwise rotation of plasma membrane F1 Fo complex unlike a proton-mediated clockwise rotation in the mitochondria, resulting in ATP hydrolysis, proton import, and apoptosis. Inhibitory peptide 1 (IF1) inhibits a unidirectional counter-clockwise rotation of F1 Fo complex without affecting ATP synthesis by a clockwise rotation. We tested the hypothesis that IF1 may antagonize the biological action of CF6 in human embryonic kidney 293 cells. We generated mature and immature IF1 expression vectors and those labeled with GFP at the C-terminus. In the immature IF1-GFP overexpressing cells, the mitochondrial network of IF1-GFP was newly found at the plasma membrane after peripheral translocation, whereas in mature IF1-GFP transfected cells, a less punctuate rather homogenous pattern was found in the cytoplasm. IF1 protein was detected in the exosome fraction of culture media, and it was enhanced by mature or immature IF1 transfection. Extracellular ATP hydrolysis was enhanced by CF6, whereas immature or mature IF1 transfection suppressed ATP hydrolysis in response to CF6. Intracellular pH was decreased by CF6 but was unchanged after immature IF1 transfection. CF6-induced increase in apoptotic cells was blocked by immature or mature IF1, being accompanied by protein kinase B (PKB) phosphorylation. IF1 antagonizes the pro-apoptotic action of CF6 by relief of intracellular acidification and resultant phosphorylation of PKB. Given the widespread biological actions of CF6, the physiological and pathological functions of IF1 may be expected to be complex. J. Cell. Biochem. 117: 1680-1687, 2016. © 2015 Wiley Periodicals, Inc.

Linagliptin Has Wide-Ranging Anti-Inflammatory Points of Action in Human Umbilical Vein Endothelial Cells

BACKGROUNDBecause of the potential anti-inflammatory effects, linagliptin, a therapeutic dipeptidyl peptidase-4 inhibitor, is used as an effective drug for diabetic patients for whom inflammation is a prognosis-related factor. We investigated the anti-inflammatory mechanism of linagliptin using seven markers.METHODSWe pretreated human umbilical vein endothelial cells (HUVECs), with linagliptin and lipopolysaccharide (LPS). The cytosolic fractions were evaluated for protein kinase A (PKA), protein kinase B (PKB), protein kinase C (PKC), ratio of reactive oxygen species (ROS) and Cu/Zn superoxide dismutase (SOD), activator protein 1 (AP-1), and adenosine 3′,5′-cyclic monophosphate (cAMP).RESULTSLinagliptin increased the PKA and PKC activities and the cAMP levels in LPS-treated cells. However, it inhibited LPS-induced PKB phosphorylation, ratio of ROS and Cu/Zn SOD, and LPS-stimulated AP-1 nuclear translocation.CONCLUSIONWe reaffirmed the anti-inflammatory and antioxidant effects of linagliptin. These effects might be related to the three protein kinases. Our findings suggest that linagliptin has a wide range of anti-inflammatory effects.

Manganese [III] Tetrakis [5,10,15,20]-Benzoic Acid Porphyrin Reduces Adiposity and Improves Insulin Action in Mice with Pre-Existing Obesity.

The superoxide dismutase mimetic manganese [III] tetrakis [5,10,15,20]-benzoic acid porphyrin (MnTBAP) is a potent antioxidant compound that has been shown to limit weight gain during short-term high fat feeding without preventing insulin resistance. However, whether MnTBAP has therapeutic potential to treat pre-existing obesity and insulin resistance remains unknown. To investigate this, mice were treated with MnTBAP or vehicle during the last five weeks of a 24-week high fat diet (HFD) regimen. MnTBAP treatment significantly decreased body weight and reduced white adipose tissue (WAT) mass in mice fed a HFD and a low fat diet (LFD). The reduction in adiposity was associated with decreased caloric intake without significantly altering energy expenditure, indicating that MnTBAP decreases adiposity in part by modulating energy balance. MnTBAP treatment also improved insulin action in HFD-fed mice, a physiologic response that was associated with increased protein kinase B (PKB) phosphorylation and expression in muscle and WAT. Since MnTBAP is a metalloporphyrin molecule, we hypothesized that its ability to promote weight loss and improve insulin sensitivity was regulated by heme oxygenase-1 (HO-1), in a similar fashion as cobalt protoporphyrins. Despite MnTBAP treatment increasing HO-1 expression, administration of the potent HO-1 inhibitor tin mesoporphyrin (SnMP) did not block the ability of MnTBAP to alter caloric intake, adiposity, or insulin action, suggesting that MnTBAP influences these metabolic processes independent of HO-1. These data demonstrate that MnTBAP can ameliorate pre-existing obesity and improve insulin action by reducing caloric intake and increasing PKB phosphorylation and expression.

Chronic ethanol treatment of human hepatocytes inhibits the activation of the insulin signaling pathway by increasing cytosolic free calcium levels.

The present study aimed to investigate the effects of ethanol treatment on the induction of intracellular calcium ([Ca(2+)](i)) levels and the inhibition of the activation of the insulin signaling pathway in human hepatocytes. L‑02 cells were treated with various concentrations of ethanol for different periods of time. Cell viability and alanine aminotransferase (ALT)/aspartate aminotransferase (AST) leakage in the culture supernatant were evaluated. Changes in [Ca(2+)](i) levels were detected by flow cytometry and confocal microscopy. Total RNA and protein were extracted to examine the mRNA and protein levels of insulin receptor substrate (IRS)1, IRS2, phosphatidylinositol 3‑kinase (PI3K) and glucose transporter 2 (GLUT2) by reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis, respectively. Furthermore, insulin was added to the ethanol‑treated L‑02 cells, and the phosphorylation levels of PI3K and protein kinase B (PKB) were determined by western blot analysis before and after Ca(2+) blockage. No significant changes were observed in cell viability, [Ca(2+)](i) levels and in the expression and phosphorylation levels of insulin signal transduction molecules when the L‑02 cells were treated with 0.5 or 1% ethanol. However, treatment with 2 or 4% ethanol resulted in a significant decrease in cell viability and in the mRNA levels of IRS1, IRS2, PI3K (p85α) and GLUT2, as well as in an increase in ALT/AST leakage and in the [Ca(2+)](i) levels (P<0.05). The expression and phosphorylation levels of PI3K (p85α) and PKB were also inhibited by treatment with 2 or 4% ethanol. These cytological effects induced by ethanol treatment were partially reversed by Ca(2+) blockage. These results suggest that ethanol treatment inhibits the activation of the insulin signal transduction pathway in a dose‑, time‑ and Ca(2+)‑dependent manner. The inhibition of IRS1/2, PI3K (p85α), PKB and GLUT2 expression and of PI3K (p85α) and PKB phosphorylation by the high concentrations of ethanol may be the core molecular mechanism of ethanol-induced insulin resistance, and may be related to the induction of [Ca(2+)](i) levels.

MnTBAP Ameliorates Obesity and Metabolic Dysfunction and Limits the TF‐PAR2 Pro‐inflammatory Pathway in White Adipose Tissue

Obesity is a prevalent metabolic disease that is associated with increased reactive oxygen species and oxidative stress. The purpose of this study was to determine if the superoxide dismutase mimetic manganese tetrakis benzoic acid porphyrin (MnTBAP) can attenuate obesity and obesity-associated metabolic dysfunction. Mice were treated with MnTBAP or vehicle during the last five weeks of a 24 week high fat diet (HFD) regimen. Energy homeostasis, insulin sensitivity, adipose macrophage infiltrates, protein kinase B (PKB) and tissue factor - protease activated receptor 2 (TF-PAR2) pathways were assessed following MnTBAP treatment. MnTBAP treatment significantly reduced body weight and adipose tissue mass. The reduction in adiposity was associated with decreased caloric intake and improved insulin sensitivity. Protein kinase B (PKB) phosphorylation and expression were significantly increased in a tissue-specific manner in skeletal muscle and adipose tissue of HFD mice treated with MnTBAP. In addition, MnTBAP treatment decreased macrophage infiltrates in adipose tissue and reduced the pro-inflammatory TF-PAR2 pathway. Our data suggests that the ability of MnTBAP to improve insulin sensitivity is due to decreased adiposity, reduced adipose tissue inflammation, and increased PKB phosphorylation and expression. Further, our findings implicate decreased caloric intake and inhibition of the TF-PAR2 pathway in the anti-obesity and insulin-sensitizing effects of MnTBAP.

Amyloid Formation Reduces Phospho-PKB Levels in Islet β-Cells Which is Restored by Blocking IL-1β Signalling

Islet transplantation provides a feasible approach for treatment of type 1 diabetes (T1D) but is currently limited by insufficient donors and islet loss during pre-transplant culture and in islet grafts. Aggregation of human islet amyloid polypeptide (hIAPP), a hallmark of pancreas in T2D, also occurs during islet culture and transplantation. Amyloid formation contributes to β-cell death in all 3 conditions. Protein kinase B (PKB) signalling pathway plays a key role in the regulation of β-cell survival, function and proliferation. In this study, we examined if: 1. hIAPP aggregates can reduce PKB phosphorylation via inducing islet IL-1β production; 2. blocking amyloid or IL-1β can restore reduced PKB phosphorylation associated with amyloid formation. hIAPP-treated INS-1 β-cells had markedly lower phospho-PKB levels, reduced proliferation and higher apoptosis than non-fibrillogenic rat IAPP-treated or untreated cells, all of which were restored by the amyloid inhibitor Congo red. Human islets and transgenic hIAPP-expressing mouse islets formed amyloid during culture associated with elevated IL-1β and reduced β-cell phospho-PKB levels, both of which were prevented by Congo red. Prevention of amyloid formation during islet culture by collagen matrix reduced islet IL-1β and restored β-cell phospho-PKB levels. Blocking IL-1 receptor (anakinra) or treatment with GLP-1 agonist (exenatide) restored β-cell phospho-PKB levels in amyloid forming islets. These data suggest that amyloid formation reduces phospho-PKB in islet β-cells likely via IL-1β signalling. Blocking amyloid or amyloid-induced IL-1β production may enhance β-cell survival by restoring phosphorylation of PKB during pre-transplant culture and in islet grafts.