12/15‐Lipoxygenase Products Induce Inflammation and Impair Insulin Signaling in 3T3‐L1 Adipocytes

Inflammation and insulin resistance associated with visceral obesity are important risk factors for the development of type 2 diabetes, atherosclerosis, and the metabolic syndrome. The 12/15‐lipoxygenase (12/15‐LO) enzyme has been linked to inflammatory changes in blood vessels that precede the development of atherosclerosis. The expression and role of 12/15‐LO in adipocytes have not been evaluated. We found that 12/15‐LO mRNA was dramatically upregulated in white epididymal adipocytes of high‐fat fed mice. 12/15‐LO was poorly expressed in 3T3‐L1 fibroblasts and was upregulated during differentiation into adipocytes. Interestingly, the saturated fatty acid palmitate, a major component of high fat diets, augmented expression of 12/15‐LO in vitro. When 3T3‐L1 adipocytes were treated with the 12/15‐LO products, 12‐hydroxyeicosatetranoic acid (12(S)‐HETE) and 12‐hydroperoxyeicosatetraenoic acid (12(S)‐HPETE), expression of proinflammatory cytokine genes, including tumor necrosis factor‐α (TNF‐α), monocyte chemoattractant protein 1 (MCP‐1), interleukin 6 (IL‐6), and IL‐12p40, was upregulated whereas anti‐inflammatory adiponectin gene expression was downregulated. 12/15‐LO products also augmented c‐Jun N‐terminal kinase 1 (JNK‐1) phosphorylation, a known negative regulator of insulin signaling. Consistent with impaired insulin signaling, we found that insulin‐stimulated 3T3‐L1 adipocytes exhibited decreased IRS‐1(Tyr) phosphorylation, increased IRS‐1(Ser) phosphorylation, and impaired Akt phosphorylation when treated with 12/15‐LO product. Taken together, our data suggest that 12/15‐LO products create a proinflammatory state and impair insulin signaling in 3T3‐L1 adipocytes. Because 12/15‐LO expression is upregulated in visceral adipocytes by high‐fat feeding in vivo and also by addition of palmitic acid in vitro, we propose that 12/15‐LO plays a role in promoting inflammation and insulin resistance associated with obesity.     

ROCK pathway participates in the processes that 15‐hydroxyeicosatetraenoic acid (15‐HETE) mediated the pulmonary vascular remodeling induced by hypoxia in rat

15‐Hydroxyeicosatetraenoic acid (15‐HETE), a product of arachidonic acid (AA) catalyzed by 15‐lipoxygenase (15‐LO), plays an essential role in hypoxic pulmonary arterial hypertension. We have previously shown that 15‐HETE inhibits apoptosis in pulmonary artery smooth muscle cells (PASMCs). To test the hypothesis that such an effect is attributable to the hypoxia‐induced pulmonary vascular remodeling (PVR), we performed these studies. We found subtle thickening of proximal media/adventitia of the pulmonary arteries (PA) in rats that had been exposed to hypoxia. This was associated with an up‐regulation of the anti‐apoptotic Bcl‐2 expression and down‐regulation of pro‐apoptotic caspase‐3 and Bax expression in PA homogenates. Nordihydroguaiaretic acid (NDGA), which inhibits the generation of endogenous 15‐HETE, reversed all the alterations following hypoxia. In situ hybridization histochemistry and immunocytochemistry showed that the 15‐LO‐1 mRNA and protein were localized in pulmonary artery endothelial cells (PAECs), while the 15‐LO‐2 mRNA and protein were localized in both PAECs and PASMCs. Furthermore, the Rho‐kinase (ROCK) pathway was activated by both endogenous and exogenous 15‐HETE, alleviating the serum deprivation (SD)‐induced PASMC apoptosis. Thus, these findings indicate that 15‐HETE protects PASMC from apoptosis, contributing to pulmonary vascular medial thickening, and the effect is, at least in part, mediated via the ROCK pathway. J. Cell. Physiol. 222:82–94, 2010. © 2009 Wiley‐Liss, Inc.     

Homocysteine modulates 5‐lipoxygenase expression level via DNA methylation

Elevated levels of homocysteinemia (Hcy), a risk factor for late‐onset Alzheimer's disease (AD), have been associated with changes in cell methylation. Alzheimer's disease is characterized by an upregulation of the 5‐lipoxygenase (5LO), whose promoter is regulated by methylation. However, whether Hcy activates 5LO enzymatic pathway by influencing the methylation status of its promoter remains unknown. Brains from mice with high Hcy were assessed for the 5LO pathway and neuronal cells exposed to Hcy implemented to study the mechanism(s) regulating 5LO expression levels and the effect on amyloid β formation. Diet‐ and genetically induced high Hcy resulted in 5LO protein and mRNA upregulation, which was associated with a significant increase of the S‐adenosylhomocysteine (SAH)/S‐adenosylmethionine ratio, and reduced DNA methyltrasferases and hypomethylation of 5‐lipoxygenase DNA. In vitro studies confirmed these results and demonstrated that the mechanism involved in the Hcy‐dependent 5LO activation and amyloid β formation is DNA hypomethylation secondary to the elevated levels of SAH. Taken together these findings represent the first demonstration that Hcy directly influences 5LO expression levels and establish a previously unknown cross talk between these two pathways, which is highly relevant for AD pathogenesis. The discovery of such a novel link not only provides new mechanistic insights in the neurobiology of Hcy, but most importantly new therapeutic opportunities for the individuals bearing this risk factor for the disease.     

Involvement of p38 MAPK in haemozoin‐dependent MMP‐9 enhancement in human monocytes

The lipid moiety of natural haemozoin (nHZ, malarial pigment) was previously shown to enhance expression and release of human monocyte matrix metalloproteinase‐9 (MMP‐9), and a major role for 15‐(S,R)‐hydroxy‐6,8,11,13‐eicosatetraenoic acid (15‐HETE), a nHZ lipoperoxidation product, was proposed. Here, the underlying mechanisms were investigated, focusing on the involvement of mitogen‐activated protein kinases (MAPKs). Results showed that nHZ promoted either early or late p38 MAPK phosphorylation; however, nHZ did not modify basal phosphorylation/expression ratios of extracellular signal‐regulated kinase‐1/2 and c‐jun N‐terminal kinase‐1/2. 15‐HETE mimicked nHZ effects on p38 MAPK, whereas lipid‐free synthetic (s)HZ and delipidized (d)HZ did not. Consistently, both nHZ and 15‐HETE also promoted phosphorylation of MAPK‐activated protein kinase‐2, a known p38 MAPK substrate; such an effect was abolished by SB203580, a synthetic p38 MAPK inhibitor. SB203580 also abrogated nHZ‐dependent and 15‐HETE‐dependent enhancement of MMP‐9 mRNA and protein (latent and activated forms) levels in cell lysates and supernatants. Collectively, these data suggest that in human monocytes, nHZ and 15‐HETE upregulate MMP‐9 expression and secretion through activation of p38 MAPK pathway. The present work provides new evidence on mechanisms underlying MMP‐9 deregulation in malaria, which might be helpful to design new specific drugs for adjuvant therapy in complicated malaria. Copyright © 2013 John Wiley & Sons, Ltd.     

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.     

Subcutaneous Fat Shows Higher Thyroid Hormone Receptor‐α1 Gene Expression Than Omental Fat

The aims of this work were to evaluate thyroid hormone receptor‐α (TRα), TRα1, and TRα2 mRNA gene expression and TRα1:TRα2 ratio, identified as candidate factors for explaining regional differences between human adipose tissue depots. TRα, TRα1, and TRα2 mRNA levels, and the gene expressions of arginine–serine‐rich, splicing factor 2 (SF2), heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), and Spot 14 (S14) were evaluated in 76 paired adipose tissue samples obtained from a population of 38 women who varied widely in terms of obesity and body fat distribution. Gene expression for these factors was also studied in stromal‐vascular cells (SVCs) and mature adipocytes (MAs) from eight paired fat depots. TRα gene and TRα1 mRNA expression were increased 1.46‐fold (P = 0.006) and 1.80‐fold (P < 0.0001), respectively, in subcutaneous (SC) vs. visceral fat. These differences in gene expression levels were most significant in the obese group, in which the TRα1:TRα2 ratio was 2.24‐fold (P < 0.0001) higher in SC vs. visceral fat. S14 gene expression was also increased by 2.42‐fold (P < 0.0001) and correlated significantly with TRα and TRα1 gene expression and with the TRα1:TRα2 ratio. In agreement with these findings, hnRNP A1:SF2 ratio was decreased by 1.39‐fold (P = 0.001). TRα and S14 levels were 2.1‐fold (P < 0.0001) and 112.4‐fold (P < 0.0001), respectively, higher in MAs than in SVCs from both fat depots. In summary, genes for TR‐α, their upstream regulators, and downstream effectors were differentially expressed in SC vs. omental (OM) adipose tissue. Our findings suggest that TRα1 could contribute to SC adipose tissue expandability in obese subjects.     

The 12‐15‐lipoxygenase is a modulator of Alzheimer's‐related tau pathology in vivo

12/15‐lipoxygenase (12‐15LO) is a lipid‐peroxidizing enzyme widely expressed in the central nervous system where it has been involved in the neurobiology of Alzheimer's disease (AD) because it modulates amyloid beta (Aβ) and APP processing. However, its biological effect on tau protein is unknown. We investigated the effect of 12‐15LO on tau levels and metabolism in vivo and in vitro and the mechanism involved by using genetic and pharmacologic approaches. While no significant differences were observed in the levels of total tau for both groups, compared with controls, Tg2576 mice overexpressing 12‐15LO had elevated levels of phosphorylated tau at two specific epitopes, Ser 202/Thr 205 and Ser 396. In vitro and in vivo studies show that 12‐15LO modulates tau metabolism specifically via the cdk5 kinase pathway. Associated with these changes were biochemical markers of synaptic pathology. Finally, 12‐15LO‐dependent alteration of tau metabolism was independent from an effect on Aβ. Our findings reveal a novel pathway by which 12‐15LO modulates endogenous tau metabolism making this protein an appealing pharmacologic target for treatment of AD and related tauopathies.     

miRNA‐221 and miRNA‐222 synergistically function to promote vascular calcification

Vascular calcification shares many similarities with skeletal mineralisation and involves the phenotypic trans‐differentiation of vascular smooth muscle cells (VSMCs) to osteoblastic cells within a calcified environment. Various microRNAs (miRs) are known to regulate cell differentiation; however, their role in mediating VSMC calcification is not fully understood. miR‐microarray analysis revealed the significant down‐regulation of a range of miRs following nine days in culture, including miR‐199b, miR‐29a, miR‐221, miR‐222 and miR‐31 (p < 0.05). Subsequent studies investigated the specific role of the miR‐221/222 family in VSMC calcification. Real‐time quantitative polymerase chain reaction data confirmed the down‐regulation of miR‐221 (32.4%; p < 0.01) and miR‐222 (15.7%; p < 0.05). VSMCs were transfected with mimics of miR‐221 and miR‐222, individually and in combination. Increased calcium deposition was observed in the combined treatment (two‐fold; p < 0.05) but not in individual treatments. Runx2 and Msx2 expression was increased during calcification, but no difference in expression was observed following transfection with miR mimics. Interestingly, miR‐221 and miR‐222 mimics induced significant changes in ectonucleotide phosphodiesterase 1 (Enpp1) and Pit‐1 expression, suggesting that these miRs may modulate VSMC calcification through cellular inorganic phosphate and pyrophosphate levels. © 2013 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

Enhanced pro‐protein convertase subtilisin/kexin type 9 expression by C‐reactive protein through p38MAPK‐HNF1α pathway in HepG2 cells

Plasma C‐reactive protein (CRP) concentration is associated positively with cardiovascular risk, including dyslipidemia. We suggested a regulating role of CRP on pro‐protein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of low‐density lipoprotein (LDL) metabolism, and demonstrated the PCSK9 as a pathway linking CRP and LDL regulation. Firstly, experiments were carried out in the presence of human CRP on the protein and mRNA expression of PCSK9 and LDL receptor (LDLR) in human hepatoma cell line HepG2 cells. Treatment with CRP (10 μg/ml) enhanced significantly the mRNA and protein expression of PCSK9 and suppressed the expression of LDLR. Of note, a late return of LDLR mRNA levels occurred at 12 hrs, while the LDLR protein continued to decrease at 24 hrs, suggesting that the late decrease in LDLR protein levels was unlikely to be accounted for the decrease in LDL mRNA. Secondly, the role of PCSK9 in CRP‐induced LDLR decrease and the underlying pathways were investigated. As a result, the inhibition of PCSK9 expression by small interfering RNA (siRNA) returned partly the level of LDLR protein and LDL uptake during CRP treatment; CRP‐induced PCSK9 increase was inhibited by the p38MAPK inhibitor, SB203580, resulting in a significant rescue of LDLR protein expression and LDL uptake; the pathway was involved in hepatocyte nuclear factor 1α (HNF1α) but not sterol responsive element‐binding proteins (SREBPs) preceded by the phosphorylation of p38MAPK. These findings indicated that CRP increased PCSK9 expression by activating p38MAPK‐HNF1α pathway, with a certain downstream impairment in LDL metabolism in HepG2 cells.     

Vitamin D metabolites and analogs induce lipoxygenase mRNA expression and activity as well as reactive oxygen species (ROS) production in human bone cell line

Vitamin D metabolites and its less-calcemic analogs (vitamin D compounds) are beneficial for bone and modulate cell growth and energy metabolism. We now analyze whether 25(OH)D(3) (25D), 1,25(OH)(2)D(3) (1,25D), 24,25(OH)(2)D(3) (24,25D), JKF1624F(2)-2 (JKF) or QW1624F(2)-2 (QW) regulate lipooxygenase (LO) mRNA expression and its products; hydroxyl-eicosatetraenoic acid (12 and 15HETE) formation, as well as reactive oxygen species (ROS) production in human bone cell line (SaOS2) and their interplay with modulation of cell proliferation and energy metabolism. All compounds except 25D increased 12LO mRNA expression and modulated 12 and 15HETE production whereas ROS production was increased by all compounds, and inhibited by NADPH oxidase inhibitors diphenyleneiodonium (DPI) and N-acetylcysteine (NAc). Baicaleine (baic) the inhibitor of 12 and 15LO activity blocked only slightly the stimulation of DNA synthesis by all compounds, whereas DPI inhibited almost completely the stimulation of DNA and CK by all compounds. Treatments of cells with 12 or 15HETE increased DNA synthesis and CK that were only slightly inhibited by DPI. These results indicate that vitamin D compounds increased oxidative stress in osteoblasts in part via induction of LO expression and activity. The increased ROS production mediates partially elevated cell proliferation and energy metabolism, whereas the LO mediation is not essential. This new feature of vitamin D compounds is mediated by intracellular and/or membranal binding sites and its potential hazard could lead to damage due to increased lipid oxidation, although the transient mediation of ROS in cell proliferation is beneficial to bone growth in a yet unknown mechanism.     

S‐ to N‐Acyl transfer in S‐acylcysteine isopeptides via 9‐, 10‐, 12‐, and 13‐membered cyclic transition states

S‐Acyl cysteine peptides containing α‐, β‐ or γ‐amino acid residues undergo long‐range S‐ to N‐acyl transfer to give analogs of native tripeptides and tetrapeptides containing additional carbon atoms in the chain. The ease of intramolecular S → N‐acyl transfer relative to intermolecular transacylation is favored increasingly for 9 < 12 < 13 ~ 10‐membered cyclic transition states; the observed order is explained on conformational and intermolecular interaction considerations. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Modification of the detrimental effect of TNF‐α on human endothelial progenitor cells by fasudil and Y27632

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor‐α (TNF‐α) reduced their number and biological activity. Yet, signal transduction events linked to TNF‐α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in‐ flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose‐dependent improvement of EPC number during exposure to TNF‐α (P < 0.05 vs. TNF‐α alone). Analysis of the signal transduction pathway activated by TNF‐α revealed that the increased expression of p‐p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF‐α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF‐α) as well as the inhibitor of Erk1/2‐specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF‐α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D‐DIGE and MALDI‐TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin‐related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:351–360, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20345     

Rutin treats myocardial damage caused by pirarubicin via regulating miR‐22‐5p‐regulated RAP1/ERK signaling pathway

Our experiments have previously demonstrated that rutin (RUT) can improve myocardial damage caused by pirarubicin (THP). However, the underlying molecular mechanisms remain uncertain. In this study, we developed an microRNA (miRNA) chip by replicating the rat model of THP‐induced myocardial injury and identified miR‐22‐5p and the RAP1‐member of RAS oncogene family/extracellular regulated protein kinases (RAP1/ERK) signaling pathway as an object of study. Also, in vivo experiments demonstrated that THP caused abnormal changes in the electrocardiogram, cardiac function, and histomorphology in rats (P < .01). THP also reduces the expression of miR‐22‐5p (P < .01) and increases the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). RUT significantly improved THP‐induced myocardial damage (P < .01), increased the expression of miR‐22‐5p (P < .01), and decreased the levels of RAP1/ERK signaling pathway‐related proteins (P < .01, P < .05). In vitro studies confirmed that Rap1a is one of the target genes of miR‐22‐5p. miR‐22‐5p overexpression in cardiomyocytes can affect the RAP1/ERK pathway and reduce reactive oxygen species production and cardiomyocyte apoptosis caused by THP (P < .01), which is consistent with the effect of RUT. Our results indicate that RUT treats THP‐induced myocardial damage, which may be achieved by upregulating miR‐22‐5p, causing changes in its target gene Rap1a and the RAP1/ERK pathway.     

Ghrelin Augments the Expressions and Secretions of Proinflammatory Adipokines,VEGF120 and MCP‐1, in Differentiated 3T3‐L1 Adipocytes

Ghrelin is a physiological‐active peptide with growth hormone‐releasing activity, orexigenic activity, etc. In addition, the recent study has also suggested that ghrelin possesses the pathophysiological abilities related with type 2 diabetes. However, the ghrelin‐direct‐effects implicated in type 2 diabetes on peripheral tissues have been still unclear, whereas its actions on the central nervous system (CNS) appear to induce the development of diabetes. Thus, to assess its peripheral effects correlated with diabetes, we investigated the regulatory mechanisms about adipokines, which play a central role in inducing peripheral insulin resistance, secreted from mature 3T3‐L1 adipocytes stimulated with ghrelin in vitro . The stimulation with 50 nmol/L ghrelin for 24 h resulted in the significant 1.9‐fold increase on vascular endothelial growth factor‐120 (VEGF₁₂₀) releases (p < 0.01) and the 1.7‐fold on monocyte chemoattractant protein‐1 (MCP‐1) (p < 0.01) from 3T3‐L1 adipocytes, respectively, while ghrelin failed to enhance tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), IL‐6, IL‐10 and adiponectin secretions. In addition, Akt phosphorylation on Ser473 and c‐Jun NH₂‐terminal protein kinase (JNK) phosphorylation on Thr183/Tyr185 were markedly enhanced 1.4‐fold (p < 0.01) and 1.6‐fold (p < 0.01) in the ghrelin‐stimulated adipocytes, respectively. Furthermore, the treatment with LY294002 (50 μmol/L) and Wortmannin (10nmol/L), inhibitors of phosphatidylinositol 3‐kinase (PI3K), significantly decreased the amplified VEGF₁₂₀ secretion by 29% (p < 0.01) and 28% (p < 0.01) relative to the cells stimulated by ghrelin alone, respectively, whereas these inhibitors had no effects on increased MCP‐1 release. On the other hand, JNK inhibitor SP600125 (10 μmol/L) clearly reduced the increased MCP‐1, but not VEGF₁₂₀, release by 35% relative to the only ghrelin‐stimulated cells (p < 0.01). In conclusion, ghrelin can enhance the secretions of proinflammatory adipokines, VEGF₁₂₀ and MCP‐1, but fails to affect IL‐10 and adiponectin which are considered to be anti‐inflammatory adipokines. Moreover, this augmented VEGF₁₂₀ release is invited through the activation of PI3K pathways and the MCP‐1 is through JNK pathways. Consequently, our results strongly suggest that ghrelin can induce the development of diabetes via its direct‐action in peripheral tissues as well as via in CNS. J. Cell. Physiol. 230: 199–209, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Expression and activity of V‐H+‐ATPase in gill and kidney of marbled eel Anguilla marmorata in response to salinity challenge

The full‐length complementary (c)DNA of vacuolar‐type‐H⁺‐ATPase B1 gene (vhab1) in marbled eel Anguilla marmorata with 1741 base pairs (bp) was identified. It contained a 1512 bp open reading frame encoding a polypeptide with 503 amino acids (55·9 kDa), an 83 bp 5′‐untranslated region (UTR) and a 146 bp 3′‐UTR. The expression levels of A. marmorata vhab1 in gill and kidney of A. marmorata were evaluated at different intervals during the exposure to various salinities (0, 10 and 25). The results indicated that the expression levels of A. marmorata vhab1 messenger (m)RNA in gill and kidney had a significant increase and reached the highest level at 1 h in brackish water (BW, salinity 10) group and 6 h in seawater (SW, salinity 25) group. Therefore, salinity did affect the relative expression level of A. marmorata vhab1 mRNA in gills, which exhibited the enhancement by c. 44 times in SW group when compared with that in fresh water. No remarkable difference in the expression of A. marmorata vhab1 mRNA was observed after 15 days of SW exposure (P > 0·05). V‐H⁺‐ATPase activity exhibited an increase by two‐ to three‐fold when compared with that in gill and kidney from the control group. The consequence primarily suggested that A. marmorata vhab1 gene product in elvers from A. marmorata plays an important role in adaptation response to SW.