Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4

Obese women have an increased risk to deliver large babies. However, the mechanisms underlying fetal overgrowth in these pregnancies are not well understood. Obese pregnant women typically have elevated circulating lipid levels. We tested the hypothesis that fatty acids stimulate placental amino acid transport, mediated via toll-like receptor 4 (TLR4) and mammalian target of rapamycin (mTOR) signaling pathways. Circulating NEFA levels and placental TLR4 expression were assessed in women with varying prepregnancy body mass index (BMI). The effects of oleic acid on system A and system L amino acid transport, and on the activation of the mTOR (4EBP1, S6K1, rpS6), TLR4 (IĸBɑ, JNK, p38 MAPK), and STAT3 signaling pathways were determined in cultured primary human trophoblast cells. Maternal circulating NEFAs (n = 33), but not placental TLR4 mRNA expression (n = 16), correlated positively with BMI (P < 0.05). Oleic acid increased trophoblast JNK and STAT3 phosphorylation (P < 0.05), whereas mTOR activity was unaffected. Furthermore, oleic acid doubled trophoblast system A activity (P < 0.05), without affecting system L activity. siRNA-mediated silencing of TLR4 expression prevented the stimulatory effect of oleic acid on system A activity. Our data suggest that maternal fatty acids can increase placental nutrient transport via TLR4, thereby potentially affecting fetal growth.     

Peroxidized unsaturated fatty acids stimulate Toll-like receptor 4 signaling in endothelial cells

AimAlthough unsaturated fatty acids are assumed to be protective against inflammatory disorders that include a pathway involving Toll-like receptor 4 (TLR4) activation, they might actually be toxic because of their high susceptibility to lipid peroxidation. Here we studied the effects of peroxidized unsaturated fatty acids on the TLR4–nuclear factor (NF)-κB pathway in endothelial cells.Main methodsConfluent cultured endothelial cells from bovine aorta were incubated for 1 h with fatty acids integrated into phosphatidylcholine vesicles. Lipopolysaccharide (LPS) or phosphatidylcholine vesicles without fatty acids were also applied as a positive control or a control for fatty acid groups, respectively. Activation of TLR4 and downstream signaling was assessed by membrane fractionation and Western blotting or immunofluorescent staining.Key findingsIn the same way as LPS, application of sufficiently peroxidized unsaturated fatty acids like oleic acid or docosahexaenoic acid, acutely caused TLR4 translocation to caveolae/raft membranes, leading to activation of NF-κB signaling in endothelial cells. In contrast, saturated fatty acids did not show such effects. Applying well-peroxidized unsaturated fatty acids, but not saturated fatty acids, acutely activates the TLR4/NF-κB pathway.SignificancePeroxidation of unsaturated fatty acid is essential for the acute activation of TLR4 by the fatty acids that follow the same pathway as the activation by LPS. Unsaturated fatty acids have been assumed to be protective against inflammatory disorders, and drugs containing unsaturated fatty acids are now developed and provided. Our result suggests that, for inflammatory disorders involving TLR4 signaling, using unsaturated fatty acids as anti-inflammatory drugs may cause contrary effects.     

Calcium supplementation relieves high-fat diet-induced liver steatosis by reducing energy metabolism and promoting lipolysis

Nonalcoholic fatty liver disease (NAFLD) is a chronic disease affecting the health of many people worldwide. Previous studies have shown that dietary calcium supplementation may alleviate NAFLD, but the underlying mechanism is not clear. In this study investigating the effect of calcium on hepatic lipid metabolism, 8-week-old male C57BL/6J mice were divided into four groups (n = 6): (1) mice given a normal chow containing 0.5% calcium (CN0.5), (2) mice given a normal chow containing 1.2% calcium (CN1.2), (3) mice given a high-fat diet (HFD) containing 0.5% calcium (HFD0.5), and (4) mice fed a HFD containing 1.2% calcium (HFD1.2). To understand the underlying mechanism, cells were treated with oleic acid and palmitic acid to mimic the HFD conditions in vitro. The results showed that calcium alleviated the increase in triglyceride accumulation induced by oleic acid and/or palmitic acid in HepG2, AML12, and primary hepatocyte cells. Our data demonstrated that calcium supplementation alleviated HFD-induced hepatic steatosis through increased liver lipase activity, proving calcium is involved in the regulation of hepatic lipid metabolism. Moreover, calcium also increased the level of glycogen in the liver, and at the same time had the effect of reducing glycolysis and promoting glucose absorption. Calcium addition increased calcium levels in the mitochondria and cytoplasm. Taken together, we concluded that calcium supplementation could relieve HFD-induced hepatic steatosis by changing energy metabolism and lipase activity.     

GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy

Background

Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress- induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.

Methodology/Principal Findings

Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.

Conclusions/Significance

GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.     

姜黄素对过度训练大鼠脾脏炎症反应的调控作用及其机制

目的:研究姜黄素调控Toll-样受体4(TLR4)-p38丝裂原活化蛋白激酶(p38 MAPK)/核因子κB(NF-κB)信号通路缓解过度训练大鼠脾脏炎症反应的作用及其机制.方法:7周龄SPF级雄性Wistar大鼠分为安静对照组(C组,n=12)、过度训练模型组(OM组,n=11)、姜黄素+模型组(COM组,n=14)...     

The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll-like receptors (TLR). Although mitogen-activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP approximately 35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF-alpha, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF-alpha, MCP-1, and KC production by Kupffer cells were significantly increased following trauma-hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG-stimulated TLR9 signaling increased TNF-alpha and IL-6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, -4, and -9 signaling. In contrast, TNF-alpha and IL-6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma-hemorrhage.     

Tripartite motif 38 alleviates the pathological process of NAFLD–NASH by promoting TAB2 degradation

Nonalcoholic fatty liver disease (NAFLD) has become the most prevalent chronic liver disease worldwide, without any Food and Drug Administration-approved pharmacological intervention in clinic. Trim38, as an important member of the TRIM (tripartite motif-containing) family, was largely reported to be involved in the regulation of innate immune and inflammatory responses. However, the functional roles of TRIM38 in NAFLD remain largely unknown. Here, the expression of TRIM38 was first detected in liver samples of both NAFLD mice model and patients diagnosed with NAFLD. We found that TRIM38 expression was downregulated in NAFLD liver tissues compared with normal liver tissues. Genetic Trim38-KO in vivo showed that TRIM38 depletion deteriorated the high-fat diet and high fat and high cholesterol diet-induced hepatic steatosis and high fat and high cholesterol diet-induced liver inflammation and fibrosis. In particular, we found that the effects of hepatocellular lipid accumulation and inflammation induced by palmitic acid and oleic acid were aggravated by TRIM38 depletion but mitigated by TRIM38 overexpression in vitro. Mechanically, RNA-Seq analysis demonstrated that TRIM38 ameliorated nonalcoholic steatohepatitis progression by attenuating the activation of MAPK signaling pathway. We further found that TRIM38 interacted with transforming growth factor-β-activated kinase 1 binding protein 2 and promoted its protein degradation, thus inhibiting the transforming growth factor-β-activated kinase 1-MAPK signal cascades. In summary, our study revealed that TRIM38 could suppress hepatic steatosis, inflammatory, and fibrosis in NAFLD via promoting transforming growth factor-β-activated kinase 1 binding protein 2 degradation. TRIM38 could be a potential target for NAFLD treatment.     

Differential modulation of Nods signaling pathways by fatty acids in human colonic epithelial HCT116 cells

Nucleotide-binding oligomerization domain-containing proteins (Nods) are intracellular pattern recognition receptors recognizing conserved moieties of bacterial peptidoglycan through their leucine-rich repeats domain. The agonists for Nods activate proinflammatory signaling pathways, including NF-kappaB pathways. The results from our previous studies showed that the activation of TLR4 and TLR2, leucine-rich repeat-containing pattern recognition receptors, were differentially modulated by saturated and n-3 polyunsaturated fatty acids in macrophages and dendritic cells. Here, we show the differential modulation of NF-kappaB activation and interleukin-8 (IL-8) expression in colonic epithelial cells HCT116 by saturated and unsaturated fatty acids mediated through Nods proteins. Lauric acid (C12:0) dose dependently activated NF-kappaB and induced IL-8 expression in HCT116 cells, which express both Nod1 and Nod2, but not detectable amounts of TLR2 and TLR4. These effects of lauric acid were inhibited by dominant negative forms of Nod1 or Nod2, but not by dominant negative forms of TLR2, TLR4, and TLR5. The effects of lauric acid were also attenuated by small RNA interference targeting Nod1 or Nod2. In contrast, polyunsaturated fatty acids, especially n-3 polyunsaturated fatty acids, inhibited the activation of NF-kappaB and IL-8 expression induced by lauric acid or known Nods ligands in HCT116. Furthermore, lauric acid induced, but docosahexaenoic acid inhibited lauric acid- or Nod2 ligand MDP-induced, Nod2 oligomerization in HEK293T cells transfected with Nod2. Together, these results provide new insights into the role of dietary fatty acids in modulating inflammation in colon epithelial cells. The results suggest that Nods may be involved in inducing sterile inflammation, one of the key etiological conditions in the development of many chronic inflammatory diseases.     

Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages

Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.     

Oxidized low density lipoprotein induces bone morphogenetic protein-2 in coronary artery endothelial cells via Toll-like receptors 2 and 4

Vascular calcification is a common complication in atherosclerosis. Bone morphogenetic protein-2 (BMP-2) plays an important role in atherosclerotic vascular calcification. The aim of this study was to determine the effect of oxidized low density lipoprotein (oxLDL) on BMP-2 protein expression in human coronary artery endothelial cells (CAECs), the roles of Toll-like receptor (TLR) 2 and TLR4 in oxLDL-induced BMP-2 expression, and the signaling pathways involved. Human CAECs were stimulated with oxLDL. The roles of TLR2 and TLR4 in oxLDL-induced BMP-2 expression were determined by pretreatment with neutralizing antibody, siRNA, and overexpression. Stimulation with oxLDL increased cellular BMP-2 protein levels in a dose-dependent manner (40-160 μg/ml). Pretreatment with neutralizing antibodies against TLR2 and TLR4 or silencing of these two receptors reduced oxLDL-induced BMP-2 expression. Overexpression of TLR2 and TLR4 enhanced the cellular BMP-2 response to oxLDL. Furthermore, oxLDL was co-localized with TLR2 and TLR4. BMP-2 expression was associated with activation of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK)1/2. Inhibition of NF-κB and ERK1/2 reduced BMP-2 expression whereas inhibition of p38 MAPK had no effect. In conclusion, oxLDL induces BMP-2 expression through TLR2 and TLR4 in human CAECs. The NF-κB and ERK1/2 pathways are involved in the signaling mechanism. These findings underscore an important role for TLR2 and TLR4 in mediating the BMP-2 response to oxLDL in human CAECs and indicate that these two immunoreceptors contribute to the mechanisms underlying atherosclerotic vascular calcification.     

Lipid-induced up-regulation of human acyl-CoA synthetase 5 promotes hepatocellular apoptosis

In the pathogenesis of nonalcoholic fatty liver disease, accumulation of lipids in hepatocytes and hepatocyte apoptosis are strongly implicated in disease progression from the potentially reversible condition of steatosis to severe acute and chronic liver injury. Acyl-CoA synthetase 5, a member of the ACSL gene family that catalyzes the activation of long-chain fatty acids for lipid biosynthesis, is the only ACSL isoform that is both, located on mitochondria and functionally involved in enterocyte apoptosis. In this study, the regulation of human ACSL5 in hepatocellular fatty acid degeneration and its involvement in hepatocyte apoptosis was investigated using models of in vitro and in vivo steatosis as well as plasmid-mediated stable gene transfer and RNAi-mediated gene silencing. ACSL5 mRNA and protein were strongly increased by uptake of dietary derived fatty acids in primary human hepatocytes, HepG2 cells and human steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL- and TNFα-, but not FAS- induced apoptosis, whereas knock down of ACSL5 reduced apoptosis susceptibility. High ACSL5 activity resulted in enhanced caspase-3/7 activity, but was not accompanied by up-regulation of death receptors, DR4, DR5 or TNF-R1. This study gives evidence that hepatocyte steatosis is associated with ACSL5 up-regulation resulting in increased susceptibility to hepatic cell death. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as important mechanism in fatty liver-related disorders.     

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4: roles for CD14, LPS-binding protein, and MD2 as targets for specificity of inhibition

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysaccharide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-alpha production, IkappaBalpha degradation, p38 MAPK phosphorylation, and NF-kappaB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I.C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from approximately 30 microm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.     

Heterotrimeric Gi/Go proteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-κB and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (Gα(i/o)) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. Gα(i/o) are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-κB. We find that Gα(i/o)-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.     

Toll-like receptor 4 contributes to uterine activation by upregulating pro-inflammatory cytokine and CAP expression via the NF-κB/P38MAPK signaling pathway during pregnancy

Evidence indicates that inflammatory response is significant during the physiological process of human parturition; however, the specific signaling pathway that triggers inflammation is undefined. Toll-like receptors (TLRs) are key upstream gatekeepers that control inflammatory activation before preterm delivery. Our previous study showed that TLR4 expression was significantly increased in human pregnancy tissue during preterm and term labor. Therefore, we explore whether TLR4 plays a role in term labor by initiating inflammatory responses, therefore promoting uterine activation. The results showed that expression of TLR4, interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), CC chemokine ligand 2 (CCL-2), and uterine contraction-associated proteins (CAPs) was upregulated in the human and mice term labor (TL) group compared with the not-in-labor (TNL) group, and the TLR4 level positively correlated with CAP expression. In pregnant TLR4-knockout (TLR4^−/−) mice, gestation length was extended by 8 hr compared with the wild-type group, and the expression of IL-1β, IL-6, TNF-α, CCL-2, and CAPs was decreased in TLR4^−/− mice. Furthermore, nuclear factor-κB (NF-κB) and P38MAPK activation is involved in the initiation of labor but was inhibited in TLR4^−/− mice. In uterine smooth muscle cells, the expression of inflammatory cytokines and CAPs decreased when the NF-κB and P38MAPK pathway was inhibited. Our data suggest that TLR4 is a key factor in regulating the inflammatory response that drives uterine activation and delivery initiation via activating the NF-κB/P38MAPK pathway.