Association between alcohol consumption and plasma fetuin-A and its contribution to incident type 2 diabetes in women

Aims/hypothesis

The benefits of moderate alcohol consumption for type 2 diabetes have been postulated to involve a mechanism of improved insulin sensitivity. Fetuin-A, which is known to inhibit insulin signalling, has emerged as a biomarker for diabetes risk. Alcohol consumption may influence circulating fetuin-A concentrations and subsequently diabetes risk by altering the insulin signal. We therefore hypothesised that moderate alcohol consumption would be associated with lower fetuin-A concentration and that fetuin-A would partly explain the association between alcohol consumption and incident type 2 diabetes.

Methods

Among diabetes-free female participants in the Nurses’ Health Study (n?=?1,331), multiple linear regression was conducted to assess the association between alcohol consumption and plasma fetuin-A. Least-squares means (lsmeans) of fetuin-A were estimated in categories of alcohol consumption (0, 0.1–4.9, 5–14.9 and ≥15 g/day). The proportion of alcohol consumption and diabetes association explained by baseline fetuin-A was assessed in 470 matched incident diabetes case–control pairs with follow-up 2000–2006.

Results

Higher alcohol consumption was associated with lower plasma fetuin-A (p for trend?=?0.009): lsmean ± SE 476.5?±?5.9 μg/ml for abstainers, 468.9?±?5.2 μg/ml for 0.1–4.9 g/day consumers, 455.9?±?7.0 μg/ml for 5.0–14.9 g/day consumers, and 450.0?±?9.4 μg/ml for ≥15.0 g/day consumers. Fetuin-A and fasting insulin explained 18.4% and 54.8%, respectively, of the inverse association between alcohol consumption and diabetes after multiple adjustment (both p for contribution <0.04).

Conclusions/interpretation

Moderate alcohol consumption is associated with lower plasma fetuin-A in diabetes-free women. Fetuin-A and insulin explain a significant proportion of the association between alcohol consumption and incident type 2 diabetes. Further studies are needed to examine potential biological mechanisms underlying this association.     

The Relationship of Circulating Fetuin-A With Liver Histology and Biomarkers of Systemic Inflammation in Nondiabetic Subjects with Nonalcoholic Fatty Liver Disease

Background/Aims:

Fetuin-A, a glycoprotein with anti-inflammatory properties, plays an important role in counter-regulating inflammatory responses. It has also been associated with insulin resistance and metabolic syndrome. We aimed to investigate circulating concentrations of fetuin-A and its possible association with hepatic and systemic inflammation in nondiabetic subjects with nonalcoholic fatty liver disease (NAFLD).

Patients and Methods:

We included 105 nondiabetic male subjects with NAFLD [nonalcoholic steatohepatitis (NASH, n = 86) and simple steatosis (SS, n = 19)]. Plasma levels of fetuin-A and markers of inflammation [high-sensitive C reactive protein (hsCRP), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and adiponectin] were measured by enzyme-linked immunosorbent assay method. Insulin sensitivity was determined by homeostasis model assessment of insulin resistance (HOMA-IR) index.

Results:

Fetuin-A was negatively correlated with age (r = −0.27, P = 0.006), however there was no association between fetuin-A and body mass index, waist circumference (WC), glucose, insulin, HOMA-IR, lipid parameters, and inflammatory markers. In addition, no significant association was observed between fetuin-A and histological findings including liver fibrosis.

Conclusion:

This study demonstrated that plasma fetuin-A levels are not correlated with the hepatic histology and systemic markers of inflammation in nondiabetic subjects with NAFLD. Our data also suggested that age is significantly associated with fetuin-A in this clinically relevant condition.     

Metabolic crosstalk between fatty pancreas and fatty liver: effects on local inflammation and insulin secretion

Aims/hypothesis

Obesity-linked ectopic fat accumulation is associated with the development of type 2 diabetes. Whether pancreatic and liver steatosis impairs insulin secretion is controversial. We examined the crosstalk of human pancreatic fat cells with islets and the role of diabetogenic factors, i.e. palmitate and fetuin-A, a hepatokine released from fatty liver.

Methods

Human pancreatic resections were immunohistochemically stained for insulin, glucagon, somatostatin and the macrophage/monocyte marker CD68. Pancreatic adipocytes were identified by Oil Red O and adiponectin staining. Primary pancreatic pre-adipocytes and differentiated adipocytes were co-cultured with human islets isolated from organ donors and the metabolic crosstalk between fatty liver and fatty pancreas was mimicked by the addition of palmitate and fetuin-A. Insulin secretion was evaluated by ELISA and RIA. Cytokine expression and secretion were assessed by RT-PCR and multiplex assay, respectively. Subcellular distribution of proteins was examined by confocal microscopy and protein phosphorylation by western blotting.

Results

In human pancreatic parenchyma, highly differentiated adipocytes were detected in the proximity of islets with normal architecture and hormone distribution. Infiltration of adipocytes was associated with an increased number of CD68-positive cells within islets. In isolated primary pancreatic pre-adipocytes and differentiated adipocytes, palmitate and fetuin-A induced IL6, CXCL8 and CCL2 mRNA expression. Cytokine production was toll-like receptor 4 (TLR4)-dependent and further accentuated in pre-adipocytes when co-cultured with islets. In islets, IL6 and CXCL8 mRNA levels were also increased by fetuin-A and palmitate. Only in macrophages within the isolated islets, palmitate and fetuin-A stimulated the production of the cytotoxic cytokine IL-1β. Palmitate, but not fetuin-A, exerted pro-apoptotic effects in islet cells. Instead, fetuin-A impaired glucose-induced insulin secretion in a TLR4-independent, but c-Jun N-terminal kinase- and Ca²⁺-dependent, manner.

Conclusions/interpretation

These results provide the first evidence that fetuin-A-mediated metabolic crosstalk of fatty liver with islets may contribute to obesity-linked glucose blindness of beta cells, while fatty pancreas may exacerbate local inflammation.

     

Serum Fetuin-A levels in obese children with biopsy proven nonalcoholic fatty liver disease

Background and aims

Fetuin-A has been proposed as a marker of liver damage in adults with obesity-related NAFLD. The aim of this study was to test serum fetuin-A concentrations in obese children with NAFLD diagnosed either by ultrasonography or by liver biopsy and to determine its applicability as predictive tool in pediatric NAFLD.

A sustained increase in plasma NEFA upregulates the Toll-like receptor network in human muscle

Aims/hypothesis

Insulin-sensitive tissues (muscle, liver) of individuals with obesity and type 2 diabetes mellitus are in a state of low-grade inflammation, characterised by increased Toll-like receptor (TLR) expression and TLR-driven signalling. However, the cause of this mild inflammatory state is unclear. We tested the hypothesis that a prolonged mild increase in plasma NEFA will increase TLR expression and TLR-driven signalling (nuclear factor κB [NFκB] and mitogen-activated kinase [MAPK]) and impair insulin action in muscle of lean healthy individuals.

Methods

Twelve lean, normal-glucose-tolerant participants were randomised to receive a 48 h infusion (30 ml/h) of saline or Intralipid followed by a euglycaemic–hyperinsulinaemic clamp. Vastus lateralis muscle biopsies were performed before and during the clamp.

Results

Lipid infusion impaired insulin-stimulated IRS-1 tyrosine phosphorylation and reduced peripheral insulin sensitivity (p?<?0.01). The elevation in circulating NEFA increased expression of TLR3, TLR4 and TLR5, and several MAPK (MAPK8, MAP4K4, MAP2K3) and inhibitor of κB kinase-NFκB (CHUK [IKKA], c-REL [REL] and p65 [RELA, NFKB3, p65]) signalling genes (p?<?0.05). The lipid infusion also increased extracellular signal-regulated kinase (ERK) phosphorylation (p?<?0.05) and tended to reduce the content of inhibitor of kappa Bα (p?=?0.09). The muscle content of most diacylglycerol, ceramide and acylcarnitine species was unaffected. In summary, insulin resistance induced by prolonged low-dose lipid infusion occurs together with increased TLR-driven inflammatory signalling and impaired insulin-stimulated IRS-1 tyrosine phosphorylation.

Conclusions/interpretation

A sustained, mild elevation in plasma NEFA is sufficient to increase TLR expression and TLR-driven signalling (NFκB and MAPK) in lean individuals. The activation of this pathway by NEFA may be involved in the pathogenesis of insulin resistance in humans. Trial registration ClinicalTrials.gov NCT01740817     

TLR2/6 and TLR4-activated macrophages contribute to islet inflammation and impair beta cell insulin gene expression via IL-1 and IL-6

Aims/hypothesis

Inflammation contributes to pancreatic beta cell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in recently diagnosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequences of a high-fat diet. Here we investigated the role of macrophages in TLR2/6- and TLR4-mediated effects on islet inflammation and beta cell function.

Methods

Genetic and pharmacological approaches were used to determine the effects of TLR2/6 and TLR4 ligands on mouse islets, human islets and purified rat beta cells. Islet macrophages were depleted and sorted by flow cytometry and the effects of TLR2/6- and TLR4-activated bone-marrow-derived macrophages (BMDMs) on beta cell function were assessed.

Results

Macrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expression in mouse and human islets and IL-1β secretion from human islets. TLR2/6 and TLR4 ligands also reduced insulin gene expression; however, this occurred in a non-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretion partly via reducing Ins1, Ins2, and Pdx1 mRNA expression. Antagonism of the IL-1 receptor and neutralisation of IL-6 completely reversed the effects of activated macrophages on beta cell gene expression.

Conclusions/interpretation

We conclude that islet macrophages are major contributors to islet IL-1β secretion in response to TLR2/6 and TLR4 ligands. BMDMs stimulated with TLR2/6 and TLR4 ligands reduce insulin secretion from pancreatic beta cells, partly via IL-1β- and IL-6-mediated decreased insulin gene expression.     

IL-8 production in response to cigarette smoke is decreased in epithelial cells from COPD patients

Cigarette smoke is the principal cause of chronic obstructive pulmonary disease (COPD), a disorder characterized by airway inflammation. As epithelial cells are the first line of defense against foreign material, the response of normal epithelial cells to smoke has been extensively studied. However, little is known about how epithelial cells derived from COPD patients respond to ongoing smoke exposure. This study was aimed at comparing the intracellular response of normal human bronchial/tracheal epithelial cells (NHBE) and COPD-diseased human bronchial/tracheal epithelial cells (DHBE) to cigarette smoke.NHBE and DHBE cells were treated with cigarette smoke condensate (CSC) for 24 h. IL-8 production was measured by ELISA and western blot was used to measure TLR4 expression. Cells were pretreated with CLI-095, a TLR4 inhibitor, or the signaling pathway inhibitors PD184352, Helenalin, or PI-103, which inhibit the ERK1/2, NF-κB and PI3K pathways, respectively.NHBE cells increased IL-8 production in a dose-dependent manner in response to CSC while DHBE cells did not show any significant difference and had a much lower production of IL-8 in response to CSC compared to NHBE cells. There was no change in TLR4 expression with CSC exposure. CLI-095 and PD184352 attenuated IL-8 secretion, indicating that CSC-induced inflammation is both TLR4- and ERK1/2-dependent.These results demonstrate that NHBE and DHBE cells differentially respond to cigarette smoke. DHBE cells exhibit a dampened IL-8 release, indicating that COPD is associated with a reduced capacity of airway epithelial cells to respond to foreign material.     

High plasma fetuin-A levels are associated with metabolic syndrome among males but not females in a Japanese general population

Aims

Fetuin-A, a protein exclusively secreted from the liver, is associated with insulin resistance and/or metabolic syndrome (MetS). However, few studies have examined this association in Japan. We investigated this issue in a Japanese general population.

Methods

We performed an epidemiological survey in a small community in Japan. The participants consisted of 659 subjects (253 males and 406 females). Fetuin-A levels were measured by a sandwich ELISA method and the modified NCEP-ATP III criteria were adopted to diagnose MetS. The homeostasis model assessment index (HOMA-IR) was calculated as a marker of insulin resistance.

Results

Statistically significant characteristics of the 659 subjects stratified by fetuin-A quartiles were male gender (inversely), age (inversely), insulin, HOMA-IR, uric acid (inversely), alcohol intake (inversely) and the prevalence of MetS. Mean fetuin-A levels were 249.7 ± 45.1 μg/ml in males and 262.7 ± 55.8 μg/ml in females. In males, the prevalence of MetS was 43.1%, and their mean HOMA-IR level was 1.1. In females, the prevalence of MetS was 17.7%, and their mean HOMA-IR level was 0.9. Multiple stepwise regression analyses showed that fetuin-A levels in males but not females were independently associated with MetS and LDL-c. Multiple logistic regression analysis of fetuin-A (quartile 1 vs. quartile 4) in males showed significant odds ratios of 1.009 (95% C.I.: 1.003–1.015) for MetS and 1.376 (95% C.I.: 1.027–1.844) for 1-SD increment increase in LDL-c.

Conclusions

High plasma fetuin-A levels were associated with MetS in community-dwelling Japanese males but not females.     

乙型肝炎病毒感染肾小管上皮细胞Toll样受体4的表达及其作用

目的 探讨HBV感染人近端肾小管上皮细胞系HK-2后对其表达Toll样受体4(TLR4)的影响,并观察TLR4抗HBV感染的生物学作用.方法 收集HBV DNA拷贝在107-102/ml的患者血清,通过显微镜及免疫荧光法观察HBV阳性血清感染HK-2前、后细胞形态及α抗平滑肌抗体(α-SMA)的变化,应用MTT法检测不同浓度TLR4刺激因子(LPS)及TLR4抑制因子(CLI-095)对HK-2细胞增殖的影响.选取10 μL/ml脂多糖(LPS)及5μg/ml CLI-095作用于HBV感染的HK-2细胞,通过细胞免疫荧光技术及免疫印迹法检测HK-2细胞内TLR4蛋白的变化,ELISA法和荧光定量PCR法观测各组细胞上清液中HBsAg、HBeAg和HBV DNA含量的变化.结果 HBV分别感染HK-2细胞12 h和24 h后,随感染时间延长,细胞形态变得不规则,数量也减少.α-SMA的表达水平与感染24 h后相比,在HBV感染12 h后表达最多.LPS浓度在小于10μg/ml范围内,HBV感染HK-2细胞24 h后其增殖程度与剂量呈正相关,与CLI-095浓度呈负相关(P＜0.05).LPS组HK-2细胞TLR4蛋白的表达高于CLI-095组,其上清液中HBV DNA水平及HBsAg、HBeAg表达水平较CLI-095组降低.结论 TLR4可能通过免疫炎症反应参与抑制HK-2细胞中的HBV复制,当HBV感染肾组织细胞时可发挥抗病毒作用.

Abstract：

Objective To explore the expression and role of Toll receptor 4(TLR4)in human proximal tubular epithelial cell line HK-2,infected by HBV. Methods The serum of HBV DNA copies between 107-108/ml was collected. Before and after infected by HBV DNA positive serum. the HK-2 cells' morphology and the expression of α-smooth muscle actin(α-SMA)were observed by microscopy and immunofluorescence, and the effects of different concentrations of lipopolysaccharides(U)S.TLR4-stimulating factor)and CLI-095(TLR4 Inhibitor)on the proliferation rate of HK-2 cells were observed by MTT assays. After HBV serum and 10μg/ml LPS and 5μl/ml CLI-095 acted on HK-2 cells,TLR4 protein expression was measured by immunofluorescence and Western-blotting assay, and HBsAg and HBeAg in cell culture medium were detected by ELISA. and HBV DNA copies by fluorescence quantitative PCR. Results The longer HBV infected HK-2 cells, the more irregular of the cells' shape, the fewer number of the cells were left. But compared with HBV infected after 24 hours, α-SMA was more expressed after HBV infected 12 hours. After infected by HBV serum in 24 hours.HK-2 cells' proliferation rate was positively correlation in a dose range of LPS, but was negatively correlated with the CLI-095(P＜0.05＝.The levels of HBsAg and HBeAg in cell culture medium were largest when the LPS concentration was at 10μg/ml and CLI-095 at 5μg/ml.The expression of TLR4 significantly increased in HK-2 cells treated with LPS compared with those with CLI-095.but HBV DNA levels and HBsAg and HBeAg expression levels were lower. Conclusions HBV infection may promote cell transdifferentiation and cell injury. The stimulation of HK-2 infected with HBV by LPS may upregulate the expression of TLR4 and reduce the copies of HBV DNA.     

Cytokines are associated with postembolization fever and survival in hepatocellular carcinoma patients receiving transcatheter arterial chemoembolization

Objective

Cytokines play important roles in angiogenesis, inflammation, and cell growth. The present study aimed to investigate the correlation between cytokine changes and clinical characteristics in hepatocellular carcinoma (HCC) patients receiving transcatheter arterial chemoembolization (TACE).

Methods

Forty-one TACE-näive HCC patients receiving 73 sessions of TACE and 30 healthy controls were studied. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin, epidermal growth factor (EGF), epidermal growth factor receptor, and transforming growth factor β1 (TGF-β1) before and at 1, 3, 5, 7, and 14 days after TACE as well as clinical parameters were analyzed.

Results

Baseline serum levels of VEGF, bFGF, IL-6, IL-8, and TNF-α in HCC patients were significantly elevated, whereas EGF and TGF-β1 levels were lower compared to those in healthy controls (p < 0.05 for all). Serum IL-6 increased rapidly and peaked on day 1 after TACE administration, whereas VEGF increased more slowly and peaked on day 14 after TACE administration. Patients with post-TACE fever had higher serum IL-6 levels on days 1, 3, and 5 (p < 0.005 for all). Patients with pre-TACE serum VEGF < 200 pg/ml had a longer survival than those with pre-TACE serum VEGF levels ≥ 200 pg/ml (22.2 months vs. 11.6 months, p = 0.014). Cox multivariate analysis showed that baseline serum VEGF significantly predicted survival for HCC patients receiving TACE.

Conclusions

TACE is associated with the modulation of serum angiogenic, inflammatory, and cell growth cytokines in HCC patients. Serum IL-6 correlates with post-TACE fever, and baseline serum VEGF independently predicts patient survival.     

Endoplasmic reticulum stress induces the expression of fetuin-A to develop insulin resistance

Fetuin-A is a biomarker reported to be important in many metabolic disorders, including obesity, diabetes, and hepatic steatosis. Although it is well known that fetuin-A is increased in diabetes and nonalcoholic fatty liver disease (NAFLD), the levels of fetuin-A in diabetic patients with NAFLD are unknown. Furthermore, the regulation of fetuin-A expression is still obscure. In this study, a total of 180 age- and sex-matched subjects with normal glucose tolerance, NAFLD, newly diagnosed diabetes (NDD), and NDD with NAFLD were recruited. We found that the levels of fetuin-A were significantly increased in NDD with NAFLD as compared with NDD or NAFLD subjects. We further used HepG2 cells to investigate the regulation of fetuin-A. Treatment with endoplasmic reticulum (ER) stress activator, thapsigargin, increased the expression of fetuin-A mRNA and protein in a time- and dose-dependent manner. Pretreatment with ER stress inhibitor, 4-phenylbutyrate, reversed high glucose or palmitate-induced fetuin-A expression. Moreover, treatment with 4-phenylbutyrate in both streptozotocin-induced and high-fat diet-induced diabetic mice not only decreased hepatic fetuin-A levels but also improved hyperglycemia. Taken together, we found that fetuin-A levels were increased in diabetes patients with NAFLD. Moreover, ER stress induced by high glucose and palmitate increased the expression of fetuin-A and further contributed to the development of insulin resistance.     

Palmitate induces a pro-inflammatory response in human pancreatic islets that mimics CCL2 expression by beta cells in type 2 diabetes

Aims/hypothesis

Beta cell failure is a crucial component in the pathogenesis of type 2 diabetes. One of the proposed mechanisms of beta cell failure is local inflammation, but the presence of pancreatic islet inflammation in type 2 diabetes and the mechanisms involved remain under debate.

Methods

Chemokine and cytokine expression was studied by microarray analysis of laser-capture microdissected islets from pancreases obtained from ten non-diabetic and ten type 2 diabetic donors, and by real-time PCR of human islets exposed to oleate or palmitate at 6 or 28 mmol/l glucose. The cellular source of the chemokines was analysed by immunofluorescence of pancreatic sections from individuals without diabetes and with type 2 diabetes.

Results

Microarray analysis of laser-capture microdissected beta cells showed increased chemokine and cytokine expression in type 2 diabetes compared with non-diabetic controls. The inflammatory response in type 2 diabetes was mimicked by exposure of non-diabetic human islets to palmitate, but not to oleate or high glucose, leading to the induction of IL-1β, TNF-α, IL-6, IL-8, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2). Interference with IL-1β signalling abolished palmitate-induced cytokine and chemokine expression but failed to prevent lipotoxic human islet cell death. Palmitate activated nuclear factor κB (NF-κB) in human pancreatic beta and non-beta cells, and chemically induced endoplasmic reticulum stress caused cytokine expression and NF-κB activation similar to that occurring with palmitate.

Conclusions/interpretation

Saturated-fatty-acid-induced NF-κB activation and endoplasmic reticulum stress may contribute to IL-1β production and mild islet inflammation in type 2 diabetes. This inflammatory process does not contribute to lipotoxicity ex vivo, but may lead to local chemokine release.     

Up-Regulation of Toll-Like Receptors 2 and 4 on Peripheral Monocytes After Major Abdominal Surgical Operation

Background

This study is to investigate the time-course expression of TLR2 and TLR4 on peripheral monocytes in patients receiving major abdominal surgical operation.

Methods

Blood samples were obtained from peripheral vein before and after an operation in 30 patients with gastrointestinal or pancreatic surgery. The mRNA expression of TLR2, TLR4, TNF-α and IL-6 on peripheral blood mononuclear cells (PBMC) was analyzed by real-time PCR. The expressions of TLR2, TLR4, HLA-DR, CD80, and CD86 on monocytes were analyzed by flow cytometry. The expressions of TLR2 and TLR4 on monocytes responding to each agonist (zymosan and lipopolysaccharide) were also measured by flow cytometry.

Results

TLR2 and TLR4 mRNA showed significant up-regulation after the completion of the operation when compared with those before the operation. TLR2 expression reached its peak level on day 1 and TLR4 on days 1 and 3. There was no significant difference between pre- and post-operation in the expressions of HLA-DR, CD80 and CD86. Stimulation with zymosan, increased the expression of TLR2 significantly after the operation and reached its highest value on day 3. Similarly, after stimulation with lipopolysaccharide, the expression of TLR4 was also increased and the highest level was observed on day 1. The expression of TNF-α and IL-6 mRNA decreased after completion of the operation and gradually returned to basal level.

Conclusions

The expressions of TLR2 and TLR4 on monocytes were up-regulated during the early period after a major abdominal surgical operation in patients, which might be related to the activation of innate immunity.     

Hepatocyte growth factor stimulates the migration of gastric epithelial cells by altering the subcellular localization of the tight junction protein ZO-1

Background

Hepatocyte growth factor (HGF) is essential for epithelial restitution, a process in which epithelial cells rapidly migrate to cover desquamated epithelium after mucosal injury in the gastrointestinal tract. In this study, we aimed to elucidate the molecular mechanisms of the HGF-mediated reconstitution of gastric epithelial structures by analyzing the expression and subcellular dynamics of tight junction proteins.

Methods

We treated human gastric epithelial MKN74 cells with HGF, and examined the effects of HGF on cell migration and proliferation, and the expression and subcellular dynamics of tight junction proteins; as well, we investigated the effect of HGF on paracellular permeability to macromolecules (using fluorescein isothiocyanate [FITC]-dextran).

Results

HGF significantly stimulated the migration of MKN74 cells, but not their proliferation, in a dose-dependent manner. HGF did not affect the expression of tight junction proteins, including claudin-1, -3, -4 and -7; occludin; and zonula occludens (ZO)-1. However, fluorescence immunostaining revealed that, in the cell membrane, the levels of ZO-1, but not those of occludin or claudin-4, were transiently decreased 1 h after HGF treatment. The results were further confirmed by western blotting: HGF reduced the amount of ZO-1 protein in the cell membrane fraction concomitantly with an increase in cytoplasmic ZO-1. Furthermore, HGF reduced the interaction between ZO-1 and occludin, and induced the tyrosine phosphorylation of occludin, whereas the phosphorylation status of ZO-1 was not affected by exposure to HGF. Despite a decrease in the ZO-1/occludin interaction, HGF did not affect paracellular permeability to macromolecules.

Conclusions

HGF alters the subcellular localization of ZO-1, probably through the tyrosine phosphorylation of occludin, which may induce cell dispersion during epithelial restitution.     

Involvement of sphingosine 1-phosphate in palmitate-induced insulin resistance of hepatocytes via the S1P2 receptor subtype

Aims/hypothesis

Enhanced plasma levels of NEFA have been shown to induce hepatic insulin resistance, which contributes to the development of type 2 diabetes. Indeed, sphingolipids can be formed via a de novo pathway from the saturated fatty acid palmitate and the amino acid serine. Besides ceramides, sphingosine 1-phosphate (S1P) has been identified as a major bioactive lipid mediator. Therefore, our aim was to investigate the generation and function of S1P in hepatic insulin resistance.

Methods

The incorporation of palmitate into sphingolipids was performed by rapid-resolution liquid chromatography-MS/MS in primary human and rat hepatocytes. The influence of S1P and the involvement of S1P receptors in hepatic insulin resistance was examined in human and rat hepatocytes, as well as in New Zealand obese (NZO) mice.

Results

Palmitate induced an impressive formation of extra- and intracellular S1P in rat and human hepatocytes. An elevation of hepatic S1P levels was observed in NZO mice fed a high-fat diet. Once generated, S1P was able, similarly to palmitate, to counteract insulin signalling. The inhibitory effect of S1P was abolished in the presence of the S1P₂ receptor antagonist JTE-013 both in vitro and in vivo. In agreement with this, the immunomodulator FTY720-phosphate, which binds to all S1P receptors except S1P₂, was not able to inhibit insulin signalling.

Conclusions/interpretation

These data indicate that palmitate is metabolised by hepatocytes to S1P, which acts via stimulation of the S1P₂ receptor to impair insulin signalling. In particular, S1P₂ inhibition could be considered as a novel therapeutic target for the treatment of insulin resistance.     

Circulating alanine transaminase (ALT) and γ-glutamyl transferase (GGT), but not fetuin-A,are associated with metabolic risk factors,at baseline and at two-year follow-up: The prospective Cyprus Metabolism Study

Objective

To comparatively evaluate traditional liver tests and fetuin A as predictors of cardiometabolic risk, we studied associations between serum alanine transaminase (ALT), γ-glutamyl transferase (GGT), aspartate aminotransferase (AST) and fetuin-A and anthropometric, metabolic, and cardiovascular parameters cross-sectionally at baseline, and prospectively, after 2-years of follow-up.

Research Design and Methods

616 randomly enrolled young healthy participants in the Cyprus Metabolism Study, including all 93 subjects who participated in the follow-up study 2 years after baseline assessment, were included in this study.

Results

In the cross-sectional study, serum ALT and GGT were strongly correlated with anthropometric, cardiovascular, and metabolic variables, while serum AST was only correlated with waist circumference and waist-to-hip ratio. Fetuin-A was correlated with anthropometric variables, systolic blood pressure (SBP), insulin, and homeostasis model of assessment-insulin resistance (HOMA-IR) in the unadjusted model. In the fully adjusted model, both serum ALT and GGT levels remained positively correlated with total and low-density lipoprotein (LDL) cholesterol. GGT levels also remained correlated with triglycerides. ALT levels remained strongly positively correlated with insulin (r = 0.17, p < .0001) and HOMA-IR (r = 0.16, p = 0.0001). Serum fetuin-A levels were no longer significantly correlated with any variables.Prospectively, ALT and GGT were predictors of anthropometric variables and LDL cholesterol, while baseline levels of AST and fetuin-A were not predictors of any variables at 2-year follow-up.

Conclusions

We confirmed associations of ALT and GGT levels but failed to demonstrate an independent association between fetuin-A and cardiometabolic risk factors in young healthy men. Traditional liver tests (LFTs) are thus better than fetuin-A predictors of metabolic risk factors cross-sectionally and prospectively in young healthy adults.     

肾衰饮对CRF大鼠TLR4/MyD88/NF-κB信号通路及炎症状态的影响

目的基于toll样受体(TLR)4/髓样细胞分化因子(MyD)88/核转录因子(NF)-κB信号通路探讨肾衰饮对慢性肾衰竭(CRF)大鼠炎症状态的干预机制。方法将60只SD大鼠,随机分成四组,分别为正常组、模型组、尿毒清组和肾衰饮组。进行4 w的干预治疗后,苏木素-伊红(HE)染色观察大鼠肾脏组织病理形态改变,检测血肌酐(Scr)、尿素氮(BUN),酶联免疫吸附试验(ELISA)检测大鼠血清白细胞介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α水平,分别采用免疫组化法和Western印迹检测大鼠肾脏组织TLR4、MyD88、NF-κB蛋白表达。结果与正常组比较,模型组HE染色显示大鼠肾脏组织发生明显病理改变,血Scr、BUN、IL-1β、IL-6、TNF-α水平均显著升高(P<0.01),肾脏组织中TLR4、MyD88、NF-κB蛋白表达均显著升高(P<0.01);与模型组比较,尿毒清组和肾衰饮组肾脏组织病理改变明显改善,血清Scr、BUN、IL-1β、IL-6、TNF-α水平均显著降低(P<0.01),肾脏组织中TLR4、MyD88、NF-κB蛋白表达均显著降低(P<0.01)。结论肾衰饮能有效改善CRF大鼠的肾功能,其机制可能是抑制TLR4/MyD88/NF-κB信号通路从而抑制炎症反应发生,达到治疗CRF的目的。     

Regulation of angiogenesis, mural cell recruitment and adventitial macrophage behavior by Toll-like receptors

The angiogenic response to injury can be studied by culturing rat or mouse aortic explants in collagen gels. Gene expression studies show that aortic angiogenesis is preceded by an immune reaction with overexpression of Toll-like receptors (TLRs) and TLR-inducible genes. TLR1, 3, and 6 are transiently upregulated at 24 h whereas TLR2, 4, and 8 expression peaks at 24 h but remains elevated during angiogenesis and vascular regression. Expression of TLR5, 7 and 9 steadily increases over time and is highest during vascular regression. Studies with isolated cells show that TLRs are expressed at higher levels in aortic macrophages compared to endothelial or mural cells with the exception of TLR2 and TLR9 which are more abundant in the aortic endothelium. LPS and other TLR ligands dose dependently stimulate angiogenesis and vascular endothelial growth factor production. TLR9 ligands also influence the behavior of nonendothelial cell types by blocking mural cell recruitment and inducing formation of multinucleated giant cells by macrophages. TLR9-induced mural cell depletion is associated with reduced expression of the mural cell recruiting factor PDGFB. The spontaneous angiogenic response of the aortic rings to injury is reduced in cultures from mice deficient in myeloid differentiation primary response 88 (MyD88), a key adapter molecule of TLRs, and following treatment with an inhibitor of the NFκB pathway. These results suggest that the TLR system participates in the angiogenic response of the vessel wall to injury and may play an important role in the regulation of inflammatory angiogenesis in reactive and pathologic processes.