Fatty acid amides as potential circulating biomarkers for sarcopenia.

BACKGROUND: Sarcopenia is characterized by a progressive decrease in skeletal muscle mass and function with age. Given that sarcopenia is associated with various metabolic disorders, effective metabolic biomarkers for its early detection are required. We aimed to investigate the metabolic biomarkers related to sarcopenia in elderly men and perform experimental studies using metabolomics. METHODS: Plasma metabolites from 142 elderly men, comprising a sarcopenia group and an age-matched control group, were measured using global metabolome profiling. Muscle and plasma samples from an aging mouse model of sarcopenia, as well as cell media and cell lysates during myoblast differentiation, were analysed based on targeted metabolome profiling. Based on these experimental results, fatty acid amides were quantified from human plasma as well as human muscle tissues. The association of fatty acid amide levels with sarcopenia parameters was evaluated. RESULTS: Global metabolome profiling showed that fatty acid amide levels were significantly different in the plasma of elderly men with sarcopenia (all Ps < 0.01). Consistent with these results in human plasma, targeted metabolome profiling in an aging mouse model of sarcopenia showed decreased levels of fatty acid amides in plasma but not in muscle tissue. In addition, the levels of fatty acid amides increased in cell lysates during muscle cell differentiation. Targeted metabolome profiling in men showed decreased docosahexaenoic acid ethanolamide (DHA EA) levels in the plasma (P = 0.016) but not in the muscle of men with sarcopenia. DHA EA level was positively correlated with sarcopenia parameters such as skeletal muscle mass index (SMI) and handgrip strength (HGS) (P = 0.001, P = 0.001, respectively). The area under the receiver-operating characteristic curve (AUC) for DHA EA level ≤ 4.60 fmol/µL for sarcopenia was 0.618 (95% confidence interval [CI]: 0.532-0.698). DHA EA level ≤ 4.60 fmol/µL was associated with a significantly greater likelihood of sarcopenia (odds ratio [OR]: 2.11, 95% CI: 1.03-4.30), independent of HGS. The addition of DHA EA level to age and HGS significantly improved the AUC from 0.620 to 0.691 (P = 0.0497). CONCLUSIONS: Our study demonstrated that fatty acid amides are potential circulating biomarkers in elderly men with sarcopenia. DHA EA, in particular, strongly related to muscle mass and strength, can be a key metabolite to become a reliable metabolic biomarker for sarcopenia. Further research on fatty acid amides will provide insights into the metabolomic changes relevant to sarcopenia from an aging perspective.

Vitreous Fatty Amides and Acyl Carnitines Are Altered in Intermediate Age-Related Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is the leading cause of visual impairment worldwide. In this study, we aimed to investigate the vitreous humor metabolite profiles of patients with intermediate AMD using untargeted metabolomics. Methods: We performed metabolomics using high-resolution liquid chromatography mass spectrometry on the vitreous humor of 31 patients with intermediate AMD and 30 controls who underwent vitrectomy for epiretinal membrane with or without cataract surgery. Univariate analyses after false discovery rate correction were performed to discriminate the metabolites and identify the significant metabolites of intermediate AMD. For biologic interpretation, enrichment and pathway analysis were conducted using MetaboAnalyst 5.0. Results: Of the 858 metabolites analyzed in the vitreous humor, 258 metabolites that distinguished patients with AMD from controls were identified (P values < 0.05). Ascorbic acid and uric acid levels increased in the AMD group (all P values < 0.05). The acyl carnitines, such as acetyl L-carnitine (1.37-fold), and fatty amides, such as anandamide (0.9-fold) and docosanamide (0.67-fold), were higher in patients with intermediate AMD. In contrast, nicotinamide (-0.55-fold), and succinic acid (-1.69-fold) were lower in patients with intermediate AMD. The metabolic pathway related oxidation of branched chain fatty acids and carnitine synthesis showed enrichment. Conclusions: Multiple metabolites related to fatty amides and acyl carnitine were found to be increased in the vitreous humor of patients with intermediate AMD, whereas succinic acid and nicotinamide were reduced, suggesting that altered metabolites related to fatty amides and acyl carnitines and energy metabolism may be implicated in the etiology of AMD.

Hypermethylation of the interferon regulatory factor 5 promoter in Epstein-Barr virus-associated gastric carcinoma.

Interferon regulatory factor-5 (IRF-5), a member of the mammalian IRF transcription factor family, is regulated by p53, type I interferon and virus infection. IRF-5 participates in virus-induced TLR-mediated innate immune responses and may play a role as a tumor suppressor. It was suppressed in various EBV-infected transformed cells, thus it is valuable to identify the suppression mechanism. We focused on a promoter CpG islands methylation, a kind of epigenetic regulation in EBV-associated Burkitt's lymphomas (BLs) and gastric carcinomas. IRF-5 is not detected in most of EBV-infected BL cell lines due to hypermethylation of IRF-5 distal promoter (promoter-A), which was restored by a demethylating agent, 5-aza-2'-deoxycytidine. Hypomethylation of CpG islands in promoter-A was observed only in EBV type III latent infected BL cell lines (LCL and Mutu III). Similarly, during EBV infection to Akata-4E3 cells, IRF-5 was observed at early time periods (2 days to 8 weeks), concomitant unmethylation of promoter-A, but suppressed in later infection periods as observed in latency I BL cell lines. Moreover, hypermethylation in IRF-5 promoter-A region was also observed in EBV-associated gastric carcinoma (EBVaGC) cell lines or primary gastric carcinoma tissues, which show type I latent infection. In summary, IRF-5 is suppressed by hypermethylation of its promoter-A in most of EBV-infected transformed cells, especially BLs and EBVaGC. EBV-induced carcinogenesis takes an advantage of proliferative effects of TLR signaling, while limiting IRF-5 mediated negative effects in the establishment of EBVaGCs.

Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1 signaling pathway.

BACKGROUND: Zinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The molecular mechanism for the inhibitory effect of zinc, however, is poorly understood. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells. RESULTS: In BMMs, zinc treatment during osteoclast differentiation decreased RANKL-induced osteoclast formation in a dose-dependent manner. We show that zinc suppressed the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Zinc also accumulated phospho-Nfatc1 (p-Nfatc1) in the cytosol in a dose-dependent manner and inhibited the translocation of Nfatc1 to the nucleus in RAW264.7 cells. Zinc suppressed the activities of Nfatc1 in the nucleus without changing the activities of NF-κB in RAW264.7 cells. In contrast, calcineurin activity decreased in response to zinc but its protein level was unchanged. RANKL-induced Ca2+ oscillations were inhibited by zinc treatment, but phospho-phospholipase Cγ1 (p-PLCγ1), the upstream signaling molecule of Ca2+ oscillations, was unaffected. Moreover, a constitutively active form of Nfatc1 obviously rescued suppression of osteoclastogenesis by zinc. CONCLUSIONS: Taken together, these results demonstrate for the first time that the inhibitory effect of zinc during osteoclastogesis is caused by suppressing the Ca2+-Calcineurin-NFATc1 signaling pathway. Thus, zinc may be a useful therapeutic candidate for the prevention of bone loss caused by NFATc1 activation in osteoclasts.

LOXL2 expression is associated with invasiveness and negatively influences survival in breast cancer patients.

Lysyl oxidase-like 2 (LOXL2) is associated with invasiveness and metastasis in breast cancer. We analyzed the prognostic impact of LOXL2 for breast cancer patients and investigated the role of LOXL2 in breast cancer cell lines. Immunohistochemical study of LOXL2 expression was done in samples from 309 patients. Survival analysis was performed using log-rank test and Cox regression hazard model. After identification of LOXL2 expression in breast cancer cell lines, we performed matrigel invasion and wound-healing assays with LOXL2-silenced cell lines. In the human study, LOXL2 was expressed in 16.2 % of patients. Comparing the LOXL2-positive versus negative groups, there was a significantly higher proportion of estrogen receptor-negative patients (54.0 vs. 37.0 %, respectively; p = 0.029) and triple-negative patients (34.0 vs. 18.0 %; p = 0.022) in the positive group. In multivariate analysis for overall survival and metastasis-free survival, positive LOXL2 was demonstrated as a poor prognostic factor (HR 2.27 and 2.10, respectively). In vitro study indicated that LOXL2 silencing induces a mesenchymal-epithelial transition-like process in basal cell lines (MDA-MB-231 and BT549) associated with decreased invasive and migratory properties. These clinical and preclinical data confirm that higher LOXL2 expression is associated with invasiveness of basal-like breast cancer cells and lower survival of breast cancer patients. Our results suggest the clinical value of LOXL2 as a therapeutic target in breast cancer.

Gallic acid suppresses lipopolysaccharide-induced nuclear factor-kappaB signaling by preventing RelA acetylation in A549 lung cancer cells.

Although multiple studies have revealed that gallic acid plays an important role in the inhibition of malignant transformation, cancer development, and inflammation, the molecular mechanism of gallic acid in inflammatory diseases is still unclear. In this study, we identified gallic acid from Rosa rugosa as a histone acetyltransferase (HAT) inhibitor with global specificity for the majority of HAT enzymes, but with no activity toward epigenetic enzymes including sirtuin (silent mating type information regulation 2 homologue) 1 (S. cerevisiae), histone deacetylase, and histone methyltransferase. Enzyme kinetic studies indicated that gallic acid uncompetitively inhibits p300/CBP-dependent HAT activities. We found that gallic acid inhibits p300-induced p65 acetylation, both in vitro and in vivo, increases the level of cytosolic IkappaBalpha, prevents lipopolysaccharide (LPS)-induced p65 translocation to the nucleus, and suppresses LPS-induced nuclear factor-kappaB activation in A549 lung cancer cells. We have also shown that gallic acid treatment inhibits the acetylation of p65 and the LPS-induced serum levels of interleukin-6 in vivo. Importantly, gallic acid generally inhibited inflammatory responses caused by other stimuli, including LPS, IFN-gamma, and interleukin-1beta, and further downregulated the expression of nuclear factor-kappaB-regulated antiapoptotic genes. These results show the crucial role of acetylation in the development of inflammatory diseases.

Epigallocatechin-3-gallate, a histone acetyltransferase inhibitor, inhibits EBV-induced B lymphocyte transformation via suppression of RelA acetylation.

Because the p300/CBP-mediated hyperacetylation of RelA (p65) is critical for nuclear factor-kappaB (NF-kappaB) activation, the attenuation of p65 acetylation is a potential molecular target for the prevention of chronic inflammation. During our ongoing screening study to identify natural compounds with histone acetyltransferase inhibitor (HATi) activity, we identified epigallocatechin-3-gallate (EGCG) as a novel HATi with global specificity for the majority of HAT enzymes but with no activity toward epigenetic enzymes including HDAC, SIRT1, and HMTase. At a dose of 100 micromol/L, EGCG abrogates p300-induced p65 acetylation in vitro and in vivo, increases the level of cytosolic IkappaBalpha, and suppresses tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. We also showed that EGCG prevents TNFalpha-induced p65 translocation to the nucleus, confirming that hyperacetylation is critical for NF-kappaB translocation as well as activity. Furthermore, EGCG treatment inhibited the acetylation of p65 and the expression of NF-kappaB target genes in response to diverse stimuli. Finally, EGCG reduced the binding of p300 to the promoter region of interleukin-6 gene with an increased recruitment of HDAC3, which highlights the importance of the balance between HATs and histone deacetylases in the NF-kappaB-mediated inflammatory signaling pathway. Importantly, EGCG at 50 micromol/L dose completely blocks EBV infection-induced cytokine expression and subsequently the EBV-induced B lymphocyte transformation. These results show the crucial role of acetylation in the development of inflammatory-related diseases.

The role of percutaneous transhepatic cholangioscopy in patients with hilar strictures.

BACKGROUND/AIMS: Various diagnostic advantages of percutaneous transhepatic cholangioscopy (PTCS) for the determination of the range of tumor and for the characterization of cholangioscopic findings have been reported. The aim of our study is to evaluate the diagnostic and therapeutic role of PTCS in patients with hilar strictures and to investigate its causes. METHODS: We retrospectively studied the medical records and cholangioscopic reports of 177 patients who received PTCS for hilar strictures between January 2000 and December 2005 at Asan Medical Center, Seoul. For each patient, cholagnioscopy, biopsy result, computed tomography (CT) and magnetic resonance cholangiopancreaticography (MRCP), operation, and pathologic reports were collected. RESULTS: Most patients had malignant hilar strictures or biliary papillomatosis while a few had benign hilar strictures. Presence of tumor vessel on PTCS was a useful diagnostic tool since direct observation of the tumor vessel strongly suggested a malignant tumor in the bile duct. The sensitivity of tumor vessel alone was 56.1%, and sensitivity of PTCS biopsy alone was 76.9%. However, sensitivity of biopsy combined with cholangioscopy of the tumor vessel was 88.4%, which was statistically significant compared with biopsy or tumor vessel alone. CONCLUSIONS: PTCS biopsy combined with cholangioscopic observation was useful in differential diagnosis of hilar strictures. PTCS also had a therapeutic role in some patients with incurable malignant hilar lesion.