Medium-chain fatty acids suppress lipotoxicity-induced hepatic fibrosis via the immunomodulating receptor GPR84.

Medium-chain triglycerides (MCTs), which consist of medium-chain fatty acids (MCFAs), are unique forms of dietary fat with various health benefits. G protein-coupled 84 (GPR84) acts as a receptor for MCFAs (especially C10:0 and C12:0); however, GPR84 is still considered an orphan receptor, and the nutritional signaling of endogenous and dietary MCFAs via GPR84 remains unclear. Here, we showed that endogenous MCFA-mediated GPR84 signaling protected hepatic functions from diet-induced lipotoxicity. Under high-fat diet (HFD) conditions, GPR84-deficient mice exhibited nonalcoholic steatohepatitis (NASH) and the progression of hepatic fibrosis but not steatosis. With markedly increased hepatic MCFA levels under HFD, GPR84 suppressed lipotoxicity-induced macrophage overactivation. Thus, GPR84 is an immunomodulating receptor that suppresses excessive dietary fat intake-induced toxicity by sensing increases in MCFAs. Additionally, administering MCTs, MCFAs (C10:0 or C12:0, but not C8:0), or GPR84 agonists effectively improved NASH in mouse models. Therefore, exogenous GPR84 stimulation is a potential strategy for treating NASH.

Short-chain fatty acid receptors and gut microbiota as therapeutic targets in metabolic, immune, and neurological diseases.

Dysbiosis is associated with various diseases. The composition and diversity of gut microbiota affect host physiology through the production of bioactive metabolites. Short-chain fatty acids are the main metabolites produced by microbial fermentation of dietary fiber. They play a crucial role in maintaining metabolic, nervous, and immune system. Short-chain fatty acids not only serve as an energy source for the host but also act as for G-protein-coupled receptor signaling molecules and histone deacetylase inhibitors. In particular, the discovery and deorphanization of free fatty acid receptors 2 and 3 (GPR43/41) have shed light on the molecular mechanisms underlying the regulation of physiological processes by short-chain fatty acids. The short-chain fatty acid receptors sense the nutrient status and transduce signals to maintain cellular homeostasis. Dysbiosis affects short-chain fatty acid production and impairs the signaling, leading to cellular dysfunction. We review the current understanding of short-chain fatty acid-mediated regulation of physiological processes and discuss the molecular pharmacology of short-chain fatty acid and the receptor. We also discuss recent advances in the use of prebiotics and probiotics in the treatment of disease.

Identification of chemical compounds as an inhibitor of mitochondrial ATP synthesis, leading to an increased stress resistance and an extended lifespan in C. elegans.

It is well known that the disruption of the mitochondrial respiratory components prolongs lifespan in many species. The mitochondrial stress response can lead to an increased survival rate through the restoration of the cellular homeostasis. Therefore, developing pharmacological interventions that induce mitochondrial stress response may be desirable to delay the onset of age-related diseases and promote a healthy life. In this study, we present chemical compounds, revealed by systematic screening of chemical libraries, which inhibit mitochondrial ATP synthesis in mammalian cells. Our study demonstrates that these compounds alter the body length and promote the oxidative stress response which leads to an increased longevity in Caenorhabditis elegans. Thus, our study identifies chemical compounds that may have potential therapeutic applications through affecting the mitochondrial function.

The MYST family histone acetyltransferase complex regulates stress resistance and longevity through transcriptional control of DAF-16/FOXO transcription factors.

The well-known link between longevity and the Sir2 histone deacetylase family suggests that histone deacetylation, a modification associated with repressed chromatin, is beneficial to longevity. However, the molecular links between histone acetylation and longevity remain unclear. Here, we report an unexpected finding that the MYST family histone acetyltransferase complex (MYS-1/TRR-1 complex) promotes rather than inhibits stress resistance and longevity in Caenorhabditis elegans Our results show that these beneficial effects are largely mediated through transcriptional up-regulation of the FOXO transcription factor DAF-16. MYS-1 and TRR-1 are recruited to the promoter regions of the daf-16 gene, where they play a role in histone acetylation, including H4K16 acetylation. Remarkably, we also find that the human MYST family Tip60/TRRAP complex promotes oxidative stress resistance by up-regulating the expression of FOXO transcription factors in human cells. Tip60 is recruited to the promoter regions of the foxo1 gene, where it increases H4K16 acetylation levels. Our results thus identify the evolutionarily conserved role of the MYST family acetyltransferase as a key epigenetic regulator of DAF-16/FOXO transcription factors.

The microRNA machinery regulates fasting-induced changes in gene expression and longevity in Caenorhabditis elegans.

Intermittent fasting (IF) is a dietary restriction regimen that extends the lifespans of Caenorhabditis elegans and mammals by inducing changes in gene expression. However, how IF induces these changes and promotes longevity remains unclear. One proposed mechanism involves gene regulation by microRNAs (miRNAs), small non-coding RNAs (∼22 nucleotides) that repress gene expression and whose expression can be altered by fasting. To test this proposition, we examined the role of the miRNA machinery in fasting-induced transcriptional changes and longevity in C. elegans We revealed that fasting up-regulated the expression of the miRNA-induced silencing complex (miRISC) components, including Argonaute and GW182, and the miRNA-processing enzyme DRSH-1 (the ortholog of the Drosophila Drosha enzyme). Our lifespan measurements demonstrated that IF-induced longevity was suppressed by knock-out or knockdown of miRISC components and was completely inhibited by drsh-1 ablation. Remarkably, drsh-1 ablation inhibited the fasting-induced changes in the expression of the target genes of DAF-16, the insulin/IGF-1 signaling effector in C. elegans Fasting-induced transcriptome alterations were substantially and modestly suppressed in the drsh-1 null mutant and the null mutant of ain-1, a gene encoding GW182, respectively. Moreover, miRNA array analyses revealed that the expression levels of numerous miRNAs changed after 2 days of fasting. These results indicate that components of the miRNA machinery, especially the miRNA-processing enzyme DRSH-1, play an important role in mediating IF-induced longevity via the regulation of fasting-induced changes in gene expression.

Where to deliver baits for deworming urban red foxes for Echinococcus multilocularis control: new protocol for micro-habitat modeling of fox denning requirements.

BACKGROUND: Deworming wild foxes by baiting with the anthelmintic praziquantel is being established as a preventive technique against environmental contamination with Echinococcus multilocularis eggs. Improvement of the cost-benefit performance of baiting treatment is required urgently to raise and maintain the efficacy of deworming. We established a spatial model of den site selection by urban red foxes, the definitive host, to specify the optimal micro-habitats for delivering baits in a new modeling approach modified for urban fox populations. METHODS: The model was established for two cities (Obihiro and Sapporo) in Hokkaido, Japan, in which a sylvatic cycle of E. multilocularis is maintained. The two cities have different degrees of urbanization. The modeling process was designed to detect the best combination of key environmental factors and spatial scale that foxes pay attention to most (here named 'heeding range') when they select den sites. All possible models were generated using logistic regression analysis, with "presence" or "absence" of fox den as the objective variable, and nine landscape categories customized for urban environments as predictor variables to detect the best subset of predictors. This procedure was conducted for each of ten sizes of concentric circles from dens and control points to detect the best circle size. Out of all models generated, the most parsimonious model was selected using Akaike's Information Criterion (AIC) inspection. RESULTS: Our models suggest that fox dens in Obihiro are located at the center of a circle with 500 m radius including low percentages of wide roads, narrow roads, and occupied buildings, but high percentages of green covered areas; the dens in Sapporo within 300 m radius with low percentages of wide roads, occupied buildings, but high percentages of riverbeds and green covered areas. The variation of the models suggests the necessity of accumulating models for various types of cities in order to reveal the patterns of the model. CONCLUSIONS: Our denning models indicating suitable sites for delivering baits will improve the cost-benefit performance of the campaign. Our modeling protocol is suitable for the urban landscapes, and for extracting the heeding range when they select the den sites.

Glomerular proteins related to slit diaphragm and matrix adhesion in the foot processes are highly tyrosine phosphorylated in the normal rat kidney.

BACKGROUND: Tyrosine phosphorylation of proteins has been a focus of extensive studies since it plays crucial roles in regulation of diverse biological reactions. To understand the involvement of tyrosine phosphorylation in kidney functions, a comprehensive proteomic study for tyrosine-phosphorylated proteins was performed in the normal rat kidney. METHODS: Two-dimensional gel electrophoresis and immunoprecipitation using anti-phosphotyrosine antibodies were employed to detect tyrosine-phosphorylated proteins. The proteins were analysed by mass spectrometry and validated by immunological analyses using specific antibodies. RESULTS: Most of tyrosine-phosphorylated proteins were confined to the glomerulus and predominantly localized along the glomerular capillary wall, especially in the foot processes of podocytes. Our systematic proteomic analysis identified nephrin, SHPS-1 (tyrosine-protein phosphatase non-receptor-type substrate 1), FAK1 and paxillin as major tyrosine-phosphorylated proteins and Neph1, talin and vinculin as minor tyrosine-phosphorylated proteins. In the present study, SHPS-1 was identified as a novel tyrosine-phosphorylated protein in the glomerulus and was also predominantly localized at the foot processes. Mass spectrometric analysis identified in vivo phosphorylation sites of SHPS-1 on Y460, Y477 and Y501. CONCLUSION: This study identified tyrosine-phosphorylated proteins in normal rat kidney, which were prominently rich in the glomerulus and localized at the podocyte foot processes. These proteins were categorized as cell-to-cell or cell-to-matrix adhesion complex-related molecules, suggesting their pivotal roles in the glomerular ultrafiltration.

Dietary supplementation with arachidonic acid but not eicosapentaenoic or docosahexaenoic acids alter lipids metabolism in C57BL/6J mice.

In order to investigate the effects of dietary supplementation rich in omega 3 and omega 6 fatty acids, we set up an experiment of twenty four C57BL/6J male mice segregated into 3 groups: normal diet (ND), omega 3 polyunsaturated fatty acid (n-3 PUFA,) and omega 6 (n-6 PUFA). At the end of the experiment that lasted for 1 month, food consumption of ND and n-3 PUFA were similar while it decreased in n-6 PUFA group. Total cholesterol, triglycerides, free fatty acids, and phospholipids profiles were increased in n-6 PUFA. LDL decreased in n-3 PUFA while increased in n-6 PUFA fed mice comparing to control group. On the other hand, there was no difference between treatments in HDL and glucose levels. Expression of leptin (ob) gene transcripts in epididymal fat were significantly elevated in n-6 PUFA mice compared to ND and n-3 PUFA groups while hypothalamic ob receptor A (obRa) mRNA did not changed in response to diet regimes. Transmission and scanning electron microscopy showed different degrees in fatty changes in the liver of both PUFA groups including lipid droplet infiltration and Ito cells with over accumulated lipids. In conclusion, under PUFA dietary supplementation, the hyperlipidemic status and elevated ob expression of n-6 PUFA but not n-3 PUFA fed mice suggests altered lipid metabolism between PUFA groups and/or different endocrine involvement. Moreover, the coincidently structural changes observed in liver of this group direct us to call for further studies to investigate the anti-obesity effect and safety of these PUFA under high supplementation condition.

Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alpha-dependent expression of thrombospondin-1.

The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF-1alpha-dependent expression of thrombospondin-1 (TSP-1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP-1, and mRNA levels of integrin beta(3) were increased with the increase in HIF-1alpha protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti-TSP-1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin beta(3). Furthermore, the migration was completely suppressed in HIF-1alpha-knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF-1alpha-overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP-1 of which induction is fully dependent on the stabilization of HIF-1alpha, in autocrine regulation. Thus we suggest that HIF-1alpha plays an important role in the pathogenesis of atherosclerosis.

[Role of thrombospondin-1 in hypoxia-induced migration of human vascular smooth muscle cells].

When the arterial wall thickens and blood-diffusion capacity is low in atherosclerotic lesions, hypoxia is a key factor for the development of atherosclerosis. Under hypoxic conditions, >100 genes, including those encoding many growth factors, are known to be induced by a transcriptional factor, hypoxia-inducible factor-1alpha (HIF-1alpha). In this study, to examine whether HIF-1alpha-dependent induction of growth factors is associated with the proliferation and migration of vascular cells in atherosclerotic lesions, we studied the role of thrombospondin-1 (TSP-1), which is induced by hypoxia, in the pathogenesis and progression of atherosclerosis in human coronary artery smooth muscle cells (CASMCs). Under hypoxic conditions, expression of HIF-1alpha increased time-dependently in human CASMCs with a concomitant increase in the proliferation and migration of cells. Under these conditions, the mRNA and protein levels of TSP-1 and the mRNA level of TSP-1 receptor, integrin beta3, were also enhanced. Neutralizing antibody against TSP-1 reduced hypoxia-induced migration, but not proliferation. Similarly, RGD peptide, which binds to integrin beta3, inhibited cell migration under hypoxia. In HIF-1alpha-knockdown CASMCs, in which expression of HIF-1alpha and TSP-1 mRNA and proteins is suppressed, hypoxia-induced migration was markedly reduced. In conclusion, hypoxia in atherosclerotic lesions induces TSP-1, which plays important roles in acceleration of the migration of human CASMCs and the progression of atherosclerosis.

VEGF-enhanced proliferation under hypoxia by an autocrine mechanism in human vascular smooth muscle cells.

AIM: Atherosclerotic lesions are reported to be hypoxic. Since hypoxia is known to induce the production of growth factors, such as vascular endothelial growth factor (VEGF), we examined the implication of hypoxia-induced VEGF in the proliferation of human coronary artery smooth muscle cells (CASMCs). METHODS: Cells were cultured under hypoxic conditions (1% O(2), 5% CO(2)) and several responses were measured. RESULTS: Under hypoxic conditions, the mRNA and protein levels of VEGF, and the mRNA level of VEGF receptor-1 (VEGFR-1) increased with an increase in hypoxia-inducible factor-1alpha (HIF-1alpha) protein, and considerable amounts of VEGF were secreted. Hypoxia enhanced the incorporation of [(3)H]-thymidine by CASMCs, which was completely inhibited by a neutralizing antibody against VEGF. A neutralizing antibody against NADPH-cytochrome P-450 reductase (NPR), which contributes to the stabilization of HIF-1alpha, also attenuated hypoxia-stimulated proliferation. In NPR-knockdown cells, the expression of VEGF, proliferation, and transcriptional activity were attenuated, whereas in NPR-overexpressing cells, they were enhanced. CONCLUSION: Hypoxia-induced proliferation of CASMCs is mediated through the expressions of VEGF and VEGFR-1 in an autocrine mechanism. Their expressions are dependent on the stabilization of HIF-1alpha, which is regulated by NPR. We suggest that hypoxia and hypoxia-induced VEGF expression are involved in the pathogenesis of progressive atherosclerosis.

Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells.

The inhibition of elicitor-induced plant defense responses by the protein kinase inhibitors K252a and staurosporine indicates that defense responses require protein phosphorylation. We isolated a cDNA clone encoding Nicotiana tabacum lectin-like receptor protein kinase 1 (NtlecRK1), an elicitor-responsive gene; in tobacco bright yellow (BY-2) cells by a differential display method. NtlecRK forms a gene family with at least three members in tobacco. All three NtlecRK genes potentially encode the N-terminal legume lectin domain, transmembrane domain and C-terminal Ser/Thr-type protein kinase domain. Green fluorescent protein (GFP) fusion showed that the NtlecRK1 protein was located on the plasma membrane. In addition, NtlecRK1 and 3 were responsive to INF1 elicitin and the bacterial elicitor harpin. These results indicate that NtlecRKs are membrane-located protein kinases that are induced during defense responses in BY-2 cells.

Acceleration of matrix metalloproteinase-1 production and activation of platelet-derived growth factor receptor beta in human coronary smooth muscle cells by oxidized LDL and 4-hydroxynonenal.

Increases in matrix metalloproteinases (MMPs) at atherosclerotic lesions are involved in the migration of smooth muscle cells (SMCs) into the intima and to the rupture of plaques, being implicated in the progression of atherosclerosis. The present study examined the mechanisms underlying the production of MMP-1, interstitial collagenase-1, induced by oxidized low-density lipoprotein (oxLDL) and 4-hydroxynonenal (4-HNE), factors proposed to play a pivotal role in atherogenesis, in human coronary SMCs. oxLDL promoted the production of MMP-1 with the preceding phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Immunoprecipitation of platelet-derived growth factor receptor beta (PDGFR-beta) revealed that oxLDL induced tyrosine phosphorylation of the receptor. Inhibition of the activation of PDGFR-beta and ERK1/2 resulted in a suppression of the production of MMP-1. Consistently, 4-HNE also elicited the production of MMP-1 with the preceding phosphorylation of PDGFR-beta and ERK1/2. The 4-HNE-induced production of MMP-1 was prevented when the activation of PDGFR-beta and ERK1/2 was inhibited. The present results suggest that the activation of PDGFR-beta and ERK1/2 is involved in the production of MMP-1 in oxLDL- and 4-HNE-stimulated human coronary SMCs.

Dietary soybean enhances Pit-1 dependent pituitary hormone production in iodine deficient rats.

Reports have shown that soybeans are goitrogenic. In the present study, we investigated the effects of a high soybean diet in rats that were fed normal or iodine-deficient chow on the regulation of anterior pituitary hormone production. Iodine deficiency alone resulted in thyroid hyperplasia, reduced serum thyroxine levels, and a tendency towards an increase in serum thyroid stimulating hormone (TSH). The combination of a high soybean and low iodine diet (ID + DS) acted synergistically to induce thyroid hypertrophy, reduce serum thyroxine and tri-iodothyronine, and markedly increase serum TSH. Immunohistochemical analysis revealed that rats fed the ID + DS diet exhibited a marked increase in their number of pituitary TSH, prolactin (PRL), and growth hormone (GH) producing cells. Pituitary transcription factor-1 (Pit-1) which is involved in the expression of the TSH, PRL, and GH genes was also increased in ID + DS fed rats. These results suggest that a diet high in soybean products modulates anterior pituitary hormone production by regulating Pit-1 induction, in iodine-deficient animals.

Pronounced synergistic promotion of N-bis(2-hydroxypropyl)nitrosamine-initiated thyroid tumorigenesis in rats treated with excess soybean and iodine-deficient diets.

We have reported that excess soybean treatment and iodine deficiency synergistically interact, resulting in remarkable induction of thyroid hyperplasias in rats. In the present study, modifying effects of excess soybean and iodine-deficient diets were investigated in the post-initiation phase of N-bis(2-hydroxypropyl)nitrosamine [DHPN]-initiated thyroid tumorigenesis in rats. AIN-93G in which casein was replaced with gluten was used as a basal diet to avoid possible iodine contamination. In Experiment 1, F-344 rats of both sexes were sc injected with DHPN at a dose of 2800 mg/kg body weight and then fed a diet containing 0%, 0.8%, 4%, or 20% defatted soybean for 12 weeks, with proportional replacement of gluten by soybean flour. Although no thyroid proliferative lesions were found in any group, the absolute thyroid weights were significantly (p < 0.01) elevated with the 20% soybean treatment. In Experiment 2, after similar sc injection of DHPN, rats were fed a basal diet or a diet containing 20% soybean under iodine normal or deficient conditions for 12 weeks. Soybean feeding to both sexes under iodine deficient but not normal conditions dramatically enhanced the development of thyroid follicular adenomas (p < 0.01) and adenocarcinomas (p < 0.05), in good agreement with decrease in thyroxine and increase in thyroid-stimulating hormone. Thus co-exposure to excess soybean and iodine deficiency results in synergistic promotion of DHPN-initiated thyroid tumorigenesis in rats, of which mechanisms appear to primarily involve effects on serum hormone levels.