Xunmei Yuan

Role of the Toll-like Receptor 4/NF-κB Pathway in Saturated Fatty Acid–Induced Inflammatory Changes in the Interaction Between Adipocytes and Macrophages

Objective—Previous studies demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they might represent an important source of inflammation. Using an in vitro coculture system composed of 3T3-L1 adipocytes and RAW264 macrophages, we previously demonstrated that saturated fatty acids (FAs) and tumor necrosis factor (TNF)-α derived from adipocytes and macrophages, respectively, play a major role in the coculture-induced inflammatory changes. Methods and Results—Coculture of adipocytes and macrophages resulted in the activation of nuclear factor-&kgr;B (NF-&kgr;B), a primary regulator of inflammatory responses, in both cell types. Pharmacological inhibition of NF-&kgr;B markedly suppressed the coculture-induced production of proinflammatory cytokines and adipocyte lipolysis. Peritoneal macrophages obtained from Toll-like receptor 4 (TLR4) mutant mice exhibited marked attenuation of TNFα production in response to saturated FAs. Notably, coculture of hypertrophied adipocytes and TLR4-mutant macrophages resulted in marked inhibition of proinflammatory cytokine production and adipocyte lipolysis. We also observed that endogenous FAs, which are released from adipocytes via the β3-adrenergic stimulation, resulted in the activation of the TLR4/NF-&kgr;B pathway. Conclusion—These findings suggest that saturated FAs, which are released in large quantities from hypertrophied adipocytes via the macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for TLR4, thereby inducing the inflammatory changes in both adipocytes and macrophages through NF-&kgr;B activation.

Increased Adiponectin Secretion by Highly Purified Eicosapentaenoic Acid in Rodent Models of Obesity and Human Obese Subjects

Objectives—Fish oil rich in n-3 polyunsaturated fatty acids (PUFAs) or n-3 PUFAs have been shown to reduce the incidence of coronary heart disease. Here we investigated the effect of highly purified eicosapentaenoic acid (EPA) on production of adiponectin, the only established antiatherogenic and antiinflammatory adipocytokine, in rodent models of obesity and human obese subjects. Methods and Results—We demonstrated that EPA increases adiponectin secretion in genetically obese ob/ob mice and high-fat diet–induced obese mice. In the in vitro coculture of adipocytes and macrophages, EPA reversed the coculture-induced decrease in adiponectin secretion at least in part through downregulation of tumor necrosis factor-&agr; in macrophages. We also showed significant increase in plasma adiponectin concentrations in human obese subjects after a 3-month treatment with EPA (1.8 g daily). Multivariate regression analysis revealed that EPA treatment is the only independent determinant of plasma adiponectin concentrations. Conclusion—This study demonstrates that EPA increases adiponectin secretion in rodent models of obesity and human obese subjects, possibly through the improvement of the inflammatory changes in obese adipose tissue. Because EPA has reduced the risk of major coronary events in a large-scale, prospective, randomized clinical trial, this study provides important insight into its therapeutic implication in obesity-related metabolic sequelae.

Activating Transcription Factor 3 Constitutes a Negative Feedback Mechanism That Attenuates Saturated Fatty Acid/Toll-Like Receptor 4 Signaling and Macrophage Activation in Obese Adipose Tissue

Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. Evidence has suggested that saturated fatty acids released via adipocyte lipolysis serve as a naturally occurring ligand that stimulates Toll-like receptor (TLR)4 signaling, thereby inducing the inflammatory responses in macrophages in obese adipose tissue. Through a combination of cDNA microarray analyses of saturated fatty acid–stimulated macrophages in vitro and obese adipose tissue in vivo, here we identified activating transcription factor (ATF)3, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, as a target gene of saturated fatty acids/TLR4 signaling in macrophages in obese adipose tissue. Importantly, ATF3, when induced by saturated fatty acids, can transcriptionally repress tumor necrosis factor-α production in macrophages in vitro. Chromatin immunoprecipitation assay revealed that ATF3 is recruited to the region containing the activator protein-1 site of the endogenous tumor necrosis factor-α promoter. Furthermore, transgenic overexpression of ATF3 specifically in macrophages results in the marked attenuation of proinflammatory M1 macrophage activation in the adipose tissue from genetically obese KKAy mice fed high-fat diet. This study provides evidence that ATF3, which is induced in obese adipose tissue, acts as a transcriptional repressor of saturated fatty acids/TLR4 signaling, thereby revealing the negative feedback mechanism that attenuates obesity-induced macrophage activation. Our data also suggest that activation of ATF3 in macrophages offers a novel therapeutic strategy to prevent or treat obesity-induced adipose tissue inflammation.

Role of DNA Methylation in the Regulation of Lipogenic Glycerol-3-Phosphate Acyltransferase 1 Gene Expression in the Mouse Neonatal Liver

The liver is a major organ of lipid metabolism, which is markedly changed in response to physiological nutritional demand; however, the regulation of hepatic lipogenic gene expression in early life is largely unknown. In this study, we show that expression of glycerol-3-phosphate acyltransferase 1 (GPAT1; Gpam), a rate-limiting enzyme of triglyceride biosynthesis, is regulated in the mouse liver by DNA methylation, an epigenetic modification involved in the regulation of a diverse range of biological processes in mammals. In the neonatal liver, DNA methylation of the Gpam promoter, which is likely to be induced by Dnmt3b, inhibited recruitment of the lipogenic transcription factor sterol regulatory element–binding protein-1c (SREBP-1c), whereas in the adult, decreased DNA methylation resulted in active chromatin conformation, allowing recruitment of SREBP-1c. Maternal overnutrition causes decreased Gpam promoter methylation with increased GPAT1 expression and triglyceride content in the pup liver, suggesting that environmental factors such as nutritional conditions can affect DNA methylation in the liver. This study is the first detailed analysis of the DNA-methylation–dependent regulation of the triglyceride biosynthesis gene Gpam, thereby providing new insight into the molecular mechanism underlying the epigenetic regulation of metabolic genes and thus metabolic diseases.

Ligand-Activated PPARα-Dependent DNA Demethylation Regulates the Fatty Acid β-Oxidation Genes in the Postnatal Liver

The metabolic function of the liver changes sequentially during early life in mammals to adapt to the marked changes in nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. However, how it is induced during the neonatal period is poorly understood. Here we show DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation does not occur in the fetal mouse liver under the physiologic condition, suggesting that it is specific to the neonatal period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator–activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveal that the DNA demethylation is PPARα dependent. We also find that DNA methylation of the fatty acid β-oxidation genes are reduced in the adult human liver relative to the fetal liver. This study represents the first demonstration that the ligand-activated PPARα-dependent DNA demethylation regulates the hepatic fatty acid β-oxidation genes during the neonatal period, thereby highlighting the role of a lipid-sensing nuclear receptor in the gene- and life-stage–specific DNA demethylation of a particular metabolic pathway.

Analysis of DNA methylation change induced by Dnmt3b in mouse hepatocytes

DNA methylation is a key epigenetic contributor to gene regulation in mammals. We have recently found that in the mouse liver, the promoter region of glycerol-3-phosphate acyltransferase 1, a rate-limiting enzyme of de novo lipogenesis, is regulated by DNA methylation, which is mediated by Dnmt3b, an enzyme required for the initiation of de novo methylation. In this study, using primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3b, we characterized Dnmt3b-dependent DNA methylation on a genome-wide basis. A genome-wide DNA methylation analysis, called microarray-based integrated analysis of methylation by isoschizomers, identified 108 genes with Dnmt3b dependent DNA methylation. In DNA expression array analysis, expression of some genes with Dnmt3b-dependent DNA methylation was suppressed. Studies with primary mouse hepatocytes overexpressing Dnmt3b or Dnmt3a revealed that many genes with Dnmt3b-dependent methylation are not methylated by Dnmt3a, whereas those methylated by Dnmt3a are mostly methylated by Dnmt3b. Bioinformatic analysis showed that the CANAGCTG and CCGGWNCSC (N denotes A, T, G, or C; W denotes A or T; and S denotes C or G) sequences are enriched in genes methylated by overexpression of Dnmt3b and Dnmt3a, respectively. We also observed a large number of genes with Dnmt3b-dependent DNA methylation in primary cultures of mouse hepatocytes with adenoviral overexpression of Dnmt3, suggesting that Dnmt3b is an important DNA methyltransferase in primary mouse hepatocytes, targets specific genes, and potentially plays a role in vivo.

Epigenetic modulation of Fgf21 in the perinatal mouse liver ameliorates diet-induced obesity in adulthood

The nutritional environment to which animals are exposed in early life can lead to epigenetic changes in the genome that influence the risk of obesity in later life. Here, we demonstrate that the fibroblast growth factor-21 gene (Fgf21) is subject to peroxisome proliferator-activated receptor (PPAR) α–dependent DNA demethylation in the liver during the postnatal period. Reductions in Fgf21 methylation can be enhanced via pharmacologic activation of PPARα during the suckling period. We also reveal that the DNA methylation status of Fgf21, once established in early life, is relatively stable and persists into adulthood. Reduced DNA methylation is associated with enhanced induction of hepatic FGF21 expression after PPARα activation, which may partly explain the attenuation of diet-induced obesity in adulthood. We propose that Fgf21 methylation represents a form of epigenetic memory that persists into adulthood, and it may have a role in the developmental programming of obesity.FGF21 exerts beneficial metabolic effects on multiple tissues. Here the authors show that the Fgf21 gene is demethylated during the postnatal suckling period, creating an epigenetic memory that determines the responsiveness of the Fgf21 gene to inducers such as PPARα activators or fasting in adulthood.

Mild Maternal Hypothyroxinemia During Pregnancy Induces Persistent DNA Hypermethylation in the Hippocampal Brain-Derived Neurotrophic Factor Gene in Mouse Offspring

BACKGROUND Thyroid hormones are essential for normal development of the central nervous system (CNS). Experimental rodents have shown that even a subtle thyroid hormone insufficiency in circulating maternal thyroid hormones during pregnancy may adversely affect neurodevelopment in offspring, resulting in irreversible cognitive deficits. This may be due to the persistent reduced expression of the hippocampal brain-derived neurotrophic factor gene Bdnf, which plays a crucial role in CNS development. However, the underlying molecular mechanisms remain unclear. METHODS Thiamazole (MMI; 0.025% [w/v]) was administered to dams from two weeks prior to conception until delivery, which succeeded in inducing mild maternal hypothyroxinemia during pregnancy. Serum thyroid hormone and thyrotropin levels of the offspring derived from dams with mild maternal hypothyroxinemia (M offspring) and the control offspring (C offspring) were measured. At 70 days after birth, several behavior tests were performed on the offspring. Gene expression and DNA methylation status were also evaluated in the promoter region of Bdnf exon IV, which is largely responsible for neural activity-dependent Bdnf gene expression, in the hippocampus of the offspring at day 28 and day 70. RESULTS No significant differences in serum thyroid hormone or thyrotropin levels were found between M and C offspring at day 28 and day 70. M offspring showed an impaired learning capacity in the behavior tests. Hippocampal steady-state Bdnf exon IV expression was significantly weaker in M offspring than it was in C offspring at day 28. At day 70, hippocampal Bdnf exon IV expression at the basal level was comparable between M and C offspring. However, it was significantly weaker in M offspring than in C offspring after the behavior tests. Persistent DNA hypermethylation was also found in the promoter region of Bdnf exon IV in the hippocampus of M offspring compared to that of C offspring, which may cause the attenuation of Bdnf exon IV expression in M offspring. CONCLUSIONS Mild maternal hypothyroxinemia induces persistent DNA hypermethylation in Bdnf exon IV in offspring as epigenetic memory, which may result in long-term cognitive disorders.