Approaches to addressing the rise in obesity levels.

Levels of obesity and overweight are increasing globally, with affected individuals often experiencing health issues and reduced quality of life. The pathogenesis of obesity is complex and multifactorial, and effective solutions have been elusive. In this Viewpoint, experts in the fields of medical therapy, adipocyte biology, exercise and muscle, bariatric surgery, genetics, and public health give their perspectives on current and future progress in addressing the rising prevalence of obesity.

Obesity related microRNA­424 is regulated by TNF­α in adipocytes.

In recent years, obesity has become a major public health concern. Obesity has been previously associated with low­grade inflammation and TNF­α induction in adipose tissue, which subsequently disrupts adipocyte metabolism. MicroRNAs (miRNAs/miRs) are important metabolic factors and their dysregulation has been associated with obesity­related metabolic syndromes. In fact, it has been directly suggested that miR­424 may be functionally associated with adipogenesis, although its exact role in this process remains unclear. The present study aimed to identify the function of miR­424 in adipogenesis. In the present study, miR­424 expression levels were analyzed during adipogenesis and the differential expression of this miRNA in the adipose tissue of obese and non­obese children was also assessed. Furthermore, the interaction between miR­424 and the adipocytokine TNF­α was determined. Finally, miR­424 target genes and downstream signaling pathways were predicted via bioinformatics and analyzed by performing a luciferase reporter assay to elucidate the functional mechanisms of miR­424 in adipogenesis of visceral adipocytes. The results revealed that the expression levels of miR­424 upregulated in the adipose tissue biopsies from obese children compared with the biopsies of non­obese children. However, in cultured adipocytes, the expression levels of miR­424 were discovered to be gradually downregulated during the adipogenesis process. TNF­α treatment significantly downregulated the expression levels of miR­424 via binding to its promoter region and reducing its transcriptional activity. Through bioinformatic prediction analysis, miR­424 target genes were analyzed, of which several were identified to be involved in signaling pathways that are known to regulate adipogenesis, such as the Wnt signaling pathway. In conclusion, the present study indicated that miR­424 was regulated by TNF­α and served an important role in adipogenesis.

Inhibition of hedgehog signaling promotes white adipose tissue browning.

White adipose tissue (WAT) browning is a potential strategy to treat obesity, and is characterized by the formation of brown adipocytes induced by cold or ß-3 adrenergic receptor (ß-3AR) agonist treatment. The hedgehog (Hh) signaling at the primary cilium is closely related to obesity, and plays a key role in the differentiation and adipogenesis of adipocytes. However, little is known about its effects on WAT browning. In this study, browning models were used to evaluate the activity and effect of Hh signaling on WAT browning using Hh antagonists, agonist, and small-interfering RNAs (siRNAs) specific for glioma-associated oncogene homologue 1 (Gli1), smoothened (Smo), and suppressor of fused (Sufu). We observed that Hh signaling activity was inhibited during the browning process both in vivo and in vitro. Further, Hh signaling inhibition enhanced WAT browning, while its activation attenuated norepinephrine-induced browning. Thus, the inhibition of Hh signaling promotes WAT browning and therefore, Hh signaling may be a therapeutic target against obesity and associated comorbidities.

PID1 in adipocytes modulates whole-body glucose homeostasis.

The novel obesity-associated protein Phosphotyrosine Interaction Domain containing 1 (PID1) inhibits insulin-PI3K/Akt signaling pathway and insulin-stimulated glucose uptake in vitro. In this study, we generated fat tissue-specific aP2-PID1 transgenic (aP2-PID1tg) mice and PID1 knockout (PID1-/-) mice to explore how PID1 affects glucose metabolism in vivo. We observed insulin resistance and impaired insulin-PI3K/Akt signaling in aP2-PID1tg mice. Consistent with these data, the PID1-/- mice displayed improved glucose tolerance and insulin sensitivity under chow diet, with increased Akt phosphorylation in white adipose tissue (WAT). We further demonstrated that PID1 could interact with low density lipoprotein receptor-related protein 1 (LRP1) but not the insulin receptor (IR) in adipocytes, and its overexpression could lead to decreased GLUT4 level. Our results thus indentify PID1 as a critical regulator of glucose metabolism in adipocytes.

Tumor necrosis factor­α and interleukin­6 suppress microRNA­1275 transcription in human adipocytes through nuclear factor­κB.

Obesity is a confirmed risk factor for hyperlipidemia, type­II diabetes, hypertension, and cardiovascular disease. MicroRNAs (miRs) have emerged as an important field of study within energy metabolism and obesity. A previous study demonstrated miR­1275 to be markedly down­regulated during maturation of human preadipocytes. It has been reported that miR­1275 dysregulates expression in several types of cancer and infections. Little is currently known about the regulation of miR­1275 transcription. The aim of the current study was to explore the mechanism underlying the expression of miR­1275 in mature human adipocytes. After differentiation, human adipocytes were incubated with tumor necrosis factor (TNF)­α and interleukin­6. The results of reverse transcription­quantitative polymerase chain reaction demonstrated that miR­1275 can be down­regulated by TNF­α and IL­6, in human mature adipocytes. Bioinformatic analysis was used to predict nuclear factor (NF)­κB binding sites of miR­1275's promoter region. Luciferase assay and rescue experiments were performed in HEK293T cells. NF­κB was involved in regulating miR­1275 transcription by binding to its promoter. In response to TNF­α, NF­κB was bound to the promoter of miR­1275 and inhibited its transcription. These results indicated that inflammatory factors could regulate miR­1275 transcription through NF­κB and influencing miR­1275 effects on obesity.

Metformin prevents LYRM1-induced insulin resistance in 3T3-L1 adipocytes via a mitochondrial-dependent mechanism.

We previously proposed that LYR motif containing 1 (LYRM1)-induced mitochondrial reactive oxygen species (ROS) production contributes to obesity-related insulin resistance. Metformin inhibits ROS production and promotes mitochondrial biogenesis in specific tissues. We assessed the effects of metformin on insulin resistance in LYRM1-over-expressing 3T3-L1 adipocytes. Metformin enhanced basal and insulin-stimulated glucose uptake and GLUT4 translocation, reduced IRS-1 and Akt phosphorylation and ROS levels, and affected the expression of regulators of mitochondrial biogenesis in LYRM1-over-expressing adipocytes. Metformin may ameliorate LYRM1-induced insulin resistance and mitochondrial dysfunction in part via a direct antioxidant effect and in part by activating the adenosine monophosphate-activated protein kinase (AMPK)-PGC1/NRFs pathway.

Knockdown of LYRM1 rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes.

LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.

TNF-α, IL-6, and leptin increase the expression of miR-378, an adipogenesis-related microRNA in human adipocytes.

Obesity has become a global public health problem associated with complications including type 2 diabetes, cardiovascular disease, and several cancers. Adipocyte differentiation (adipogenesis) plays an important role in obesity and energy homeostasis. Adipose tissue secretes multiple cytokines and adipokines which can cause the complications of obesity, especially insulin resistance. TNF-α, IL-6, leptin, and resistin have been identified as the main regulators of obesity and insulin activity. miR-378 is highly induced during adipogenesis and has been reported to be positively regulated in adipogenesis. In the current study, matured human adipocytes were treated with TNF-α, IL-6, leptin, or resistin on the 15th day after the induction of human pre-adipocyte differentiation. We demonstrated that TNF-α, IL-6, and leptin upregulated miR-378 expression indicating that miR-378 probably is a novel mediator in the development of insulin resistance related to obesity.

Molecular cloning, expression and functional analysis of B-cell activating factor (BAFF) in yellow grouper, Epinephelus awoara.

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation and immunoglobulin secretion. In this study, the yellow grouper (Epinephelus awoara) BAFF (designated EaBAFF) gene was cloned using RT-PCR and RACE (rapid amplification of cDNA ends) techniques. The full-length EaBAFF was 1442bp and contained an open reading frame of 780bp encoding a putative protein of 259 amino acids. Amino acids sequence comparison indicated that EaBAFF possessed the TNF signature. The soluble BAFF (EasBAFF) had been cloned into pET28a. SDS-PAGE and Western blotting analysis confirmed that the soluble fusion protein His-EasBAFF was efficiently expressed in Escherichia coli BL21 (DE3). In vitro, the WST-8 assay indicated that EasBAFF was not only able to promote the survival/proliferation of yellow grouper splenic lymphocytes but also able to promote the survival/proliferation of mouse splenic B cells. Our findings may provide valuable information for research into the immune system of E. awoara and EasBAFF may serve as a potential immunologic factor for enhancing immunological efficacy in fish.

Downregulation of miR-183 inhibits apoptosis and enhances the invasive potential of endometrial stromal cells in endometriosis.

Endometriosis is a common gynecological disease, yet its pathogenesis remains poorly understood. Recent studies have demonstrated that the aberrant expression of certain microRNAs (miRNAs) may correlate with the development and progression of endometriosis. In this study, we profiled several differentially expressed miRNAs in the normal, eutopic and ectopic endometrium by miRNA microarray screening analysis, among which, miR-183 was found to be downregulated in the ectopic and eutopic tissues, and the result was further confirmed by real-time PCR (qPCR). Functional analysis indicated that miR-183 plays a promotional role in endometrial stromal cell (ESC) apoptosis and has a negative regulatory impact on the invasive ability of cells, although it has no effect on ESC proliferation. Ovarian steroids (17ß-estradiol and progesterone) and inflammatory factors (tumor necrosis factor-α and interleukin-6) decreased the expression of miR-183 in the ESCs. This regulatory function may further manifest the growth and invasive potential of ESCs by altering the expression of miR-183. These findings suggest that the downregulation of miR-183 expression is involved in the development and progression of endometriosis.

MiR-335, an adipogenesis-related microRNA, is involved in adipose tissue inflammation.

During the development of obesity, adipose tissue releases a host of different adipokines and inflammatory cytokines, such as leptin, resistin, tumor necrosis factor α (TNF-α), Interleukin-6 (IL-6), and adiponectin, which mediate insulin resistance. Recently, some microRNAs (miRNAs) regulated by adiponectin were identified as novel targets for controlling adipose tissue inflammation. Therefore, the relationship between adipokines and miRNA is worth studying. MiR-335 is an adipogenesis-related miRNA and implicated in both fatty acid metabolism and lipogenesis. In this study, we focused on the association of miR-335 and adipokines, and examined the expression trend of miR-335 during human adipocyte differentiation. Our results showed that miR-335 is significantly upregulated with treatment of leptin, resistin, TNF-α, and IL-6 in human mature adipocytes, and its expression elevated in the process of adipocyte differentiation. Interestingly, the transcriptional regulation of miR-335 by these adipokines seems independent of its host gene (mesoderm-specific transcript homolog, MEST). Thus, we cloned and identified potential promoter of miR-335 within the intron of MEST. As a result, a fragment about 600-bp length upstream sequences of miR-335 had apparent transcription activity. These findings indicated a novel role for miR-335 in adipose tissue inflammation, and miR-335 might play an important role in the process of obesity complications via its own transcription mechanism.

Aberrant hepatic microRNA expression in nonalcoholic fatty liver disease.

BACKGROUND/AIM: Emerging evidence suggests that microRNA (miRNA) mediated gene regulation influences the maintenance of metabolic homeostasis, particularly the states of obesity and insulin resistance, thereby providing a potential link between miRNAs and nonalcoholic fatty liver disease (NAFLD). METHODS: Sprague-Dawley rats fed a high-fat diet (HFD) were used to establish a rat model of NAFLD. The miRNA expression profile of liver tissues was evaluated using Illumina HiSeq deep sequencing. Selected miRNAs were then validated by real-time PCR at both 4- and 12-week time points. Furthermore, the expression levels of these miRNAs were assessed in HepG2 cells and human hepatocytes treated with free fatty acids (FFAs) and proinflammatory factors (tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). RESULTS: Our results showed that consumption of a HFD for 4 weeks caused simple steatosis, which progressed to steatohepatitis at 12 weeks. miRNA deep sequencing analysis identified 44 known up-regulated miRNAs (fold change >1.5) and 12 down-regulated miRNAs (fold change <0.5). Among the abnormally expressed miRNAs, miR-200a, miR-200b, miR-200c, miR-146a, miR-146b and miR-152 were up-regulated both in vitro and vivo. Interestingly, the expression levels of these six miRNAs were increased in HepG2 cells and human hepatocytes after treatment with FFAs and proinflammatory factors. CONCLUSION: These findings suggest a critical role for miRNAs in the pathogenesis of NAFLD.

MicroRNA-106b induces mitochondrial dysfunction and insulin resistance in C2C12 myotubes by targeting mitofusin-2.

MicroRNA-106b (miR-106b) is reported to correlate closely with skeletal muscle insulin resistance and type 2 diabetes. The aim of this study was to identify an mRNA targeted by miR-106b which regulates skeletal muscle insulin sensitivity. MiR-106b was found to target the 3' untranslated region (3' UTR) of mitofusin-2 (Mfn2) through miR-106b binding sites and to downregulate Mfn2 protein abundance at the post-transcriptional level by luciferase activity assay combined with mutational analysis and immunoblotting. Overexpression of miR-106b resulted in mitochondrial dysfunction and insulin resistance in C2C12 myotubes. MiR-106b was increased in insulin-resistant cultured C2C12 myotubes induced by TNF-α, and accompanied by increasing Mfn2 level, miR-106b loss of function improved mitochondrial function and insulin sensitivity impaired by TNF-α in C2C12 myotubes. In addition, both overexpression and downregulation of miR-106b upregulated peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and estrogen-related receptor (ERR)-α expression. MiR-106b targeted Mfn2 and regulated skeletal muscle mitochondrial function and insulin sensitivity. Therefor, Inhibition of miR-106b may be a potential new strategy for treating insulin resistance and type 2 diabetes.

Overexpression of TFAM protects 3T3-L1 adipocytes from NYGGF4 (PID1) overexpression-induced insulin resistance and mitochondrial dysfunction.

NYGGF4, also known as phosphotyrosine interaction domain containing 1(PID1), is a recently discovered gene which is involved in obesity-related insulin resistance (IR) and mitochondrial dysfunction. We aimed to further elucidate the effects and mechanisms underlying NYGGF4-induced IR by investigating the effect of overexpressing mitochondrial transcription factor A (TFAM), which is essential for mitochondrial DNA transcription and replication, on NYGGF4-induced IR and mitochondrial abnormalities in 3T3-L1 adipocytes. Overexpression of TFAM increased the mitochondrial copy number and ATP content in both control 3T3-L1 adipocytes and NYGGF4-overexpressing adipocytes. Reactive oxygen species (ROS) production was enhanced in NYGGF4-overexpressing adipocytes and reduced in TFAM-overexpressing adipocytes; co-overexpression of TFAM significantly attenuated ROS production in NYGGF4-overexpressing adipocytes. However, overexpression of TFAM did not affect the mitochondrial transmembrane potential (ΔΨm) in control 3T3-L1 adipocytes or NYGGF4-overexpressing adipocytes. In addition, co-overexpression of TFAM-enhanced insulin-stimulated glucose uptake by increasing Glucose transporter type 4 (GLUT4) translocation to the PM in NYGGF4-overexpressing adipocytes. Overexpression of NYGGF4 significantly inhibited tyrosine phosphorylation of Insulin receptor substrate 1 (IRS-1) and serine phosphorylation of Akt, whereas overexpression of TFAM strongly induced phosphorylation of IRS-1 and Akt in NYGGF4-overexpressing adipocytes. This study demonstrates that NYGGF4 plays a role in IR by impairing mitochondrial function, and that overexpression of TFAM can restore mitochondrial function to normal levels in NYGGF4-overexpressing adipocytes via activation of the IRS-1/PI3K/Akt signaling pathway.

NYGGF4 (PID1) effects on insulin resistance are reversed by metformin in 3T3-L1 adipocytes.

NYGGF4 (also called PID1) is a recently discovered gene that is involved in obesity-related insulin resistance (IR). We aimed in the present study to further elucidate the effects of NYGGF4 on IR and the underlying mechanisms through using metformin treatment in 3T3-L1 adipocytes. Our data showed that the metformin pretreatment strikingly enhanced insulin-stimulated glucose uptake through increasing GLUT4 translocation to the PM in NYGGF4 overexpression adipocytes. NYGGF4 overexpression resulted in significant inhibition of tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, whereas incubation with metformin strongly activated IRS-1 and Akt phosphorylation in NYGGF4 overexpression adipocytes. The reactive oxygen species (ROS) levels in NYGGF4 overexpression adipocytes were strikingly enhanced, which could be decreased by the metformin pretreatment. Our data also showed that metformin increased the expressions of PGC1-α, NRF-1, and TFAM, which were reduced in the NYGGF4 overexpression adipocytes. These results suggest that NYGGF4 plays a role in IR and its effects on IR could be reversed by metformin through activating IRS-1/PI3K/Akt and AMPK-PGC1-α pathways.

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway.

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.

Knockdown of NYGGF4 increases glucose transport in C2C12 mice skeletal myocytes by activation IRS-1/PI3K/AKT insulin pathway.

NYGGF4, an obesity-related gene, is proposed to be involved in the development of insulin resistance. Skeletal muscle is a primary target organ for insulin and NYGGF4 showed a relatively high expression level in skeletal muscle. Therefore, this study aimed to explore the effect of NYGGF4 on insulin sensitivity of skeletal muscle cells. RNA interference (RNAi) was adopted to silence NYGGF4 expression in mice C2C12 skeletal myocytes. A remarkably increased insulin-stimulated glucose uptake and GLUT4 translocation was observed in NYGGF4 silencing C2C12 cells. Importantly, the enhanced glucose uptake induced by NYGGF4 silencing could be abrogated by the PI3K inhibitor LY294002. In addition, the crucial molecules involved in PI3K insulin signaling pathway were detected by western blotting. The results showed that NYGGF4 knockdown dramatically activate the insulin-stimulated phosphorylation of IRS-1 and AKT. Taken together, these data demonstrate that NYGGF4 knockdown increases glucose transport in myocytes by activation of the IRS-1/PI3K/AKT insulin pathway.

α-Lipoic acid protects 3T3-L1 adipocytes from NYGGF4 (PID1) overexpression-induced insulin resistance through increasing phosphorylation of IRS-1 and Akt.

NYGGF4 (also called PID1) was demonstrated that it may be related to the development of obesity-related IR. We aimed in the present study to further elucidate the effects of NYGGF4 on IR and the underlying mechanisms through using α-Lipoic acid (LA) treatment, which could facilitate glucose transport and utilization in fully differentiated adipocytes. Our data showed that the LA pretreatment strikingly enhanced insulin-stimulated glucose uptake through increasing GLUT4 translocation to the PM in NYGGF4 overexpression adipocytes. The reactive oxygen species (ROS) levels in NYGGF4 overexpression adipocytes were strikingly enhanced, which could be decreased by the LA pretreatment. NYGGF4 overexpression resulted in significant inhibition of tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, whereas incubation with LA strongly activated IRS-1 and Akt phosphorylation in NYGGF4 overexpression adipocytes. These results suggest that LA protects 3T3-L1 adipocytes from NYGGF4-induced IR partially through increasing phosphorylation of IRS-1 and Akt and provide evidence that NYGGF4 may be a potential target for the treatment of obesity and obesity-related IR.

Effects of Lyrm1 knockdown on mitochondrial function in 3 T3-L1 murine adipocytes.

To explore the effects of Lyrm1 knockdown on the mitochondrial function of 3 T3-L1 adipocytes using small interfering RNA (siRNA). 3 T3-L1 preadipocytes were infected with either a negative control (NC) expression lentivirus or a Lyrm1-shRNA expression lentivirus and induced to differentiate. The knockdown efficiency of Lrym1-specific shRNA in 3 T3-L1 cells was evaluated by real-time PCR. The ultrastructure of the mitochondria in adipocytes was visualized using transmission electron microscopy after differentiation. The levels of mitochondrial DNA copy numbers and Ucp2 mRNA were detected by real-time quantitative PCR. The levels of ATP production was detected using a photon-counting luminometer. The mitochondrial membrane potential and ROS levels of cells were analyzed with a FACScan flow cytometer using Cell Quest software. Cells transfected with lentiviral-Lyrm1-shRNA showed a significantly reduced transcription of Lyrm1 mRNA compared with NC cells. The size and ultrastructure of mitochondria in Lyrm1 knockdown adipocytes was similar to those of the NC cells. There was no significant difference in mtDNA copy number between the two groups. The total level of ATP production, mitochondrial membrane potential and Ucp2 mRNA expression levels were dramatically increased in adipocytes transfected with Lyrm1 RNAi. Furthermore, the level of ROS was dramatically decreased in Lyrm1 knockdown adipocytes. Knockdown of the Lyrm1 gene in adipocytes resulted in dramatically increased cellular ATP production, mitochondrial membrane potentials and levels Ucp2 mRNA, while ROS levels were significantly decreased. These results imply that mitochondrial function is improved in adipocytes after the knockdown of Lyrm1.

Differential DNA methylation status between human preadipocytes and mature adipocytes.

Obesity is a multifactorial disease resulting from interactions between susceptibility genes, psychosocial, and environmental factors. However, it is becoming evident that interindividual differences in obesity susceptibility depend also on epigenetic factors, although the mechanisms have not been fully elucidated. We have undertaken a genome-wide analysis of DNA methylation of human preadipocytes and mature adipocytes to examine the differences in methylation between them. We found hypomethylation occurring in 2,701 genes and hypermethylation in 1,070 genes after differentiation. Meanwhile, Gene Ontology analysis and Ingenuity Pathway Analysis showed many significant gene functions and pathways with altered methylation status after adipocyte differentiation. In addition, Signal-Net analysis showed that tumor necrosis factor-α, mitogen-activated protein kinase, and interleukin-8 were important to the formation of this network. Our results suggest that DNA methylation mechanisms may be involved in regulating the differentiation process of human preadipocytes.