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.

A novel peptide RIFV suppresses human adipocyte differentiation through the inhibition of C/EBP-ß expression.

BACKGROUND: Obesity is a global epidemic disease that increases the risk of metabolic syndrome. However, therapeutic drugs for obesity are still scarce. In recent years, peptides have been identified as new biological regulators. RIFV (R-I-F-V-P-I-K-G-R-P-A-P), a novel active peptide from our peptide database. METHODS: We performed oil red O staining and triglyceride measurement to analyze the influence of RIFV on white preadipocytes differentiation. Then the effects of RIFV on cell proliferation, apoptosis and cell cycle were determined by using CCK-8 assay and flow cytometry. The mRNA and protein levels of adipogenesis-related genes were respectively detected by qRT-PCR and western blot. Rescue experiment was conducted to confirm whether RIFV could regulate adipocytes differentiation via targeting C/EBP-ß. Finally, the luciferase reporter gene assay was performed to verify the regulation of RIFV on C/EBP-ß gene. RESULTS: RIFV was revealed to inhibit the differentiation of human white adipocytes without affecting their proliferation. Additionally, RIFV could also suppress the differentiation of mouse primary white preadipocytes isolated from inguinal fat tissues. Furthermore, RIFV may have an inhibitory effect on adipogenesis by inhibiting the regulation of the adipogenic gene C/EBP-ß. CONCLUSIONS: Our results indicated that RIFV may be a novel essential regulator of adipocyte differentiation and represents a therapeutic strategy for obesity and related complications.

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.

Transcriptome analysis reveals the potential contribution of long noncoding RNAs to brown adipocyte differentiation.

Brown adipose tissue (BAT) functions to dissipate energy in response to cold exposure or overfeeding. Counteracting obesity has been extensively considered as a promising target. Long noncoding RNAs (lncRNAs) are an important class of pervasive genes involved in a variety of biological functions. However, the potential biological functions of lncRNAs during mouse brown fat cell differentiation have not been fully understood. Here, we performed lncRNA and mRNA expression profile analysis using microarray technology and identified 1064 lncRNAs with differential expression (fold change| ≥4, p ≤ 0.01) on day 0 and day 8 during differentiation. Furthermore, candidate lncRNAs were characterized by comprehensive examination of their genomic context, gene ontology (GO) enrichment of their associated protein-coding genes and pathway analysis. We identified three lncRNAs (Gm15051, Tmem189 and Cebpd) associated with their flanking coding genes (Hoxa1, C/EBPß and C/EBPδ), which participated in adipose commitment. Collectively, our findings indicated lncRNAs are involved in mouse BAT development and provide potential targets for obesity therapy.

The biological effects of hsa-miR-1908 in human adipocytes.

MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of gene expression. MiR-1908 is a recently identified miRNA that is highly expressed in human adipocytes. However, it is not known what role of miR-1908 is involved in the regulation of human adipocytes. In this study, we demonstrate that the level of miR-1908 increases during the adipogenesis of human multipotent adipose-derived stem (hMADS) cells and human preadipocytes-visceral. Overexpression of miR-1908 in hMADS cells inhibited adipogenic differentiation and increased cell proliferation, suggesting that miR-1908 is involved in the regulation of adipocyte cell differentiation and metabolism, and, thus, may have an effect on human obesity.

MiR-146b is a regulator of human visceral preadipocyte proliferation and differentiation and its expression is altered in human obesity.

Visceral obesity is an independent risk factor for metabolic syndrome, and abnormal fat accumulation is linked to increases in the number and size of adipocytes. MiR-146b was a miRNA highly expressed in mature adipocytes while very lowly expressed in human mesenchymal stem cells (hMSCs) and human visceral preadipocytes (vHPA). In this paper, we mainly focused on the roles of miR-146b in adipogenesis. We found miR-146b could inhibit the proliferation of visceral preadipocytes and promote their differentiation. MiR-146b in human visceral adipocytes inhibited the expression of KLF7, a member of the Kruppel-like transcription factors, as demonstrated by a firefly luciferase reporter assay, indicating that KLF7 is a direct target of the endogenous miR-146b. MiR-146b expression was significantly altered in visceral and subcutaneous adipose tissues in human overweight and obese subjects, and in the epididymal fat tissues and brown fat tissues of diet-induced obese mice. Our data indicates that miR-146b may be a new therapeutic target against human visceral obesity and metabolic dysfunction.

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.

Measurement of fetal abdominal and subscapular subcutaneous tissue thickness during pregnancy to predict macrosomia: a pilot study.

This study assessed the growth trends and reference ranges of the ultrasound parameters, fetal abdominal subcutaneous tissue thickness (ASTT) and subscapular subcutaneous tissue thickness (SSTT), in the last two trimesters of normal pregnancy in a Chinese population. We recruited 744 healthy women with singleton pregnancies. The ASTT and SSTT were evaluated at different times between 21 and 36 weeks of gestation. The correlations between these parameters and fetal gestational weeks were assessed using linear regression analysis. Both ASTT and SSTT increased with gestation, and both parameters showed a strong correlation with gestation (ASTT vs. GA, R(2) = 0.792; P<0.0001; SSTT vs. GA, R(2) = 0.302; P<0.0001). Time-specific reference ranges, including 5th, 50th and 95th percentiles and means ± SD, were constructed for ASTT and SSTT. These results provide a preliminary reference range to evaluate whether fetal development and maternal metabolic health is normal or not in a Chinese population.

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.

Characterization of microRNA expression profiles in 3T3-L1 adipocytes overexpressing C10orf116.

Our data in the previous report demonstrated that C10orf116 (AFRO) is an adipocyte lineage-specific nuclear factor that can modulate the master adipogenesis transcription factors early during differentiation. However, more precise functional properties of this gene need to be clarified and await further investigation. Therefore, in this study, we performed an expression profile of cellular MicroRNAs (miRNAs) in the C10orf116 overexpression 3T3-L1 adipocytes and performed target prediction and functional enrichment of the differentially expressed miRNAs. Our study identified 34 miRNAs up-regulated in the 3T3-L1 adipocytes stably overexpressing C10orf116, whereas 43 miRNAs up-regulated in the control cells. The target genes of differentially expressed miRNAs were found to be involved in multiple signalling pathways, such as Wnt, TGF-beta, MAPK, Jak-STAT, insulin signalling pathway, et al. Our data provided novel information for the identification of C10orf116.

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.

Peptidome analysis of human skim milk in term and preterm milk.

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. Due to the difficulties associated with detection technology of the peptides, the expression of the peptides present in human milk is not known widely. In recent years, peptidome analysis has received increasing attention. In this report, the analysis of endogenous peptides in human milk was done by mass spectrometry. A method was also developed by our researchers, which can be used in the extraction of peptide from human milk. Analysis of the extracts by LC-MS/MS resulted in the detection of 1000-3000Da peptide-like features. Out of these, 419 peptides were identified by MS/MS. The identified peptides were found to originate from 34 proteins, of which several have been reported. Analysis of the peptides' cleavage sites showed that the peptides are cleaved with regulations. This may reflect the protease activity and distribution in human body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery. Isotope dimethyl labeling analysis was also used to test the effects of premature delivery on milk protein composition in this study. Differences in peptides expression between breast milk in term milk (38-41weeks gestation) and preterm milk (28-32weeks gestation) were investigated in this study. 41 Peptides in these two groups were found expressed differently. 23 Peptides were present at higher levels in preterm milk, and 18 were present at higher levels in term milk.