Search

FAM129B is one of Niban-like proteins described in neoplastic cells and implicated in melanoma cell invasion, but no reports have been published on FAM129B and cell differentiation. We show that FAM129B is early and transiently expressed and crucial for 3T3-F442A adipogenesis. Fam129b is expressed downstream of the early genes Cebpb, Klf4, Klf5 and Srebf1a, but upstream of Pparg2 since knockdown of Fam129b blocked Pparg2 expression and adipose differentiation. Glycogen synthase kinase 3 beta activity, a crucial kinase for adipogenesis, and the ERK1/2 are involved in FAM129B phosphorylation as part of the adipogenic program. Phosphorylated FAM129B is crucial for Pparg2 expression and the lipogenic gene expression downstream of Pparg2, and hence for adipogenesis. Fam129b knockdown reduced adipocyte cluster formation and size, regulating commitment and clonal amplification. In vivo, BAT, inguinal and epidydimal fat expressed Fam129b, suggesting a role in adipose tissue development. We conclude that FAM129B is a cooperative protein that regulates differentiation during the early stages of adipogenesis.

3-hydroxymorphinan (3-HM), a metabolite of dextromethorphan, has previously been reported to have anti-inflammatory, anti-oxidative stress, and neuroprotective effects. However, its effect on energy metabolism in adipocytes remains unclear. Herein, we investigated 3-hydroxymorphinan (3-HM) effects on mitochondrial biogenesis, oxidative stress, and lipid accumulation in 3T3-L1 adipocytes. Further, we explored 3-HM-associated molecular mechanisms. Mouse adipocyte 3T3-L1 cells were treated with 3-HM, and various protein expression levels were determined by western blotting analysis. Mitochondria accumulation and lipid accumulation were measured by staining methods. Cell toxicity was assessed by cell viability assay. We found that treatment of 3T3-L1 adipocytes with 3-HM increased expression of brown adipocyte markers, such as uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α). 3-HM promotes mitochondrial biogenesis and its-mediated gene expression. Additionally, 3-HM treatment suppressed mitochondrial ROS generation and superoxide along with improved mitochondrial complex I activity. We found that treatment of 3-HM enhanced AMPK phosphorylation. siRNA-mediated suppression of AMPK reversed all these changes in 3T3-L1 adipocytes. In sum, 3-HM promotes mitochondrial biogenesis and browning and attenuates oxidative stress and lipid accumulation in 3T3-L1 adipocytes via AMPK signaling. Thus, 3-HM-mediated AMPK activation can be considered a therapeutic approach for treating obesity and related diseases.

Artemisinin derivatives could inhibit adipogenic differentiation of 3T3-L1 preadipocytes and prevent obesity in mice. However, the molecular mechanism remains largely unclear. Our research was designed to investigate the specific molecular target of artemisinin derivatives in adipogenic differentiation of 3T3-L1 preadipocytes. Here, we revealed that in response to dihydroartemisinin (DHA) or artesunate (ATS), intracellular lipid was decreased in a concentration dependent manner as shown by BODIPY staining. Quantitative PCR analysis showed that expression of Cebpa, Pparg, Fabp4 and Plin was significantly decreased by DHA treatment in a concentration and time dependent manner. Also, DHA treatment remarkably downregulated expression of CCAAT/enhancer-binding protein α (C/EBPα) and nuclear receptor peroxisome proliferation-activated receptor γ (PPARγ) of adipogenic induced 3T3-L1 cells as assayed by western blotting. RNA-seq analysis identified thousands of differential expression genes (DEGs), among which CHOP expression was significantly improved in DHA treated cells. Upregulation of CHOP was verified by quantitative PCR and western blotting, respectively. Knockdown of CHOP by the specific shRNA revealed that the inhibition of adipogenesis by DHA was strongly blocked, resulting in restored lipid accumulation and expression of adipogenic molecules. In conclusions, the inhibitory effect of DHA on adipogenic differentiation of 3T3-L1 preadipocytes was exerted in a concentration and time dependent manner, which was mediated by expression of CHOP.

Adipose tissue, a key regulator of systemic energy homeostasis, can synthesize and store triglycerides to meet long-term energy demands. In response to nutrient overload, adipose tissue expands by hypertrophy or hyperplasia. As an oncogene, MDM2 has exerted diverse biological activities including human development, tissue regeneration, and inflammation, in addition to major oncogenic activities. Recently, some studies indicated that MDM2 plays an important role in adipose tissue function. However, the role of MX69, a MDM2 inhibitor, in adipose tissue function has not been fully elucidated. Here, we administered MX69 intraperitoneally to high-fat diet-induced obesity (DIO) wild type C57BL/6 mice and found that MX69 could promote the body weight and white adipose tissue weight of DIO mice. Moreover, MX69 had no effects on glucose tolerance and insulin sensitivity in DIO mice. And MX69 treatment decreased the size of adipocytes and fat deposition in adipose tissue and inhibited 3T3-L1 preadipocytes differentiation. Mechanistically, MX69 inhibited the protein levels of MDM2 and the mRNA levels of genes related to adipogenesis and differentiation. In summary, our results indicated that MDM2 has a crucial and complex role in regulating adipose tissue function.

We previously demonstrated that kaempferol, a flavonoid present in various herbs, inhibits adipogenesis by repressing peroxisome proliferator-activated receptor γ (PPARγ) activity. Here, we focused on elucidation of the underlying mechanism using genome-wide tools. First, RNA sequencing (RNA-seq) analysis showed downregulation of genes involved in adipogenesis in response to kaempferol. Subsequent ChIP assays revealed that kaempferol regulates the expression of adipogenic (Adipoq, Fabp4, Lpl) genes by modulating enrichment of active H3K4me3 and repressive H3K27me3 histone codes on target promoters. Second, we performed ChIP sequencing analysis of active H3K4me3, and co-analysis with RNA-seq identified PPARγ responsive sites in genes downregulated by kaempferol, in terms of expression and H3K4me3 deposition. Third, direct kaempferol binding to PPARγ, for which the K

In the current work we have investigated the cellular and molecular regulation of resistin secretion in cultured and primary mouse adipocytes. Resistin is an adipose tissue hormone proposed to contribute to metabolic disease. In rodents, resistin is secreted from white adipocytes whereas it is in humans synthesised and released from other cell types within white adipose tissue. The metabolic importance of resistin has been studied in both mouse and man, but the regulation of its release remains poorly investigated. Here we define that, in mouse adipocytes, resistin secretion is triggered by an intracellular elevation of cAMP and/or Ca2+. Resistin release is stimulated via activation of beta 3 adrenergic receptors (β3ARs) and the downstream signalling protein exchange protein activated by cAMP (Epac). The secretion of resistin is markedly abrogated in adipocytes isolated from obese and diabetic mice. Immunocytochemical staining demonstrates a significant overlap between signals for resistin and the adipocyte hormone adiponectin. Our data propose that resistin and adiponectin are contained within the same vesicles in mouse adipocytes and that the two hormones are co-secreted in response to the same exocytosis-triggering signals.

Skeletal muscle and white adipose tissue are important organs of glucose-lipid metabolism. However, excessive lipolysis and free fatty acids (FFA) release in adipocytes elevate plasma FFA, leading to insulin resistance in skeletal muscle. Here, we investigated effects of insulin-resistant adipocytes on skeletal muscle in vitro by simulating body environment using a transwell coculture method. Insulin-resistant 3T3-L1 adipocytes increased lipolysis and FFA release, which reduced insulin sensitivity in the cocultured C2C12 myotubes. Rosiglitazone (RSG) decreased excessive lipolysis by reducing expression of adipose triglyceride lipase (ATGL) and activity of hormone-sensitive lipase (HSL), which led to decrease of FFA release from insulin-resistant 3T3-L1 adipocytes. Meanwhile, insulin resistance in C2C12 myotubes cocultured with insulin-resistant 3T3-L1 adipocytes was ameliorated after RSG treatment. Taken together, our present study provided direct evidence to better understand insulin resistance between skeletal muscle and adipose tissue in type 2 diabetes.

Substantial evidence has linked dehydroepiandrosterone (DHEA) levels to the anti-obesity and anti-diabetic effects of exercise. While 5'-adenosine monophosphate-activated protein kinase (AMPK) is a negative regulator of adipocyte differentiation and lipid accumulation, activation of mammalian target of rapamycin complex 1 (mTORC1), which is inhibited by AMPK, is required for adipocyte differentiation and positively regulates lipid accumulation. DHEA treatment activates the AMPK pathway in C2C12 myotubes. Hence, DHEA addition to preadipocytes and adipocytes might activate AMPK and inhibit mTORC1, resulting in the inhibition of adipogenesis and lipid accumulation. Therefore, we investigated the effect of DHEA on the AMPK pathway, mTORC1 activity, adipocyte differentiation, and lipid accumulation in 3T3-L1 cells. DHEA suppressed lipid accumulation and adipogenic marker expression during differentiation. It also activated AMPK signaling in preadipocytes and adipocytes and suppressed mTORC1 activity during differentiation. These results suggest that the activation of the AMPK pathway and inhibition of mTORC1 activity may mediate the anti-obesity effect of DHEA, providing novel molecular-level insights into its physiological functions.

Farnesoid X receptor (FXR), a bile acid receptor, is known to be involved in the promotion of adipogenesis. However, the regulation mechanism of FXR-promoted adipogenesis remains unclear. In this study, we investigated the regulation mechanism of FXR-mediated activation of adipogenesis in murine adipocyte 3T3-L1 cells. Chenodeoxycholic acid (CDCA), a potent FXR agonist, enhanced the accumulation of intracellular triglycerides and the expression of the adipogenic and lipogenic genes, while guggulsterone, an FXR antagonist, suppressed CDCA-activated adipogenesis. Moreover, troglitazone, a peroxisome proliferator-activated receptor (PPAR) γ agonist, elevated the expression of the FXR gene during adipogenesis, similar to that of the PPARγ gene. Chromatin immunoprecipitation assay demonstrated that PPARγ bound to the PPAR-responsive element of the FXR gene in a PPARγ agonist-dependent manner. Furthermore, FXR activation induced the expression of the stearoyl-CoA desaturase (SCD) gene in adipocytes. The FXR-response element (FXRE) was found in the SCD gene promoter, and FXR bound to the FXRE of the SCD gene promoter, and its binding efficiency was enhanced by CDCA in adipocytes. These results indicate that FXR assisted lipogenesis through the enhanced expression of SCD in a PPARγ-dependent manner in adipocytes, making this study the first to identify the role of FXR in the promotion of lipogenesis by PPARγ activation.

A decline in intracellular nicotinamide adenine mononucleotide (NAD+) causes adipose tissue dysfunction. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in the NAD+ biosynthesis pathway. However, the molecular mechanism that mediates regulation of NAMPT expression in adipocytes is yet to be elucidated. This study found that intracellular cAMP regulates NAMPT expression and promoter activity in 3T3-L1 adipocytes. cAMP-mediated Nampt promoter activity was suppressed by protein kinase A inhibitor H89, whereas AMP-activated protein kinase inhibitor compound C did not affect cAMP-mediated Nampt promoter activity. Intracellular cAMP induced CCAAT/enhancer-binding protein β (C/EBPβ) expression. Knockdown of C/EBPβ suppressed NAMPT expression and promoter activity. Furthermore, the Nampt promoter was activated by C/EBPβ, while LIP activated the dominant-negative form of C/EBPβ. Promoter sequence analysis revealed that the region from -96 to -76 on Nampt was required for C/EBPβ-mediated promoter activity. Additionally, chromatin immunoprecipitation assay demonstrated that C/EBPβ was bound to the promoter sequences of Nampt. Finally, NAMPT inhibitor FK866 suppressed adipogenesis in 3T3-L1 cells, and this suppressive effect was restored by nicotinamide mononucleotide treatment. These findings showed that intracellular cAMP increased NAMPT levels by induction of C/EBPβ expression and indicated that the induction of NAMPT expression was important for adipogenesis., Article, Biochemical and Biophysical Research Communications. 522(3): 770-775. (2020)

CeRNA effect was an important regulation mode of miRNA mediated bio-activities, however, most of the researches of ceRNA were on ncRNAs synergetic with mRNAs, the exploration of ceRNA effect regulated mRNA interaction was still lack of. Besides, C/EBPα was one of the most crucial adipogenic regulators, which has been demonstrated to form a protein complex with FOXO1 to mediate AdipoQ expression. So that, we try to explore whether the ceRNA effect mediated the interaction of C/EBPα and FOXO1, and identified the key miRNAs of their ceRNA effect. In this paper, we found the ceRNA effect of C/EBPα and FOXO1 mediated their protein complex formation, furthermore regulated its transcriptional role for AdipoQ, thereby influencing pre-adipocytes adipogenesis. More importantly, we demonstrated that the miR-144 was the decisive factor that mediated the ceRNA effect of C/EBPα and FOXO1 to influence AdipoQ, thus regulated pre-adipocytes adipogenesis. This research will provide a new supplementary idea of the miRNA role in mediating coding RNA interaction that regulates pre-adipocyte adipogenesis.

Luffa cylindrica stem sap (LuCS) has been ethnopharmacologically used as a cosmetic ingredients to improve the facial condition in Asians, but there is no scientific proof about the advantages of LuCS as a supplement for skin elasticity inducer.Presently, we have validated the beneficial effect of LuCS in human preadipocyte and fibroblast.In vitro activities of LuCS on expression of cellular elastin and collagen type I were validated using Western blot analysis in human fibroblasts. Effect of LuCS on preadipocyte development was performed using MDI medium containing isobutyl-methylxanthine, dexamethasone, and insulin and then evaluated using oil red O staining.Treatment of LuCS stimulated the expression of cellular elastin and type I procollagen in human skin fibroblasts. Exposure to LuCS induced lipid accumulation of preadipocytes via activation of CEBP/α signaling pathway in preadipocytes. Expression of collagen I, elastin, or CEBP/α mRNA was decreased by age. 3-bromo-3-methylisoxazol-5-amine enhanced the synthesis of cellular lipid in preadipocytes.Collectively, these results suggest the rationale of LuCS treatment in enhancing the skin condition.

Long non-coding RNAs (lncRNAs) have gained extensive attentions due to their significant roles in diverse biological process. However, the potential functions of lncRNAs participation in adipocyte differentiation have not been fully explored. In the present study, we globally profiled lncRNA expression using lncRNA microarray and identified 1745 lncRNA probes with differential expression on day 0 and day 4 post-induction in both C3H10T1/2 mesenchymal stem cells and 3T3-L1 preadipocytes. Furthermore, we showed that stable shRNA knockdown (KD) of NR_015556, a novel lncRNA that was significantly down-regulated in adipocyte differentiation, promoted adipocyte differentiation by increasing the number of lipid droplets and adipocyte markers such as Fabp4, Adipsin and Fasn. Mechanistically, NR_015556 KD attenuated the expression of Wnt signaling components Wnt10b and non-phospho (active) β-catenin, and elevated adipocyte master factors Ppar-γ and C/EBPα levels. Conversely, pharmacological activation of Wnt10b-β-catenin signaling by LiCl suppressed NR_015556 KD-induced enhancement of adipocyte differentiation and Ppar-γ and C/EBPα expression levels. Taken together, these results indicate that down-regulation of NR_015556 promotes adipocyte differentiation through inhibiting Wnt10b-β-catenin signaling pathway and then elevating Ppar-γ and C/EBPα triggered transcriptional cascades.

Glutamine is the most abundant amino acid in the body, and adipose tissue is one of the glutamine-producing organs. Glutamine has important and unique metabolic functions; however, its effects in adipocytes are still unclear. 3T3-L1 adipocytes produced and secreted glutamine dependent on glutamine synthetase, but preadipocytes did not. The inhibition of glutamine synthetase by methionine sulfoximine (MSO) impaired the differentiation of preadipocytes to mature adipocytes, and this inhibitory effect of MSO was rescued by exogenous glutamine supplementation. Glutamine concentrations were low, and Atgl gene expression was high in epididymal white adipose tissues of fasting mice in vivo. In 3T3-L1 adipocytes, glutamine deprivation induced Atgl expression and increased glycerol concentration in culture medium. Atgl expression is regulated by FoxO1, and glutamine deprivation reduced FoxO1 phosphorylation (Ser256), indicating the activation of FoxO1. These results demonstrate that glutamine is necessary for the differentiation of preadipocytes and regulates lipolysis through FoxO1 in mature adipocytes.

The ovariectomy would induce the occurrence of obesity, but its regulatory mechanism is not clear. This study aimed to elucidate the regulation on fat accumulation for ovariectomy in mouse. In the current study, the abdominal fat mass dramatically increased in OVX mice compared with sham mice at eighth week after ovariectomy, accompanied with the higher GnRH level in blood and abdominal fat tissue. Also, a decrease of the abdominal fat mass was occurred in OVX mice with a GnRH-antagonist injection. Furthermore, the results in vivo and in vitro confirmed that GnRH promoted the transition of G1/S phase by upregulating CCND1 and CCNE1 mRNA levels by the mediation of GnRHR via the PKA-CREB pathway. Meanwhile, the higher FSH secretion was induced by increase GnRH and accelerate fat deposition in abdominal fat tissue. Our findings are the first to elucidate the effect mechanism of ovariectomy on obesity in mouse. GnRH stimulates fat accumulation in adipocytes via PKA-CREB pathway by directly promoting cell proliferation for driving the cell cycle and simultaneously accelerating differentiation for improving the FSH secretion.

There is growing evidence in support of an independent association between obstructive sleep apnea (OSA) and type 2 diabetes, and in which hypoxia may play an important role. Hypoxia is the hallmark feature and the most important pathophysiologic pathway of OSA. Recently, receptor for advanced glycation end products (RAGE) was found to be involved in the pathogenesis of insulin resistance (IR). However, whether RAGE contributes to the IR of adipocytes under hypoxia remains unknown. In the present study, we found that hypoxia reduced glucose consumption and upregulated RAGE expression in 3T3-L1 adipocytes in a time-dependent manner. RAGE knockdown efficiently attenuated hypoxia-induced IR, including inhibiting serine phosphorylation of insulin receptor substrate-1 (IRS-1), increasing the expression of phosphorylated Akt (Ser473), and improving insulin-stimulated glucose uptake. In addition, hypoxia activated nuclear transcription factor κB (NF-κB). However, RAGE knockdown inhibited hypoxia-induced NF-κB activity in adipocytes. Finally, an NF-κB inhibitor (PDTC) significantly reduced the hypoxia-induced upregulation of RAGE expression and IR. Therefore, this study indicates that the RAGE/NF-κB pathway mediates hypoxia-induced IR in 3T3-L1 adipocytes, and suggests that RAGE may be a potential therapeutic target for suppressing IR in OSA.

Obesity that is critically correlated with the initiation and development of metabolic syndrome and cardiovascular diseases has increased worldwide. Adipogenesis is coordinated through multi-steps involving adipogenic commitment, mitotic clonal expansion (MCE) and differentiation. Recently, protein arginine methyltransferase 4 (PRMT4) and PRMT5 have been implicated in modulation of adipogenesis via regulation of C/EBP-β activity or PPAR-γ2 expression. In the current study, we demonstrate a suppressive role of PRMT7 in adipogenesis. PRMT7-depleted preadipocytes or PRMT7-/- mouse embryonic fibroblasts (MEFs) displayed increased adipogenesis while PRMT7 overexpression attenuated it. PRMT7 depletion in preadipocytes promoted MCE, an initial step of adipogenesis. Furthermore, we found that PRMT7 interacted with and methylated a key adipogenic factor C/EBP-β upon adipogenic induction and modulated the accumulation of C/EBP-β at its target sites in the PPAR-γ2 promoter. Taken together, our data suggest that PRMT7 suppresses adipogenesis through modulation of C/EBP-β activity.

Wnt3a is established as an important regulator of various developmental processes, especially in osteogenesis, adipogenesis and mitochondrial biogenesis. Numerous studies reported Wnt3a regulates osteogenesis and adipogenesis, but the mechanisms by which Wnt3a regulates mitochondrial biogenesis are not well understood. In this study, results suggested that Wnt3a stimulates mitochondrial biogenesis by increasing the expression of mitochondrial biogenesis genes and regulators, as well as mitochondrial copy number in adipocytes. As a key mediator of canonical Wnt/β-catenin pathway, β-catenin knockdown had no effect on basal or Wnt3a-mediated mitochondrial biogenesis in adipocytes, which suggested that Wnt3a-mediated mitochondrial biogenesis was independent of β-catenin-dependent canonical Wnt/β-catenin pathway. However, Wnt3a inhibited p38/CREB (p38 mitogen-activated protein kinase/cAMP response element-binding protein) signaling activation and p38 inhibitor impaired Wnt3a-stimulated mitochondrial biogenesis, indicating p38/CREB pathway could be involved in the regulation of Wnt3a-mediated mitochondrial biogenesis in adipocytes. In conclusion, our data showed that Wnt3a stimulates mitochondrial biogenesis in adipocytes, which is at least partially through activation of p38/CREB pathway.

Adipogenesis is an organized process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes. miR-16–5p has been reported to be involved in cell proliferation, apoptosis, differentiation and angiogenesis. However little is known about miR-16–5p functional role in 3T3-L1 adipocyte differentiation. In this study, we found that miRNA-16–5p was significantly upregulated during 3T3-L1 preadipocytes differentiation towards mature adipocytes. Over-expression of miRNA-16–5p promoted mature adipocytes specific genes expression and fat droplet accumulation in vitro and in vivo. Meanwhile we have identified EPT1 as the target gene of miRNA-16–5p. Taken together, our data provided evidence to support that miRNA-16–5p promotes adipocyte differentiation by suppressing EPT1.

Adipogenesis, which directly control body fat mass, plays a crucial role in lipid metabolism and obesity-related diseases. Hedgehog interacting protein (Hhip) belongs to Hedgehog (Hh) signaling pathway. The Hh signaling pathway was already linked with adipogenesis in previous reports, however, the physiological functions of Hhip on lipid deposition are still poorly understood. In this study, the level of Hhip was down-regulated during the development of porcine adipose tissues. Recombinant Hedgehog interacting protein (rHhip) could down-regulate cell cycle related genes and cell numbers in S phage to inhibit cell proliferation. Moreover, rHhip could increase adipocytes differentiation by targeting canonical Hh signaling, indicated by the increase of lipid accumulation and up-regulation of Glut4 and PPARγ expression. Collectively, these findings illustrated the essential role of Hhip in the proliferation and differentiation of adipocytes, and provided a potential novel target for preventing obesity.

Aims/introduction Caloric restriction (CR) promotes longevity and exerts anti-aging effects by increasing Sirtuin production and activation. Gastric inhibitory polypeptide (GIP), a gastrointestinal peptide hormone, exerts various effects on pancreatic β-cells and extra-pancreatic tissues. GIP promotes glucose-dependent augmentation of insulin secretion and uptake of nutrients into the adipose tissue. Materials and methods Gipr-/- and Gipr+/+ mice were used for lifespan analysis, behavior experiments and gene expression of adipose tissue and muscles. 3T3-L1 differentiated adipocytes were used for Sirt1 and Nampt expression followed by treatment with GIP and α-lipoic acid. Results We observed that GIP receptor-knockout (Gipr-/-) mice fed normal diet showed an extended lifespan, increased exploratory and decreased anxiety-based behaviors, which are characteristic behavioral changes under CR. Moreover, Gipr-/- mice showed increased Sirt1 and Nampt expression in the adipose tissue. GIP suppressed α-lipoic acid-induced Sirt1 expression and activity in differentiated adipocytes. Conclusions Although maintenance of CR is difficult, food intake and muscle endurance of Gipr-/- mice were similar to those of wild-type mice. Inhibition of GIP signaling may be a novel strategy to extend the lifespan of diabetic patients.

Recently, there is a rapid increase in the incidence of obesity, a condition for which there are no effective therapeutic agents. Capmatinib (CAP), a novel mesenchymal-to-epithelial transition inhibitor, is reported to attenuate pro-inflammatory mediators and oxidative stress. In this study, the effects of CAP on lipogenesis in the adipocytes were examined. Treatment with CAP dose-dependently suppressed lipid accumulation in, and differentiation of, and increased lipolysis in, 3T3-L1 adipocytes. Additionally, CAP treatment augmented adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and FNDC5 expression in the adipocytes. Transfection with si-AMPK or si-FNDC5 mitigated the CAP-induced suppression of lipogenesis and enhanced lipolysis. Furthermore, transfection with si-FNDC5 mitigated the CAP-induced phosphorylation of AMPK. These results suggest that the anti-obesity effect of CAP is mediated through the irisin/AMPK pathway and that CAP is a novel therapeutic agent for obesity.

In the past decades, remarkable efforts have been made to unravel the regulation of adipose tissue metabolism, given the increasing prevalence of obesity and its huge impact on human health. Wnt signaling pathway is closely involved in this entity. As extracellular inhibitors to Wnt signaling, secreted protein Dickkopfs (Dkks) may be potential targets to combat obesity and related metabolic disorders. In this study, we showed that Dkk2 was a beige fat-enriched adipokine to regulate adipogenesis. Dkk2 was strikingly expressed in beige fat depot compared to classic white, brown, and subcutaneous fat. Dkk2 treatment inhibited adipogenesis in 3T3-L1 pre-adipocytes, C3H10T1/2 mesenchymal stem cells, and primary bone marrow mesenchymal stromal cells. Activation of the master adipogenic factor PPARγ by the synthetic Thiazolidinedione ligand rosiglitazone largely rescued the inhibition of adipogenesis by Dkk2. Furthermore, adenoviral overexpression of Dkk2 in the liver to mimic its gain-of-function showed minimal effect on whole-body metabolism. These results collectively suggest that Dkk2 is a first-in-class beige fat adipokine and functions mainly through a paracrine manner to inhibit adipogenesis rather than as an endocrine factor. Our findings aid a better understanding of beige fat function and regulation and further, provide a potential therapeutic target for treating obesity.

SPOCK1 is a calcium-binding matricellular proteoglycan that has been extensively studied in several cancer cells. Previously, we generated a mouse line overexpressing SPOCK1 (Spock1-Tg mouse) and showed that SPOCK1 might play an important role in drug-induced gingival overgrowth, indicating that it possesses physiological functions in non-cancer diseases as well. Although SPOCK1 was reported to be secreted from human adipocytes, its role in adipocyte physiology has not been addressed yet. In this study, SPOCK1 protein expression was confirmed in pancreas, adipose tissues, spleen, and liver of normal diet (ND)-fed mice. Interestingly, SPOCK1 was up-regulated in the pancreas and adipose tissues of the high-fat diet (HFD)-fed mice. Spock1-Tg mice fed with ND showed increased maturation in epididymal and inguinal adipose tissues. In addition, Spock1 overexpression strongly decreased expression of UCP-1 in adipose tissues, suggesting that SPOCK1 might regulate thermogenic function through suppression of UCP-1 expression. Finally, exogenous SPOCK1 treatment directly accelerated the differentiation of 3T3-L1 adipocytes, accompanied by the up-regulation of adipocyte differentiation-related gene expression. In conclusion, we demonstrated for the first time that SPOCK1 induced adipocyte differentiation via the up-regulation of adipogenesis-related genes.

Obesity causes the development of insulin resistance and type 2 diabetes. Phosphatidylcholine (PPC) has been reported to increase hepatic insulin sensitivity and lipolysis in adipose tissue to resolve local obesity. In this study, we proposed 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), the main active species of PPC, as an effective substance for the treatment of obesity-mediated disorders such as impaired fat metabolism and insulin resistance. Therefore, we investigated the potential lipolytic effects of DLPC on adipocytes and insulin signaling in muscle cells. In this study, DLPC-treated 3T3-L1 adipocytes showed enhanced tumor necrosis factor α (TNF-α) release. Suppression of TNF-α by short interfering RNA (siRNA) mitigated DLPC-induced lipolysis and apoptosis. DLPC treatment increased peroxisome proliferator-activated receptor α (PPARα) expression levels in C2C12 myocytes. siRNA-mediated suppression of PPARα abrogated the suppressive effects of DLPC on palmitate-induced inflammation and insulin resistance. In conclusion, DLPC enhanced lipolysis and apoptosis via a TNFα-dependent pathway in adipocytes and attenuated palmitate-induced insulin resistance through PPARα-mediated suppression of inflammation in myocytes.

Obesity is associated with metabolic disorders. Fibroblast growth factor 21 (FGF21) has been recognized as important in metabolism. Glucosamine (GLN) has been demonstrated to perform diverse beneficial functions. This study aimed to reveal whether and how GLN would modulate FGF21 production in relation to metabolism. With in vivo model of normal diet (ND) and high-fat diet (HFD) mice receiving GLN injection and in vitro model of mouse AML12 liver cells and differentiated 3T3L1 adipocytes challenged with GLN, GLN appeared to improve the glucose metabolism in HFD and ND mice and to elevate FGF21 protein expression in HFD liver and to increase both FGF21 protein and mRNA levels in WAT from HFD and ND mice and it also upregulated FGF21 expression in both AML12 and differentiated 3T3L1 cells. By using inhibitors against various signaling pathways, p38, Akt, NF-κB, and PKA appeared potentially involved in GLN-mediated FGF21 production in AML12 cells; GLN was able to mediate activation of NF-κB, p38 or PKA/CREB signaling. Our accumulated findings suggest that GLN may potentially improve the metabolic performance by inducing FGF21 production in liver and adipose tissues and such induction in liver cells may act in part due to GLN induction of the NF-κB, p38 and PKA pathways.

Obesity is a disease characterized by chronic inflammation and increases the risk of comorbidities such as type 2 diabetes mellitus, non-alcoholic fatty liver disease and cardiovascular dysfunction. In obese individuals, elevated levels of plasma circulating saturated fatty acids (e.g. palmitate) contribute to subclinical chronic inflammation. Genetic variants play a major role in determining susceptibility or resistance to obesity. Some recently described variant in genetically Lean mice that leads to upregulated expression selectively in adipose tissue of the nuclear encoded mitochondrial gene thiosulfate sulfurtransferase, Tst, has been causally linked to reduced adiposity and insulin sensitisation. Here we show that macrophages derived from the high-TST expressing Lean line of mice have a reduced inflammatory response to TLR-ligand activation than those from the low-TST expressing Fat line of mice. Furthermore, using the mouse clonal 3T3-L1 adipocytes we describe a positive effect of TST upregulation via its substrates, sodium thiosulfate (STS) and the garlic compound diallyl disulfide (DADS), on palmitate-induced inflammation resulting in significant reduction of inflammatory cytokines and inhibition of reactive oxygen species. Additionally, using an IL1β-induced insulin resistance 3T3-L1 cell model, upregulation of TST associated with antidiabetic effects. Therefore, we demonstrate that TST-mediated suppression of the inflammatory response protects in vitro cell model of white adipocytes from insulin resistance-induced inflammation. These results encourage the development of potent medicines in the future that specifically target TST in the fat tissue, which could lead to new ways of treating diabetes associated with obesity.

Browning of white adipose tissue is a novel mechanism to counteract obesity in view of its thermogenic activity. Activation of G-protein-coupled receptor 120 (GPR120) can promote the browning of white fat. 9-PAHSA, an endogenous mammalian lipid, which is acting as the ligand of GPR120 to enhance glucose uptake and exert anti-inflammatory effect. In the study, we would like to investigate the biological effects of 9-PAHSA on adipocyte browning. Here, we show that 9-PAHSA induces browning of 3T3-L1 adipocytes via enhanced expression of brown fat specific genes. 9-PAHSA-induced browning in white adipocytes of WT mice and ob/ob mice was investigated by determining expression levels of brown adipocyte-specific genes/proteins by quantitative real-time polymerase chain reaction analysis, immunoblot analysis and immunochemical staining. The effects of 9-PAHSA on brown fat markers in 3T3-L1 cells were decreased when GPR120 gene was silenced. To investigate the molecular mechanism of 9-PAHSA on adipocyte browning, lipopolysaccharide (LPS)-induced inflammatory model was conducted. 9-PAHSA treatment abolished LPS-induced NF-kappa B (NF-κB) activation and inflammatory cytokine secretion. But these anti-inflammatory effects of 9-PAHSA were attenuated by GPR120 knockdown. Our finding demonstrated that the browning of adipocyte was induced by 9-PAHSA through activating GPR120 and inhibiting the LPS/NF-κB pathway. This promising result will help to reveal the potential pathogenesis of obesity.

Adipose tissue (AT) contains well-developed vascular networks. Pathological AT expansion often accompany the reduction in AT blood vessels, which further exacerbates adipocyte dysfunction due to hypoxia; however, it remains unclear whether AT vascular rarefaction is simply secondary to adipocyte hypertrophy, or if there is an actively regulated pathway that mediates impaired AT angiogenesis in obesity. We searched for growth factors whose expression in AT is down-regulated in obesity; accordingly, we identified neuregulin-4 (Nrg4), a member of the EGF family of proteins. Nrg4 is highly and preferentially expressed in healthy adipocytes, while its expression was substantially reduced in obesity. Nrg4 activated endothelial angiogenic functions and angiogenesis both in vitro and in vivo. Genetic loss of Nrg4 caused reduction in brown and white AT blood vessels, and induced overweight even while consuming normal chow. Conditional knockout of Nrg4 in brown adipocytes caused blood vessel reduction in brown but not in white AT, and was sufficient to induce obese phenotype. Our data demonstrated that Nrg4 plays a critical role in maintaining AT vasculature and its metabolic functions. Considering the substantial reduction of Nrg4 in obesity, disruption of Nrg4-mediated angiogenesis could be an active mechanism for the obesity-associated vascular rarefaction in AT, and thus Nrg4 is an attracting pharmacotherapeutic target in the prevention and/or treatment of obesity-related metabolic disorders.

Browning of white adipose tissue is a promising strategy to tackle obesity. Recently, Janus kinase (JAK) inhibition was shown to induce white-to-brown metabolic conversion of adipocytes in vitro; however effects of JAK inhibition on browning and systemic metabolic health in vivo remain to be elucidated. Here, we report that systemic administration of JAK inhibitor (JAKi) ameliorated obesity-related metabolic disorders. Administration of JAKi in mice fed a high-fat diet increased UCP-1 and PRDM16 expression in white adipose tissue, indicating the browning of white adipocyte. Food intake was increased in JAKi-treated mice, while the body weight and adiposity was similar between the JAKi- and vehicle-treated mice. In consistent with the browning, thermogenic capacity was enhanced in mice treated with JAKi. Chronic inflammation in white adipose tissue was not ameliorated by JAKi-treatment. Nevertheless, insulin sensitivity was well preserved in JAKi-treated mice comparing with that in vehicle-treated mice. Serum levels of triglyceride and free fatty acid were significantly reduced by JAKi-treatment, which is accompanied by ameliorated hepatosteatosis. Our data demonstrate that systemic administration of JAKi has beneficial effects in preserving metabolic health, and thus inhibition of JAK signaling has therapeutic potential for the treatment of obesity and its-related metabolic disorders.

Although Fibronectin Type III Domain Containing 4 (FNDC4) has been reported to be involved in the modulation of inflammation in macrophages, its effects on inflammation and insulin resistance in adipose tissue are unknown. In the current study, we investigated the effects of FNDC4 on hyperlipidemia-mediated endoplasmic reticulum (ER) stress, inflammation, and insulin resistance in adipocytes via the AMP-activated protein kinase (AMPK)/heme oxygenase-1 (HO-1)-mediated pathway. Hyperlipidemia-induced nuclear factor κB (NFκB), inhibitory κBα (IκBα) phosphorylation, and pro-inflammatory cytokines such as TNFα and MCP-1 were markedly mitigated by FNDC4. Furthermore, FNDC4 treatment attenuated impaired insulin signaling in palmitate-treated differentiated 3T3-L1 cells and in subcutaneous adipose tissue of HFD-fed mice. FNDC4 administration ameliorated glucose intolerance and reduced HFD-induced body weight gain in mice. However, FNDC4 treatment did not affect calorie intake. Additionally, treatment with FNDC4 attenuated hyperlipidemia-induced phosphorylation or expression of ER stress markers such as IRE-1, eIF2α, and CHOP in 3T3-L1 adipocytes and in subcutaneous adipose tissue of mice. FNDC4 treatment stimulated AMPK phosphorylation and HO-1 expression in 3T3-L1 adipocytes and in subcutaneous adipose tissue of mice. siRNA-mediated suppression of AMPK and HO-1 abrogated the suppressive effects of FNDC4 on palmitate-induced ER stress, inflammation, and insulin resistance. In conclusion, our results show that FNDC4 ameliorates insulin resistance via AMPK/HO-1-mediated suppression of inflammation and ER stress, indicating that FNDC4 may be a novel therapeutic agent for treating insulin resistance and type 2 diabetes.

Ca2+ impacts a large array of cellular processes in every known cell type. In the white adipocyte, Ca2+ is involved in regulation of metabolic processes such as lipolysis, glucose uptake and hormone secretion. Although the importance of Ca2+ in control of white adipocyte function is clear, knowledge is still lacking regarding the control of dynamic Ca2+ alterations within adipocytes and mechanisms inducing intracellular Ca2+ changes remain elusive. Own work has recently demonstrated the existence of store-operated Ca2+ entry (SOCE) in lipid filled adipocytes. We defined stromal interaction molecule 1 (STIM1) and the calcium release-activated calcium channel protein 1 (ORAI1) as the key players involved in this process and we showed that the transient receptor potential (TRP) channel TRPC1 contributed to SOCE. Here we have aimed to further characterised SOCE in the white adipocyte by use of single cell whole-cell patch clamp recordings. The electrophysiological measurements show the existence of a seemingly constitutively active current that is inhibited by known store-operated Ca2+ channel (SOCC) blockers. We demonstrate that the mechanical force applied to the plasma membrane upon patching leads to an elevation of the cytoplasmic Ca2+ concentration and that this elevation can be reversed by SOCC antagonists. We conclude that a mechanically activated current with properties similar to TRPC1 is present in white adipocytes. Activation of TRPC1 by membrane tension/stretch may be specifically important for the function of this cell type, since adipocytes can rapidly increase or decrease in size.

Lactate contains a chiral carbon and thus has two optical isomers-d-lactate and l-lactate. l-Lactate is the predominant form that is produced by the body and can be delivered to the organs. On the other hand, gut microbiota produce both isomers, which can then flow into the body. Although both d-lactate and l-lactate can contribute to energy metabolism, their potential roles in adipocyte differentiation remain to be elucidated. Here, we investigated the effects of l-lactate and d-lactate on the differentiation of 3T3-L1 preadipocytes. Both lactate enantiomers were demonstrated to enhance triglyceride accumulation by stimulating the early phase of adipocyte differentiation. Notably, d-lactate was more potent than l-lactate in inducing triglyceride accumulation. The degree of triglyceride accumulation induced by l-lactate was similar to that induced by pyruvate. d-Lactate was more potent than l-lactate in increasing the activity of glycerol-3-phosphate dehydrogenase. Both lactate enantiomers did not affect cell viability. Moreover, both enantiomers upregulated the expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, sterol regulatory element-binding protein-1c, and fatty acid synthase, with d-lactate exerting stronger effects than l-lactate. By contrast, lactate did not influence the expression of C/EBPβ and C/EBPδ. d-Lactate significantly increased and l-lactate tended to increase p38 MAPK phosphorylation, and the p38 MAPK inhibitor SB203580 inhibited the stimulation of adipocyte differentiation by d-lactate and l-lactate. These findings showed that both lactate enantiomers stimulate preadipocyte differentiation, with d-lactate showing more potent effects than l-lactate. In addition, our study demonstrated that d-lactate and l-lactate exert different effects on physiological events.

Adipogenesis is the process of differentiation from preadipocytes to adipocytes and is orchestrated by various transcription factors, such as the peroxisome proliferator-activated receptor gamma (PPARγ) and the CCAAT-enhancer-binding protein alpha (C/EBPα). Oxidative stress is also a crucial factor in adipogenesis, and adipocyte differentiation is affected by the cellular redox status. The nuclear factor E2-related factor 2 (Nrf2), which is a basic leucine zipper (bZIP) transcription factor, acts as a regulator of cellular oxidative stress. Although several previous studies examined the function of Nrf2 in adipogenesis, their results were controversial. In this study, we investigated whether the suppression of Nrf2 in 3T3-L1 cells affected adipogenesis. We found that adipogenesis master regulator genes, such as PPARγ and C/EBPα, were downregulated during the differentiation stage in Nrf2-knockdown 3T3-L1 cells. Moreover, the fibroblast growth factor 21 (FGF21) and manganese superoxide dismutase (MnSOD) were markedly downregulated in Nrf2-knockdown 3T3-L1 cells. Taken together, the results of the present study suggest that the suppression of Nrf2 attenuates adipogenesis and decreases FGF21 expression through PPARγ in 3T3-L1 cells.

Recent studies have highlighted recruiting and activating brite adipocytes in WAT (so-called "browning") would be an attractive anti-obesity strategy. Zinc alpha2 glycoprotein (ZAG) as an important adipokine, is reported to ameliorate glycolipid metabolism and lose body weight in obese mice. However whether the body reducing effect mediated by browning programme remains unclear. Here, we show that overexpression of ZAG in 3T3-L1 adipocytes enhanced expression of brown fat-specific markers (UCP-1, PRDM16 and CIDEA), mitochondrial biogenesis genes (PGC-1α, NRF-1/2 and mtTFA) and the key lipid metabolism lipases (ATGL, HSL, CPT1-A and p-acyl-CoA carboxylase). Additionally, those effects were dramaticlly abolished by H89/SB203580, revealing ZAG-induced browning depend on PKA and p38 MAPK signaling. Overall, our findings suggest that ZAG is a candidate therapeutic agent against obesity via induction of brown fat-like phenotype in white adipocytes.