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T1R3 is a class C G protein-coupled receptor family member that forms heterodimeric umami and sweet taste receptors with T1R1 and T1R2, respectively, in the taste cells of taste buds. T1R3 is expressed in 3T3-L1 cells in homomeric form and negatively regulates adipogenesis in a Gαs-dependent but cAMP-independent manner. Although T1R3 expression is markedly upregulated during adipogenesis, its physiological role in mature adipocytes remains obscure. Here, we show that stimulation of T1R3 with sucralose or saccharin induces microtubule disassembly in differentiated 3T3-L1 adipocytes. The effect was reproduced by treatment with cholera toxin or isoproterenol but not with forskolin. Treatment with sucralose or saccharin for 3 h inhibited insulin-stimulated glucose uptake by 32% and 45% in differentiated adipocytes, respectively, similar to the inhibitory effect of nocodazole (by 33%). Isoproterenol treatment inhibited insulin-stimulated glucose transport by 26%, whereas sucralose did not affect the intrinsic activity of the glucose transporter, indicating that it inhibited insulin-induced GLUT4 translocation to the plasma membrane. Immunostaining analysis showed that insulin-stimulated GLUT4 accumulation on the plasma membrane was abrogated in sucralose-treated cells, in association with depolymerization of microtubules. Sucralose-mediated inhibition of GLUT4 translocation was reversed by the overexpression of dominant-negative Gαs (Gαs-G226A) or knockdown of Gαs. Additionally, membrane fractionation analysis showed that sucralose treatment reduced GLUT4 levels in the plasma membrane fraction from insulin-stimulated adipocytes. We have identified a novel non-gustatory role for homomeric T1R3 in adipocytes, and activation of the T1R3 receptor negatively regulates insulin action of glucose transport via Gαs-dependent microtubule disassembly.

Obesity significantly impacts living a normal life by increasing morbidity. Additionally, obesity has been shown to be closely associated with severe inflammation in adipocytes. It is widely reported that berberine (BBR) has an anti-inflammatory effect and can reduce glucose and lipid accumulation, whereas ginsenoside Rb1 (Rb1) has been shown to have a significant inhibitory effect on insulin resistance and lipid peroxidation. In this study, we aimed to explore the synergetic effect of BBR and Rb1 on tumor necrosis factor alpha (TNF-α)-treated adipocytes and the mechanisms underlying it. We found that TNF-α reduced cell viability, facilitated the production of inflammatory factors, induced adipogenesis, activated the nuclear factor kappa B (NF-κB) pathway, and increased the expression of peroxisome proliferator-activated receptor gamma, CCAAT enhancer-binding protein alpha, and sterol regulatory element-binding protein-1 c in adipocytes. However, these effects were significantly alleviated by BBR or Rb1. Additionally, a synergetic effect was observed when BBR and Rb1 were used in combination. The effects of BBR in combination with Rb1 on cell proliferation, inflammation, adipogenesis, and the NF-κB pathway in TNF-α-treated adipocytes were significantly abolished by receptor activator of nuclear factor kappa-Β ligand, which is an activator of the NF-κB pathway. Collectively, the results revealed that BBR and Rb1 have a synergetic protective effect against TNF-α-induced inflammation in adipocytes. The mechanism underlying this synergetic effect was found to be inhibition of the NF-κB signaling pathway.

Background: Clopidogrel has been commonly prescribed as a selective P2Y12 receptor antagonist to reduce heart attack and stroke risk. Nearly 10% of absorbed clopidogrel is metabolized to active forms by cytochrome P450 (CYP) enzymes in the liver and 90% to inactive clopidogrel carboxylate by esterases. Objective: Since different forms of clopidogrel have cytotoxic potential, our aim was to determine the effect of 7.5, 40, and 75μM clopidogrel over DNA damage in adipocytes and hepatocytes. Methods: In the present study, DNA damage was investigated by Comet analysis using 3T3-L1 adipocytes and Alpha Mouse 12 (AML-12) hepatocytes. Results: DNA fragmentation was found to be increased as a response to 7.5 μM, 40 μM, and 75 μM clopidogrel treatment compared to non-treated control groups in AML-12 hepatocytes (p Conclusion: According to our results, clopidogrel results in more DNA damage in adipocytes than in hepatocytes. The molecular mechanism of clopidogrel genotoxicity needs to be further investigated especially in adipose tissue.

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.

The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, glucose intolerance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid [non-esterified fatty acid (NEFA) 16:1] concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a hitherto unknown interplay between MDM2 and MORC2/LIPIN1 involved in balancing adipocyte function.

The objective of the study was to investigate the effect of nobiletin on adipogenesis in 3T3-L1 cells. Here, we found that nobiletin could promote adipogenesis in 3T3-L1 cells in the absence of adipogenic inducers such as insulin, 3-Isobutyl-1-methylxanthine and dexamethasone. In addition, Real time quantitative PCR showed that the expression of adipogenic genes such as CCAAT/enhancer binding proteins, peroxisome proliferator-activated receptors-γ and fatty acid-binding protein aP2 were up-regulated in 3T3-L1 cells in the presence of nobiletin. Next, we investigated the role of Akt in the effect of nobiletin on the adipogenesis in 3T3-L1 cells by LY294002 blocking PI3K/Akt pathway. The experimental results showed that the promotive effect of nobiletin on adipogenesis was attenuated, accompanied by the down-regulation of adipogenic genes expression, after the activity of Akt was inhibited, suggesting that Akt plays an important role in the effect of nobiletin on promoting adipogenesis in 3T3-L1 cells. Still, the inhibition of Akt pathway by LY294002 was attenuated in the presence of nobiletin. These findings provide a possibility that nobiletin may be a potential candidate agent for stimulating Akt pathway in 3T3-L1 cells.

Background: Astaxanthin is a natural active substance with a plurality of biological activities, such as anti-oxidation, anti-inflammatory and anti-cardio-cerebrovascular diseases. However, there is less research on the effects of astaxanthin on obesity. Astaxanthin with different structural forms affect the corresponding biological activity. Objective: The study aimed to compare Astaxanthin-octanoic acid diester (C8-AST) and Free Astaxanthin (F-AST) to explore the effect on lipogenesis in vitro. Methods: 3T3-L1 preadipocytes were cultured under astaxanthin treatment. Cell proliferation and differentiation were evaluated by MTT assay and oil red O staining, respectively. The synthesis of metabolic mechanism of intracellular fatty acids and triglycerides was examined by qRT-PCR and Western blotting. Results: C8-AST and F-AST had no effect on adipocyte proliferation at low concentration, but inhibited adipocyte differentiation. The treatment of astaxanthin could inhibit the expression of PPARγ, C/EBPα and SREBP-1c in adipocytes, up-regulate the expression of Wnt10b, LRP6, FZ in Wnt/ β-catenin signaling pathway and increase the β-catenin entry into nucleus, suggesting that the activation of Wnt/β-catenin signaling was involved in axtaxanthin-regulated lipogenesis. Notably, inhibition effect of lipogenesis on C8-AST was better than F-AST overall. Conclusion: At the cellular level, both two kinds of astaxanthins inhibit the synthesis of intracellular fatty acids and triglycerides. Notably, the inhibition effect of C8-AST is better than F-AST overall.

In this study, we evaluated a detailed molecular mechanism of anti-adipogenic activity of vitexin, apigenin flavone glucoside, present in germinated fenugreek seeds, in differentiating human mesenchymal stem cells (hMSCs). The lipid content of differentiated adipocytes was estimated by ORO staining. Effect on mitotic clonal expansion was checked by cell cycle analysis. Expression of early and terminal adipocyte differentiation markers, anti- and pro-adipogenic transcription factors and signalling intermediates regulating them was evaluated at RNA and protein level. We found vitexin to be non-cytotoxic up to 20 μM at which intracellular lipid accumulation was significantly decreased. Cell cycle analysis suggested that vitexin does not affect mitotic clonal expansion. Expression of early and late differentiation markers, such as CEBPα, CEBPβ, PPARγ, FABP4, perilipin, adiponectin and Glut4 was significantly reduced in the presence of vitexin. Expression of KLF4 and KLF15, positive regulators of PPARγ, was decreased, whereas that of negative regulators, namely KLF2, GATA2, miR20a, miR27a, miR27b, miR128, miR130a, miR130b, miR182 and miR548 increased with vitexin treatment. This effect was mediated by the activation of the AMP-activated protein kinase (AMPK) pathway via the activation of LepR and additionally by inhibiting ROS. Thus, our results showed that vitexin regulates the expression of PPARγ and inhibits adipogenesis of hMSCs at an early stage of differentiation.

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor playing an important role in immune response and inflammation. Lipopolysaccharide (LPS) stimulation can significantly induce PTX3 expression and secretion in adipocytes. Appropriate regulation of PTX3 secretion is critical for inflammatory homeostasis. Using chemical inhibitors of conventional and unconventional protein secretion, we explored the mechanisms that control LPS-stimulated PTX3 secretion in 3T3-L1 adipocytes. Inhibiting the conventional protein secretion blocked LPS-stimulated PTX3 secretion, resulting in cellular PTX3 accumulation in adipocytes. We also detected PTX3 in exosomes from LPS-treated adipocytes; inhibiting exosome trafficking attenuated PTX3 secretion. However, only 4.3% of secreted PTX3 was detected in exosomes compared to 95.7% in the non-exosomal fractions. The fractionation of isolated exosomes by the iodixanol density gradient centrifugation confirmed that a small portion of secreted PTX3 overlapped with exosomal markers in small extracellular-vesicle fractions. We conclude that PTX3 is secreted mainly through conventional protein secretion, and a small percentage of PTX3 is released in exosomes from LPS-stimulated adipocytes.

Adipocyte-rich omentum offers "good soil" for disseminating ovarian cancer (OvCa), contributing to therapeutic difficulty. However, little is understood about the association between adipocytes and tumor growth at peritoneal dissemination site. Herein, we report the induction of adipocyte dedifferentiation by OvCa cells and pro-tumorigenic effects of resulted adipocyte-derived fibroblasts. We confirmed that malignant ascites promoted the dedifferentiation of the primary human adipocytes obtained from surgical omental specimen into omental adipocyte-derived fibroblast (O-ADF) that possess both mesenchymal stem cell and myofibroblast-like features. This promotion of dedifferentiation by malignant ascites was blocked by addition of Wnt signaling inhibitor. The effects of dedifferentiated adipocytes in proliferation and migration of OvCa cells were analyzed with in vitro co-culturing experimental models and in vivo mice model, and we demonstrated that OvCa cell lines showed enhanced proliferative characteristics, as well as increased migratory abilities upon co-culturing with O-ADF. Additionally, exogenous transforming growth factor-β1 augmented desmoplastic morphological change of O-ADF, leading to higher proliferative ability. Our results suggest that OvCa cells promote dedifferentiation of peritoneal adipocytes by activating Wnt/β-catenin signaling, and generated O-ADFs exhibit pro-tumoral hallmarks. This article is protected by copyright. All rights reserved.

Phthalates, a plasticizer group, are used extensively in many of the products we use every day. Public health concerns are growing as recent studies have implicated butyl benzyl phthalate (BBP) as an obesogen. However, BBP-induced epigenetic regulation during adipogenesis is still unknown. We investigated if BBP altered miR-34a-5p, a key miRNA involved in obesity, and regulated its downstream pathway. Differentiating 3T3-L1 cells were exposed to various doses of BBP without exogenous adipogenic stimuli, tested for adipogenesis markers (PPARγ and aP2), and stained for lipid accumulation with Oil Red O staining. We then measured the expression of miR-34a-5p and its target genes, Nampt and Sirt1, along with another significant epigenetic modulator, Sirt3. Furthermore, using antagomiR, we examined whether miR-34a-5p knockdown decreased adipogenesis. BBP exposure resulted in augmented expression levels of miR-34a-5p with an associated increase in adipogenesis. BBP significantly decreased the Nampt, Sirt1, and Sirt3 gene expression levels. However, a decrease in the protein expression was observed only for Nampt, indicating that miR-34a-5p under BBP exposure may regulate Sirt1/Sirt3 only at the transcriptional level. Interestingly, in the presence of BBP, knockdown of miR-34a-5p decreased adipogenesis in the differentiating 3T3-L1 cells. Furthermore, miR-34a-5p knockdown increased the Nampt protein expression levels as well as NAD+ levels, indicating that miR-34a-5p regulates Nampt during BBP exposure. Additionally, the NAD+-dependent sirtuin activity decreased in BBP-treated cells and increased in miR-34a-5p knockdown cells with BBP treatment. BBP exposure demonstrated the involvement of epigenetic regulation by altering the expression patterns of miR-34a-5p and its target Nampt, which may perturb the energy homeostasis of the differentiating adipocytes by altering NAD+ levels and sirtuin activity, resulting in increased adipogenesis.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master transcription factor in adipocyte differentiation, while distal-less homeobox 5 (Dlx5) is essential for initiating osteoblast differentiation by driving Runt-related transcription factor 2 expression. Considering that adipocytes and osteoblasts share common progenitors, there is a reciprocal correlation between bone and fat formation. However, the mechanism by which Dlx5 controls PPARγ remains unclear. We elucidated that Dlx5 physically binds to PPARγ during immunoprecipitation; in particular, the ligand-binding and DNA-binding domains of PPARγ were involved in the interaction. Transcriptional activity of PPARγ was significantly decreased by Dlx5 overexpression, whereas the opposite results were detected with Dlx5 knockdown. Rosiglitazone, a PPARγ agonist, further enhanced the PPARγ-induced transcriptional activity; however, Dlx5 overexpression effectively repressed the rosiglitazone-mediated increase in activity. Finally, DNA-binding affinity assay revealed that Dlx5 interrupts the interaction of PPARγ with the PPARγ response element promoter. In conclusion, our findings indicate that Dlx5 impedes PPARγ-induced activity, and it may be useful for managing diabetes drug-mediated obesity.

Obesity is now recognized as a disease. This study revealed a novel role for pyruvate dehydrogenase kinase (PDK) in diet-induced hypertrophic obesity. Mice with global or adipose tissue-specific PDK2 deficiency were protected against diet-induced obesity. The weight of adipose tissues and the size of adipocytes were reduced. Adipocyte-specific PDK2 deficiency slightly increased insulin sensitivity in HFD-fed mice. In studies with 3T3-L1 preadipocytes, PDK2 and PDK1 expression was strongly increased during adipogenesis. Evidence was found for epigenetic induction of both PDK1 and PDK2. Gain- and loss-of-function studies with 3T3-L1 cells revealed a critical role for PDK1/2 in adipocyte differentiation and lipid accumulation. PDK1/2 induction during differentiation was also accompanied by increased expression of hypoxia-inducible factor-1α (HIF1α) and enhanced lactate production, both of which were absent in the context of PDK1/2 deficiency. Exogenous lactate supplementation increased the stability of HIF1α and promoted adipogenesis. PDK1/2 overexpression-mediated adipogenesis was abolished by HIF1α inhibition, suggesting a role for the PDK-lactate-HIF1α axis during adipogenesis. In human adipose tissue, the expression of PDK1/2 was positively correlated with that of the adipogenic marker PPARγ and inversely correlated with obesity. Similarly, PDK1/2 expression in mouse adipose tissue was decreased by chronic high-fat diet feeding. We conclude that PDK1 and 2 are novel regulators of adipogenesis that play critical roles in obesity., Obesity: Effects of different forms of key enzyme The discovery that two forms of a key enzyme appear to play a critical role in fat production triggered by overeating might lead to new approaches to prevent and treat obesity. Hyeon-Ji Kang at Kyungpook National University, Daegu, South Korea, and colleagues in South Korea and the USA examined the role of the enzymes pyruvate dehydrogenase kinase types 1 and 2 (PDK1/2). PDK enzymes regulate the activity of a multi-enzyme complex that catalyzes a key step in the use of glucose to provide energy stores for cells. Mice deficient in PDK2 were protected from diet-induced obesity, and PDK 1 and 2 activity was increased during the generation of fat cells. Studies using mice and human fat tissue confirmed that the enzymes regulate the development and growth of fat cells. Drugs inhibiting PDK enzymes might combat obesity.

Objective The present study was conducted to elucidate the in-vitro anti-oxidant and anti-adipogenic effect of the flavone, chrysin in comparison with the citrus bioflavonoid, hesperidin during adipogenic differentiation in 3T3-L1 mouse preadipocytes. Methods The effect of chrysin and hesperidin on adipogenic differentiation was evaluated using Oil red-O staining, triglyceride estimation, free glycerol release, and ROS accumulation. The expression of adipogenesis-related genes was evaluated in real time-polymerase chain reaction. Results 50 µmol chrysin or hesperidin did not affect the cell viability of 3T3-L1 preadipocytes and adipocytes, but significantly reduced preadipocyte clonal population, accumulation of intracellular lipid and ROS and consequently increased lipolysis and antioxidant enzyme defence. It also decreased the expression of major adipogenic transcription factors, CCAAT/enhancer-binding protein-β, peroxisome proliferator activated receptor-γ, sterol regulatory element binding protein 1c, fatty acid synthase and hormone sensitive lipase. Conclusion(s) Herein we have indicated, for the first time, the effective anti-adipogenic mechanism of chrysin by down-regulating adipogenesis, lipogenesis and ROS and up-regulating lipolysis and antioxidant enzyme in differentiated 3T3-L1 adipocytes. As a nutritional bioflavonoid, chrysin with its more effective inhibition on adipogenesis than hesperidin has the potential to be developed as an anti-adipogenic nutraceutical agent.

Injectable hydrogel with multifunctional tunable properties comprising biocompatibility, anti-oxidative, anti-bacterial, and/or anti-infection are highly preferred to efficiently promote diabetic wound repair and its development remains a challenge. In this study, we report hyaluronic acid and Pullulan-based injectable hydrogel loaded with curcumin that could potentiate reepithelization, increase angiogenesis, and collagen deposition at wound microenvironment to endorse healing cascade compared to other treatment groups. The physical interaction and self-assembly of hyaluronic acid-Pullulan-grafted-pluronic F127 injectable hydrogel were confirmed using nuclear magnetic resonance (1H NMR) and Fourier transformed infrared spectroscopy (FT-IR), and cytocompatibility was confirmed by fibroblast viability assay. The CUR-laden hyaluronic acid-Pullulan-g-F127 injectable hydrogel promptly undergoes a sol-gel transition and has proved to potentiate wound healing in a streptozotocin-induced diabetic rat model by promoting 93% of wound closure compared to other groups having 35%, 38%, and 62%. The comparative in vivo study and histological examination was conducted which demonstrated an expeditious recovery rate by significantly reducing the wound healing days i.e. 35 days in a control group, 33 days in the CUR suspension group, 21 days in unloaded injectable, and 13 days was observed in CUR loaded hydrogel group. Furthermore, we suggest that the injectable hydrogel laden with CUR showed a prompt wound healing potential by increasing the cell proliferation and serves as a drug delivery platform for sustained and targeted delivery of hydrophobic moieties.

Tetranectin (TN), an adipogenic serum protein, enhances adipocyte differentiation, however, its functional mechanism has yet to be elucidated. In the present study, we investigated the adipogenic function of TN by using medium containing TN-depleted fetal bovine serum (TN-del-FBS) and recombinant mouse TN (mTN). The adipocyte differentiation of 3T3-L1 cells was significantly enhanced by mTN supplementation essentially at differentiation induction, which indicated a potential role of the protein in the early differentiation phase. The adipogenic effect of mTN was more significant with insulin in the differentiation induction cocktail, implicating their close functional relationship. mTN enhanced not only the proliferation of growing cells, but also mitotic clonal expansion (MCE) that is a prerequisite for adipocyte differentiation in the early phase. Consistently, mTN increased the phosphorylation of ERK in the early phase of adipocyte differentiation. Results of this study demonstrate that the adipogenic function of mTN is mediated by enhancing MCE via ERK signaling. [BMB Reports 2021; 54(7): 374-379].

Alendronate, a bisphosphonate used to prevent osteoporosis, stimulates osteogenesis but impairs adipogenesis. Different clinical trials suggest that the incidence of diabetes may be lower in patients treated with alendronate. Taking into account the importance of adipocytes and macrophages of adipose tissue in insulin resistance and type 2 diabetes, it is necessary to evaluate the effect of alendronate in both cell types. In this paper, we investigated the effect of alendronate on the differentiation to adipocytes of 3T3-L1 fibroblasts, the cell line most used to study adipogenesis, and also its effect on lipid content and oxidative stress in mature adipocytes as well as on the inflammatory response of macrophages. We found that alendronate inhibits differentiation of 3T3-L1 fibroblasts to adipocytes in keeping with reports in other cell lines. On the other hand, treatment of 3T3-L1 adipocytes with alendronate was able to decrease triglyceride content and to prevent H2O2-induced lipid peroxidation which was evaluated as an indicator of oxidative stress. In addition, it was found that activation of RAW 264.7 macrophages to a pro-inflammatory M1 type is inhibited by this bisphosphonate. These results suggest that alendronate may contribute to prevent adipocyte excessive enlargement and the induction of oxidative stress in 3T3-L1 adipocytes as well as the activation of macrophages to a pro-inflammatory M1 type, which are events associated with adipose tissue dysfunction and insulin resistance. In this study, we unraveled the underlying mechanisms of events that were previously observed in clinical trials.

Background and purpose Obesity is becoming a major global health issue and is mainly induced by the accumulation of adipose tissues mediated by adipogenesis, which is reported to be regulated by peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα). Trichostatin A (TSA) is a novel histone deacetylase inhibitor (HDACI) that was recently reported to exert multiple pharmacological functions. The present study will investigate the inhibitory effect of TSA on adipogenesis, as well as the underlying mechanism. Methods The adipogenesis of 3T3-L1 cells was induced by stimulation with a differentiation cocktail (DMI) medium for 8 days. MTT assay was used to measure the cell viability and Oil Red O staining was used to evaluate the adipogenesis of 3T3-L1 cells. The total level of triglyceride and released glycerol were detected to evaluate the lipolysis during 3T3-L1 adipogenesis. The expression levels of Leptin, fatty acid-binding protein 4 (FABP4), and sterol regulatory element-binding protein (SREBP1C) were determined by qRT-PCR. qRT-PCR assay was utilized to detect the expression levels of PPARγ and C/EBPα in 3T3-L1 cells. A high-fat diet (HFD) was used to construct an obese mice model, followed by the treatment with TSA. HE staining was conducted to evaluate the pathological state of adipose tissues. Body weights and the weights of adipose tissues were recorded to evaluate the anti-obesity property of TSA. Results Firstly, the promoted lipid accumulation induced by DMI incubation was significantly reversed by the treatment with TSA in a dose-dependent manner. The elevated expression levels of Leptin, FABP4, SREBP1C, PPARγ, and C/EBPα induced by the stimulation with DMI incubation were dramatically inhibited by the introduction of TSA, accompanied by the upregulation of phosphorylated AMP-activated protein kinase (p-AMPK). Secondly, the inhibitory effect of TSA against the expression level of PPARγ and lipid accumulation was greatly abolished by an AMPK inhibitor. Lastly, the increased body weights and visceral adipocyte tissue weight, as well as the enlarged size of adipocytes induced by HFD were pronouncedly reversed by the administration of TSA. Conclusion TSA inhibited adipogenesis in 3T3-L1 preadipocytes by activating the AMPK pathway. Background and purpose Obesity is becoming a major global health issue and is mainly induced by the accumulation of adipose tissues mediated by adipogenesis, which is reported to be regulated by peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα). Trichostatin A (TSA) is a novel histone deacetylase inhibitor (HDACI) that was recently reported to exert multiple pharmacological functions. The present study will investigate the inhibitory effect of TSA on adipogenesis, as well as the underlying mechanism. Methods The adipogenesis of 3T3-L1 cells was induced by stimulation with a differentiation cocktail (DMI) medium for 8 days. MTT assay was used to measure the cell viability and Oil Red O staining was used to evaluate the adipogenesis of 3T3-L1 cells. The total level of triglyceride and released glycerol were detected to evaluate the lipolysis during 3T3-L1 adipogenesis. The expression levels of Leptin, fatty acid-binding protein 4 (FABP4), and sterol regulatory element-binding protein (SREBP1C) were determined by qRT-PCR. qRT-PCR assay were utilized to detect the expression levels of PPARγ and C/EBPα in 3T3-L1 cells. A high-fat diet (HFD) was used to construct an obese mice model, followed by the treatment with TSA. HE staining was conducted to evaluate the pathological state of adipose tissues. Body weights and the weights of adipose tissues were recorded to evaluate the anti-obesity property of TSA. Results Firstly, the promoted lipid accumulation induced by DMI incubation was significantly reversed by the treatment with TSA in a dose-dependent manner. The elevated expression levels of Leptin, FABP4, SREBP1C, PPARγ, and C/EBPα induced by the stimulation with DMI incubation were dramatically inhibited by the introduction of TSA, accompanied by the upregulation of phosphorylated AMP-activated protein kinase (p-AMPK). Secondly, the inhibitory effect of TSA against the expression level of PPARγ and lipid accumulation was greatly abolished by an AMPK inhibitor. Lastly, the increased body weights and visceral adipocyte tissue weight, as well as the enlarged size of adipocytes induced by HFD were pronouncedly reversed by the administration of TSA. Conclusion TSA inhibited adipogenesis in 3T3-L1 preadipocytes by activating the AMPK pathway.

Aims/Introduction It was reported previously that N4bp2l1 expression increases in 3T3‐L1 cells in a differentiation‐dependent manner and N4bp2l1 knockdown suppresses adipocyte differentiation. However, the physiological function of N4BP2L1 in adipocytes remains unknown. This study aimed to elucidate the physiological mechanism of N4bp2l1 expression and the role of N4BP2L1 in the physiological function of adipocytes. Materials and Methods Analysis of gene expression levels of N4bp2l1 in adipose tissue during feeding in mice was conducted. Identification of transcription factors that regulate N4bp2l1 expression was conducted using a reporter assay. Investigation of N4BP2L1‐interacting proteins was carried out using immunoprecipitation. A GLUT4 translocation assay and a glucose uptake assay in 3T3‐L1 adipocytes were performed using N4bp2l1 overexpression and knockdown adenovirus. Results The results indicated that N4bp2l1 is a novel FoxO1 target gene and its expression is controlled by the insulin‐mediated regulation of FoxO1. N4BP2L1 interacts with dynactin, which binds to the microtubule motor dynein, indicating that N4BP2L1 is involved in GLUT4 trafficking and glucose uptake in 3T3‐L1 adipocytes. Conclusions Our results suggest that N4BP2L1 is involved in adipocyte homeostasis by interacting with dynein–dynactin and affecting GLUT4‐mediated glucose uptake and the insulin signaling pathway., N4bp2l1 is a novel FoxO1 target gene that is controlled by insulin‐mediated regulation of FoxO1. N4BP2L1 interacts with dynactin, which binds to the microtubule motor dynein, suggesting that N4BP2L1 is involved in GLUT4 trafficking and glucose uptake.

It has been previously shown that chronic ethanol administration-induced increase in adipose tissue lipolysis and reduction in the secretion of protective adipokines collectively contribute to alcohol-associated liver disease (ALD) pathogenesis. Further studies have revealed that increased adipose S-adenosylhomocysteine (SAH) levels generate methylation defects that promote lipolysis. Here, we hypothesized that increased intracellular SAH alone causes additional related pathological changes in adipose tissue as seen with alcohol administration. To test this, we used 3-deazaadenosine (DZA), which selectively elevates intracellular SAH levels by blocking its hydrolysis. Fully differentiated 3T3-L1 adipocytes were treated in vitro for 48 h with DZA and analysed for lipolysis, adipokine release and differentiation status. DZA treatment enhanced adipocyte lipolysis, as judged by lower levels of intracellular triglycerides, reduced lipid droplet sizes and higher levels of glycerol and free fatty acids released into the culture medium. These findings coincided with activation of both adipose triglyceride lipase and hormone sensitive lipase. DZA treatment also significantly reduced adipocyte differentiation factors, impaired adiponectin and leptin secretion but increased release of pro-inflammatory cytokines, IL-6, TNF and MCP-1. Together, our results demonstrate that elevation of intracellular SAH alone by DZA treatment of 3T3-L1 adipocytes induces lipolysis and dysregulates adipokine secretion. Selective elevation of intracellular SAH by DZA treatment mimics ethanol’s effects and induces adipose dysfunction. We conclude that alcohol-induced elevations in adipose SAH levels contribute to the pathogenesis and progression of ALD.

Culturing cells on bio-gels are believed to provide a more in vivo-like extracellular matrix. 3T3-L1 cells cultured on Matrigel® significantly alteregd their proliferation and differentiation as compared to growth on tissue culture-coated polystyrene surfaces. Growth on a 250-μm thick layer of Matrigel® facilitated the formation of cellular aggregates of 3T3-L1 cells. Differentiation of 3T3-L1 cells cultured on Matrigel® demonstrated increased levels of mRNA levels for key adipogenic transcription factors (PPARγ, C/EBPα, SREBP1), lipogenic markers (FAS, FABP4, LPL, PLIN1) and markers of adipocyte maturity (LEP), compared to cells cultured directly on a polystyrene tissue culture surface. The gene expression of extracellular matrix proteins (FN1, COL1A1, COL4A1, COL6, LAM) was decreased in 3T3-L1 cells cultured on Matrigel®. Furthermore, growth on Matrigel® increased lipid accumulation in 3T3-L1 cells in the presence and absence of rosiglitazone, a thiazolidinedione routinely used to optimize differentiation in these cells. These changes in adipocyte gene expression and lipid accumulation patterns may be a result of the increased cell–cell and cell–ECM interactions occurring on the Matrigel®, a scenario that is more reflective of an in vivo model. Taken together, our data advance the understanding of the value of culturing 3T3-L1 cells on Matrigel®.

BECN1, a protein essential for autophagy, is involved in adipocyte differentiation, lipolysis and insulin resistance. The discovery of new mechanisms for modifying BECN1 in adipocytes may provide novel therapeutic targets for obesity. This study aimed to investigate the impact of mutations at the acetylation sites of BECN1 on adipocyte differentiation and lipolysis. We found that Ace‐BECN1 levels were increased in 3T3‐L1 adipocyte differentiation and isoproterenol‐/TNF‐α‐stimulated lipolysis and in subcutaneous and visceral adipose tissues of high‐fat diet mice. K414 was identified as an acetylation site of BECN1, which affects the stability of the BECN1 protein. Mutation at K414 of BECN1 affected autophagy, differentiation and lipolysis in 3T3‐L1 adipocytes. These data indicated the potential of BECN1 K414 as a key molecule and a drug target for regulating autophagy and lipid metabolism in adipocytes.

Recent findings have revealed that exposure to environmental contaminants may result in obesity and pose a health threat to the general public. As the activity of transient receptor potential channels (TRPs) plays a permissive role in adipogenesis, the interactions between TRPs and some food pollutants, i.e. bisphenol A, di (2-ethylhexyl) phthalate, zearalenone, and zeranol at 10 μM were investigated in the present study. TRP-V1,-V3, -C4 and -C6 are reported to be differentially expressed in the adipocyte differentiation, and immunoblotting was performed to quantify changes in these TRPs affected by the pollutants. Our result indicated that the mycoestrogen zeranol or α-zearalanol suppressed the expression of the V1 and C6 isoforms. Subsequently, confocal microscopy was used to measure the calcium inflow repressed by zeranol from 0.1 μM to 10 μM. Oil Red O staining was used to determine the differentiation of 3T3 L1 preadipocytes. Zeranol could suppress the expression of TRP-V1 and -C6 protein and inhibit the associated flow of calcium into the cytosol of 3T3 L1 cells. Its IC50 value for inhibiting calcium inflow stimulated by 40 μM capsaicin or 10 μM GSK1702934A was estimated to be around 6 μM. Reduced TRP-V1 or -C6 activity might result in promoting adipogenesis. In conclusion, this study demonstrated that zeranol could potentiate fat cell differentiation through antagonizing TRP-V1 and –C6 activities.

The anti-inflammatory adipokine CTRP-3 might affect innate immune reactions such as NOD1. The impact of CTRP-3 on NOD1-mediated inflammation in adipocytes and monocytic cells as well as on NOD1 expression was investigated. Murine 3T3-L1 pre-adipocytes and adipocytes as well as human THP-1 monocyte-like cells were co-stimulated with the synthetic NOD1 agonist Tri-DAP and recombinant CTRP-3. Gonadal adipose tissue and primary adipocytes were obtained from a murine model carrying a knockout (KO) of CTRP-3 in adipocytes but not in stroma-vascular cells. Wildtype mice with lipopolysaccharide (LPS)-induced elevated NOD1 expression were treated with CTRP-3. Secreted inflammatory cytokines in cell supernatants were measured by ELISA and mRNA levels were quantified by RT-PCR. Pro-inflammatory chemokine and cytokine secretion (MCP-1, RANTES, TNFα) was induced by NOD1 activation in adipocytes and monocyte-like cells, and MCP-1 and RANTES release was effectively inhibited by pre-incubation of cells with CTRP-3. CTRP-3 also antagonized LPS-triggered induction of NOD1 gene expression in murine adipose tissue, whereas adipocyte CTRP-3 deficiency upregulated NOD1 expression in adipose tissue. CTRP-3 is an effective antagonist of peptidoglycan-induced, NOD1-mediated inflammation and of LPS-induced NOD1 expression. Since basal NOD1 expression is increased by adipocyte CTRP-3 deficiency, there have to be also inflammation-independent mechanisms of NOD1 expression regulation by CTRP-3.

The brown rice vinegar was prepared by adding 10% of vinegar starter, and incubated using the strain Acetobacter pasteurianus JBA172705. The alcohol concentration of brown rice liquor was adjusted to 6%, and seed vinegar was added to 10%. Then, it was cultured by shaking for 16 days (30°C, 120 rpm). After fermentation, brown rice vinegar was extracted with 70% ethanol to evaluate the anti-obesity effect using 3T3-L1 adipocyte cells. The total acidity of brown rice vinegar increased to 6.43% after 16 days of fermentation, and acetic acid increased 15 times and succinic acid 2.5 times or more. The total phenolics and free amino acid contents decreased during the acetic acid fermentation period, and were 48.53 mg GAE/mL and 18.79 mg% (w/v) at the 16th day of fermentation. At a concentration of 100 μg/mL of brown rice vineagar extract (BRV 16d), the lipid accumulation rate was 72.2%. The leptin expression level was reduced by more than 50% according to the treatment of brown rice vinegar extract. It was confirmed that the PPARγ and SREBP-1c expression levels decreased at a concentration of 100 μg/mL. As a result, brown rice vinegar is expected to be effective in anti-obesity by suppressing the expression of transcription factors that induce obesity.

Abnormal adipocyte growth, distinguished by an increase in cell numbers and cellular differentiation, is regarded as a major pathological characteristic of obesity. Thus, inhibition of adipogenic differentiation in adipocytes could prevent obesity. Recently, certain probiotic stains have been reported to regulate lipid metabolism in vitro and/or in vivo. In this backdrop, this study aimed to investigate basic probiotic properties and potential antiobesity characteristics of mouse 3T3-L1 preadipocytes. Six lactic acid bacteria (LAB) strains were prescreened for their cholesterol-lowering activity, antioxidant activity, and survival at low pH and in a solution containing bile salts. These six strains were investigated for antiadipogenic activity by employing 3T3-L1 mouse preadipocytes. 3T3-L1 cells were treated with selected strains during the differentiation process. Lactobacillus johnsonii 3121 and Lactobacillus rhamnosus 86 were found to be more capable of reducing triglyceride and lipid accumulation, as compared to control group, which are fully differentiated 3T3-L1 adipocytes. These strains also inhibited adipocyte differentiation by downregulating the adipogenic transcription factor in 3T3-L1 adipocytes. Taken together, these results indicate that L. johnsonni 3121 and L. rhamnosus 86 could potentially act as probiotic bacteria and prevent fat accumulation by regulating adipogenesis-related markers.

Zbtb7c is a proto-oncoprotein that controls the cell cycle and glucose, glutamate, and lipid metabolism. Zbtb7c expression is increased in the liver and white adipose tissues of aging or high-fat diet-fed mice. Knockout or knockdown of Zbtb7c gene expression inhibits the adipocyte differentiation of 3T3-L1 cells and decreases adipose tissue mass in aging mice. We found that Zbtb7c was a potent transcriptional repressor of SIRT1 and that SIRT1 was derepressed in various tissues of Zbtb7c-KO mice. Mechanistically, Zbtb7c interacted with p53 and bound to the proximal promoter p53RE1 and p53RE2 to repress the SIRT1 gene, in which p53RE2 was particularly critical. Zbtb7c induced p53 to interact with the corepressor mSin3A-HADC1 complex at p53RE. By repressing the SIRT1 gene, Zbtb7c increased the acetylation of Pgc-1α and Pparγ, which resulted in repression or activation of Pgc-1α or Pparγ target genes involved in lipid metabolism. Our study provides a molecular target that can overexpress SIRT1 protein in the liver, pancreas, and adipose tissues, which can be beneficial in the treatment of diabetes, obesity, longevity, etc., Metabolic disease: SIRT1 gene, a potential therapeutic target Targeting a regulatory DNA sequence linked to the repression of a critical enzyme during metabolic diseases could prove valuable for future therapies. The SIRT1 enzyme is involved in metabolic processes and stress resistance, and its dysregulation is linked to obesity and diabetes development. SIRT1 expression also decreases with aging and stress, but the precise regulation mechanisms are unclear. In experiments on aging mice and mice fed a high-fat diet, Man-Wook Hur at Yonsei University in Seoul, South Korea, and co-workers demonstrated that SIRT1 expression is repressed by a protein called Zbtb7c, which is highly expressed in fat and liver tissues. Aging mice without the Zbtb7c-encoding gene had less fatty tissue than controls. Zbtb7c represses the SIRT1 gene by interacting with protein p53. A sequence critical to this repression mechanism may provide a therapeutic target.

Peroxisome proliferator-activated receptor γ (PPARγ) modulators are expected to exert anti-diabetic effects without PPARγ-related adverse effects, such as fluid retention, weight gain, and bone loss. The present study showed that the novel tetrazole derivative KY-903 exerted similar selective PPARγ partial agonist properties to INT-131, a known PPARγ modulator, in transactivation assays, and decreased plasma glucose and triglyceride levels with increases in adiponectin levels in diabetic KK-Ay mice. These effects were similar to those of pioglitazone. Pioglitazone, but not KY-903, increased adipose tissue and heart weights. In pre-adipocytes (3T3-L1), KY-903, in contrast to pioglitazone, increased adiponectin mRNA levels without adipocyte differentiation, indicating anti-diabetic effects via adiponectin without adipogenesis. In ovariectomized rats fed a high-fat diet (OVX/HFD), KY-903 and pioglitazone decreased plasma triglyceride and non-esterified fatty acid levels and increased adiponectin levels, indicating insulin sensitization via adiponectin. KY-903 reduced body weight gain and adipose tissue weight, while pioglitazone increased heart weight and markedly reduced bone mineral density. In mesenchymal stem cell-like ST2 cells, KY-903 slightly reduced osteoblast differentiation without adipocyte differentiation, while pioglitazone markedly reduced it with adipocyte differentiation. In conclusion, KY-903 is a novel PPARγ modulator that exerts anti-diabetic effects without body weight gain or cardiac hypertrophy in diabetic mice and anti-obesity effects with minor bone loss in OVX/HFD, possibly due to increases in adiponectin levels without adipogenesis.

The effects of three B-type proanthocyanidin (PA) dimers covering procyanidin B2 (B-0g), procyanidin B2 3'-O-gallate (B-1g), and procyanidin B2 3,3'-di-O-gallate (B-2g) on 3T3-L1 preadipocyte differentiation and the underlying mechanisms were investigated. The results showed that digalloylated B-type PA dimers (B-2g) strongly inhibited 3T3-L1 preadipocyte differentiation through disrupting the integrity of the lipid raft structure and inhibiting the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) and then downregulating the expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) factors, followed by B-1g, while B-0g had little effect. The different inhibitory effects were mainly due to the difference in the B-type PA dimer structure and the ability to interfere with lipid rafts. The greater the galloylation degree of B-type PA dimers, the stronger the ability to disrupt the lipid raft structure and oppose 3T3-L1 preadipocyte differentiation. In addition, galloylated B-type PA dimers had greater molecular hydrophobicity and topological polarity surface area and could penetrate into the lipid rafts to form multiple hydrogen bonds with the rafts by molecular dynamics simulation. These findings highlighted that the strong lipid raft-perturbing potency of galloylated B-type PA dimers was responsible for inhibition of 3T3-L1 preadipocyte differentiation.

Soyasaponins are triterpenoid glycosides found in soybean. We investigated whether soyasaponin ameliorates lipid metabolism and its possible mechanisms. In C57BL/6J mice fed a high-fat diet (HFD), soyasaponin (SAP) was orally administered for 9 weeks. Additionally, we evaluated the effect of soyasapogenols on 3T3-L1 adipocytes. In HFD-fed mice, the SAP significantly reduced body weight by 7% and relative adipose tissue weight by 35%. X-ray computed tomography demonstrated that the SAP reduced visceral and subcutaneous adipose tissue weights during week 3 of feeding. The SAP reduced sterol regulatory element-binding protein-1c (SREBP-1c) mRNA levels by 32% in the epididymal adipose tissue, significantly decreasing the triacylglycerol (TAG) content by 37% and SREBP-1c and fatty acid synthase mRNA levels by 52% and 61%, respectively, in the liver. In 3T3-L1 adipocytes, soyasapogenol B significantly decreased lipid droplets. The SAP containing soyasaponin A and B as conjugates demonstrate anti-obesity effects by suppressing adipocyte differentiation and lipogenesis, with a preventive effect on hepatic TAG accumulation by suppressing lipogenesis. PRACTICAL APPLICATION: Soyasaponin is one of the oleanane triterpenoids in soybeans. We have demonstrated that soyasaponin potently reduces body weight and white adipose tissue weight, and hepatic triacylglycerol accumulation in high-fat diet-fed mice. Thus, soyasaponin is a beneficial compound to prevent obesity and fatty liver.

Biotransformation, the structural modification of chemical compounds, has proved to be an indispensable tool in providing beneficial health effects. Although the health benefits of biotransformation using plant sources has been widely studied, the anti-adipogenic effect of biotransformed dairy products, such as whey, have not yet been demonstrated. Here, we investigated the anti-adipogenic effect of whey biotransformed by Weissella cibaria in 3T3-L1 adipocytes. Weissella cibaria-biotransformed whey considerably reduced the accumulation of lipid droplets and intracellular triglycerides in 3T3-L1 cells. In the presence of W. cibaria-biotransformed whey, the mRNA and protein expression of a key transcription factor, peroxisome proliferator-activated receptor γ (PPARγ), for adipogenesis was markedly suppressed in 3T3-L1 cells. Additionally, W. cibaria-biotransformed whey also decreased the mRNA and protein expressions of lipoprotein lipase and adipocyte fatty acid-binding protein, which are regulated by PPARγ. Moreover, W. cibaria-biotransformed whey inhibited the expression of adipokines, resistin, and leptin. Collectively, these results suggest that whey biotransformed by W. cibaria has the potential to exert anti-adipogenic effects by inhibiting intracellular signaling events of adipogenic-related transcription factors and target genes.

Obesity is a chronic disease that increases the risk of type II diabetes, heart diseases and nonalcoholic fatty liver disease. Unfortunately, to date, only a handful of drugs are approved for clinical use. This study aims at the discovery of anti-obesity agents based on naturally sourced oleanolic acid (OA) derivatives. 3T3-L1 preadipocytes were differentiated into mature adipocytes for in vitro assays, and a high-fat diet (HFD)-induced obesity mice model was established for in vivo studies. The screening of the OA derivatives was performed with 3T3-L1 cell, and resulted in a discovery of a novel compound HA-20 with a potent inhibitory activity on 3T3-L1 adipogenesis. In vitro data demonstrated that HA-20 markedly suppressed the adipogenesis in 3T3-L1 at the early stage without cytotoxicity. In vivo research using HFD mice revealed that HA-20 lowered the body weight, and possessed a lipid-lowering effect. Transcriptome analysis discovered that the mainly adipogenesis/lipogenesis genes regulated by HA-20 were Pparg, Cebpa, Fas, Acc, and Fabp4/aP2. Mechanism study revealed that HA-20 played its bioactive roles at least via downregulating PPARγ-FABP4/aP2 pathway in 3T3-L1, which was further confirmed in HFD-induced obesity mice. Our findings provided a new insight into fighting fat accumulation based on OA derivatives, and demonstrated that HA-20 may sever as a worthy leading compound for the further development of anti-obesity agents.

Liver fibrosis, with the characterization of progressive accumulation of extracellular matrix (ECM), is the common pathologic feature in the process of chronic liver disease. Hepatic stellate cells (HSCs) which are activated and differentiate into proliferative and contractile myofibroblasts are recognized as the main drivers of fibrosis. Obesity-related adipocytokine dysregulation is known to accelerate liver fibrosis progression, but the direct fibrogenic effect of mature adipocytes on HSCs has been rarely reported. Therefore, the purpose of this study was to explore the fibrogenic effect of adipocyte 3T3-L1 cells on hepatic stellate LX-2 cells. The results showed that incubating LX-2 cells with the supernatant of 3T3-L1 adipocytes triggered the expression of ECM related proteins, such as α-smooth muscle actin (α-SMA), type I collagen (CO-I), and activated TGF β/Smad2/3 signaling pathway in LX-2 cells. In addition, 3T3-L1 cells inhibited insulin sensitivity, activated endoplasmic reticulum stress and autophagy to promote the development of fibrosis. These results supported the notion that mature adipocytes can directly activate hepatic stellate cells, and the establishment of an in vitro model of adipocytes on HSCs provides an insight into screening of drugs for liver diseases, such as nonalcoholic fatty liver disease.

Obesity is a major contributing factor to the pathogenesis of Type 2 diabetes. Multiple human genetics studies suggest that high activity of the low molecular weight protein tyrosine phosphatase (LMPTP) promotes metabolic syndrome in obesity. We reported that LMPTP is a critical promoter of insulin resistance in obesity by regulating liver insulin receptor signaling and that inhibition of LMPTP reverses obesity-associated diabetes in mice. Since LMPTP is expressed in adipose tissue but little is known about its function, here we examined the role of LMPTP in adipocyte biology. Using conditional knockout mice, we found that selective deletion of LMPTP in adipocytes impaired obesity-induced subcutaneous adipocyte hypertrophy. We assessed the role of LMPTP in adipogenesis in vitro, and found that LMPTP deletion or knockdown substantially impaired differentiation of primary preadipocytes and 3T3-L1 cells into adipocytes, respectively. Inhibition of LMPTP in 3T3-L1 preadipocytes also reduced adipogenesis and expression of proadipogenic transcription factors peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha. Inhibition of LMPTP increased basal phosphorylation of platelet-derived growth factor receptor alpha (PDGFRα) on activation motif residue Y849 in 3T3-L1, resulting in increased activation of the mitogen-associated protein kinases p38 and c-Jun N-terminal kinase and increased PPARγ phosphorylation on inhibitory residue S82. Analysis of the metabolome of differentiating 3T3-L1 cells suggested that LMPTP inhibition decreased cell glucose utilization while enhancing mitochondrial respiration and nucleotide synthesis. In summary, we report a novel role for LMPTP as a key driver of adipocyte differentiation via control of PDGFRα signaling.

Persimmon peels, though usually discarded, are useful sources of nutraceuticals. In this study, persimmon peel–derived pomolic acid was found to suppress the increase in the activity of glycerol-3 phosphate dehydrogenase, a neutral fat synthesis-related enzyme, in 3T3-L1 adipocytes, whereas oleanolic and ursolic acids did not exert this effect. Therefore, persimmon peel may be an effective functional food material.

Aims/hypothesis Levels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model. Methods miR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic–hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue. Results The proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance. Conclusions/interpretation Concurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target. Graphical abstract

The antiadipogenic activity of conjugated linoleic acids (CLA) in the form of phosphatidylcholine-bound (CLA-PC) or free fatty acids (FFA; CLA-FFA) was evaluated using 3T3-L1 adipocytes. Phosphatidylcholine from soya (soy-PC) was used as the comparison of PC form. Both the lipid accumulation and activity of glycerol-3-phosphate dehydrogenase were measured to determine lipogenesis, whereas the glycerol content was measured to evaluate lipolysis. The CLA uptake also measured to find out the utilization of CLA by the cells. As a results, lipid accumulation in 3T3-L1 adipocytes was inhibited in a dose-dependent manner following treatment with CLA-PC (50-400 μM). Both CLA-PC and soy-PC significantly suppressed lipid accumulation compared with CLA-FFA, even though the amount of CLA in CLA-PC was a half than CLA-FFA. The CLA uptake of PC form was superior to FFA form, however, no difference was noted between CLA-PC and soy-PC. These forms exerted their antiadipogenic activity via the suppression of lipogenesis, and not by increasing lipolysis. Short-term treatment, especially in the middle stage of differentiation, was more effective than long-term treatment; especially for CLA-FFA. The antiadipogenic effect of CLA-PC was partially attributed to the chemical structure of the PC molecule. These results provide important information for the utilization of physiologically functional fatty acids and particularly CLA in the food and medical fields.

Obesity is a pathological state related to various lifestyle-related diseases, such as diabetes and dyslipidemia, that may be prevented through the development of anti-obesity treatments. Lipid accumulation in cells could be affected by vitamin E ester α-tocopheryl succinate (TS), which has various biological activities, such as anti-cancer effect, via activation of cell signaling pathways, although the antioxidative activity of TS is lost due to esterification of the phenolic OH group. In this study, we found for the first time that TS significantly suppressed lipid accumulation in mouse 3T3-L1 adipocytes. TS treatment reduced the amount of triglycerides in the culture medium, and inhibited activity of glycerol-3-phosphate dehydrogenase, a marker of lipid synthesis. Furthermore, TS accelerated lipolysis. Treatment of adipocytes with TS for 24 h induced no significant cytotoxicity. In TS-treated cells, phosphorylation of Akt, which is involved in fatty acid synthesis via sterol regulatory element-binding proteins (SREBP), was prevented, while levels of phosphorylated protein kinase A (PKA) did not change. Taken together, these results suggest that vitamin E ester TS can suppress lipid accumulation in adipocytes by regulating lipid metabolic cell signaling.

Background/objectives The steamed ginger has been shown to have antioxidative effects and a protective effect against obesity. In the present study, we investigated the effects of ethanolic extract of steamed ginger (SGE) on adipogenesis in 3T3-L1 preadipocytes and diet-induced obesity (DIO) mouse model. Materials/methods The protective effects of SGE on adipogenesis were examined in 3T3-L1 adipocytes by measuring lipid accumulations and genes involved in adipogenesis. Male C57BL/6J mice were fed a normal diet (ND, 10% fat w/w), a high-fat diet (HFD, 60% fat w/w), and HFD supplemented with either 40 mg/kg or 80 mg/kg of SGE for 12 weeks. Serum chemistry was measured, and the expression of genes involved in lipid metabolism was determined in the adipose tissue. Histological analysis and micro-computed tomography were performed to identify lipid accumulations in epididymal fat pads. Results In 3T3-L1 cells, SGE significantly decreased lipid accumulation, with concomitant decreases in the expression of adipogenesis-related genes. SGE significantly attenuated the increase in body, liver, and epididymal adipose tissue weights by HFD. Serum total cholesterol and triglyceride levels were significantly lower in SGE fed groups compared to HFD. In adipose tissue, SGE significantly decreased adipocyte size than that of HFD and altered adipogenesis-related genes. Conclusions In conclusion, steamed ginger exerted anti-obesity effects by regulating genes involved in adipogenesis and lipogenesis in 3T3-L1 cell and epididymal adipose tissue of DIO mice.

Acute organophosphorus pesticide poisoning (AOPP) is a worldwide health concern that has threatened human lives for decades, which attacks acetylcholinesterase (AChE) and causes nervous system disorders. Classical treatment options are associated with short in vivo half-life and side effects. As a potential alternative, delivery of mammalian-derived butyrylcholinesterase (BChE) offers a cost-effective way to block organophosphorus attack on acetylcholinesterase, a key enzyme in the neurotransmitter cycle. Yet the use of exotic BChE as a prophylactic or therapeutic agent is compromised by short plasma residence, immune response and unfavorable biodistribution. To overcome these obstacles, BChE nanodepots (nBChE) composed of a BChE core/polymorpholine shell structure were prepared via in situ polymerization, which showed enhanced stability, prolonged plasma circulation, attenuated antigenicity and reduced accumulation in non-targeted tissues. In vivo administration of nBChE pre- or post-organophosphorus exposure in a BALB/C mouse model resulted in potent prophylactic and therapeutic efficiency. To our knowledge, this is the first systematic delivery of non-human BChE to tackle AOPP. In addition, this work also opens up a new avenue for real applications in both research and clinical settings to cope with acute intoxication-related diseases.

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.

Trehalose is a disaccharide reported to ameliorate adipocyte hypertrophy and improve metabolic state. The present study demonstrated that trehalose augments the levels of SQSTM1, lysosome‐related factors and antioxidative factors, which induces lysosomal activity and antioxidative capacity in adipocytes. These observations reveal a mechanism for the above effects of trehalose., Adipocytes, which comprise the majority of white adipose tissue (WAT), are involved in obesity‐related pathology via various mechanisms, including disturbed lysosomal enzymatic activity and accumulation of oxidative stress. Sequestosome 1 (SQSTM1/p62) is an autophagy marker that participates in antioxidative responses via the activation of nuclear factor erythroid‐derived 2‐like 2 (NRF2). Trehalose is a non‐reducing disaccharide reported to suppress adipocyte hypertrophy in obese mice and improve glucose tolerance in humans. We recently revealed that trehalose increases SQSTM1 levels and enhances antioxidative capacity in hepatocytes. Here, to further evaluate the mechanism behind the beneficial effects of trehalose on metabolism, we examined SQSTM1 levels, autophagy, and oxidative stress in trehalose‐treated adipocytes. We initially confirmed that trehalose increases SQSTM1 transcription and protein levels without affecting autophagy in adipocytes. Trehalose also elevated transcription of several lysosomal genes and the activity of cathepsin L, a lysosomal enzyme, independently of the transcription factor EB. In agreement with our data from hepatocytes, trehalose induced the nuclear translocation of NRF2 and the transcription of its downstream antioxidative genes, resulting in reduced cellular reactive oxygen species levels. Moreover, some cellular trehalose was detected in trehalose‐treated adipocytes, implying that extracellular trehalose is taken into cells. These observations reveal the mechanism behind the beneficial effects of trehalose on metabolism and suggest its potential for preventing or treating obesity‐related pathology.

Obesity is growing at an alarming rate, which is characterized by increased adipose tissue. It increases the probability of many health complications, such as diabetes, arthritis, cardiac disease, and cancer. In modern society, with a growing population of obese patients, several individuals have increased insulin resistance. Herbal medicines are known as the oldest method of health care treatment for obesity-related secondary health issues. Several traditional medicinal plants and their effective phytoconstituents have shown anti-diabetic and anti-adipogenic activity. Adipose tissue is a major site for lipid accumulation as well as the whole-body insulin sensitivity region. 3T3-L1 cell line model can achieve adipogenesis. Adipocyte characteristics features such as expression of adipocyte markers and aggregation of lipids are chemically induced in the 3T3-L1 fibroblast cell line. Differentiation of 3T3-L1 is an efficient and convenient way to obtain adipocyte like cells in experimental studies. Peroxisome proliferation activated receptor γ (PPARγ) and Cytosine-Cytosine-Adenosine-Adenosine-Thymidine/Enhancer-binding protein α (CCAAT/Enhancer-binding protein α or C/EBPα) are considered to be regulating adipogenesis at the early stage, while adiponectin and fatty acid synthase (FAS) is responsible for the mature adipocyte formation. Excess accumulation of these adipose tissues and lipids leads to obesity. Thus, investigating adipose tissue development and the underlying molecular mechanism is important in the therapeutical approach. This review describes the cellular mechanism of 3T3-L1 fibroblast cells on potential anti-adipogenic herbal bioactive compounds.

Objective Expression of zinc finger protein 423 (ZFP423), a key proadipogenic transcription factor in adipocyte precursor cells, is regulated by interaction of the proadipogenic early B-cell factor 1 (EBF1) and antiadipogenic ZFP521. The ubiquitin ligase seven-in-absentia homolog 2 (SIAH2) targets ZFP521 for degradation. This study asked whether SIAH2 is expressed in adipocyte precursor cells and whether SIAH2 interacts with ZFP521 and EBF1 to regulate ZFP521 protein levels during adipogenesis. Methods SIAH2 expression in precursor cells was assessed in primary cells and tissues from wild-type and SIAH2 null mice fed a control or high-fat diet. Primary cells, 3T3-L1 preadipocytes, and HEK293T cells were used to analyze Siah2, Ebf1, and Zfp521 expression and SIAH2-mediated changes in ZFP521 and EBF1 protein levels. Results Siah2 is expressed in platelet-derived growth factor receptor α (PDGFRα)+ and stem cell antigen-1 (SCA1)+ adipocyte precursor cells. SIAH2 depletion reduces Ebf1 gene expression and increases EBF1 protein levels in early but not late adipogenesis. In early adipogenesis, SIAH2 forms a protein complex with EBF1 and ZFP521 to enhance SIAH2-mediated ubiquitylation and degradation of ZFP521 while increasing EBF1 protein levels. Conclusions Siah2 is expressed in PDGFRα+ adipocyte precursor cells and is linked to precursor cell commitment to adipogenesis by interacting with EBF1 and ZFP521 proteins to target the antiadipogenic ZFP521 for degradation.

Obesity, which has become a major public health problem, can arise from complex dyslipidemia, insulin resistance, and immune responses, among other mechanisms. Some Lactobacillus strains effectively ameliorate obesity; however, the beneficial effects of Lactococcus spp., which are often used as dairy starters, remain unclear. In the present study, we evaluated the efficacy of Lactococcus chungangensis CAU 28 using the 3T3-L1 cell line and obese mice fed a high-fat diet. Overall, administration of Lc. chungangensis CAU 28 effectively resolved obesity associated with weight gain and lipid accumulation. In differentiated 3T3-L1 cells, Lc. chungangensis CAU 28 treatment significantly diminished the total lipid quantity, inhibited triglyceride formation, and prevented the proliferation of adipogenic transcription factors (fatty acid synthase, adiponectin, peroxisome proliferator-activated receptor-gamma, and CCAAT-enhancer-binding protein-α) associated with lipid accumulation. In the obesity mouse model, wherein the intake of Lc. chungangensis CAU 28 effectively reduced body weight gain, along with fat differentiation and accumulation (white fat; abdominal and subcutaneous). Furthermore, Lc. chungangensis CAU 28 increased serum adiponectin levels, decreased serum leptin levels, and effectively regulated adipokine secretion. It also increased the high-density lipoprotein:cholesterol ratio, reduced total cholesterol and triglyceride levels, reduced the low-density lipoprotein:cholesterol ratio, and affected obesity-regulated inflammatory cytokines IL-6, tumor necrosis factor-α, IFN-γ, and IL-1β. Additionally, Lc. chungangensis CAU 28 was associated with an increase in the CD3+CD4+CD8- phenotype among obese mice. Thus, the administration of Lc. chungangensis CAU 28 induced antiobesity effects, suggesting potential applications of this species as a supplement for obesity mitigation.

Obesity is strongly associated with insulin resistance (IR). IR at the molecular level may be defined as a diminished activation of insulin signaling-related molecules (IRS-1/Akt/AS160) as well as reduced glucose uptake. Subject with obesity have elevated plasma levels of saturated fatty acids, such as palmitic acid (PA), which triggers insulin signaling disruption in vivo and in vitro. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in folk medicine of Northern Chile to counteract inflammatory diseases. Hydroethanolic extracts of lampaya (HEL) contain considerable amounts of flavonoids that may explain the biological activity of the plant. The aim of this study was to assess whether HEL exposure protects against PA-disrupted insulin signaling and glucose uptake in adipocytes.Cytotoxicity of a range of HEL concentrations (0.01-10 μg/mL) was evaluated in 3T3-L1 adipocytes. Cells were exposed or not to 0.1 μg/mL of HEL before adding 0.65 mM PA or vehicle and incubated with 100 nM insulin (or vehicle) for 15 min. Phosphorylation of Tyr-IRS-1, Ser-Akt, Thr-AS160 was evaluated by Western blot. Glucose uptake was assessed using the 2-NBDG analogue.HEL was not cytotoxic at any concentration assessed. PA-induced reduction in insulin-stimulated phosphorylation of IRS-1, Akt and AS160 and glucose uptake were abolished by co-treatment with HEL.These findings give new insights about the effect of HEL ameliorating PA- impaired IRS-1/Akt/AS160 pathway and glucose uptake in adipocytes. More studies should focus on lampaya, since might represent a preventive approach in individuals whose circulating PA levels contribute to IR.

The incretin hormone glucose-dependent insulinotropic polypeptide (GIP), released postprandially from K-cells, has established actions on adipocytes and lipid metabolism. In addition, xenin, a related peptide hormone also secreted from K-cells after a meal, has postulated effects on energy regulation and lipid turnover. The current study has probed direct individual and combined effects of GIP and xenin on adipocyte function in 3T3-L1 adipocytes, using enzyme-resistant peptide analogues, (d-Ala2)GIP and xenin-25-Gln, and knockdown (KD) of receptors for both peptides. (d-Ala2)GIP stimulated adipocyte differentiation and lipid accumulation in 3T3-L1 adipocytes over 96 h, with xenin-25-Gln evoking similar effects. Combined treatment significantly countered these individual adipogenic effects. Individual receptor KD impaired lipid accumulation and adipocyte differentiation, with combined receptor KD preventing differentiation. (d-Ala2)GIP and xenin-25-Gln increased glycerol release from 3T3-L1 adipocytes, but this lipolytic effect was significantly less apparent with combined treatment. Key adipogenic and lipolytic genes were upregulated by (d-Ala2)GIP or xenin-25-Gln, but not by dual peptide culture. Similarly, both (d-Ala2)GIP and xenin-25-Gln stimulated insulin-induced glucose uptake in 3T3-L1 adipocytes, but this effect was annulled by dual treatment. In conclusion, GIP and xenin possess direct, comparable, lipogenic and lipolytic actions in 3T3-L1 adipocytes. However, effects on lipid metabolism are significantly diminished by combined administration.

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2'-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.

Insulin stimulates glucose uptake via the translocation of the glucose transporter GLUT4 to the plasma membrane in adipocytes. Several lines of evidence suggest that the small GTPase Rac1 plays an important role in insulin-stimulated glucose uptake in skeletal muscle and adipocytes. The purpose of this study is to investigate the mechanisms whereby Rac1 is regulated in adipocyte insulin signaling. Here, we show that knockdown of the guanine nucleotide exchange factor FLJ00068 inhibits Rac1 activation and GLUT4 translocation by insulin and a constitutively activated form of the protein kinase Akt2. Furthermore, constitutively activated FLJ00068 induced Rac1 activation and Rac1-dependent GLUT4 translocation. Collectively, these results suggest the involvement of FLJ00068 downstream of Akt2 in insulin-stimulated glucose uptake signaling in adipocytes.