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Objective: The study was designed to test solvent extracts of Rubus suavissimus (RS) for compounds that regulate lipid metabolism and to measure their relative activity as quantitative markers of lipid metabolism-regulating activities based on the spectrum-effect relationship. Method: RS was extracted with 95% ethanol, the different extraction parts were successively extracted with petroleum ether, methylene chloride, ethyl acetate and n-butanol, respectively. The solution of different extraction parts was applied to the induced differentiated 3T3-L1 preadipocytes, and the oil red O staining and triglyceride release from the cells were determined to screen the best metabolism-regulating site. Analysis of different extraction sited by UPLC-Q-TOF-MS/MS technique to separate and identify the components. Based on specific assays, image intensity analysis, grey correlation and partial least squares regressions, the spectrum-effect relationship of the most abundant active components and their ability to regulate lipid metabolism were determined, and the quality markers were screened. Results: Ethyl acetate and n-butanol extraction site was the best metabolism-regulating site. Four components, including ellagic acid, centaurin-3-O-rutinoside, rubusoside and enantio-kauri-16-ene-19-carboxylic acid-13-O-β-D-glucoside were highly associated with metabolism-regulating effects. It could be used as quality markers for regulating lipid metabolism. Conclusion: Investigation of the quality markers of RS extracts based on the spectrum-effect relationship is of great importance for elucidating the pharmacodynamics, screening the core quality markers for therapeutic activity, and ensuring the safety and rational application of traditional medicines. [ABSTRACT FROM AUTHOR]

Objective: The study was designed to test solvent extracts of Rubus suavissimus (RS) for compounds that regulate lipid metabolism and to measure their relative activity as quantitative markers of lipid metabolism-regulating activities based on the spectrum-effect relationship. Method: RS was extracted with 95% ethanol, the different extraction parts were successively extracted with petroleum ether, methylene chloride, ethyl acetate and n-butanol, respectively. The solution of different extraction parts was applied to the induced differentiated 3T3-L1 preadipocytes, and the oil red O staining and triglyceride release from the cells were determined to screen the best metabolism-regulating site. Analysis of different extraction sited by UPLC-Q-TOF-MS/MS technique to separate and identify the components. Based on specific assays, image intensity analysis, grey correlation and partial least squares regressions, the spectrum-effect relationship of the most abundant active components and their ability to regulate lipid metabolism were determined, and the quality markers were screened. Results: Ethyl acetate and n-butanol extraction site was the best metabolism-regulating site. Four components, including ellagic acid, centaurin-3-O-rutinoside, rubusoside and enantio-kauri-16-ene-19-carboxylic acid-13-O-β-D-glucoside were highly associated with metabolism-regulating effects. It could be used as quality markers for regulating lipid metabolism. Conclusion: Investigation of the quality markers of RS extracts based on the spectrum-effect relationship is of great importance for elucidating the pharmacodynamics, screening the core quality markers for therapeutic activity, and ensuring the safety and rational application of traditional medicines.

Abstract Interleukin-33 (IL-33), an emerging cytokine within the IL-1 family, assumes a pivotal function in the control of obesity. However, the specific mechanism of its regulation of obesity formation remains unclear. In this study, we found that the expression level of IL-33 increased in visceral adipose tissue in mice fed with a high-fat diet (HFD) compared with that in mice fed with a normal diet (ND). In vitro, we also found the expression level of IL-33 was upregulated during the adipogenesis of 3T3-L1 cells. Functional test results showed that knockdown of IL-33 in 3T3-L1 cells differentiation could promote the accumulation of lipid droplets, the content of triglyceride and the expression of adipogenic–related genes (i.e. PPAR-γ, C/EBPα, FABP4, LPL, Adipoq and CD36). In contrast, overexpression of IL-33 inhibits adipogenic differentiation. Meanwhile, the above tests were repeated after over-differentiation of 3T3-L1 cells induced by oleic acid, and the results showed that IL-33 played a more significant role in the regulation of adipogenesis. To explore the mechanism, transcriptome sequencing was performed and results showed that IL-33 regulated the PPAR signaling pathway in 3T3-L1 cells. Further, Western blot and confocal microscopy showed that the inhibition of IL-33 could promote PPAR-γ expression by inhibiting the Wnt/β-catenin signal in 3T3-L1 cells. This study demonstrated that IL-33 was an important regulator of preadipocyte differentiation and inhibited adipogenesis by regulating the Wnt/β-catenin/PPAR-γ signaling pathway, which provided a new insight for further research on IL-33 as a new intervention target for metabolic disorders.

Interleukin-33 (IL-33), an emerging cytokine within the IL-1 family, assumes a pivotal function in the control of obesity. However, the specific mechanism of its regulation of obesity formation remains unclear. In this study, we found that the expression level of IL-33 increased in visceral adipose tissue in mice fed with a high-fat diet (HFD) compared with that in mice fed with a normal diet (ND). In vitro, we also found the expression level of IL-33 was upregulated during the adipogenesis of 3T3-L1 cells. Functional test results showed that knockdown of IL-33 in 3T3-L1 cells differentiation could promote the accumulation of lipid droplets, the content of triglyceride and the expression of adipogenic–related genes (i.e. PPAR-γ, C/EBPα, FABP4, LPL, Adipoq and CD36). In contrast, overexpression of IL-33 inhibits adipogenic differentiation. Meanwhile, the above tests were repeated after over-differentiation of 3T3-L1 cells induced by oleic acid, and the results showed that IL-33 played a more significant role in the regulation of adipogenesis. To explore the mechanism, transcriptome sequencing was performed and results showed that IL-33 regulated the PPAR signaling pathway in 3T3-L1 cells. Further, Western blot and confocal microscopy showed that the inhibition of IL-33 could promote PPAR-γ expression by inhibiting the Wnt/β-catenin signal in 3T3-L1 cells. This study demonstrated that IL-33 was an important regulator of preadipocyte differentiation and inhibited adipogenesis by regulating the Wnt/β-catenin/PPAR-γ signaling pathway, which provided a new insight for further research on IL-33 as a new intervention target for metabolic disorders. [ABSTRACT FROM AUTHOR]

The global prevalence of obesity is rising year by year, which has become a public health problem worldwide. Many animal and clinical studies have shown that Lactiplantibacillus plantarum is considered an ideal probiotic and potential supplement for the treatment of obesity. In this study, we aimed to complete the genome sequence of L. plantarum HOM2217, which was isolated from human milk, and study its physiological characteristics and anti-obesity effects in 3T3-L1 cells and rats fed a high-fat diet (HFD) to determine its potential as a starter for functional food products. Whole-genome analysis demonstrated that HOM2217 contained a single circular chromosome of 3,267,529 bp with a GC content of 44.5% and one plasmid (62,350 bp) with a GC content of 38.5%. Compared to the reference strains, HOM2217 demonstrated superior tolerance to gastrointestinal conditions, higher adhesion to intestinal epithelial cell lines, potent antimicrobial activity against Enterobacter cloacae ATCC 13047, and effective cholesterol removal ability in vitro. Treatment with heat-killed HOM2217 significantly reduced lipid accumulation and intracellular triglyceride production in 3T3-L1 adipocytes. Daily treatment of HFD-fed rats with HOM2217 for 7 weeks decreased body weight, body weight gain, and body fat without changes in food intake. HOM2217 also significantly increased the serum high-density lipoprotein cholesterol (HDL-C) level, decreased the serum tumor necrosis factor (TNF-α) and increased short-chain fatty acid (SCFA) (formic acid, acetic acid, and butyric acid) levels in the cecum. Thus, HOM2217 could potentially prevent obesity in rats by inhibiting inflammatory responses and regulating lipid metabolism and SCFAs expression. Therefore, HOM2217 has potential as an alternative treatment for obesity.

Objective: To investigate the active components, protein targets, and mechanisms underlying the anti-obesity effects of Hippophae fructus using network pharmacology and molecular docking techniques, and to validate its in vitro anti-obesity efficacy. Methods: The TCMSP platform was utilized to retrieve the active components and targets of Hippophae fructus, and disease targets were collected. Venny 2.0.2 was used to identify the intersection of targets between Hippophae fructus and obesity-related targets. The STRING database was used to establish a drug-target-disease protein interaction (PPI) network. The intersecting targets were analyzed using the David database to perform GO enrichment analysis and KEGG pathway analysis. Cytoscape 3.9.1 was used to construct a network diagram of the components of Hippophae fructus, anti-obesity targets, and related signaling pathways. Autodock Dock 1.5.7 and Pymol 2.2.0 were used to carry out molecular docking between the core targets of Hippophae fructus and its components, followed by visualization. The in vitro anti-obesity effect of Hippophae fructus extract was evaluated through cell experiments using 3T3-L1 cells. Results: A total of 33 active components, 2820 disease targets, and 151 intersection targets of Hippophae fructus were identified. The main active components included flavonoids, vitamins, and sterols, while key targets involved AKT1, TNF, IL6, TP53, VEGFA, CASP3, and others. KEGG pathway enrichment analysis revealed 131 signaling pathways, including those related to malignant tumors, lipid and atherosclerosis, and AGE-RAGE signaling. Molecular docking results demonstrated favorable binding interactions between the core targets and the corresponding active components of Hippophae fructus. The in vitro experiments indicated that Hippophae fructus extract exhibited inhibitory effects on the proliferation of 3T3-L1 pre-adipocytes. Conclusion: This study reveals that Hippophae fructus exerts anti-obesity effects through multiple components, targets, and pathways, providing valuable insights for its clinical research and product development.

Objective: To investigate the active components, protein targets, and mechanisms underlying the anti-obesity effects of Hippophae fructus using network pharmacology and molecular docking techniques, and to validate its in vitro anti-obesity efficacy. Methods: The TCMSP platform was utilized to retrieve the active components and targets of Hippophae fructus, and disease targets were collected. Venny 2.0.2 was used to identify the intersection of targets between Hippophae fructus and obesity-related targets. The STRING database was used to establish a drug-target-disease protein interaction (PPI) network. The intersecting targets were analyzed using the David database to perform GO enrichment analysis and KEGG pathway analysis. Cytoscape 3.9.1 was used to construct a network diagram of the components of Hippophae fructus, anti-obesity targets, and related signaling pathways. Autodock Dock 1.5.7 and Pymol 2.2.0 were used to carry out molecular docking between the core targets of Hippophae fructus and its components, followed by visualization. The in vitro anti-obesity effect of Hippophae fructus extract was evaluated through cell experiments using 3T3-L1 cells. Results: A total of 33 active components, 2820 disease targets, and 151 intersection targets of Hippophae fructus were identified. The main active components included flavonoids, vitamins, and sterols, while key targets involved AKT1, TNF, IL6, TP53, VEGFA, CASP3, and others. KEGG pathway enrichment analysis revealed 131 signaling pathways, including those related to malignant tumors, lipid and atherosclerosis, and AGE-RAGE signaling. Molecular docking results demonstrated favorable binding interactions between the core targets and the corresponding active components of Hippophae fructus. The in vitro experiments indicated that Hippophae fructus extract exhibited inhibitory effects on the proliferation of 3T3-L1 pre-adipocytes. Conclusion: This study reveals that Hippophae fructus exerts antiobesity effects through multiple components, targets, and pathways, providing valuable insights for its clinical research and product development. [ABSTRACT FROM AUTHOR]

Obesity is the excessive accumulation of body fat resulting from impairment in energy balance mechanisms. In this study, we aimed to investigate the mechanism whereby GABA (γ-aminobutyric acid) prevents high-fat diet-induced obesity, and whether it induces lipolysis and browning in white adipose tissue (WAT), using high-fat diet (HFD)-fed obese mice and 3T3-L1 adipocytes. We demonstrated that GABA substantially inhibits the body mass gain of mice by suppressing adipogenesis and lipogenesis. Consistent with this result, histological analysis of WAT demonstrated that GABA decreases adipocyte size. Moreover, we show that GABA administration decreases fasting blood glucose and improves serum lipid profiles and hepatic lipogenesis in HFD-fed obese mice. Furthermore, Western blot and immunofluorescence analyses showed that GABA activates protein kinase A (PKA) signaling pathways that increase lipolysis and promote uncoupling protein 1 (UCP1)-mediated WAT browning. Overall, these results suggest that GABA exerts an anti-obesity effect via the regulation of lipid metabolism. [ABSTRACT FROM AUTHOR]

Abstract Background Gremlin-1 (GREM1) and Gremlin-2 (GREM2) are bone morphogenetic protein antagonists that play important roles in organogenesis, tissue differentiation, and tissue homeostasis. Although GREM1 has been reported to be involved in promoting various cancers, little has been reported about effects of GREM2 on cancer. Recently, it has been reported that GREM2 can inhibit adipogenesis in adipose-derived stromal/stem cells. However, as an inhibitor of adipogenesis, the role of GREM2 in cancer progression is not well understood yet. Methods Pre-adipocyte 3T3-L1 cells overexpressing mock or Grem2 were established using a lentiviral transduction system and differentiated into adipocytes-mock and adipocytes-Grem2, respectively. To investigate the effect of adipocyte-Grem2 on breast cancer cells, we analyzed the proliferative and invasion abilities of spheroids using a 3D co-culture system of breast cancer cells and adipocytes or conditioned medium (CM) of adipocytes. An orthotopic breast cancer mouse model was used to examine the role of adipocytes-Grem2 in breast cancer progression. Results Grem2 overexpression suppressed adipogenesis of 3T3-L1 cells. Proliferative and invasion abilities of spheroids formed by co-culturing MTV/TM-011 breast cancer cells and adipocytes-Grem2 were significantly reduced compared to those of spheroids formed by co-culturing MTV/TM-011 cells and adipocytes-mock. Compared to adipocytes-mock, adipocytes-Grem2 showed decreased mRNA expression of several adipokines, notably IL-6. The concentration of IL-6 in the CM of these cells was also decreased. Proliferative and invasive abilities of breast cancer cells reduced by adipocytes-Grem2 were restored by IL-6 treatment. Expression levels of vimentin, slug, and twist1 in breast cancer cells were decreased by treatment with CM of adipocytes-Grem2 but increased by IL-6 treatment. In orthotopic breast cancer mouse model, mice injected with both MTV/TM-011 cells and adipocytes-Grem2 showed smaller primary tumors and lower lung metastasis than controls. However, IL-6 administration increased both the size of primary tumor and the number of metastatic lung lesions, which were reduced by adipocytes-Grem2. Conclusions Our study suggests that GREM2 overexpression in adipocytes can inhibit adipogenesis, reduce the expression and secretion of several adipokines, including IL-6, and ultimately inhibit breast cancer progression.

There is increasing evidence of a role for environmental contaminants in disrupting metabolic health in both humans and animals. Despite a growing need for well-understood models for evaluating adipogenic and potential obesogenic contaminants, there has been a reliance on decades-old in vitro models that have not been appropriately managed by cell line providers. There has been a quick rise in available in vitro models in the last ten years, including commercial availability of human mesenchymal stem cell and preadipocyte models; these models require more comprehensive validation but demonstrate real promise in improved translation to human metabolic health. There is also progress in developing three-dimensional and co-culture techniques that allow for the interrogation of a more physiologically relevant state. While diverse rodent models exist for evaluating putative obesogenic and/or adipogenic chemicals in a physiologically relevant context, increasing capabilities have been identified for alternative model organisms such as Drosophila, C. elegans, zebrafish, and medaka in metabolic health testing. These models have several appreciable advantages, including most notably their size, rapid development, large brood sizes, and ease of high-resolution lipid accumulation imaging throughout the organisms. They are anticipated to expand the capabilities of metabolic health research, particularly when coupled with emerging obesogen evaluation techniques as described herein.

Fat browning, which converts white adipose tissue to brown, has attracted attention as a promising strategy for the treatment of obesity. Betanin (BT) has been reported to have potential anti-obesity activity. 3T3-L1 cells were differentiated for 7 days during BT treatment. The BT concentration range for the study was determined using an MTT assay, and lipid accumulation was evaluated by Oil-Red-O staining. The expression of protein level was analyzed by Western blot. Immunofluorescence images were performed with confocal microscopy to visually show the amount and location of thermogenesis factor uncoupling protein1 (UCP1) and mitochondria. qRT-PCR was performed to evaluate mRNA expression. BT inhibited lipid accumulation and increased the expression of UCP1, peroxisome-proliferator-activated receptor gamma (PPARγ), and PPARγ coactivator-1 alpha (PGC-1α). In addition, the increases in beige adipocyte-specific markers were observed, supporting BT-mediated browning of the fat tissue. The UCP1 was localized in the inner membrane of the mitochondria, and its expression was associated with mitochondrial activation. Consistent with this, the mRNA expression of mitochondrial biogenesis markers increased in 3T3-L1 cells after BT treatment. Immunofluorescence staining also indicated an increased number of mitochondria and UCP1, respectively. Moreover, BT inhibited lipogenesis and enhanced lipolysis and fatty acid oxidation. This mechanism has been suggested to be mediated by an adenosine monophosphate-activated protein kinase (AMPK) pathway. BT induces fat browning and regulates lipid metabolism via the AMPK-mediated pathway in 3T3-L1 cells, suggesting that BT can be a promising candidate for controlling obesity. [ABSTRACT FROM AUTHOR]

Natural polyphenol-rich plant resources, such as agricultural waste, were proven to diminish insulin resistance and weight gain in rats on a high-fat diet. To test whether date seed polyphenol pills (DSPPs) might lower adipose tissue accumulation by precisely affecting adipocytes, we explored the impacts of DSPPs on cell proliferation, differentiation, and lipolysis in 3T3-L1 cells. We utilized tablets made commercially from date seed polyphenols that were mostly composed of epicatechin (45.9 g/kg). The total polyphenol and antioxidant capacities of the digested and non-digested DSPPs were also evaluated. DSPPs at doses of 25, 50, and 100 µg/mL hindered the proliferation of both pre-confluent preadipocytes and mature post-confluent adipocytes. DSPPs decreased the quantity of viable cells in completely developed adipocytes. Treatment with 100 µg/mL of DSPPs decreased the basal lipolysis of completely differentiated adipocytes but modestly boosted epinephrine-induced lipolysis. A significant transcription factor for the adipogenic gene, the peroxisome proliferator-activated receptor (PPAR), was repressed by DSPPs, which significantly decreased lipid buildup. The total polyphenol and antioxidant capacities were also increased after digestion with a good bubble Pearson correlation between both. DSPPs may have anti-obesity and anti-diabetic characteristics by inhibiting adipocyte development and basal lipolysis, which could be commercially industrialized. [ABSTRACT FROM AUTHOR]

Background and Objectives: This study evaluated the in vitro anti-adipogenic and anti-inflammatory properties of black cumin (Nigella sativa L.) seed extract (BCS extract) as a potential candidate for developing herbal formulations targeting metabolic disorders. Materials and Methods: We evaluated the BCS extract by assessing its 2,2-diphenyl-1-picrohydrazyl (DPPH) radical scavenging activity, levels of prostaglandin E2 (PGE2) and nitric oxide (NO), and mRNA expression levels of key pro-inflammatory mediators. We also quantified the phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) signaling molecules. To assess anti-adipogenic effects, we used differentiated 3T3-L1 cells and BCS extract in doses from 10 to 100 μg/mL. We also determined mRNA levels of key adipogenic genes, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/BEPα), adipocyte protein 2 (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), and sterol-regulated element-binding protein 1c (SREBP-1c) using real-time quantitative polymerase chain reaction (qPCR). Results: This study showed a concentration-dependent DPPH radical scavenging activity and no toxicity at concentrations up to 30 μg/mL in Raw264.7 cells. BCS extract showed an IC50 of 328.77 ± 20.52 μg/mL. Notably, pre-treatment with BCS extract (30 μg/mL) significantly enhanced cell viability in lipopolysaccharide (LPS)-treated Raw264.7 cells. BCS extract treatment effectively inhibited LPS-induced production of PGE2 and NO, as well as the expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), interleukin (IL)-1β and IL-6, possibly by limiting the phosphorylation of p38, p65, inhibitory κBα (I-κBα), and c-Jun N-terminal kinase (JNK). It also significantly attenuated lipid accumulation and key adipogenic genes in 3T3-L1 cells. Conclusions: This study highlights the in vitro anti-adipogenic and anti-inflammatory potential of BCS extract, underscoring its potential as a promising candidate for managing metabolic disorders. [ABSTRACT FROM AUTHOR]

Background: Gremlin-1 (GREM1) and Gremlin-2 (GREM2) are bone morphogenetic protein antagonists that play important roles in organogenesis, tissue differentiation, and tissue homeostasis. Although GREM1 has been reported to be involved in promoting various cancers, little has been reported about effects of GREM2 on cancer. Recently, it has been reported that GREM2 can inhibit adipogenesis in adipose-derived stromal/stem cells. However, as an inhibitor of adipogenesis, the role of GREM2 in cancer progression is not well understood yet. Methods: Pre-adipocyte 3T3-L1 cells overexpressing mock or Grem2 were established using a lentiviral transduction system and differentiated into adipocytes-mock and adipocytes-Grem2, respectively. To investigate the effect of adipocyte-Grem2 on breast cancer cells, we analyzed the proliferative and invasion abilities of spheroids using a 3D co-culture system of breast cancer cells and adipocytes or conditioned medium (CM) of adipocytes. An orthotopic breast cancer mouse model was used to examine the role of adipocytes-Grem2 in breast cancer progression. Results: Grem2 overexpression suppressed adipogenesis of 3T3-L1 cells. Proliferative and invasion abilities of spheroids formed by co-culturing MTV/TM-011 breast cancer cells and adipocytes-Grem2 were significantly reduced compared to those of spheroids formed by co-culturing MTV/TM-011 cells and adipocytes-mock. Compared to adipocytes-mock, adipocytes-Grem2 showed decreased mRNA expression of several adipokines, notably IL-6. The concentration of IL-6 in the CM of these cells was also decreased. Proliferative and invasive abilities of breast cancer cells reduced by adipocytes-Grem2 were restored by IL-6 treatment. Expression levels of vimentin, slug, and twist1 in breast cancer cells were decreased by treatment with CM of adipocytes-Grem2 but increased by IL-6 treatment. In orthotopic breast cancer mouse model, mice injected with both MTV/TM-011 cells and adipocytes-Grem2 showed smaller primary tumors and lower lung metastasis than controls. However, IL-6 administration increased both the size of primary tumor and the number of metastatic lung lesions, which were reduced by adipocytes-Grem2. Conclusions: Our study suggests that GREM2 overexpression in adipocytes can inhibit adipogenesis, reduce the expression and secretion of several adipokines, including IL-6, and ultimately inhibit breast cancer progression. [ABSTRACT FROM AUTHOR]

Squalene (SQ) is a natural compound with anti-inflammatory, anti-cancer, and anti-oxidant effects, but due to its low solubility, its biological properties have been greatly underestimated. This study aims to explore the differences in gene expression patterns of four newly synthesized amphipathic ethylene glycol (EG) derivatives of SQ by whole-genome transcriptomics analysis using DNA microarray to examine the mRNA expression profile of adipocytes differentiated from 3T3-L1 cells treated with SQ and its EG derivatives. Enrichment analyses of the transcriptional data showed that compared with SQ, its EG derivatives exerted different, in most cases desirable, biological responses. EG derivatives showed increased enrichment of mitochondrial functions, lipid and glucose metabolism, and inflammatory response. Mono-, di-, and tetra-SQ showed higher enrichment of the cellular component-ribosome. Histological staining showed EG derivatives prevented excessive lipid accumulation. Additionally, mitochondrial transcription factors showed upregulation in tetra-SQ-treated cells. Notably, EG derivatives showed better anti-inflammatory effects. Further, gene-disease association analysis predicted substantial improvement in the bioactivities of SQ derivatives in metabolic diseases. Cluster analyses revealed di- and tetra-SQ had more functional similarities than others, reflected in their scanning electron microscopy images; both di- and tetra-SQ self-organized into similar sizes and shapes of vesicles, subsequently improving their cation binding activities. Protein-protein interaction networks further revealed that cation binding activity might explain a major part, if not all, of the differences observed in functional analyses. Altogether, the addition of EG derivatives may improve the biological responses of SQ and thus may enhance its health-promoting potential.

The dysregulation of energy homeostasis in obesity involves multihormone resistance. Although leptin and insulin resistance have been well characterized, catecholamine resistance remains largely unexplored. Murine β3-adrenergic receptor expression in adipocytes is orders of magnitude higher compared with that of other isoforms. While resistant to classical desensitization pathways, its mRNA (Adrb3) and protein expression are dramatically downregulated after ligand exposure (homologous desensitization). β3-Adrenergic receptor downregulation also occurs after high-fat diet feeding, concurrent with catecholamine resistance and elevated inflammation. This downregulation is recapitulated in vitro by TNF-α treatment (heterologous desensitization). Both homologous and heterologous desensitization of Adrb3 were triggered by induction of the pseudokinase TRIB1 downstream of the EPAC/RAP2A/PI-PLC pathway. TRIB1 in turn degraded the primary transcriptional activator of Adrb3, CEBPα. EPAC/RAP inhibition enhanced catecholamine-stimulated lipolysis and energy expenditure in obese mice. Moreover, adipose tissue expression of genes in this pathway correlated with body weight extremes in a cohort of genetically diverse mice and with BMI in 2 independent cohorts of humans. These data implicate a signaling axis that may explain reduced hormone-stimulated lipolysis in obesity and resistance to therapeutic interventions with β3-adrenergic receptor agonists.

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.

The present study explores the molecular mechanism of anti-obesity effect of total lutein oxidized products (LOPs) in high-fat diet-induced obese C57BL/6 mice. Total LOPs (50, 100, 200 mg/kg b.wt.) were intubated for 22 weeks. Lipid profile, peroxides, AST, ALT, GST activities, lipid metabolizing enzymes/molecules, 3T3-L1 cell differentiation, TGF-β1 and p38 MAPK expressions were studied. Total LOPs at 100 mg/kg b.wt. reduced plasma and hepatic cholesterol, triglycerides, phospholipids, ALT and AST activities. FAS and CPT activities were inhibited in the liver homogenate, and epididymal WAT (eWAT). Total LOPs at 200 mg/kg b.wt. reduced HMGCoA:Mevalonate in eWAT, lipid peroxides in eWAT and liver. GST activity was high in the liver and eWAT. Total LOPs (100μg/ml) suppressed adipogenesis and decreased the accumulation of lipid droplets from two to eight days. Adiponectin was upregulated, PPAR γ, lipoprotein lipase, TGF-β1, and p38 MAPK were down-regulated. The results have shown that total LOPs are potent anti-obese molecules.ABBREVIATIONS: AIN 93G: American institute of nutrition 93 for growth; ALT: alanine transaminase; AST: aspartate transaminase; CEBPα: ccaat/enhancer binding protein α; C-DNA: complementary deoxyribonucleic acid; CPT: carnitine palmitoyl transferase; GST: glutathione-S-transferase; ELISA: enzyme-linked immune sorbent assay; FAS: fatty acid synthase; HDL: high-density lipoprotein; HFD: high-fat diet; HMG CoA: β-Hydroxy β-methylglutaryl-Coenzyme A; LDL: low-density lipoprotein; LOPs: total lutein oxidized products; LPL: lipoprotein lipase; MDA-Malonaldehyde; MTT: 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide; MVA: mevalonate; p38MAPK: p38 mitogen-activated protein kinases; RNA: ribonucleic acid; RTq-PCR: quantitative reverse transcription polymerase chain reaction; PPAR-γ: peroxisome proliferator-activated receptor-gamma; TGF-β1: transforming growth factor beta 1; WAT: white adipose tissue.

The signaling pathway of fatty acids in the context of obesity is an extensively explored topic, yet their primary mechanism of action remains incompletely understood. This study aims to examine the effect of docosahexaenoic acid (DHA) on some crucial aspects of adipogenesis in differentiating 3T3-L1 cells, using palmitic acid-treated (PA), standard differentiated, and undifferentiated adipocytes as controls. Employing 60 µM DHA or PA, 3T3-L1 preadipocytes were treated from the onset of adipogenesis, with negative and positive controls included. After eight days, we performed microscopic observations, cell viability assays, the determination of adiponectin concentration, intracellular lipid accumulation, and gene expression analysis. Our findings demonstrated that DHA inhibits adipogenesis, lipolysis, and glucose uptake by suppressing peroxisome proliferator-activated receptor gamma (Pparg) and G-protein coupled receptor 120 (Gpr120) gene expression. Cell cytotoxicity was ruled out as a causative factor, and β-oxidation involvement was suspected. These results challenge the conventional belief that omega-3 fatty acids, acting as Pparg and Gpr120 agonists, promote adipogenesis and enhance insulin-dependent glucose cell flux. Moreover, we propose a novel hypothesis suggesting the key role of the co-repressor G protein pathway suppressor 2 in mediating this process. Additional investigations are required to elucidate the molecular mechanisms driving DHA's anti-adipogenic effect and its broader health implications. [ABSTRACT FROM AUTHOR]

Obesity is a major risk factor for a variety of diseases and contributes to chronic inflammation. Resveratrol is a naturally occurring antioxidant that can reduce adipogenesis. In this study, the antiadipogenic and anti-inflammatory activities of resveratrol-enriched rice were investigated in 3T3-L1 adipocyte cells. Cotreatment of dexamethasone and isobutylmethylxanthin upregulated adipogenic transcription factors and signaling pathways. Subsequent treatment of adipocytes with rice seed extracts suppressed the differentiation of 3T3-L1 by downregulating adipogenic transcription factors (peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α) and signaling pathways (extracellular signal-regulated kinase 1/2 and protein kinase B Akt), this was especially observed in cells treated with germinated resveratrol-enriched rice seed extract (DJ526_5). DJ526_5 treatment also markedly reduced lipid accumulation in the cells and expression of adipogenic genes. Lipopolysaccharide (LPS)-induced inflammatory cytokines (prostaglandin-endoperoxide synthase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) decreased in cells treated with DJ526_5. Collectively, DJ526_5 exerts antiadipogenic effects by suppressing the expression of adipogenesis transcription factors. Moreover, DJ526_5 ameliorates anti-inflammatory effects in 3T3-L1 adipocytes by inhibiting the activation of phosphorylation NF-κB p65 and ERK ½ (MAPK). These results highlight the potential of resveratrol-enriched rice as an alternative obesity-reducing and anti-inflammatory agent. [ABSTRACT FROM AUTHOR]

The contribution of adipose-derived FGF21 to energy homeostasis is unclear. Here we show that browning of inguinal white adipose tissue (iWAT) by β-adrenergic agonists requires autocrine FGF21 signaling. Adipose-specific deletion of the FGF21 co-receptor β-Klotho renders mice unresponsive to β-adrenergic stimulation. In contrast, mice with liver-specific ablation of FGF21, which eliminates circulating FGF21, remain sensitive to β-adrenergic browning of iWAT. Concordantly, transgenic overexpression of FGF21 in adipocytes promotes browning in a β-Klotho-dependent manner without increasing circulating FGF21. Mechanistically, we show that β-adrenergic stimulation of thermogenic gene expression requires FGF21 in adipocytes to promote phosphorylation of phospholipase C-γ and mobilization of intracellular calcium. Moreover, we find that the β-adrenergic-dependent increase in circulating FGF21 occurs through an indirect mechanism in which fatty acids released by adipocyte lipolysis subsequently activate hepatic PPARα to increase FGF21 expression. These studies identify FGF21 as a cell-autonomous autocrine regulator of adipose tissue function.

Adipose tissue mass and adiposity change throughout the lifespan. During aging, while visceral adipose tissue (VAT) tends to increase, peripheral subcutaneous adipose tissue (SAT) decreases significantly. Unlike VAT, which is linked to metabolic diseases, including type 2 diabetes, SAT has beneficial effects. However, the molecular details behind the aging-associated loss of SAT remain unclear. Here, by comparing scRNA-seq of total stromal vascular cells of SAT from young and aging mice, we identify an aging-dependent regulatory cell (ARC) population that emerges only in SAT of aged mice and humans. ARCs express adipose progenitor markers but lack adipogenic capacity; they secrete high levels of pro-inflammatory chemokines, including Ccl6, to inhibit proliferation and differentiation of neighboring adipose precursors. We also found Pu.1 to be a driving factor for ARC development. We identify an ARC population and its capacity to inhibit differentiation of neighboring adipose precursors, correlating with aging-associated loss of SAT.

Infections are a major complication of obesity, but the mechanisms responsible for impaired defense against microbes are not well understood. Here, we found that adipocyte progenitors were lost from the dermis during diet-induced obesity (DIO) in humans and mice. The loss of adipogenic fibroblasts from mice resulted in less antimicrobial peptide production and greatly increased susceptibility to Staphylococcus aureus infection. The decrease in adipocyte progenitors in DIO mice was explained by expression of transforming growth factor-β (TGFβ) by mature adipocytes that then inhibited adipocyte progenitors and the production of cathelicidin in vitro. Administration of a TGFβ receptor inhibitor or a peroxisome proliferator-activated receptor-γ agonist reversed this inhibition in both cultured adipocyte progenitors and in mice and subsequently restored the capacity of obese mice to defend against S. aureus skin infection. Together, these results explain how obesity promotes dysfunction of the antimicrobial function of reactive dermal adipogenesis and identifies potential therapeutic targets to manage skin infection associated with obesity.

Bile acids have recently emerged as key metabolic hormones with beneficial impacts in multiple metabolic diseases. We previously discovered that hepatic bile acid overload distally modulates glucose and fatty acid metabolism in adipose tissues to exert anti-obesity effects. However, the detailed mechanisms that explain the salutary effects of serum bile acid elevation remain unclear. Here, proteomic profiling identified a new hepatokine, Orosomucoid (ORM) that governs liver-adipose tissue crosstalk. Hepatic ORMs were highly induced by both genetic and dietary bile acid overload. To address the direct metabolic effects of ORM, purified ORM proteins were administered during adipogenic differentiation of 3T3-L1 cells and mouse stromal vascular fibroblasts. ORM suppressed adipocyte differentiation and strongly inhibited gene expression of adipogenic transcription factors such as C/EBPβ, KLF5, C/EBPα, and PPARγ. Taken together, our data clearly suggest that bile acid-induced ORM secretion from the liver blocks adipocyte differentiation, potentially linked to anti-obesity effect of bile acids.

Obesity is the excessive accumulation of body fat resulting from impairment in energy balance mechanisms. In this study, we aimed to investigate the mechanism whereby GABA (γ-aminobutyric acid) prevents high-fat diet-induced obesity, and whether it induces lipolysis and browning in white adipose tissue (WAT), using high-fat diet (HFD)-fed obese mice and 3T3-L1 adipocytes. We demonstrated that GABA substantially inhibits the body mass gain of mice by suppressing adipogenesis and lipogenesis. Consistent with this result, histological analysis of WAT demonstrated that GABA decreases adipocyte size. Moreover, we show that GABA administration decreases fasting blood glucose and improves serum lipid profiles and hepatic lipogenesis in HFD-fed obese mice. Furthermore, Western blot and immunofluorescence analyses showed that GABA activates protein kinase A (PKA) signaling pathways that increase lipolysis and promote uncoupling protein 1 (UCP1)-mediated WAT browning. Overall, these results suggest that GABA exerts an anti-obesity effect via the regulation of lipid metabolism.

Recent studies have demonstrated the existence of a discrete pool of cholesterol in the plasma membranes (PM) of mammalian cells-referred to as the accessible cholesterol pool-that can be detected by the binding of modified versions of bacterial cytolysins (e.g., anthrolysin O). When the amount of accessible cholesterol in the PM exceeds a threshold level, the excess cholesterol moves to the endoplasmic reticulum (ER), where it regulates the SREBP2 pathway and undergoes esterification. We reported previously that the Aster/Gramd1 family of sterol transporters mediates nonvesicular movement of cholesterol from the PM to the ER in multiple mammalian cell types. Here, we investigated the PM pool of accessible cholesterol in cholesterol-loaded fibroblasts with a knockdown of Aster-A and in mouse macrophages from Aster-B and Aster-A/B-deficient mice. Nanoscale secondary ion mass spectrometry (NanoSIMS) analyses revealed expansion of the accessible cholesterol pool in cells lacking Aster expression. The increased accessible cholesterol pool in the PM was accompanied by reduced cholesterol movement to the ER, evidenced by increased expression of SREBP2-regulated genes. Cosedimentation experiments with liposomes revealed that the Aster-B GRAM domain binds to membranes in a cholesterol concentration-dependent manner and that the binding is facilitated by the presence of phosphatidylserine. These studies revealed that the Aster-mediated nonvesicular cholesterol transport pathway controls levels of accessible cholesterol in the PM, as well as the activity of the SREBP pathway.

G protein-coupled receptor 120 (GPR120) and PPARγ agonists each have insulin sensitizing effects. But whether these two pathways functionally interact and can be leveraged together to markedly improve insulin resistance has not been explored. Here, we show that treatment with the PPARγ agonist rosiglitazone (Rosi) plus the GPR120 agonist Compound A leads to additive effects to improve glucose tolerance and insulin sensitivity, but at lower doses of Rosi, thus avoiding its known side effects. Mechanistically, we show that GPR120 is a PPARγ target gene in adipocytes, while GPR120 augments PPARγ activity by inducing the endogenous ligand 15d-PGJ2 and by blocking ERK-mediated inhibition of PPARγ. Further, we used macrophage- (MKO) or adipocyte-specific GPR120 KO (AKO) mice to show that GRP120 has anti-inflammatory effects via macrophages while working with PPARγ in adipocytes to increase insulin sensitivity. These results raise the prospect of a safer way to increase insulin sensitization in the clinic.

Small GTPases are essential signaling molecules for regulating glucose uptake in adipose tissues upon insulin stimulation, and this regulation maintains an appropriate range of glycemia. The involvement of small GTPases in adipogenesis, however, has not been systemically investigated. In this study, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled peptides, to identify differentially expressed small GTPase proteins during adipogenesis of cultured murine cells. We were able to quantify the relative levels of expression of 55 and 49 small GTPases accompanied by adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells, respectively. When compared with analysis conducted in the data-dependent acquisition (DDA) mode, the MRM-based proteomic platform substantially increased the coverage of the small GTPase proteome. Western blot analysis further corroborated the MRM quantification results for selected small GTPases. Interestingly, overall a significant number of small GTPases were down-regulated during adipogenesis. Among them, the expression levels of Rab32 protein were consistently lower in differentiated adipocytes than the corresponding undifferentiated precursors in both cell lines. Overexpression of Rab32 in 3T3-L1 and C3H10T1/2 cells prior to adipogenesis induction suppressed their differentiation. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome during adipogenesis, and we reveal a previously unrecognized role of Rab32 in adipogenic differentiation.

Our understanding of adipose tissue biology has steadily evolved. While structural and energy storage functionalities have been in the forefront, a key endocrine role for adipocytes was revealed only over the last few decades. In contrast to the wealth of information on dynamic function of other endocrine tissues, few studies have focused on dynamic adipose tissue function or on tool development toward that end. Here, we apply our unique droplet-based microfluidic devices to culture, perfuse, and sample secretions from primary murine epididymal white adipose tissue (eWAT), and from predifferentiated clusters of 3T3-L1 adipocytes. Through automated control, oil-segmented aqueous droplets (∼2.6 nL) were sampled from tissue or cells at 3.5 second temporal resolution (including sample and reference droplets), with integrated enzyme assays enabling real-time quantification of glycerol (down to 1.9 fmol per droplet). This high resolution revealed previously unreported oscillations in secreted glycerol at frequencies of 0.2 to 2.0 min-1 (∼30-300 s periods) present in the primary tissue but not in clustered cells. Low-level bursts (∼50 fmol) released in basal conditions were contrasted with larger bursts (∼300 fmol) during stimulation. Further, both fold changes and burst magnitudes were decreased in eWAT of aged and obese mice. These results, combined with immunostaining and photobleaching analyses, suggest that gap-junctional coupling or nerve cell innervation within the intact ex vivo tissue explants play important roles in this apparent tissue-level, lipolytic synchronization. High-resolution, quantitative sampling by droplet microfluidics thus permitted unique biological information to be observed, giving an analytical framework poised for future studies of dynamic oscillatory function of adipose and other tissues.

Adult mesenchymal stem cells, including preadipocytes, possess a cellular sensory organelle called the primary cilium. Ciliated preadipocytes abundantly populate perivascular compartments in fat and are activated by a high-fat diet. Here, we sought to understand whether preadipocytes use their cilia to sense and respond to external cues to remodel white adipose tissue. Abolishing preadipocyte cilia in mice severely impairs white adipose tissue expansion. We discover that TULP3-dependent ciliary localization of the omega-3 fatty acid receptor FFAR4/GPR120 promotes adipogenesis. FFAR4 agonists and ω-3 fatty acids, but not saturated fatty acids, trigger mitosis and adipogenesis by rapidly activating cAMP production inside cilia. Ciliary cAMP activates EPAC signaling, CTCF-dependent chromatin remodeling, and transcriptional activation of PPARγ and CEBPα to initiate adipogenesis. We propose that dietary ω-3 fatty acids selectively drive expansion of adipocyte numbers to produce new fat cells and store saturated fatty acids, enabling homeostasis of healthy fat tissue.

Insulin resistance is a key feature of obesity and type 2 diabetes. PU.1 is a master transcription factor predominantly expressed in macrophages but after HFD feeding PU.1 expression is also significantly increased in adipocytes. We generated adipocyte specific PU.1 knockout mice using adiponectin cre to investigate the role of PU.1 in adipocyte biology, insulin and glucose homeostasis. In HFD-fed obese mice systemic glucose tolerance and insulin sensitivity were improved in PU.1 AKO mice and clamp studies indicated improvements in both adipose and liver insulin sensitivity. At the level of adipose tissue, macrophage infiltration and inflammation was decreased and glucose uptake was increased in PU.1 AKO mice compared with controls. While PU.1 deletion in adipocytes did not affect the gene expression of PPARg itself, we observed increased expression of PPARg target genes in eWAT from HFD fed PU.1 AKO mice compared with controls. Furthermore, we observed decreased phosphorylation at serine 273 in PU.1 AKO mice compared with fl/fl controls, indicating that PPARg is more active when PU.1 expression is reduced in adipocytes. Therefore, in obesity the increased expression of PU.1 in adipocytes modifies the adipocyte PPARg cistrome resulting in impaired glucose tolerance and insulin sensitivity.

Background: Blue honeysuckle (BH; Lonicera caerulea L.), which is a traditional medicinal plant, is known to be a rich source of anthocyanins and phenolic acids due to its strong antioxidant and anti-inflammatory activities. Its anti-obesity effects, which are a result of attenuating abnormal lipid and glucose metabolisms, have also been reported. Aim: The purpose of this study is to investigate the effect of BH on genes and proteins that are involved in the adipocyte differentiation using 3T3-L1 cells. Methods: The effects of the water extracts of the BH were examined on adipogenesis and lipolysis using a biochemical and molecular analysis of the 3T3-L1 cells. Results: Cyanidin-3-O-glucoside (C3G) from the BH extract was determined in order to contain 1.67 mg/g by the high-performance liquid chromatography analysis. The lipid accumulation in the adipocytes was reduced, which ranged from 58 to 26% in the BH (500 and 1,000 µg/mL) compared to the control group. The lipolysis that was measured by the glycerol content was not affected by the BH at 1,000 μg/mL. The BH downregulated the expression of the main transcription factors of the adipogenesis pathway, such as peroxisome proliferator-activated receptor γ, 1, 2, adipose differentiation-related protein, CCAAT/enhancer binding protein α, and acetyl CoA carboxylase, while increasing the expression of the Adenosine monophosphate (AMP)-activated protein kinase α. Conclusion: These findings suggest that the BH is a good source of C3G, and it could be effective in regard to inhibiting the adipogenesis as opposed to the lipolysis, which indicates the potential for natural anti-obesity ingredients.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. However, the effect of APE1/Ref-1 on adipogenic transcription factor regulation remains unknown. In this study, we investigated the effect of APE1/Ref-1 on the regulation of adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression significantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-α and peroxisome proliferator-activated receptor (PPAR)-γ, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-α, PPAR-γ, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-α, PPAR-γ, and aP2 during adipocyte differentiation. These results suggest that APE1/Ref-1 inhibits adipocyte differentiation by regulating adipogenic transcription factors, suggesting that APE1/Ref-1 is a potential therapeutic target for regulating adipocyte differentiation. [ABSTRACT FROM AUTHOR]

Obesity is a disease in which fat is abnormally or excessively accumulated in the body, and many studies have been conducted to overcome it with various techniques. In this study, we evaluated whether micro-current stimulation (MCS) can be applied to prevent obesity by regulating the adipogenesis through 3T3-L1 cells and ob/ob mice. To specify the intensity of MCS, Oil Red O staining was conducted with various intensities of MCS. Based on these, subsequent experiments used 200 and 400 μA for the intensity of MCS. The expressions of insulin signaling pathway-related proteins, including phosphorylation of IGF-1 and IR, were decreased in all MCS groups, and in turn, downstream signals such as Akt and ERK were decreased. In addition, MCS reduced the nucleus translocation of PPAR-γ and decreased the protein expression of C/EBP-α. In the ob/ob mouse model, MCS reduced body weight gain and abdominal adipose tissue volume. In particular, the concentration of triglycerides in serum was also decreased. Taken together, our findings showed that MCS inhibited lipid accumulation by regulating insulin signaling in 3T3-L1, and it was effective at reducing body weight and adipose tissue volume in ob/ob mice. These suggest that MCS may be a useful treatment approach for obesity. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are one of the most prevalent classes of environmental pollutants. Some evidence shows that PAHs could be involved in human obesity. However, little is known about the distribution patterns of PAHs in human adipose tissue (AT) and the role of PAHs on adipogenesis/lipogenesis. The aims of this pilot study were to determine concentrations of 16 PAHs defined as high-priority pollutants in the plasma and adipose tissue of French and Polish bariatric patients, as well as their correlation with body mass index (BMI), plasma and AT adipokines expression levels. We finally investigated the role of naphthalene on cell proliferation, viability, and differentiation in 3T3-L1 preadipocytes. The concentration of most PAHs was similar in the three types of AT and it was significantly higher in AT as compared to plasma, suggesting bioaccumulation. Polish patients had higher PAH levels in AT than French ones. Only the concentration of naphthalene in AT was positively correlated with the BMI and serum or adipose chemerin, adiponectin and resistin expression, in French but not in Polish patients, who had significantly higher BMIs. Moreover, naphthalene exposure increased the cell proliferation of 3T3-L1 preadipocytes and lipogenesis, and increased the expression of genes involved in adipogenesis after cell differentiation. Taken together, PAHs and more particularly naphthalene could be an obesogenic molecule and increase the risk of obesity. [ABSTRACT FROM AUTHOR]

The present study explores the molecular mechanism of anti-obesity effect of total lutein oxidized products (LOPs) in high-fat diet-induced obese C57BL/6 mice. Total LOPs (50, 100, 200 mg/kg b.wt.) were intubated for 22 weeks. Lipid profile, peroxides, AST, ALT, GST activities, lipid metabolizing enzymes/molecules, 3T3-L1 cell differentiation, TGF-β1 and p38 MAPK expressions were studied. Total LOPs at 100 mg/kg b.wt. reduced plasma and hepatic cholesterol, triglycerides, phospholipids, ALT and AST activities. FAS and CPT activities were inhibited in the liver homogenate, and epididymal WAT (eWAT). Total LOPs at 200 mg/kg b.wt. reduced HMGCoA:Mevalonate in eWAT, lipid peroxides in eWAT and liver. GST activity was high in the liver and eWAT. Total LOPs (100μg/ml) suppressed adipogenesis and decreased the accumulation of lipid droplets from two to eight days. Adiponectin was upregulated, PPAR γ, lipoprotein lipase, TGF-β1, and p38 MAPK were down-regulated. The results have shown that total LOPs are potent anti-obese molecules. ABBREVIATIONS: AIN 93G: American institute of nutrition 93 for growth; ALT: alanine transaminase; AST: aspartate transaminase; CEBPα: ccaat/enhancer binding protein α; C-DNA: complementary deoxyribonucleic acid; CPT: carnitine palmitoyl transferase; GST: glutathione-S-transferase; ELISA: enzyme-linked immune sorbent assay; FAS: fatty acid synthase; HDL: high-density lipoprotein; HFD: high-fat diet; HMG CoA: β-Hydroxy β-methylglutaryl-Coenzyme A; LDL: low-density lipoprotein; LOPs: total lutein oxidized products; LPL: lipoprotein lipase; MDA-Malonaldehyde; MTT: 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide; MVA: mevalonate; p38MAPK: p38 mitogen-activated protein kinases; RNA: ribonucleic acid; RTq-PCR: quantitative reverse transcription polymerase chain reaction; PPAR-γ: peroxisome proliferator-activated receptor-gamma; TGF-β1: transforming growth factor beta 1; WAT: white adipose tissue. [ABSTRACT FROM AUTHOR]

The study aimed to compare the effect of 100 µM docosahexaenoic acid and oleic acid alone, and as a combination in immature and already mature 3T3-L1. The cells were subjected to adipogenic induction for 2 days and then were maintained in a high glucose culture medium supplemented with 10 µg/mL of insulin for additional 16 days. The treatment with docosa-hexaenoic acid and oleic acid lasted 9 days in total, as half of the cells were treated during induction (from day 1 to 9), and the rest--after reaching maturity (day 9--18). The palmitic fatty acid was used as a positive control. Applied in immature adipocytes, docosahexaenoic acid prevented intracellular neutral lipid deposition and reduced lipolysis upon well-preserved glucose utilization. At the same time, oleic acid alone or combined with docosahexaenoic acid increased lipid accumulation and slightly attenuated lipolysis only related to the palmitic fatty acid-treated group. However, free fatty acids application in mature adipocytes did not cause any beneficial effect. They elevated lipolysis rate compared to the induced control, which in vivo might be a predisposing factor for ectopic fat accumulation. The results emphasized that free fatty acids' impact on adipocyte metabolism was highly affected by their maturation stage and should be considered in anti-obesity strategies based on free fatty acids signaling modulations. [ABSTRACT FROM AUTHOR]

Fat browning, which converts white adipose tissue to brown, has attracted attention as a promising strategy for the treatment of obesity. Betanin (BT) has been reported to have potential anti-obesity activity. 3T3-L1 cells were differentiated for 7 days during BT treatment. The BT concentration range for the study was determined using an MTT assay, and lipid accumulation was evaluated by Oil-Red-O staining. The expression of protein level was analyzed by Western blot. Immunofluorescence images were performed with confocal microscopy to visually show the amount and location of thermogenesis factor uncoupling protein1 (UCP1) and mitochondria. qRT-PCR was performed to evaluate mRNA expression. BT inhibited lipid accumulation and increased the expression of UCP1, peroxisome-proliferator-activated receptor gamma (PPARγ), and PPARγ coactivator-1 alpha (PGC-1α). In addition, the increases in beige adipocyte-specific markers were observed, supporting BT-mediated browning of the fat tissue. The UCP1 was localized in the inner membrane of the mitochondria, and its expression was associated with mitochondrial activation. Consistent with this, the mRNA expression of mitochondrial biogenesis markers increased in 3T3-L1 cells after BT treatment. Immunofluorescence staining also indicated an increased number of mitochondria and UCP1, respectively. Moreover, BT inhibited lipogenesis and enhanced lipolysis and fatty acid oxidation. This mechanism has been suggested to be mediated by an adenosine monophosphate-activated protein kinase (AMPK) pathway. BT induces fat browning and regulates lipid metabolism via the AMPK-mediated pathway in 3T3-L1 cells, suggesting that BT can be a promising candidate for controlling obesity.

Natural polyphenol-rich plant resources, such as agricultural waste, were proven to diminish insulin resistance and weight gain in rats on a high-fat diet. To test whether date seed polyphenol pills (DSPPs) might lower adipose tissue accumulation by precisely affecting adipocytes, we explored the impacts of DSPPs on cell proliferation, differentiation, and lipolysis in 3T3-L1 cells. We utilized tablets made commercially from date seed polyphenols that were mostly composed of epicatechin (45.9 g/kg). The total polyphenol and antioxidant capacities of the digested and non-digested DSPPs were also evaluated. DSPPs at doses of 25, 50, and 100 µg/mL hindered the proliferation of both pre-confluent preadipocytes and mature post-confluent adipocytes. DSPPs decreased the quantity of viable cells in completely developed adipocytes. Treatment with 100 µg/mL of DSPPs decreased the basal lipolysis of completely differentiated adipocytes but modestly boosted epinephrine-induced lipolysis. A significant transcription factor for the adipogenic gene, the peroxisome proliferator-activated receptor (PPAR), was repressed by DSPPs, which significantly decreased lipid buildup. The total polyphenol and antioxidant capacities were also increased after digestion with a good bubble Pearson correlation between both. DSPPs may have anti-obesity and anti-diabetic characteristics by inhibiting adipocyte development and basal lipolysis, which could be commercially industrialized.

Background and Objectives: This study evaluated the in vitro anti-adipogenic and anti-inflammatory properties of black cumin (Nigella sativa L.) seed extract (BCS extract) as a potential candidate for developing herbal formulations targeting metabolic disorders. Materials and Methods: We evaluated the BCS extract by assessing its 2,2-diphenyl-1-picrohydrazyl (DPPH) radical scavenging activity, levels of prostaglandin E2 (PGE2) and nitric oxide (NO), and mRNA expression levels of key pro-inflammatory mediators. We also quantified the phosphorylation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) signaling molecules. To assess anti-adipogenic effects, we used differentiated 3T3-L1 cells and BCS extract in doses from 10 to 100 μg/mL. We also determined mRNA levels of key adipogenic genes, including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/BEPα), adipocyte protein 2 (aP2), lipoprotein lipase (LPL), fatty acid synthase (FAS), and sterol-regulated element-binding protein 1c (SREBP-1c) using real-time quantitative polymerase chain reaction (qPCR). Results: This study showed a concentration-dependent DPPH radical scavenging activity and no toxicity at concentrations up to 30 μg/mL in Raw264.7 cells. BCS extract showed an IC50 of 328.77 ± 20.52 μg/mL. Notably, pre-treatment with BCS extract (30 μg/mL) significantly enhanced cell viability in lipopolysaccharide (LPS)-treated Raw264.7 cells. BCS extract treatment effectively inhibited LPS-induced production of PGE2 and NO, as well as the expression of monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), interleukin (IL)-1β and IL-6, possibly by limiting the phosphorylation of p38, p65, inhibitory κBα (I-κBα), and c-Jun N-terminal kinase (JNK). It also significantly attenuated lipid accumulation and key adipogenic genes in 3T3-L1 cells. Conclusions: This study highlights the in vitro anti-adipogenic and anti-inflammatory potential of BCS extract, underscoring its potential as a promising candidate for managing metabolic disorders.

A subset of dermal fibroblasts undergo rapid differentiation into adipocytes in response to infection and acutely produce the cathelicidin antimicrobial peptide gene Camp Vitamin A and other retinoids inhibit adipogenesis yet can show benefit to skin disorders, such as cystic acne, that are exacerbated by bacteria. We observed that retinoids potently increase and sustain the expression of Camp in preadipocytes undergoing adipogenesis despite inhibition of markers of adipogenesis, such as Adipoq, Fabp4, and Rstn Retinoids increase cathelicidin in both mouse and human preadipocytes, but this enhancement of antimicrobial peptide expression did not occur in keratinocytes or a sebocyte cell line. Preadipocytes undergoing adipogenesis more effectively inhibited growth of Staphylococcus aureus when exposed to retinoic acid. Whole transcriptome analysis identified hypoxia-inducible factor 1-α (HIF-1α) as a mechanism through which retinoids mediate this response. These observations uncouple the lipid accumulation element of adipogenesis from the innate immune response and uncover a mechanism, to our knowledge previously unsuspected, that may explain therapeutic benefits of retinoids in some skin disorders.

We previously demonstrated that indoor house dust extracts could induce adipogenesis in pre-adipocytes, suggesting a potential role for indoor contaminant mixtures in metabolic health. Herein, we investigated the potential role of thyroid receptor beta (TRβ) antagonism in adipogenic effects (dust-induced triglyceride accumulation and pre-adipocyte proliferation) following exposure to environmental mixtures (indoor house dust extracts). Concentrations of specific flame retardants were measured in extracts, and metabolic health information was collected from residents (n = 137). 90% of dust extracts exhibited significant adipogenic activity, >60% via triglyceride accumulation, and >70% via pre-adipocyte proliferation. Triglyceride accumulation was positively correlated with concentrations of each of twelve flame retardants, despite most being independently inactive; this suggests a putative role for co-exposures or mixtures. We further reported a positive correlation between dust-induced triglyceride accumulation and serum thyroid stimulating hormone concentrations, negative correlations with serum free triiodothyronine and thyroxine concentrations, and a positive and significant association between dust-induced triglyceride accumulation and residents' body mass index (BMI). We hypothesized that inhibition of TR antagonism might counteract these effects, and both addition of a TR agonist and siRNA knock-down of TR resulted in decreased dust-induced triglyceride accumulation in a subset of samples, bolstering this as a contributory mechanism. These results highlight a contributory role of environmental TR antagonism as a putative factor in metabolic health, suggesting that further research should evaluate this mechanism and determine whether in vitro adipogenic activity could have utility as a biomarker for metabolic health in residents.

Abstract Background MicroRNAs (MiRNAs) are known to participate in preadipocyte differentiation, but the manner in which miR-146a-5p participates in this process remains unclear. This study was performed to examine the participation of miR-146a-5p in 3T3-L1 cell differentiation. Material and Methods miR-146a-5p expression was upregulated and down-regulated to examine effects on 3T3-L1 cell differentiation. Bioinformatics analysis was performed to predict its target genes, and the signaling pathway it regulates was identified by qRT-PCR and Western blotting. The expression of miR-146a-5p in epididymal adipose tissue from obese mice and in an obese mouse adipose cell model was examined by qRT-PCR. Results 3T3-L1 cells differentiated into mature adipocytes successfully, as verified by increased areas of intracellular lipid droplets and elevated expression of mature adipocyte markers, and these cells had elevated miR-146a-5p expression. The intracellular lipid droplet and triglyceride contents and the expression of mature adipocyte markers were significantly increased in miR-146a-5p–overexpressing 3T3-L1 cells and markedly decreased in miR-146a-5p–inhibited 3T3-L1 cells. ErbB4 was a predicted target gene of miR-146a-5p. In miR-146a-5p–overexpressing 3T3-L1 cells, ErbB4 expression and ERK1/2 phosphorylation were decreased and the expression of PPAR-γ was increased; the opposite was observed in miR-146a-5p–inhibited 3T3-L1 cells. In addition, miR-146a-5p expression was significantly increased in the mouse epididymal adipose tissue and adipose cell model. Conclusions Upregulated miR-146a-5p expression was related to 3T3-L1 cell differentiation. MiR-146a-5p promoted 3T3-L1 cell differentiation by targeting ErbB4 and via the ERK1/2/PPAR-γ signaling pathway.

Emerging evidence indicates that epigenetic mechanisms like DNA methylation directly contribute to metabolic regulation. For example, we previously demonstrated that de novo DNA methyltransferase Dnmt3a plays a causal role in the development of adipocyte insulin resistance. Recent studies suggest that DNA demethylation plays an important role in the developmental process of adipocytes. However, little is known about whether DNA demethylase ten-eleven translocation (TET) proteins regulate the metabolic functions of adipocytes.MethodsThe expression of Tet genes was assessed in the fractionated adipocytes of chow- and high fat diet-fed C57/Bl6 mice using qPCR and western blotting. The effect of Tet2 gain- or loss-of-function in fully mature 3T3-L1 adipocytes in the presence/absence of Rosiglitazone (Rosi) and TNF-α on insulin sensitivity was using the insulin-stimulated glucose uptake and insulin signaling assays. Gene expression and DNA methylation analyses of PPARγ target genes was performed in the same setting. In addition, PPARγ reporter assays, co-immunoprecipitation assays, PPARγ ChIP-PCR analyses were performed.ResultsWe found that adipose expression of TET2, alone among its family members, was significantly reduced in diet-induced insulin resistance. TET2 gain-of-function was sufficient to promote insulin sensitivity while loss-of-function was necessary to facilitate insulin sensitization in response to the PPARγ agonist Rosiglitazone (Rosi) in cultured adipocytes. Consistent with this, TET2 was required for Rosi-dependent gene activation of certain PPARγ targets accompanied by changes in DNA demethylation at the promoter regions. Furthermore, TET2 was necessary to sustain PPARγ binding to target loci upon activation with Rosi via physical interaction with PPARγ.ConclusionsOur data demonstrate that TET2 works as an epigenetic regulator of Rosi-mediated insulin sensitization and transcriptional regulation in adipocytes.

Tetrabromobisphenol A (TBBPA) is the most common flame retardant used in electrical housings, circuit boards, and automobiles. High-throughput screening and binding assays have identified TBBPA as an agonist for human peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipogenesis. TBBPA has been suggested to be an obesogen based on in vitro cellular assays and zebrafish data. We hypothesized that exposing preadipocytes to TBBPA could influence adipogenesis via genes other than those in the PPARγ pathway due to its structural similarity to bisphenol A, which demonstrates varied endocrine disrupting activities. Mouse-derived 3T3-L1 preadipocytes were induced to differentiate and continually treated with TBBPA for 8 days. High-content imaging of adipocytes displayed increased adipocyte number and lipid accumulation when treated with TBBPA. TBBPA exhibited weak induction of mPPARγ, with an AC50 of 397 µM. Quantitative PCR revealed that TBBPA exposure increased early expression of genes involved in glucocorticoid receptor (GR) signaling and PPARγ transcriptional activation, as well as upregulating downstream genes needed for adipocyte maintenance and nontraditional ER signaling, such as Gpr30. Additionally, Pref1 and Thy1, inhibitors of differentiation, were downregulated by some concentrations of TBBPA. Furthermore, proliferating preadipocytes treated with TBBPA, only prior to differentiation, exhibited increased adipocyte number and lipid accumulation after 8 days in normal culture conditions. In conclusion, TBBPA influenced gene expression changes in GR, nontraditional ER, and known adipogenic regulatory genes, prior to PPARγ expression; effects suggesting early programming of adipogenic pathways.

The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R7 and R8 A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors. Analogues display IC50 values as low as 210 nM and reveal A-ring substituents that enhance selectivity toward either kinase. In cell assays, selected analogues display enhanced phosphorylation of p38 or TBK1 and elicited IL-6 secretion in 3T3-L1 adipocytes better than amlexanox. An analogue bearing a R7 cyclohexyl modification demonstrated robust IL-6 production in 3T3-L1 cells as well as a phosphorylation marker of efficacy and was tested in obese mice where it promoted serum IL-6 response, weight loss, and insulin sensitizing effects comparable to amlexanox. These studies provide impetus to expand the SAR around the amlexanox core toward uncovering analogues with development potential.

Chronic low-grade inflammation is a hallmark of obesity, which is a risk factor for the development of type 2 diabetes. The drug amlexanox inhibits IκB kinase ε (IKKε) and TANK binding kinase 1 (TBK1) to promote energy expenditure and improve insulin sensitivity. Clinical studies have demonstrated efficacy in a subset of diabetic patients with underlying adipose tissue inflammation, albeit with moderate potency, necessitating the need for improved analogs. Herein we report crystal structures of TBK1 in complex with amlexanox and a series of analogs that modify its carboxylic acid moiety. Removal of the carboxylic acid or mutation of the adjacent Thr156 residue significantly reduces potency toward TBK1, whereas conversion to a short amide or ester nearly abolishes the inhibitory effects. IKKε is less affected by these modifications, possibly due to variation in its hinge that allows for increased conformational plasticity. Installation of a tetrazole carboxylic acid bioisostere improved potency to 200 and 400 nM toward IKKε and TBK1, respectively. Despite improvements in the in vitro potency, no analog produced a greater response in adipocytes than amlexanox, perhaps because of altered absorption and distribution. The structure-activity relationships and cocrystal structures described herein will aid in future structure-guided inhibitor development using the amlexanox pharmacophore for the treatment of obesity and type 2 diabetes.

Adipocytes arise from the commitment and differentiation of adipose precursors in white adipose tissue (WAT). In studying adipogenesis, precursor markers, including Pref-1 and PDGFRα, are used to isolate precursors from stromal vascular fractions of WAT, but the relation among the markers is not known. Here, we used the Pref-1 promoter-rtTA system in mice for labeling Pref-1+ cells and for inducible inactivation of the Pref-1 target Sox9. We show the requirement of Sox9 for the maintenance of Pref-1+ proliferative, early precursors. Upon Sox9 inactivation, these Pref-1+ cells become PDGFRα+ cells that express early adipogenic markers. Thus, we show that Pref-1+ cells precede PDGFRα+ cells in the adipogenic pathway and that Sox9 inactivation is required for WAT growth and expansion. Furthermore, we show that in maintaining early adipose precursors, Sox9 activates Meis1, which prevents adipogenic differentiation. Our study also demonstrates the Pref-1 promoter-rtTA system for inducible gene inactivation in early adipose precursor populations.

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Salix pseudolasiogyne (Salicaceae), the "weeping willow," has been used in traditional Korean medicine to treat pain and fever due to its high concentrations of salicylic acid and salicin. The present study investigated bioactive compounds from S. pseudolasiogyne twigs to discover bioactive natural products. Phytochemical investigation of the ethanol (EtOH) extract of S. pseudolasiogyne twigs followed by liquid chromatography–mass spectrometry (LC/MS)-based analysis led to the isolation of two salicin derivatives, salicortinol and salicortin, the structures of which were determined by interpretation of their NMR spectra and data from the LC/MS analysis. To the best of our knowledge, this is the first report of salicortinol isolated from S. pseudolasiogyne. The isolated compounds were evaluated for their anti-adipogenic effects in 3T3-L1 cells. Both salicortinol and salicortin were found to significantly inhibit adipocyte differentiation in 3T3-L1 cells. In particular, salicortin exhibited a strong inhibitory effect on lipid accumulation. Furthermore, salicortin inhibited the expression of lipogenic and adipogenic transcription factors, including FASN, FABP4, C/EBPα, C/EBPβ, and PPARγ, without inducing cytotoxicity. These results suggest that salicortin could be a potential therapeutic compound for the prevention or treatment of metabolic disorders such as obesity. [ABSTRACT FROM AUTHOR]