Differentiated 3T3-L1 Adipocytes Research Articles

Inhibition of γ-secretase in adipocytes leads to altered IL-6 secretion and adipose inflammation

ABSTRACT Adipocyte-mediated inflammatory signalling has been proposed to alter adipose physiology in obesity and Type 2 diabetes mellitus. Novel targets for alteration of inflammatory signalling are needed to improve obesity-related outcomes. The γ-secretase enzyme complex has been suggested to play a role both in adipocyte function as well as in immune regulation. We hypothesized that adipocyte-specific γ-secretase inhibition could alter the inflammatory makeup of adipose tissue. We found that genetic blockade of γ-secretase in adipocytes leads to a decrease in EMR1 (F4/80) expression, as a marker of macrophage presence, in adipose tissue without changes in expression of markers of other inflammatory cell types. To explore the mechanism by which adipocytes can alter macrophage function in vitro, fully differentiated 3T3-L1 adipocytes were treated with a γ-secretase inhibitor in the presence of lipopolysaccharide (LPS) and transcription of IL6 and ccl2 (MCP1) were quantified. IL-6 expression and secretion were significantly inhibited by γ-secretase blockade, with little effect on MCP1. Preconditioned media from 3T3-L1 adipocytes treated with a γ-secretase inhibitor also alters macrophage activation but did not affect macrophage translocation in vitro. Therefore, γ-secretase inhibition in fully differentiated adipocytes can alter IL-6 signalling to macrophages, consistent with our hypothesis that that γ-secretase is involved in adipocyte-initiated inflammatory signalling cascades.

Bio-transformation of green tea infusion with tannase and its improvement on adipocyte metabolism

Catechins in green tea possess various health benefits. Enzymatic treatment improves physiological activities by inducing bioconversion of catechins. Here, we investigated the effect of green tea infusion (GT) after tannase treatment, which transforms (-)-epigallocatechin gallate (EGCG) to gallic acid (GA) and (-)-epigallocatechin (EGC), on adipocyte differentiation and mature adipocyte metabolism. The optimal conditions for tannase-mediated improvement in GA and EGC yields in GT were investigated using response surface methodology. Yields of GA and EGC were 43-fold (0.43 mg/mL) and 1.66-fold higher (1.11 mg/mL), respectively, compared to GT without tannase treatment. The optimal reaction conditions for tannase-mediated biotransformation were observed on 0.54 mg mL−1 of tannase, reaction time (86.79 min), and reaction temperature at 22.59 °C. GT and tannase-treated GT (TANs) upregulated adiponectin, uncoupling protein 1, adipose triglyceride lipase, and hormone-sensitive lipase gene expression in differentiated 3T3-L1 adipocytes, with TAN inducing better effects than GT, which implies that tannase treatment improved the beneficial effect of GT on adipocyte metabolism. Thus, tannase-mediated bio-transformation is an attractive candidate for preparing GT with enhanced anti-obesity properties.

Exogenous FABP4 interferes with differentiation, promotes lipolysis and inflammation in adipocytes.

Fatty acid binding protein 4 (FABP4) has been demonstrated to be secreted from adipocytes in an unconventional pathway associated with lipolysis. Circulating FABP4 is elevated in metabolic disorders and has been shown to affect various peripheral cells such as pancreatic β-cells, hepatocytes and macrophages, but its effects on adipocytes remains unclear. The aim of this study was to investigate the effects of exogenous FABP4 (eFABP4) on adipocyte differentiation and function. 3T3-L1 pre-adipocytes or mature adipocytes were treated with recombinant FABP4 in the absence or presence of FABP4 inhibitor I-9/p38 MAPK inhibitor SB203580; Meanwhile male C57BL/6J mice were subcutaneously injected twice a day with recombinant FABP4 (0.35 mg/kg) with or without I-9 (50 mg/kg) for 2 weeks. The effects of eFABP4 on differentiation, lipolysis and inflammation were determined by triglyceride measurement or lipolysis assay, western blotting, or RT-qPCR analysis. eFABP4 treatment significantly reduced intracellular triglyceride content and decreased expression of adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα), intracellular FABP4, and adiponectin in 3T3-L1 cells. Besides, eFABP4 promoted lipolysis and inflammation in differentiated 3T3-L1 adipocytes as well as in adipose tissue of eFABP4-treated C57BL/6J mice, with elevated gene expression of monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, and elevated protein expression of adipose triglyceride lipase (ATGL), phosphorylation of hormone-sensitive lipase (HSL) (Ser-660), p38, and nuclear factor-kappa B (NF-κB). The pro-inflammatory and pro-lipolytic effects of eFABP4 could be reversed by SB203580/I-9. These findings indicate that eFABP4 interferes with adipocyte differentiation, induces p38/HSL mediated lipolysis and p38/NF-κB mediated inflammation in adipocytes in vitro and in vivo.

New Antidiabetic Targets of α-Glucosidase Inhibitory Peptides, SVPA, SEPA, STYV and STY: Inhibitory Effects on Dipeptidyl Peptidase-IV and Lipid Accumulation in 3T3-L1 Differentiated Adipocytes with Scavenging Activities Against Methylglyoxal and Reactive Oxygen Species

Type 2 diabetes mellitus (T2DM) is a multifactorial disease that requires multiple therapeutic strategies for its management. Bioactive peptides with multiple anti-diabetic targets are attractive therapeutic molecules. The present study was conducted to identify additional anti-diabetic targets of α-glucosidase inhibitory peptides, SVPA, SEPA, STYV, and STY. The α-glucosidase inhibitory activity of the peptides was in the order STYV > STY > SEPA > SVPA while molecular docking against human dipeptidyl peptidase IV (DPP-IV) showed that SVPA had the best binding affinity. In contrast, in vitro studies indicated that SEPA had a significantly higher (P < 0.05) DPP-IV inhibitory activity (IC50 = 5.78 ± 0.19 mM) than other peptides. SVPA and SEPA showed mixed inhibition pattern while STYV and STY were uncompetitive inhibitors of the enzyme. IPI (diprotin A), STYV and STY were not cytotoxic while SEPA displayed some cytotoxicity. In differentiated 3T3-L1 adipocytes, SVPA and STYV were found to induce a significant (P < 0.05) decrease in intracytoplasmic lipid accumulation when added during the differentiation process while STY, GSH and IPI caused significant reduction (P < 0.05) in the lipid accumulation when added after the differentiation. The SVPA, SEPA and STYV were better scavengers of methylglyoxal than STY but STYV had the best scavenging activities toward reactive oxygen species and nitric oxide. It was concluded that the four α-glucosidase inhibitory peptides including IPI have multiple targets against type T2DM but, overall, of the four peptides evaluated, STYV tends to have better potential for application as a multifunctional anti-diabetic peptide.

Anti-adipogenic Effect of β-Carboline Alkaloids from Garlic (Allium sativum).

Garlic (Allium sativum L.) is utilized worldwide for culinary and medicinal use and has diverse health benefits. As part of our ongoing research to identify bioactive components from natural resources, phytochemical analysis of the methanolic extract of garlic led to the isolation and characterization of six compounds: Three eugenol diglycosides (1–3) and three β-carboline alkaloids (4–6). In particular, the absolute configurations of β-carboline alkaloids (5 and 6) were established by gauge-including atomic orbital nuclear magnetic resonance chemical shift calculations, followed by DP4+ analysis. Here, we evaluated the effects of compounds 1–6 on 3T3-L1 preadipocyte adipogenesis and lipid metabolism. 3T3-L1 adipocyte differentiation was evaluated using Oil Red O staining; the expression of adipogenic genes was detected using RT-qPCR. Among compounds 1–6, (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (6) inhibited 3T3-L1 preadipocyte adipogenesis and reduced the expression of adipogenic genes (Fabp4, PPARγ, C/EBPβ, Adipsin, and Adipoq). Moreover, it markedly decreased the actylation of α-tubulin, which is crucial for cytoskeletal remodeling during adipogenesis. Anti-adipogenic effects were observed upon treatment with compound 6, not only during the entire process, but also on the first two days of adipogenesis. Additionally, treatment with compound 6 regulated the expression of genes involved in adipocyte lipid metabolism, decreasing the lipogenic gene (SREBP1) and increasing lipolytic genes (ATGL and HSL). We provide experimental evidence of the health benefits of using (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid obtained from garlic to prevent excessive adipogenesis in obesity.

WDR76 mediates obesity and hepatic steatosis via HRas destabilization

Ras/MAPK (mitogen active protein kinase) signaling plays contradictory roles in adipocyte differentiation and is tightly regulated during adipogenesis. However, mechanisms regulating adipocyte differentiation involving Ras protein stability regulation are unknown. Here, we show that WD40 repeat protein 76 (WDR76), a novel Ras regulating E3 linker protein, controls 3T3-L1 adipocyte differentiation through HRas stability regulation. The roles of WDR76 in obesity and metabolic regulation were characterized using a high-fat diet (HFD)-induced obesity model using Wdr76−/− mice and liver-specific Wdr76 transgenic mice (Wdr76Li−TG). Wdr76−/− mice are resistant to HFD-induced obesity, insulin resistance and hyperlipidemia with an increment of HRas levels. In contrast, Wdr76Li-TG mice showed increased HFD-induced obesity, insulin resistance with reduced HRas levels. Our findings suggest that WDR76 controls HFD-induced obesity and hepatic steatosis via HRas destabilization. These data provide insights into the links between WDR76, HRas, and obesity.

Catechins and Caffeine Promote Lipid Metabolism and Heat Production Through the Transformation of Differentiated 3T3-L1 Adipocytes from White to Beige Adipocytes.

To elucidate effects of catechins and caffeine on lipid metabolism in adipocytes and identify the mechanism of action, differentiated 3T3-L1 adipocytes were incubated in culture media containing catechins at 1, 2.5, 5, and 10 µg/mL and caffeine at 50 and 100 µg/mL, singly or in combination, for 8 days. Intracellular lipid accumulation and glycerol-3-phosphate dehydrogenase activity were strongly suppressed by catechins and caffeine combination treatment. The mRNA expression of PPARɤ, GLUT4, HSL, UCP-1, and TMEM26 were significantly increased in the combined groups. These findings suggest that the combined treatment inhibited lipid synthesis and improved lipid metabolism in adipocytes. Moreover, it was indicated that the differentiated 3T3-L1 adipocytes could be transformed from white adipocytes to beige-like adipocytes by catechins and caffeine, and accordingly that this transformation could promote calorigenic action. PRACTICAL APPLICATION: In this study, we revealed that the combined treatment of catechins and caffeine inhibited lipid synthesis and improved lipid metabolism in adipocytes. Moreover, the treatment may contribute to the transforming from white adipocytes to beige-like adipocytes, which could strongly promote calorigenic action.

PipeNig®-FL, a Fluid Extract of Black Pepper (Piper Nigrum L.) with a High Standardized Content of Trans-β-Caryophyllene, Reduces Lipid Accumulation in 3T3-L1 Preadipocytes and Improves Glucose Uptake in C2C12 Myotubes.

Trans-β-caryophyllene (BCP) is a natural sesquiterpene hydrocarbon with several important pharmacological activities, including antioxidant, anti-inflammatory, anticancer, and cardioprotective functions. These properties are mainly due to its selective interaction with the peripherally expressed cannabinoid receptor 2. In addition, BCP activates peroxisome proliferated activator receptors α and γ and inhibits the Toll-like receptor signaling pathway. Given the growing scientific interest in BCP, the aim of our study was to investigate the metabolic effects of a black pepper extract (PipeNig®-FL), containing a high standardized content of BCP. In particular our interest was focused on its potential activity on lipid accumulation and glucose uptake. The extract PipeNig®-FL was chemically characterized by gas chromatography–mass spectrometry (GC–MS) and gas chromatography with flame-ionization detection (GC–FID), confirming a high content (814 mg/g) of BCP. Experiments were performed on 3T3-L1 preadipocytes and on C2C12 myotubes. Lipid content following 3T3-L1 adipogenic differentiation was quantified with AdipoRed fluorescence staining. Glucose uptake and GLUT4 membrane translocation were studied in C2C12 myotubes with the fluorescent glucose analog 2-NBDG and by immunofluorescence analysis. Here we show that PipeNig®-FL reduces 3T3-L1 adipocyte differentiation and lipid accumulation. Moreover, acute exposure of C2C12 myotubes to PipeNig®-FL improves glucose uptake activity and GLUT4 migration. Taken together, these results reveal interesting and novel properties of BCP, suggesting potential applications in the prevention of lipid accumulation and in the improvement of glucose uptake.

N,N-Dimethlyacetamide Prevents the High-Fat Diet-Induced Increase in Body Weight.

Increased body weight caused by visceral fat accumulation is on the rise and is reaching epidemic proportions worldwide. Hence, means and ways to tackle the problem of increased adiposity is of utmost importance. In this work, we report the effect of a water-soluble small molecule N,N-Dimethlyacetamide (DMA) on weight gain and adiposity in vitro and in vivo. To monitor the in vitro effect of DMA on adipogenesis, 3T3-L1 preadipocytes and pluripotent C2C12 cells were differentiated to adipocytes in the presence of DMA (5 mM and 10 mM). Oil red O staining and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to evaluate the differentiation to adipocytes. To study the in vivo effect of DMA on body weight, experiments were done with C57BL/6J male mice (6 weeks old). The mice were randomly assigned to receive either high-fat diet (HFD; 45% fat) or a normal diet (7% fat) and were either intraperitoneally injected with DMA or phosphate-buffered saline (PBS) once a week for 20 weeks. Glucose tolerance test was performed on living mice. Post-experiment, the epididymal and subcutaneous adipose tissue were excised from the sacrificed animal, and histology, RT-PCR and plasma triglyceride assay were performed. DMA had no inhibitory effect on adipocyte differentiation when applied only once. However, sustained treatment with DMA inhibited the adipocyte differentiation in both 3T3-L1 and C2C12 cells, and significantly lowered the expression of adipocyte markers, in particular, fatty acid-binding protein 4 (fabp4). Under HFD, C57BL/6J mice treated with DMA had lower body weight compared with PBS treatment. Moreover, the HFD-induced higher body weight was controlled when the mice were switched from PBS to DMA treatment. Further, the HFD-mediated adipocyte hypertrophy from epididymal and subcutaneous adipose tissue was significantly reduced with DMA treatment. Interestingly, the glucose clearance and triglyceride levels in the plasma were improved in mice when DMA treatment was initiated early. Taken together, our results show that DMA exhibits a clear potential to prevent weight gain and restricts adiposity in response to high-fat feeding.

Berberine improves insulin resistance in adipocyte models by regulating the methylation of hypoxia-inducible factor-3α.

Methylation of hypoxia-inducible factor-3α (HIF3A) was previously demonstrated to be highly associated with insulin resistance (IR) in patients with gestational diabetes mellitus (GDM). We aimed to study the therapeutic effects of Berberine (BBR) on GDM and the possible mechanisms. The expressions and methylated states of HIF3A in pregnant women with GDM were compared with that in healthy controls. The IR cell models of 3T3-L1 adipocytes was constructed by 1 μmol/l dexamethasone (Dex) and 1 μmol/l insulin (Ins). To evaluate the effects of BBR on IR adipocyte models, cells were subjected to BBR treatment at different concentrations. Transfection of HIF3A siRNA further confirmed the role of HIF3A in the BBR-induced improving effects. Low expression and high methylation of HIF3A gene were frequent in the GDM pregnancies. BBR treatment noticeably increased the glucose usage rates, adiponectin secretion and cell differentiation of IR 3T3-L1 adipocytes. Increased HIF3A expression and decreased methylated state of HIF3A were also found in IR adipocytes. Furthermore, HIF3A silencing not only reversed the effects of BBR on improving insulin sensibility, but also partially abolished the expression alterations of insulin-related genes in IR adipocytes induced by BBR treatment. Our results suggest that BBR improves insulin sensibility in IR adipocyte models, and the improving effects of BBR are possibly realized through the inhibition of HIF3A methylation.

Preadipocyte factor 1 regulates adipose tissue browning via TNF-α-converting enzyme-mediated cleavage

BackgroundIncreasing adaptive thermogenesis in adipose tissue may be a potential therapeutic target for overcoming obesity and obesity-related disorders. Preadipocyte factor 1 (Pref-1), a preadipocyte secreted protein, plays an inhibitory role in adipogenic differentiation. However, the role of Pref-1 in adipose tissue browning remains unknown. We investigated whether Pref-1 regulates thermogenic program and beige fat biogenesis. MethodsPref-1 expression levels were examined in inguinal white adipose tissue (iWAT) and differentiated 3T3-L1 adipocytes in thermogenic conditions induced by cold exposure or a beta-adrenergic stimulus (CL316,243). Overexpression and knockdown studies were performed both in vivo and in vitro to clarify the role of Pref-1 in iWAT browning. ResultsCold exposure or CL316,243 induced a thermogenic program in adipose tissue of C57BL/6N mice and in 3T3-L1 adipocytes. Notably, Pref-1 levels were down-regulated in iWAT and adipocytes under these conditions. Overexpressing Pref-1 showed reduced thermogenic gene expressions in response to CL316,243 treatment, whereas depletion of Pref-1 augmented thermogenic program in 3T3-L1 adipocytes. Correspondingly, treating C57BL/6N mice with Pref-1 resulted in reduced expression of thermogenic and beige fat markers, a reduced rate of oxygen consumption, blunting of UCP1 expression and beige fat formation in iWAT in response to cold exposure or CL316,243 injection compared to the untreated mice. The opposite phenotype was observed in mice with inducible fat-specific knock-out of Pref-1. Mechanistically, these effects were regulated by modulation of TNF-α-converting enzyme activity and Pref-1 cleavage. ConclusionOur findings establish a novel role of Pref-1 that regulates adaptive thermogenesis. This offers a unique target for improving energy homeostasis and treating obesity.

Proteomic Analysis of 3T3-L1 Adipocytes Treated with Insulin and TNF-α.

Insulin resistance is an indication of early stage Type 2 diabetes (T2D). Insulin resistant adipose tissues contain higher levels of insulin than the physiological level, as well as higher amounts of intracellular tumor necrosis factor-α (TNF-α) and other cytokines. However, the mechanism of insulin resistance remains poorly understood. To better understand the roles played by insulin and TNF-α in insulin resistance, we performed proteomic analysis of differentiated 3T3-L1 adipocytes treated with insulin (Ins), TNF-α (TNF), and both (Ins + TNF). Out of the 693 proteins identified, the abundances of 78 proteins were significantly different (p < 0.05). Carnitine parmitoyltransferase-2 (CPT2), acetyl CoA carboxylase 1 (ACCAC-1), ethylmalonyl CoA decarboxylase (ECHD1), and methylmalonyl CoA isomerase (MCEE), enzymes required for fatty acid β-oxidation and respiratory electron transport, and β-glucuronidase, an enzyme responsible for the breakdown of complex carbohydrates, were down-regulated in all the treatment groups, compared to the control group. In contrast, superoxide dismutase 2 (SOD2), protein disulfide isomerase (PDI), and glutathione reductase, which are the proteins responsible for cytoskeletal structure, protein folding, degradation, and oxidative stress responses, were up-regulated. This suggests higher oxidative stress in cells treated with Ins, TNF, or both. We proposed a conceptual metabolic pathway impacted by the treatments and their possible link to insulin resistance or T2D.

Macrophage-Derived microRNA-155 Increases in Obesity and Influences Adipocyte Metabolism by Targeting Peroxisome Proliferator-Activated Receptor Gamma.

This study aimed to investigate cellular sources of microRNAs (miRNA) within adipose tissue and the impact of obesity on miRNA expression, as well as to examine targets of miRNAs. miRNA expression by quantitative polymerase chain reaction was examined in adipocytes, adipose tissue macrophages (ATM), and peripheral blood mononuclear cells from and individuals withnormal weight and with obesity. Differentiated 3T3-L1 adipocytes were cocultured with macrophages, and 3T3-L1 and differentiated human mesenchymal stem cells were transfected with miR-155, with peroxisome proliferator-activated receptor gamma (PPAR-γ) and solute carrier family 2 member 4 (GLUT4) abundance measured via Western blot analysis. Abundance of miR-155 and miR-210 was increased in ATM of participants with obesity by 6.7-fold and 2.9-fold (P = 0.002 and P = 0.013, respectively). miR-130b expression was increased 1.8-fold in ATM and 4.3-fold in adipocytes from participants with obesity (P = 0.007 and P = 0.02, respectively). PPARG mRNA expression decreased 32% (P = 0.044) in adipocytes from individuals with obesity. In 3T3-L1 cells exposed to macrophages, PPARG expression decreased 99.4% (P = 0.02). PPAR-γ protein content declined 75% (P = 0.001) in 3T3-L1 cells transfected with miR-155. GLUT4 protein levels were reduced by 55% (P = 0.021) in differentiated human mesenchymal stem cells exposed to miR-155. Adipose tissue miRNAs are influenced in a cell type-specific fashion by obesity, with macrophage miR-155 potentially impacting neighboring adipocytes.

Effects of (+)-catechin on the differentiation and lipid metabolism of 3T3-L1 adipocytes

Abstract Catechins are highly bioactive tea flavanols, which have demonstrated anti-inflammatory and anti-cancer activities. This study follows recent attempts to discover and evaluate anti-obesity agents occurring in food materials, with the focus on the effects of (+)-catechin on 3T3-L1 preadipocyte differentiation and adipogenesis in mature adipocytes. The results showed that (+)-catechin treatments remarkably inhibited the differentiation of 3T3-L1 preadipocytes and lipid decomposition of mature adipocytes. On the molecular level, (+)-catechin reduced the expression of C/EBPα and PPARγ and affected the transcription level of downstream functional genes (FAS) during differentiation of preadipocytes. Furthermore, (+)-catechin intervened lipid decomposition through the classic cAMP/PKA signaling pathway, not only reducing the expression of HSL, ATGL and PLIN, but also lowering the phosphorylation level of HSL during lipid decomposition of mature adipocytes. In summary, (+)-catechin had strong anti-differentiation and anti-adipogenesis effects on 3T3-L1 preadipocytes and mature adipocytes, via regulating the C/EBPs/PPARγ/SREBP1C and cAMP/PKA signaling pathways, respectively.

Deep Lipidomics and Molecular Imaging of Unsaturated Lipid Isomers: A Universal Strategy Initiated by mCPBA Epoxidation.

Cellular lipidome is highly regulated through lipogenesis, rendering diverse double-bond positional isomers (C═C isomer) of a given unsaturated lipid species. In recent years, there are increasing reports indicating the physiological roles of C═C isomer compositions associated with diseases, while the biochemistry has not been broadly investigated due to the challenge in characterizing lipid isomers inherent to conventional mass spectrometry-based lipidomics. To address this challenge, we reported a universal, user-friendly, derivatization-based strategy, MELDI (mCPBA Epoxidation for Lipid Double-bond Identification), which enables both large-scale identification and spatial mapping of biological C═C isomers using commercial mass spectrometers without any instrument modification. With the developed liquid-chromatography mass spectrometry (LC-MS) lipidomics workflow, we elucidated more than 100 isomers among monounsaturated and polyunsaturated fatty acids and glycerophospholipids in human serum, where uncommon isomers of low abundance were quantified for the first time. The capability of MELDI-LC-MS in lipidome analysis was further demonstrated using the differentiated 3T3-L1 adipocytes, providing an insight into the cellular lipid reprogramming upon stearoyl-coenzyme A desaturase 1 (SCD1) inhibition. Finally, we highlighted the versatility of MELDI coupled with ambient mass spectrometry imaging to spatially resolve cancer-associated alteration of lipid isomers in a metastatic mouse tissue section. Our results suggested that MELDI will contribute to current lipidomics pipelines with a deeper level of structural information, allowing us to investigate the underlying lipid biochemistry.

Abstract 277: Neonatal Cardiomyocyte-Released Signaling Protein Reduces Adipocyte Differentiation

The heart continuously cycles through periods of muscle contraction and relaxation to pump blood throughout the body, which requires a constant supply of metabolic substrates to the heart to power these contractile cycles. To ensure adequate energy availability, the heart must communicate with other organ systems to maintain global metabolic energy homeostasis. In addition to the role of the sympathetic nervous system in global metabolic regulation, mounting evidence suggests cardiomyocytes can release signaling factors that alter global energy balance through communication with other organ systems, including the liver and adipose tissue. To identify potential cardiomyocyte-released circulating factors, we collected media conditioned by neonatal rat ventricular myocytes (NRVM-CM) and screened for released-factor activity using an in vitro adipocyte differentiation assay. Treatment of differentiating 3T3-L1 adipocytes reduced lipid accumulation and decreased expression of adipocyte proteins, demonstrating NRVM-released factor activity. Released factor activity was also under the control of regulators of GPCR signaling as overexpression of G-protein coupled receptor kinase 2 (GRK2) further enhanced the NRVM-released factor activity. We further determined that NRVM released-factor activity was abolished by trypsin treatment and could be segregated into &gt;30kDa CM fractions using centrifugal filter units. To identify proteins responsible for the NRVM-released factor activity, NRVM-CM retained in the 30kDA centrifugal filter units (containing all proteins &gt;30kDa) was analyzed by mass spectrometry. This revealed a number of signaling proteins with known adipocyte regulatory roles that could potentially regulate adipocyte physiology. We confirmed these results by examining NRVM cell lysate and concentrated NRVM-CM by Western blot to test for the presence of decorin, one of the proteins identified by mass spectrometry known to have adipocyte regulatory roles. These results provide a framework to identify potential in vivo cardiomyocyte-released factors that regulate global energy balance.

Isoimperatorin enhances 3T3-L1 preadipocyte differentiation by regulating PPARγ and C/EBPα through the Akt signaling pathway.

Lipodystrophic patients have an adipose tissue triglyceride storage defect that causes ectopic lipid accumulation, leading to severe insulin resistance. The present study investigated the potential role of isoimperatorin on 3T3-L1 adipocyte differentiation. mRNA and protein levels of differentiation- and lipid accumulation-associated genes, as well as the adipogenesis-related signaling pathway were analyzed in control and isoimperatorin-treated differentiated 3T3-L1 adipocytes using reverse transcription-quantitative PCR and western blot analysis. Results determined that isoimperatorin promoted 3T3-L1 fibroblast adipogenesis in a dose-dependent manner compared with standard differentiation inducers. Isoimperatorin significantly increased mRNA and protein expression of the crucial adipogenic transcription factors peroxisome proliferator activated receptor-γ (PPARγ) and CCAAT enhancer binding protein-α (C/EBPα). mRNA expression of the downstream adipogenesis-related genes sterol regulatory element-binding transcription factor 1c, adipocyte protein 2, fatty acid synthase, adiponectin and diacylglycerol O-acyltransferase 2 were also significantly increased following isoimperatorin treatment. The underlying mechanism likely involved activation of the Akt signaling pathway. Taken together, the present findings indicated that isoimperatorin may alter PPARγ and C/EBPα expression via the Akt signaling pathway, resulting in promotion of adipogenesis. The results highlighted the potential use of isoimperatorin as a therapeutic agent for preventing diabetes.

Khellactone Derivatives and Other Phenolics of Phlojodicarpus sibiricus (Apiaceae): HPLC-DAD-ESI-QQQ-MS/MS and HPLC-UV Profile, and Antiobesity Potential of Dihydrosamidin.

With obesity, the consumption of phenolic-enriched food additives as a part of traditional nutrition avoids the negative implications of eating high-calorie products. This study investigated the new herbal food additive, Phlojodicarpus sibiricus roots and herb, ubiquitously used in Siberia as a spice. Chromatographic techniques such as HPLC-DAD-ESI-QQQ-MS/MS and microcolumn HPLC-UV were the basic instruments for component profiling and quantification, and antiobesity potential was investigated using a differentiated 3T3-L1 adipocytes assay. We found that the roots and herb of P. sibiricus were high-coumarin-containing additives inhibiting triacylglycerol accumulation in 3T3-L1 preadipocytes. Forty-one phenolics were detected in P. sibiricus extracts, and 35 were coumarins, including 27 khellactone derivatives present as esters and glucosides. Total coumarin content varied from 36.16 mg/g of herb to 98.24 mg/g of roots, and from 0.32 mg/mL to 52.91 mg/mL in P. sibiricus preparations. Moreover, Siberian populations of P. sibiricus were characterised by a different HPLC-based coumarin profile. The most pronounced inhibiting effect on triacylglycerol accumulation in 3T3-L1 preadipocytes was shown for dihydrosamidin (khellactone 3′-O-isovaleroyl-4′-O-acetyl ester), which was more active than other khellactone esters and glucosides. The results demonstrated that if used as a food additive Phlojodicarpus sibiricus could be a source of bioactive coumarins of the khellactone group with high antiobesity potential.

Cellular retinoic acid binding protein 1 protects mice from high-fat diet-induced obesity by decreasing adipocyte hypertrophy.

ObjectivesObesity, an emerging global health issue, involves numerous factors; understanding its underlying mechanisms for prevention and therapeutics is urgently needed. Cellular retinoic acid binding protein 1 (Crabp1) knockout (CKO) mice exhibit an obese phenotype under normal diet feedings, which prompted us to propose that Crabp1 could play a role in modulating adipose tissue development/homeostasis. Studies were designed to elucidate the underlying mechanism of Crabp1’s action in reducing obesity.Subjects/methodsIn animal studies, 6 weeks old male wild type and CKO mice were fed with normal diet (ND) or high fat diet (HFD) for 10 weeks. Body weight and food intake were regularly monitored. Glucose tolerance test and biological parameters of plasma (glucose and insulin levels) were measured after 10 weeks of ND vs. HFD feedings. Visceral adipose tissues were collected for histological and molecular analyses to determine affected signaling pathways. In cell culture studies, the 3T3L1 adipocyte differentiation model was used to examine and validate relevant signaling pathways.ResultsCKO mice, compared to WT mice, gained more body weight, exhibited more elevated fasting plasma glucose levels, and developed more severe impaired glucose tolerance under both ND and HFD. Histological examination revealed readily increased adipocyte hypertrophy and adipose tissue inflammation under HFD feedings. In 3T3L1 adipocytes, Crabp1 silencing enhanced extracellular signal-regulated kinase 1/2 (ERK1/2) activation, accompanied by elevated markers and signaling pathways of lipid accumulation and adipocyte hypertrophy.ConclusionsThis study identifies Crabp1’s physiological role against the development of obesity. The protective function of CRABP1 is likely attributed to its classically proposed (canonical) activity as a trap for RA, which will reduce RA availability, thereby dampening RA-stimulated ERK1/2 activation and adipocyte hypertrophy. The results suggest Crabp1 as a potentially new therapeutic target in managing obesity and metabolic diseases.

Patchouli Alcohol from Pogostemon Cablin Prevents Development of Obesity and Improves Glucose Tolerance in High Fat Diet-induced Obese Mice (P06-107-19)

ObjectivesPatchouli alcohol is a sesquiterpene alcohol found in Pogostemon cablin. Recently, we observed that patchouli alcohol reduced lipid accumulation in differentiated 3T3-L1 adipocytes and increased glucose uptake in differentiated C2C12 myocytes. This study was designed to investigate anti-obese and anti-diabetic activities of patchouli alcohol using high fat diet-induced obese mouse model. MethodsForty-eight 5-week old C57BL/6 J male mice were assigned into four groups and fed with 1) normal diet (control), 2) high fat diet, 3) high fat diet with gavaging 25 mg of patchouli alcohol/kg body weight and 4) high fat diet with gavaging 50 mg of patchouli alcohol/kg body weight. High fat diet or control diets were provided to each treatment group for four weeks and then different doses of patchouli alcohol (0, 25 or 50 mg/kg body weight) was orally administered for following 8 weeks with the diet. At age of week 17, all animals were sacrificed, fat tissues were collected, and tissue weight was measured. In addition, twenty C57BL/6 J male mice were assigned into the same treatment groups above. At the end of the 8 weeks (age of week 17), the mice were fasted for 12 h and the oral glucose tolerance test was performed after intraperitoneal injection of 2 g of anhydrous glucose/kg body weight. The blood was collected from tail at 0, 15, 30, 90 and 120 min after injection and blood glucose level was analyzed using glucose meter. ResultsTreatment of patchouli alcohol (50 mg/kg body weight) significantly reduced body weight and accumulation of body fat pads which was highly induced by feeding of high fat diet. An analysis of individual fat pad weights (expressed as mg weight of fat pad/g body weight) revealed a significant decrease of epididymal and retroperitoneal fat pad in patchouli alcohol-treated mice whereas brown adipose tissue were not significantly altered. And, slightly improved glucose tolerance was observed at 90 and 120 minutes after glucose injection in mice treated with patchouli alcohol (50 mg/kg body weight) compared to those fed with high fat diet alone. ConclusionsWe propose a potential use of patchouli alcohol as an anti-obesity compound in obese population. Funding SourcesNIFA Hatch grant. Supporting Tables, Images and/or Graphs▪