α-Cedrene protects rodents from high-fat diet-induced adiposity via adenylyl cyclase 3.

OBJECTIVE: The increasing global prevalence of obesity and its associated disorders points to an urgent need for the development of novel and effective strategies for the prevention of weight gain. Here, we investigated the potential of α-cedrene, a volatile sesquiterpene compound derived from cedarwood oil, in regulation of obesity and delineated the mechanisms involved. METHODS: For the prevention of obesity, C57BL/6 N mice were fed a high-fat diet (HFD) and were orally administered either with vehicle or α-cedrene for 8 weeks. For the therapy of obesity, obese Sprague Dawley rats, induced by a HFD for 8 weeks, were orally treated either with vehicle or α-cedrene for 12 weeks. To determine whether the action of α-cedrene was Adcy3 dependent, Adcy3 heterozygous null mice (Adcy3+/-) and wild-type controls were fed either HFD or α-cedrene supplemented HFD for 17 weeks. RESULTS: Oral α-cedrene administration prevented or reversed HFD-induced obesity and abnormal metabolic aberrations in rodents, without affecting their food intake. Downregulation of Adcy3 expression by small interfering RNA abrogated the beneficial effects of α-cedrene on the oxygen consumption rate and intracellular lipid accumulation in 3T3-L1 adipocytes. Similarly, in Adcy3+/- mice, the α-cedrene-driven suppression of body weight gain observed in wild-type mice was substantially (~50%) attenuated. Expression of thermogenic and lipid oxidation genes was increased in adipose tissues of α-cedrene-treated mice, with concomitant downregulation of adipogenic gene expression. These beneficial molecular changes elicited by α-cedrene were blunted in adipose tissues of Adcy3+/- mice. CONCLUSIONS: Our results highlight the potential of α-cedrene for antiobesity interventions and suggest that the antiobesity effect of α-cedrene is mediated by Adcy3 in adipose tissues.

Embelin attenuates adipogenesis and lipogenesis through activating canonical Wnt signaling and inhibits high-fat diet-induced obesity.

BACKGROUND: Recent studies suggest that Embelin, a natural plant extract might have the potential to prevent body weight gain in rats. However, the mechanisms involved remain to be elucidated. METHODS: Effects of Embelin on adipocyte differentiation and lipogenesis were studied in murine ST2 stromal cells and C3H10T1/2 mesenchymal cells. The mechanisms through which Embelin regulates adipogenic differentiation and lipogenesis were explored. The in vivo anti-obesity effects of Embelin in high-fat diet (HFD)-induced obesity mice and possible transcriptional impact were investigated. RESULTS: Embelin treatment suppressed ST2 and C3H10T1/2 cells to proliferate, and differentiate into mature adipocytes, along with the inhibition of adipogenic factors peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein-α, adipocyte protein 2 and adipsin. Embelin treatment also decreased the expression levels of lipogenic factors sterol regulatory element-binding protein 1, fatty acid synthase, acetyl-CoA carboxylase 1 and stearoyl-Coenzyme A desaturase 1. Embelin promoted the translocation of ß-catenin from the cytoplasm into the nucleus in C3H10T1/2. The nuclear protein levels of ß-catenin and TCF-4 were increased following Embelin treatment. Furthermore, Dickkopf-1 (Dkk1) expression was downregulated by Embelin, and overexpression of Dkk1 in C3H10T1/2 reversed the inhibition of adipogenesis and lipogenesis by Embelin. In vivo studies showed that Embelin treatment reduced the gain of body weight and fat, decreased the serum level of triglycerides, free fatty acid and total cholesterol, and improved glucose tolerance and insulin resistance in HFD-fed mice. Moreover, Embelin blocked induction of adipogenic and lipogenic factors and Dkk1 in adipose tissue in HFD-fed mice. CONCLUSIONS: The present work provides evidences that Embelin is effective in inhibiting adipogenesis and lipogenesis in vitro and the mechanisms may involve canonical Wnt signaling. Embelin has the potential to prevent body weight gain and fat accumulation, and to improve obesity-related glucose tolerance impairment and insulin resistance in the HFD-fed mice.

Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach.

BACKGROUND: From ancient times, marine algae have emerged as alternative medicine and foods, contains the rich source of natural products like proteins, vitamins, and secondary metabolites, especially Chlorella vulgaris (C. vulgaris) contains numerous anti-inflammatory, antioxidants and wound healing substances. Type 2 diabetes mellitus is closely associated with adipogenesis and their factors. Hence, we aimed to investigate the chemical constituents and adipogenic modulatory properties of C. vulgaris in 3T3-L1 pre-adipocytes. RESULTS: We analysed chemical constituents in ethanolic extract of C. vulgaris (EECV) by LC-MS. Results revealed that the EECV contains few triterpenoids and saponin compounds. Further, the effect of EECV on lipid accumulation along with genes and proteins expressions which are associated with adipogenesis and lipogenesis were evaluated using oil red O staining, qPCR and western blot techniques. The data indicated that that EECV treatment increased differentiation and lipid accumulation in 3T3-L1 cells, which indicates positive regulation of adipogenic and lipogenic activity. These increases were associated with up-regulation of PPAR-γ2, C/EBP-α, adiponectin, FAS, and leptin mRNA and protein expressions. Also, EECV treatments increased the concentration of glycerol releases as compared with control cells. Troglitazone is a PPAR-γ agonist that stimulates the PPAR-γ2, adiponectin, and GLUT-4 expressions. Similarly, EECV treatments significantly upregulated PPAR-γ2, adiponectin, GLUT-4 expressions and glucose utilization. Further, EECV treatment decreased AMPK-α expression as compared with control and metformin treated cells. CONCLUSION: The present research findings confirmed that the EECV effectively modulates the lipid accumulation and differentiation in 3T3-L1 cells through AMPK-α mediated signalling pathway.

Complete Membrane Fractionation of 3T3-L1 Adipocytes.

Fractionation techniques can facilitate the isolation of intracellular organelles containing insulin-sensitive glucose transporter isoform 4 (GLUT4), which is mobilized from GLUT4 storage vesicles in fat and muscle cells in response to insulin. This protocol for the full membrane fractionation of 3T3-L1 adipocytes results in five distinct fractions. A heavy membrane-containing pellet is produced and then further separated into the plasma membrane, mitochondrial and nuclear, and high-density membrane fractions. The initial supernatant is subjected to a series of centrifugation steps to isolate the low-density membrane fraction, which contains the majority of the insulin-sensitive pool of GLUT4; the supernatant produced in this step is the soluble fraction. The distribution of GLUT4 in fractions from insulin-stimulated versus untreated cells is assessed via immunoblotting.

Iodixanol Gradient Centrifugation to Separate Components of the Low-Density Membrane Fraction from 3T3-L1 Adipocytes.

We optimized a set of fractionation techniques to facilitate the isolation of subcellular compartments containing insulin-sensitive glucose transporter isoform 4 (GLUT4), which is mobilized from GLUT4 storage vesicles (GSVs) in fat and muscle cells in response to insulin. In the absence of insulin, GLUT4 undergoes a continuous cycle of GSV formation and fusion with other compartments. Full membrane fractionation of 3T3-L1 adipocytes produces a low-density membrane fraction that contains both the constitutive recycling pool (the endosomal recycling compartments) and the insulin-sensitive pool (the GSVs). These two pools can be separated based on density using iodixanol gradient centrifugation, described here.

Effects and Molecular Mechanism of GST-Irisin on Lipolysis and Autocrine Function in 3T3-L1 Adipocytes.

Irisin, which was recently identified as a myokine and an adipokine, transforms white adipose tissue to brown adipose tissue and has increasingly caught the attention of the medical and scientific community. However, the signaling pathway of irisin and the molecular mechanisms responsible for the lipolysis effect remain unclear. In this study, we established an efficient system for the expression and purification of GST-irisin in Escherichia coli. The biological activity of GST-irisin was verified using the cell counting kit-8 assay and by detecting the mRNA expression of uncoupling protein 1. Our data showed that GST-irisin regulates mRNA levels of lipolysis-related genes such as adipose triglyceride lipase and hormone-sensitive lipase and proteins such as the fatty acid-binding protein 4, leading to increased secretion of glycerol and decreased lipid accumulation in 3T3-L1 adipocytes. In addition, exogenous GST-irisin can increase its autocrine function in vitro by regulating the expression of fibronectin type III domain-containing protein 5. GST-irisin could regulate glucose uptake in 3T3-L1 adipocytes. Hence, we believe that recombinant GST-irisin could promote lipolysis and its secretion in vitro and can potentially prevent obesity and related metabolic diseases.

Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach

BACKGROUND: From ancient times, marine algae have emerged as alternative medicine and foods, contains the rich source of natural products like proteins, vitamins, and secondary metabolites, especially Chlorella vulgaris (C. vulgaris) contains numerous anti-inflammatory, antioxidants and wound healing substances. Type 2 diabetes mellitus is closely associated with adipogenesis and their factors. Hence, we aimed to investigate the chemical constituents and adipo-genic modulatory properties of C. vulgaris in 3T3-L1 pre-adipocytes. RESULTS: We analysed chemical constituents in ethanolic extract of C. vulgaris (EECV) by LC-MS. Results revealed that the EECV contains few triterpenoids and saponin compounds. Further, the effect of EECV on lipid accumulation along with genes and proteins expressions which are associated with adipogenesis and lipogenesis were evaluated using oil red O staining, qPCR and western blot techniques. The data indicated that that EECV treatment increased differentiation and lipid accumulation in 3T3-L1 cells, which indicates positive regulation of adipogenic and lipogenic activity. These increases were associated with up-regulation of PPAR-γ2, C/EBP-α, adiponectin, FAS, and leptin mRNA and protein expressions. Also, EECV treatments increased the concentration of glycerol releases as compared with control cells. Troglitazone is a PPAR-γ agonist that stimulates the PPAR-y2, adiponectin, and GLUT-4 expressions. Similarly, EECV treatments significantly upregulated PPAR-γ, adiponectin, GLUT-4 expressions and glucose utilization. Further, EECV treatment decreased AMPK-α expression as compared with control and metformin treated cells. CONCLUSION: The present research findings confirmed that the EECV effectively modulates the lipid accumulation and differentiation in 3T3-L1 cells through AMPK-α mediated signalling pathway.

Differentiation of 3T3-L1 preadipocytes is inhibited under a modified ceiling culture.

Ceiling culture is an inverted and closed cell culture system which represents a novel method for exploring adipocyte characteristics and function. Although the role of ceiling culture in mature adipocyte dedifferentiation has been extensively studied, its potential effects on preadipocyte differentiation remain unclear. In this study, we established a simplified dish ceiling culture method for 3T3-L1 preadipocytes and showed that our novel ceiling culture method could reproduce the function of the traditional flask ceiling culture. Then, we investigated the effects of ceiling culture on 3T3-L1 preadipocyte differentiation by Oil red O staining and RT-qPCR. The results showed that ceiling culture significantly impaired triglyceride accumulation and adipogenic marker genes expression in 3T3-L1 preadipocytes. These findings suggest that ceiling culture inhibited 3T3-L1 preadipocyte differentiation while inducing mature adipocytes dedifferentiation. Taken together, our data facilitate the understanding of the property of ceiling culture and promote the study of revealing the underlying mechanisms of mature adipocytes dedifferenatiation.

Corticotrophin-Releasing Factor (CRF) and the urocortins are potent regulators of the inflammatory phenotype of human and mouse white adipocytes and the differentiation of mouse 3T3L1 pre-adipocytes.

Chronic activation of innate immunity takes place in obesity and initiated by the hypertrophic adipocytes which obtain a pro-inflammatory phenotype. The corticotrophin-releasing factor (CRF) family of neuropeptides and their receptors (CRF1 and CRF2) affect stress response and innate immunity. Adipose tissue expresses a complete CRF system. The aim of this study was to examine the role of CRF neuropeptides in the immune phenotype of adipocytes assessed by their expression of the toll-like receptor-4 (TLR4), the production of inflammatory cytokines IL-6, TNF-α and IL-1ß, chemokines IL-8, monocyte attractant protein-1 (MCP-1) and of the adipokines adiponectin, resistin and leptin. Our data are as follows: (a) CRF, UCN2 and UCN3 are expressed in human white adipocytes as well as CRFR1a, CRFR2a and CRFR2b but not CRFR2c. 3T3L1 pre-adipocytes and differentiated adipocytes expressed both CRF1 and CRF2 receptors and UCN3, while UCN2 was detected only in differentiated adipocytes. CRF2 was up-regulated in mouse mature adipocytes. (b) CRF1 agonists suppressed media- and LPS-induced pre-adipocyte differentiation while CRF2 receptor agonists had no effect. (c) In mouse pre-adipocytes, CRF2 agonists suppressed TLR4 expression and the production of IL-6, CXCL1 and adiponectin while CRF1 agonists had no effect. (d) In mature mouse adipocytes LPS induced IL-6 and CXCL1 production and suppressed leptin. (e) In human visceral adipocytes LPS induced IL-6, TNF-α, IL-8, MCP-1 and leptin production and suppressed adiponectin and resistin. (f) In mouse mature adipocytes CRF1 and CRF2 agonists suppressed basal and LPS-induced production of inflammatory cytokines, TLR4 expression and adiponectin production, while in human visceral adipocytes CRF and UCN1 suppressed basal and LPS-induced IL-6, TNF-α, IL-8 and MCP-1 production. In conclusion, the effects of the activation of CRF1 and CRF2 may be significant in ameliorating the pro-inflammatory activity of adipocytes in obesity.

Apigenin isolated from Daphne genkwa Siebold et Zucc. inhibits 3T3-L1 preadipocyte differentiation through a modulation of mitotic clonal expansion.

AIMS: Obesity develops when energy intake chronically exceeds total energy expenditure. We sought to assess whether the flavonoid-rich fraction of crude extracts from Daphne genkwa Siebold et Zuccarini (GFF) might inhibit adipogenesis of 3T3-L1 cells. MAIN METHODS: Cell viability of 3T3-L1 preadipocytes was assessed by MTT assays, and lipid accumulation was measured by Oil Red O. Adipogenesis related factors were checked by Western blot analysis. Flow cytometry was used to analyze the mitotic cell cycle during the mitotic clonal expansion phase. KEY FINDINGS: Among five flavonoids isolated from GFF, only apigenin potently inhibited the differentiation of 3T3-L1 cells. Apigenin reduced CCAAT/enhancer binding protein (C/EBP) α and peroxisome proliferator-activated receptor γ levels. Apigenin-treated 3T3-L1 cells failed to undergo clonal expansion during the early phase of adipocyte differentiation. Apigenin arrested cell cycle progression at the G0/G1 phase. This effect was associated with a marked decrease in cyclin D1 and cyclin-dependent kinase 4 expression, with the concomitant and sustained expression of p27(Kip1). In addition, apigenin inhibited the DNA-binding activity of C/EBPß in differentiating 3T3-L1 cells by down-regulating the 35kDa isoform of C/EBPß (liver-enriched activating protein) and up-regulating the expression of two different sets of C/EBP inhibitors: C/EBP homologous protein and the phospho-liver-enriched inhibitory protein isoform of C/EBPß. SIGNIFICANCE: These findings suggest that apigenin can prevent 3T3-L1 preadipocyte differentiation by the inhibition of the mitotic clonal expansion and the adipogenesis related factors and upregulation of the expression of multiple C/EBPß inhibitors.

Echinacea purpurea root extract enhances the adipocyte differentiation of 3T3-L1 cells.

Echinacea purpurea has been shown to have anti-diabetic activities; for example, it activates peroxisome proliferator-activated receptor γ (PPARγ) and increases insulin-stimulated glucose uptake. Adipogenesis has been used to study the insulin signaling pathway and to screen anti-diabetic compounds. The present study was conducted to investigate the effects of an ethanol extract of E. purpurea (EEEP) and its constituents on the insulin-induced adipocyte differentiation of 3T3-L1 preadipocytes. When adipocyte differentiation was induced with insulin plus 3-isobutyl-1-methylxanthine and dexamethasone, the accumulation of lipid droplets and the cellular triglyceride content were significantly increased by EEEP. The expressions of PPARγ and C/EBPα in adipocytes treated with EEEP were gradually increased as compared with control cells. Fat accumulation and triglyceride content of adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly increased as compared with control cells. The expressions of PPARγ and C/EBPα in adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly higher than in control cells. These results suggest EEEP promotes the adipogenesis that is partially induced by insulin and that dodeca-2(E),4(E)-dienoic acid isobutylamide appears to be responsible for EEEP-enhanced adipocyte differentiation.

Anti-adipogenic activity of the edible brown alga Ecklonia stolonifera and its constituent fucosterol in 3T3-L1 adipocytes.

Fucosterol is a sterol metabolite of brown algae and regulates genes involved with cholesterol homeostasis. As a part of our continuous search for anti-obesity agents from natural marine sources, the anti-adipogenic activities of Ecklonia stolonifera and its sterol, fucosterol, were evaluated for the inhibition of adipocyte differentiation and lipid formation. Oil Red O staining was used to evaluate triglyceride contents in 3T3-L1 pre-adipocytes primed by differentiation medium (DM) I and DM II. The methanolic extract of E. stolonifera showed strong anti-adipogenic activity, and was thus fractionated with several solvents. Among the tested fractions, the dichloromethane (CH2Cl2) fraction was found to be the most active fraction, with significant inhibition (40.5 %) of intracellular lipid accumulation at a non-toxic concentration, followed by the ethyl acetate fraction (30.2 %) at the same concentration, while the n-butanol and water fractions did not show inhibitory activity within the tested concentrations. The strong anti-adipogenic CH2Cl2-soluble fraction was further purified by a repeated chromatography to yield fucosterol. Fucosterol reduced lipid contents in a concentration-dependent manner without showing any cytotoxicity. Fucosterol treatment also yielded a decrease in the expression of the adipocyte marker proteins peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) in a concentration-dependent manner. Taken together, these results suggest that fucosterol inhibits expression of PPARγ and C/EBPα, resulting in a decrease of lipid accumulation in 3T3-L1 pre-adipocytes, indicating that the potential use of E. stolonifera and its bioactive fucosterol as an anti-obesity agent.

The use of Transwells™ improves the rates of differentiation and growth of cultured 3T3L1 cells.

The use of Transwells™ for routine cultures of 3T3L1 cells results in a much improved rate of differentiation of fibroblasts to adipocytes (100% in 9 of 10 tests) compared with bottom-well layer cultures. Mean size of cells was not different, but the cell number and overall cell mass was 3× larger in transwell in spite of a smaller surface area. The difference between both models was the accessibility in transwells of both sides of the cells to the medium (and oxygen). Cells were counted, and their size estimated using a handheld cell counter, Scepter™, designed for blood cells, but adjusted to the larger size of adipocytes. Finally, the effect of nitric oxide was tested using spermineNONOate, a nitric oxide (NO·) donor. The product was released to cultures at a constant 1 µl/h rate for up to 3 days using osmotic Alzet™ minipumps held in wells with water and discharging their contents to the cultured cell-laden wells through a short capillary tube. A rate of 0.3 pmol/min/ml of medium did not affect the cells' size, but 0.4 pmol/min/ml significantly increased cell mass. The methodological improvements presented here allow for more uniform cultured cell yields and a more flexible environment for control of cell size and administration of signaling agents.

Functional ion channels and cell proliferation in 3T3-L1 preadipocytes.

Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K(+) current (I(Kir), recorded in 15% of cells) inhibited by Ba(2+), a Ca(2+)-activated intermediate K(+) current (IK(Ca), recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (I(Cl)) inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8 T) insult, implicating a volume-sensitive I(Cl) (I(Cl.vol)). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IK(Ca)), Kir2.1 (for I(Kir)), and Clcn3 (for I(Cl.vol)). Blockade of IK(Ca) with TRAM-34 or I(Cl.vol) with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, I(KCa), I(Cl.vol), and I(Kir), are heterogeneously present in 3T3-L1 preadipocytes. I(KCa) and I(Cl.vol) participate in the regulation of cell proliferation.

Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3-L1 preadipocytes.

Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndromes, a condition with impaired preadipocytes differentiation. In the present study, we tested the hypothesis that AOPPs disturb preadipocyte differentiation. Exposure of 3T3-L1 preadipocytes to increased levels of AOPPs inhibited accumulation of intracellular triglyceride and decreased the expression of the essential markers of matured adipocytes, such as adipocyte fatty-acid-binding protein (aP2), CAAT/enhancer-binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma, in response to standard adipogenic induction. Inhibitory effects of AOPPs on preadipocytes differentiation was time sensitive, which occurred at the early stage of differentiation. In the presence of AOPPs, induction of preadipocytes differentiation resulted in upregulated expression of C/EBP homologous protein (CHOP) and CUG-Triplet repeat-binding protein (CUGBP), two important inhibitors of preadipocytes differentiation. In addition, treatment with AOPPs increased abundance of C/EBP-beta-liver enriched inhibitory protein (C/EBP-beta-LIP), a truncated C/EBP-beta isoform without adipogenic activity. Moreover, AOPPs-treated preadipocytes expressed a macrophage marker F4/80 and overexpressed tumor necrosis factor-alpha and interleukin-6 via nuclear factor-kappaB (NF-kappaB)-dependent pathway. However, blocking inflammation with NF-kappaB inhibitor failed to improve AOPPs-induced inhibition of preadipocytes differentiation. These data suggest that accumulation of AOPPs may inhibit differentiation of preadipocytes and activate inflammation in these cells. This information might have implication for understanding the impairment of preadipocytes differentiation and fat inflammation seen in metabolic syndrome.

CNP/GC-B system: a new regulator of adipogenesis.

Adipogenesis is regulated by a wide variety of compounds. An adipogenic cocktail containing insulin (INS), dexamethasone (DEX) and 3-isobutyl-1-methyl xanthine (IBMX) is routinely used to induce adipogenesis in 3T3-L1 preadipocytes, but the biochemical actions in adipogenesis of IBMX, a non-specific phosphodiesterase inhibitor, are not completely understood. In this study we show that C-type natriuretic peptide (CNP) is an endogenous adipogenesis regulator which can largely replace the function of IBMX. In 3T3-L1 preadipocytes, CNP potently elevated cGMP production through guanylyl cyclase-B (GC-B). Lipid droplets were evident in these cells upon stimulation with CNP for 12 days in the presence of INS and DEX, and their adiposity, evaluated by Oil Red O, was significantly higher than in cells stimulated with INS and DEX only. Membrane-permeable cGMP analogue also enhanced adiposity when cells were cultured together with INS and DEX, and KT5823, a non-specific cGMP-dependent kinase (cGK) inhibitor, suppressed the stimulatory effect of IBMX on adipogenesis, revealing that IBMX-stimulated adipogenesis is mediated through cGK. The enhancement of adiposity elicited by CNP was accompanied by increased mRNA levels of adipocyte-specific genes including those encoding peroxisome proliferator-activated receptor gamma and glucose transporter 4. Interestingly, the mRNA level of CNP itself was markedly enhanced in 3T3-L1 cells upon stimulation with INS, DEX and IBMX, reaching a maximum at 8h incubation with the cocktail. These observations suggest that the CNP/GC-B system participates in regulation of adipogenesis, particularly at an early stage in the process.

Inhibition of thrombin action ameliorates insulin resistance in type 2 diabetic db/db mice.

The binding of thrombin to its receptor stimulates inflammatory cytokines including IL-6 and monocyte chemoattractant protein-1 (MCP-1); both are associated with the development of insulin resistance. Because increased adiposity enhanced the expression of coagulation factor VII that stimulates the coagulation pathway in adipose tissue, we tested whether the inhibition of thrombin action ameliorates insulin resistance in obese diabetic (Lpr(-/-):db/db) mice. The 4-wk administration of argatroban, a selective thrombin inhibitor, reduced fasting plasma glucose and ameliorated insulin resistance in these mice. It also reduced adipocyte size and macrophage infiltration into adipose tissue. The aberrant gene expression of MCP-1, IL-6, adiponectin, and factor VII and suppressed insulin receptor substrate-1-Akt signaling in adipose tissue of db/db mice were reversed by argatroban treatment. These results demonstrate that increased adiposity enhances the production of thrombin in adipose tissue by stimulating factor VII expression and suggest that increased thrombin activity in adipose tissue plays an important role in the development of insulin resistance via enhancing MCP-1 production, leading to macrophage infiltration and insulin receptor substrate-1-Akt pathway inactivation.

Mechanisms regulating repression of haptoglobin production by peroxisome proliferator-activated receptor-gamma ligands in adipocytes.

Obesity leads to inflammation of white adipose tissue involving enhanced secretion of cytokines and acute-phase proteins in response in part to the accumulation of excess lipids in adipocytes. Haptoglobin is an acute-phase reactant secreted by white adipose tissue and induced by inflammatory cytokines such as TNFalpha. In this study, we investigated the mechanisms regulating haptoglobin expression in adipocytes. Peroxisome proliferator-activated receptor (PPAR)-gamma agonists such as thiazolidinediones (TZDs) as well as non-TZD ligands can repress in vitro and in vivo haptoglobin expression in adipocytes and also prevent its induction by TNFalpha. This action requires direct involvement of PPAR gamma in regulating haptoglobin gene transcription because mutation of critical amino acids within helix 7 of the ligand-binding domain of PPAR gamma prevents repression of the haptoglobin gene by the synthetic ligands. Chromatin immunoprecipitation analysis shows active binding of PPAR gamma to a distal region of the haptoglobin promoter, which contains putative PPAR gamma binding sites. Additionally, PPAR gamma induces transcription of a luciferase reporter gene when driven by the distal promoter region of the haptoglobin gene, and TZD treatment significantly reduces the extent of this induction. Furthermore, the mutated PPAR gamma is incapable of enhancing luciferase activity in these in vitro reporter gene assays. In contrast to other adipokines repressed by TZDs such as resistin and chemerin, repression of haptoglobin does not require either CCAAT/enhancer-binding protein C/EBP alpha or the corepressors C-terminal binding protein 1 or 2. These data are consistent with a model in which synthetic PPAR gamma ligands selectively activate PPAR gamma bound to the haptoglobin gene promoter to arrest haptoglobin gene transcription.

Characterization of GLUT4-containing vesicles in 3T3-L1 adipocytes by total internal reflection fluorescence microscopy.

Insulin-responsive GLUT4 (glucose transporter 4) translocation plays a major role in regulating glucose uptake in adipose tissue and muscle. Whether or not there is a specialized secretory GSV (GLUT4 storage vesicle) pool, and more importantly how GSVs are translocated to the PM (plasma membrane) under insulin stimulation is still under debate. In the present study, we systematically analyzed the dynamics of a large number of single GLUT4-containing vesicles in 3T3-L1 adipocytes by TIRFM (total internal reflection fluorescence microscopy). We found that GLUT4-containing vesicles can be classified into three groups according to their mobility, namely vertical, stable, and lateral GLUT4-containing vesicles. Among these groups, vertical GLUT4-containing vesicles exclude transferrin receptors and move towards the PM specifically in response to insulin stimulation, while stable and lateral GLUT4-containing vesicles contain transferrin receptors and show no insulin responsiveness. These data demonstrate that vertical GLUT4-containing vesicles correspond to specialized secretory GSVs, which approach the PM directly and bypass the constitutive recycling pathway.

Endoplasmic reticulum stress regulates adipocyte resistin expression.

OBJECTIVE: Resistin is a secreted polypeptide that impairs glucose metabolism and, in rodents, is derived exclusively from adipocytes. In murine obesity, resistin circulates at elevated levels but its gene expression in adipose tissue is paradoxically reduced. The mechanism behind the downregulation of resistin mRNA is poorly understood. We investigated whether endoplasmic reticulum (ER) stress, which is characteristic of obese adipose tissue, regulates resistin expression in cultured mouse adipocytes. RESEARCH DESIGN AND METHODS: The effects of endoplasmic stress inducers on resistin mRNA and secreted protein levels were examined in differentiated 3T3-L1 adipocytes, focusing on the expression and genomic binding of transcriptional regulators of resistin. The association between downregulated resistin mRNA and induction of ER stress was also investigated in the adipose tissue of mice fed a high-fat diet. RESULTS: ER stress reduced resistin mRNA in 3T3-L1 adipocytes in a time- and dose-dependent manner. The effects of ER stress were transcriptional because of downregulation of CAAT/enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma transcriptional activators and upregulation of the transcriptional repressor CAAT/enhancer binding protein homologous protein-10 (CHOP10). Resistin protein was also substantially downregulated, showing a close correspondence with mRNA levels in 3T3-L1 adipocytes as well as in the fat pads of obese mice. CONCLUSIONS: ER stress is a potent regulator of resistin, suggesting that ER stress may underlie the local downregulation of resistin mRNA and protein in fat in murine obesity. The paradoxical increase in plasma may be because of various systemic abnormalities associated with obesity and insulin resistance.