Design,synthesis, and biological evaluation of biotin-labeled (−)-ternatin,a potent fat-accumulation inhibitor against 3T3-L1 adipocytes

The design, synthesis, and biological activity of biotin-labeled (?)-ternatin are reported. Chemical modification, that is, biotinylation, was conducted using Click chemistry at the 6-position (NMe-d-ProGly moiety), which was a plausible location selected on the basis of our SAR studies. The compound displayed sufficient fat-accumulation inhibitory effect against 3T3-L1 adipocytes for further bio-organic studies.     

Osthol inhibits fatty acid synthesis and release via PPARα/γ-mediated pathways in 3T3-L1 adipocytes

Osthol is an active constituent isolated from the fruit of Cnidium monnieri (L.) Cusson (Apiaceae), and has obvious therapeutic effect on fatty liver, but its mechanisms are not yet understood completely. One potential link between adipose tissue and fatty liver may be circulating fatty acids. In the present study, the effect of osthol on fatty acid synthesis and release in cultured 3T3-L1 adipocytes was observed. Following treatment of adipocytes with osthol, the intracellular levels of free fatty acids (FFA) and triacylglycerols as well as cultured supernatant level of FFA were decreased, and some lipogenic gene and protein expressions were also decreased, while the peroxisome proliferator-activated receptor (PPAR) α/γ mRNA expressions were increased. Osthol-reduced lipogenic gene expressions were decreased or abrogated after pretreatment with specific inhibitor(s) of PPARα and/or PPARγ.     

Design,synthesis and SAR of new-di-substituted pyridopyrimidines as ATP-competitive dual PI3Kα/mTOR inhibitors

PI3Kα/mTOR ATP-competitive inhibitors are considered as one of the promising molecularly targeted cancer therapeutics. Based on lead compound A from the literature, two similar series of 2-substituted-4-morpholino-pyrido[3,2-d]pyrimidine and pyrido[2,3-d]pyrimidine analogs were designed and synthesized as PI3Kα/mTOR dual inhibitors. Interestingly, most of the series gave excellent inhibition for both enzymes with IC₅₀ values ranging from single to double digit nM. Unlike many PI3Kα/mTOR dual inhibitors, our compounds displayed selectivity for PI3Kα. Based on its potent enzyme inhibitory activity, selectivity for PI3Kα and good therapeutic index in 2D cell culture viability assays, compound 4h was chosen to be evaluated in 3D culture for its IC₅₀ against MCF7 breast cancer cells as well as for docking studies with both enzymes.     

Effects of 17β-estradiol and progesterone on the production of adipokines in differentiating 3T3-L1 adipocytes: Role of Rho-kinase

Effect of female sex hormones on the production/release of adipocyte-derived cytokines has been debatable. Furthermore, whether the cellular signaling triggered by these hormones involve Rho-kinase has not been investigated yet. Therefore, in this study, effects of 17β-estradiol and progesterone as well as the Rho-kinase inhibitor, Y-27632 on the level of adipokines such as resistin, adiponectin, leptin, TNF-α and IL-6 were investigated in 3T3-L1-derived adipocytes. Differentiation was induced in the post-confluent preadipocytes by the standard differentiation medium (Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum together with the mixture of isobutylmethylxanthine, dexamethasone and insulin) in the presence of 17β-estradiol (10⁻⁸–10⁻⁷ M), progesterone (10⁻⁶–10⁻⁵ M), the Rho-kinase inhibitor, Y-27632 (10⁻⁵ M) and their combination for 8 days. Measurements of the adipokines were performed in the culturing medium by ELISA kits using specific monoclonal antibodies. 17β-estradiol elevated resistin but decreased adiponectin and IL-6 levels; however, it did not alter the concentration of leptin and TNF-α. Y-27632 pretreatment inhibited the rise of resistin and the fall of adiponectin by 17β-estradiol without any effects by its own. Progesterone did not change resistin, leptin and TNF-α level; however, it elevated adiponectin and decreased IL-6 production. Neither 17β-estradiol nor Y-27632 was able to antagonize the increase of adiponectin and the reduction of IL-6 levels by progesterone. While Y-27632 alone lowered IL-6 level, it increased leptin and TNF-α concentration without altering resistin and adiponectin. In conclusion, 17β-estradiol could modify adipokine production in 3T3-L1 adipocytes with the actions some of which involve Rho-kinase mediation.     

Hsp90 chaperones PPARγ and regulates differentiation and survival of 3T3-L1 adipocytes

Adipose tissue dysregulation has a major role in various human diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) is a key regulator of adipocyte differentiation and function, as well as a target of insulin-sensitizing drugs. The Hsp90 chaperone stabilizes a diverse set of signaling ‘client'' proteins, thereby regulates various biological processes. Here we report a novel role for Hsp90 in controlling PPARγ stability and cellular differentiation. Specifically, we show that the Hsp90 inhibitors geldanamycin and novobiocin efficiently impede the differentiation of murine 3T3-L1 preadipocytes. Geldanamycin at higher concentrations also inhibits the survival of both developing and mature adipocytes, respectively. Further, Hsp90 inhibition disrupts an Hsp90-PPARγ complex, leads to the destabilization and proteasomal degradation of PPARγ, and inhibits the expression of PPARγ target genes, identifying PPARγ as an Hsp90 client. A similar destabilization of PPARγ and a halt of adipogenesis also occur in response to protein denaturing stresses caused by a single transient heat-shock or proteasome inhibition. Recovery from stress restores PPARγ stability and adipocyte differentiation. Thus, our findings reveal Hsp90 as a critical stress-responsive regulator of adipocyte biology and offer a potential therapeutic target in obesity and the metabolic syndrome.     

Regulation and roles of PI3Kβ, a major actor in platelet signaling and functions

Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.     

Dehydroabietic acid activates peroxisome proliferator-activated receptor-γ and stimulates insulin-dependent glucose uptake into 3T3-L1 adipocytes

Dehydroabietic acid (DAA) is a food-derived terpenoid with various bioactivities. Our previous study has revealed that DAA activates peroxisome proliferator-activated receptor-γ (PPARγ) in luciferase assay and suppresses chronic inflammation in obese adipose tissues. In this study, we examined the effects of DAA on adipocyte differentiation. DAA treatment stimulated the adipocyte differentiation of 3T3-L1 preadipocytes. The DAA treatment increased the mRNA expression levels of adipocyte differentiation marker genes such as aP2, lipoprotein lipase (LPL), and PPARγ. In particular, the expression level of adiponectin, which is an adipocytokine with stimulatory effects on insulin sensitivity, was increased at both the mRNA and protein levels by the DAA treatment. Moreover, the DAA treatment stimulated insulin-dependent glucose uptake into differentiated 3T3-L1 adipocytes. These findings indicate that DAA stimulates adipocyte differentiation and insulin sensitivity in 3T3-L1 cells, suggesting that DAA is a valuable food-derived compound for the management of metabolic syndrome.     

Flavanone exhibits PPARγ ligand activity and enhances differentiation of 3T3-L1 adipocytes

Flavanones are class of polyphenolic compounds, some of which are found in foods and provide health benefits. In this study, we show that flavanone significantly enhances differentiation of 3T3-L1 preadipocytes. During adipogenesis, flavanone enhanced expression of genes and accumulation of proteins that are involved in adipocyte function. Some reports have indicated that flavanone inhibits proliferation of mammalian cells, and down-regulates expression of growth-related proteins. Such proteins include phosphorylated ERK1/2, cyclins, and Cdks that are important for an early event in adipogenesis, mitotic clonal expansion (MCE). We demonstrated that flavanone did not inhibit MCE or expression of MCE-related proteins, except for a modest inhibition of cyclin D1 expression. Using luciferase reporter assays, we found that flavanone acted as a peroxisome proliferator-activated receptor γ (PPARγ) ligand in a dose-dependent manner. Together, our results suggest that flavanone enhances adipogenesis, at least in part, through its PPARγ ligand activity.     

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.     

Developmentally spliced PKCβII provides a possible link between mTORC2 and Akt kinase to regulate 3T3-L1 adipocyte insulin-stimulated glucose transport

Functional adipocyte glucose disposal is a key component of global glucose homeostasis. PKCβII is involved in rat skeletal muscle cell ISGT. Western blot analysis and real-time PCR revealed 3T3-L1 cells developmentally regulated PKCβ splicing such that PKCβI was downregulated and PKCβII was upregulated during the course of differentiation. An initial glucose uptake screen using PKC inhibitor LY379196 pointed to a PKC isozyme other than PKCζ mediating 3T3-L1 adipocyte ISGT. Subsequent use of PKCβII inhibitor CGP53353 pointed to a role for PKCβII in ISGT. Western blot analysis showed that CGP53353 specifically inhibited phosphorylation of PKCβII Serine 660. Subcellular fractionation and immunofluorescence demonstrated that PKCβII regulates GLUT4 translocation. Further Western blot, immunofluorescence and co-immunoprecipitation analysis reveal that PKCβII inhibition does not affect mTORC2 activity yet abrogates phosphorylation of Akt Serine 473. PKCβII regulates GLUT4 translocation by regulating Akt phosphorylation and thus activity.     

Nr4a1 siRNA expression attenuates α-MSH regulated gene expression in 3T3-L1 adipocytes

Several recent investigations have underscored the growing role of melanocortin signaling in the peripheral regulation of lipid, glucose, and energy homeostasis. In addition, the melanocortins play a critical role in the central control of satiety. These observations, and the latest reports highlighting the emerging role of the nuclear hormone receptor (NR) 4A subgroup in metabolism, have prompted us to investigate the cross talk between [Nle(4), d-Phe(7)] (NDP)-α-MSH and Nr4a signaling in adipose. We have shown that NDP-MSH strikingly and preferentially induces the expression of the NR4A subgroup (but not any other members of the NR superfamily) in differentiated 3T3-L1 adipocytes. Utilization of quantitative PCR on custom-designed metabolic TaqMan low-density arrays identified the concomitant and marked induction of the mRNAs encoding Il-6, Cox2, Pdk4, and Pck-1 after NDP-MSH treatment. Similar experiments demonstrated that the mRNA expression profile induced by cAMP and NDP-MSH treatment displayed unique but also overlapping properties and suggested that melanocortin-mediated induction of gene expression involves cAMP-dependent and -independent signaling. Nr4a1/Nur77 small interfering RNA (siRNA) expression suppressed NDP-MSH-mediated induction of Nr4a1/Nur77 and Nr4a3/Nor-1 (but not Nr4a2/Nurr1). Moreover, expression of the siRNA-attenuated NDP-MSH mediated induction of the mRNAs encoding Il-6, Cox2/Ptgs2, and Pck-1 expression. In addition, Nur77 siRNA expression attenuated NDP-MSH-mediated glucose uptake. In vivo, ip administration of NDP-MSH to C57 BL/6J (male) mice significantly induced the expression of the mRNA encoding Nur77 and increased IL-6, Cox2, Pck1, and Pdk4 mRNA expression in (inguinal) adipose tissue. We conclude that Nur77 expression is necessary for MSH-mediated induction of gene expression in differentiated adipocytes. Furthermore, this study demonstrates cross talk between MSH and Nr4a signaling in adipocytes.     

Anti‐adipogenic Effects of αAL14 Mediated by Modulation of PI3K/Akt Pathways in 3T3-L1 Cells

International Journal of Peptide Research and Therapeutics - A model peptide, αAL14, was designed from the primary structure of a novel antimicrobial peptide purified form the abalone....     

Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-κB pathway in 3T3-L1 adipocytes

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-κB) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-κB DNA binding activity (NF-κBp50 and NF-κBp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-κB pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-κB (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-κB (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-κB signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals.