Fetal Rat Brown Adipocytes Research Articles

Rosiglitazone and Retinoic Acid Induce Uncoupling Protein-1 (UCP-1) in a p38 Mitogen-activated Protein Kinase-dependent Manner in Fetal Primary Brown Adipocytes

Brown adipose tissue expresses the thermogenic uncoupling protein-1 (UCP-1), which is positively regulated by peroxisome proliferator-activated receptor (PPAR) agonists and retinoids through the activation of the heterodimers PPAR/retinoid X receptor (RXR) and retinoic acid receptor (RAR)/RXR and binding to specific elements in the ucp-1 enhancer. In this study we show that in fetal rat brown adipocyte primary cultures the PPARgamma agonist rosiglitazone (Rosi), as well as retinoic acids 9-cis-retinoic acid and all-trans-retinoic acid also have "extragenic" effects and induce p44/p42 and p38 mitogen-activated protein kinase (p38MAPK) activation. The latter is involved in UCP-1 gene expression, because inhibition of p38MAPK activity with PD169316 impairs the ability of Rosi and retinoids for UCP-1 induction. The inhibitory effects of PD169316 are mimicked by the antioxidant GSH, suggesting a role for reactive oxygenated species (ROS) generation in the increase of UCP-1 expression in response either to Rosi or 9-cis-retinoic acid. Thus, we propose that Rosi and retinoids act as PPAR/RXR and RAR/RXR agonists and also activate p38MAPK. These two coordinated actions could result in a high increase of transcriptional activity on the ucp-1 enhancer and hence on thermogenesis. PPARalpha and gamma agonists but not retinoids also increase UCP-3 expression in fetal brown adipocytes. However, the regulation of UCP-3, which is not involved in thermogenesis, seems to differ from UCP-1 given the fact that is not affected by p38MAPK inhibition.

Synergistic Activation of UCP-3 Expression in Cultured Fetal Rat Brown Adipocytes by PPARα and PPARγ Ligands

Rat brown adipocytes express mRNAs for Uncoupling Proteins (UCP) 1, 2 and 3 and the Peroxisome Proliferator Activated Receptors (PPAR) α and γ. We have examined the effects of selective PPARα or -γ activation on changes in UCP-1 and UCP-3 mRNA levels in cultured fetal rat brown adipocytes (FBA). Rosiglitazone (1.0 μM), a selective PPARγ agonist, elicited 5- and 3-fold increases in UCP-1 and UCP-3, respectively. The PPARα ligand, Wy14643 (10.0 μM) increased UCP-3 tenfold, but decreased UCP-1. A synergistic effect on UCP-3 expression (30-fold increase; P < 0.05) was observed when FBA were exposed to a combination of Wy14643 (10.0 μM) and rosiglitazone (10.0 μM). Thus, activation of PPARγ increases UCP-1 and UCP-3 levels which are differentially regulated by PPARα. A synergistic interaction occurs between PPARα and PPARγ in the regulation of UCP-3 in FBA, probably via co-activator recruitment, suppression of co-repressor proteins or through a direct interaction at the level of the PPRE.

PPARα Activation by Wy 14643 Induces Transactivation of the Rat UCP-1 Promoter without Increasing UCP-1 mRNA Levels and Attenuates PPARγ-Mediated Increases in UCP-1 mRNA Levels Induced by Rosiglitazone in Fetal Rat Brown Adipocytes

Rodent brown adipocytes express peroxisome proliferator activated receptor-α (PPARα) and PPARγ and while the rodent uncoupling protein-1 (UCP-1) gene contains a putative peroxisome proliferator response element (PPRE), only PPARγ activation by thiazolidinediones increase UCP-1 mRNA levels. We have investigated this phenomenon in foetal rat brown adipocytes (FBA) and show that although transactivation occurs in FBA containing a plasmid encoding 4.5kb of the 5′-flanking region of the rat UCP-1 promoter ((−4551)-UCP-1-CAT) treated with either the selective PPARγ agonist rosiglitazone (1.0 μM) or the selective PPARα agonist Wy 14643 (10.0 μM), only rosiglitazone induced transcription of UCP-1 mRNA. Furthermore, Wy 14643 (10 and 100.0 μM) abolished rosiglitazone-induced UCP-1 mRNA induction in spite of a transactivation event occurring with the combination treatment. Thus in FBA PPARα-activation with Wy 14643 elicits a “blind” transactivation of the UCP-1 promoter which can prevent PPARγ-mediated UCP-1 mRNA transcription either by competition for the PPRE or by an unidentified post-transcriptional event.

Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes.

In the present study we have investigated the contribution of the insulin receptor substrate proteins (IRS-1 and IRS-2) to the insulin/insulin like growth factor I (IGF-I)-signaling pathways in fetal rat brown adipocytes, a model that expresses both insulin and IGF-I receptors. Insulin/IGF-I rapidly stimulated IRS-1 and IRS-2 tyrosine phosphorylation, their association with p85alpha, and IRS-1- and IRS-2-associated phosphatidylinositol (PI) 3-kinase activation to the same extent, the effect of insulin being stronger than the effect of IGF-I at the same physiological dose (10 nM). Furthermore, insulin/IGF-I stimulated IRS-1-associated Grb-2 phosphorylation. However, IRS-2-associated Grb-2 phosphorylation was barely detected. Pull-down experiments with glutathione-S-transferase-fusion proteins containing SH2-domains of p85alpha revealed a strong association between IRS-1 and IRS-2 with p85alpha in response to insulin/IGF-I, the insulin effect being stronger than IGF-I. However, the Grb-2-SH2 domain showed functional differences. While a strong association between IRS-1/Grb-2 was found, IRS-2/Grb-2 association was virtually absent in response to insulin/IGF-I, as also demonstrated in competition studies with a phosphopeptide containing the phosphotyrosine 895 residue within the putative Grb-2-binding domain. Finally, insulin/IGF-I stimulated tyrosine phosphorylation of the three SHC proteins (46, 52, and 66 kDa). Moreover, insulin/IGF-I markedly increased the amount of Grb-2-associated SHC proteins by the same extent. Our results suggest that both IRS-1 and IRS-2 are required for phosphatidylinositol 3-kinase activation that leads to adipogenic and thermogenic differentiation of fetal brown adipose tissue; meanwhile, IRS-1 and SHC, but not IRS-2, associate with Grb-2 leading to the ras-mitogen-activated protein kinase-signaling pathway required for fetal brown adipocyte proliferation.

Phosphatidylinositol 3-Kinase Is a Requirement for Insulin-Like Growth Factor I-Induced Differentiation, but not for Mitogenesis, in Fetal Brown Adipocytes

In the present study we have examined the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the insulin-like growth factor I (IGF-I)-signaling pathways involved in differentiation and in mitogenesis in fetal rat brown adipocytes. Activation of PI 3-kinase in response to IGF-I was markedly inhibited by two PI 3-kinase inhibitors (wortmannin and LY294002) in a dose-dependent manner. IGF-I-stimulated glucose uptake was also inhibited by both compounds. The expression of adipogenic-related genes such as fatty acid synthase, malic enzyme, glycerol 3-phosphate dehydrogenase, and acetylcoenzyme A carboxylase induced by IGF-I was totally prevented in the presence of IGF-I and any of those inhibitors, resulting in a marked decrease of the cytoplasmic lipid content. Moreover, the expression of the thermogenic marker uncoupling protein induced by IGF-I was also down-regulated in the presence of wortmannin/LY294002. IGF-I-induced adipogenic- and thermogenic-related gene expression was only partly inhibited by the p70S6k inhibitor rapamycin. In addition, pretreatment of brown adipocytes with either wortmannin or LY294002, but not with rapamycin, blocked protein kinase C ζ activation by IGF-I. In contrast, IGF-I-induced fetal brown adipocyte proliferation was PI 3-kinase-independent. Our results show for the first time an essential requirement of PI 3-kinase in the IGF-I-signaling pathways leading to fetal brown adipocyte differentiation, but not leading to mitogenesis. In addition, protein kinase C ζ seems to be a signaling molecule also involved in the IGF-I differentiation pathways downstream from PI 3-kinase.

Phosphatidylinositol 3-kinase is a requirement for insulin-like growth factor I-induced differentiation, but not for mitogenesis, in fetal brown adipocytes.

In the present study we have examined the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the insulin-like growth factor I (IGF-I)-signaling pathways involved in differentiation and in mitogenesis in fetal rat brown adipocytes. Activation of PI 3-kinase in response to IGF-I was markedly inhibited by two PI 3-kinase inhibitors (wortmannin and LY294002) in a dose-dependent manner. IGF-I-stimulated glucose uptake was also inhibited by both compounds. The expression of adipogenic-related genes such as fatty acid synthase, malic enzyme, glycerol 3-phosphate dehydrogenase, and acetylcoenzyme A carboxylase induced by IGF-I was totally prevented in the presence of IGF-I and any of those inhibitors, resulting in a marked decrease of the cytoplasmic lipid content. Moreover, the expression of the thermogenic marker uncoupling protein induced by IGF-I was also down-regulated in the presence of wortmannin/LY294002. IGF-I-induced adipogenic- and thermogenic-related gene expression was only partly inhibited by the p70S6k inhibitor rapamycin. In addition, pretreatment of brown adipocytes with either wortmannin or LY294002, but not with rapamycin, blocked protein kinase C zeta activation by IGF-I. In contrast, IGF-I-induced fetal brown adipocyte proliferation was PI 3-kinase-independent. Our results show for the first time an essential requirement of PI 3-kinase in the IGF-I-signaling pathways leading to fetal brown adipocyte differentiation, but not leading to mitogenesis. In addition, protein kinase C zeta seems to be a signaling molecule also involved in the IGF-I differentiation pathways downstream from PI 3-kinase.

Insulin-like growth factor I and insulin induce adipogenic-related gene expression in fetal brown adipocyte primary cultures.

Fetal rat brown adipocytes show high-affinity binding sites for both insulin-like growth factor I (IGF-I) and insulin. Cell culture for 24 h in the presence of IGF-I or insulin, independently, up-regulated the mRNA expression of adipogenic-related genes, such as fatty acid synthase (FAS), glycerol-3-phosphate de-hydrogenase and insulin-regulated glucose transporter Glut4, and down-regulated the expression of phosphoenolpyruvate carboxykinase mRNA in a dose-dependent manner. Moreover, both IGF-I and insulin increased the FAS gene transcription rate at 2 h, producing a time-dependent accumulation of FAS mRNA. Furthermore IGF-I or insulin increased glucose uptake and lipid content throughout the 24 h culture period. Our results suggest that both IGF-I and insulin are major signals involved in initiating and/or maintaining the expression of adipogenic-related genes in fetal rat brown adipocytes.

Involvement of Raf-1 kinase and protein kinase C zeta in insulin-like growth factor I-induced brown adipocyte mitogenic signaling cascades: inhibition by cyclic adenosine 3',5'-monophosphate.

In the present study we have examined the signaling cascades involved in insulin-like growth factor I (IGF-I)-induced mitogenesis in fetal rat brown adipocyte primary cultures, a model that constitutively expresses a high number of IGF-I receptors, where IGF-I is a complete mitogen at physiological concentrations. IGF-I rapidly stimulated beta-chain IGF-I receptor autophosphorylation, which peaked at a physiological/mitogenic concentration (1.4 nM) and also stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Tyrosine-phosphorylated IRS-1 bound and subsequently activated phosphatidylinositol 3-kinase by 3.5-fold, whereas the tyrosine-phosphorylated IGF-I receptor was not directly associated with the p85 subunit of the phosphatidylinositol 3-kinase. Moreover, mitogenic concentrations of IGF-I enhanced glucose transport by 2.5-fold. In addition, tyrosine phosphorylation of the 46- and 52-kDa SHC proteins was high in the basal state and doubled after IGF-I treatment, whereas IGF-I enhanced by 4-fold tyrosine phosphorylation of the 66-kDa SHC band. Furthermore, a 2-fold increase in the Ras. GTP active form was induced upon IGF-I stimulation. Downstream from Ras, IGF-I increased both Raf kinase and protein kinase C (PKC) zeta activities by 3.5-fold. (Bu)2cAMP, an inhibitor of IGF-I-induced mitogenesis in fetal brown adipocyte primary cultures, did not block the very early steps of the IGF-I-induced mitogenic cascade, such as IGF-I receptor autophosphorylation, IRS-1 or SHC tyrosine phosphorylation, and Ras activation to its GTP active form. However, (Bu)2cAMP disrupted IGF-I-Raf and IGF-I-PKC zeta signaling pathways by preventing IGF-I-induced Raf-1 kinase and PKC zeta enzymatic activities, respectively. Our results show the first characterization in situ of an IGF-I mitogenic signaling cascade that downstream Ras diverges to the nucleus through two different serine/threonine kinases (Raf-1 kinase and PKC zeta) in mammalian fetal primary cells under physiological conditions. Both kinases represent a point of regulation primarily described for IGF-I-induced, cAMP-inhibited mitogenic pathways.

Transforming growth factor beta 1 induces mitogenesis in fetal rat brown adipocytes.

The presence of transforming growth factor beta 1 (TGF-beta 1) for 24 or 48 h stimulated DNA synthesis, the percentage of cells in the S + G2/M phases of the cell cycle, and cell number, as compared to quiescent cells. The mitogenic capacity of TGF-beta 1 (1 pM) was similar to that shown by 10% fetal calf serum (FCS). TGF-beta 1 for 48 h increased by 5-fold the percentage of cells containing (3H)thymidine-labeled nuclei as compared to quiescent cels. In addition, single fetal brown adipocytes, showing their typical multilocular fat droplets phenotype, become positive for (3H)thymidine-labeled nuclei in response to TGF-beta 1. Moreover, TGF-beta 1 induced the mRNA expression of a complete set of proliferation-related genes, such as c-fos (30 min), c-myc and beta-actin (2 h), and H-ras, cdc2 kinase, and glucose 6-phosphate dehydrogenase (G6PD) at 4 and 8 h, as compared to quiescent cells. Concurrently, TGF-beta 1 for 12 h increased the protein content of proliferating cellular nuclear antigen (PCNA) by 6-fold and p21-ras by 2-fold. Although our results demonstrate that TGF-beta 1 induces the expression of very early genes related to cell proliferation, TGF-beta 1 could be acting either as a mitogen or as a survival factor in induce proliferation to fetal brown adipocytes.

Triiodothyronine Induces the Transcription of the Uncoupling Protein Gene and Stabilizes Its mRNA in Fetal Rat Brown Adipocyte Primary Cultures

Confluent fetal rat brown adipocytes in primary culture showed an almost undetectable level of uncoupling protein (UCP) mRNA and a low mitochondrial content of functional UCP. Treatment of confluent cells with 10 nM triiodothyronine in a serum-free medium, in the absence of noradrenergic stimulation, increased the amount of UCP mRNA in a time-dependent manner. This effect was due to an increased UCP gene transcription rate and UCP mRNA stabilization, resulting in a higher content of immunoreactive mitochondrial UCP and functional UCP (detected by its ability to bind GDP). Thus, triiodothyronine might play a significant physiological role in the UCP expression throughout fetal development, when brown adipose tissue starts to differentiate and UCP is primarily expressed.

Transforming growth factor β1 induces differentiation-specific gene expression in fetal rat brown adipocytes

Fetal rat brown adipocytes show a low number of transforming growth factor β 1 (TGF- β 1) binding sites of high affinity, revealing the presence of type I, II and IIITGF- β 1 receptors and a minor-labeled species of approximately 140 kDa. The culture of cells in the presence ofTGF- β 1 induced the expression of the tissue-specific gene uncoupling protein in a dose- and time-dependent manner. In addition,TGF- β 1 up-regulates the expression of genes involved in adipogenesis such as fatty acid synthase, glycerol 3-phosphate dehydrogenase, malic enzyme and glucose 6-phosphate dehydrogenase, as well as induces the expression of fibronectin (specific target gene forTGF- β 1). Our results suggest thatTGF- β 1 is a major signal involved in initiating and/or maintaining the thermogenic and adipogenic differentiation of rat fetal brown adipocytes.

CAMP Inhibits IGF-I-Induced Mitogenesis in Fetal Rat Brown Adipocytes: Role of p21 ras

Dibutyryl cAMP (DBcAMP) inhibited insulin-like growth factor I (IGF-I)-induced DNA synthesis of rat fetal brown adipocyte primary cultures. Its mitogenic inhibitory capacity, measured as [ 3H]thymidine incorporation into acid-precipitable material, was dose-dependent, being maximal at 0.5 m M. The entry of cells into S + G 2/M phases of the cell cycle and the increase in cell number induced by IGF-I were partially inhibited by DBcAMP at 24 h and totally prevented at 48 h. DBcAMP inhibited the mRNA expression of c- myc induced by IGF-I at 2 h. Moreover, DBcAMP inhibited in parallel H- ras mRNA expression, p21 ras, and proliferating cellular nuclear antigen protein content induced by IGF-I at 24 and 48 h of culture, respectively, suggesting the involvement of p21 ras in the progression of fetal brown adipocytes through the S phase of the cell cycle and DNA synthesis.

IGF-I Induces the Uncoupling Protein Gene Expression in Fetal Rat Brown Adipocyte Primary Cultures: Role of C/EBP Transcription Factors

Fetal rat brown adipocytes proliferate and reach confluence when cultured in the presence of serum, almost losing their expression of the tissue-specific gene uncoupling protein (UCP). Confluent cells in a serum-free medium induced the expression of UCP in response to 1.4 nM IGF-I at 72 h, in a time-dependent manner. This effect was not produced by 1 nM insulin. However, insulin but not IGF-I induced the expression of the lipogenic marker malic enzyme, suggesting that IGF-I but not insulin is involved in the thermogenic differentiation process of fetal brown adipocytes. Furthermore, the expression of C/EBP transcription factors was present in fetal brown adipocytes prior culture, decreasing during cell proliferation throughout culture. After confluence, insulin induced the expression of C/EBPα and δ, whereas the expression of C/EBPβ remained essentially unmodified. IGF-I induced C/EBPα but decreased C/EBPδ, this might indicate that both transcription factors play a reciprocal role in the expression of the tissue specific UCP gene.

Triiodothyronine induces the expression of the uncoupling protein in long term fetal rat brown adipocyte primary cultures: role of nuclear thyroid hormone receptor expression.

Fetal rat brown adipocytes at the beginning of culture showed a minimal T3 nuclear binding capacity and very low expression of the c-erbA genes, with low steady state levels of the mRNA forms beta-type and 5.5- and 2.6-kilobase (kb) alpha-types. The levels of these mRNA species increased after 7 days in culture in the presence of thyroid hormone-depleted serum; however, no significant increase in nuclear T3-binding capacity was observed. The addition of T3 incremented the abundance of the c-erbA beta-mRNA, induced the appearance of the 6.6-kb c-erbA alpha-mRNA and increased the nuclear T3-binding capacity by 30-fold. Parallel analysis of the uncoupling protein (UCP) mRNA, immunoreactive UCP, and functional UCP (detected by its ability to bind GDP) demonstrated that T3 induced the expression of the UCP gene in long term treated fetal brown adipocytes in culture. The presence of noradrenaline, a positive control for UCP expression, increased only the level of expression of the 5.5-kb c-erbA alpha-mRNA, but values for nuclear T3-binding capacity similar to those obtained in T3-treated cells were observed. Simultaneous addition of both hormones gave a pattern of expression of the c-erbA-mRNA forms similar to those obtained with these agents individually, and no further increment in their separate effects was observed on the nuclear T3-binding capacity and UCP expression.

Triiodothyronine induces the expression of the uncoupling protein in long term fetal rat brown adipocyte primary cultures: role of nuclear thyroid hormone receptor expression

Triiodothyronine induces the expression of the uncoupling protein in long term fetal rat brown adipocyte primary cultures: role of nuclear thyroid hormone receptor expression

IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes.

Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF-I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early-response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H-ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes.

Fetal rat brown adipocyte primary cultures: characterization of a system for lipid synthesis studies

Conference Article| June 01 1988 Fetal rat brown adipocyte primary cultures: characterization of a system for lipid synthesis studies MARGARITA LORENZO; MARGARITA LORENZO 1Departamento de Bioquímica y Biología Molecular, Centro Mixto C.S.I.C/U.C.M., Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain Search for other works by this author on: This Site PubMed Google Scholar ISABEL FABREGAT; ISABEL FABREGAT 1Departamento de Bioquímica y Biología Molecular, Centro Mixto C.S.I.C/U.C.M., Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain Search for other works by this author on: This Site PubMed Google Scholar CÉSAR RONCERO; CÉSAR RONCERO 1Departamento de Bioquímica y Biología Molecular, Centro Mixto C.S.I.C/U.C.M., Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain Search for other works by this author on: This Site PubMed Google Scholar MANUEL BENITO MANUEL BENITO * 1Departamento de Bioquímica y Biología Molecular, Centro Mixto C.S.I.C/U.C.M., Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain *To whom correspondence and requests for reprints should be addressed Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1988) 16 (3): 274–275. https://doi.org/10.1042/bst0160274 Article history Received: November 26 1987 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation MARGARITA LORENZO, ISABEL FABREGAT, CÉSAR RONCERO, MANUEL BENITO; Fetal rat brown adipocyte primary cultures: characterization of a system for lipid synthesis studies. Biochem Soc Trans 1 June 1988; 16 (3): 274–275. doi: https://doi.org/10.1042/bst0160274 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1988 Biochemical Society1988 Article PDF first page preview Close Modal You do not currently have access to this content.