Glycogen Dynamics Drives Lipid Droplet Biogenesis during Brown Adipocyte Differentiation.

Browning induction or transplantation of brown adipose tissue (BAT) or brown/beige adipocytes derived from progenitor or induced pluripotent stem cells (iPSCs) can represent a powerful strategy to treat metabolic diseases. However, our poor understanding of the mechanisms that govern the differentiation and activation of brown adipocytes limits the development of such therapy. Various genetic factors controlling the differentiation of brown adipocytes have been identified, although most studies have been performed using in vitro cultured pre-adipocytes. We investigate here the differentiation of brown adipocytes from adipose progenitors in the mouse embryo. We demonstrate that the formation of multiple lipid droplets (LDs) is initiated within clusters of glycogen, which is degraded through glycophagy to provide the metabolic substrates essential for de novo lipogenesis and LD formation. Therefore, this study uncovers the role of glycogen in the generation of LDs.

Breast hibernomas: Questioning the embryological origin?

This paper aims to review the concept of hibernomas, with focus on their occurrence, in the breast. It will make reference to a specific case from the Helen Joseph Hospital's Breast Clinic situated in Johannesburg, South Africa. We describe the clinical, radiological and pathological findings in a patient as well as the final diagnosis and treatment (in the form of surgery). This report emphasizes the distinguishable features of hibernomas, and gives guidance as to the surgical approach in large hibernomas stressing the ease of achieving cosmesis without major oncoplastic techniques. More specifically, discussion as whether these rapidly growing, nonrecurring, usually encapsulated growths consisting of brown fat tissue similar to that found in hibernating animals arise from the breast or the underlying muscle is considered.

HMGA1 overexpression in adipose tissue impairs adipogenesis and prevents diet-induced obesity and insulin resistance.

High-Mobility-Group-A1 (HMGA1) proteins are non-histone proteins that regulate chromatin structure and gene expression during embryogenesis, tumourigenesis and immune responses. In vitro studies suggest that HMGA1 proteins may be required to regulate adipogenesis. To examine the role of HMGA1 in vivo, we generated transgenic mice overexpressing HMGA1 in adipose tissues. HMGA1 transgenic mice showed a marked reduction in white and brown adipose tissue mass that was associated with downregulation of genes involved in adipogenesis and concomitant upregulation of preadipocyte markers. Reduced adipogenesis and decreased fat mass were not associated with altered glucose homeostasis since HMGA1 transgenic mice fed a regular-chow diet exhibited normal glucose tolerance and insulin sensitivity. However, when fed a high-fat diet, overexpression of HMGA1 resulted in decreased body-weight gain, reduced fat mass, but improved insulin sensitivity and glucose tolerance. Although HMGA1 transgenic mice exhibited impaired glucose uptake in adipose tissue due to impaired adipogenesis, the increased glucose uptake observed in skeletal muscle may account for the improved glucose homeostasis. Our results indicate that HMGA1 plays an important function in the regulation of white and brown adipogenesis in vivo and suggests that impaired adipocyte differentiation and decreased fat mass is not always associated with impaired whole-body glucose homeostasis.

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source.

Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes.

Expression and subcellular localization of estrogen receptors α and ß in human fetal brown adipose tissue.

CONTEXT: Brown adipose tissue (BAT) has the unique ability of generating heat due to the expression of mitochondrial uncoupling protein 1 (UCP1). A recent discovery regarding functional BAT in adult humans has increased interest in the molecular pathways of BAT development and functionality. An important role for estrogen in white adipose tissue was shown, but the possible role of estrogen in human fetal BAT (fBAT) is unclear. OBJECTIVE: The objective of this study was to determine whether human fBAT expresses estrogen receptor α (ERα) and ERß. In addition, we examined their localization as well as their correlation with crucial proteins involved in BAT differentiation, proliferation, mitochondriogenesis and thermogenesis including peroxisome proliferator-activated receptor γ (PPARγ), proliferating cell nuclear antigen (PCNA), PPARγ-coactivator-1α (PGC-1α), and UCP1. DESIGN: The fBAT was obtained from 4 human male fetuses aged 15, 17, 20, and 23 weeks gestation. ERα and ERß expression was assessed using Western blotting, immunohistochemistry, and immunocytochemistry. Possible correlations with PPARγ, PCNA, PGC-1α, and UCP1 were examined by double immunofluorescence. RESULTS: Both ERα and ERß were expressed in human fBAT, with ERα being dominant. Unlike ERß, which was present only in mature brown adipocytes, we detected ERα in mature adipocytes, preadipocytes, mesenchymal and endothelial cells. In addition, double immunofluorescence supported the notion that differentiation in fBAT probably involves ERα. Immunocytochemical analysis revealed mitochondrial localization of both receptors. CONCLUSION: The expression of both ERα and ERß in human fBAT suggests a role for estrogen in its development, primarily via ERα. In addition, our results indicate that fBAT mitochondria could be targeted by estrogens and pointed out the possible role of both ERs in mitochondriogenesis.

Brown adipose tissue: development, metabolism and beyond.

Obesity represents a major risk factor for the development of several of our most common medical conditions, including Type 2 diabetes, dyslipidaemia, non-alcoholic fatty liver, cardiovascular disease and even some cancers. Although increased fat mass is the main feature of obesity, not all fat depots are created equal. Adipocytes found in white adipose tissue contain a single large lipid droplet and play well-known roles in energy storage. By contrast, brown adipose tissue is specialized for thermogenic energy expenditure. Owing to its significant capacity to dissipate energy and regulate triacylglycerol (triglyceride) and glucose metabolism, and its demonstrated presence in adult humans, brown fat could be a potential target for the treatment of obesity and metabolic syndrome. Undoubtedly, fundamental knowledge about the formation of brown fat and regulation of its activity is imperatively needed to make such therapeutics possible. In the present review, we integrate the recent advancements on the regulation of brown fat formation and activity by developmental and hormonal signals in relation to its metabolic function.

Vascular endothelial growth factor is important for brown adipose tissue development and maintenance.

Vascular endothelial growth factor (VEGF) is critical for angiogenesis, but also has pleiotropic effects on several nonvascular cells. Our aim was to investigate the role of VEGF in brown adipose tissue (BAT). We show that VEGF expression increases 2.5-fold during differentiation of cultured murine brown adipocytes and that VEGF receptor-2 is phosphorylated, indicating VEGF signaling. VEGF increased proliferation in brown preadipocytes in vitro by 70%, and blockade of VEGF signaling using anti-VEGFR2 antibody DC101 increased brown adipocyte apoptosis, as determined by cell number and activation of caspase 3. Systemic VEGF neutralization in mice, accomplished by adenoviral expression of soluble Flt1, resulted in 7-fold increase in brown adipocyte apoptosis, mitochondrial degeneration, and increased mitophagy compared to control mice expressing a null adenovirus. Absence of the heparan sulfate-binding VEGF isoforms, VEGF164 and VEGF188, resulted in abnormal BAT development in mice at E15.5, with fewer brown adipocytes and lower mitochondrial protein compared to wild-type littermates. These results suggest a role for VEGF in brown adipocytes and preadipocytes to promote survival, proliferation, and normal mitochondria and development.

The implication of brown adipose tissue for humans.

We here discuss the role of brown adipose tissue on energy homeostasis and assess its potential as a target for body weight management. Because of their high number of mitochondria and the presence of uncoupling protein 1, brown fat adipocytes can be termed as energy inefficient for adenosine-5'-triphosphate (ATP) production but energy efficient for heat production. Thus, the energy inefficiency of ATP production, despite high energy substrate oxidation, allows brown adipose tissue to generate heat for body temperature regulation. Whether such thermogenic property also plays a role in body weight regulation is still debated. The recent (re)discovery of brown adipose tissue in human adults and a better understanding of brown adipose tissue development have encouraged the quest for new alternatives to treat obesity since obese individuals seem to have less brown adipose tissue mass/activity than do their lean counterparts. In this review, we discuss the physiological relevance of brown adipose tissue on thermogenesis and its potential usefulness on body weight control in humans.

CARM1 promotes adipocyte differentiation by coactivating PPARgamma.

The coactivator-associated arginine methyltransferase 1 (CARM1) is recruited to gene promoters by many transcription factors. To identify new pathways that use CARM1, we carried out a comprehensive transcriptome analysis of CARM1-knockout embryos. By using complementary DNA microarrays and serial analysis of gene expression, we identified various genes involved in lipid metabolism that were underrepresented in CARM1-knockout embryos, indicating an important role for this coactivator in adipose tissue biology. We also observed that the amount of brown fat in CARM1-knockout embryos is reduced. Furthermore, cells lacking CARM1 have a severely curtailed potential to differentiate into mature adipocytes. Reporter experiments and chromatin immunoprecipitation analysis show that CARM1 regulates these processes by acting as a coactivator for peroxisome proliferator-activated receptor gamma (PPARgamma). Together, these results show that CARM1 promotes adipocyte differentiation by coactivating PPARgamma-mediated transcription and thus might be important in energy balance.

Liver X receptor agonists ameliorate TNFalpha-induced insulin resistance in murine brown adipocytes by downregulating protein tyrosine phosphatase-1B gene expression.

AIMS/HYPOTHESIS: The nuclear receptors, including nuclear receptor subfamily 1, group H, member 3 (NR1HR, also known as liver X receptor [LXR]), are sensors of cholesterol metabolism and lipid biosynthesis that have recently been proposed as insulin sensitisers. TNFalpha has been described as a link between obesity and the development of insulin resistance, an important contributor to the pathogenesis of type 2 diabetes. Therefore, we decided to investigate the ability of NR1HR agonists to ameliorate TNFalpha-induced insulin resistance in brown adipocytes. METHODS: Primary brown adipocytes from rat fetuses, and from wild-type neonate mice and neonate mice deficient in the gene encoding protein tyrosine phosphatase-1B (Ptpn1, also known as Ptp1b) were cultured in the absence or presence of TNFalpha and different nuclear receptor agonists. Among them, the unrelated NR1HR ligands T0901317, GW3965 and (22R)-hydroxycholesterol were tested. After insulin stimulation, glucose uptake and solute carrier family 2 (facilitated glucose transporter), member 4 (SLC2A4, formerly known as GLUT4) translocation were measured. Next the insulin signalling cascade was determined by submitting cells to lysis, immunoprecipitation and immunoblotting. RESULTS: NR1HR agonists ameliorate TNFalpha-induced insulin resistance restoring completely insulin-stimulated glucose uptake and SLC2A4 translocation to plasma membrane. This effect is parallel to the recovery of the insulin cascade insulin receptor/IRS-2/phosphatidylinositol 3-kinase/protein kinase B, and could be due to the fact that T0901317 prevents the increase of PTPN1 production and phosphatase activity produced by TNFalpha. In this regard, Ptpn1-deficient brown adipocytes showed protection against insulin resistance by TNFalpha. Moreover, we observed that T0901317 produced in itself a significant increase over basal glucose uptake consistent with an increase of SLC2A4 protein content in plasma membrane, attributable to the activation of protein kinase zeta and/or the increase of Slc2a4 expression. CONCLUSIONS/INTERPRETATION: Nuclear receptors NR1HR are interesting potential targets for drug treatment of insulin resistance.

Essential role of insulin-like growth factor I receptor in insulin-induced fetal brown adipocyte differentiation.

To define the specific role of IGF-I receptor (IGF-IR) in adipogenic and thermogenic differentiation of brown adipocytes during late fetal life, we have established immortalized brown adipocyte cell lines from fetuses of IGF-IR-deficient mice (IGF-IR(-/-)) as well as from wild-type mice (IGF-IR(+/+)). IGF-IR(-/-) cells showed an increased insulin sensitivity regarding insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation despite a substantial reduction in IRS-1 protein content. Furthermore, insulin-induced total and IRS-1-associated phosphatidylinositol 3-kinase activities were augmented in IGF-IR-deficient cells compared with wild-type cells. Downstream phosphatidylinositol 3-kinase activation of Akt, but not p70s6 kinase, were elicited at lower doses of insulin in IGF-IR(-/-) brown adipocytes. Activation of protein kinase Czeta by insulin was similar in both cell types as was insulin-induced glucose uptake. Treatment of wild-type brown adipocytes with insulin for 12 h up-regulated fatty acid synthase (FAS) and adipocyte determination and differentiation (ADD1/SREBP) mRNAs; this effect was impaired in the absence of IGF-IR. At the protein level, insulin increased FAS content and the amount of the mature form of adipocyte determination and differentiation (ADD1/SREBP) in the nucleus in wild-type cells, but not in IGF-IR(-/-) cells. Furthermore, 24 h of insulin stimulation induced the expression of both uncoupling protein-1 and CCAAT/enhancer-binding protein alpha (C/EBPalpha) in wild-type brown adipocytes; these effects were abolished in IGF-I-R(-/-) cells. Retrovirus-mediated reexpression of peroxisomal proliferator-activated receptor gamma (PPARgamma) in IGF-IR(-/-) brown adipocytes could overcome FAS mRNA impairment, bypassing insulin signaling. However, insulin further increased FAS mRNA expression in C/EBPalpha-IGF-IR(-/-) cells, but not in PPARgamma-IGF-IR(-/-) cells. In addition, fetal brown adipocytes lacking IGF-IR up-regulated uncoupling protein-1 expression in the absence of insulin when PPARgamma, but not C/EBPalpha, was overexpressed. These data provide strong evidence for a critical role of IGF-IR in the differentiation of the brown adipocyte phenotype in fetal life; this effect is mimicked by PPARgamma in an insulin-independent manner.

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.

Increased insulin sensitivity in IGF-I receptor--deficient brown adipocytes.

Immortalized brown adipocyte cell lines have been generated from fetuses of mice deficient in the insulin-like growth factor I receptor gene (IGF-IR(-/-)), as well as from fetuses of wild-type mice (IGF-IR(+/+)). These cell lines maintained the expression of adipogenic- and thermogenic-differentiation markers and show a multilocular fat droplets phenotype. IGF-IR(-/-) brown adipocytes lacked IGF-IR protein expression; insulin receptor (IR) expression remained unchanged as compared with wild-type cells. Insulin-induced tyrosine autophosphorylation of the IR beta-chain was augmented in IGF-IR--deficient cells. Upon insulin stimulation, tyrosine phosphorylation of (insulin receptor substrate-1) IRS-1 was much higher in IGF-IR(-/-) brown adipocytes, although IRS-1 protein content was reduced. In contrast, tyrosine phosphorylation of IRS-2 decreased in IGF-IR--deficient cells; its protein content was unchanged as compared with wild-type cells. Downstream, the association IRS-1/growth factor receptor binding protein-2 (Grb-2) was augmented in the IGF-IR(-/-) brown adipocyte cell line. However, SHC expression and SHC tyrosine phosphorylation and its association with Grb-2 were unaltered in response to insulin in IGF-IR--deficient brown adipocytes. These cells also showed an enhanced activation of mitogen-activated protein kinase (MAPK) kinase (MEK1/2) and p42/p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. In addition, the lack of IGF-IR in brown adipocytes resulted in a higher mitogenic response (DNA synthesis, cell number, and proliferating cell nuclear antigen expression) to insulin than wild-type cells. Finally, cells lacking IGF-IR showed a much lower association between IR or IRS-1 and phosphotyrosine phosphatase 1B (PTP1B) and also a decreased PTP1B activity upon insulin stimulation. However, PTP1B/Grb-2 association remained unchanged in both cell types, regardless of insulin stimulation. Data presented here provide strong evidence that IGF-IR--deficient brown adipocytes show an increased insulin sensitivity via IRS-1/Grb-2/MAPK, resulting in an increased mitogenesis in response to insulin.

TNF-alpha inhibits UCP-1 expression in brown adipocytes via ERKs. Opposite effect of p38MAPK.

Tumor necrosis factor-alpha (TNF-alpha) activates extracellular-regulated kinases (ERKs) and p38 mitogen-activated protein kinase (p38MAPK), and inhibits the expression of uncoupling protein-1 (UCP-1) and adipocyte-specific genes in rat fetal brown adipocytes. MEK inhibition with PD98059 abolished the inhibitory effect of TNF-alpha on UCP-1, but not on adipogenic genes. In contrast, inhibition of p38MAPK with SB203580 potentiated the negative effect of TNF-alpha on UCP-1 and adipogenic genes. The inhibitory action of TNF-alpha was partially correlated with changes in C/EBPalpha and beta protein levels and in their DNA binding activity, suggesting a role for these transcription factors. However, other transcription factors might explain the different regulation of UCP-1 and adipogenic genes by ERKs.

p38 mitogen-activated protein kinase mediates tumor necrosis factor-alpha-induced apoptosis in rat fetal brown adipocytes.

Tumor necrosis factor-alpha (TNFalpha) induces apoptosis and cell growth inhibition in primary rat fetal brown adipocytes. Here, we examine the role played by some members of the mitogen-activated protein kinase (MAPK) superfamily. TNFalpha activates extracellular regulated kinase-1/2 (ERK1/2) and p38MAPK. Inhibition of p38MAPK by either SB203580 or SB202190 highly reduces apoptosis induced by TNFalpha, whereas ERK inhibition potentiates it. Moreover, cotransfection of an active MKK3 mutant and p38MAPK induces apoptosis. p38MAPK inhibition also prevents TNFalpha-induced cell cycle arrest, whereas MEK1 inhibition enhances this effect, which correlates with changes in proliferating cell nuclear antigen expression, but not in cyclin D1. c-Jun and activating transcription factor-1 are potential downstream effectors of p38MAPK and ERKs upon TNFalpha treatment. Thus, TNFalpha-induced c-Jun messenger RNA expression requires ERKs activation, whereas p38MAPK inhibition enhances its expression. In addition, TNFalpha-induced activating transcription factor-1 phosphorylation is extensively decreased by SB203580. However, TNFalpha-induced NF-kappaB DNA-binding activity is independent of p38MAPK and ERK activation. On the other hand, C/EBP homology protein does not appear to mediate the actions of TNFalpha, because its expression is almost undetectable and even reduced by TNFalpha. Finally, although TNFalpha induces c-Jun N-terminal kinase (JNK) activation, transfection of a dominant negative of either JNK1 or JNK2 had no effect on TNFalpha-induced apoptosis. These results suggest that p38MAPK mediates TNFalpha-induced apoptosis and cell cycle arrest, whereas ERKs do the opposite, and JNKs play no role in this process of apoptosis.

Activation of the thyroid hormone receptor alpha gene promoter by the orphan nuclear receptor ERR alpha.

The superfamily of nuclear receptors comprises transcription factors that depend on a ligand for their activity. In addition, the superfamily includes a number of orphan receptors, for which no ligand is known. We report here that the orphan receptor estrogen receptor related alpha receptor (ERR alpha) stimulates the expression of the thyroid hormone receptor alpha (TR alpha) gene promoter. We characterized a responsive site that is both necessary and sufficient for ERR alpha-induced transactivation. In addition, we show that both TR alpha and ERR alpha are coexpressed in embryonic intestine, brown fat and heart as well as in the adult gonads. In the testis, expression of both receptors can be found in the seminiferous tubes where it is totally restricted to spermatocytes I. Altogether this suggests that TR alpha is an in vivo target of ERR alpha.

A comparative study of the expression patterns for vegf, vegf-b/vrf and vegf-c in the developing and adult mouse.

With the goal of better understanding the function and regulation of the different members of the VEGF family this study reports mapping of vegf, vegf-b and vegf-c mRNA expression in developing and adult mice. On embryonic day 14 (E14) there is a high expression of vegf and vegf-b, vegf-b being exceptionally high in heart and CNS. The vegf-c expression is lower with distinct signals in CNS and heart. Prior to birth (E17), vegf and vegf-b expression is moderately downregulated. Overlapping expression is present in intrascapular fat and heart. vegf dominates in thyroid and lung, while vegf-b appears to be the only VEGF member expressed at detectable levels in the CNS. In young adult mouse vegf and vegf-b show partly overlapping expression patterns particularly in kidney, heart and in the thymus, vegf displays higher levels in lung and liver, vegf-b appears to be dominating in brain, heart, testis and kidney. In brain the highest levels of vegf-b is present in the hippocampus. No vegf-c mRNA expression could be detected in the adult. Taken together, these results illustrate, in detail, the different regulations of the members of the VEGF gene family. There are at present at least three specific effectors of vascular proliferation with clear differences in their expression.

Akt2 mRNA is highly expressed in embryonic brown fat and the AKT2 kinase is activated by insulin.

Akt2 encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling molecules. Although there has been extensive interest in the mechanism by which the closely-related Akt kinase participates in phosphatidylinositol 3-kinase-mediated signaling, comparatively little is known regarding the expression and function of Akt2. This manuscript is the first to describe Akt2 mRNA expression in the developing mouse and the activation of AKT2 by insulin. These studies demonstrate that Akt2 is especially abundant in brown fat and, to a lesser extent, skeletal muscle and liver, tissues which are highly insulin-responsive and play a role in glucose metabolism. Endogenous Akt2 expression also is upregulated in fully-differentiated C2C12 myotubes and 3T3-L1 adipocytes, suggesting that these murine cell lines represent useful in vitro models for studies of Akt2 function. We show that HA-tagged AKT2 is activated in response to insulin stimulation in vitro and that activation of AKT2 is not induced in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase. These data suggest that Akt2 expression is fundamental to the differentiated state of fat and muscle cells and that activation of AKT2 kinase by insulin is mediated through the phosphatidylinositol 3-kinase signaling pathway.

Alterations in the insulin signaling pathway induced by immortalization and H-ras transformation of brown adipocytes.

In fetal brown adipocyte primary cultures, insulin rapidly (at 5 min) induced tyrosine phosphorylation of the insulin receptor beta-subunit; this effect was maximal at physiological concentrations (1 nM). Insulin also stimulated insulin receptor substrate-1 tyrosine phosphorylation and subsequently activated phosphatidylinositol 3-kinase. Moreover, a 3-fold increase in the Ras.GTP active form and a 6-fold increase in Raf-1 kinase activity were induced after insulin stimulation. An immortalized brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo cotransfection) showed a reduced maximal responsiveness to insulin in the same range of insulin concentrations studied (1-100 nM). Transformed brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo H-ras(lys12) cotransfection) developed insulin resistance upstream from Ras, showing an impairment in the insulin receptor autophosphorylation, and in insulin receptor substrate-1 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase upon treatment with 1 nM insulin, although insulin receptor number and affinity (Kd) remained unaltered. This lack of effect was ameliorated upon treatment with higher insulin concentrations, in a dose-dependent manner. However, downstream from Ras, events such as formation of the Ras.GTP active form, and Raf-1 kinase and 12-O-tetradecanoylphorbol-13-acetate response element-chloramphenicol transferase (transiently transfected) activities were overstimulated, compared with those in primary and immortalized cells, in an insulin-independent manner. Wheat-germ lectin-purified receptors from H-ras(lys12)-transformed brown adipocytes showed a marked phosphorylation in the basal state, which was suppressed by serine-threonine phosphatase pretreatment. Moreover, alkaline phosphatase pretreatment restored the tyrosine kinase activity of the receptor in response to insulin. We conclude that the decreased tyrosine autophosphorylation rate of the insulin receptor from H-ras(lys12)-transformed brown adipocytes is a consequence of its basal serine/threonine phosphorylation, resulting in severe insulin resistance.

p21ras induced differentiation-related gene expression in fetal brown adipocyte primary cells and cell lines.

Transfection of primary rat fetal brown adipocytes with constructs of SV40 large T antigen, alone and together with lys12-mutated H-ras gene, gave permanent cell lines showing an immortalized or transformed phenotype, respectively, all of them selected by the expression of the uncoupling protein (UCP), a tissue-specific marker. Primary brown adipocytes and immortalized cell lines respond to insulin-like growth factor I (IGF-I) by increasing their lipid content and the mRNA expression of both the adipogenic marker fatty acid synthase (FAS) and the thermogenic marker UCP. IGF-I-induced differentiation-related gene expression at 24 h in both primary and immortalized brown adipocytes was mediated by an increase in p21ras.GTP active protein content. Transformed cell lines overexpressing exogenous p21ras (mainly in its ras.GTP active form) constitutively showed a higher lipid content and a higher FAS and UCP mRNA expression compared to primary and immortalized cells. These transformed cells were IGF-I independent with respect to their studied differentiation-related parameters. Additionally, transient transfection of primary brown adipocytes with the transforming ras gene induced UCP and FAS mRNA expression as well as cotransactivated UCP-chloramphenicol acetyltransferase fusion gene. Moreover, IGF-I transactivation of UCP promoter was partially precluded by cotransfection with the dominant-negative ras gene. Our results strongly suggest that IGF-I/p21ras induces adipogenic- and thermogenic-related gene expression in brown adipocytes.