HIB1B Brown Adipocytes Research Articles

Revealing the contribution of iron overload-brown adipocytes to iron overload cardiomyopathy: Insights from RNA-seq and exosomes coculture technology

Iron overload has been demonstrated to be associated with insulin resistance, iron overload cardiomyopathy (IOC). Brown adipose tissue (BAT) is emerging as a novel therapeutic target for the treatment of various diseases, not only because of its capacity for dissipating excess energy via non-shivering thermogenesis, but also because of its implication in physiological and pathophysiological processes. However, little attention has been devoted to the precise alterations and impacts of iron overload-BAT. We conducted RNA-Seq analysis on BAT samples obtained from mice subjected to a high iron diet (HID) or a normal chow diet (CON), respectively. The RNA-seq transcriptomic analysis revealed that 1,289 differentially expressed RNAs (DEGs) were identified, with a higher number of the downregulated genes (910 genes) compared to the upregulated genes (379 genes). The results of Gene Ontology (GO) and The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the downregulated DEGs were primarily involved in hypertrophic cardiomyopathy, dilated cardiomyopathy, which were defined as IOC under the iron overload condition. The association between iron overload-BAT with cardiomyopathy was further investigated using exosome coculture technology. Our results demonstrated that the exosomes derived from ferric citrate treated-mature HIB 1B brown adipocytes, could be internalized by HL-1 cardiomyocytes, and contributed to the dysfunction in these cells. The present study has revealed the alterations and impacts of iron overload-BAT, particularly on the onset of IOC via not only RNA-seq but also exosomes coculture technology. The outputs might shed light on the novel therapeutic strategies for the treatment of IOC.

Regulation of iron homeostasis to support differentiation and thermogenic function of adipocyte

The development of thermogenic adipocytes concurs with mitochondrial biogenesis, an iron-dependent pathway. Iron regulatory protein (IRP) 1 and 2 are RNA-binding proteins that regulate intracellular iron homeostasis. IRPs bind to the iron-response element (IRE) of their target mRNAs, balancing iron uptake and deposition at the post-transcriptional levels. However, IRP/IRE-dependent of iron regulation in adipocytes is largely unknown. We hypothesized that iron demands are higher in brown/beige adipocytes than white adipocytes to maintain the thermogenic mitochondrial capacity. To test this hypothesis, we investigated the IRP/IRE regulatory system in different depots of adipose tissue. Our results revealed that 1) IRP/IRE interaction was increased in proportional to the thermogenic function of the adipose depot, 2) adipose iron content was increased in adipose tissue browning upon b3-adrenoceptor stimulation, while decreased in thermoneutral conditions, and 3) modulation of iron content was linked with mitochondrial biogenesis. Moreover, the iron requirement was higher in HIB1B brown adipocytes than 3T3-L1 white adipocytes during differentiation. The reduction of the labile iron pool (LIP) suppressed the differentiation of brown/beige adipocytes and mitochondrial biogenesis. Using the 59Fe-Tf, we also demonstrated that thermogenic stimuli triggered cell-autonomous iron uptake and mitochondrial compartmentalization as well as enhanced mitochondrial respiration. Collectively, our work demonstrated that IRP/IRE signaling and subsequent adaptation in iron metabolism is a critical determinant for the thermogenic function of adipocytes.

Loss of family with sequence similarity 107, member A (FAM107A) induces browning in 3T3-L1 adipocytes

Induction of white fat browning (beiging) and activation of brown fat has been considered a promising strategy to treat obesity and associated metabolic complications. However, the molecular mechanisms regulating brown and beige fat-mediated thermogenesis remains unclear. Our study aimed to identify genes with a hitherto unknown mechanism in the metabolic functions of adipocytes and identified family with sequence similarity 107, member A (FAM107A) as a factor that interferes with fat browning in white adipocytes. We explored physiological roles of FAM107A in cultured 3T3-L1 white adipocytes and HIB1B brown adipocytes by using FAM107A-deficient adipocytes. Significant loss in FAM107A gene functionality induced fat browning was evidenced by evaluating the gene and protein expression level of brown fat-associated markers through real-time qRT-PCR and immunoblot analysis, respectively. Deficiency of FAM107A promoted mitochondrial biogenesis and significantly upregulated core fat-browning marker proteins (PGC-1α, PRDM16, and UCP1) and beige-specific genes (Cd137, Cited1, Tbx1, and Tmem26). Furthermore, FAM107A increased adipogenesis and negatively regulated lipid metabolism in 3T3-L1 adipocytes. In addition, in-silico analysis revealed a strong interaction between FAM107A and β3-AR based on their energy binding score. Next, mechanistic study revealed that specific knockdown of FAM107A induces browning in white adipocytes via activation of β3-AR, AMPK and p38 MAPK-dependent signaling pathways. Our data unveiled a previously unknown mechanism of FAM107A in the regulation of lipid metabolism and identified its significant role in metabolic homeostasis. This highlighted the potential of FAM107A as a pharmacotherapeutic target in treating obesity and related metabolic disorders.

BMP11 regulates thermogenesis in white and brown adipocytes.

Bone morphogenetic protein-11 (BMP11), also known as growth differentiation factor-11 (GDF11), is implicated in skeletal development and joint morphogenesis in mammals. However, its functions in adipogenesis and energy homeostasis are mostly unknown. The present study investigates crucial roles of BMP11 in cultured 3T3-L1 white and HIB1B brown adipocytes, using Bmp11 gene depletion and pharmacological inhibition of BMP11. The silencing of Bmp11 markedly decreases the expression levels of brown-fat signature proteins and beige-specific genes in white adipocytes and significantly down-regulates the expression levels of brown fat-specific genes in brown adipocytes. The deficiency of Bmp11 reduces the expressions of lipolytic protein markers in white and brown adipocytes. Moreover, BMP11 induces browning of 3T3-L1 adipocytes via coordination of multiple signalling pathways, including mTORC1-COX2 and p38MAPK-PGC-1α as non-canonical pathways, as well as Smad1/5/8 as a canonical pathway. We believe this study is the first to provide evidence of the potential roles of BMP11 for improvement of lipid catabolism in both cultured white and brown adipocytes, as well as the effect on browning of white adipocytes. Taken together, these results demonstrate the therapeutic potential for the treatment of obesity.

The thermogenic characteristics of adipocytes are dependent on the regulation of iron homeostasis

The development of thermogenic adipocytes concurs with mitochondrial biogenesis, an iron-dependent pathway. Iron regulatory proteins (IRP) 1 and 2 are RNA-binding proteins that regulate intracellular iron homeostasis. IRPs bind to the iron-response element (IRE) of their target mRNAs, balancing iron uptake and deposition at the posttranscriptional levels. However, IRP/IRE-dependent iron regulation in adipocytes is largely unknown. We hypothesized that iron demands are higher in brown/beige adipocytes than white adipocytes to maintain the thermogenic mitochondrial capacity. To test this hypothesis, we investigated the IRP/IRE regulatory system in different depots of adipose tissue. Our results revealed that 1) IRP/IRE interaction was increased in proportional to the thermogenic function of the adipose depot, 2) adipose iron content was increased in adipose tissue browning upon β3-adrenoceptor stimulation, while decreased in thermoneutral conditions, and 3) modulation of iron content was linked with mitochondrial biogenesis. Moreover, the iron requirement was higher in HIB1B brown adipocytes than 3T3-L1 white adipocytes during differentiation. The reduction of the labile iron pool (LIP) suppressed the differentiation of brown/beige adipocytes and mitochondrial biogenesis. Using the 59Fe-Tf, we also demonstrated that thermogenic stimuli triggered cell-autonomous iron uptake and mitochondrial compartmentalization as well as enhanced mitochondrial respiration. Collectively, our work demonstrated that IRP/IRE signaling and subsequent adaptation in iron metabolism are a critical determinant for the thermogenic function of adipocytes.

Secreted protein acidic and rich in cysteine (SPARC) regulates thermogenesis in white and brown adipocytes

SPARC, also known as osteonectin, is well known for its physiological roles in bone formation and tissue remodeling, as well as in cancer pathology; however, evidence regarding its function in adipocytes is lacking. The present study explored the physiological role of SPARC in cultured 3T3-L1 white and HIB1B brown adipocytes of murine cell lines. Treatment of recombinant SPARC upregulated the fat browning marker proteins and genes in white adipocytes and activated brown adipocytes. Conversely, knockdown of Sparc markedly reduced these genes and proteins in both cell lines. In addition, recombinant SPARC inhibited expression of adipogenic and lipogenic proteins but elevated lipolytic and fatty acid oxidation proteins. Furthermore, in silico analysis revealed that SPARC directly interacted and regulated VEGF in adipocytes. In conclusion, SPARC acts as a regulatory protein in both white and brown adipocytes by controlling thermogenesis and is thus regarded as a possible therapeutic target for treatment of obesity.

Serotonin Prevents Differentiation of Brown Adipocytes by Interfering with Their Clock.

Serotonin was shown to interfere with the differentiation of brown adipocytes. In addition, clock components inhibit brown adipogenesis through direct transcriptional control of key components of the transforming growth factor β pathway. The aim of this study was to investigate whether serotonin abrogates brown adipogenesis by affecting clock functionality. Nondifferentiated and differentiated HIB1B brown adipocytes were treated with serotonin, and their clock expression and functionality and differentiation state were examined. Nondifferentiated HIB1B brown adipocytes treated with serotonin showed increased brown adipocyte markers alongside increased brain-muscleArnt-like protein1 (Bmal1) andRAR related orphan receptor A (Rora) but decreasednuclear receptor Rev-erbα mRNA levels. BMAL1 overexpression together with serotonin led to significantly lower brown adipocyte markers. Serotonin in the differentiation cocktail led to reduced brown adipocyte markers as well as clock gene expression. After differentiation, serotonin treatment significantly decreased brown adipocyte markers and reduced BMAL1 and RORα but increased REV-ERBα protein levels. Addition of serotonin to the differentiation medium or addition after differentiation reduced activity of calcium/calmodulin-dependent protein kinasetype IIsubunit gamma, which interferes with circadian locomoter output cycles protein kaput (CLOCK):BMAL1 dimerization and transactivation. Clock expression is required at the early stages of differentiation to brown adipocytes, and serotonin interferes with this process by modulating clock functionality. Serotonin interferes with clock functionality by reducing the levels of the active form of calcium/calmodulin-dependent protein kinase typeIIsubunit gamma.

Trans-Cinnamic Acid Stimulates White Fat Browning and Activates Brown Adipocytes.

Recently, pharmacological activation of brown fat and induction of white fat browning (beiging) have been considered promising strategies to treat obesity. To search for natural products that could stimulate the process of browning in adipocytes, we evaluated the activity of trans-cinnamic acid (tCA), a class of cinnamon from the bark of Cinnamomum cassia, by determining genetic expression using real time reverse transcription polymerase chain reaction (RT-PCR) and protein expression by immunoblot analysis for thermogenic and fat metabolizing markers. In our study tCA induced brown like-phenotype in 3T3-L1 white adipocytes and activated HIB1B brown adipocytes. tCA increased protein content of brown-fat-specific markers (UCP1, PRDM16, and PGC-1α) and expression levels of beige-fat-specific genes (Cd137, Cidea, Cited1, Tbx1, and Tmen26) in 3T3-L1 white adipocytes, as well as brown-fat-specific genes (Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. Furthermore, tCA reduced expression of key adipogenic transcription factors C/EBPα and PPARγ in white adipocytes, but enhanced their expressions in brown adipocytes. In addition, tCA upregulates lipid catabolism. Moreover, mechanistic study revealed that tCA induced browning in white adipocytes by activating the β3-AR and AMPK signaling pathways. tCA can induce browning, increase fat oxidation, reduce adipogenesis and lipogenesis in 3T3-L1 adipocytes, and activate HIB1B adipocytes, suggesting its potential to treat obesity.

Functional Secretion of Granulocyte Colony Stimulating Factor in Bacillus subtilis and Its Thermogenic Activity in Brown Adipocytes

Granulocyte colony-stimulating factor (G-CSF) is involved in generation of colonies hematopoietic cells by stimulation of bone marrow cells, resulting in the survival, proliferation, differentiation, and function of neutrophil precursors and mature neutrophils. To express and secrete G-CSF in Bacillus system, the G-CSF gene was amplified using gene-specific primers and template DNA (pUNO1-hGCSFb). As a result, the PCR product of 530 bp was obtained, sequenced, and analyzed using the BLAST program. The open reading frame encoded a 204-amino acid polypeptide precursor consisting of the 30-residue signal sequence and a 174-residue mature polypeptide. Recombinant secretion vector (pRBAS-CSF3, 6.96 kb) was constructed using pRBAS vector which contains promoter (0.45 kb) and signal sequence (87 bp) of alkaline protease gene (aprE), ribosome binding site (GGAGAGGG) and transferred to Bacillus subtilis LKS. G-CSF protein was secreted and optimized to increase the productivity (19% of total secreted proteins, 0.12 mg/ml). The culture broth was harvested and followed by metal chelating, ethanol precipitation, and gel chromatography. The purified G-CSF was tested for thermogenic activity in cultured brown adipocytes. G-CSF activated HIB1B brown adipocytes by increasing expression levels of brown fat-specific marker proteins (UCP1 and PRDM16) and genes (Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. This result suggests that G-CSF per se or G-CSF-containing foodstuff can be used to treat obesity.

Theobromine, a Methylxanthine in Cocoa Bean, Stimulates Thermogenesis by Inducing White Fat Browning and Activating Brown Adipocytes

Natural medicinal compounds to treat obesity have recently attracted a great deal of attention because of the serious side effects of synthetic anti-obesity drugs. Recent advances have been made to identify natural products showing thermogenic activity, which is responsible for energy expenditure in brown or brown-like (beige) adipocytes. Here, we explored the thermogenic effects of theobromine, one of the most abundant methylxanthines in cocoa, on 3T3-L1 white adipocytes and HIB1B brown adipocytes. Theobromine markedly increased the expression levels of brown-fat signature proteins (PGC-1α, PRDM16, and UCP1) and beige-specific genes (Cd137, Cidea, Cited1, Tbx1, and Tmen26) in 3T3-L1 white adipocytes and remarkably elevated the expression levels of brown fatspecific genes (Cidea, Lhx8, Ppargc1, Prdm16, Ucp1, and Zic1) in HIB1B brown adipocytes. Theobromine also reduced the expression of the key adipogenic transcription factors, C/EBPα and PPARγ, in white adipocytes, while enhancing their expression in HIB1B cells. In addition, theobromine regulated lipolytic events and fat oxidation by upregulating the expression of pACC, ATGL, pHSL, ACOX, and CPT1. Additional mechanistic study revealed that theobromine activates β3-AR and AMPK. In summary, our results provide evidence for the first time indicating that theobromine has a potential beneficial effect on browning of white adipocytes and improves lipid catabolic metabolism in both cultured white and brown adipocytes via β-adrenergic signaling and AMPK activation. Consumption of theobromine may be a feasible way to activate thermogenesis and improve systematic lipid metabolism to protect against obesity and other metabolic disorders.

L-rhamnose induces browning in 3T3-L1 white adipocytes and activates HIB1B brown adipocytes.

Induction of the brown adipocyte-like phenotype in white adipocytes (browning) is considered as a novel strategy to fight obesity due to the ability of brown adipocytes to increase energy expenditure. Here, we report that L-rhamnose induced browning by elevating expression levels of beige-specific marker genes, including Cd137, Cited1, Tbx1, Prdm16, Tmem26, and Ucp1, in 3T3-L1 adipocytes. Moreover, L-rhamnose markedly elevated expression levels of proteins involved in thermogenesis both in 3T3-L1 white and HIB1B brown adipocytes. L-rhamnose treatment in 3T3-L1 adipocytes also significantly elevated protein levels of p-HSL, p-AMPK, ACOX, and CPT1 as well as reduced levels of ACC, FAS, C/EBPα, and PPARγ, suggesting its possible role in enhancement of lipolysis and lipid catabolism as well as reduced adipogenesis and lipogenesis, respectively. The quick technique of efficient molecular docking provided insight into the strong binding of L-rhamnose to the fat-digesting glycine residue of β3 -adrenergic receptor (AR), indicating strong involvement of L-rhamnose in fat metabolism. Further examination of the molecular mechanism of L-rhamnose revealed that it induced browning of 3T3-L1 adipocytes via coordination of multiple signaling pathways through β3 -AR, SIRT1, PKA, and p-38. To the best of our knowledge, this is the first study to demonstrate that L-rhamnose plays multiple modulatory roles in the induction of white fat browning, activation of brown adipocytes, as well as promotion of lipid metabolism, thereby demonstrating its therapeutic potential for treatment of obesity. © 2018 IUBMB Life, 70(6):563-573, 2018.

Nobiletin induces brown adipocyte-like phenotype and ameliorates stress in 3T3-L1 adipocytes

Browning of white adipocytes (beiging) is an attractive therapeutic strategy against obesity and its associated metabolic complications. Nobiletin (NOB) is a polymethoxylated flavone present in citrus fruits and has been reported to have anti-obesity effects. Here, we report that nobiletin exerts dual modulatory effects on adipocytes via induction of browning in 3T3-L1 white adipocytes and amelioration of stress in adipocytes. Nobiletin-induced beiging was investigated by determining expression levels of beige-specific genes and proteins by RT-PCR and immunoblot analysis, respectively. Nobiletin treatment rapidly elevated the expression levels of beige-specific genes such as Cd137, Cidea, Tbx1, and Tmem26. Further, nobiletin enhanced expression of the key transcription factors C/EBPβ, PPARδ, and PPARα, which are responsible for remodeling of white adipocytes. Nobiletin also strikingly activated HIB1B brown adipocytes and induced mitochondrial biogenesis in 3T3-L1 white adipocytes. In addition, nobiletin altered the expression of several lipid metabolism-related proteins such as ACOX1, CPT1, FAS, p-PLIN, SREBP and SIRT1. Moreover, nobiletin ameliorated stress in adipocytes by inhibiting expression levels of key stress molecules such as JNK and c-JUN. Nobiletin-induced browning could be mediated by tight regulation of kinases, as nobiletin induced PKA and p-AMPK at the protein expression level, and inhibition of PKA and p-AMPK by H-89 and dorsomorphin, respectively, abolished expression of the thermogenic markers PGC-1α and UCP1. Taken together, our findings suggest that nobiletin plays a modulatory role in adipocytes via induction of browning in 3T3-L1 white adipocytes and activation of HIB1B brown adipocytes combined with amelioration of stress in adipocytes, thereby exhibiting therapeutic potential against obesity.

Serotonin prevents differentiation into brown adipocytes and induces transdifferentiation into white adipocytes.

Serotonin is synthesized by many cells in the periphery to affect vasoconstriction, intestinal motility, and glucose and lipid metabolism. It has recently been shown that serotonin leads to fat accumulation in white adipose tissue (WAT). However, the direct effect of serotonin on brown adipose tissue differentiation and metabolism is limited. Therefore, our aim was to investigate the effect of serotonin on brown adipocyte metabolism and differentiation. Non-differentiated HIB1B cells and differentiated HIB1B brown adipocytes were treated with serotonin and their metabolism and differentiation examined. Differentiated HIB1B brown adipocytes treated with serotonin had reduced levels of the thermogenic markers uncoupling protein 1 (UCP1) and fibroblast growth factor 21 (FGF21) and increased levels of UCP2. In parallel, serotonin led to 3-6-fold reduction in the gene expression of brown adipocyte differentiation markers, that is, Prdm16 (positive regulatory domain 16), Bmp7 (bone morphogenic protein 7) and Pparγ (peroxisome-proliferator-activated receptor γ). Serotonin treatment reduced catabolism and mitochondrial activity shifting metabolism towards fatty acid synthesis rather than oxidation. Strikingly, non-differentiated HIB1B preadipocytes incubated with serotonin failed to differentiate into brown adipocytes. Moreover, although BMP6-treated myoblasts can readily differentiate into brown adipocytes, serotonin interfered with this process. Serotonin leads to whitening of brown adipocytes, shifting their metabolism to fat accumulation rather than oxidation. In addition, serotonin interferes with the differentiation process into brown adipocytes.

Honokiol exerts dual effects on browning and apoptosis of adipocytes.

Induction of brown adipocyte-like phenotype (browning) in white adipocytes and promotion of apoptosis by dietary and pharmacological compounds is considered a novel strategy against obesity. Here, we show that honokiol exerts dual modulatory effects on adipocytes via induction of browning in 3T3-L1 white adipocytes and apoptosis as well as activation of HIB1B brown adipocytes combined with inhibition of apoptosis. Honokiol-induced browning and apoptosis were investigated by determining expression levels of brown adipocyte-specific genes and proteins by RT-PCR and immunoblot analysis, respectively. Apoptotic data were validated by immunofluorescence and ROS levels were measured by FACS analysis. Honokiol treatment induced browning by elevating expression levels of brown adipocyte-specific genes such as Cidea, Cox8, Fgf21, Pgc-1α, and Ucp1. Honokiol promoted apoptosis of 3T3-L1 white adipocytes and inhibited apoptosis of HIB1B brown adipocytes via opposite regulation of the pro-apoptotic protein BAX and anti-apoptotic protein Bcl-2. Honokiol also significantly increased protein expression levels of ACOX1, CPT1, p-HSL, and p-PLIN and reduced ROS levels, suggesting its possible role in fat oxidation and lipid catabolism. Honokiol-induced browning could be mediated by activation of ERK, as inhibition of ERK by FR180204 abolished expression of PGC-1α and UCP1. Our findings suggest that honokiol exhibits a modulatory role in adipocytes via induction of browning and apoptosis in white adipocytes, promotion of catabolic lipid metabolism, as well as activation and inhibition of apoptosis in HIB1B brown adipocytes, thereby exhibiting therapeutic potential against obesity.

Rhein Protects against Obesity and Related Metabolic Disorders through Liver X Receptor-Mediated Uncoupling Protein 1 Upregulation in Brown Adipose Tissue

Liver X receptors (LXRs) play important roles in regulating cholesterol homeostasis, and lipid and energy metabolism. Therefore, LXR ligands could be used for the management of metabolic disorders. We evaluated rhein, a natural compound from Rheum palmatum L., as an antagonist for LXRs and investigated its anti-obesity mechanism in high-fat diet-fed mice. Surface plasmon resonance assays were performed to examine the direct binding of rhein to LXRs. LXR target gene expression was assessed in 3T3-L1 adipocytes and HepG2 hepatic cells in vitro. C57BL/6J mice fed a high-fat diet were orally administered with rhein for 4 weeks, and then the expression levels of LXR-related genes were analyzed. Rhein bound directly to LXRs. The expression levels of LXR target genes were suppressed by rhein in 3T3-L1 and HepG2 cells. In white adipose tissue, muscle and liver, rhein reprogrammed the expression of LXR target genes related to adipogenesis and cholesterol metabolism. Rhein activated uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) in wild-type mice, but did not affect UCP1 expression in LXR knockout mice. In HIB-1B brown adipocytes, rhein activated the UCP1 gene by antagonizing the repressive effect of LXR on UCP1 expression. This study suggests that rhein may protect against obesity and related metabolic disorders through LXR antagonism and regulation of UCP1 expression in BAT.

Non-transactivational, dual pathways for LPA-induced Erk1/2 activation in primary cultures of brown pre-adipocytes

In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G i-protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.

SIRT3, a Mitochondrial Sirtuin Deacetylase, Regulates Mitochondrial Function and Thermogenesis in Brown Adipocytes

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and Caenorhabditis elegans. Because adipose tissue is essential in energy homeostasis and also plays a role in life span determination, we decided to investigate the function of sirtuin members in fat. We report here that murine SIRT3 is expressed in brown adipose tissue and is localized on the mitochondria inner membrane. Caloric restriction activates SIRT3 expression in both white and brown adipose. Additionally, cold exposure up-regulates SIRT3 expression in brown fat, whereas elevated climate temperature reduces the expression. Enforced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of the uncoupling protein PGC-1alpha, UCP1, and a series of mitochondria-related genes. Both ADP-ribosyltransferase and deacetylase activities of SIRT3 are required for this action. Furthermore, the SIRT3 deacetylase mutant exhibits a dominant negative effect by inhibiting UCP1 expression. This inhibitive effect can be abolished by the coexpression of PGC-1alpha, indicating a major role of PGC-1alpha in the SIRT3 action. In addition, SIRT3 stimulates CREB phosphorylation, which reportedly activates PGC-1alpha promoter directly. Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species production while increasing cellular respiration. Finally, SIRT3, along with genes related to mitochondrial function, is down-regulated in the brown adipose tissue of several genetically obese mice. In summary, our results demonstrate that SIRT3 activates mitochondria functions and plays an important role in adaptive thermogenesis in brown adipose.

Opposing effects of glucocorticoids on β 3-adrenergic receptor expression in HIB-1B brown adipocytes

Glucocorticoids (GC) have been reported to promptly repress β 3-adrenergic receptor (β 3-AR) gene transcription in a white adipose tissue cell line. However, the effect of these hormones on β 3-AR expression in brown adipose tissue in vivo suggests a more complex mechanism of action. To avoid potential in vivo confounding variables, we investigated the effect of GC on the β 3-AR of HIB-1B brown adipocytes. While β 3-AR mRNA had same rapid turnover as in white fat cells, 1.5–2 h, the time course of its descent following dexamethasone was complex. A rapid initial descent β 3-AR mRNA with t 1/2≈1.6 h was consistent with a prompt, complete inhibition of transcription. Such rapid initial phase was followed ≈2 h later by a plateau or even an increase of β 3-AR mRNA, to descend thereafter following a slower single exponential ( t 1/2≈10 h). The change in the time course was abrogated by cycloheximide, and was not due to dexamethasone degradation or stabilization of β 3-AR mRNA at later times after dexamethasone. In vivo, a sufficiently large dose of dexamethasone was associated with a transient ≈70% reduction of brown adipose tissue β 3-AR mRNA by 4 h and full recovery by 24 h. These findings suggest that GC have two opposing effects on β 3-AR gene expression: they rapidly and directly inhibit transcription but also induce a rapidly turned-over protein (C/EBPβ?) that stimulates gene transcription. The relative magnitude of these two effects may vary explaining apparently discrepant observations.

Thiazolidinediones inhibit the expression of beta3-adrenergic receptors at a transcriptional level.

The effect of the thiazolidinediones (TZDs) darglitazone and troglitazone on beta3-adrenergic receptor (AR) expression was studied in cultured cell lines representing several tissues. After 24 h of exposing HIB-1B brown adipocytes to 30 micromol/l darglitazone or 20 micromol/l troglitazone, beta3-AR mRNA levels were reduced by 75%. This effect was significant within 1 h of exposure to a maximal dose of these drugs, with the full effect obtained within 10 h. The darglitazone ID50 was approximately 10 nmol/l, similar to the Kd of TZDs binding to peroxisome proliferator-activated receptor-gamma (PPAR-gamma). These drugs also decreased beta3-AR mRNA in 3T3-F442A white adipocytes, but not in SK-N-MC cells, which lack PPAR-gamma2. A luciferase reporter gene containing 1.4 kb of 5' flanking sequence of the mouse beta3-AR was transiently transfected, with or without PPAR-gamma2, in SK-N-MC cells. The vigorous expression of luciferase driven by the beta3-AR gene sequence was inhibited by TZDs in a PPAR-gamma2-dependent manner. The half-lives of gamma3-AR precursor RNA and mRNA were short, approximately 40 and approximately 100 min, respectively, and remained unaffected by TZD treatment. Exposure of HIB-1B cells to 30 micromol/l darglitazone was associated with reduced beta3-AR mRNA levels, as well as decreased response of uncoupling protein 1 to norepinephrine + propranolol (a beta1 beta2-AR antagonist) or the specific beta3-AR agonist CL 316, 243. Both the beta3-AR mRNA level and response to these stimuli fully recovered by 24 h of removing the drug, indicating that the beta3-AR protein and its coupling to adenylyl cyclase rapidly followed the changes in mRNA. Thus, TZDs can rapidly reduce beta3-AR expression at the transcriptional level, acting through PPAR-gamma2. The rapid turnover and responses of beta3-AR to perturbations, along with numerous other factors reported to regulate its expression, suggest a tight control of beta3-AR and function. Lastly, leptin being the only other known gene suppressed by TZDs, the present studies support a concerted lipogenic effect of these drugs.

Regulation of mitochondrial biogenesis in brown adipose tissue: nuclear respiratory factor-2/GA-binding protein is responsible for the transcriptional regulation of the gene for the mitochondrial ATP synthase beta subunit.

The regulation of transcription of the gene for the beta subunit of the FoF1 ATP synthase (ATPsynbeta) in brown adipose tissue has been studied as a model to determine the molecular mechanisms for mitochondrial biogenesis associated with brown adipocyte differentiation. The expression of the ATPsynbeta mRNA is induced during the brown adipocyte differentiation that occurs during murine prenatal development or when brown adipocytes differentiate in culture. This induction occurs in parallel with enhanced gene expression for other nuclear and mitochondrially-encoded components of the respiratory chain/oxidative phosphorylation system (OXPHOS). Transient transfection assays indicated that the expression of the ATPsynbeta gene promoter is higher in differentiated HIB-1B brown adipocytes than in non-differentiated HIB-1B cells. A major transcriptional regulatory site was identified between nt -306 and -266 in the ATPsynbeta promoter. This element has a higher enhancer capacity in differentiated brown adipocyte HIB-1B cells than in non-differentiated cells. Electrophoretic shift analysis indicated that Sp1and nuclear respiratory factor-2/GA-binding protein (NRF2/GABP) were the main nuclear proteins present in brown adipose tissue that bind this site. Double-point mutant analysis indicated a major role for the NRF2/GABP site in the enhancer capacity of this element in brown fat cells. It is proposed that NRF2/GABP plays a pivotal role in the co-ordinated enhancement of OXPHOS gene expression associated with mitochondrial biogenesis in brown adipocyte differentiation.