Latent TGFß-binding proteins regulate UCP1 expression and function via TGFß2.

OBJECTIVE: Activation of brown adipose tissue (BAT) in humans has been proposed as a new treatment approach for combating obesity and its associated diseases, as BAT participates in the regulation of energy homeostasis as well as glucose and lipid metabolism. Genetic contributors driving brown adipogenesis in humans have not been fully understood. METHODS: Profiling the gene expression of progenitor cells from subcutaneous and deep neck adipose tissue, we discovered new secreted factors with potential regulatory roles in white and brown adipogenesis. Among these, members of the latent transforming growth factor beta-binding protein (LTBP) family were highly expressed in brown compared to white adipocyte progenitor cells, suggesting that these proteins are capable of promoting brown adipogenesis. To investigate this potential, we used CRISPR/Cas9 to generate LTBP-deficient human preadipocytes. RESULTS: We demonstrate that LTBP2 and LTBP3 deficiency does not affect adipogenic differentiation, but diminishes UCP1 expression and function in the obtained mature adipocytes. We further show that these effects are dependent on TGFß2 but not TGFß1 signaling: TGFß2 deficiency decreases adipocyte UCP1 expression, whereas TGFß2 treatment increases it. The activity of the LTBP3-TGFß2 axis that we delineate herein also significantly correlates with UCP1 expression in human white adipose tissue (WAT), suggesting an important role in regulating WAT browning as well. CONCLUSIONS: These results provide evidence that LTBP3, via TGFß2, plays an important role in promoting brown adipogenesis by modulating UCP1 expression and mitochondrial oxygen consumption.

Pharmacological modulation of LMNA SRSF1-dependent splicing abrogates diet-induced obesity in mice.

Bakground/Objectives:Intense drug discovery efforts in the metabolic field highlight the need for novel strategies for the treatment of obesity. Alternative splicing (AS) and/or polyadenylation enable the LMNA gene to express distinct protein isoforms that exert opposing effects on energy metabolism and lifespan. Here we aimed to use the splicing factor SRSF1 that contribute to the production of these different isoforms as a target to uncover new anti-obesity drug. SUBJECTS/METHODS: Small molecules modulating SR protein activity and splicing were tested for their abilities to interact with SRSF1 and to modulate LMNA (AS). Using an LMNA luciferase reporter we selected molecules that were tested in diet-induced obese (DIO) mice. Transcriptomic analyses were performed in the white adipose tissues from untreated and treated DIO mice and mice fed a chow diet. RESULTS: We identified a small molecule that specifically interacted with the RS domain of SRSF1. ABX300 abolished DIO in mice, leading to restoration of adipose tissue homeostasis. In contrast, ABX300 had no effect on mice fed a standard chow diet. A global transcriptomic analysis revealed similar profiles of white adipose tissue from DIO mice treated with ABX300 and from untreated mice fed a chow diet. Mice treated with ABX300 exhibited an increase in O2 consumption and a switch in fuel preference toward lipids. CONCLUSIONS: Targeting SRSF1 with ABX300 compensates for changes in RNA biogenesis induced by fat accumulation and consequently represents a novel unexplored approach for the treatment of obesity.

White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways.

OBJECTIVE: Fat depots with thermogenic activity have been identified in humans. In mice, the appearance of thermogenic adipocytes within white adipose depots (so-called brown-in-white i.e., brite or beige adipocytes) protects from obesity and insulin resistance. Brite adipocytes may originate from direct conversion of white adipocytes. The purpose of this work was to characterize the metabolism of human brite adipocytes. METHODS: Human multipotent adipose-derived stem cells were differentiated into white adipocytes and then treated with peroxisome proliferator-activated receptor (PPAR)γ or PPARα agonists between day 14 and day 18. Gene expression profiling was determined using DNA microarrays and RT-qPCR. Variations of mRNA levels were confirmed in differentiated human preadipocytes from primary cultures. Fatty acid and glucose metabolism was investigated using radiolabelled tracers, Western blot analyses and assessment of oxygen consumption. Pyruvate dehydrogenase kinase 4 (PDK4) knockdown was achieved using siRNA. In vivo, wild type and PPARα-null mice were treated with a ß3-adrenergic receptor agonist (CL316,243) to induce appearance of brite adipocytes in white fat depot. Determination of mRNA and protein levels was performed on inguinal white adipose tissue. RESULTS: PPAR agonists promote a conversion of white adipocytes into cells displaying a brite molecular pattern. This conversion is associated with transcriptional changes leading to major metabolic adaptations. Fatty acid anabolism i.e., fatty acid esterification into triglycerides, and catabolism i.e., lipolysis and fatty acid oxidation, are increased. Glucose utilization is redirected from oxidation towards glycerol-3-phophate production for triglyceride synthesis. This metabolic shift is dependent on the activation of PDK4 through inactivation of the pyruvate dehydrogenase complex. In vivo, PDK4 expression is markedly induced in wild-type mice in response to CL316,243, while this increase is blunted in PPARα-null mice displaying an impaired britening response. CONCLUSIONS: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

Oxytocin and bone status in men: analysis of the MINOS cohort.

UNLABELLED: Oxytocin, a neurohypophysial hormone, regulates bone metabolism in animal studies and postmenopausal women. In men, oxytocin is not associated with bone mineral density, bone turnover markers, or prevalent fractures, but weakly negatively with incident fragility fracture requiring further studies. INTRODUCTION: We previously showed that serum oxytocin (OT) level is associated with bone mineral density (BMD) and bone turnover rate in postmenopausal women. The aim of our study was to assess the relationship between circulating OT levels and bone status in men. METHODS: In 552 men aged 50 and older from the MINOS cohort, we measured serum levels of OT. We assessed the association of serum OT levels with BMD (lumbar, femoral neck, total hip), bone turnover markers (BTM) (serum N-terminal propeptide of type I procollagen (PINP), bone-specific alkaline phosphatase (bone ALP), and C-terminal telopeptide of type I collagen (CTX-I)) and fracture risk. RESULTS: In the univariate analysis, serum OT level was not associated with BMD at any site, BTM levels, or with prevalent or incident fracture. OT was significantly correlated with body mass index (BMI) (r = 0.17, p < 0.001), total or bioavalaible 17ß-estradiol (r = 0.09, p = 0.04 and r = 0.20, p < 0.001, respectively), free testosterone (r = 0.17, p < 0.001), and leptin (r = 0.16, p < 0.001). Multivariate analysis did not show significant relationship between serum OT and BMD. After adjustment for age, BMI, interaction BMI/age, history of fall in the last year, and BMD, OT and prevalent fracture were not associated. By contrast, the same analysis with additional adjustment for prevalent fracture showed a weakly significant negative association between OT and incident fracture, e.g., after adjustment for femoral neck BMD, HR = 0.73, 95 %CI 0.55-0.99, p = 0.04. CONCLUSION: In men, serum OT levels are not associated with BMD, bone turnover rate, or prevalent fractures. The weak negative relationship with fracture risk requires further studies.

Mesoderm-specific transcript (MEST) is a negative regulator of human adipocyte differentiation.

BACKGROUND: A growing body of evidence suggests that many downstream pathologies of obesity are amplified or even initiated by molecular changes within the white adipose tissue (WAT). Such changes are the result of an excessive expansion of individual white adipocytes and could potentially be ameliorated via an increase in de novo adipocyte recruitment (adipogenesis). Mesoderm-specific transcript (MEST) is a protein with a putative yet unidentified enzymatic function and has previously been shown to correlate with adiposity and adipocyte size in mouse. OBJECTIVES: This study analysed WAT samples and employed a cell model of adipogenesis to characterise MEST expression and function in human. METHODS AND RESULTS: MEST mRNA and protein levels increased during adipocyte differentiation of human multipotent adipose-derived stem cells. Further, obese individuals displayed significantly higher MEST levels in WAT compared with normal-weight subjects, and MEST was significantly correlated with adipocyte volume. In striking contrast to previous mouse studies, knockdown of MEST enhanced human adipocyte differentiation, most likely via a significant promotion of peroxisome proliferator-activated receptor signalling, glycolysis and fatty acid biosynthesis pathways at early stages. Correspondingly, overexpression of MEST impaired adipogenesis. We further found that silencing of MEST fully substitutes for the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) as an inducer of adipogenesis. Accordingly, phosphorylation of the pro-adipogenic transcription factors cyclic AMP responsive element binding protein (CREB) and activating transcription factor 1 (ATF1) were highly increased on MEST knockdown. CONCLUSIONS: Although we found a similar association between MEST and adiposity as previously described for mouse, our functional analyses suggest that MEST acts as an inhibitor of human adipogenesis, contrary to previous murine studies. We have further established a novel link between MEST and CREB/ATF1 that could be of general relevance in regulation of metabolism, in particular obesity-associated diseases.

Comparative analysis of two 14-3-3 homologues and their expression pattern in the root-knot nematode Meloidogyne incognita.

14-3-3 proteins are highly conserved ubiquitous proteins found in all eukaryotic organisms. They are involved in various cellular processes including signal transduction, cell-cycle control, apoptosis, stress response and cytoskeleton organisation. We report here the cloning of two genes encoding 14-3-3 isoforms from the plant parasitic root-knot nematode Meloidogyne incognita, together with an analysis of their expression. Both genes were shown to be transcribed in unhatched second stage larvae, infective second stage larvae, adult males and females. The Mi-14-3-3-a gene was shown to be specifically transcribed in the germinal primordium of infective larvae, whereas Mi-14-3-3-b was transcribed in the dorsal oesophageal gland in larvae of this stage. The MI-14-3-3-B protein was identified by mass spectrometry in in vitro-induced stylet secretions from infective larvae. The stability and distribution of MI-14-3-3 proteins in host plant cells was assessed after stable expression of the corresponding genes in tobacco BY2 cells.

PPARgamma-dependent and PPARgamma-independent effects on the development of adipose cells from embryonic stem cells.

Peroxisome proliferator-activated receptor (PPAR) gamma was shown to be required for adipocyte formation both in vivo and in vitro. However, the role of PPARgamma in the initial steps of adipose cell development was not distinguished from its role in the terminal steps. We now show that PPARgamma is expressed early in embryoid bodies (EBs) derived from embryonic stem cells and in E.8.5 mouse embryos. Addition of a specific ligand for PPARgamma in developing EBs over-expressing PPARgamma did not commit stem cells towards the adipose lineage. In differentiated PPARgamma(-/-) EBs, only markers characteristic of preadipocytes were found to be expressed. PPARdelta is present in EBs but did not compensate for the lack of PPARgamma in terminal differentiation. Taken together, these results favor a critical PPARgamma-independent phase culminating in preadipocyte formation that precedes a PPARgamma-dependent phase in the development of adipose cells from pluripotent stem cells.

Structural and functional characterization of the mouse fatty acid translocase promoter: activation during adipose differentiation.

Fatty acid translocase (FAT/CD36) is a cell-surface glycoprotein that functions as a receptor/transporter for long-chain fatty acids (LCFAs), and interacts with other protein and lipid ligands. FAT/CD36 is expressed by various cell types, including platelets, monocytes/macrophages and endothelial cells, and tissues with an active LCFA metabolism, such as adipose, small intestine and heart. FAT/CD36 expression is induced during adipose cell differentiation and is transcriptionally up-regulated by LCFAs and thiazolidinediones in pre-adipocytes via a peroxisome-proliferator-activated receptor (PPAR)-mediated process. We isolated and analysed the murine FAT/CD36 promoter employing C(2)C(12)N cells directed to differentiate to either adipose or muscle. Transient transfection studies revealed that the 309 bp upstream from the start of exon 1 confer adipose specific activity. Sequence analysis of this DNA fragment revealed the presence of two imperfect direct repeat-1 elements. Electrophoretic mobility-shift assay demonstrated that these elements were peroxisome-proliferator-responsive elements (PPREs). Mutagenesis and transfection experiments indicated that both PPREs co-operate to drive strong promoter activity in adipose cells. We conclude that murine FAT/CD36 expression in adipose tissue is dependent upon transcriptional activation via PPARs through binding to two PPREs located at -245 to -233 bp and -120 to -108 bp from the transcription start site.

Inhibition of myogenesis enables adipogenic trans-differentiation in the C2C12 myogenic cell line.

C2C12 cells are a well-established model system for studying myogenesis. We examined whether inhibiting the process of myogenesis via expression of dominant negative (DN) mitogen-activated protein kinase kinase-3 (MKK3) facilitated the trans-differentiation of these cells into adipocytes. Cells expressing DN MKK3 respond to rosiglitazone, resulting in adipocyte formation. The effects of rosiglitazone appear to be potentiated through peroxisome proliferator activating receptor-gamma. This trans-differentiation is inhibited by the use of the phosphoinositide-3 (PI3) kinase inhibitor, LY294002. These results indicate that preventing myogenesis through expression of DN MKK3 facilitates adipocytic trans-differentiation, and involves PI3 kinase signalling.

Long chain fatty acids as modulators of gene transcription in preadipose cells.

During the last years, it has been clearly established that long-chain fatty acids act as modulators of gene expression in various tissues, such as adipose tissue, intestine and liver. This transcriptional action of fatty acids explains in part adaptation mechanisms of tissues to nutritional changes and especially to high-fat diets by increasing expression of proteins involved in lipid catabolism in liver and fatty acid uptake and utilization in other tissues. It is now clearly demonstrated that some of these transcriptional effects of fatty acids are mediated by activation of specific nuclear hormone receptors, called peroxisome proliferator-activated receptors (PPARs). These findings will be discussed with a special reference to control of gene expression in preadipocytes and adipose tissue development.

Trans-differentiation of myoblasts to adipoblasts: triggering effects of fatty acids and thiazolidinediones.

Long-chain fatty acids (LCFA) and thiazolidinediones are potent activators of differentiation of preadipose cells. These adipogenic effects are, at least in part, mediated by nuclear receptors of the peroxisome proliferator-activated receptor (PPAR) subfamily. This report describes the effects of these agents on the differentiation pathway of myoblasts. Exposure of C2C12 myoblasts to LCFA or thiazolidinediones prevents the formation of multinucleated myotubes and the expression of specific muscle markers, leading in parallel to the expression of a typical adipose differentiation program. Similar transdifferentiation also occurs in mouse muscle satellite cells maintained in primary cell culture. These observations indicate that PPAR activators, such as LCFA or thiazolidinediones, convert the differentiation pathway of myoblasts into that of adipoblasts. This phenomenon could explain the appearance of adipocytes into muscle which occurs in some pathological states characterized by an increase of fatty acid disposal, such as obesity or mitochondrial myopathy.

Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport.

An adipocyte membrane glycoprotein, (FAT), homologous to human CD36, has been previously implicated in the binding/transport of long-chain fatty acids. It bound reactive derivatives of long-chain fatty acids and binding was specific and associated with significant inhibition of fatty acid uptake. Tissue distribution of the protein and regulation of its expression were also consistent with its postulated role. In this report, we have examined the effects of FAT expression on rates and properties of fatty acid uptake by Ob17PY fibroblasts lacking the protein. Three clones (P21, P22, and P25) were selected based on FAT mRNA and protein levels. Cell surface labeling could be demonstrated with the anti-CD36 antibody FITC-OKM5. In line with this, the major fraction of immunoreactive FAT was associated with the plasma membrane fraction. Assays of oleate and/or palmitate uptake demonstrated higher rates in the three FAT-expressing clones, compared to cells transfected with the empty vector. Clone P21, which had the highest protein levels on Western blots, exhibited the largest increase in transport rates. Fatty acid uptake in FAT-expressing P21 cells reflected two components, a phloretin-sensitive high-affinity saturable component with a Km of 0.004 microM and a basal phloretin-insensitive component that was a linear function of unbound fatty acid. P21 cells incorporated more exogenous fatty acid into phospholipids, indicating that binding of fatty acids was followed by their transfer into the cell and that both processes were increased by FAT expression. The data support the interpretation that FAT/CD36 functions as a high-affinity membrane receptor/transporter for long-chain fatty acids.

Fatty acids regulate the expression of lipoprotein lipase gene and activity in preadipose and adipose cells.

During fasting, a reduction in lipoprotein lipase (LPL) activity has been observed in rat fat pad with no change in enzyme mass, whereas LPL mRNA and synthesis are increased, suggesting that insulin and/or fatty acids (FA) regulate LPL activity post-translationaly [Doolittle, Ben-Zeev, Elovson, Martin and Kirchgessner (1990) J. Biol. Chem. 265, 4570-4577]. To examine the role of FA, either preadipose Ob1771 cells or Ob1771 and 3T3-F442A adipose cells were exposed to long-chain FA and to 2-bromopalmitate, a non-metabolized FA. A rapid (2-8 h) and dose-dependent increase (up to 6-fold) in LPL mRNA occurred, primarily due to increased transcription, which is accompanied by a decrease (down to 4-fold) in LPL cellular activity. Under these conditions, secretion of active LPL was nearly abolished. Removal of FA led to full recovery of LPL activity. LPL gene expression in 3T3-C2 fibroblasts was not affected by FA treatment. However fatty acid-activated receptor transfected-3T3-C2 cells, which show FA responsiveness, had increased LPL gene expression upon FA addition. LPL synthesis and cellular content appeared unaffected by FA treatment, whereas secretion of LPL was inhibited. These results indicate that FA regulate the post-translational processing of LPL. It is proposed that the regulation of LPL activity by FA is important with regard to the fine-tuning of FA entry into adipocytes during fasting/feeding periods.

Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells.

Fatty acids and thiazolidinediones act as potent activators of the adipose differentiation program in established preadipose cell lines. In this report, the effects of these agents on the differentiation pathway of myoblasts have been investigated. Exposure of C2C12N myoblasts (a subclone of the C2C12 cell line) to thiazolidinediones or fatty acids prevents the expression of myogenin, alpha-actin, and creatine kinase, thus abolishing the formation of multinucleated myotubes. These treatments lead in parallel to the expression of a typical adipose differentiation program including acquisition of adipocyte morphology and activation of adipose-related genes. A similar transition toward the adipose differentiation pathway also occurs in mouse muscle satellite cells maintained in primary culture. Thiazolidinediones exert their adipogenic effects only in non-terminally differentiated myoblasts; myotubes are insensitive to the compounds. Continuous exposure to inducers after growth arrest is not required to maintain the adipose phenotype, but proliferation of adipose-like C2C12N cells leads to a complete reversion toward undifferentiated cells able to undergo either myogenic or adipogenic differentiation depending on the composition of culture medium. These results indicate that adipogenic inducers, such as thiazolidinediones or fatty acids, specifically convert the differentiation pathway of myoblasts into that of adipoblasts.

Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. Homology to peroxisome proliferator-activated receptors.

Exposure of preadipocytes to long chain fatty acids induces expression of several gene markers of adipocyte differentiation. This report describes the cloning, from a preadipocyte library, of a cDNA encoding a fatty acid-activated receptor, FAAR. The cDNA had the characteristics and ligand-binding domains of nuclear hormone receptors and encoded a 440 amino acid protein related to peroxisome proliferator-activated receptors, PPAR. The deduced protein sequence was 88% homologous to that of hNUC I, isolated from human osteosarcoma cells. FAAR mRNA was abundant in adipose tissue, intestine, brain, heart, and skeletal muscles and less abundant in kidney, liver, testis, and spleen. The mRNA was undetectable in growing Ob1771 and 3T3-F442A preadipocytes, was strongly induced early during differentiation, and was increased by fatty acid. Transcription assays using hybrid receptor showed strong stimulation by fatty acid and weaker induction by fibrates. Transfection of 3T3-C2 fibroblasts, with a FAAR expression vector, conferred fatty acid inducibility of the adipocyte lipid-binding protein and the fatty acid transporter. Transcriptional induction of these genes exhibited inducer specificity identical to that described in preadipocytes. In summary, the data indicate that FAAR is likely a mediator of fatty acid transcriptional effects in preadipocytes.

Fatty acids and adipose cell differentiation.

Fatty acids are important metabolic substrates for adipose tissue. In preadipose cells, fatty acids are also potent inducers of various genes encoding proteins directly involved in fatty acid metabolism. On a longer-term basis, fatty acids induce the terminal differentiation of preadipose to adipose cells. Fatty acids act primarily at a transcriptional level. A member of the steroid/thyroid hormone receptor superfamily has been identified by cDNA cloning from a mouse Ob1771 preadipose cell library. This receptor is likely the fatty acid-activated receptor implicated in the transcriptional effects of fatty acids in adipose cells. Thus fatty acids appear to play a new role as signal transducing molecules which are involved in adipose cell differentiation.

Evidence for a common mechanism of action for fatty acids and thiazolidinedione antidiabetic agents on gene expression in preadipose cells.

In diabetic rodents, thiazolidinediones are able to improve insulin sensitivity of target tissues and to reverse, at least partially, the diabetic state. The effects of these drugs on phenotypic expression in various tissues, including adipose tissue, have been reported. We report here that a new thiazolidinedione compound, BRL 49653, exerts, in preadipose cells, potent effects on the expression of genes encoding proteins involved in fatty acid metabolism. These effects of BRL 49653 in Ob 1771 preadipose cells are similar, in terms of kinetics, reversibility, specificity of genes affected, and requirement for protein synthesis, to those already described for natural or nonmetabolizable fatty acids. Moreover, when used at submaximally effective concentrations, BRL49653 and 2-bromopalmitate act in an additive manner to induce gene expression in preadipose cells, but this additivity of effects is lost when one of the compounds is used at a maximally effective concentration. These observations, suggesting similar mechanisms of action for thiazolidinediones and fatty acids, are strongly supported by the demonstration that (i) both molecules activate, in a heterogolous trans-activation assay, the same nuclear receptor of the steroid/thyroid hormone nuclear receptor superfamily and (ii) transfection of 3T3-C2 fibroblasts with an expression vector for this nuclear receptor confers thiazolidinedione inducibility of adipocyte lipid-binding protein gene expression.

Retinoids are positive effectors of adipose cell differentiation.

Retinoids, especially all-trans retinoic acid (t-RA), have been reported in the last decade to inhibit the differentiation of preadipose cells. In those studies, however, the concentrations of t-RA were supraphysiological (0.1-10 microM range). In contrast we show that, when present at concentrations below or close to the Kd values of retinoic acid receptors, retinoids behave as potent adipogenic hormones (1 pM to 10 nM range). As shown by the use of specific ligands for each RAR subtype, these positive effects on adipose differentiation involve in particular the RAR alpha subtype, and have been observed in Ob17 cells exposed to serum-supplemented or serum-free medium, and in rat preadipocytes exposed to serum-free medium. Among the two classes of retinoid acid receptors (RARs) and retinoid X receptors (RXRs), RAR alpha, RAR gamma, RXR alpha and RXR beta mRNAs could be detected in growing adipoblasts and were found to be increased in committed preadipocytes and differentiated cells upon retinoid treatment. Like other adipogenic hormones, retinoids were only effective in the terminal differentiation process leading from preadipocytes to adipocytes.

Expression cloning in K+ transport defective yeast and distribution of HBP1, a new putative HMG transcriptional regulator.

The rat HBP1 cDNA was cloned by its capacity to suppress the potassium transport-defective phenotype of mutant Saccharomyces cerevisiae cells. HBP1 cDNA encodes a 513 amino acids protein which, unexpectedly, does not share any homology with K+ transporters or K+ channels. However, a search in protein databases reveals that HBP1 contains a putative DNA-binding domain called HMG-box. Northern blot analysis shows that HBP1 is expressed in a variety of tissues and that in adipocyte and myogenic cell lines, its expression is directly related to differentiation. Taken together, the results suggest that the rat HBP1 is a new member of the HMG class of transcriptional regulators involved in cell differentiation pathways.