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.

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.

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.

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.