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.

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.

The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation.

DNA topoisomerase I (Topo1) contributes to vital biological functions, but its regulation is not clearly understood. The BTBD1 protein was recently cloned on the basis of its interaction with the core domain of Topo1 and is expressed particularly in skeletal muscle. To determine BTBD1 functions in this tissue, the in vitro model used was the C2C12 mouse muscle cell line, which expresses BTBD1 mainly after myotube differentiation. We studied the effects of a stably overexpressed BTBD1 protein truncated of the 108 N-terminal amino-acid residues and harbouring a C-terminal FLAG tag (Delta-BTBD1). The proliferation speed of Delta-BTBD1 C2C12 cells was significantly decreased and no myogenic differentiation was observed, although these cells maintained their capacity to enter adipocyte differentiation. These alterations could be related to Topo1 deregulation. This hypothesis is further supported by the decrease in nuclear Topo1 content in Delta-BTBTD1 proliferative C2C12 cells and the switch from the main peripheral nuclear localization of Topo1 to a mainly nuclear diffuse localization in Delta-BTBTD1 C2C12 cells. Finally, this study demonstrated that BTBD1 is essential for myogenic differentiation.

Self-renewal gene tracking to identify tumour-initiating cells associated with metastatic potential.

Tumour-initiating cells (TICs) are rare cancer cells isolated from tumours of different origins including high-grade tumours that sustain neoplasic progression and development of metastatic disease. They harbour deregulated stem cells pathways and exhibit an unchecked ability to self-renew, a property essential for tumour progression. Among the essential factors maintaining embryonic stem (ES) cells properties, OCT-4 (also known as POU5F1) has been detected in tumours of different origins. Although ectopic expression results in dysplasic growth restricted to epithelial tissues, overexpression expands the proportion of immature cells in teratomas. However, OCT-4-expressing cells have not been purified from spontaneously occurring tumours, thus information concerning their properties is rather scant. Here, using p53-/- mice expressing green fluorescent protein and the puromycin resistance gene under the control of the Oct-4 promoter, we show that OCT-4 is expressed in 5% onwards of the undifferentiated tumour cell populations derived from different organs. OCT-4 expression was low as compared with ES cells, but was associated with a 'stemness' signature and expression of the chemokine receptor CXCR4. These cells displayed cancer stem cell features, including increased self-renewal and differentiation ability in vitro and in vivo. They not only formed allografts containing immature bone regions but also disseminated into different organs, including lung, liver and bone. Experiments based on RNA interference revealed that Oct-4 expression drives both their engraftment and metastasis formation. This work points out the crucial contribution of Oct-4-expressing TICs in the hierarchical organization of the malignant potential, leading to metastasis formation. Consequently, it provides an appropriate model to develop novel therapies aiming to strike down TICs by targeting self-renewal genes, therefore efficient to reduce tumour growth and metastatic disease.

Oncogenic mechanisms in myeloproliferative disorders.

Myeloproliferative disorders (MPDs) are clonal haematopoietic malignancies involving the abnormal proliferation of myeloid lineages. The World Health Organisation (WHO) classification of haematopoietic malignancies distinguishes MPDs from myelodysplastic/ myeloproliferative disorders and systemic mastocytosis. These malignancies frequently involve constitutive tyrosine kinase activity, resulting from either oncogenic fusion protein production or from point mutations. Chronic myelogenous leukaemia is the model used for studies of the consequences of such molecular defects. However, the heterogeneity of the clinical course of MPDs should be seen in a more rationale conceptual framework, including the many molecular events associated with these diseases. This review focuses on the various tyrosine kinase-related molecular mechanisms underlying both MPDs and rare diseases with myeloproliferative features. We pay particular attention to the newly identified JAK2 V617F mutation in polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis and deal with disease heterogeneity and putative additional molecular mechanisms.

Analysis of altered gene expression in rat soleus muscle atrophied by disuse.

The present study involved a global analysis of genes whose expression was modified in rat soleus muscle atrophied after hindlimb suspension (HS). HS muscle unloading is a common model for muscle disuse that especially affects antigravity slow-twitch muscles such as the soleus muscle. A cDNA cloning strategy, based on suppression subtractive hybridization technology, led to the construction of two normalized soleus muscle cDNA libraries that were subtracted in opposite directions, i.e., atrophied soleus muscle cDNAs subtracted by control cDNAs and vice versa. Differential screening of the two libraries revealed 34 genes with altered expression in HS soleus muscle, including 11 novel cDNAs, in addition to the 2X and 2B myosin heavy chain genes expressed only in soleus muscles after HS. Gene up- and down-regulations were quantified by reverse Northern blot and classical Northern blot analysis. The 25 genes with known functions fell into seven important functional categories. The homogeneity of gene alterations within each category gave several clues for unraveling the interplay of cellular events implied in the muscle atrophy phenotype. In particular, our results indicate that modulations in slow- and fast-twitch-muscle component balance, the protein synthesis/secretion pathway, and the extracellular matrix/cytoskeleton axis are likely to be key molecular mechanisms of muscle atrophy. In addition, the cloning of novel cDNAs underlined the efficiency of the chosen technical approach and gave novel possibilities to further decipher the molecular mechanisms of muscle atrophy.