Sophie Rome

Weight loss regulates inflammation-related genes in white adipose tissue of obese subjects

Adipose tissue produces inflammation and immunity molecules suspected to be involved in obesity‐related complications. The pattern of expression and the nutritional regulation of these molecules in humans are poorly understood. We analyzed the gene expression profiles of subcutaneous white adipose tissue from 29 obese subjects during very low calorie diet (VLCD) using cDNA microarray and reverse transcription quantitative PCR. The patterns of expression were compared with that of 17 non‐obese subjects. We determined whether the regulated genes were expressed in adipocytes or stromavascular fraction cells. Gene expression profiling identified 100 inflammation‐related transcripts that are regulated in obese individuals when eating a 28 day VLCD but not a 2 day VLCD. Cluster analysis showed that the pattern of gene expression in obese subjects after 28 day VLCD was closer to the profile of lean subjects than to the pattern of obese subjects before VLCD. Weight loss improves the inflammatory profile of obese subjects through a decrease of proinflammatory factors and an increase of anti‐inflammatory molecules. The genes are expressed mostly in the stromavascular fraction of adipose tissue, which is shown to contain numerous macrophages. The beneficial effect of weight loss on obesity‐related complications may be associated with the modification of the inflammatory profile in adipose tissue.— Clément, K., Viguerie, N., Poitou, C., Carette, C., Pelloux, V., Curat, C. A., Sicard, A., Rome, S., Benis, A., Zucker, J.‐D., Vidal, H., Laville, M., Barsh, G. S., Basdevant, A., Stich, V., Cancello R., Langin, D. Weight loss regulates inflammation‐related genes in white adipose tissue of obese subjects. FASEB J. 18, 1657–1669 (2004)

Profiling of Circulating MicroRNAs Reveals Common MicroRNAs Linked to Type 2 Diabetes That Change With Insulin Sensitization

OBJECTIVE This study sought to identify the profile of circulating microRNAs (miRNAs) in type 2 diabetes (T2D) and its response to changes in insulin sensitivity. RESEARCH DESIGN AND METHODS The circulating miRNA profile was assessed in a pilot study of 12 men: 6 with normal glucose tolerance (NGT) and 6 T2D patients. The association of 10 circulating miRNAs with T2D was cross-sectionally validated in an extended sample of 45 NGT vs. 48 T2D subjects (65 nonobese and 28 obese men) and longitudinally in 35 T2D patients who were recruited in a randomized, double-blinded, and placebo-controlled 3-month trial of metformin treatment. Circulating miRNAs were also measured in seven healthy volunteers before and after a 6-h hyperinsulinemic-euglycemic clamp and insulin plus intralipid/heparin infusion. RESULTS Cross-sectional studies disclosed a marked increase of miR-140-5p, miR-142-3p, and miR-222 and decreased miR-423-5p, miR-125b, miR-192, miR-195, miR-130b, miR-532-5p, and miR-126 in T2D patients. Multiple linear regression analyses revealed that miR-140-5p and miR-423-5p contributed independently to explain 49.5% (P < 0.0001) of fasting glucose variance after controlling for confounders. A discriminant function of four miRNAs (miR-140-5p, miR-423-5p, miR-195, and miR-126) was specific for T2D with an accuracy of 89.2% (P < 0.0001). Metformin (but not placebo) led to significant changes in circulating miR-192 (49.5%; P = 0.022), miR-140-5p (−15.8%; P = 0.004), and miR-222 (−47.2%; P = 0.03), in parallel to decreased fasting glucose and HbA1c. Furthermore, while insulin infusion during clamp decreased miR-222 (−62%; P = 0.002), the intralipid/heparin mixture increased circulating miR-222 (163%; P = 0.015) and miR-140-5p (67.5%; P = 0.05). CONCLUSIONS This study depicts the close association between variations in circulating miRNAs and T2D and their potential relevance in insulin sensitivity.

Endometrial Exosomes/Microvesicles in the Uterine Microenvironment: A New Paradigm for Embryo-Endometrial Cross Talk at Implantation

Exosomes are nanoparticles (∼100 nm diameter) released from cells, which can transfer small RNAs and mRNA via the extracellular environment to cells at distant sites. We hypothesised that exosomes or the slightly larger microvesicles (100–300 nm) are released from the endometrial epithelium into the uterine cavity, and that these contain specific micro (mi)RNA that could be transferred to either the trophectodermal cells of the blastocyst or to endometrial epithelial cells, to promote implantation. The aim of this study was to specifically identify and characterise exosomes/microvesicles (mv) released from endometrial epithelial cells and to determine whether exosomes/mv are present in uterine fluid. Immunostaining demonstrated that the tetraspanins, CD9 and CD63 used as cell surface markers of exosomes are present on the apical surfaces of endometrial epithelial cells in tissue sections taken across the menstrual cycle: CD63 showed cyclical regulation. Exosome/mv pellets were prepared from culture medium of endometrial epithelial cell (ECC1 cells) and from uterine fluid and its associated mucus by sequential ultracentifugation. Exosomes/mv were positively identified in all preparations by FACS and immunofluorescence staining following exosome binding to beads. Size particle analysis confirmed the predominance of particles of 50–150 nm in each of these fluids. MiRNA analysis of the ECC1 cells and their exosomes/mv demonstrated sorting of miRNA into exosomes/mv: 13 of the 227 miRNA were specific to exosomes/mv, while a further 5 were not present in these. The most abundant miRNA in exosomes/mv were hsa-miR-200c, hsa-miR-17 and hsa-miR-106a. Bioinformatic analysis showed that the exosome/mv-specific miRNAs have potential targets in biological pathways highly relevant for embryo implantation. Thus exosomes/mv containing specific miRNA are present in the microenvironment in which embryo implantation occurs and may contribute to the endometrial-embryo cross talk essential for this process.

The microRNA Signature in Response to Insulin Reveals Its Implication in the Transcriptional Action of Insulin in Human Skeletal Muscle and the Role of a Sterol Regulatory Element–Binding Protein-1c/Myocyte Enhancer Factor 2C Pathway

OBJECTIVE Factors governing microRNA expressions in response to changes of cellular environment are still largely unknown. Our aim was to determine whether insulin, the major hormone controlling whole-body energy homeostasis, is involved in the regulation of microRNA expressions in human skeletal muscle. RESEARCH DESIGN AND METHODS We carried out comparative microRNA (miRNA) expression profiles in human skeletal muscle biopsies before and after a 3-h euglycemic-hyperinsulinemic clamp, with TaqMan low-density arrays. Then, using DNA microarrays, we determined the response to insulin of the miRNA putative target genes in order to determine their role in the transcriptional action of insulin. We further characterized the mechanism of action of insulin on two representative miRNAs, miR-1 and miR-133a, in human muscle cells. RESULTS Insulin downregulated the expressions of 39 distinct miRNAs in human skeletal muscle. Their potential target mRNAs coded for proteins that were mainly involved in insulin signaling and ubiquitination-mediated proteolysis. Bioinformatic analysis suggested that combinations of different downregulated miRNAs worked in concert to regulate gene expressions in response to insulin. We further demonstrated that sterol regulatory element–binding protein (SREBP)-1c and myocyte enhancer factor 2C were involved in the effect of insulin on miR-1 and miR-133a expression. Interestingly, we found an impaired regulation of miRNAs by insulin in the skeletal muscle of type 2 diabetic patients, likely as consequences of altered SREBP-1c activation. CONCLUSIONS This work demonstrates a new role of insulin in the regulation of miRNAs in human skeletal muscle and suggests a possible implication of these new modulators in insulin resistance.

MicroRNAs contribute to compensatory β cell expansion during pregnancy and obesity

Pregnancy and obesity are frequently associated with diminished insulin sensitivity, which is normally compensated for by an expansion of the functional β cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory β cell mass expansion is largely unknown. We found in rodents that β cell mass expansion during pregnancy and obesity is associated with changes in the expression of several islet microRNAs, including miR-338-3p. In isolated pancreatic islets, we recapitulated the decreased miR-338-3p level observed in gestation and obesity by activating the G protein-coupled estrogen receptor GPR30 and the glucagon-like peptide 1 (GLP1) receptor. Blockade of miR-338-3p in β cells using specific anti-miR molecules mimicked gene expression changes occurring during β cell mass expansion and resulted in increased proliferation and improved survival both in vitro and in vivo. These findings point to a major role for miR-338-3p in compensatory β cell mass expansion occurring under different insulin resistance states.

Acute Hyperglycemia Induces a Global Downregulation of Gene Expression in Adipose Tissue and Skeletal Muscle of Healthy Subjects

To define the effects of acute hyperglycemia per se (i.e., without the confounding effect of hyperinsulinemia) in human tissues in vivo, we performed global gene expression analysis using microarrays in vastus lateralis muscle and subcutaneous abdominal adipose tissue of seven healthy men during a hyperglycemic-euinsulinemic clamp with infusion of somatostatin to inhibit endogenous insulin release. We found that doubling fasting blood glucose values while maintaining plasma insulin in the fasting range modifies the expression of 316 genes in skeletal muscle and 336 genes in adipose tissue. More than 80% of them were downregulated during the clamp, indicating a drastic effect of acute high glucose, in the absence of insulin, on mRNA levels in human fat and muscle tissues. Almost all the biological pathways were affected, suggesting a generalized effect of hyperglycemia. The induction of genes from the metallothionein family, related to detoxification and free radical scavenging, indicated that hyperglycemia-induced oxidative stress could be involved in the observed modifications. Because the duration and the concentration of the experimental hyperglycemia were close to what is observed during a postprandial glucose excursion in diabetic patients, these data suggest that modifications of gene expression could be an additional effect of glucose toxicity in vivo.

Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation

It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as “endocrine signals” during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube–exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis.

The ubiquitin-proteasome pathway is a new partner for the control of insulin signaling

Purpose of reviewInsulin signaling is a transitory effect that has to be tightly controlled in magnitude and duration in order to maintain cell homeostasis. Recent reports have demonstrated that members of the ubiquitin-proteasome pathway represent new partners that have to be taken into account for the regulation of insulin action. Recent findingsThe protein amounts of the different signaling molecules involved in insulin action are regulated by their rates of synthesis and degradation. The ubiquitin-proteasome system is involved in the internalization of the insulin receptor, in the control of the amount of insulin receptor substrates 1 and 2, and in insulin degradation. Finally, ubiquitination and sumoylation regulate transcription factors and nuclear receptors that mediate insulin-induced gene expression. SummaryIt is well known from transgenic models that inappropriate levels of signaling molecules strongly affect insulin action. In humans also, several reports have provided evidence of altered levels of key proteins involved in insulin action in pathologies such as type 2 diabetes. The relationship between these abnormalities and the ubiquitin-proteasome pathway has yet to be clarified, but clarifying the role of ubiquitination in insulin action will certainly lead to a better understanding of insulin resistance.

A New Role for Sterol Regulatory Element Binding Protein 1 Transcription Factors in the Regulation of Muscle Mass and Muscle Cell Differentiation

ABSTRACT The role of the transcription factors sterol regulatory element binding protein 1a (SREBP-1a) and SREBP-1c in the regulation of cholesterol and fatty acid metabolism has been well studied; however, little is known about their specific function in muscle. In the present study, analysis of recent microarray data from muscle cells overexpressing SREBP1 suggested that they may play a role in the regulation of myogenesis. We then demonstrated that SREBP-1a and -1c inhibit myoblast-to-myotube differentiation and also induce in vivo and in vitro muscle atrophy. Furthermore, we have identified the transcriptional repressors BHLHB2 and BHLHB3 as mediators of these effects of SREBP-1a and -1c in muscle. Both repressors are SREBP-1 target genes, and they affect the expression of numerous genes involved in the myogenic program. Our findings identify a new role for SREBP-1 transcription factors in muscle, thus linking the control of muscle mass to metabolic pathways.

Microarray analyses of SREBP-1a and SREBP-1c target genes identify new regulatory pathways in muscle

In this study we have identified the target genes of sterol regulatory element binding protein (SREBP)-1a and SREBP-1c in primary cultures of human skeletal muscle cells, using adenoviral vectors expressing the mature nuclear form of human SREBP-1a or SREBP-1c combined with oligonucleotide microarrays. Overexpression of SREBP-1a led to significant changes in the expression of 1,315 genes (655 upregulated and 660 downregulated), whereas overexpression of SREBP-1c modified the mRNA level of 514 genes (310 upregulated and 204 downregulated). Gene ontology analysis indicated that in human muscle cells SREBP-1a and -1c are involved in the regulation of a large number of genes that are at the crossroads of different functional pathways, several of which are not directly connected with cholesterol and lipid metabolism. Six hundred fifty-two of all genes identified to be differentially regulated on SREBP overexpression had a sterol regulatory element (SRE) motif in their promoter sequences. Among these, 429 were specifically regulated by SREBP-1a, 69 by SREBP-1c, and 154 by both 1a and 1c. Because both isoforms recognize the same binding motif, we determined whether some of these functional differences could depend on the environment of the SRE motifs in the promoters. Results from promoter analysis showed that different combinations of transcription factor binding sites around the SRE binding motifs may determine regulatory networks of transcription that could explain the superposition of lipid and cholesterol metabolism with various other pathways involved in adaptive responses to stress like hypoxia and heat shock, or involvement in the immune response.