M1-derived extracellular vesicles polarize recipient macrophages into M2-like macrophages and alter skeletal muscle homeostasis in a hyper-glucose environment.

BACKGROUND: Macrophages release not only cytokines but also extracellular vesicles (EVs). which are small membrane-derived nanovesicles with virus-like properties transferring cellular material between cells. Until now, the consequences of macrophage plasticity on the release and the composition of EVs have been poorly explored. In this study, we determined the impact of high-glucose (HG) concentrations on macrophage metabolism, and characterized their derived-EV subpopulations. Finally, we determined whether HG-treated macrophage-derived EVs participate in immune responses and in metabolic alterations of skeletal muscle cells. METHODS: THP1-macrophages were treated with 15mM (MG15) or 30mM (MG30) glucose. Then, M1/M2 canonical markers, pro- and anti-inflammatory cytokines, activities of proteins involved in glycolysis or oxidative phosphorylation were evaluated. Macrophage-derived EVs were characterized by TEM, NTA, MRSP, and 1H-Nuclear magnetic resonance spectroscopy for lipid composition. Macrophages or C2C12 muscle cells were used as recipients of MG15 and MG30-derived EVs. The lipid profiles of recipient cells were determined, as well as proteins and mRNA levels of relevant genes for macrophage polarization or muscle metabolism. RESULTS: Untreated macrophages released small and large EVs (sEVs, lEVs) with different lipid distributions. Proportionally to the glucose concentration, glycolysis was induced in macrophages, associated to mitochondrial dysfunction, triacylglycerol and cholesterol accumulation. In addition, MG15 and MG30 macrophages had increased level of CD86 and increase release of pro-inflammatory cytokines. HG also affected macrophage sphingolipid and phospholipid compositions. The differences in the lipid profiles between sEVs and lEVs were abolished and reflected the lipid alterations in MG15 and MG30 macrophages. Interestingly, MG15 and MG30 macrophages EVs induced the expression of CD163, Il-10 and increased the contents of triacylglycerol and cholesterol in recipient macrophages. MG15 lEVs and sEVs induced insulin-induced AKT hyper-phosphorylation and accumulation of triacylglycerol in myotubes, a state observed in pre-diabetes. Conversely, MG30 lEVs and sEVs induced insulin-resistance in myotubes. CONCLUSIONS: As inflammation involves first M1 macrophages, then the activation of M2 macrophages to resolve inflammation, this study demonstrates that the dialog between macrophages through the EV route is an intrinsic part of the inflammatory response. In a hyperglycemic context, EV macrophages could participate in the development of muscle insulin-resistance and chronic inflammation.

Blood-derived miRNA levels are not correlated with metabolic or anthropometric parameters in obese pre-diabetic subjects but with systemic inflammation.

As blood-derived miRNAs (c-miRNAs) are modulated by exercise and nutrition, we postulated that they might be used to monitor the effects of a lifestyle intervention (LI) to prevent diabetes development. To challenge this hypothesis, obese Asian Indian pre-diabetic patients were submitted to diet modifications and physical activity for 4 months (LI group) and compared to a control group which was given recommendations only. We have considered 2 periods of time to analyze the data, i.e.; a first one to study the response to the intervention (4 months), and a second one post-intervention (8 months). At basal, 4 months and 8 months post-intervention the levels of 17 c-miRNAs were quantified, selected either for their relevance to the pathology or because they are known to be modulated by physical activity or diet. Their variations were correlated with variations of 25 metabolic and anthropometric parameters and cytokines. As expected, fasting-glycaemia, insulin-sensitivity, levels of exercise- and obesity-induced cytokines were ameliorated after 4 months. In addition, the levels of 4 miRNAs (i.e.; miR-128-3p, miR-374a-5p, miR-221-3p, and miR-133a-3p) were changed only in the LI group and were correlated with metabolic improvement (insulin sensitivity, cytokine levels, waist circumference and systolic blood pressure). However, 8 months post-intervention almost all ameliorated metabolic parameters declined indicating that the volunteers did not continue the protocol on their own. Surprisingly, the LI positive effects on c-miRNA levels were still detected, and were even more pronounced 8 months post-intervention. In parallel, MCP-1, involved in tissue infiltration by immune cells, and Il-6, adiponectin and irisin, which have anti-inflammatory effects, continued to be significantly and positively modified, 8 months post-intervention. These data demonstrated for the first time, that c-miRNA correlations with metabolic parameters and insulin sensitivity are in fact only indirect and likely associated with the level systemic inflammation. More generally speaking, this important result explains the high variability between the previous studies designed to identify specific c-miRNAs associated with the severity of insulin-resistance. The results of all these studies should take into account the level of inflammation of the patients. In addition, this finding could also explain why, whatever the pathology considered (i.e.; cancers, diabetes, neurodegenerative disorders, inflammatory diseases) the same subset of miRNAs is always found altered in the blood of patients vs healthy subjects, as these pathologies are all associated with the development of inflammation.

Adipocyte-derived extracellular vesicles in health and diseases: Nano-packages with vast biological properties.

As the largest human energy reservoir, adipocytes drive an intense dialog with other cells/organs throughout the body to regulate the size of adipose tissue and to communicate with other metabolic tissues and the brain to regulate energy supply. Adipokines have long been described as mediators of this crosstalk, participating in obesity-associated complications. Recently, adipocyte-derived extracellular vesicles (Ad-EVs) have emerged as new key actors in this communication due to their powerful capacity to convey complex messages between cells. Ad-EVs convey specific subpopulations of RNA, proteins, and lipids from their parental cells, and can transfer these cargoes into various recipient cells, modulating their metabolism and cell cycle. In healthy individuals, Ad-EVs actively participate in adipose tissue remodeling to compensate energy supply variations by exchanging information between adipocytes or stroma-vascular cells, including immune cells. Besides this, recent evidence points out that Ad-EV secretion and composition from dysfunctional adipocytes are strongly impacted within adipose tissue where they modulate local intercellular communication, contributing to inflammation, fibrosis, abnormal angiogenesis, and at distance with other cells/tissues intrinsically linked to fat (muscle, hepatocytes and even cancer cells). Additionally, some data even suggests that Ad-EVs might have a systemic action. In this review, we will describe the particular properties of Ad-EVs and their involvement in health and diseases, with a particular focus on metabolic and cardiovascular diseases as well as cancer.

Use of Nanovesicles from Orange Juice to Reverse Diet-Induced Gut Modifications in Diet-Induced Obese Mice.

We have determined whether orange juice-derived nanovesicles (ONVs) could be used for the treatment of obesity-associated intestinal complications. ONVs were characterized by lipidomic, metabolomic, electron microscopy. In vitro, intestinal barriers (IBs = Caco-2+HT-29-MTX) were treated with ONVs and co-cultured with adipocytes to monitor IB fat release. In vivo, obesity was induced with a high-fat, high-sucrose diet (HFHSD mice) for 12 weeks. Then, half of HFHSD mice were gavaged with ONVs. One-month ONV treatment did not modify HFHSD-induced insulin resistance but reversed diet-induced gut modifications. In the jejunum, ONVs increased villi size, reduced triglyceride content, and modulated mRNA levels of genes involved in immune response (tumor necrosis factor [TNF]-α and interleukin [IL]-1ß), barrier permeability (CLDN1, OCLN, ZO1), fat absorption, and chylomicron release. ONVs targeted microsomal triglyceride transfer protein (MTP) and angiopoietin-like protein-4 (ANGPTL4), two therapeutic targets to reduce plasma lipids and inflammation in gastrointestinal diseases. Interestingly, ONV treatment did not aggravate liver steatosis, as MTP mRNA was increased in the liver. Therefore, ONVs protected both intestine and the liver from fat overload associated with the HFHSD. As ONVs concentrated amino acids and bioactive lipids versus orange juice, which are deficient in obese patients, the use of ONVs as a dietary supplement could bring physiological relevant compounds in the jejunum to accelerate the restoration of intestinal functions during weight loss in obese patients.

Mycoplasma hyopneumoniae J elicits an antioxidant response and decreases the expression of ciliary genes in infected swine epithelial cells.

Mycoplasma hyopneumoniae is the most costly pathogen for swine production. Although several studies have focused on the host-bacterium association, little is known about the changes in gene expression of swine cells upon infection. To improve our understanding of this interaction, we infected swine epithelial NPTr cells with M. hyopneumoniae strain J to identify differentially expressed mRNAs and miRNAs. The levels of 1,268 genes and 170 miRNAs were significantly modified post-infection. Up-regulated mRNAs were enriched in genes related to redox homeostasis and antioxidant defense, known to be regulated by the transcription factor NRF2 in related species. Down-regulated mRNAs were enriched in genes associated with cytoskeleton and ciliary functions. Bioinformatic analyses suggested a correlation between changes in miRNA and mRNA levels, since we detected down-regulation of miRNAs predicted to target antioxidant genes and up-regulation of miRNAs targeting ciliary and cytoskeleton genes. Interestingly, most down-regulated miRNAs were detected in exosome-like vesicles suggesting that M. hyopneumoniae infection induced a modification of the composition of NPTr-released vesicles. Taken together, our data indicate that M. hyopneumoniae elicits an antioxidant response induced by NRF2 in infected cells. In addition, we propose that ciliostasis caused by this pathogen is partially explained by the down-regulation of ciliary genes.

Bis(monoacylglycero)phosphate, a new lipid signature of endosome-derived extracellular vesicles.

Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), is a phospholipid specifically enriched in the late endosome-lysosome compartment playing a crucial role for the fate of endocytosed components. Due to its presence in extracellular fluids during diseases associated with endolysosomal dysfunction, it is considered as a possible biomarker of disorders such as genetic lysosomal storage diseases and cationic amphiphilic drug-induced phospholipidosis. However, there is no true validation of this biomarker in human studies, nor a clear identification of the carrier of this endolysosome-specific lipid in biofluids. The present study demonstrates that in absence of any sign of renal failure, BMP, especially all docosahexaenoyl containing species, are significantly increased in the urine of patients treated with the antiarrhythmic drug amiodarone. Such urinary BMP increase could reflect a generalized drug-induced perturbation of the endolysosome compartment as observed in vitro with amiodarone-treated human macrophages. Noteworthy, BMP was associated with extracellular vesicles (EVs) isolated from human urines and extracellular medium of human embryonic kidney HEK293 cells and co-localizing with classical EV protein markers CD63 and ALIX. In the context of drug-induced endolysosomal dysfunction, increased BMP-rich EV release could be useful to remove excess of undigested material. This first human pilot study not only reveals BMP as a urinary biomarker of amiodarone-induced endolysosomal dysfunction, but also highlights its utility to prove the endosomal origin of EVs, also named as exosomes. This peculiar lipid already known as a canonical late endosome-lysosome marker, may be thus considered as a new lipid marker of urinary exosomes.

Obesity paradox in cancer: Is bigger really better?

While obesity is widely recognized as a risk factor for cancer, survival among patients with cancer is often higher for obese than for lean individuals. Several hypotheses have been proposed to explain this "obesity paradox," but no consensus has yet emerged. Here, we propose a novel hypothesis to add to this emerging debate which suggests that lean healthy persons present conditions unfavorable to malignant transformation, due to powerful natural defenses, whereby only rare but aggressive neoplasms can emerge and develop. In contrast, obese persons present more favorable conditions for malignant transformation, because of several weight-associated factors and less efficient natural defenses, leading to a larger quantity of neoplasms comprising both nonaggressive and aggressive ones to regularly emerge and progress. If our hypothesis is correct, testing would require the consideration of the raw quantity, not the relative frequency, of aggressive cancers in obese patients compared with lean ones. We also discuss the possibility that in obese persons, nonaggressive malignancies may prevent the subsequent progression of aggressive cancers through negative competitive interactions between tumors.

Biological properties of plant-derived extracellular vesicles.

Identification of active constituents of our diet is crucial to understand the impact of food on health, and disease development, and for the formulation of functional food and nutraceuticals. Until now research into the pharmacological properties of the components of our diet has focused on vitamins, sterols, polyphenols, fiber, etc. But very recently, it has been found that plants contain various types of vesicles which are in contact with the intestinal tract throughout our lives. They participate in intestinal tissue renewal processes and modulate gut microbiota in healthy subjects and have important biological functions against inflammatory diseases (e.g.; colitis injury, liver steatosis) or cancers associated with their specific lipid and miRNA content. In addition, recent data have suggested that plant-derived nanovesicles would be excellent candidates for the delivery of therapeutic agents (e.g.; anti-cancerous drugs, siRNAs) or poorly soluble natural compounds (e.g.; curcumin), as they are able to cross mammalian barriers without inducing either an inflammatory response or necrosis, conversely to conventional liposomes. It is thus important to consider these plant-derived vesicles as new components of our food in order to evaluate their potential for health benefit and food-derived technology.

Analysis of the microRNA signature in left atrium from patients with valvular heart disease reveals their implications in atrial fibrillation.

BACKGROUND: Among the potential factors which may contribute to the development and perpetuation of atrial fibrillation, dysregulation of miRNAs has been suggested. Thus in this study, we have quantified the basal expressions of 662 mature human miRNAs in left atrium (LA) from patients undergoing cardiac surgery for valve repair, suffering or not from atrial fibrillation (AF) by using TaqMan® Low Density arrays (v2.0). RESULTS: Among the 299 miRNAs expressed in all patients, 42 miRNAs had altered basal expressions in patients with AF. Binding-site predictions with Targetscan (conserved sites among species) indicated that the up- and down-regulated miRNAs controlled respectively 3,310 and 5,868 genes. To identify the most relevant cellular functions under the control of the altered miRNAs, we focused on the 100 most targeted genes of each list and identified 5 functional protein-protein networks among these genes. Up-regulated networks were involved in synchronisation of circadian rythmicity and in the control of the AKT/PKC signaling pathway (i.e., proliferation/adhesion). Down-regulated networks were the IGF-1 pathway and TGF-beta signaling pathway and a network involved in RNA-mediated gene silencing, suggesting for the first time that alteration of miRNAs in AF would also perturbate the whole miRNA machinery. Then we crossed the list of miRNA predicted genes, and the list of mRNAs altered in similar patients suffering from AF and we found that respectively 44.5% and 55% of the up- and down-regulated mRNA are predicted to be conserved targets of the altered miRNAs (at least one binding site in 3'-UTR). As they were involved in the same biological processes mentioned above, these data demonstrated that a great part of the transcriptional defects previously published in LA from AF patients are likely due to defects at the post-transcriptional level and involved the miRNAs. CONCLUSIONS: Our stringent analysis permitted us to identify highly targeted protein-protein networks under the control of miRNAs in LA and, among them, to highlight those specifically affected in AF patients with altered miRNA signature. Further studies are now required to determine whether alterations of miRNA levels in AF pathology are causal or represent an adaptation to prevent cardiac electrical and structural remodeling.

Changes in diet associated with cancer: An evolutionary perspective.

Changes in diet are frequently correlated with the occurrence and progression of malignant tumors (i.e., cancer) in both humans and other animals, but an integrated conceptual framework to interpret these changes still needs to be developed. Our aim is to provide a new perspective on dietary changes in tumor-bearing individuals by adapting concepts from parasitology. Dietary changes may occur alongside tumor progression for several reasons: (i) as a pathological side effect with no adaptive value, (ii) as the result of self-medication by the host to eradicate the tumor and/or to slow down its progression, (iii) as a result of host manipulation by the tumor that benefits its progression, and finally (iv) as a host tolerance strategy, to alleviate and repair damages caused by tumor progression. Surprisingly, this tolerance strategy can be beneficial for the host even if diet changes are beneficial to tumor progression, provided that cancer-induced death occurs sufficiently late (i.e., when natural selection is weak). We argue that more data and a unifying evolutionary framework, especially during the early stages of tumorigenesis, are needed to understand the links between changes in diet and tumor progression. We argue that a focus on dietary changes accompanying tumor progression can offer novel preventive and therapeutic strategies against cancer.

Diagnostic Value of Cell-free Circulating MicroRNAs for Obesity and Type 2 Diabetes: A Meta-analysis.

Type 2 diabetes mellitus (T2DM) is the most common metabolic disorder worldwide. Because of population aging and increasing trends toward obesity and sedentary lifestyles, the number of affected individuals is increasing at worrisome rates. While both environmental and genetic factors are known to contribute to the development of T2DM, continuous research is needed to identify specific biomarkers that could aid both in prevention of the disease and development of newer therapeutic options. Circulating miRNAs are considered as potential biomarkers because they are stable and resistant to degradation by blood RNAses and are modified under different pathophysiological conditions. In this study we carried out a systematic electronic search on PubMed to retrieve all articles that have investigated circulating miRNAs for diagnosing obesity andT2DM in human. We also included lifestyle intervention studies known to be highly effective in delaying onset of diabetes, and studies analyzing the effect of bariatric surgery and anti-diabetic treatment. A total of 26 studies were enrolled in the global meta-analysis. Candidate miRNAs were defined as those reported in at least 2 studies with same direction of differential expression. Ten miRNAs altered in blood of patients suffering fromT2DM were identified (increased: miR-320a, miR-142-3p, miR-222, miR-29a, miR-27a, miR-375; decreased: miR-197, miR-20b, miR-17, miR-652) and 7 miRNAs in blood of obese subjects were identified (increased: miR-142-3p, miR-140-5p, miR-222; decreased:miR-21-5p, miR-221-3p, miR-125-5p, mir-103-5p). Both obese and T2DM patients had elevated concentrations of miR-142-3p and miR-222. MiRNAs target genes were predicted and their cellular functions are discussed in relation with the pathologies. Although a significant number of studies were taken into account in this review, we found a strong discrepancy between miRNA detection and quantification indicating that many of pre-analytical variables have yet to be normalized. Pre-analytical and analytical challenges are also discussed.

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.

MicroRNAs contribute to compensatory ß 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 ß cell mass that prevents chronic hyperglycemia and development of diabetes mellitus. The molecular basis underlying compensatory ß cell mass expansion is largely unknown. We found in rodents that ß 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 ß cells using specific anti-miR molecules mimicked gene expression changes occurring during ß 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 ß cell mass expansion occurring under different insulin resistance states.

αv integrin processing interferes with the cross-talk between αvß5/ß6 and α2ß1 integrins.

BACKGROUND INFORMATION: Previous studies have reported that cross-talk between integrins may be an important regulator of integrin-ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvß5/ß6 integrin represses α2ß1-dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. RESULTS: Inhibition of convertases by the convertase inhibitor α1-PDX (α1-antitrypsin Portland variant), leading to the cell-surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2ß1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen-activated protein kinase). This outside-in signalling stimulation was associated with increased levels of activated ß1 integrin located in larger than usual focal-adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3-kinase)/Akt (also called protein kinase B) pathway. CONCLUSIONS: The increase in cell migration observed upon convertases inhibition appears to be due to the up-regulation of ß1 integrins and to their location in larger focal-adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvß5/ß6 integrin to control α2ß1 function and could thus play an essential role in colon cancer cell migration.

Transcriptome profiling in response to adiponectin in human cancer-derived cells.

The adipocyte-derived hormone adiponectin exerts protective actions in several disorders, including some cancers. However, while growing data suggest that adiponectin could be an effective anticancer agent, its mechanism of action in cancer cells is still poorly known. Here, using microarrays, we identified a set of 1,301 genes commonly modulated in three cancer-derived cell lines in response to short-term stimulation with full-length recombinant human adiponectin. Most of these genes are involved in translation regulation, immune or stress responses, and cell proliferation. Furthermore, among genes linked to disease that were retrieved by functional enrichment tests using text mining based on PubMed analysis, we found that 66% are involved in malignant neoplasms, further supporting the link between adiponectin and cancer mechanisms. Bioinformatic analysis demonstrated the diversity of signaling pathways and transcription factors potentially mediating adiponectin effects on gene expression, illustrating the complexity of adiponectin mechanisms of action in cancer cells.

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.

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.

Changes in gene expression in skeletal muscle in response to fat overfeeding in lean men.

OBJECTIVE: The adaptive mechanisms in response to excess energy supply are still poorly known in humans. Our aims were to define metabolic responses and changes in gene expression in skeletal muscle of healthy volunteers during fat overfeeding. RESEARCH METHODS AND PROCEDURES: Eight lean young healthy men were given a diet rich in saturated fat with an excess of approximately 550 kcal/d for 4 weeks. Using oligonucleotide microarrays, gene expression changes in skeletal muscle were analyzed at Day 0, Day 14, and Day 28. RESULTS: Fat overfeeding led to an increase in body weight (1.0 +/- 0.3 kg) and waist circumference (2.2 +/- 0.5 cm, p = 0.005) and a significant decrease in fasting non-esterified fatty acid plasma levels (-29 +/- 5%, p = 0.028). Respiratory quotient was significantly increased (0.84 +/- 0.01 to 0.88 +/- 0.02, p = 0.034) and lipid oxidation rate tended to decrease. The expression of 55 genes was modified in skeletal muscle. The main pathways indicated a coordinated stimulation of triacylglycerol synthesis, inhibition of lipolysis, reduction of fatty acid oxidation, and development of adipocytes. Promoter analysis of the regulated genes suggests that sterol regulatory element binding proteins might be important players of the short-term adaptation to fat overfeeding in human skeletal muscle. DISCUSSION: This combined metabolic and genomic investigation shows that fat overfeeding for 28 days promotes the storage of the excess energy in lean men and demonstrates the usefulness of a transcriptomic approach to a better understanding of the metabolic adaptation to changes in nutritional behavior in human.

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

PURPOSE OF REVIEW: Insulin 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 FINDINGS: The 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. SUMMARY: It 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.

Microarray profiling of human skeletal muscle reveals that insulin regulates approximately 800 genes during a hyperinsulinemic clamp.

Insulin action in target tissues involved precise regulation of gene expression. To define the set of insulin-regulated genes in human skeletal muscle, we analyzed the global changes in mRNA levels during a 3-h hyperinsulinemic euglycemic clamp in vastus lateralis muscle of six healthy subjects. Using 29,308 cDNA element microarrays, we found that the mRNA expression of 762 genes, including 353 expressed sequence tags, was significantly modified during insulin infusion. 478 were up-regulated and 284 down-regulated. Most of the genes with known function are novel targets of insulin. They are involved in the transcriptional and translational regulation (29%), intermediary and energy metabolisms (14%), intracellular signaling (12%), and cytoskeleton and vesicle traffic (9%). Other categories consisted of genes coding for receptors, carriers, and transporters (8%), components of the ubiquitin/proteasome pathways (7%) and elements of the immune response (5.5%). These results thus define a transcriptional signature of insulin action in human skeletal muscle. They will help to better define the mechanisms involved in the reduction of insulin effectiveness in pathologies such as type 2 diabetes mellitus, a disease characterized by defective regulation of gene expression in response to insulin.