Maud Robert

Adipose Tissue–Derived Stem Cells From Obese Subjects Contribute to Inflammation and Reduced Insulin Response in Adipocytes Through Differential Regulation of the Th1/Th17 Balance and Monocyte Activation

Obesity, through low-grade inflammation, can drive insulin resistance and type 2 diabetes. While infiltration of adipose tissue (AT) with mononuclear cells (MNCs) is well established in obesity, the functional consequences of these interactions are less understood. Herein, we cocultured human adipose-derived stem cells (ASCs) from obese individuals with MNCs and analyzed their reciprocal behavior. Presence of ASCs 1) enhanced interleukin (IL)-17A secretion by Th17 cells, 2) inhibited γ-interferon and tumor necrosis factor α secretion by Th1 cells, and 3) increased monocyte-mediated IL-1β secretion. IL-17A secretion also occurred in stromal vascular fractions issued from obese but not lean individuals. Th17 polarization mostly depended on physical contacts between ASCs and MNCs—with a contribution of intracellular adhesion molecule-1—and occurred through activation of the inflammasome and phosphatidylinositol 3-kinase pathways. ASCs favored STAT3 over STAT5 transcription factor binding on STAT binding sites within the IL-17A/F gene locus. Finally, conditioned media from activated ASC-MNC cocultures inhibited adipocyte differentiation mRNA markers and impaired insulin-mediated Akt phosphorylation and lipolysis inhibition. In conclusion, we report that obese- but not lean-derived ASCs induce Th17 promotion and monocyte activation. This proinflammatory environment, in turn, inhibits adipogenesis and adipocyte insulin response. The demonstration of an ASC-Th17-monocyte cell axis reveals a novel proinflammatory process taking place in AT during obesity and defines novel putative therapeutic targets.

Human skeletal myotubes display a cell- autonomous circadian clock implicated in basal myokine secretion

Objective Circadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion. Methods We established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock. Results Bioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption. Conclusions Our study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes.

Results of laparoscopic Heller myotomy without anti-reflux procedure in achalasia. Monocentric prospective study of 106 cases

BackgroundHeller myotomy (HM) combined with an anti-reflux procedure has been shown to be effective for the treatment of achalasia, as postoperative gastro-esophageal reflux (GER) is observed in about 10% of the cases. Laparoscopy has brought an undeniable benefit in providing excellent visualisation of the gastro-esophageal junction (GEJ) without lateral and posterior dissection. Respecting the anatomical fixation of the GEJ seems to permit the performing of HM without an anti-reflux procedure, the need for which is therefore debatable. The purpose of this study was to analyse the results of this controversial procedure.MethodsA monocentric prospective study was carried out on 106 patients who underwent HM without an anti-reflux procedure. The postoperative assessment consisted of a manometry and a 24-hour pH study two months after surgery, and a yearly clinical examination for a minimum of five years. The data capture was done using a statistical analysis.ResultsThere was no mortality, one conversion to an open procedure, and four mucosal perforations. Postoperative morbidity was 2%. The average follow-up period was 55 months (range, 2 to 166), with 10 patients lost to follow-up. Good functional results were observed in 91.4% of patients at one year, and 78.6% at five years. Two months after surgery, a 9.4% prevalence of GER was detected in the pH study, and the lower esophageal sphincter pressure had significantly decreased. After a long term follow-up we observed an 11.3% global rate of GER. No repeat surgery was necessary to control postoperative GER.ConclusionsLaparoscopic HM without anti-reflux procedure gives good functional results provided the anatomical fixation of the GOJ is respected.

Achalasia-Like Disorder After Laparoscopic Adjustable Gastric Banding: a Reversible Side Effect?

Literature data concerning the effect of laparoscopic adjustable gastric banding (LAGB) on esophageal motility are conflicting. Achalasia-like disorder involving the absence of esophageal peristalsis and impaired esophago-gastric junction (EGJ) is probably under-estimated and can result in failure and band removal. The aim of our study was to focus on cases of achalasia-like disorder and study its evolution after band deflating or removal. LAGB patients with food intolerance and whose esophageal manometry confirmed dysmotility were selected from our database. Achalasia-like disorder was defined as the absence of esophageal peristalsis (< 20 % contraction waves) with impairment of EGJ relaxation. Manometric control was performed after removal or band deflating; functional results were assessed. Eleven patients among 20 (55 %) with esophageal motility disorders (EMD) fitted the manometric criteria of achalasia-like disorder with a mean EGJ resting pressure of 32.1 cmH2O and a EGJ relaxation pressure of 24.2. Nine patients out of 11 underwent band removal which resulted in the resolution of their symptoms. The other two underwent band deflation. Manometric control after band removal showed both a decrease in resting and relaxation EGJ pressures (mean of 9.5 and 6.5 cmH2O) and a recovery of wave contractions in 87.5 % of cases. Four patients underwent revision surgery due to weight regain with a successful outcome. Achalasia-like disorder is a manometric diagnosis and accounts for a significant part of symptomatic EMD after LAGB. It often results in band removal, allowing some reversibility of the disorders.

Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro

Significance Our experiments provide the analysis of lipid metabolite circadian oscillations in a cellular system synchronized in vitro, suggesting cell-autonomous diurnal changes in lipid profiles independent of feeding. Moreover, our work represents a comprehensive comparison between the lipid composition of human skeletal muscle derived from sedentary healthy adults, receiving hourly isocaloric solutions, and human primary skeletal myotubes cultured in vitro. A substantial number of lipid metabolites, in particular membrane lipids, exhibited oscillatory patterns in muscle tissue and in myotube cells, where they were blunted upon cell-autonomous clock disruption. As lipid oscillations in skeletal muscle membrane lipids may impact on insulin signaling and on the development of insulin resistance, studying the temporal lipid composition of human muscle is therefore of utmost importance. Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest–activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.

Adipocytes, like their progenitors, contribute to inflammation of adipose tissues through promotion of Th-17 cells and activation of monocytes, in obese subjects

ABSTRACT Recently, we have reported that adipose tissue-derived stem cells (ASC) harvested from obese donors induce a pro-inflammatory environment when co-cultured with peripheral blood mononuclear cells (MNC), with a polarization of T cells toward the Th17 cell lineage, increased secretion of IL-1β and IL-6 pro-inflammatory cytokines, and down-regulation of Th1 cytokines, such as IFNγ and TNFα. However, whether differentiated adipocytes, like the aforementioned ASC, are pro-inflammatory in obese subject AT remained to be investigated. Herein, we isolated ASC from AT of obese donors and differentiated them into adipocytes, for either 8 or 14 d. We analyzed their capacity to activate blood MNC after stimulation with phytohemagglutinin A (PHA), or not, in co-culture assays. Our results showed that co-cultures of MNC with adipocytes, like with ASC, increased IL-17A, IL-1β, and IL-6 pro-inflammatory cytokine secretion. Moreover, like ASC, adipocytes down-regulated TNFα secretion by Th1 cells. As adipocytes differentiated from ASC of lean donors also promoted IL-17A secretion by MNC, an experimental model of high-fat versus chow diet mice was used and supported that adipocytes from obese, but not lean AT, are able to mediate IL-17A secretion by PHA-activated MNCs. In conclusion, our results suggest that, as ASC, adipocytes in obese AT might contribute to the establishment of a low-grade chronic inflammation state.

Transcriptomic analyses reveal rhythmic and CLOCK-driven pathways in human skeletal muscle

Circadian regulation of transcriptional processes has a broad impact on cell metabolism. Here, we compared the diurnal transcriptome of human skeletal muscle conducted on serial muscle biopsies in vivo with profiles of human skeletal myotubes synchronized in vitro. More extensive rhythmic transcription was observed in human skeletal muscle compared to in vitro cell culture as a large part of the in vivo mRNA rhythmicity was lost in vitro. siRNA-mediated clock disruption in primary myotubes significantly affected the expression of ~8% of all genes, with impact on glucose homeostasis and lipid metabolism. Genes involved in GLUT4 expression, translocation and recycling were negatively affected, whereas lipid metabolic genes were altered to promote activation of lipid utilization. Moreover, basal and insulin-stimulated glucose uptake were significantly reduced upon CLOCK depletion. Our findings suggest an essential role for the circadian coordination of skeletal muscle glucose homeostasis and lipid metabolism in humans.

Proteolysis inhibition by hibernating bear serum leads to increased protein content in human muscle cells

Muscle atrophy is one of the main characteristics of human ageing and physical inactivity, with resulting adverse health outcomes. To date, there are still no efficient therapeutic strategies for its prevention and/or treatment. However, during hibernation, bears exhibit a unique ability for preserving muscle in conditions where muscle atrophy would be expected in humans. Therefore, our objective was to determine whether there are components of bear serum which can control protein balance in human muscles. In this study, we exposed cultured human differentiated muscle cells to bear serum collected during winter and summer periods, and measured the impact on cell protein content and turnover. In addition, we explored the signalling pathways that control rates of protein synthesis and degradation. We show that the protein turnover of human myotubes is reduced when incubated with winter bear serum, with a dramatic inhibition of proteolysis involving both proteasomal and lysosomal systems, and resulting in an increase in muscle cell protein content. By modulating intracellular signalling pathways and inducing a protein sparing phenotype in human muscle cells, winter bear serum therefore holds potential for developing new tools to fight human muscle atrophy and related metabolic disorders.

Laparoscopic Revision of Roux-en-Y Gastric Bypass to Biliopancreatic Diversion with Duodenal Switch: Technical Points

We present here the case of a 50-year-old man with a residual body mass index (BMI) of 47.4 kg/m after failure of gastric band followed by revisional Roux-en-Y gastric bypass (RYGB). The initial BMI was 51.2 at the time of gastric banding in 1998 and reached 62.3 with the band in place. A revisional RYGB was then performed in 2005 with a nadir BMI of 38.8, followed by weight regain (+36 kg) and weight stabilization at BMI 47.4. Main comorbidities were arterial hypertension, glucose intolerance, and lumbosacral sciatica. Considering the high residual BMI, young age, metabolic syndrome, and reduced ability, revisional biliopancreatic diversion with duodenal switch was decided after multidisciplinary evaluation and agreement.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity. In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.