14-color flow cytometry to determine the contribution of mitochondrial mass to differences in glycolytic capacity in human immune cell subsets

Mitochondrial metabolism controls immune cell function, but comprehensive tools to assess human primary immune cell metabolic capacity remain rudimentary. We previously demonstrated that CD19+ B cells rely more heavily on anaerobic glycolysis (i.e. are more glycolytic) than CD4+ T cells. Furthermore, both PBMCs and CD4+ T cells from subjects with type 2 diabetes (T2D) are more glycolytic than their counterparts from BMI-matched non-T2D controls. The contribution of mitochondrial mass, an indicator of non-glycolytic metabolism, to the various metabolic phenotypes is untested. To assess the contribution of immune cell subset identity and mitochondrial mass to the enhanced glycolytic capacity of resting B cells and PBMCs from T2D subjects, we designed a 13-color panel based on standard immune cell subset markers and chemokine receptors, and included MitoTracker Green FM (MTG), which quantitatively indicates mitochondrial mass. We used this novel panel to phenotype 63 total samples from BMI-matched subjects in three groups: non-T2D, pre-T2D, and fulminant T2D, as defined by American Diabetes Association guidelines. The panel was built in several iterations to accommodate spillover of MTG fluorescence into neighboring channels and includes, besides MTG and live-dead discriminator, the following surface markers: CD4, CD8, CD19, CD45RA, CD25, CD127, CD14, CCR4, CCR5, CCR6, CXCR3, and CD161. The PBMC samples were run on a 4-laser BD FACSARIA II SORP with pre-established panel-specific PMT voltages tracked using 6-peak Ultrarainbow beads. To normalize MTG fluorescence intensity and thus minimize batch effects, each of 5 total batches included a reference donor PBMC sample that was frozen in multiple aliquots from one blood draw. Using this approach, we quantified the percentages of immune cell populations (CD19+ B cells, CD8+ naïve and memory/effector T cells, and CD4+ cells including Tregs and populations enriched in Th1, Th2 and Th17) along with the relative mitochondrial mass in each subset. We found that CD19+ B cells in PBMCs from both ND and T2D subjects had significantly less mitochondrial mass than CD4+ cells, supporting the demonstration that B cells are more glycolytic than CD4+ T cells. Of all the CD4+ T cell subsets, Th17 cells consistently had the lowest mitochondrial mass, consistent with the interpretation that Th17s are more dependent on glycolysis than previously appreciated. Our results validate the utility of our 13-color panel to simultaneously quantify relative mitochondrial mass in numerous immune cell subsets and thereby provide a new tool to explore metabolism in human primary cells

Aspiration therapy for the treatment of obesity: 4-year results of a multicenter randomized controlled trial.

BackgroundThe AspireAssist is the first Food and Drug Administration-approved endoluminal device indicated for treatment of class II and III obesity.ObjectivesWe earlier reported 1-year results of the PATHWAY study. Here, we report 4-year outcomes.SettingUnited States-based, 10-center, randomized controlled trial involving 171 participants with the treatment arm receiving Aspiration Therapy (AT) plus Lifestyle Therapy and the control arm receiving Lifestyle Therapy (2:1 randomization).MethodsAT participants were permitted to continue in the study for an additional year up to a maximum of 5 years providing they maintained at least 10% total weight loss (TWL) from baseline at each year end. For AT participants who continued the study, 5 medical monitoring visits were provided at weeks 60, 68, 76, 90, and 104 and thereafter once every 13 weeks up to week 260. Exclusion criteria were a history of eating disorder or evidence of eating disorder on a validated questionnaire. Follow-up weight, quality of life, and co-morbidities were compared with the baseline levels. In addition, rates of serious adverse event, persistent fistula, withdrawal, and A-tube replacement were reported. All analyses were performed using a per-protocol analysis.ResultsOf the 82 AT participants who completed 1 year, 58 continued to this phase of the trial. Mean baseline body mass index of these 58 patients was 41.6 ± 4.5 kg/m2. At the end of first year (at the beginning of the follow-up study), these 58 patients had a body mass index of 34.1 ± 5.4 kg/m2 and had achieved an 18.3 ± 8.0% TWL. On a per protocol basis, patients experienced 14.2%, 15.3%, 16.6%, and 18.7% TWL at 1, 2, 3, and 4 years, respectively (P < .01 for all). Forty of 58 patients (69%) achieved at least 10% TWL at 4 years or at time of study withdrawal. Improvements in quality of life scores and select cardiometabolic parameters were also maintained through 4 years. There were 2 serious adverse events reported in the second through fourth years, both of which resolved with removal or replacement of the A tube. Two persistent fistulas required surgical repair, representing approximately 2% of all tube removals. There were no clinically significant metabolic or electrolytes disorders observed, nor any evidence for development of any eating disorders.ConclusionsThe results of this midterm study have shown that AT is a safe, effective, and durable weight loss alternative for people with class II and III obesity and who are willing to commit to using the therapy and adhere to adjustments in eating behavior

Fatty Acid Metabolites Combine with Reduced β Oxidation to Activate Th17 Inflammation in Human Type 2 Diabetes

Mechanisms that regulate metabolites and downstream energy generation are key determinants of T cell cytokine production, but the processes underlying the Th17 profile that predicts the metabolic status of people with obesity are untested. Th17 function requires fatty acid uptake, and our new data show that blockade of CPT1A inhibits Th17-associated cytokine production by cells from people with type 2 diabetes (T2D). A low CACT:CPT1A ratio in immune cells from T2D subjects indicates altered mitochondrial function and coincides with the preference of these cells to generate ATP through glycolysis rather than fatty acid oxidation. However, glycolysis was not critical for Th17 cytokines. Instead, β oxidation blockade or CACT knockdown in T cells from lean subjects to mimic characteristics of T2D causes cells to utilize 16C-fatty acylcarnitine to support Th17 cytokines. These data show long-chain acylcarnitine combines with compromised β oxidation to promote disease-predictive inflammation in human T2D. Although glycolysis generally fuels inflammation, Nicholas, Proctor, and Agrawal et al. report that PBMCs from subjects with type 2 diabetes use a different mechanism to support chronic inflammation largely independent of fuel utilization. Loss- and gain-of-function experiments in cells from healthy subjects show mitochondrial alterations combine with increases in fatty acid metabolites to drive chronic T2D-like inflammation

Galectin-3 is associated with stage B metabolic heart disease and pulmonary hypertension in young obese patients

Background Obesity is a precursor to heart failure with preserved ejection fraction. Biomarkers that identify preclinical metabolic heart disease ( MHD ) in young obese patients would help identify high-risk individuals for heart failure prevention strategies. We assessed the predictive value of GAL3 (galectin-3), FSTL3 (follistatin-like 3 peptide), and NT-proBNP (N-terminal pro-B-type natriuretic peptide) to identify stage B MHD in young obese participants free of clinically evident cardiovascular disease. Methods and Results Asymptomatic obese patients (n=250) and non-obese controls (n=21) underwent echocardiographic cardiac phenotyping. Obese patients were classified as MHD positive ( MHD - POS ; n=94) if they had abnormal diastolic function or left ventricular hypertrophy and had estimated pulmonary artery systolic pressure ≥35 mm Hg. Obese patients without such abnormalities were classified as MHD negative (MHD-NEG; n=52). Serum biomarkers timed with echocardiography. MHD - POS and MHD-NEG individuals were similarly obese, but MHD - POS patients were older, with more diabetes mellitus and metabolic syndrome. Right ventricular coupling was worse in MHD - POS patients ( P<0.001). GAL 3 levels were higher in MHD - POS versus MHD -NEG patients (7.7±2.3 versus 6.3±1.9 ng/mL, respectively; P<0.001). Both GAL 3 and FSTL 3 levels correlated with diastolic dysfunction and increased pulmonary artery systolic pressure but not with left ventricular mass. In multivariate models including all 3 biomarkers, only GAL 3 remained associated with MHD (odds ratio: 1.30; 95% CI , 1.01-1.68; P=0.04). Conclusions In young obese individuals without known cardiovascular disease, GAL 3 is associated with the presence of preclinical MHD . GAL 3 may be useful in screening for preclinical MHD and identifying individuals with increased risk of progression to obesity-related heart failure with preserved ejection fraction.Deepa M. Gopal, Nir Ayalon, Yi, Chih Wang, Deborah Siwik, Aaron Sverdlov, Courtney Donohue ... et al

Metformin Enhances Autophagy and Normalizes Mitochondrial Function to Alleviate Aging-Associated Inflammation

Age is a non-modifiable risk factor for the inflammation that underlies age-associated diseases; thus, anti-inflammaging drugs hold promise for increasing health span. Cytokine profiling and bioinformatic analyses showed that Th17 cytokine production differentiates CD4+ T cells from lean, normoglycemic older and younger subjects, and mimics a diabetes-associated Th17 profile. T cells from older compared to younger subjects also had defects in autophagy and mitochondrial bioenergetics that associate with redox imbalance. Metformin ameliorated the Th17 inflammaging profile by increasing autophagy and improving mitochondrial bioenergetics. By contrast, autophagy-targeting siRNA disrupted redox balance in T cells from young subjects and activated the Th17 profile by activating the Th17 master regulator, STAT3, which in turn bound IL-17A and F promoters. Mitophagy-targeting siRNA failed to activate the Th17 profile. We conclude that metformin improves autophagy and mitochondrial function largely in parallel to ameliorate a newly defined inflammaging profile that echoes inflammation in diabetes

Joint international consensus statement for ending stigma of obesity

People with obesity commonly face a pervasive, resilient form of social stigma. They are often subject to discrimination in the workplace as well as in educational and healthcare settings. Research indicates that weight stigma can cause physical and psychological harm, and that affected individuals are less likely to receive adequate care. For these reasons, weight stigma damages health, undermines human and social rights, and is unacceptable in modern societies. To inform healthcare professionals, policymakers, and the public about this issue, a multidisciplinary group of international experts, including representatives of scientific organizations, reviewed available evidence on the causes and harms of weight stigma and, using a modified Delphi process, developed a joint consensus statement with recommendations to eliminate weight bias. Academic institutions, professional organizations, media, public-health authorities, and governments should encourage education about weight stigma to facilitate a new public narrative about obesity, coherent with modern scientific knowledge

TGFbeta Family Members Are Key Mediators in the Induction of Myofibroblast Phenotype of Human Adipose Tissue Progenitor Cells by Macrophages

International audienceOBJECTIVE: The present study was undertaken to characterize the remodeling phenotype of human adipose tissue (AT) macrophages (ATM) and to analyze their paracrine effects on AT progenitor cells. RESEARCH DESIGN AND METHODS: The phenotype of ATM, immunoselected from subcutaneous (Sc) AT originating from subjects with wide range of body mass index and from paired biopsies of Sc and omental (Om) AT from obese subjects, was studied by gene expression analysis in the native and activated states. The paracrine effects of ScATM on the phenotype of human ScAT progenitor cells (CD34(+)CD31(-)) were investigated. RESULTS: Two main ATM phenotypes were distinguished based on gene expression profiles. For ScAT-derived ATM, obesity and adipocyte-derived factors favored a pro-fibrotic/remodeling phenotype whereas the OmAT location and hypoxic culture conditions favored a pro-angiogenic phenotype. Treatment of native human ScAT progenitor cells with ScATM-conditioned media induced the appearance of myofibroblast-like cells as shown by expression of both α-SMA and the transcription factor SNAIL, an effect mimicked by TGFβ1 and activinA. Immunohistochemical analyses showed the presence of double positive α-SMA and CD34 cells in the stroma of human ScAT. Moreover, the mRNA levels of SNAIL and SLUG in ScAT progenitor cells were higher in obese compared with lean subjects. CONCLUSIONS: Human ATM exhibit distinct pro-angiogenic and matrix remodeling/fibrotic phenotypes according to the adiposity and the location of AT, that may be related to AT microenvironment including hypoxia and adipokines. Moreover, human ScAT progenitor cells have been identified as target cells for ScATM-derived TGFβ and as a potential source of fibrosis through their induction of myofibroblast-like cells

Tailoring pharmacotherapy to specific eating behaviours in obesity: Can recommendations for personalised therapy be made from the current data?

Pharmacotherapy provides an adjunct to behaviour modification in the management of obesity. There are a number of new drug therapies purportedly targeting appetite; liraglutide, and bupropion/naltrexone, which are European Medicines Agency and US Food and Drug Administration (FDA) approved, and lorcaserin and phentermine/topiramate, which have FDA approval only. Each of the six drugs, used singly or in combination, has distinct pharmacological, and presumably distinct behavioural, mechanisms of action, thus the potential to provide defined therapeutic options to personalise the management of obesity. Yet, with regard to pharmacotherapy for obesity, we are far from true personalised medicine. We review the limited mechanistic data with four mono and combination pharmacotherapies, to assess the potential for tailoring their use to target specific obesogenic behaviours. Potential treatment options are considered, but in the absence of adequate research in respect to effects of these drugs on eating behaviour, neural activity and psychological substrates that underlie poorly controlled eating, we are far from definitive therapeutic recommendations. Specific mechanistic studies and broader behavioural phenotyping, possibly in conjunction with pharmacogenetic research, are required to characterise responders for distinct pharmacotherapeutic options

Endothelial Dysfunction and Specific Inflammation in Obesity Hypoventilation Syndrome

BACKGROUND: Obesity hypoventilation syndrome (OHS) is associated with increased cardiovascular morbidity. What moderate chronic hypoventilation adds to obesity on systemic inflammation and endothelial dysfunction remains unknown. QUESTION: To compare inflammatory status and endothelial function in OHS versus eucapnic obese patients. METHODOLOGY: 14 OHS and 39 eucapnic obese patients matched for BMI and age were compared. Diurnal blood gazes, overnight polysomnography and endothelial function, measured by reactive hyperemia peripheral arterial tonometry (RH-PAT), were assessed. Inflammatory (Leptin, RANTES, MCP-1, IL-6, IL-8, TNFalpha, Resistin) and anti-inflammatory (adiponectin, IL-1Ra) cytokines were measured by multiplex beads immunoassays. PRINCIPAL FINDINGS: OHS exhibited a higher PaCO(2), a lower forced vital capacity (FVC) and tended to have a lower PaO(2) than eucapnic obese patients. (HS)-CRP, RANTES levels and glycated haemoglobin (HbA1c) were significantly increased in OHS (respectively 11.1+/-10.9 vs. 5.7+/-5.5 mg x l(-1) for (HS)-CRP, 55.9+/-55.3 vs 23.3+/-15.8 ng/ml for RANTES and 7.3+/-4.3 vs 6.1+/-1.7 for HbA1c). Serum adiponectin was reduced in OHS (7606+/-2977 vs 13,660+/-7854 ng/ml). Endothelial function was significantly more impaired in OHS (RH-PAT index: 0.22+/-0.06 vs 0.51+/-0.11). CONCLUSIONS: Compared to eucapnic obesity, OHS is associated with a specific increase in the pro-atherosclerotic RANTES chemokine, a decrease in the anti-inflammatory adipokine adiponectin and impaired endothelial function. These three conditions are known to be strongly associated with an increased cardiovascular risk. TRIAL REGISTRATION: ClinicalTrials.gov NCT00603096