White Adipose Tissue Biopsies Research Articles

White adipose tissue remodeling in Little Brown Myotis (Myotis lucifugus) with white-nose syndrome.

White-nose syndrome (WNS) is a fungal wildlife disease of bats that has caused precipitous declines in certain Nearctic bat species. A key driver of mortality is premature exhaustion of fat reserves, primarily white adipose tissue (WAT), that bats rely on to meet their metabolic needs during winter. However, the pathophysiological and metabolic effects of WNS have remained ill-defined. To elucidate metabolic mechanisms associated with WNS mortality, we infected a WNS susceptible species, the Little Brown Myotis (Myotis lucifugus), with Pseudogymnoascus destructans (Pd) and collected WAT biopsies for histology and targeted lipidomics. These results were compared to the WNS-resistant Big Brown Bat (Eptesicus fuscus). A similar distribution in broad lipid class was observed in both species, with total WAT primarily consisting of triacylglycerides. Baseline differences in WAT chemical composition between species showed that higher glycerophospholipids (GPs) levels in E. fuscus were dominated by unsaturated or monounsaturated moieties and n-6 (18:2, 20:2, 20:3, 20:4) fatty acids. Conversely, higher GP levels in M. lucifugus WAT were primarily compounds containing n-3 (20:5 and 22:5) fatty acids. Following Pd-infection, we found that perturbation to WAT reserves occurs in M. lucifugus, but not in the resistant E. fuscus. A total of 66 GPs (primarily glycerophosphocholines and glycerophosphoethanolamines) were higher in Pd-infected M. lucifugus, indicating perturbation to the WAT structural component. In addition to changes in lipid chemistry, smaller adipocyte sizes and increased extracellular matrix deposition was observed in Pd-infected M. lucifugus. This is the first study to describe WAT GP composition of bats with different susceptibilities to WNS and highlights that recovery from WNS may require repair from adipose remodeling in addition to replenishing depot fat during spring emergence.

Interindividual differences of dietary fat-inducible Mest in white adipose tissue of C57BL/6J mice are not heritable.

Differences in white adipose tissue (WAT) expression of mesoderm-specific transcript (Mest) in C57BL6/J mice fed a high-fat diet (HFD) are concomitant with and predictive for the development of obesity. However, the basis for differences in WAT Mest among mice is unknown. This study investigated whether HFD-inducible WAT Mest, as well as susceptibility to obesity, is transmissible from parents to offspring. WAT biopsies of mice fed an HFD for 2 weeks identified parents with low and high WAT Mest for breeding. Obesity phenotypes, WAT Mest, hepatic gene expression, and serum metabolites were determined in offspring fed an HFD for 2 weeks. Offspring showed no heritability of obesity or WAT Mest phenotypes from parents but did show hepatic and serum metabolite changes consistent with their WAT Mest. Importantly, retired male breeders showed WAT Mest expression congruent with initial WAT biopsies even though HFD exposure occurred early in life. Disparity of HFD-induced Mest in mice is not heritable but, rather, is reestablished during each generation and remains fixed from an early age to adulthood. Short-term HFD feeding reveals variation of WAT Mest expression within isogenic mice that is positively associated with the development of obesity.

Inhibition of VEGF-B signaling prevents non-alcoholic fatty liver disease development by targeting lipolysis in the white adipose tissue

Hepatic steatosis is a hallmark of non-alcoholic fatty liver disease (NAFLD), a common comorbidity in type 2 diabetes mellitus (T2DM). The pathogenesis of NAFLD is complex and involves the crosstalk between the liver and the white adipose tissue (WAT). Vascular endothelial growth factor B (VEGF-B) has been shown to control tissue lipid accumulation by regulating the transport properties of the vasculature. The role of VEGF-B signaling and the contribution to hepatic steatosis and NAFLD in T2DM is currently not understood. C57BL/6J mice treated with a neutralizing antibody against VEGF-B, or mice with adipocyte-specific overexpression or under-expression of VEGF-B (AdipoqCre+/VEGF-BTG/+ mice and AdipoqCre+/Vegfbfl/+mice) were subjected to a 6-month high-fat diet (HFD), or chow-diet, whereafter NAFLD development was assessed. VEGF-B expression was analysed in WAT biopsies from patients with obesity and NAFLD in a pre-existing clinical cohort (n= 24 patients with NAFLD and n= 24 without NAFLD) and correlated to clinicopathological features. Pharmacological inhibition of VEGF-B signaling in diabetic mice reduced hepatic steatosis and NAFLD by blocking WAT lipolysis. Mechanistically we show, by using HFD-fed AdipoqCre+/VEGF-BTG/+ mice and HFD-fed AdipoqCre+/Vegfbfl/+mice, that inhibition of VEGF-B signaling targets lipolysis in adipocytes. Reducing VEGF-B signaling ameliorated NAFLD by decreasing WAT inflammation, resolving WAT insulin resistance, and lowering the activity of the hormone sensitive lipase. Analyses of human WAT biopsies from individuals with NAFLD provided evidence supporting the contribution of VEGF-B signaling to NAFLD development. VEGF-B expression levels in adipocytes from two WAT depots correlated with development of dysfunctional WAT and NAFLD in humans. Taken together, our data from mouse models and humans suggest that VEGF-B antagonism may represent an approach to combat NAFLD by targeting hepatic steatosis through suppression of lipolysis. Non-alcoholic fatty liver disease (NAFLD) is a common comorbidity in type 2 diabetes mellitus (T2DM) and has a global prevalence of between 25-29%. There are currently no approved drugs for NAFLD, and given the scale of the ongoing diabetes epidemics, there is an urgent need to identify new treatment options. Our work suggests that VEGF-B antagonism may represent an approach to combat NAFLD by targeting hepatic steatosis through suppression of lipolysis. The neutralizing anti-VEGF-B antibody, which was used in this study, has already entered clinical trials for patients with diabetes. Therefore, we believe that our results are of great general interest to a broad audience, including patients and patient organizations, the medical community, academia, the life science industry and the public.

Sex-specific regulation of IL-10 production in human adipose tissue in obesity.

BackgroundObesity-associated metabolic complications display sexual dimorphism and can be impacted by cytokines. We previously showed that interleukin-10 (IL-10) was upregulated in white adipose tissue (WAT) of obese women with type 2 diabetes (T2D). Whether this pertains to men is unknown. The aim of this study was to compare the impact of obesity and T2D on WAT IL-10 levels in men versus women.MethodsPlasma and subcutaneous WAT biopsies were obtained from 108 metabolically well-characterized individuals. WAT IL10 expression/secretion and WAT-resident IL-10-secreting macrophage number were measured. Circulating sex hormone levels were correlated to WAT IL10 expression in 22 individuals and sex hormone effects on macrophage IL10 expression were investigated in vitro.ResultsObese women with T2D showed increased IL10 expression/secretion and IL-10-secreting WAT macrophage number compared to other female groups. This difference was absent in men. Non-obese women and men with T2D showed similar IL-10 levels compared to healthy controls, indicating that T2D alone does not regulate IL-10. Although WAT IL10 expression correlated with serum estrone (E1) concentrations, recombinant E1 did not affect macrophage IL10 expression in vitro. ConclusionWAT IL-10 levels are higher in women with obesity and T2D, but not in men and this effect is primarily attributed to obesity per se. This is less likely to be driven by circulating sex hormones. We propose that the WAT IL-10 might exert protective effects in obesity-associated chronic inflammation in women which could be one of the contributing factors for the decreased morbidity observed in women during obesity than men.

Increased plasma ANGPTL7 levels with increased obstructive sleep apnea severity.

BackgroundWeight-loss surgery is one of the recommended methods for treating obstructive sleep apnea (OSA) in obese patients. While weight reduction is critical to relieve symptoms of OSA, the biochemical factors involved in post-surgery improvement are still unknown. We aimed to explore the link between ANGPTL7 and OSA in patients with different OSA severity. Furthermore, we examined the effect of treating OSA with bariatric surgery on ANGPTL7 level.MethodsWe quantified levels of circulating ANGPTL7 in fasting plasma and adipose tissue samples of 88 participants before and after bariatric surgery. Confocal microscopy analyses were also performed to assess the ANGPTL7 expression in subcutaneous white adipose tissue biopsies obtained from people with moderate-to-severe OSA compared to those with none or mild OSA. The study involved 57 individuals with none or mild OSA and 31 patients with moderate-to-severe OSA.ResultsLevels of circulating ANGPTL7 were significantly higher in people with moderate-to-severe OSA (1440 ± 1310 pg/ml) compared to the none or mild OSA group (734 ± 904 pg/ml, p = 0.01). The increase in ANGPTL7 correlated significantly and positively with the apnea-hypopnea index (AHI, r = .226, p = .037), and AHI-supine (r = .266, p = .019) in participants with moderate-to-severe OSA. Multivariate logistic regression analysis demonstrated an association between ANGPTL7 and OSA severity (log2 ANGPTL7; OR =1.24, p = 0.024). ANGPTL7 levels exhibited significant positive correlations with the levels of TG and oxLDL (p-value = 0.002 and 0.01 respectively). Bariatric surgery reduced the levels of both ANGPTL7 and AHI significantly.ConclusionHere we report significantly increased levels of ANGPTL7 both in the circulation and in adipose tissue of patients with OSA, which concurred with increased inflammation and OSA severity. Levels of ANGPTL7 decreased significantly as OSA showed a significant improvement post-surgery supporting a potential role for ANGPTL7 in either OSA progression or a role in an OSA-related mechanism.

77-OR: Native LDL Upregulate the NLRP3 Inflammasome in Human Adipose Tissue: A Mechanism for Higher Risk for Type 2 Diabetes in Subjects with HyperapoB

Background and Hypothesis: Elevated plasma apoB-lipoproteins (hyperapoB, mostly LDL) predicts the incidence of type 2 diabetes (T2D) and its risk factors including systemic activation of the interleukin-1 system (measured as plasma IL-1 receptor antagonist, IL-1Ra) . Moreover, native LDL induce white adipose tissue (WAT) dysfunction. Underlying mechanisms for these findings remain unclear. Activation of WAT NLRP3 inflammasome and secretion of IL-1β promotes WAT dysfunction and related risk factors for T2D. We hypothesized that native LDL upregulate WAT NLRP3 inflammasome/IL-1β pathway. Methods: This data represents baseline analysis of a 3-month clinical trial with omega-3 fatty acid supplementation in nondiabetic men (N=13) and women (N=27) . We assessed insulin secretion and sensitivity by Botnia clamps, postprandial fat metabolism after a high-fat-meal (68% fat) , and priming and/or activation of NLRP3 inflammasome in subject hip WAT biopsies ex vivo using native LDL, lipopolysaccharides (LPS) (priming control) and/or ATP (activation control) . Results: Incubation of WAT with native LDL (1.2 g apoB/L) increased IL-1β secretion in ATP- but not LPS-treated WAT. Subjects with higher than median plasma apoB (1.23±0.15 g/L) had higher WAT IL-1β-secretion than those with lower apoB, using multiple combination of LDL, LPS and/or ATP, independent of adiposity, macronutrient intake or plasma lipids. WAT NLRP3 mRNA expression and IL-1β secretion correlated positively with hyperinsulinemia, insulin resistance, postprandial hypertriglyceridemia and hyperchylomicronemia and plasma IL-1Ra. Adjusting for sex and WAT IL-1β-secretion or sex and plasma IL-1Ra eliminated the association of plasma apoB with these risk factors (except hypertriglyceridemia) . Conclusion: Native LDL are priming signals of WAT NLRP3 inflammasome, which may explain higher risk for T2D in subjects with hyperapoB. ClinicalTrials.gov (NCT04496154) . Disclosure S.Bissonnette: None. V.Lamantia: None. Y.Cyr: None. M.Devaux: None. R.Rabasa-lhoret: Consultant; HLS Therapeutics Inc., Pfizer Inc., Other Relationship; Abbott Diabetes, AstraZeneca, Boehringer Ingelheim International GmbH, Dexcom, Inc., Eli Lilly and Company, Insulet Corporation, Janssen Pharmaceuticals, Inc., Medtronic, Merck & Co., Inc., Novo Nordisk Canada Inc., Sanofi, Vertex Pharmaceuticals Incorporated, Research Support; Canadian Institutes of Health Research, Cystic Fibrosis Canada, Diabetes Canada, Fondation Francophone pour la Recherche en Diabète (FFRD) , JDRF, National Institutes of Health, Société Francophone du Diabète (SFD) , Speaker's Bureau; Canadian Medical & Surgical Knowledge Translation Research Group (CMS) , CPD Network, Tandem Diabetes Care, Inc. M.Chretien: None. M.Saleh: None. M.Faraj: None. Funding Canadian Institutes of Health Research (CIHR) (FRN# 123409)

Ameliorating Effects of Lifelong Physical Activity on Healthy Aging and Mitochondrial Function in Human White Adipose Tissue.

Growing old is patently among the most prominent risk factors for lifestyle-related diseases and deterioration in physical performance. Aging in particular affects mitochondrial homeostasis, and maintaining a well-functioning mitochondrial pool is imperative in order to avoid age-associated metabolic decline. White adipose tissue (WAT) is a key organ in energy balance, and impaired mitochondrial function in adipocytes has been associated with increased low-grade inflammation, altered metabolism, excessive reactive oxygen species (ROS) production, and an accelerated aging phenotype. Exercise training improves mitochondrial health but whether lifelong exercise training can sufficiently maintain WAT mitochondrial function is currently unknown. Therefore, to dissect the role and dose-dependence of lifelong exercise training on aging WAT metabolic parameters and mitochondrial function, young and older untrained, as well as moderately and highly exercise trained older male subjects were recruited and abdominal subcutaneous (s)WAT biopsies and venous blood samples were obtained to measure mitochondrial function and key metabolic factors in WAT and plasma. Mitochondrial intrinsic respiratory capacity was lower in sWAT from older than from young subjects. In spite of this, maximal mitochondrial respiration per wet weight, markers of oxidative capacity, and mitophagic capacity were higher in sWAT from the lifelong highly exercise trained group than all other groups. Furthermore, ROS emission was generally lower in sWAT from lifelong highly exercise trained subjects than older untrained subjects. Taken together, aging reduces intrinsic mitochondrial respiration in human sWAT, but lifelong high-volume exercise training increases oxidative capacity by increasing mitochondrial volume likely contributing to healthy aging.

Oncostatin M suppresses browning of white adipocytes via gp130-STAT3 signaling

ObjectiveObesity is associated with low-grade adipose tissue inflammation and locally elevated levels of several glycoprotein 130 (gp130) cytokines. The conversion of white into brown-like adipocytes (browning) may increase energy expenditure and revert the positive energy balance that underlies obesity. Although different gp130 cytokines and their downstream targets were shown to regulate expression of the key browning marker uncoupling protein 1 (Ucp1), it remains largely unknown how this contributes to the development and maintenance of obesity. Herein, we aim to study the role of gp130 cytokine signaling in white adipose tissue (WAT) browning in the obese state. MethodsProtein and gene expression levels of UCP1 and other thermogenic markers were assessed in a subcutaneous adipocyte cell line, adipose tissue depots from control or adipocyte-specific gp130 knockout (gp130Δadipo) mice fed either chow or a high-fat diet (HFD), or subcutaneous WAT biopsies from a human cohort of lean and obese subjects. WAT browning was modeled in vitro by exposing mature adipocytes to isoproterenol after stimulation with gp130 cytokines. ERK and JAK-STAT signaling were blocked using the inhibitors U0126 and Tofacitinib, respectively. ResultsInguinal WAT of HFD-fed gp130Δadipo mice exhibited significantly elevated levels of UCP1 and other browning markers such as Cidea and Pgc-1α. In vitro, treatment with the gp130 cytokine oncostatin M (OSM) lowered isoproterenol-induced UCP1 protein and gene expression levels in a dose-dependent manner. Mechanistically, OSM mediated the inhibition of Ucp1 via the JAK-STAT but not the ERK pathway. As with mouse data, OSM gene expression in human WAT positively correlated with BMI (r = 0.284, p = 0.021, n = 66) and negatively with UCP1 expression (r = −0.413, p < 0.001, n = 66). ConclusionsOur data support the notion that OSM negatively regulates thermogenesis in WAT and thus may be an attractive target for treating obesity.

Dysregulation of endocannabinoid concentrations in human subcutaneous adipose tissue in obesity and modulation by omega-3 polyunsaturated fatty acids.

Obesity is believed to be associated with a dysregulated endocannabinoid system which may reflect enhanced inflammation. However, reports of this in human white adipose tissue (WAT) are limited and inconclusive. Marine long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) have anti-inflammatory actions and therefore may improve obesity-associated adipose tissue inflammation. Therefore, fatty acid (FA) concentrations, endocannabinoid concentrations, and gene expression were assessed in subcutaneous WAT (scWAT) biopsies from healthy normal weight individuals (BMI 18.5-25 kg/m2) and individuals living with metabolically healthy obesity (BMI 30-40 kg/m2) prior to and following a 12-week intervention with 3 g fish oil/day (1.1 g eicosapentaenoic acid (EPA) + 0.8 g DHA) or 3 g corn oil/day (placebo). WAT from individuals living with metabolically healthy obesity had higher n-6 PUFAs and EPA, higher concentrations of two endocannabinoids (anandamide (AEA) and eicosapentaenoyl ethanolamide (EPEA)), higher expression of phospholipase A2 Group IID (PLA2G2D) and phospholipase A2 Group IVA (PLA2G4A), and lower expression of CNR1. In response to fish oil intervention, WAT EPA increased to a similar extent in both BMI groups, and WAT DHA increased by a greater extent in normal weight individuals. WAT EPEA and docosahexaenoyl ethanolamide (DHEA) increased in normal weight individuals only and WAT 2-arachidonyl glycerol (2-AG) decreased in individuals living with metabolically healthy obesity only. Altered WAT fatty acid, endocannabinoid, and gene expression profiles in metabolically healthy obesity at baseline may be linked. WAT incorporates n-3 PUFAs when their intake is increased which affects the endocannabinoid system; however, effects appear greater in normal weight individuals than in those living with metabolically healthy obesity.

Effects of High-Intensity Exercise Training on Adipose Tissue Mass, Glucose Uptake and Protein Content in Pre- and Post-menopausal Women.

The menopausal transition is accompanied by changes in adipose tissue storage, leading to an android body composition associated with increased risk of type 2 diabetes and cardiovascular disease in post-menopausal women. Estrogens probably affect local adipose tissue depots differently. We investigated how menopausal status and exercise training influence adipose tissue mass, adipose tissue insulin sensitivity and adipose tissue proteins associated with lipogenesis/lipolysis and mitochondrial function. Healthy, normal-weight pre- (n = 21) and post-menopausal (n = 20) women participated in high-intensity exercise training three times per week for 12 weeks. Adipose tissue distribution was determined by dual-energy x-ray absorptiometry and magnetic resonance imaging. Adipose tissue glucose uptake was assessed by positron emission tomography/computed tomography (PET/CT) by the glucose analog [18F]fluorodeoxyglucose ([18F]FDG) during continuous insulin infusion (40 mU·m−2·min−1). Protein content associated with insulin signaling, lipogenesis/lipolysis, and mitochondrial function were determined by western blotting in abdominal and femoral white adipose tissue biopsies. The mean age difference between the pre- and the post-menopausal women was 4.5 years. Exercise training reduced subcutaneous (~4%) and visceral (~6%) adipose tissue masses similarly in pre- and post-menopausal women. Insulin-stimulated glucose uptake, assessed by [18F]FDG-uptake during PET/CT, was similar in pre- and post-menopausal women in abdominal, gluteal, and femoral adipose tissue depots, despite skeletal muscle insulin resistance in post- compared to pre-menopausal women in the same cohort. Insulin-stimulated glucose uptake in adipose tissue depots was not changed after 3 months of high-intensity exercise training, but insulin sensitivity was higher in visceral compared to subcutaneous adipose tissue depots (~139%). Post-menopausal women exhibited increased hexokinase and adipose triglyceride lipase content in subcutaneous abdominal adipose tissue. Physical activity in the early post-menopausal years reduces abdominal obesity, but insulin sensitivity of adipose tissue seems unaffected by both menopausal status and physical activity.

1712-P: HIV-1 Vpr Couples Metabolic Inflexibility with White Adipose Tissue Thermogenesis

The HIV accessory protein Vpr contributes to cardinal metabolic defects noted in persons living with HIV (PLWH), including accelerated lipolysis, impaired fatty acid oxidation, slowed fatty acid export, and steatohepatitis (Agarwal et al, Sci Trans Med 2017). PLWH also manifest “beiging” and markers of thermogenesis within some white adipose tissue (WAT) depots, but paradoxically these changes correlate with decreased insulin sensitivity and hyperlipidemia. We sought to determine whether Vpr activity affects thermogenesis and the dynamics of beiging in two Vpr mouse models. UCP1 expression was strongly increased in subcutaneous fat of Vpr mice and showed equivalent copy number in WAT biopsies from PLWH. Histology confirmed browning of subcutaneous but not visceral WAT in the Vpr mouse models. Adipocytes derived from these depots showed increased oxygen consumption rate in subcutaneous but not visceral fat cells, also strongly indicative of beiging. Measurements of energy balance indicated Vpr mice display metabolic inflexibility and cannot shift efficiently from carbohydrates to fat during day-night cycles. Furthermore, Vpr mice mice showed a marked inability to defend body temperature when exposed to 4oC for 6 hours. Thus HIV-1 Vpr disrupts metabolic efficiency coupled with maladaptive subcutaneous WAT beiging. These data suggest an expanded role for Vpr in the pathogenesis of HIV-associated metabolic disorders. Disclosure N. Agarwal: None. P. Saha: None. S.M. Hartig: None. A. Balasubramanyam: None. Funding National Institutes of Health (R21/R33AI116208)

Short-Term Caloric Restriction Attenuates Obesity-Induced Pro-Inflammatory Response in Male Rhesus Macaques.

White adipose tissue (WAT) hypertrophy is an essential hallmark of obesity and is associated with the activation of resident immune cells. While the benefits of caloric restriction (CR) on health span are generally accepted, its effects on WAT physiology are not well understood. We previously demonstrated that short-term CR reverses obesity in male rhesus macaques exposed to a high-fat Western-style diet (WSD). Here, we analyzed subcutaneous WAT biopsies collected from this cohort of animals before and after WSD and following CR. This analysis showed that WSD induced adipocyte hypertrophy and inhibited β-adrenergic-simulated lipolysis. CR reversed adipocyte hypertrophy, but WAT remained insensitive to β-adrenergic agonist stimulation. Whole-genome transcriptional analysis revealed that β3-adrenergic receptor and de novo lipogenesis genes were downregulated by WSD and remained downregulated after CR. In contrast, WSD-induced pro-inflammatory gene expression was effectively reversed by CR. Furthermore, peripheral blood monocytes isolated during the CR period exhibited a significant reduction in the production of pro-inflammatory cytokines compared to those obtained after WSD. Collectively, this study demonstrates that short-term CR eliminates an obesity-induced pro-inflammatory response in WAT and peripheral monocytes.

Short-term dietary reduction of branched-chain amino acids reduces meal-induced insulin secretion and modifies microbiome composition in type 2 diabetes: a randomized controlled crossover trial

ABSTRACTBackgroundEpidemiological studies have shown that increased circulating branched-chain amino acids (BCAAs) are associated with insulin resistance and type 2 diabetes (T2D). This may result from altered energy metabolism or dietary habits.ObjectiveWe hypothesized that a lower intake of BCAAs improves tissue-specific insulin sensitivity.MethodsThis randomized, placebo-controlled, double-blinded, crossover trial examined well-controlled T2D patients receiving isocaloric diets (protein: 1 g/kg body weight) for 4 wk. Protein requirements were covered by commercially available food supplemented ≤60% by an AA mixture either containing all AAs or lacking BCAAs. The dietary intervention ensured sufficient BCAA supply above the recommended minimum daily intake. The patients underwent the mixed meal tolerance test (MMT), hyperinsulinemic-euglycemic clamps (HECs), and skeletal muscle and white adipose tissue biopsies to assess insulin signaling.ResultsAfter the BCAA− diet, BCAAs were reduced by 17% during fasting (P < 0.001), by 13% during HEC (P < 0.01), and by 62% during the MMT (P < 0.001). Under clamp conditions, whole-body and hepatic insulin sensitivity did not differ between diets. After the BCAA− diet, however, the oral glucose sensitivity index was 24% (P < 0.01) and circulating fibroblast-growth factor 21 was 21% higher (P < 0.05), whereas meal-derived insulin secretion was 28% lower (P < 0.05). Adipose tissue expression of the mechanistic target of rapamycin was 13% lower, whereas the mitochondrial respiratory control ratio was 1.7-fold higher (both P < 0.05). The fecal microbiome was enriched in Bacteroidetes but depleted of Firmicutes.ConclusionsShort-term dietary reduction of BCAAs decreases postprandial insulin secretion and improves white adipose tissue metabolism and gut microbiome composition. Longer-term studies will be needed to evaluate the safety and metabolic efficacy in diabetes patients.This trial was registered at clinicaltrials.gov as NCT03261362.

Adipose tissue mitochondrial dysfunction in human obesity is linked to a specific DNA methylation signature in adipose-derived stem cells

BackgroundA functional population of adipocyte precursors, termed adipose-derived stromal/stem cells (ASCs), is crucial for proper adipose tissue (AT) expansion, lipid handling, and prevention of lipotoxicity in response to chronic positive energy balance. We previously showed that obese human subjects contain a dysfunctional pool of ASCs. Elucidation of the mechanisms underlying abnormal ASC function might lead to therapeutic interventions for prevention of lipotoxicity by improving the adipogenic capacity of ASCs.MethodsUsing epigenome-wide association studies, we explored the impact of obesity on the methylation signature of human ASCs and their differentiated counterparts. Mitochondrial phenotyping of lean and obese ASCs was performed. TBX15 loss- and gain-of-function experiments were carried out and western blotting and electron microscopy studies of mitochondria were performed in white AT biopsies from lean and obese individuals.ResultsWe found that DNA methylation in adipocyte precursors is significantly modified by the obese environment, and adipogenesis, inflammation, and immunosuppression were the most affected pathways. Also, we identified TBX15 as one of the most differentially hypomethylated genes in obese ASCs, and genetic experiments revealed that TBX15 is a regulator of mitochondrial mass in obese adipocytes. Accordingly, morphological analysis of AT from obese subjects showed an alteration of the mitochondrial network, with changes in mitochondrial shape and number.ConclusionsWe identified a DNA methylation signature in adipocyte precursors associated with obesity, which has a significant impact on the metabolic phenotype of mature adipocytes.

OP-18 - Analytical strategies for the identification of oxidized lipids from adipose tissue

Obesity has reached epidemic proportions in modern societies with a prevalence of more than 20% of the population and has been recognized as a risk factor for numerous metabolic disorders including type 2 diabetes, cardiometabolic, liver and renal diseases. Onset and disease progression is closely associated with metabolic reconfiguration of white adipose tissue (WAT) in which a constant low-level inflammation in obese WAT may lead to protein and lipid oxidation altering cellular signaling. Oxidized lipids represent a complex class of cellular signaling mediators that are, due to their complexity and wide dynamic range, difficult to analyze. In order to understand the role of oxidized lipids in WAT pathology several lipid extraction and fractionation methods were used on WAT biopsies followed by untargeted LC-QTOF-MS/MS analysis. With the help of in-house developed LPPtiger software ( https://bitbucket.org/SysMedOs/lpptiger ), WAT native lipidome is further used to predict and identify oxidized lipid species formed under pro-inflammatory conditions associated with obesity. Identified oxidized lipids will be further implemented into WAT specific metabolic networks to understand the role of lipid redox mediators in adipose tissue dysfunction.

Effects of insulin and exercise training on FGF21, its receptors and target genes in obesity and type 2 diabetes.

Pharmacological doses of FGF21 improve glucose tolerance, lipid metabolism and energy expenditure in rodents. Induced expression and secretion of FGF21 from muscle may increase browning of white adipose tissue (WAT) in a myokine-like manner. Recent studies have reported that insulin and exercise increase FGF21 in plasma. Obesity and type 2 diabetes are potentially FGF21-resistant states, but to what extent FGF21 responses to insulin and exercise training are preserved, and whether FGF21, its receptors and target genes are altered, remains to be established. The effects of insulin during euglycaemic-hyperinsulinaemic clamps and 10week endurance training on serum FGF21 were examined in individuals with type 2 diabetes and in glucose tolerant overweight/obese and lean individuals. Gene expression of FGF21, its receptors and target genes in muscle and WAT biopsies was evaluated by quantitative real-time PCR (qPCR). Insulin increased serum and muscle FGF21 independent of overweight/obesity or type 2 diabetes, and there were no effects associated with exercise training. The insulin-induced increases in serum FGF21 and muscle FGF21 expression correlated tightly (p<0.001). In WAT, overweight/obesity with and without type 2 diabetes led to reduced expression of KLB, but increased FGFR1c expression. However, the expression of most FGF21 target genes was unaltered except for reduced CIDEA expression in individuals with type 2 diabetes. Insulin-induced expression of muscle FGF21 correlates strongly with a rise in serum FGF21, and this response appears intact in overweight/obesity and type 2 diabetes. FGF21 resistance may involve reduced KLB expression in WAT. However, increased FGFR1c expression or other mechanisms seem to ensure adequate expression of most FGF21 target genes in WAT.

The impact of protein quantity during energy restriction on genome-wide gene expression in adipose tissue of obese humans.

Overweight and obesity is a growing health problem worldwide. The most effective strategy to reduce weight is energy restriction (ER). ER has been shown to be beneficial in disease prevention and it reduces chronic inflammation. Recent studies suggest that reducing the protein quantity of a diet contributes to the beneficial effects by ER. The organ most extensively affected during ER is white adipose tissue (WAT). The first objective was to assess changes in gene expression between a high-protein diet and a normal protein diet during ER. Second, the total effect of ER on changes in gene expression in WAT was assessed. In a parallel double-blinded controlled study, overweight older participants adhered to a 25% ER diet, either combined with high-protein intake (HP-ER, 1.7 g kg-1 per day), or with normal protein intake (NP-ER, 0.9 g kg-1 per day) for 12 weeks. From 10 HP-ER participants and 12 NP-ER participants subcutaneous WAT biopsies were collected before and after the diet intervention. Adipose tissue was used to isolate total RNA and to evaluate whole-genome gene expression changes upon a HP-ER and NP-ER diet. A different gene expression response between HP-ER and NP-ER was observed for 530 genes. After NP-ER, a downregulation in expression of genes linked to immune cell infiltration, adaptive immune response and inflammasome was found, whereas no such effect was found after HP-ER. HP-ER resulted in upregulation in expression of genes linked to cell cycle, GPCR signalling, olfactory signalling and nitrogen metabolism. Upon 25% ER, gene sets related to energy metabolism and immune response were decreased. Based on gene expression changes, we concluded that consumption of normal protein quantity compared with high-protein quantity during ER has a more beneficial effect on inflammation-related gene expression in WAT.

Erythropoietin does not activate erythropoietin receptor signaling or lipolytic pathways in human subcutaneous white adipose tissue in vivo.

BackgroundErythropoietin (Epo) exerts direct effects on white adipose tissue (WAT) in mice in addition to its erythropoietic effects, and in humans Epo increases resting energy expenditure and affect serum lipid levels, but direct effects of Epo in human WAT have not been documented. We therefore investigated the effects of acute and prolonged Epo exposure on human WAT in vivo.MethodData were obtained from two clinical trials: 1) acute Epo exposure (rHuEpo, 400 IU/kg) followed by WAT biopsies after 1 h and 2) 10 weeks treatment with the erythropoiesis-stimulating agent (ESA) Darbepoietin-alpha. Biopsies were analyzed by PCR for Epo receptor (Epo-R) mRNA. A new and highly specific antibody (A82, Amgen) was used to evaluate the presence of Epo-R by western blot analysis in addition to Epo-R signaling proteins (Akt, STAT5, p70s6k, LYN, and p38MAPK), activation of lipolytic pathways (ATGL, HSL, CGI-58, G0S2, Perilipin, Cidea, Cidec, AMPK, and ACC), and mitochondrial biogenesis (VDAC, HSP90, PDH, and SDHA).ResultsNo evidence of in vivo activation of the Epo-R in WAT could be documented despite detectable levels of Epo-R mRNA.ConclusionThus, in contradiction to animal studies, Epo treatment within a physiological relevant range in humans does not exert direct effects in a subcutaneous WAT.

Isolation and Culture of Human Adipose-derived Stem Cells from Subcutaneous and Visceral White Adipose Tissue Compartments

Human Adipose-derived Stem/Stromal Cells (ASCs) have been widely used in stem cell and obesity research, as well as clinical applications including cell-based therapies, tissue engineering and reconstruction. Compared with mesenchymal stem cells (MSCs) derived from other tissues such as umbilical cord and bone marrow, isolation of ASCs from human white adipose tissue (WAT) has great advantages due to its rich tissue source and simple surgical procedure. In this detailed protocol we describe a protocol to isolate and characterize ASCs from human WAT. Molecular characterization of isolated ASCs was performed through surface marker expression profiling using flow cytometry. Adipogenic capacity of the isolated ASCs was confirmed through inducing adipogenic differentiation and Oil Red O staining of lipid. This protocol provides researchers with the tools to culture and assess purity and adipogenic differentiation capacity of human ASCs, which can then be utilized for required downstream in vitro applications. This protocol has been modified from Baglioni et al. (2009), Baglioni et al. (2012), and van Harmelen et al. (2005) to describe in detail a complete technique to isolate and subsequently characterize human ASCs from human WAT biopsies. This protocol has been utilized to isolate and characterize human ASCs from both subcutaneous and visceral WAT. The isolated human ASCs show high purity and demonstrate adipogenic differentiation capacity in vitro.

The apoB-to-PCSK9 ratio: A new index for metabolic risk in humans

Proprotein convertase subtilisin/kexin type 9 (PCSK9) shuttles low-density lipoprotein (LDL) receptors for degradation, thus upregulates LDL plasma clearance. Although PCSK9 loss of function is cardioprotective, its role in metabolic risks remains unknown. Increased apoB-lipoproteins uptake into nonhepatic tissues such as white adipose tissue (WAT) induces their dysfunction, which may be favored by lower plasma PCSK9. We hypothesized that lower plasma PCSK9 relative to apoB, or higher apoB-to-PCSK9 ratio, is a better predictor of metabolic disturbances than PCSK9 alone in humans. Thirty-three men and 48 postmenopausal women (>27 kg/m(2), aged 45-74 years, normoglycemic) underwent in-depth assessment of glucose and fat metabolism using high-fat meals, WAT biopsies, intravenous glucose-tolerance tests, and hyperinsulinemia clamps. Plasma apoB correlated positively with fasting and postprandial triglycerides and chylomicron clearance (R = 0.44-0.66) and glucose-stimulated insulin secretion (R = 0.24) and negatively with insulin sensitivity (R = -0.28) and gynoid WAT in situ lipoprotein lipase activity (ie, ex vivo WAT function, R(2) = 0.34). Neither PCSK9 nor LDL cholesterol associated with these risks. In regression analysis that adjusted for body mass index, lower plasma PCSK9 strengthened the association of apoB to WAT dysfunction and insulin resistance. Moreover, plasma apoB-to-PCSK9 ratio correlated positively with all these metabolic risks and further associated positively with android-to-gynoid fat ratio (R = 0.41) and negatively with gynoid fat mass (R = -0.23, all P ≤ .05). No significant sex differences existed in these associations. Lower plasma PCSK9 relative to apoB associates with metabolic risks and WAT dysfunction in normoglycemic obese subjects. We hypothesize that the plasma apoB-to-PCSK9 ratio provides a better clinical index than PCSK9 alone for monitoring early metabolic disturbances that may be promoted by reduction in plasma PCSK9.