Human White Adipocytes Research Articles

Atrial Natriuretic Peptide Orchestrates a Coordinated Physiological Response to Fuel Non-shivering Thermogenesis

Atrial natriuretic peptide (ANP) is a cardiac hormone controlling blood volume and pressure in mammals. It is still unclear whether ANP controls cold-induced thermogenesis invivo. Here, we show that acute cold exposure induces cardiac ANP secretion in mice and humans. Genetic inactivation of ANP promotes cold intolerance and suppresses half of cold-induced brown adipose tissue (BAT) activation in mice. While white adipocytes are resistant to ANP-mediated lipolysis at thermoneutral temperature in mice, cold exposure renders white adipocytes fully responsive to ANP to activate lipolysis and a thermogenic program, a physiological response that is dramatically suppressed in ANP null mice. ANP deficiency also blunts liver triglycerides and glycogen metabolism, thus impairing fuel availability for BAT thermogenesis. ANP directly increases mitochondrial uncoupling and thermogenic gene expression in human white and brown adipocytes. Together, these results indicate that ANP is a major physiological trigger of BAT thermogenesis upon cold exposure in mammals.

208-LB: Stimulation of the ß3 Adrenergic Receptors Prime Human White and Brown Adipocytes for Increased Lipolysis and Thermogenesis

Recent clinical trials show that chronic activation of the β3-adrenergic receptor (AR) by mirabegron leads to numerous beneficial physiological responses, including increased energy expenditure, insulin sensitivity, and improved lipoprotein profile. Preliminary evidence suggests that they are mediated in part through adaptations by adipose tissue, but little is known about how mirabegron affects the cellular metabolism of human white or brown adipocytes (hWA and BA, respectively). We therefore treated paired immortalized hWA and hBA for 6 hours with a range of mirabegron doses (1 nM - 10 µM), as well as the β1- and β2-AR selective agonists, dobutamine and terbutaline, at 10 µM. We found that in untreated cells, ADRB1 and ADRB2 were higher in hWA than hBA, but ADRB3 was higher in hBA. However, mirabegron at all concentrations increased the expression of ADRB3 in hWA, and the highest dose of mirabegron boosted expression above that in hBA. While an increase in ADRB3 was detected in hWA with β1- and β2-agonist treatment, no changes occurred to the other two β-ARs when exposed to their selective agonists. Downstream, the β1- and β2-agonists increased thermogenic UCP1 expression in hWA, but mirabegron raised UCP1 expression only in hBA. Among the genes associated with lipolysis, glucose metabolism, and insulin signaling, mirabegron 10 µM promoted the expression of ATGL and HSL encoding genes, as well as GLUT4 in hBA only. In contrast, no changes occurred in hWA. In summary, chronic exposure to a β3-AR agonist primes human white adipocytes to respond more potently to adrenergic-mediated lipolysis and enables human brown adipocytes to increase their thermogenic capacity. Disclosure H.J. Lea: None. K.Y. Zhu: None. C. Cero: None. A.M. Cypess: None.

Indomethacin promotes browning and brown adipogenesis in both murine and human fat cells.

Indomethacin (Indo), a nonsteroidal antiinflammatory drug, has been shown to promote murine brown adipogenesis both in vitro and in vivo, possibly due to its peroxisome proliferator‐activated receptor gamma (PPARγ)‐agonist activities. However, it is unclear whether Indo induces browning of white adipocytes from both murine and human origins or induces human brown adipogenesis. To bridge the gap, this study investigated the effects of increasing concentrations of Indo on murine 3T3‐L1, human primary subcutaneous white adipocytes (HPsubQ), and human brown (HBr) adipocytes. The results show that Indo dose‐dependently enhanced 3T3‐L1 adipocyte differentiation and upregulated both mRNA and protein expression of brown and beige adipocyte markers, while simultaneously suppressing white adipocyte‐specific marker mRNA expression. mRNA and protein expression of mitochondrial biogenesis and structural genes were dose‐dependently enhanced in Indo treated 3T3‐L1 adipocytes. This was accompanied by augmented mitochondrial DNA, enhanced oxygen consumption, proton leak, and maximal and spare respiratory capacity. Dose‐dependent transactivation of PPARγ confirmed Indo's PPARγ‐agonist activity in 3T3‐L1 cells. Knockdown of PPARγ significantly attenuated Indo's activities in selective browning genes, demonstrating PPARγ dependence of these effects. Moreover, Indo enhanced mRNA and protein expression of brown markers in HPsubQ adipocytes. Interestingly, Indo‐induced differential effects on individual PPARγ isoforms with significant dose‐dependent induction of PPARγ‐2 and suppression of PPARγ‐1 protein expression. Finally, Indo significantly promoted brown adipogenesis in HBr cells. Taken together, these results demonstrate Indo to be a potent thermogenic compound in both murine and human fat cells and may be explored as a therapeutic agent for obesity treatment and prevention.

MON-596 Effects of Angiotensin Type 1 Receptor Blockers (ARBs) on the Expression and Secretion of Adiponectin and Leptin in Human White Adipocytes

[Introduction]Adiponectin and leptin are adipokines that are mainly produced in adipocytes and exert various functions. Adiponectin decreases atherosclerosis, oxidative stress, angiogenesis, inflammation, and apoptosis, whereas leptin works oppositely. Angiotensin type-1 receptor (AT1R) blockers (ARBs) are widely used as antihypertensive drugs. Some ARBs are known to activate peroxisome proliferator-activated receptor (PPAR) γ, which is a key regulator of fatty acid metabolism. It is reported that adiponectin secretion increases by pioglitazone, a full agonist of PPARγ, and some ARBs via PPAR γ activation. However, the effects of ARBs on leptin secretion are controversial. The present study aimed to examine the effects of ARBs on the expression and secretion of adiponectin and leptin in human white adipocytes.[Materials and Methods]Human white preadipocytes (Promo Cell) were differentiated into mature adipocytes in the medium containing insulin, dexamethasone, thyroxin and isobutylmethylxanthine. Pioglitazone and ARBs including telmisartan, irbesartan, azilsartan, candesartan, losartan, olmesartan and valsartan (1µM) were administered in the culture medium on day 4 and 8. The medium was collected on day 12 and the concentrations of adiponectin and leptin were measured by enzyme immunoassay. Real time PCR was performed to quantitate the mRNA expression of adiponectin and leptin in adipocytes. The experiments were performed in quadruplicate.[Results]Pioglitazone significantly increased adiponectin secretion (386.7 ± 133.7 vs. 7.3 ± 1.9 ng/ml in control) from human adipocytes. Among ARBs, adiponectin secretion significantly increased by telmisartan (136.7 ± 16.3 ng/ml) and irbesartan (69.7 ± 23.1 ng/ml), while the other 5 ARBs did not have any influence on adiponectin secretion. Real-time PCR also showed that mRNA expression increased 5.1-fold, 3.8-fold and 1.5-fold by pioglitazone, telmisartan and irbesartan, respectively. Leptin secretion significantly decreased by pioglitazone (27.7 ± 5.0 vs. 82.5 ± 3.8 ng/ml in control). Among ARBs, only telmisartan (38.7 ± 4.2 ng/ml) decreased leptin secretion. Real-time PCR also showed that mRNA expression decreased to be 0.5-fold and 0.7-fold by pioglitazone and telmisartan, respectively. GW9662, a selective antagonist of PPARγ, potently blocked pioglitazone-induced changes of adiponectin and leptin expression and secretion. On the other hand, GW9662 did not reverse telmisartan and irbesartan induced changes.[Conclusion]The changes in adiponectin and leptin secretion by pioglitazone are via PPARγ activation, while those by telmisartan and irbesartan may occur in PPARγ-independent manner.

SUN-746 Thyroid Hormone Receptor Alpha Sumoylation Is Important for the Development of Adipose Tissue

Thyroid hormone (TH) plays an essential role in normal development. TH action requires binding to its receptor. There are two types of thyroid hormone receptor, alpha (THRA) and beta (THRB). THRB is important for regulation of the Hypothalamic-Pituitary-Thyroid axis and regulating cholesterol metabolism. THRA is involved in the development and the function of brain, adipose tissue, small intestine, bone and heart. Both THRA and THRB are post-translationally modified by small ubiquitin-like modifier (SUMO). Previously, we showed in an in vitro model that mutation of a THRA sumoylation motif impairs proliferation and differentiation of human primary white adipocytes. This is due to interference of the desumolyated THRA in the cell cycle at G1/S phase and disruption of PPAR signaling. To determine the in vivo effects of a desumoylated THRA, we generated a mouse model carrying a mutation of the THRA gene, which eliminates SUMO conjugation at lysine (K) 283. At 12 weeks of age, there was no difference in body composition (body weight, fat mass, and lean mass) in THRA mutant mice compared to wild-type (WT). From 12 weeks onward mutant mice and WT did not have significant change body weight compared to Wt mice. However, THRA sumoylation mutant mice had much lower percent body fat. Dissected WAT fat pads from mutant mice were significantly smaller compared to WT mice. Histological analysis showed that the cell size of white adipose tissue in mutant mice was significantly smaller than that in Wt mice as well. Serum leptin levels in the mutant mice were significantly lower than that in mice, consistent with reduced fat mass. We isolated fat stem cells from stromal vesicular fraction of 6 week old mice and found that the number of fat stem cells was significant less in mutant mice compared to those in WT mice. This data suggest that de-sumoylated THRA is associated with reduced fat stem cells at a young age, resulting in reduced adipose tissue mass in adult. THRA sumoylation is important for thyroid hormone modulation of fat mass. Reference: (1) Yan-Yun Liu et al, JBC 2012 (2) Yan-Yun Liu et al., JBC 2015

WAT-on-a-chip integrating human mature white adipocytes for mechanistic research and pharmaceutical applications

Obesity and its numerous adverse health consequences have taken on global, pandemic proportions. White adipose tissue (WAT) – a key contributor in many metabolic diseases – contributes about one fourth of a healthy human’s body mass. Despite its significance, many WAT-related pathophysiogical mechanisms in humans are still not understood, largely due to the reliance on non-human animal models. In recent years, Organ-on-a-chip (OoC) platforms have developed into promising alternatives for animal models; these systems integrate engineered human tissues into physiological microenvironment supplied by a vasculature-like microfluidic perfusion. Here, we report the development of a novel OoC that integrates functional mature human white adipocytes. The WAT-on-a-chip is a multilayer device that features tissue chambers tailored specifically for the maintenance of 3D tissues based on human primary adipocytes, with supporting nourishment provided through perfused media channels. The platform’s capability to maintain long-term viability and functionality of white adipocytes was confirmed by real-time monitoring of fatty acid uptake, by quantification of metabolite release into the effluent media as well as by an intact responsiveness to a therapeutic compound. The novel system provides a promising tool for wide-ranging applications in mechanistic research of WAT-related biology, in studying of pathophysiological mechanisms in obesity and diabetes, and in R&D of pharmaceutical industry.

PPARγ and PPARα synergize to induce robust browning of white fat in vivo

ObjectivePeroxisome proliferator-activated receptors (PPARs) are key transcription factors that regulate adipose development and function, and the conversion of white into brown-like adipocytes. Here we investigated whether PPARα and PPARγ activation synergize to induce the browning of white fat. MethodsA selection of PPAR activators was tested for their ability to induce the browning of both mouse and human white adipocytes in vitro, and in vivo in lean and obese mice. ResultsAll dual PPARα/γ activators tested robustly increased uncoupling protein 1 (Ucp1) expression in both mouse and human adipocytes in vitro, with tesaglitazar leading to the largest Ucp1 induction. Importantly, dual PPARα/γ activator tesaglitazar strongly induced browning of white fat in vivo in both lean and obese male mice at thermoneutrality, greatly exceeding the increase in Ucp1 observed with the selective PPARγ activator rosiglitazone. While selective PPARγ activation was sufficient for the conversion of white into brown-like adipocytes in vitro, dual PPARα/γ activation was superior to selective PPARγ activation at inducing white fat browning in vivo. Mechanistically, the superiority of dual PPARα/γ activators is mediated at least in part via a PPARα-driven increase in fibroblast growth factor 21 (FGF21). Combined treatment with rosiglitazone and FGF21 resulted in a synergistic increase in Ucp1 mRNA levels both in vitro and in vivo. Tesaglitazar-induced browning was associated with increased energy expenditure, enhanced insulin sensitivity, reduced liver steatosis, and an overall improved metabolic profile compared to rosiglitazone and vehicle control groups. ConclusionsPPARγ and PPARα synergize to induce robust browning of white fat in vivo, via PPARγ activation in adipose, and PPARα-mediated increase in FGF21.

The roles of triiodothyronine and irisin in improving the lipid profile and directing the browning of human adipose subcutaneous cells

Triiodothyronine (T3) and irisin (I) can modulate metabolic status, increase heat production, and promote differentiation of white adipose tissue (WAT) into brown adipose tissue (BAT). Herein, human subcutaneous white adipocytes were treated with 10 nM T3 or 20 nM I for 24 h to evaluate intracellular lipid accumulation, triglyceride, and glycerol levels, oxidative stress, DNA damage, and protein levels of uncoupling protein 1 (UCP1), adiponectin, leptin, peroxisome proliferator-activated receptor gamma (PPARγ), and fibronectin type III domain-containing protein 5 (FNDC5). T3 and irisin improved UCP1 production, lipid profile, oxidative stress, and DNA damage. T3 elevated adiponectin and leptin levels with a concomitant decrease in PPARy and FNDC5 levels. However, irisin did not alter adipokine, PPARy, and FNDC5 levels. The results indicate that T3 may be used to increase leptin and adiponectin levels to improve insulin sensitivity, and irisin may be used to prevent obesity or maintain weight due to its impact on the lipid profile without altering adipokine levels.

Depot-specific UCP1 expression in human white adipose tissue and its association with obesity-related markers.

This study investigated depot-specific mRNA expression of uncoupling protein 1 (UCP1) in human white adipose tissue (WAT) and its association with obesity-related markers. We recruited 39 normal-weight, 41 nondiabetic obese, and 22 diabetic obese women. We measured UCP1 mRNA expression in abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), and investigated the associations between UCP1 mRNA expression in VAT and SAT, and obesity-related markers including mRNA expression of leptin, adiponectin, CCAAT-enhancer-binding protein homologous protein (CHOP), and positive regulatory domain-containing protein 16 (PRDM16). We also evaluated UCP1 mRNA expression in differentiated human white adipocytes after treatment with various stressors and metabolic improvement agents in vitro. UCP1 mRNA in VAT was significantly higher than in SAT in all groups. UCP1 mRNA in SAT was negatively correlated with BMI, total abdominal fat area, visceral fat area, blood pressure, fasting glucose, insulin, HOMA-IR score, triglyceride, hsCRP, fasting leptin levels, and adipocyte size. UCP1 mRNA in SAT was positively correlated with fasting adiponectin levels. UCP1 mRNA in VAT was negatively correlated with visceral-to-subcutaneous fat ratio (VSR), fasting glucose, and triglyceride levels. In SAT, UCP1 mRNA was negatively correlated with mRNA expression of leptin and CHOP, and positively correlated with mRNA expression of adiponectin. The expression of PRDM16 was positively correlated with UCP1 mRNA in both VAT and SAT. UCP1 mRNA expression in differentiated human white adipocytes was significantly reduced after incubation with thapsigargin, tunicamycin, homocysteine, TNF-α, or IL-β, and significantly increased after incubation with exendin 4, dapagliflozin, and telmisartan. This study demonstrated depot-specific mRNA expression of UCP1 and its association with obesity-related markers in human WAT. UCP1 mRNA in human white adipocytes was suppressed by inflammatory agents and enhanced by metabolic improvement agents. UCP1 in human WAT might participate in the pathogenesis of obesity-related metabolic diseases.

Associations of IL1RAP and IL1RL1 gene polymorphisms with obesity and inflammation mediators.

Obesity is attributable to high free fatty acids, ER stress, oxidative stress and inflammation. The expression of IL-33, IL-1RL1 and IL-1RAP gene was observed in human visceral white fats, pre-adipocytes and adipocytes. The aim of this study was to determine whether IL1RAP and IL1RL1 gene variants were associated with obesity and inflammation mediators. 3 SNPs of IL1RAP (rs9990107, rs3836449 and rs9290936) and 11 SNPs of IL1RL1 (rs3771180, rs13431828, rs3214363, rs1420101, rs12905, rs3771175, rs3821204, rs12712142, rs10204137, rs4988958, and rs10206753) were genotyped for 175 obesity (BMI ≥ 25) and 358 non-obesity (BMI < 25.0) subjects. The genotype of SNPs was determined by the Axiom Genome-Wide Human Assay. The allele and genotype frequencies of 2 SNPs in the IL1RAP gene (rs9990107 and rs3836449) and 11 SNPs in the IL1RL1 gene (rs3771180, rs13431828, rs3214363, rs1420101, rs12905, rs3771175, rs3821204, rs12712142, rs10204137, rs4988958 and rs10206753) were significantly associated between the obesity and non-obesity groups. The two haplotypes (GCTTATGAATT and TT-CGACCGCC) in block1 were associated with obesity. In the non-obesity group, genotype frequencies of rs3771180, rs13431828, rs3214363, rs10204137, rs4988958 and rs10206753 SNPs of IL1RL1 showed significant differences in the dominant models in lymphatic cell percentage. The genotype frequencies of rs1420101, rs21905, rs3821024 and rs12712142 SNPs of IL1RL1 showed significant differences in the dominant models in eosinophil percentage. Our results suggest that IL1RAP and IL1RL1 gene polymorphisms may be associated with obesity and inflammation mediators.

Fattening chips: hypertrophy, feeding, and fasting of human white adipocytes in vitro.

Adipose is a distributed organ that performs vital endocrine and energy homeostatic functions. Hypertrophy of white adipocytes is a primary mode of both adaptive and maladaptive weight gain in animals and predicts metabolic syndrome independent of obesity. Due to the failure of conventional culture to recapitulate adipocyte hypertrophy, technology for production of adult-size adipocytes would enable applications such as in vitro testing of weight loss therapeutics. To model adaptive adipocyte hypertrophy in vitro, we designed and built fat-on-a-chip using fiber networks inspired by extracellular matrix in adipose tissue. Fiber networks extended the lifespan of differentiated adipocytes, enabling growth to adult sizes. By micropatterning preadipocytes in a native cytoarchitecture and by adjusting cell-to-cell spacing, rates of hypertrophy were controlled independent of culture time or differentiation efficiency. In vitro hypertrophy followed a nonlinear, nonexponential growth model similar to human development and elicited transcriptomic changes that increased overall similarity with primary tissue. Cells on the chip responded to simulated meals and starvation, which potentiated some adipocyte endocrine and metabolic functions. To test the utility of the platform for therapeutic development, transcriptional network analysis was performed, and retinoic acid receptors were identified as candidate drug targets. Regulation by retinoid signaling was suggested further by pharmacological modulation, where activation accelerated and inhibition slowed hypertrophy. Altogether, this work presents technology for mature adipocyte engineering, addresses the regulation of cell growth, and informs broader applications for synthetic adipose in pharmaceutical development, regenerative medicine, and cellular agriculture.

A novel peptide RIFV suppresses human adipocyte differentiation through the inhibition of C/EBP-\u03b2 expression

BackgroundObesity is a global epidemic disease that increases the risk of metabolic syndrome. However, therapeutic drugs for obesity are still scarce. In recent years, peptides have been identified as new biological regulators. RIFV (R-I-F-V-P-I-K-G-R-P-A-P), a novel active peptide from our peptide database.MethodsWe performed oil red O staining and triglyceride measurement to analyze the influence of RIFV on white preadipocytes differentiation. Then the effects of RIFV on cell proliferation, apoptosis and cell cycle were determined by using CCK-8 assay and flow cytometry. The mRNA and protein levels of adipogenesis-related genes were respectively detected by qRT-PCR and western blot. Rescue experiment was conducted to confirm whether RIFV could regulate adipocytes differentiation via targeting C/EBP-β. Finally, the luciferase reporter gene assay was performed to verify the regulation of RIFV on C/EBP-β gene.ResultsRIFV was revealed to inhibit the differentiation of human white adipocytes without affecting their proliferation. Additionally, RIFV could also suppress the differentiation of mouse primary white preadipocytes isolated from inguinal fat tissues. Furthermore, RIFV may have an inhibitory effect on adipogenesis by inhibiting the regulation of the adipogenic gene C/EBP-β.ConclusionsOur results indicated that RIFV may be a novel essential regulator of adipocyte differentiation and represents a therapeutic strategy for obesity and related complications.

Biological Effects of Ciliary Neurotrophic Factor on hMADS Adipocytes.

Administration of ciliary neurotrophic factor (CNTF) to experimental animals exerts anti-obesity effects by acting on multiple targets. In white adipose tissue CNTF reduces lipid content, promotes fatty acid (FA) oxidation and improves insulin sensitivity. This study was performed to establish whether CNTF exerts similar effects on human white adipocytes. To this end, adipose differentiation was induced in vitro in human multipotent adipose-derived stem (hMADS) cells. CNTF receptor α (CNTFRα) expression was assessed in hMADS cells and adipocytes by qRT-PCR, Western blotting, and immunocytochemistry. After administration of human recombinant CNTF, signaling pathways and gene expression were evaluated by Western blotting and qRT-PCR. Glucose uptake was assessed by measuring 2-nitrobenzodeoxyglucose uptake with a fluorescence plate reader. Lastly, CNTF-induced anti-inflammatory responses were evaluated in hMADS adipocytes stressed with tumor necrosis factor α (TNFα) for 24 h. Results showed that CNTFRα protein expression was higher in undifferentiated hMADS cells than in hMADS adipocytes, where it was however clearly detectable. In hMADS adipocytes, 1 nM CNTF strongly activated the JAK-STAT3 (Janus kinase-signaling transducer and activator of transcription 3) pathway and acutely and transiently activated the AMPK (AMP-activated protein kinase) and AKT (protein kinase B) pathways. Acute CNTF treatment for 20 min significantly increased basal glucose uptake and was associated with increased AKT phosphorylation. Longer-term (24 and 48 h) treatment reduced the expression of lipogenic markers (FA synthase and sterol regulatory element-binding protein-1) and increased the expression of lipolytic [hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL)] and mitochondrial (peroxisome proliferator-activated receptor γ coactivator-1α and carnitine palmitoyltransferase 1) markers. In TNFα-treated hMADS adipocytes, CNTF significantly reduced the expression of monocyte chemoattractant protein 1 and TNFα-induced AKT inhibition. Collectively, these findings demonstrate for the first time that CNTF plays a role also in human adipocytes, driving their metabolism toward a less lipid-storing and more energy-consuming phenotype.

Proteomics-Based Comparative Mapping of the Secretomes of Human Brown and White Adipocytes Reveals EPDR1 as a Novel Batokine

Adipokines secreted from white adipose tissue play arole in metabolic crosstalk and homeostasis, whereas the brown adipose secretome is less explored. We performed high-sensitivity mass-spectrometry-based proteomics on the cell media of human adipocytes derived from the supraclavicular brown adipose and from the subcutaneous white adipose depots of adult humans. We identified 471 potentially secreted proteins covering interesting categories such as hormones, growth factors, extracellular matrix proteins, and proteins of the complement system, which were differentially regulated between brown and white adipocytes. A total of 101 proteins were exclusively quantified in brown adipocytes, and among these was ependymin-related protein 1 (EPDR1). EPDR1 was detected in human plasma, and functional studies suggested a role for EPDR1 in thermogenic determination during adipogenesis. In conclusion, we report substantial differences between the secretomes of brown and white human adipocytes and identify novel candidate batokines that can be important regulators of human metabolism.

Bisphenol AF promotes inflammation in human white adipocytes.

Endocrine-disrupting chemicals interact with transcription factors essential for adipocyte differentiation. Exposure to endocrine-disrupting chemicals corresponds with elevated risks of obesity, but the effects of these compounds on human cells remain largely undefined. Widespread use of bisphenol AF (BPAF) as a bisphenol A (BPA) alternative in the plastics industry presents unknown health risks. To this end, we discovered that BPAF interferes with the metabolic function of mature human adipocytes. Although 4-day exposures to BPAF accelerated adipocyte differentiation, we observed no effect on mature fat cell marker genes. Additional gene and protein expression analysis showed that BPAF treatment during human adipocyte differentiation failed to suppress the proinflammatory transcription factor STAT1. Microscopy and respirometry experiments demonstrated that BPAF impaired mitochondrial function and structure. To test the hypothesis that BPAF fosters vulnerabilities to STAT1 activation, we treated mature adipocytes previously exposed to BPAF with interferon-γ (IFNγ). BPAF increased IFNγ activation of STAT1 and exposed mitochondrial vulnerabilities that disrupt adipocyte lipid and carbohydrate metabolism. Collectively, our data establish that BPAF activates inflammatory signaling pathways that degrade metabolic activity in human adipocytes. These findings suggest how the BPA alternative BPAF contributes to metabolic changes that correspond with obesity.

Elevated UCP1 levels are sufficient to improve glucose uptake in human white adipocytes

Brown adipose tissue (BAT) has been considered beneficial for metabolic health by participating in the regulation of glucose homoeostasis. The browning factors that improve glucose uptake beyond normal levels are still unknown but glucose uptake is not affected in UCP1 knockout mice. Here, we demonstrate in human white adipocytes that basal/resting glucose uptake is improved by solely elevating UCP1 protein levels. Generating human white Simpson-Golabi-Behmel syndrome (SGBS) adipocytes with a stable knockout and overexpression of UCP1, we discovered that UCP1 overexpressing adipocytes significantly improve glucose uptake by 40%. Mechanistically, this is caused by higher glycolytic flux, seen as increased oxygen consumption, extracellular acidification and lactate secretion rates. The improvements in glucose handling are comparable to white-to-brown transitions, as judged by, for the first time, directly comparing in vitro differentiated mouse brown vs white adipocytes. Although no adipogenic, metabolic and mitochondrial gene expressions were significantly altered in SGBS cells, pharmacological inhibition of GLUT1 completely abrogated differences between UCP1+ and control cells, thereby uncovering GLUT1-mediated uptake as permissive gatekeeper. Collectively, our data demonstrate that elevating UCP1 levels is sufficient to improve human white adipocytes as a glucose sink without adverse cellular effects, thus not requiring the adrenergic controlled, complex network of browning which usually hampers translational efforts.

Resveratrol and HIV-protease inhibitors control UCP1 expression through opposite effects on p38 MAPK phosphorylation in human adipocytes.

Brown and brown-like adipocytes (BBAs) control thermogenesis and are detected in adult humans. They express UCP1, which transforms energy into heat. They appear as promising cells to fight obesity. Deciphering the molecular mechanisms leading to the browning of human white adipocytes or the whitening of BBAs represents a goal to properly and safely control the pathways involved in these processes. Here, we analyzed how drugs endowed with therapeutic potential affect the differentiation of human adipose progenitor-cells into BBAs and/or their phenotype. We showed that HIV-protease inhibitors (PI) reduced UCP1 expression in BBAs modifying their metabolic profile and the mitochondria functionality. Lopinavir (LPV) was more potent than darunavir (DRV), a last PI generation. PPARγ and PGC-1α were decreased in a PI or cell-specific manner, thus altering UCP1's constitutive expression. In addition, LPV altered p38 MAPK phosphorylation, blunting then the β-adrenergic responses. In contrast, low doses of resveratrol stimulated the activatable expression of UCP1 in a p38 MAPK-dependent manner and counteracted the LPV induced loss of UCP1. This effect was independent of the resveratrol-induced sirtuin-1 expression. Altogether our results uncover how drugs impact crucial components of the networks regulating the expression of the thermogenic signature. They provide important information to control the relevant pathways involved in energy expenditure.

Olaparib induces browning of in vitro cultures of human primary white adipocytes

Mitochondrial biogenesis is a key feature of energy expenditure and organismal energy balance. Genetic deletion of PARP1 or PARP2 was shown to induce mitochondrial biogenesis and energy expenditure. In line with that, PARP inhibitors were shown to induce energy expenditure in skeletal muscle. We aimed to investigate whether pharmacological inhibition of PARPs induces brown or beige adipocyte differentiation. SVF fraction of human pericardial adipose tissue was isolated and human adipose-derived mesenchymal stem cells (hADMSCs) were differentiated to white and beige adipocytes. A subset of hADMSCs were differentiated to white adipocytes in the presence of Olaparib, a potent PARP inhibitor currently in clinical use, to induce browning. Olaparib induced morphological changes (smaller lipid droplets) in white adipocytes that is a feature of brown/beige adipocytes. Furthermore, Olaparib induced mitochondrial biogenesis in white adipocytes and enhanced UCP1 expression. We showed that Olaparib treatment inhibited nuclear and cytosolic PAR formation, induced NAD+/NADH ratio and consequently boosted SIRT1 and AMPK activity and the downstream transcriptional program leading to increases in OXPHOS. Olaparib treatment did not induce the expression of beige adipocyte markers in white adipocytes, suggesting the formation of brown or brown-like adipocytes.PARP1, PARP2 and tankyrases are key players in the formation of white adipose tissue. Hereby, we show that PARP inhibition induces the transdifferentiation of white adipocytes to brown-like adipocytes suggesting that PARP activity could be a determinant of the differentiation of these adipocyte lineages.

137-OR: The Selective Human Beta 3 Adrenergic Receptor Mirabegron Potently Activates Lipolysis in Human White Adipocytes

Human obesity is an increasing burden worldwide. Lipids stored as metabolic fuel in white adipose tissue (WAT) can be rapidly mobilized through lipolysis. In rodent adipocytes, activation of the β3-adrenergic receptors (β3-ARs) leads to lipolysis. The physiological role of the β3-AR in human adipocytes remains controversial due to its reported low expression and previous access only to partial β3-AR agonists. Mirabegron, an FDA-approved drug for overreactive bladder, is a highly-selective human β3-AR agonist. In clinical trials, mirabegron treatment led to activation of human brown adipose tissue (BAT) thermogenesis and increased plasma NEFA’s. Therefore, we investigated if mirabegron directly mediates lipolysis in human white adipocytes. We found similar gene expression of the three β-ARs in human immortalized and primary white adipocytes, with β2-AR being the most expressed, followed by β1-AR and β3-AR. Next, to assess the functionally of the β3-AR in human adipocytes, we established dose-response curves and found that mirabegron increased the release of glycerol, a lipolytic product, in the picomolar range. To identify the molecular mechanism controlling mirabegron-induced lipolysis, we assessed the regulation of key downstream lipolytic proteins. Mirabegron mediated lipolysis by rapidly phosphorylating hormone sensitive lipase (HSL), as well as the mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). Moreover, acute inhibition of either the PKA or MAPK/ERK pathways decreased lipolysis. In summary, we identify a major role played by the β3-AR in the regulation of human WAT lipolysis. Mirabegron acts as a potent pro-lipolytic effector, signaling through the canonical adrenergic receptor pathway as well as the MAPK/ERK pathway. Due to the comparatively minimal effects previously reported of mirabegron on the cardiovascular system, mirabegron may be the optimal choice for targeted lipid mobilization in human WAT. Disclosure C. Cero: None. J.W. Johnson: None. A. O’Mara: None. J.D. Linderman: None. A.M. Cypess: None. Funding National Institutes of Health

1970-P: Epinephrine and Insulin Stimulates White Adipocyte Secretion of Diverse Adiponectin Forms

The white adipocyte hormone adiponectin is released in different molecular forms and a reduction of high-molecular weight (HMW) adiponectin has specifically been associated with the development of metabolic disease. We have recently shown that adiponectin is released via cAMP/epinephrine-stimulated exocytosis, involving activation of beta 3 adrenergic receptors (beta3ARs). However, a number of studies have, seemingly incompatible with the catecholamine-triggered release, reported that insulin (lowers cAMP) increases adiponectin secretion. Here we have pinpointed the differences between insulin- and catecholamine-stimulated short-term adiponectin secretion. Using a combination of biophysical and biochemical techniques applied to mouse and human adipocytes as well as whole animal studies, we show that catecholamines specifically stimulate the rapid exocytosis of high-molecular weight (HMW) adiponectin. In contrast, insulin induces secretion of smaller adiponectin forms only, with slower time kinetics. The catecholamine-stimulated secretion of HMW adiponectin is blunted in adipocytes isolated from obese and diabetic mice (8 weeks high-fat diet) and this secretory defect is paralleled by a ∼50% reduction of serum HMW adiponectin. The insulin-induced release of smaller adiponectin forms is also abrogated i adipocytes from obese/diabetic mice. However, the total serum adiponectin level remains unaltered, likely due to enhanced basal (unstimulated) release of smaller adiponectin forms. Our findings suggest that insulin and catecholamines have entirely different roles in the regulation of adiponectin secretion and demonstrate a previously unknown role of beta3ARs for human white adipocyte physiology. We propose that the reduced levels of HMW adiponectin observed in diabetic individuals, and that have been linked to the development of metabolic disease, is a result of a secretory disturbance largely depending on disrupted adrenergic signaling. Disclosure S. Musovic: None. A.M. Komai: None. E. Banke Nordbeck: None. U.A. Noor: None. I. Wernstedt Asterholm: None. C.S. Olofsson: None. Funding Swedish Diabetes Foundation; Knut and Alice Wallenberg Foundation; Swedish Medical Research Council