White Preadipocytes Research Articles

1\u03b1,25(OH)2D3 attenuates IL-6 and IL-1\u03b2-mediated inflammatory responses in macrophage conditioned medium-stimulated human white preadipocytes by modulating p44/42 MAPK and NF-\u03baB signaling pathways

BackgroundMetabolic syndrome is characterized by macrophage infiltration and inflammatory responses—metaflammation in adipose tissue. IL-6 and IL-1β could mediate the inflammatory responses in macrophage stimulated-preadipocytes by modulating MAPK and NF-κB pathways. To test this hypothesis we used antibodies to block IL-6 and IL-1β action in macrophage conditioned medium (MacCM)-stimulated human white preadipocytes. Moreover, as interventions that prevent this could potentially be used to treat or prevent metabolic syndrome, and 1α,25(OH)2D3 has previously been reported to exert an anti-inflammatory action on macrophage-stimulated adipocytes, in this study we also investigated whether 1α,25(OH)2D3 could attenuate inflammatory responses in MacCM-stimulated preadipocytes, and explored the potential anti-inflammatory mechanisms.MethodsHuman white preadipocytes were cultured with 25% MacCM for 24 h to elicit inflammatory responses. This was confirmed by measuring the concentrations and mRNA levels of major pro-inflammatory factors [IL-1β, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1 and regulated on activation, normal T cell expressed and secreted (RANTES)] by ELISA and qPCR, respectively. IL-6 and IL-1β actions were blocked using IL-6 antibody (300 ng/ml) and IL-1β antibody (15 μg/ml), respectively. Potential anti-inflammatory effects of 1α,25(OH)2D3 were investigated by pre-treatment and treatment of 1α,25(OH)2D3 (0.01 to 10 nM) for 48 h in MacCM-stimulated preadipocytes. In parallel, western blotting was used to determine inflammatory signaling molecules including relA of the NF-κB pathway and p44/42 MAPK modified during these processes.ResultsMacCM enhanced the secretion and gene expression of IL-1β, IL-6, IL-8, MCP-1 and RANTES by increasing the phosphorylation levels of relA and p44/42 MAPK in preadipocytes, whereas blocking IL-6 and IL-1β action inhibited the inflammatory responses by decreasing p44/42 MAPK and relA phosphorylation, respectively. Furthermore, 10 nM of 1α,25(OH)2D3 generally inhibited the IL-6 and IL-1β-mediated inflammatory responses, and reduced both p44/42 MAPK and relA phosphorylation in MacCM-stimulated preadipocytes.Conclusions1α,25(OH)2D3 attenuates IL-6 and IL-1β-mediated inflammatory responses, probably by inhibiting p44/42 MAPK and relA phosphorylation in MacCM-stimulated human white preadipocytes.

Alpha-glucosidase inhibitor 1-Deoxynojirimycin promotes beige remodeling of 3T3-L1 preadipocytes via activating AMPK

Obesity is a serious health challenge in the world, and searching effective drugs to cure obesity is of great importance. 1-Deoxynojirimycin (DNJ) is extracted from mulberry leaves and acts as an α-glucosidase inhibitor to lower blood glucose. Recent studies demonstrated that it also has anti-obesity effect, but the mechanisms remain unknown. In our present study, we mainly examined the effects of DNJ on beige remodeling of 3T3-L1 preadipocytes. We observed that DNJ didn't affect the mRNA levels of fatty acid binding protein 4 (aP2), peroxisome proliferator-activated receptor γ (PPARγ), preadipocyte factor-1 (Pref-1) as well as the mitochondrial uncoupling protein 1 (UCP1), PR domain containing protein 16 (PRDM16), transmembrane protein 26 (TMEM26) in undifferentiated preadipocytes. But after inducing 3T3-L1 preadipocytes to differentiation with white or beige adipogenic medium, DNJ significantly reduced aP2, PPARγ and Pref-1 expressions, while up-regulated the expressions of UCP1, PRDM16 and TMEM26, accompanying with decreased lipid deposition. The ratio of p-AMPK/AMPK was up-regulated by DNJ (10 μM) treatment for 10 days, and the effects of DNJ on p-AMPK/AMPK, UCP1 and PRDM16 could be blocked by AMPK inhibitor Compound C. These results demonstrated that hypoglycemic agent DNJ could suppress the adipogenesis during the differentiation of white preadipocytes, and promote the switch of white preadipocytes to beige adipocytes via activating AMPK, which provided new mechanisms for explaining the benefits of DNJ on obesity-related disorders.

Role of estradiol and testosterone in Ucp1 expression in brown/beige adipocytes.

Activity of brown/beige adipocytes is higher in women than in men. The expression level of uncoupling protein 1 (UCP1) is largely consistent with the thermogenic activity in brown/beige adipocytes. The present study examined the direct effects of sex hormones on Ucp1 expression in brown adipocytes and beige adipocytes, which were differentiated from HB2 brown preadipocytes and 3T3-L1 white preadipocytes, respectively; treatment with estradiol or testosterone was used during the early (days 0-8) or late stage (days 8-12) of brown adipogenesis and beige adipogenesis. On day 8 or day 12, cells were treated with or without isoproterenol (Iso), an agonist for the β-adrenergic receptor, for 4hours. Furthermore, the sex of cells was examined; the sex-determining region y gene, which is located on the y chromosome, was present in HB2 cells, but not in 3T3-L1 cells, suggesting that HB2 cells and 3T3-L1 cells are male and female cells, respectively. Treatment with 17β-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to Iso on Ucp1 induction, whereas treatment during the late stage of brown adipogenesis decreased Ucp1 expression in unstimulated brown adipocytes. Estradiol decreased Iso-induced Ucp1 expression during the early stage of beige adipogenesis. Treatment with testosterone during the early stage of brown adipogenesis did not affect Ucp1 expression but increased the responsiveness to Iso on Ucp1 induction by the treatment during the late stage of brown adipogenesis. The present results suggest that sex hormones modulate the expression level of Ucp1 in brown/beige adipocytes in a stage-dependent manner. Direct effects of sex hormones in brown/beige adipogenesis were evaluated. Treatment with 17β-estradiol during the early stage of brown adipogenesis enhanced the responsiveness to isoproterenol (Iso), an agonist for the β-adrenergic receptor, on Ucp1 induction, whereas treatment during the late stage of brown adipogenesis decreased Ucp1 expression in unstimulated brown adipocytes. Estradiol decreased Iso-induced Ucp1 expression during the early stage of beige adipogenesis. Testosterone during the late stage of brown adipogenesis increased the responsiveness to Iso on Ucp1 induction. Sex hormones modulate the expression level of Ucp1 in brown/beige adipocytes in a stage-dependent manner.

MiR-199a-3p regulates brown adipocyte differentiation through mTOR signaling pathway.

Recent discoveries of functional brown adipocytes in mammals illuminates their therapeutic potential for combating obesity and its associated diseases. However, on account of the limited amount and activity in adult humans of brown adipocyte depots, identification of miRNAs and characterization their regulatory roles in human brown adipogenesis are urgently needed. This study focused on the role of microRNA (miR)-199a-3p in human brown adipocyte differentiation and thermogenic capacity. A decreased expression pattern of miR-199a-3p was consistently observed during the differentiation course of brown adipocytes in mice and humans. Conversely, its level was induced during the differentiation course of human white pre-adipocytes derived from visceral fat. miR-199a-3p expression was relatively abundant in interscapular BAT (iBAT) and differentially regulated in the activated and aging BAT in mice. Additionally, miR-199a-3p expression level in human brown adipocytes was observed decreased upon thermogenic activation and increased by aging-related stimuli. Using primary pre-adipocytes, miR-199a-3p over-expression was capable of attenuating lipid accumulation and adipogenic gene expression as well as impairing brown adipocytes' metabolic characteristics as revealed by decreased mitochondrial DNA content and respiration. Suppression of miR-199a-3p by a locked nucleic acid (LNA) modified-anti-miR led to increased differentiation and thermogenesis in human brown adipocytes. By combining target prediction and examination, we identified mechanistic target of rapamycin kinase (mTOR) as a direct target of miR-199a-3p that affected brown adipogenesis and thermogenesis. Our results point to a novel role for miR-199a-3p and its downstream effector mTOR in human brown adipocyte differentiation and maintenance of thermogenic characteristics, which can be manipulated as therapeutic targets against obesity and its related metabolic disorders.

Vitamin D receptor ligands attenuate the inflammatory profile of IL-1β-stimulated human white preadipocytes via modulating the NF-κB and unfolded protein response pathways

Metaflammation in adipose tissue, which is characterized by increased local gene expression and secretion of pro-inflammatory factors, may contribute to the increased risk of metabolic complications in obesity. It has been suggested that IL-1β might induce metaflammation in adipose tissue via modulating the NF-κB signaling pathway. In our study, the mRNA and secretion levels of major pro-inflammatory factors including IL (interleukin)-1β, IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and RANTES (regulated on activation, normal T cell expressed and secreted) were measured as indicators of the inflammatory profile. We herein showed that IL-1β could induce adipose tissue metaflammation by enhancing the inflammatory profile of preadipocytes. Moreover, IL-1β could enhance pro-inflammatory gene expression by increasing the phosphorylation or decreasing the methylation of relA (NF-κB p65) of the NF-κB signaling pathway. VDR (vitamin D receptor) ligands have been shown to have anti-inflammatory properties in adipocytes. Likewise, our study demonstrated that the inflammatory profile of IL-1β-stimulated preadipocytes is significantly attenuated by VDR ligands 1α,25(OH)2D3, ZK159222 and ZK191785. Importantly, we showed that ZK159222 and ZK191785 could inhibit pro-inflammatory gene expression by decreasing the phosphorylation or increasing the methylation of relA. Furthermore, pro-inflammatory secretion could be reduced by 1α,25(OH)2D3, ZK159222 and ZK191785 via increasing the phosphorylation of eIF (eukaryotic translation initiation factor)-2α of the UPR (unfolded protein response) pathway. These observations suggest that the VDR ligands may be considered potential anti-inflammatory treatments for obesity associated metabolic complications.

Ostreolysin induces browning of adipocytes and ameliorates hepatic steatosis.

Non-alcoholic fatty liver disease (NAFLD) is associated with all features of the metabolic syndrome. Deposition of excess triglycerides in liver cells, a hallmark of NAFLD, is associated with loss of insulin sensitivity. Ostreolysin (Oly) is a 15-kDa fungal protein known to interact with cholesterol-enriched raft-like membrane domains. We aim to test whether a recombinant version of Oly (rOly) can induce functional changes in vitro in adipocytes or in vivo in mice fed a high-fat diet (HFD). White preadipocyte 3T3-L1 cells or mouse primary adipocytes treated with rOly. Male C57BL/6 mice were fed a control or HFD and treated with saline or with rOly (1mg/kg BW) every other day for 4weeks. White preadipocyte 3T3-L1 cells or mouse primary adipocytes treated with rOly acquire a browning phenotype through activation of 5' adenosine monophosphate-activated protein kinase and downregulation of tumor necrosis factor α-mediated activation of IκB kinase ε and TANK-binding kinase 1. HFD-fed mice treated with rOly showed a 10% reduction in BW and improved glucose tolerance, which paralleled improved expression of liver and adipose functionality, metabolism, and inflammation status, mimicking the in vitro findings. This study provides first evidence of rOly's prevention of HFD-induced NAFLD by stimulating liver and adipose muscle tissue functionality and oxidative potential, improving glucose tolerance, and ameliorating the metabolic profile of diet-induced obese mice.

The Autism-Related Protein CHD8 Cooperates with C/EBPβ to Regulate Adipogenesis

The gene encoding the chromatin remodeler CHD8 isthe most frequently mutated gene in individuals with autism spectrum disorder (ASD). Heterozygous mutations in CHD8 give rise to ASD that is often accompanied by macrocephaly, gastrointestinal complaints, and slender habitus. Whereas most phenotypes of CHD8 haploinsufficiency likely result from delayed neurodevelopment, the mechanism underlying slender habitus has remained unknown. Here, weshow that CHD8 interacts with CCAAT/enhancer-binding protein β (C/EBPβ) and promotes its transactivation activity during adipocyte differentiation. Adipogenesis was impaired in Chd8-deleted preadipocytes, with the upregulation of C/EBPα and peroxisome-proliferator-activated receptor γ (PPARγ), two master regulators of this process, being attenuated in mutant cells. Furthermore, mice with CHD8 ablation in white preadipocytes had a markedly reduced white adipose tissue mass. Our findings reveal a mode of C/EBPβ regulation by CHD8 during adipogenesis, with CHD8 deficiency resulting in a defect in the development of white adipose tissue.

Abstract 604: Anti-apoptotic Role of Leptin in Adipose Tissue

Background: Leptin contributes to obesity-related cardiometabolic pathology. While the role of adipose tissue dysfunction in altering metabolic profile in obesity is recognized, the direct effects of leptin signaling in adipose tissue are not completely understood. Among factors impacting adipose tissue function in obesity, adipocyte death is associated with increased inflammation with consequent adipose tissue insulin-resistance. Furthermore, apoptosis of adipocyte progenitor cells (preadipocytes) is important as it lowers adipose tissue capacity to maintain and expand fat storage. Objective: To determine the effects of leptin on regulation of proteins important to apoptotic pathways in cultured human white preadipocytes (HWP) and differentiated HWP (dHWP). Methods: Cells were exposed to increasing concentrations of leptin to evaluate the role of leptin on regulation of anti-apoptotic Bcl2, and pro-apoptotic Bax and CD95 expression. Next, we examined if pre-exposure to leptin attenuated TNF-α and FasL induced apoptosis. Results: Leptin upregulated transcription of Bcl2 (p=0.02) and down-regulated transcription of Bax (p=0.02) and CD95 (p=0.04) in a leptin-concentration dependent manner in dHWP. Leptin dependent increases in Bcl2 (p=0.004) and decreases in Bax (p=0.003) and CD95 (P=0.03) protein expression were also observed. Furthermore, 24 hour pre-exposure to leptin protected cells from TNF-α (p=0.01; p=0.006) and FasL (p=0.03; p=0.007) induced apoptosis in dHWP and HWP respectively. Additionally, leptin attenuated TNF-mediated activation of the p53 pathway, suggesting another mechanism by which leptin mediates its inhibitory effects on TNF-induced apoptosis. Conclusion: Leptin regulates the expression of proteins involved in the apoptotic pathway such that it decreases the apoptotic potential of adipose tissue by increasing anti-apoptotic Bcl2 and decreasing pro-apoptotic Bax and CD95 protein expression. In contrast to our in-vitro findings, prior studies in adipose tissue from obese humans report decreased Bcl2 and increased Bax protein expression, along with high leptin expression. This suggests that increased adipose tissue apoptosis in obesity may at least in part be secondary to altered leptin signaling.

Label-free quantitative chemical imaging and classification analysis of adipogenesis using mouse embryonic stem cells.

Stem cells have received much attention recently for their potential utility in regenerative medicine. The identification of their differentiated progeny often requires complex staining procedures, and is challenging for intermediary stages which are a priori unknown. In this work, the ability of label-free quantitative coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy to identify populations of intermediate cell states during the differentiation of murine embryonic stem cells into adipocytes is assessed. Cells were imaged at different days of differentiation by hyperspectral CARS, and images were analysed with an unsupervised factorization algorithm providing Raman-like spectra and spatially resolved maps of chemical components. Chemical decomposition combined with a statistical analysis of their spatial distributions provided a set of parameters that were used for classification analysis. The first 2 principal components of these parameters indicated 3 main groups, attributed to undifferentiated cells, cells differentiated into committed white pre-adipocytes, and differentiating cells exhibiting a distinct protein globular structure with adjacent lipid droplets. An unsupervised classification methodology was developed, separating undifferentiated cell from cells in other stages, using a novel method to estimate the optimal number of clusters. The proposed unsupervised classification pipeline of hyperspectral CARS data offers a promising new tool for automated cell sorting in lineage analysis.

Simpson–Golabi–Behmel syndrome human adipocytes reveal a changing phenotype throughout differentiation

The Simpson-Golabi-Behmel syndrome (SGBS) cell strain is widely considered to be a representative in vitro model of human subcutaneous white pre-adipocytes. These cells achieve a transient expression of classical brown markers, such as uncoupling protein 1, peaking at day 14 of differentiation and decreasing thereafter. Adipocyte browning process involves dynamic changes in lipid droplet (LD) dimension, in mitochondria morphology, and in the expression of brown-specific marker genes. This study analyzes SGBS transient phenotypic transformation by quantifying the heterogeneity of LDs, mitochondrial dynamics, and a panel of genes involved in adipocyte differentiation and browning. LDs at 21days of differentiation were larger than in the previous stages, without any change in the number per cell. The expression of genes such as peroxisome peroxisome proliferator-activated receptor γ, leptin, and lipase E significantly raised from 0 to 21days. Adiponectin was significantly upregulated at 14days of differentiation. Brown-specific marker PR domain containing 16 was highly expressed at D0. The variability of mitochondrial shape and interconnectivity reflects differences in the relative rates of fusion and fission, resulting in a significant shift from a networked shape at D7 to a fragmented and swollen one at D14 and D21. The transient phenotype experienced by this cellular model should be considered whether used in studies involving the stimulation of adipocyte browning and could be an interesting human model to further elucidate the browning process in the absence of any stimulation.

A novel brown adipocyte-enriched long non-coding RNA that is required for brown adipocyte differentiation and sufficient to drive thermogenic gene program in white adipocytes

The thermogenic activities of brown and beige adipocytes can be exploited to reduce energy surplus and counteract obesity. Recent RNA sequencing studies have uncovered a number of long noncoding RNAs (lncRNAs) uniquely expressed in white and brown adipose tissues (WAT and BAT), but whether and how these lncRNAs function in adipogenesis remain largely unknown. Here, we report the identification of a novel brown adipocyte-enriched LncRNA (AK079912), and its nuclear localization, function and regulation. The expression of AK079912 increases during brown preadipocyte differentiation and in response to cold-stimulated browning of white adipocytes. Knockdown of AK079912 inhibits brown preadipocyte differentiation, manifested by reductions in lipid accumulation and down-regulation of adipogenic and BAT-specific genes. Conversely, ectopic expression of AK079912 in white preadipocytes up-regulates the expression of genes involved in thermogenesis. Mechanistically, inhibition of AK079912 reduces mitochondrial copy number and protein levels of mitochondria electron transport chain (ETC) complexes, whereas AK079912 overexpression increases the levels of ETC proteins. Lastly, reporter and pharmacological assays identify Pparγ as an upstream regulator of AK079912. These results provide new insights into the function of non-coding RNAs in brown adipogenesis and regulating browning of white adipocytes.

Adipogenesis in Primary Cell Culture.

Obesity involves a contrasting expansion of the energy-storing white fat and loss of functionally competent brown fat, an energy-consuming thermogenic adipose. Leveraging our understanding of white and brown adipocyte recruitment and investigating factors that regulate these processes might reveal novel targets for counteracting obesity. In vitro differentiation of primary preadipocytes mimics many of the morphological and transcriptional events occurring during adipogenesis in vivo. Moreover, preadipocytes isolated from a specific depot maintain features of their originating niche. This makes in vitro adipogenesis a valuable model for identifying differential regulation patterns between brown and white adipogenesis. In this chapter, we describe step-by-step how to isolate brown and white preadipocytes from human tissue biopsies and how to culture and differentiate them in vitro. We discuss this process, what to consider, and how this in vitro system can be used to model in vivo adipogenesis.

Differential Patterns of Secreted Frizzled-Related Protein 4 (SFRP4) in Adipocyte Differentiation: Adipose Depot Specificity

Background/Aims: Secreted frizzled-related protein 4 (SFRP4) is a member of the SFRP family that acts as soluble modulators of Wnt signaling. Given the substantial rise in obesity, depot-specific fat accumulation and its associated diseases like diabetes, it is important to understand the molecular basis of depot-specific adipocyte differentiation. In the current study, we investigated the expression of SFRP4 in both subcutaneous and visceral adipose tissue in terms of their differentiation. Methods: White preadipocytes were isolated from the inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) from C57BL/6J mice (age: 8-week-old, male). SFRP4 expression in iWAT and eWAT preadipocytes was silenced by siRNA transfection and harvested cells for gene and protein expression analysis was performed during the differentiation. Furthermore, iWAT and eWAT preadipocytes treated with or without IL-1β were harvested for gene and protein expression analysis. Results: SFRP4 expression levels were gradually increased and proportionally associated with eWAT adipocyte differentiation toward maturation at 14 days, while iWAT adipocyte just showed an opposite tendency. Moreover, genetic (adiponectin, C/EBPα, C/EBPβ, FABP4, GLUT4 and PPARγ) analysis demonstrated that depot-specific adipogenesis in response to SFRP4 silencing in eWAT and iWAT preadipocytes. Upon IL-1β treatment, SFRP4 mRNA expression decreased significantly in iWAT adipocyte, but the expression was no significant difference in eWAT adipocyte. Conclusion: These results suggest that SFRP4 expression differentially mediates adipocyte differentiation and may play an important role in adipogenesis.

ZBTB16 Overexpression Enhances White Adipogenesis and Induces Brown-Like Adipocyte Formation of Bovine White Intramuscular Preadipocytes

Background/Aims: Our study aims to characterize functions of ZBTB16 gene in the process of intramuscular fat (IMF) deposition and metabolism of bovine, thereby providing insights into mechanisms for the use of ZBTB16 in fat management. Methods: Primary preadipocytes derived from bovine IMF tissue were isolated and used as the in vitro cell model. An adenovirus Ad-ZBTB16 was transfected into bovine preadipocytes to overexpress the ZBTB16 gene. By using real-time quantitative PCR (RT-qPCR), western blotting, Oil Red-O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity assay, and cell counting kit-8 (CCK-8) test, adipogenic and proliferative signals in adipocytes were monitored to investigate effects of ZBTB16 on adipogenesis of bovine preadipocytes. Results: After transfection, mRNA and protein levels of ZBTB16 gene were significantly increased. Enhanced ZBTB16 significantly promoted preadipocyte differentiation, as evidenced by accelerated lipid accumulation, enhanced GPDH activity, consistently increased mRNA expressions of adipogenic key transcription factors PPARγ, C/EBPα, FABP4, and ADIPOQ, and markedly increased protein expressions of PPARγ and FABP4. No difference was observed concerning proliferation of preadipocytes after treatment with Ad-ZBTB16. Furthermore, relative mRNA levels of brown adipocyte selective genes (PRDM16, UCP1, Cidea, Cox8b, and PGC-1α) and beige adipocyte selective genes (CD137, TMEM26, and Tbx1) as well as UCP1 protein expression were significantly increased by Ad-ZBTB16. Meanwhile, Ad-ZBTB16 treatment remarkably induced mitochondrial biogenesis and increased relative mitochondrial DNA (mtDNA) copy number in bovine adipocytes. Conclusion: These results suggest that ZBTB16 overexpression can promote white adipogenesis and induce brown-like adipocyte formation for bovine white intramuscular preadipocytes.

Tbx15 Defines a Glycolytic Subpopulation and White Adipocyte Heterogeneity.

Tbx15 is a member of the T-box gene family of mesodermal developmental genes. We have recently shown that Tbx15 plays a critical role in the formation and metabolic programming of glycolytic myofibers in skeletal muscle. Tbx15 is also differentially expressed among white adipose tissue (WAT) in different body depots. In the current study, using three independent methods, we show that even within a single WAT depot, high Tbx15 expression is restricted to a subset of preadipocytes and mature white adipocytes. Gene expression and metabolic profiling demonstrate that the Tbx15Hi preadipocyte and adipocyte subpopulations of cells are highly glycolytic, whereas Tbx15Low preadipocytes and adipocytes in the same depot are more oxidative and less glycolytic. Likewise, in humans, expression of TBX15 in subcutaneous and visceral WAT is positively correlated with markers of glycolytic metabolism and inversely correlated with obesity. Furthermore, overexpression of Tbx15 is sufficient to reduce oxidative and increase glycolytic metabolism in cultured adipocytes. Thus, Tbx15 differentially regulates oxidative and glycolytic metabolism within subpopulations of white adipocytes and preadipocytes. This leads to a functional heterogeneity of cellular metabolism within WAT that has potential impact in the understanding of human metabolic diseases.

SGBS cells as a model of human adipocyte browning: A comprehensive comparative study with primary human white subcutaneous adipocytes

The Simpson Golabi Behmel Syndrome (SGBS) pre-adipocyte cell strain is widely considered to be a representative in vitro model of human white pre-adipocytes. A recent study suggested that SGBS adipocytes exhibit an unexpected transient brown phenotype. Here, we comprehensively examined key differences between SGBS adipocytes and primary human white subcutaneous (PHWSC) adipocytes. RNA-Seq analysis revealed that extracellular matrix (ECM)-receptor interaction and metabolic pathways were the top two KEGG pathways significantly enriched in SGBS adipocytes, which included positively enriched mitochondrial respiration and oxidation pathways. Compared to PHWSC adipocytes, SGBS adipocytes showed not only greater induction of adipogenic gene expression during differentiation but also increased levels of UCP1 mRNA and protein expression. Functionally, SGBS adipocytes displayed higher ISO-induced basal leak respiration and overall oxygen consumption rate, along with increased triglyceride accumulation and insulin-stimulated glucose uptake. In conclusion, we confirmed that SGBS adipocytes, which are considered of white adipose tissue origin can shift towards a brown/beige adipocyte phenotype. These differences indicate SGBS cells may help to identify mechanisms leading to browning, and inform our understanding for the use of SGBS vis-à-vis primary human subcutaneous adipocytes as a human white adipocyte model, guiding the selection of appropriate cell models in future metabolic research.

Mycobacterium canettii Infection of Adipose Tissues.

Adipose tissues were shown to host Mycobacterium tuberculosis which is persisting inside mature adipocytes. It remains unknown whether this holds true for Mycobacterium canettii, a rare representative of the M. tuberculosis complex responsible for lymphatic and pulmonary tuberculosis. Here, we infected primary murine white and brown pre-adipocytes and murine 3T3-L1 pre-adipocytes and mature adipocytes with M. canettii and M. tuberculosis as a positive control. Both mycobacteria were able to infect 18–22% of challenged primary murine pre-adipocytes; and to replicate within these cells during a 7-day experiment with the intracellular inoculums being significantly higher in brown than in white pre-adipocytes for M. canettii (p = 0.02) and M. tuberculosis (p = 0.03). Further in-vitro infection of 3T3-L1 mature adipocytes yielded 9% of infected cells by M. canettii and 17% of infected cells by M. tuberculosis (p = 0.001). Interestingly, M. canettii replicated and accumulated intra-cytosolic lipid inclusions within mature adipocytes over a 12-day experiment; while M. tuberculosis stopped replicating at day 3 post-infection. These results indicate that brown pre-adipocytes could be one of the potential targets for M. tuberculosis complex mycobacteria; and illustrate differential outcome of M. tuberculosis complex mycobacteria into adipose tissues. While white adipose tissue is an unlikely sanctuary for M. canettii, it is still an open question whether M. canettii and M. tuberculosis could persist in brown adipose tissues.

A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3D culture of human and murine adipocytes.

The lack of relevant in vitro models for adipose tissue makes necessary the development of a more physiological environment providing spatial and chemical cues for the effective maturation of adipocytes. We developed a biofunctionalized hydrogel with components of adipose extracellular matrix: collagen I, collagen VI, and the cell binding domain of fibronectin and we compared it to usual 2D cultures on plastic plates. This scaffold allowed 3D culture of mature adipocytes from the preadipocytes cell lines 3T3-L1 and 3T3-F442A, as well as primary Human White Preadipocytes (HWP), acquiring in vivo-like organization, with spheroid shaped adipocytes forming multicellular aggregates. The size of these aggregates increased with time up to 120 μm in diameter after 4 weeks of maturation, with good viability. Significantly higher lipogenic activity (up to 20-fold at day 28 for HWP cultures) and differentiation rates were also observed compared to 2D. Gene expression analyses highlighted earlier differentiation and complete maturation of 3D HWP compared to 2D, reinforced by the expression of Perilipin protein after 21 days of nutrition. This increase in adipocytes phenotypic and genotypic markers made this scaffold-driven culture as a robust adipose 3D model. Retinoic acid inhibition of lipogenesis in HWP or isoprenalin and caffeine induction of lipolysis performed on mouse 3T3-F442A cells, showed higher doses of molecules than typically used in 2D, underlying the physiologic relevance of this 3D culture system. Biotechnol. Bioeng. 2017;114: 1813-1824. © 2017 Wiley Periodicals, Inc.

GM13133 is a negative regulator in mouse white adipocytes differentiation and drives the characteristics of brown adipocytes.

Obesity is tightly associated with the disturbance of white adipose tissue storing excess energy. Thermogenic adipocytes (brown and beige) exert a critical role of oxidizing nutrients at the high rates through non-shivering thermogenesis. The recruitment of brown characteristics in white adipocytes, termed browning, has been considered as a promising strategy for treating obesity and associated metabolic complications. Recently, long noncoding RNAs play a crucial role in regulating tissue development and participating in disease pathogenesis, yet their effects on the conversion of white into brown-like adipocytes and thermogenic function were not totally understood. Here, we identified a mouse brown adipose specific expressed lncRNA, termed GM13133. Moreover, a considerable amount of GM13133 is expressed in adipocytes and actively modulated by cold, β3 -adrenergic agonist and cAMP stimuli, implying a potential role in the conversion from white to brown adipocytes. Overexpression of GM13133 did not affect the proliferation of mouse white pre-adipocytes, but inhibited white adipocyte differentiation by decreasing lipid accumulation. The forced expression of GM13133 also significantly drove the conversion of white into brown-like adipocytes with the enhanced mitochondrial biogenesis and the induced expression of brown adipocytes specific markers. A global mRNA analysis further indicated the possible regulatory role of cAMP signaling pathway in GM13133 mediated white-to-brown adipocytes conversion. Our results identified a lncRNA-mediated modulation in primary mouse white adipocyte differentiation and indicate the functional significance of GM13133 in promoting browning of white adipocytes and maintenance of thermogenesis, further providing a potential strategy to treating obesity.

Metformin Decreases Caveolin‐1 Expression in Differentiated Human White Preadipocytes But Not in Adipose Tissue From Obese Individuals

BackgroundCaveolin‐1 is a structural protein of caveolae which forms a signaling platform for several cell surface receptors including leptin and insulin receptor. Caveolin‐1 expression is increased with obesity which is implicated in impaired adipose tissue leptin signaling. Considering the overlap between insulin and leptin cellular signaling pathways, we investigated known insulin‐sensitizing drugs which may reduce caveolin‐1 expression in adipose tissue and consequently improve leptin signaling.MethodsWe used an in‐vitro approach to identify insulin‐sensitizing drugs which may reduce caveolin‐1 expression. Differentiated human white preadipocytes (dHWP) were treated with pioglitazone (5–30 μM), troglitazone (10–30 μM), metformin (1–5 mM), or glimepiride (20–100 μM) for 24 hours. Caveolin‐1 expression was determined by Western blot. We further tested the responses in human adipose tissue from obese individuals (n=12, age: 45.4 ± 9.2 years, BMI: 48.9 ± 7.3 kg/m2) undergoing surgery. Tissue was incubated with drugs for 24 h and caveolin‐1 expression was determined. Furthermore, the effect of drugs on leptin‐dependent activation of ERK and STAT3 was examined by western blot.ResultsAmong the drugs investigated, only treatment with increasing concentrations of metformin resulted in a dose‐dependent decease in caveolin‐1 protein (p=0.01) expression in dHWP. Treatment with increasing concentrations of troglitazone upregulated caveolin‐1 (p=0.01), while pioglitazone (p=0.50) and glimepiride (p=0.09) had no effect of caveolin‐1 protein expression. Given these findings, only metformin was tested in human adipose tissue. Overall, metformin did not alter caveolin‐1 expression in adipose tissue from obese individuals (n=12, control: 0.040 ± 0.056, metformin: 0.024 ± 0.030, p=0.57), although a metformin dependent decrease in caveolin‐1 was evident in adipose tissue from 7 of the 12 obese subjects (control: 0.062 ± 0.066, metformin: 0.014 ± 0.013, p=0.02). Notably, changes in caveolin‐1 expression induced by metformin treatment were positively correlated with BMI (p=0.05, r2=0.33, n=12). Also, leptin dependent activation of ERK (control: 0.26 ± .098, leptin: 0.28 ± .08) and STAT3 (control: 0.028 ± 0.027, leptin: 0.032 ± 0.023) was impaired in obese adipose tissue and did not change with metformin treatment even in the 7 individuals with metformin associated decreases in caveolin‐1.ConclusionsMetformin reduces caveolin‐1 expression in differentiated HWP. However, metformin decreases caveolin‐1 expression in adipose tissue of some but not all obese subjects, and does not have any effects on leptin signaling. The heterogeneous effects of metformin on caveolin‐1 expression in adipose tissue from obese individuals may be related to BMI and needs further investigation.Support or Funding InformationMAP and KRS were supported by the American Physiological Society Stride Summer Undergraduate Research Fellowship NHLBI 1 R25 HL115473‐01. PS was supported by AHA scientist development grant 11SDG7260046.