Fetuin B in white adipose tissue induces inflammation and is associated with peripheral insulin resistance in mice and humans.

OBJECTIVE: Fetuin B is a steatosis-responsive hepatokine that causes glucose intolerance in mice, but the underlying mechanisms remain incompletely described. This study aimed to elucidate the mechanisms of action of fetuin B by investigating its putative effects on white adipose tissue metabolism. METHODS: First, fetuin B gene and protein expression was measured in multiple organs in mice and in cultured adipocytes. Next, the authors performed a hyperinsulinemic-euglycemic clamp in mice and in humans to examine the link between white adipose tissue fetuin B content and indices of insulin sensitivity. Finally, the effect of fetuin B on inflammation was investigated in cultured adipocytes by quantitative polymerase chain reaction and full RNA sequencing. RESULTS: This study demonstrated in adipocytes and mice that fetuin B was produced and secreted by the liver and taken up by adipocytes and adipose tissue. There was a strong negative correlation between white adipose tissue fetuin B content and peripheral insulin sensitivity in mice and in humans. RNA sequencing and polymerase chain reaction analysis revealed that fetuin B induced an inflammatory response in adipocytes. CONCLUSIONS: Fetuin B content in white adipose tissue strongly associated with peripheral insulin resistance in mice and humans. Furthermore, fetuin B induced a proinflammatory response in adipocytes, which might drive peripheral insulin resistance.

A simple preparation step to remove excess liquid lipids in white adipose tissue enabling improved detection of metabolites via MALDI-FTICR imaging MS.

Matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) imaging mass spectrometry (MS) is a powerful technology used to analyze metabolites in various tissues. However, it faces significant challenges in studying adipose tissues. Poor matrix distribution and crystallization caused by excess liquid lipids on the surface of tissue sections hamper m/z species detection, an adverse effect that particularly presents in lipid-rich white adipose tissue (WAT). In this study, we integrated a simple and low-cost preparation step into the existing MALDI-FTICR imaging MS pipeline. The new method-referred to as filter paper application-is characterized by an easy sample handling and high reproducibility. The aforementioned filter paper is placed onto the tissue prior to matrix application in order to remove the layer of excess liquid lipids. Consequently, MALDI-FTICR imaging MS detection was significantly improved, resulting in a higher number of detected m/z species and higher ion intensities. After analyzing various durations of filter paper application, 30 s was found to be optimal, resulting in the detection of more than 3700 m/z species. Apart from the most common lipids found in WAT, other molecules involved in various metabolic pathways were detected, including nucleotides, carbohydrates, and amino acids. Our study is the first to propose a solution to a specific limitation of MALDI-FTICR imaging MS in investigating lipid-rich WAT. The filter paper approach can be performed quickly and is particularly effective for achieving uniform matrix distribution on fresh frozen WAT while maintaining tissue integrity. It thus helps to gain insight into the metabolism in WAT.

Comprehensive Sex Steroid Profiling in Multiple Tissues Reveals Novel Insights in Sex Steroid Distribution in Male Mice.

A comprehensive atlas of sex steroid distribution in multiple tissues is currently lacking, and how circulating and tissue sex steroid levels correlate remains unknown. Here, we adapted and validated a gas chromatography tandem mass spectrometry method for simultaneous measurement of testosterone (T), dihydrotestosterone (DHT), androstenedione, progesterone (Prog), estradiol, and estrone in mouse tissues. We then mapped the sex steroid pattern in 10 different endocrine, reproductive, and major body compartment tissues and serum of gonadal intact and orchiectomized (ORX) male mice. In gonadal intact males, high levels of DHT were observed in reproductive tissues, but also in white adipose tissue (WAT). A major part of the total body reservoir of androgens (T and DHT) and Prog was found in WAT. Serum levels of androgens and Prog were strongly correlated with corresponding levels in the brain while only modestly correlated with corresponding levels in WAT. After orchiectomy, the levels of the active androgens T and DHT decreased markedly while Prog levels in male reproductive tissues increased slightly. In ORX mice, Prog was by far the most abundant sex steroid, and, again, WAT constituted the major reservoir of Prog in the body. In conclusion, we present a comprehensive atlas of tissue and serum concentrations of sex hormones in male mice, revealing novel insights in sex steroid distribution. Brain sex steroid levels are well reflected by serum levels and WAT constitutes a large reservoir of sex steroids in male mice. In addition, Prog is the most abundant sex hormone in ORX mice.

Extracellular vesicle secretion is tissue-dependent ex vivo and skeletal muscle myofiber extracellular vesicles reach the circulation in vivo.

Extracellular vesicles (EVs) are biomarkers and modifiers of human disease. EVs secreted by insulin-responsive tissues like skeletal muscle (SkM) and white adipose tissue (WAT) contribute to metabolic health and disease but the relative abundance of EVs from these tissues has not been directly examined. Human Protein Atlas data and directly measuring EV secretion in mouse SkM and WAT using an ex vivo tissue explant model confirmed that SkM tissue secretes more EVs than WAT. Differences in EV secretion between SkM and WAT were not due to SkM contraction but may be explained by differences in tissue metabolic capacity. We next examined how many EVs secreted from SkM tissue ex vivo and in vivo are myofiber-derived. To do this, a SkM myofiber-specific dual fluorescent reporter mouse was created. Spectral flow cytometry revealed that SkM myofibers are a major source of SkM tissue-derived EVs ex vivo and EV immunocapture indicates that ∼5% of circulating tetraspanin-positive EVs are derived from SkM myofibers in vivo. Our findings demonstrate that 1) SkM secretes more EVs than WAT, 2) many SkM tissue EVs are derived from SkM myofibers, and 3) SkM myofiber-derived EVs reach the circulation in vivo. These findings advance our understanding of EV secretion between metabolically active tissues and provide direct evidence that SkM myofibers secrete EVs that can reach the circulation in vivo.

Role of Glucose-6-Phosphate in Metabolic Adaptation of Staphylococcus aureus in Diabetes.

Diabetic foot ulcer (DFU) is the most common and costly sequela of diabetes mellitus, often leading to lower-extremity amputation with poor 5-year survival rates. Staphylococcus aureus is the most prevalent pathogen isolated from DFU, suggesting adaptation of S. aureus to the unique metabolic conditions of diabetes. Diabetes is a complex metabolic disorder with increases not only in serum glucose levels but also in levels of other sugars, including fructose, mannose, and glucose-6-phosphate (G6P). However, the effect of metabolism of these sugars on the pathogenesis of S. aureus is not fully understood. In this study, we demonstrated that metabolism of G6P, fructose, and mannose induced greater expression of staphylococcal virulence factors than did glucose metabolism, but only G6P effects were independent of glucose-mediated carbon catabolite repression, suggesting a physiologically relevant role in diabetes. Our in vivo studies further demonstrated that G6P was highly present in skin adipose tissues of diabetic TALLYHO/JngJ mice, and subcutaneous infection with S. aureus caused significantly greater tissue necrosis and bacterial burden, compared to nondiabetic SWR/J mice. Finally, enhanced pathogenesis of S. aureus in diabetic TALLYHO/JngJ mice was significantly attenuated by deletion of the hexose phosphate transport (HPT) system. These results suggest that G6P is an important metabolic signal for S. aureus, enhancing the virulence in diabetes. A better understanding of how G6P metabolism is linked to the virulence of S. aureus will lead to the development of novel alternative therapeutics. IMPORTANCE Sugars are essential nutrients for S. aureus to survive and proliferate within the host. Because elevated serum glucose levels are a hallmark of diabetes, most studies have focused on the effect of glucose metabolism, and very little is known regarding the effects of metabolism of other sugars on the pathogenesis of S. aureus in diabetes. In this study, we demonstrated that G6P, which is highly present in diabetes, can induce expression of staphylococcal virulence factors that cause severe tissue necrosis and bacterial burden in skin infections. Our results highlight the importance of nutritional control of blood sugar levels, not only glucose but also other highly metabolizable sugars such as G6P. A better understanding of how activation of the HPT system is linked to the virulence of S. aureus will guide development of novel alternative therapeutics.

An RNAi Screening of Clinically Relevant Transcription Factors Regulating Human Adipogenesis and Adipocyte Metabolism.

CONTEXT: Healthy hyperplasic (many but smaller fat cells) white adipose tissue (WAT) expansion is mediated by recruitment, proliferation and/or differentiation of new fat cells. This process (adipogenesis) is controlled by transcriptional programs that have been mostly identified in rodents. OBJECTIVE: A systemic investigation of adipogenic human transcription factors (TFs) that are relevant for metabolic conditions has not been revealed previously. METHODS: TFs regulated in WAT by obesity, adipose morphology, cancer cachexia, and insulin resistance were selected from microarrays. Their role in differentiation of human adipose tissue-derived stem cells (hASC) was investigated by RNA interference (RNAi) screen. Lipid accumulation, cell number, and lipolysis were measured for all screened factors (148 TFs). RNA (RNAseq), protein (Western blot) expression, insulin, and catecholamine responsiveness were examined in hASC following siRNA treatment of selected target TFs. RESULTS: Analysis of TFs regulated by metabolic conditions in human WAT revealed that many of them belong to adipogenesis-regulating pathways. The RNAi screen identified 39 genes that affected fat cell differentiation in vitro, where 11 genes were novel. Of the latter JARID2 stood out as being necessary for formation of healthy fat cell metabolic phenotype by regulating expression of multiple fat cell phenotype-specific genes. CONCLUSION: This comprehensive RNAi screening in hASC suggests that a large proportion of WAT TFs that are impacted by metabolic conditions might be important for hyperplastic adipose tissue expansion. The screen also identified JARID2 as a novel TF essential for the development of functional adipocytes.

Beige Adipose Tissue Identification and Marker Specificity-Overview.

Adipose tissue (AT) is classified based on its location, physiological and functional characteristics. Although there is a clear demarcation of anatomical and molecular features specific to white (WAT) and brown adipose tissue (BAT), the factors that uniquely differentiate beige AT (BeAT) remain to be fully elaborated. The ubiquitous presence of different types of AT and the inability to differentiate brown and beige adipocytes because of similar appearance present a challenge when classifying them one way or another. Here we will provide an overview of the latest advances in BeAT, BAT, and WAT identification based on transcript markers described in the literature. The review paper will highlight some of the difficulties these markers pose and will offer new perspectives on possible transcript-specific identification of BeAT. We hope that this will advance the understanding of the biology of different ATs. In addition, concrete strategies to distinguish different types of AT may be relevant to track the efficacy and mechanisms around interventions aimed to improve metabolic health and thwart excessive weight gain.

Subcutaneous adipose tissue composition and function are unaffected by liraglutide-induced weight loss in adults with type 1 diabetes.

Adipose tissue is the primary energy reservoir of the human body, which also possesses endocrine functions. The glucagon-like peptide agonist liraglutide produces weight loss, although the specific effects on adipose tissue are unknown. We aimed to characterize the white adipose tissue composition and pericellular fibrosis of subcutaneous adipose tissue in response to liraglutide treatment. Furthermore, we explored the level of circulating free fatty acids, cluster of differentiation 163 (CD163) macrophage marker, leptin and adiponectin. Thirty-nine adults with type 1 diabetes and polyneuropathy were randomly assigned to 26 weeks of liraglutide or placebo treatment. Biopsies of subcutaneous tissue were formalin-fixed stained with picrosirius red to visualize collagen or immunohistochemically stained for CD163. Serum concentrations of free fatty acids, CD163, leptin and adiponectin were assessed with immunoassays or multiplex panels. In comparison with placebo, liraglutide induced weight loss (3.38 kg, 95% CI -5.29; -1.48, P < 0.001), but did not cause any differences in cell size, distribution of CD163-positive cells, pericellular fibrosis and serum levels of free fatty acids, CD163, leptin or adiponectin (all P < 0.1). Additionally, no associations between weight loss, cell size and serum markers were found (all P > 0.08). In conclusion, despite liraglutide's effect on weight loss, sustained alterations in subcutaneous adipose tissue did not seem to appear.

White adipose tissue-infiltrated CD11b+ myeloid cells are a source of S100A4, a new potential marker of hepatic damage.

CONTEXT: The endocrine and immunological properties of subcutaneous vs visceral adipose tissue (sWAT and vWAT, respectively) have turned a milestone in the study of metabolic diseases. The cytokine S100A4 is increased in obesity and has a role in adipose tissue dysfunction. However, the cellular source and its potential role in hepatic damage in obesity has not been elucidated. OBJECTIVE: We aim to study the regulation of S100A4 in immune cells present in sWAT and vWAT, as well as its potential role as a circulating marker of hepatic inflammation and steatosis. DESIGN: A cohort of 60 patients with obesity and distinct metabolic status was analyzed. CD11b+ myeloid cells and T cells were isolated from sWAT and vWAT by magnetic-activating cell sorting, and RNA was obtained. S100A4 gene expression was measured, and correlation analysis with clinical data was performed. Liver biopsies were obtained from 20 patients, and S100A4 circulating levels were measured to check the link with hepatic inflammation and steatosis. RESULTS: S100A4 gene expression was strongly upregulated in sWAT- vs vWAT-infiltrated CD11b+ cells, but this modulation was not observed in T cells. S100A4 mRNA levels from sWAT (and not from vWAT) CD11b+ cells positively correlated with glycemia, triglycerides, TNF-α gene expression and proliferation markers. Finally, circulating S100A4 directly correlated with liver steatosis and hepatic inflammatory markers. CONCLUSION: Our data suggest that sWAT-infiltrated CD11b+ cells could be a major source of S100A4 in obesity. Moreover, our correlations identify circulating S100A4 as a potential novel biomarker of hepatic damage and steatosis.

Anti-obesity effects of DHA and EPA in high fat-induced insulin resistant mice.

Docosahexaenoic acid (DHA, 22:6) and eicosapentaenoic acid (EPA, 20:5) have been reported to improve metabolic disorders, but their differential effects on anti-obesity under insulin resistance (IR) are still unclear. We fed IR mice with high-fat diet with added 1%, 2%, 4% (w/w) DHA or EPA for 12 weeks. Changes in weight, food intake, white adipose tissue (WAT), liver and blood lipids were assessed. GPR120 and PPARγ of WAT were evaluated to explore the related molecular mechanisms of DHA and EPA for anti-obesity in IR mice. 1%DHA and 1%EPA inhibit adipogenesis by down-regulating GPR120; 4%DHA stimulates browning of WAT and improves IR and inflammatory infiltration by up-regulating PPARγ; 4%EPA exerts its anti-obesity effect by mechanisms independent of PPARγ and GPR120 signaling.

Assessment of mitochondrial physiology of murine white adipose tissue by mechanical permeabilization and lipid depletion.

White adipose tissue (WAT) represents a major site of triacylglycerol energy storage and is directly associated with metabolic disorders. Mitochondria regulate cellular energy expenditure and are active in WAT. Although isolated mitochondria have been classically used to assess their functions, several artifacts can be introduced by this approach. Furthermore, important limitations exist in the available methods to determine mitochondrial physiology in permeabilized WAT. Here, we established and validated a method for functional evaluation of mice mesenteric WAT (mWAT) mitochondria by using MEchanical Permeabilization and LIpid DEpletion (MEPLIDE) coupled to high-resolution respirometry. We observed that mild stirring of mWAT for 20 min at room temperature with 4% fatty acid-free albumin (FAF-BSA) followed by 50 min without FAF-BSA selectively permeabilized white adipocytes plasma membrane. In these conditions, mWAT mitochondria were intact, exhibiting succinate-induced respiratory rates that were sensitive to classical oxidative phosphorylation modulators. Finally, the respiratory capacity of mWAT in female mice was significantly higher than in males, an observation that agrees with reported data. Therefore, the functional assessment of mWAT mitochondria through MEPLIDE coupled to high resolution respirometry proposed here will contribute to a better understanding of WAT biology in several pathophysiological contexts.

Targeted lipidomics and transcriptomics profiling reveal the heterogeneity of visceral and subcutaneous white adipose tissue.

AIMS: The depot-specific differences in lipidome of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) reflect heterogeneity of white adipose tissue (WAT), which plays a central role in its distinct response to outside stimuli. However, the detailed lipidome of depot-specific WAT is largely unknown, especially the minor constitutes including phospholipid and sphingolipid. MATERIALS AND METHODS: To investigate this field, we applied a high-coverage targeted lipidomics approach of VAT and SAT in male C57BL/6J mice to compare the basal level of their lipid profiles. Applying microarray and quantitative real-time polymerase chain reaction, we analyzed the transcriptome of twodepot-specific WAT and verified the differences in individual genes. KEY FINDINGS: In total, 342 lipid species from 19 lipid classes were identified. Our results showed the composition of TAG and FFA were different in length of chain and saturation. Interestingly, low abundance phospholipid, sphingolipid and cardiolipin were significantly higher in SAT. Lipid correlation network analysis vindicated that TAG and phospholipid formed distinct subnet and had more connections with other lipid species. Enriched ontology analysis of gene screened from LIPID MAPS and microarray suggested the differences were mainly involved in lipid metabolism, insulin resistance and inflammatory response. SIGNIFICANCE: Our comprehensive lipidomics and transcriptomics analyses revealed differences in lipid composition and lipid metabolism of two depot-specific WAT, which would offer new insights into the investigation of heterogeneity of visceral and subcutaneous white adipose tissue.

Healthy Obese Subjects Differ in Chronotype, Sleep Habits, and Adipose Tissue Fatty Acid Composition from Their Non-Healthy Counterparts.

Obesity is not the same in all individuals and two different phenotypes have been described: metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUO). The aim of this study was to identify factors that explain metabolic health status in a rigorously matched Spanish population. Subcutaneous and visceral fat, adipocyte size and fatty acid composition, cardiometabolic markers in serum, and lifestyle habits were assessed. Higher physical activity in the mornings (Odds Ratio (95% Confidence Interval) (OR (95% CI) = 1.54 (1.09-2.18), p = 0.01)), earlier bedtimes (8:30-10:30 pm) (OR = 2.11 (1.02-4.36), p = 0.04), a complete breakfast (OR = 1.59 (1.07-2.36), p = 0.02), and a greater number of meals per day (4.10 ± 0.05 vs. 3.93 ± 0.05, p < 0.01), were associated with the MHO phenotype. Concentrations of 20:5 n-3 eicosapentaenoic acid (0.26 ± 0.46 vs. 0.10% ± 0.11%, p = 0.04) and 18:3 n-6 gamma-linolenic acid (0.37 ± 0.24 vs. 0.23% ± 0.22%, p = 0.04) in subcutaneous adipocytes were higher and omental adipocyte size (187 094 ± 224 059 µm3 vs. 490 953 ± 229 049 µm3, p = 0.02) was lower in MHO subjects than in those with MUO. Visceral fat area differed between MHO and MUO subjects (135 ± 60 cm2 vs. 178 ± 85 cm2, p = 0.04, respectively). The study highlights specific lifestyle habits that could form part of obesity therapies, not only involving healthier eating habits but also earlier sleeping and exercise patterns.

Potential Role of Leptin in Cardiac Steatosis Induced by Highly Saturated Fat Intake during Adolescence.

SCOPE: To identify the age-dependent effect of diets containing elevated amounts of either saturated or unsaturated fatty acids on cardiac steatosis in mice. METHODS AND RESULTS: Five- and eight-week-old C57BL/6J mice cohorts are given free access to either a saturated or an unsaturated fatty-acid-enriched diet during 8 weeks. Body weight (BW) and food intake are monitored during this period. Cardiac lipid content, carnitine palmitoyltransferase-I (CPT-I) activity, and the amount of uncoupling proteins 2 and 3 (UCP2 and UCP3) are analyzed and correlated with blood leptin concentration. Leptin and PPARγ gene expression is quantified in white adipose tissue (WAT). Both diets have a similar effect on food intake, BW, and adiposity, independently of the age. Nevertheless, cardiac steatosis is specifically identified in adolescent mice consuming the saturated diet. These animals also display lower activity of cardiac CPT-I, a down-regulation of cardiac UCP2, together with lower concentration of plasma leptin. Accordingly, leptin gene expression is reduced in the visceral WAT. CONCLUSION: Consumption of diets containing elevated amounts of saturated fat during adolescence and early adult life promotes cardiac steatosis in mice. An insufficient endocrine activity of WAT, in terms of leptin production, may account for such an effect.

Cachexia-related long noncoding RNA, CAAlnc1, suppresses adipogenesis by blocking the binding of HuR to adipogenic transcription factor mRNAs.

Cancer-associated cachexia (CAC) is a devastating syndrome characterized by progressive losses of adipose tissue and skeletal muscle. CAC-related adipose tissue loss (CAL) occurs early and is associated with a shorter survival time. To explore potential regulatory long noncoding RNAs (lncRNAs) of CAL, RNA microarrays were used to analyze the transcriptomes of white adipose tissue from CAC mice vs. control mice. A set of differentially expressed lncRNAs was identified, and among them was CAAlnc1, which suppressed adipogenesis of C3H10 cells as demonstrated by gain-of-function and loss-of-function experiments. RNA immunoprecipitation and pull-down assays revealed Hu antigen R (HuR) was an important binding partner of CAAlnc1. The interaction between CAAlnc1 and HuR blocked the binding of HuR to adipogenic transcription factor mRNAs and further downregulated the expression of these transcription factors. This study generated a list of CAL-related lncRNAs and provided details of a functional lncRNA which may play an important role in CAL.

Maternal high-fat diet during suckling programs visceral adiposity and epigenetic regulation of adipose tissue stearoyl-CoA desaturase-1 in offspring.

OBJECTIVE: The lactation-suckling period is critical for white adipose tissue (WAT) development. Early postnatal nutrition influences later obesity risk but underlying mechanisms remain elusive. Here, we tested whether altered postnatal nutrition specifically during suckling impacts epigenetic regulation of key metabolic genes in WAT and alter long-term adiposity set point. METHODS: We analyzed the effects of maternal high-fat (HF) feeding in rats exclusively during lactation-suckling on breast milk composition and its impact on male offspring visceral epidydimal (eWAT) and subcutaneous inguinal (iWAT) depots during suckling and in adulthood. RESULTS: Maternal HF feeding during lactation had no effect on mothers' body weight (BW) or global breast milk composition, but induced qualitative changes in breast milk fatty acid (FA) composition (high n-6/n-3 polyunsaturated FA ratio and low medium-chain FA content). During suckling, HF neonates showed increased BW and mass of both eWAT and iWAT depot but only eWAT displayed an enhanced adipogenic transcriptional signature. In adulthood, HF offspring were predisposed to weight gain and showed increased hyperplastic growth only in eWAT. This specific eWAT expansion was associated with increased expression and activity of stearoyl-CoA desaturase-1 (SCD1), a key enzyme of FA metabolism. SCD1 converts saturated FAs, e.g. palmitate and stearate, to monounsaturated FAs, palmitoleate and oleate, which are the predominant substrates for triglyceride synthesis. Scd1 upregulation in eWAT was associated with reduced DNA methylation in Scd1 promoter surrounding a PPARγ-binding region. Conversely, changes in SCD1 levels and methylation were not observed in iWAT, coherent with a depot-specific programming. CONCLUSIONS: Our data reveal that maternal HF feeding during suckling programs long-term eWAT expansion in part by SCD1 epigenetic reprogramming. This programming events occurred with drastic changes in breast milk FA composition, suggesting that dietary FAs are key metabolic programming factors in the early postnatal period.

Analysis of fatty acids in mouse tissue via in situ transmethylation with biochar.

Lipid derivatization technology-mediated fatty acid profiling studies have been suggested to dissect the contents of lipids in white fat and brown fat tissue. The focus of this study is to profile fatty acid lipidomics in brown adipose tissue and white adipose tissue of mice by derivatizing their lipids into fatty acid methyl esters via in situ transmethylation using a rice husk-derived biochar as porous media. The in situ transmethylation using biochar is advantageous in biological analysis because there was no loss of samples inevitably occurring in the loss of lipid in solvent extraction and purification steps.

Role of cAMP and cGMP Signaling in Brown Fat.

Cold-induced activation of brown adipose tissue (BAT) is mediated by norepinephrine and adenosine that are released during sympathetic nerve activation. Both signaling molecules induce an increase in intracellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) in murine and human BAT. In brown adipocytes, cAMP plays a central role, because it activates lipolysis, glucose uptake, and thermogenesis. Another well-studied intracellular second messenger is 3',5'-cyclic guanosine monophosphate (cGMP), which closely resembles cAMP. Several studies have shown that intact cGMP signaling is essential for normal adipogenic differentiation and BAT-mediated thermogenesis in mice. This chapter highlights recent observations, demonstrating the physiological significance of cyclic nucleotide signaling in BAT as well as their potential to induce browning of white adipose tissue (WAT) in mice and humans.

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining.

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

9-PAHSA promotes browning of white fat via activating G-protein-coupled receptor 120 and inhibiting lipopolysaccharide / NF-kappa B pathway.

Browning of white adipose tissue is a novel mechanism to counteract obesity in view of its thermogenic activity. Activation of G-protein-coupled receptor 120 (GPR120) can promote the browning of white fat. 9-PAHSA, an endogenous mammalian lipid, which is acting as the ligand of GPR120 to enhance glucose uptake and exert anti-inflammatory effect. In the study, we would like to investigate the biological effects of 9-PAHSA on adipocyte browning. Here, we show that 9-PAHSA induces browning of 3T3-L1 adipocytes via enhanced expression of brown fat specific genes. 9-PAHSA-induced browning in white adipocytes of WT mice and ob/ob mice was investigated by determining expression levels of brown adipocyte-specific genes/proteins by quantitative real-time polymerase chain reaction analysis, immunoblot analysis and immunochemical staining. The effects of 9-PAHSA on brown fat markers in 3T3-L1 cells were decreased when GPR120 gene was silenced. To investigate the molecular mechanism of 9-PAHSA on adipocyte browning, lipopolysaccharide (LPS)-induced inflammatory model was conducted. 9-PAHSA treatment abolished LPS-induced NF-kappa B (NF-κB) activation and inflammatory cytokine secretion. But these anti-inflammatory effects of 9-PAHSA were attenuated by GPR120 knockdown. Our finding demonstrated that the browning of adipocyte was induced by 9-PAHSA through activating GPR120 and inhibiting the LPS/NF-κB pathway. This promising result will help to reveal the potential pathogenesis of obesity.