Brown Preadipocytes Research Articles

Hepatic SerpinA1 improves energy and glucose metabolism through regulation of preadipocyte proliferation and UCP1 expression.

Lipodystrophy and obesity are associated with insulin resistance and metabolic syndrome accompanied by fat tissue dysregulation. Here, we show that serine protease inhibitor A1 (SerpinA1) expression in the liver is increased during recovery from lipodystrophy caused by the adipocyte-specific loss of insulin signaling in mice. SerpinA1 induces the proliferation of white and brown preadipocytes and increases the expression of uncoupling protein 1 (UCP1) to promote mitochondrial activation in mature white and brown adipocytes. Liver-specific SerpinA1 transgenic mice exhibit increased browning of adipose tissues, leading to increased energy expenditure, reduced adiposity and improved glucose tolerance. Conversely, SerpinA1 knockout mice exhibit decreased adipocyte mitochondrial function, impaired thermogenesis, obesity, and systemic insulin resistance. SerpinA1 forms a complex with the Eph receptor B2 and regulates its downstream signaling in adipocytes. These results demonstrate that SerpinA1 is an important hepatokine that improves obesity, energy expenditure and glucose metabolism by promoting preadipocyte proliferation and activating mitochondrial UCP1 expression in adipocytes.

Impaired energy expenditure following exposure to either DDT or DDE in mice may be mediated by DNA methylation changes in brown adipose.

The insecticide dichlorodiphenyltrichloroethane (DDT) and its persistent metabolite, dichlorodiphenyldichloroethylene (DDE), have been associated with increased adiposity and obesity in multiple generations of rodents and humans. These lipophilic pollutants accumulate in adipose tissue and appear to decrease energy expenditure through the impairment of thermogenesis in brown adipose tissue (BAT). We hypothesized that impaired thermogenesis is due to persistent epigenetic modifications of BAT. To address this, we exposed C57BL/6 J mice to DDT or DDE from gestational day (GD) 11.5 to postnatal day (PND) 5, evaluated longitudinal body temperature, and performed reduced representation bisulfite sequencing and RNA sequencing of BAT from infant and adult offspring. Exposure to DDT or DDE reduced core body temperature in adult mice, and differential methylation at the pathway and gene level was persistent from infancy to adulthood. Furthermore, thermogenesis and biological pathways essential for thermogenic function, such as oxidative phosphorylation and mechanistic target of rapamycin kinase (mTOR) signaling, were enriched with differential methylation and RNA transcription in adult mice exposed to DDT or DDE. PAZ6 human brown preadipocytes were differentiated in the presence of DDT or DDE to understand the brown adipocyte-autonomous effect of these pollutants. In vitro exposure led to limited changes in RNA expression; however, mitochondrial membrane potential was decreased in vitro with 0.1 µM and 1 µM doses of DDT or DDE. These results demonstrate that concentrations of DDT and DDE relevant to human exposure have a significant effect on thermogenesis, the transcriptome, and DNA methylome of mouse BAT and the mitochondrial function of human brown adipocytes.

UCP1 expression in human brown adipose tissue is inversely associated with cardiometabolic risk factors.

Brown adipose tissue (BAT) is a therapeutic target for obesity. 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) is commonly used to quantify human BAT mass and activity. Detectable 18F-FDG uptake by BAT is associated with reduced prevalence of cardiometabolic disease. However, 18F-FDG uptake may not always be a reliable marker of BAT thermogenesis, for example, insulin resistance may reduce glucose uptake. Uncoupling protein 1 (UCP1) is the key thermogenic protein in BAT. Therefore, we hypothesised that UCP1 expression may be altered in individuals with cardiometabolic risk factors. We quantified UCP1 expression as an alternative marker of thermogenic capacity in BAT and white adipose tissue (WAT) samples (n = 53) and in differentiated brown and white pre-adipocytes (n = 85). UCP1 expression in BAT, but not in WAT or brown/white differentiated pre-adipocytes, was reduced with increasing age, obesity, and adverse cardiometabolic risk factors such as fasting glucose, insulin, and blood pressure. However, UCP1 expression in BAT was preserved in obese subjects of <40 years of age. To determine if BAT activity was also preserved in vivo, we undertook a case-control study, performing 18F-FDG scanning during mild cold exposure in young (mean age ∼22 years) normal weight and obese volunteers. 18F-FDG uptake by BAT and BAT volume were similar between groups, despite increased insulin resistance. 18F-FDG uptake by BAT and UCP1 expression are preserved in young obese adults. Older subjects retain precursor cells with the capacity to form new thermogenic adipocytes. These data highlight the therapeutic potential of BAT mass expansion and activation in obesity.

CTRP9 Promotes Brown Adipose Tissue Lipolysis in Mice Fed a High-Fat Diet.

This study aimed to elucidate the molecular mechanism through which C1q/tumor necrosis factor (TNF)-related protein 9 (CTRP9) acts in the formation and differentiation of brown adipose tissue (BAT). Adenovirus particles encoding CTRP9 and green fluorescent protein were inoculated into the scapula of C57BL/6J mice and fed a high-fat diet for 8 weeks; the body weight, lipid droplet morphology, glucose tolerance, insulin tolerance, and protein expression levels were analyzed. In addition, CTRP9 adenovirus was transfected into brown preadipocytes, and differentiation was induced to identify the effect of CTRP9 overexpression on adipocyte differentiation. CTRP9 overexpression significantly increased the weight gain of mice. Additionally, the CTRP9 overexpression group exhibited significantly increased adipose tissue weight and glucose clearance rates and decreased insulin sensitivity and serum triglyceride levels compared to the control group. Furthermore, CTRP9 overexpression significantly upregulated the adipose triglyceride lipase (ATGL) and perilipin 1 protein expression levels in BAT. The cell experiment results confirmed that CTRP9 overexpression significantly inhibited the adipogenesis of brown adipocytes as evidenced by the downregulation of uncoupling protein 1, beta-3 adrenergic receptor, ATGL, and hormone-sensitive lipase mRNA levels and the significant suppression of uncoupling protein 1, ATGL, and perilipin 1 protein levels in brown adipocytes. The finding of this study demonstrated that CTRP9 promotes lipolysis by upregulating ATGL expression in vivo and inhibits the differentiation of brown preadipocytes in vitro.

1699-P: The Conversion and Potential Mechanism of Conditionally Immortalized Human Classical Brown Preadipocytes from Proliferative to Adipogenic States

1699-P: The Conversion and Potential Mechanism of Conditionally Immortalized Human Classical Brown Preadipocytes from Proliferative to Adipogenic States

Neuronostatin regulates proliferation and differentiation of rat brown primary preadipocytes.

Neuronostatin suppresses the differentiation of white preadipocytes. However, the role of neuronostatin in brown adipose tissue remains elusive. Therefore, we investigated the impact of neuronostatin on the proliferation and differentiation of isolated rat brown preadipocytes. We report that neuronostatin and its receptor (GPR107) are synthesized in brown preadipocytes and brown adipose tissue. Furthermore, neuronostatin promotes the replication of brown preadipocytes via the AKT pathway. Notably, neuronostatin suppresses the expression of markers associated with brown adipogenesis (PGC-1α, PPARγ, PRDM16, and UCP1) and reduces cellular mitochondria content. Moreover, neuronostatin impedes the differentiation of preadipocytes by activating the JNK signaling pathway. These effects were not mimicked by somatostatin. Our results suggest that neuronostatin is involved in regulating brown adipogenesis.

Cell-autonomous Action of the FGF19 Promotes UCP1 Expression and Function in Human Brown Adipocytes

Fibroblast growth factor 19 (FGF19) is an intestine-derived hormone that regulates glucose and energy metabolism. Central and systemic administration of human FGF19 in mice promotes increased energy expenditure and body weight loss through mechanisms that are not fully understood. In this study, we investigated whether FGF19 can activate thermogenesis in brown adipocytes (BA) in a cell-autonomous manner. We differentiated immortalized human brown preadipocytes into mature adipocytes with adipogenic induction media in the presence and absence of FGF19 for 18 days to evaluate the effects of FGF19 on adipogenesis and thermogenesis. Through qPCR and RNA-sequencing analyses, we observed that FGF19 receptors were expressed in both brown preadipocytes and mature BA. However, the obligatory FGF19 co-receptor, β-klotho, was not expressed in preadipocytes, suggesting that the effects of FGF19 must occur preferentially in mature BA. Lipid staining with Oil Red O as well as gene expression of the adipogenic markers fatty acid binding protein (FABP4) and peroxisome proliferator activated receptor-γ (PPAR-γ) showed that adipogenesis was not affected by FGF19. We then treated mature BA with FGF19 (100 ng/mL) acutely and chronically to evaluate FGF19-induced changes in signaling, gene transcription, and thermogenic function. FGF19 signaling was assessed by measuring ERK1/2 phosphorylation levels, which increased within 5 min and remained elevated for at least 30 min after treatment. Using Seahorse assays, we observed that 24-hour FGF19 treatment promoted increased basal respiration, ATP production, and proton leak in human BA, indictive of enhanced mitochondrial function. Since proton leak across the inner membrane through the uncoupling protein 1 (UCP1) generates heat, we assessed the expression of UCP1 and other thermogenic genes. Interestingly, FGF19 treatment specifically increased UCP1 gene expression without affecting other classical adrenergic-mediated thermogenic genes, such as deiodinase 2 (DIO2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), PR domain containing 16 (PRDM16), and cell death-inducing DNA fragmentation factor A (CIDEA). These findings suggest that FGF19 regulates UCP1 expression through non-canonical pathways. Future work will elucidate the mechanism by which FGF19 promotes UCP1 gene expression in mature BA. Taken together, FGF19 stimulates ERK1/2 phosphorylation and UCP1 expression in human BA, leading to enhanced mitochondrial respiration. These results provide new mechanistic insights into FGF19’s effects on metabolic regulation. Topic subcategories: Adipocyte biology. Funding sources: Sao Paulo Research Foundation (FAPESP): 2022/15148-2 and NIH R01 DK102898. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gene Regulation and Mitochondrial Activity During White and Brown Adipogenesis Are Modulated by KDM5 Histone Demethylase.

Body fat accumulation differs between males and females and is influenced by both gonadal sex (ovaries vs testes) and chromosomal sex (XX vs XY). We previously showed that an X chromosome gene, Kdm5c, is expressed at higher levels in females compared to males and correlates with adiposity in mice and humans. Kdm5c encodes a KDM5 histone demethylase that regulates gene expression by modulating histone methylation at gene promoters and enhancers. Here, we use chemical inhibition and genetic knockdown to identify a role for KDM5 activity during early stages of white and brown preadipocyte differentiation, with specific effects on white adipocyte clonal expansion, and white and brown adipocyte gene expression and mitochondrial activity. In white adipogenesis, KDM5 activity modulates H3K4 histone methylation at the Dlk1 gene promoter to repress gene expression and promote progression from preadipocytes to mature adipocytes. In brown adipogenesis, KDM5 activity modulates H3K4 methylation and gene expression of Ucp1, which is required for thermogenesis. Unbiased transcriptome analysis revealed that KDM5 activity regulates genes associated with cell cycle regulation and mitochondrial function, and this was confirmed by functional analyses of cell proliferation and cellular bioenergetics. Using genetic knockdown, we demonstrate that KDM5C is the likely KDM5 family member that is responsible for regulation of white and brown preadipocyte programming. Given that KDM5C levels are higher in females compared to males, our findings suggest that sex differences in white and brown preadipocyte gene regulation may contribute to sex differences in adipose tissue function.

Mineralocorticoid Receptor Antagonism Prevents Type 2 Familial Partial Lipodystrophy Brown Adipocyte Dysfunction.

Type-2 Familial Partial Lipodystrophy (FPLD2), a rare lipodystrophy caused by LMNA mutations, is characterized by a loss of subcutaneous fat from the trunk and limbs and excess accumulation of adipose tissue in the neck and face. Several studies have reported that the mineralocorticoid receptor (MR) plays an essential role in adipose tissue differentiation and functionality. We previously showed that brown preadipocytes isolated from a FPLD2 patient's neck aberrantly differentiate towards the white lineage. As this condition may be related to MR activation, we suspected altered MR dynamics in FPLD2. Despite cytoplasmic MR localization in control brown adipocytes, retention of MR was observed in FPLD2 brown adipocyte nuclei. Moreover, overexpression of wild-type or mutated prelamin A caused GFP-MR recruitment to the nuclear envelope in HEK293 cells, while drug-induced prelamin A co-localized with endogenous MR in human preadipocytes. Based on in silico analysis and in situ protein ligation assays, we could suggest an interaction between prelamin A and MR, which appears to be inhibited by mineralocorticoid receptor antagonism. Importantly, the MR antagonist spironolactone redirected FPLD2 preadipocyte differentiation towards the brown lineage, avoiding the formation of enlarged and dysmorphic lipid droplets. Finally, beneficial effects on brown adipose tissue activity were observed in an FPLD2 patient undergoing spironolactone treatment. These findings identify MR as a new lamin A interactor and a new player in lamin A-linked lipodystrophies.

OR07-03 UCP1 Expression In Human Brown Adipose Tissue Is Inversely Associated With Cardiometabolic Risk Factors

Abstract Disclosure: T. Kwok: None. L.E. Ramage: None. K.J. Suchacki: None. C. Gray: None. K. Alexandra: None. L.D. Boyle: None. R.K. Semple: None. S.I. Semple: None. T. MacGillivray: None. E.J. van Beek: None. S. Wakelin: None. R.H. Stimson: None. Brown adipose tissue (BAT) is a promising therapeutic target for obesity. 18F-Fluorodeoxyglucose positron emission tomography (18F-FDG PET) is the most commonly used imaging modality to quantify human BAT activity. 18F-FDG uptake may be reduced in obesity but insulin resistance has been suggested as a potential confounding factor. Uncoupling protein 1 (UCP1) is the key thermogenic protein in BAT and thus serves as a surrogate measure of BAT thermogenic capacity. We quantified UCP1 expression in paired BAT and white adipose tissue (WAT) samples from 141 patients undergoing elective neck surgery. UCP1 mRNA was quantified in whole tissue (n=53) and differentiated pre-adipocytes (n=88). Data are presented as mean ± SEM. Individuals with high UCP1 expression in whole BAT (&amp;gt;2 arbitrary units) were younger (44.2 ± 3.5 vs 55.7 ± 2.0 years) with lower BMI (26.5 ± 1.2 vs 30.1 ± 1.0kg/m2), waist circumference (87.9 ± 2.9 vs 101.1 ± 2.6cm), waist-hip ratio (0.86 ± 0.01 vs 0.92 ± 0.01), fat percentage (29.1 ± 2.6 vs 35.3 ± 1.7%), insulin resistance (HOMA-IR 1.44 ± 0.18 vs 2.69 ± 0.35), systolic (131 ± 6 vs 143 ± 4mmHg) and diastolic blood pressure (79 ± 3 vs 86 ± 2mmHg) (all P&amp;lt;0.05). BAT UCP1 expression was lower in subjects on beta-blockers or with existing hypertension. BAT (but not WAT) UCP1 expression correlated negatively with age (r=−0.305), weight (r=−0.374), waist circumference (r=−0.296), fat mass (r=−0.388) and BMI (r=−0.282) (all P&amp;lt;0.05). However, UCP1 levels in differentiated brown adipocytes were not associated with any of the above measurements. In multiple regression, age was the only independent predictor of high BAT UCP1 expression. Interestingly, BAT UCP1 levels were not reduced in obese subjects aged &amp;lt;40 years. To determine whether BAT activity was preserved in young obese subjects in vivo, we performed 18F-FDG-PET/MR scanning during mild cold exposure (16-17°C) in 6 normal weight (BMI 22 ± 1kg/m2) and 6 obese (BMI 32 ± 1kg/m2) age-matched (22 ± 1 years) subjects. BAT 18F-FDG uptake (standard uptake value 5.1 ± 0.5 vs 4.2 ± 0.6 g/mL) and volume (69 ± 14 vs 72 ± 32 cm3) were similar between weight groups, despite greater insulin resistance in obese subjects (fasting glucose 5.5 ± 0.2 vs 4.3 ± 0.1mmol/L, insulin 11.9 ± 1.3 vs 5.8 ± 0.9mU/L, HOMA-IR 2.9 ± 0.3 vs 1.1 ± 0.2, free fatty acids 465 ± 78 vs 240 ± 34µM, all P&amp;lt;0.05), suggesting that 18F-FDG uptake by BAT is not significantly affected by insulin resistance. In conclusion, BAT UCP1 expression is reduced in individuals with adverse cardiometabolic risk factors, but these subjects retain brown pre-adipocytes with the capacity to form new thermogenic adipocytes after appropriate stimulation. UCP1 expression and cold-induced 18F-FDG uptake by BAT is preserved in young obese subjects. These data highlight the therapeutic potential of BAT mass expansion and activation as a therapeutic strategy to ameliorate the cardiometabolic consequences of obesity. Presentation: Thursday, June 15, 2023

SAT662 Bone Morphogenetic Protein 7 Induces Transdifferentiation Of Human White Preadipocytes Into Thermogenic Beige Adipocytes

Abstract Disclosure: S.L. Ali: None. J. Katz: None. A.M. Cypess: None. Chronic treatment of women with mirabegron, which activates the β3-adrenergic receptor, increases the mass and energy expenditure of thermogenic adipocytes and leads to metabolic benefits including improvement of glucose and lipid homeostasis. In humans, thermogenic adipose tissue is composed of brown and beige/brite (from “brown-in-white”) cells that have different lineages and potentially different functions. Beige cells are of therapeutic interest because they are derived from white adipose tissue, which is present at a considerably higher volume compared to brown adipose tissue. Identifying processes that can transdifferentiate white into beige cells could lead to treatments for obesity-related metabolic disease. We hypothesized that short-term treatment with bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-β (TGF-β) family of cell regulatory proteins, prior to differentiation could be sufficient to induce white to beige transition in human white preadipocytes. We established paired clonal immortalized human white and brown preadipocyte lines and differentiated the white adipocytes ± BMP7 (3.3 nM) for 7 days prior to differentiation. Cells were collected at d0 (undifferentiated) and d30 (fully mature adipocytes) for gene expression analyses. Short-term exposure to BMP7 in white preadipocytes significantly decreased the expression of white-specific markers TCF21 and HOXC8. The beige-specific markers TBX1 and TMEM26, and the thermogenic marker UCP1 were significantly higher in BMP7-treated white adipocytes. However, long term, 30-day exposure to BMP7 treatment reversed the thermogenic transdifferentiation both human white and brown adipocytes. Basal and drug-induced maximal respiration measured via Seahorse XFe96 Analyzer were significantly higher in short-term BMP7-treated white adipocytes. Our data indicate that BMP7 is capable of transdifferentiating human white preadipocytes into thermogenic beige adipocytes. Given that extended treatment reduced the impact of BMP7 indicates that precise timing of interventions will be needed to utilize white-to-beige transdifferentiation to treat obesity-associated metabolic disease. Presentation: Saturday, June 17, 2023

Abstract P3203: Characterization Of Nrac, A Novel Cardiac And Adipose Enriched Microprotein

Objective: Nrac ( n utritionally- r egulated a dipose and c ardiac-enriched gene) was previously identified as a microprotein encoded by the long noncoding RNA A530016L24Rik (human C14orf180) with 2 conserved transmembrane domains. Nrac is highly expressed in the heart as well as in brown and white adipose tissue and its expression is decreased in response to both fasting and obesity. To date, the biological function of Nrac is unknown, however a previous study indicated it may localize to the plasma membrane in white adipocytes. The goal of this study was to examine the molecular function of Nrac in adipocytes and the heart. Methods and Results: Using a brown adipocyte cell culture system, we differentiated pre-adipocytes to mature adipocytes and analyzed Nrac expression levels by qPCR. Nrac expression was undetectable in pre-adipocytes but markedly increased during differentiation and was robustly expressed in mature adipocytes. To determine the subcellular localization of Nrac, we transfected brown pre-adipocytes with a HA-V5-eptipe tagged Nrac fusion protein (HA-V5-Nrac), differentiated these cells to mature brown adipocytes, and performed immunostaining. These experiments showed that Nrac localizes specifically to the endoplasmic reticulum, and that overexpression of Nrac led to a striking increase in intracellular lipid droplet size as determined by BODIPY staining. To analyze Nrac in vivo, we generated a novel Nrac knockout (KO) mouse line using CRISPR/Cas9. Histological analysis of brown adipose tissue (BAT) from these mice revealed that intracellular lipid droplet size was markedly decreased in Nrac KO tissue compared to BAT from WT mice. RNA-sequencing analysis of heart tissue from WT and Nrac KO mice revealed a striking downregulation of genes related to adipogenesis and fatty acid metabolism in Nrac KO hearts and an upregulation in endoplasmic reticulum stress related genes. Conclusions: Our data indicate that Nrac is an endoplasmic reticulum membrane-localized microprotein that is highly expressed in the heart and adipose tissue. Our studies indicate that Nrac is involved in adipogenesis and lipid droplet formation. Future studies will aim to define the molecular mechanisms that contribute to Nrac function in heart and adipose tissue.

310-OR: FBF1 Deficiency Promotes Ciliary Cold-Sensing TRP Channel Activation in Thermogenesis

Brown adipose tissue (BAT) dissipates energy as heat to maintain optimal thermogenesis and promotes metabolic health. Exposure to cold conditions strongly induces both hyperplasia and activity of brown fat by distinct but poorly understood mechanisms. The proliferation of preadipocytes and their subsequent differentiation is proposed to be a major contributor to BAT hyperplasia. Primary cilia are sensory organelles emanating from the surface of most eukaryotic cells. We previously discovered that primary cilia mediate hyperplasia and beiging of the white fat tissue in mice by FBF1-dependent manner. WAT preadipocytes transiently form primary cilia during induced differentiation. FBF1 deficiency promotes proliferation, differentiation, and beiging of WAT preadipocytes and thus leads to expansion of metabolically healthy fat tissue. Here, we discovered that BAT preadipocytes dynamically produce sensory cilia during differentiation. FBF1 deficiency enhances brown preadipocyte differentiation and expansion by stimulating PPARγ transcriptional activity. Consistently, Fbf1tm1a/tm1a mice exhibit BAT hyperplasia. We further discovered that FBF1 deficiency promotes the ciliary recruitment of cold-sensing TRP channels upon cold exposure, which likely explains the mechanism underlying cilia-mediated BAT hyperplasia. Collectively, our data provide molecular insights into coldness-induced BAT hyperplasia and highlight the importance of cilia-mediated TRP pathway in thermogenesis regulation. Disclosure Y.Zhang: None. B.Liu: None. J.Hao: None. Y.Huang: None. K.Ling: None. J.Hu: None.

Angiotensin II type 2 receptor as a novel activator of brown adipose tissue in obesity.

The angiotensin II type 2 receptor (AT2R) exerts vasorelaxant, anti-inflammatory, and antioxidant properties. In obesity, its activation counterbalances the adverse cardiovascular effects of angiotensin II mediated by the AT1R. Preliminary results indicate that it also promotes brown adipocyte differentiation in vitro. Our hypothesis is that AT2R activation could increase BAT mass and activity in obesity. Five-week-old male C57BL/6J mice were fed a standard or a high-fat (HF) diet for 6 weeks. Half of the animals were treated with compound 21 (C21), a selective AT2R agonist, (1 mg/kg/day) in the drinking water. Electron transport chain (ETC), oxidative phosphorylation, and UCP1 proteins were measured in the interscapular BAT (iBAT) and thoracic perivascular adipose tissue (tPVAT) as well as inflammatory and oxidative parameters. Differentiation and oxygen consumption rate (OCR) in the presence of C21 was tested in brown preadipocytes. In vitro, C21-differentiated brown adipocytes showed an AT2R-dependent increase of differentiation markers (Ucp1, Cidea, Pparg) and increased basal and H+ leak-linked OCR. In vivo, HF-C21 mice showed increased iBAT mass compared to HF animals. Both their iBAT and tPVAT showed higher protein levels of the ETC protein complexes and UCP1, together with a reduction of inflammatory and oxidative markers. The activation of the AT2R increases BAT mass, mitochondrial activity, and reduces markers of tissue inflammation and oxidative stress in obesity. Therefore, insulin reduction and better vascular responses are achieved. Thus, the activation of the protective arm of the renin-angiotensin system arises as a promising tool in the treatment of obesity.

Mapping the transcriptional landscape of human white and brown adipogenesis using single-nuclei RNA-seq

Adipogenesis is key to maintaining organism-wide energy balance and healthy metabolic phenotype, making it critical to thoroughly comprehend its molecular regulation in humans. By single-nuclei RNA-sequencing (snRNA-seq) of over 20,000 differentiating white and brown preadipocytes, we constructed a high-resolution temporal transcriptional landscape of human white and brown adipogenesis. White and brown preadipocytes were isolated from a single individual's neck region, thereby eliminating inter-subject variability across two distinct lineages. These preadipocytes were also immortalized to allow for controlled, in vitro differentiation, allowing sampling of distinct cellular states across the spectrum of adipogenic progression. Pseudotemporal cellular ordering revealed the dynamics of ECM remodeling during early adipogenesis, and lipogenic/thermogenic response during late white/brown adipogenesis. Comparison with adipogenic regulation in murine models Identified several novel transcription factors as potential targets for adipogenic/thermogenic drivers in humans. Among these novel candidates, we explored the role of TRPS1 in adipocyte differentiation and showed that its knockdown impairs white adipogenesis in vitro. Key adipogenic and lipogenic markers revealed in our analysis were applied to analyze publicly available scRNA-seq datasets; these confirmed unique cell maturation features in recently discovered murine preadipocytes, and revealed inhibition of adipogenic expansion in humans with obesity. Overall, our study presents a comprehensive molecular description of both white and brown adipogenesis in humans and provides an important resource for future studies of adipose tissue development and function in both health and metabolic disease state.

Optical diffraction tomography and Raman spectroscopy reveal distinct cellular phenotypes during white and brown adipocyte differentiation

White adipose tissue (WAT) and brown adipose tissue (BAT) are the primary types of fats in humans, and they play prominent roles in energy storage and thermogenesis, respectively. While the mechanisms of terminal adipogenesis are well understood, much remains unknown about the early stages of adipogenic differentiation. Label-free approaches, such as optical diffraction tomography (ODT) and Raman spectroscopy, offer the ability to retrieve morphological and molecular information at the single cell level without the negative effects of photobleaching and system perturbation due to introduction of fluorophores. In this study, we employed 3D ODT and Raman spectroscopy to gain deeper insights into the early stages of differentiation of human white preadipocytes (HWPs) and human brown preadipocytes (HBPs). We utilized ODT to retrieve morphological information, including cell dry mass and lipid mass, and Raman spectroscopy to obtain molecular information about lipids. Our findings reveal that HWPs and HBPs undergo dynamic and differential changes during the differentiation process. Notably, we found that HBPs accumulated lipids more rapidly and had a higher lipid mass than HWPs. Additionally, both cell types experienced an increase and subsequent decrease in cell dry mass during the first seven days, followed by an increase after day 7, which we attribute to the transformation of adipogenic precursors in the early stages. Finally, HBPs had higher lipid unsaturation levels than HWPs for the same differentiation timepoints. The insights gained from our study provide crucial contributions towards the advancement of new therapies for obesity and related diseases.

U0126 Compound Triggers Thermogenic Differentiation in Preadipocytes via ERK-AMPK Signaling Axis.

In recent years, thermogenic differentiation and activation in brown and white adipose tissues have been regarded as one of the major innovative and promising strategies for the treatment and amelioration of obesity. However, the pharmacological approach towards this process has had limited and insufficient commitments, which presents a greater challenge for obesity treatment. This research evaluates the effects of U0126 compound on the activation of thermogenic differentiation during adipogenesis. The results show that U0126 pretreatment primes both white and brown preadipocytes to upregulate thermogenic and mitochondrial genes as well as enhance functions during the differentiation process. We establish that U0126-mediated thermogenic differentiation induction occurs partially via AMPK activation signaling. The findings of this research suggest U0126 as a promising alternative ligand in pursuit of a pharmacological option to increase thermogenic adipocyte formation and improve energy expenditure. Thus it could pave the way for the discovery of therapeutic drugs for the treatment of obesity and its related complications.

The Effects of Neuropeptide B on Proliferation and Differentiation of Porcine White Preadipocytes into Mature Adipocytes

Neuropeptide B (NPB) affects energy homeostasis and metabolism by binding and activating NPBWR1 and NPBWR2 in humans and pigs. Recently, we reported that NPB promotes the adipogenesis of rat white and brown preadipocytes as well as 3T3-L1 cells. In the present study, we evaluated the effects of NPB on the proliferation and differentiation of white porcine preadipocytes into mature adipocytes. We identified the presence of NPB, NPBWR1, and NPBWR2 on the mRNA and protein levels in porcine white preadipocytes. During the differentiation process, NPB increased the mRNA expression of PPARγ, C/EBPβ, C/EBPα, PPARγ, and C/EBPβ protein production in porcine preadipocytes. Furthermore, NPB stimulated lipid accumulation in porcine preadipocytes. Moreover, NPB promoted the phosphorylation of the p38 kinase in porcine preadipocytes, but failed to induce ERK1/2 phosphorylation. NPB failed to stimulate the expression of C/EBPβ in the presence of the p38 inhibitor. Taken together, we report that NPB promotes the differentiation of porcine preadipocytes via a p38-dependent mechanism.

Identification and characterization of the long non-coding RNA NFIA-AS2 as a novel locus for body mass index in American Indians

BackgroundGenome-wide association studies have shown that body mass index (BMI), an estimate of obesity, is highly polygenic. Individual variants typically have small effect sizes, making it challenging to identify unique loci in under-represented ethnic groups which lack statistical power due to their small sample size. Yet obesity is a major health disparity and is particularly prevalent in southwestern American Indians. Here, we identify and characterize a new locus for BMI that was detected by analyzing moderate associations with BMI obtained in a population-based sample of southwestern American Indians together with the well-powered GIANT dataset.MethodsGenotypes for 10.5 million variants were tested for association with BMI in 5870 American Indians and 2600 variants that showed an association P < 10−3 in the American Indian sample were combined in a meta-analysis with the BMI data reported in GIANT (N = 240,608). The newly identified gene, NFIA-AS2 was functionally characterized, and the impact of its lead associated variant rs1777538 was studied both in-silico and in-vitro.ResultsRs1777538 (T/C; C allele frequency = 0.16 in American Indians and 0.04 in GIANT, meta-analysis P = 5.0 × 10−7) exhibited a large effect in American Indians (1 kg/m2 decrease in BMI per copy of C allele). NFIA-AS2 was found to be a nuclear localized long non-coding RNA expressed in tissues pertinent to human obesity. Analysis of this variant in human brown preadipocytes showed that NFIA-AS2 transcripts carrying the C allele had increased RNA degradation compared to the T allele transcripts (half-lives = 9 h, 13 h respectively). During brown adipogenesis, NFIA-AS2 featured a stage-specific regulation of nearby gene expression where rs1777538 demonstrated an allelic difference in regulation in the mature adipocytes (the strongest difference was observed for L1TD1, P = 0.007).ConclusionOur findings support a role for NFIA-AS2 in regulating pathways that impact BMI.

The ABA/LANCL1/2 Hormone/Receptor System Controls Adipocyte Browning and Energy Expenditure

The abscisic acid (ABA)/LANC-like protein 1/2 (LANCL1/2) hormone/receptor system regulates glucose uptake and oxidation, mitochondrial respiration, and proton gradient dissipation in myocytes. Oral ABA increases glucose uptake and the transcription of adipocyte browning-related genes in rodent brown adipose tissue (BAT). The aim of this study was to investigate the role of the ABA/LANCL system in human white and brown adipocyte thermogenesis. Immortalized human white and brown preadipocytes, virally infected to overexpress or silence LANCL1/2, were differentiated in vitro with or without ABA, and transcriptional and metabolic targets critical for thermogenesis were explored. The overexpression of LANCL1/2 increases, and their combined silencing conversely reduces mitochondrial number, basal, and maximal respiration rates; proton gradient dissipation; and the transcription of uncoupling genes and of receptors for thyroid and adrenergic hormones, both in brown and in white adipocytes. The transcriptional enhancement of receptors for browning hormones also occurs in BAT from ABA-treated mice, lacking LANCL2 but overexpressing LANCL1. The signaling pathway downstream of the ABA/LANCL system includes AMPK, PGC-1α, Sirt1, and the transcription factor ERRα. The ABA/LANCL system controls human brown and "beige" adipocyte thermogenesis, acting upstream of a key signaling pathway regulating energy metabolism, mitochondrial function, and thermogenesis.