Brown Adipocyte Cell Line Research Articles

Human Genetic Variation at rs10071329 Correlates With Adiposity-Related Traits, Modulates PPARGC1B Expression, and Alters Brown Adipocyte Function.

Human genetic variation in PPARGC1B has been associated with adiposity, but the genetic variants that affect PPARGC1B expression have not been experimentally determined. Here, guided by previous observational data, we used CRISPR/Cas9 to scarlessly edit the alleles of the candidate causal genetic variant rs10071329 in a human brown adipocyte cell line (hBAs). Switching the rs10071329 genotype from A/A to G/G enhanced PPARGC1B expression throughout the adipogenic differentiation, identifying rs10071329 as a cis-eQTL. The higher PPARGC1B expression in G/G cells coincided with greater accumulation of triglycerides, and higher expression of mitochondria-encoded genes, but without significant effects on adipogenic marker expression. Furthermore, G/G cells had improved basal- and norepinephrine-stimulated mitochondrial respiration, possibly relating to enhanced mitochondrial gene expression. The G/G cells also exhibited increased norepinephrine-stimulated glycerol release, indicating improved lipolysis. Altogether, our results showed that rs10071329 is a cis-eQTL, with the G/G genotype conferring enhanced PPARGC1B expression, with consequent improved mitochondrial function and response to norepinephrine in brown adipocytes. This genetic variant, and as yet undetermined eQTLs, at PPARGC1B could prove useful in genotype-based precision medicine for obesity treatment.

Glibenclamide promotes FGF21 secretion in interscapular BAT and attenuates depression-like behaviors in male mice with HFD-induced obesity

AimsEpidemiological evidence suggests that comorbidity of obesity and depression is extremely common and continues to grow in prevalence. However, the mechanisms connecting these two conditions are unknown. In this study, we explored how treatment with KATP channel blocker glibenclamide (GB) or the well-known metabolic regulator FGF21 impact male mice with high-fat diet (HFD)-induced obesity and depressive-like behaviors. Materials and methodsMice were fed with HFD for 12 weeks and then treated with recombinant FGF21 protein by infusion for 2 weeks, followed by intraperitoneal injection of 3 mg/kg recombinant FGF21 once per day for 4 days. Measurements were made of catecholamine levels, energy expenditure, biochemical endpoints and behavior tests, including sucrose preference and forced swim tests were. Alternatively, animals were infused with GB into brown adipose tissue (BAT). The WT-1 brown adipocyte cell line was used for molecular studies. Key findingsCompared to HFD controls, HFD + FGF21 mice exhibited less severe metabolic disorder symptoms, improved depressive-like behaviors, and more extensive mesolimbic dopamine projections. FGF21 treatment also rescued HFD-induced dysregulation of FGF21 receptors (FGFR1 and co-receptor β-klotho) in the ventral tegmental area (VTA), and it altered dopaminergic neuron activity and morphology in HFD-fed mice. Importantly, we also found that FGF21 mRNA level and FGF21 release were increased in BAT after administration of GB, and GB treatment to BAT reversed HFD-induced dysregulation of FGF21 receptors in the VTA. SignificanceGB administration to BAT stimulates FGF21 production in BAT, corrects HFD-induced dysregulation of FGF21 receptor dimers in VTA dopaminergic neurons, and attenuates depression-like symptoms.

Olanzapine-induced decreases of FGF21 in brown adipose tissue via histone modulations drive UCP1-dependent thermogenetic impairment

Long-term olanzapine treatment has been associated with serious metabolism disorders, such as abnormal body weight gain, hyperglycemia, and dyslipidemia. Recently, accumulated evidence points to a link between the metabolic disorders caused by olanzapine and thermogenetic impairment. Fibroblast growth factor 21 (FGF21), a pleiotropic protein, is a potent stimulator of thermogenesis in brown adipose tissue (BAT). However, the relationship between autocrine FGF21 in BAT and thermogenetic impairment induced by olanzapine has not been investigated. In this study, C57BL/6 mice and C3H10T1/2 (a brown adipocyte cell line) were used to investigate the role of FGF21 in modulating thermogenetic impairments caused by olanzapine. Our data found a fall in BAT temperature, with a decrease in the protein levels of uncoupling protein 1 (UCP1) and FGF21 in olanzapine-treatment mice. Olanzapine-induced deficits of mitochondrial activity and the expression of UCP1 and related thermogenetic factors could be improved by FGF21-overexpression in brown adipocytes. Furthermore, ChIP-sequencing showed the H3K9me3 modification in Fgf21 was dramatically increased in BAT of mice with olanzapine treatment. Lysine-specific demethylase 4a (KDM4a), a histone demethylase responsible for site-specific erasure of H3K9me3, was decreased in olanzapine-treated C3H10T1/2 cells, whereas FGF21 and UCP1 expression and thermogenesis were upregulated in KMD2a-overexpressing brown adipocyte. We concluded that FGF21 was a crucial regulator mediating UCP1-dependent thermogenetic impairments by olanzapine-modulating histone methylations. Our results also provide novel insights into identifying a new therapeutic target for treating metabolic side effects caused by the antipsychotic drug.

NanoSPECT imaging reveals the uptake of 123I-labelled oxidized low-density lipoprotein in the brown adipose tissue of mice via CD36.

The liver is the major organ shown to remove oxidized low-density lipoprotein (oxLDL) from the circulation. Given increased evidence that thermogenic adipose tissue has anti-effects, we used 123I-labelled oxLDL as a tracer to reveal oxLDL accumulation in the brown adipose tissue (BAT) of mice. We also explored the mechanisms of oxLDL accumulation in BAT. We used high-resolution nanoSPECT/CT to investigate the tissue distribution of 123I-oxLDL and 123I-LDL (control) following intravenous injection into conscious mice. 123I-oxLDL distribution was discovered in BAT at an intensity equivalent to that in the liver, whereas 123I-LDL was detected mostly in the liver. Consistent with the function of BAT related to sympathetic nerve activity, administering anaesthesia in mice almost completely eliminated the accumulation of 123I-oxLDL in BAT, and this effect was reversed by administering β3-agonist. Furthermore, exposing mice to cold stress at 4°C enhanced 123I-oxLDL accumulation in BAT. Because in 123I-oxLDL, the protein of oxLDL was labelled, we performed additional experiments with DiI-oxLDL in which the lipid phase of oxLDL was fluorescently labelled and observed similar results, suggesting that the whole oxLDL particle was taken up by BAT. To identify the receptor responsible for oxLDL uptake in BAT, we analysed the expression of known oxLDL receptors (e.g. SR-A, CD36, and LOX-1) in cultured brown adipocyte cell line and primary brown adipocytes and found that CD36 was the major receptor expressed. Treatment of cells with CD36 siRNA or CD36 neutralizing antibody significantly inhibited DiI-oxLDL uptake. Finally, CD36 deletion in mice abolished the accumulation of 123I-oxLDL and DiI-oxLDL in BAT, indicating that CD36 is the major receptor for oxLDL in BAT. We show novel evidence for the CD36-mediated accumulation of oxLDL in BAT, suggesting that BAT may exert its anti-atherogenic effects by removing atherogenic LDL from the circulation.

Nobiletin and 3'-Demethyl Nobiletin Activate Brown Adipocytes upon β-Adrenergic Stimulation.

Brown adipose tissue (BAT) specifically regulates energy expenditure via heat production. Nobiletin (NOB), a natural polymethoxylated flavone present in citrus fruits, can activate thermogenesis in the BAT of high-fat diet-induced obese mice. The activity of BAT is directly regulated by β-adrenergic stimulation. In this study, we report the effects of NOB on BAT activation using β-adrenergic agonists. We observed that when HB2 brown adipocyte cell lines are stimulated with β-adrenergic agonists, NOB enhances the expression of uncoupling protein 1 (UCP1), which is associated with the mitochondrial energy metabolism in these cells. Moreover, NOB increases the mRNA expression of the brown adipokines neuregulin-4 (Nrg4) and fibroblast growth factor-21 (FGF-21) and the secretion of FGF-21. These results suggest that NOB can enhance the thermogenic functions of brown adipocytes and promote brown adipokine secretion due to enhanced β-adrenergic stimulation. In addition, 3'-demethyl nobiletin (3'-DMN), an NOB CYP-enzyme metabolite, can increase UCP1 mRNA expression. Both NOB and 3'-DMN significantly promoted mitochondrial membrane potential in HB2 adipocytes following β-adrenergic stimulation. Therefore, we believe that NOB could be a promising candidate for activating BAT under β-adrenergic stimulation and preventing the onset of obesity.

Defective insulin-stimulated GLUT4 translocation in brown adipocytes induces systemic glucose homeostasis dysregulation independent of thermogenesis in female mice

ObjectiveRecent studies indicate that brown adipose tissue, in addition to its role in thermogenesis, has a role in the regulation of whole-body metabolism. Here we characterize the metabolic effects of deleting Rab10, a protein key for insulin stimulation of glucose uptake into white adipocytes, solely from brown adipocytes. MethodsWe used a murine brown adipocyte cell line and stromal vascular fraction-derived in vitro differentiated brown adipocytes to study the role of Rab10 in insulin-stimulated GLUT4 translocation to the plasma membrane and insulin-stimulated glucose uptake. We generated a brown adipocyte-specific Rab10 knockout for in vivo studies of metabolism and thermoregulation. ResultsWe demonstrate that deletion of Rab10 from brown adipocytes results in a two-fold reduction of insulin-stimulated glucose transport by reducing translocation of the GLUT4 glucose transporter to the plasma membrane, an effect linked to whole-body glucose intolerance and insulin resistance in female mice. This effect on metabolism is independent of the thermogenic function of brown adipocytes, thereby revealing a metabolism-specific role for brown adipocytes in female mice. The reduced glucose uptake induced by Rab10 deletion disrupts ChREBP regulation of de novo lipogenesis (DNL) genes, providing a potential link between DNL in brown adipocytes and whole-body metabolic regulation in female mice. However, deletion of Rab10 from male mice does not induce systemic insulin resistance, although ChREBP regulation is disrupted. ConclusionsOur studies of Rab10 reveal the role of insulin-regulated glucose transport into brown adipocytes in whole-body metabolic homeostasis of female mice. Importantly, the contribution of brown adipocytes to whole-body metabolic regulation is independent of its role in thermogenesis. It is unclear whether the whole-body metabolic sexual dimorphism is because female mice are permissive to the effects of Rab10 deletion from brown adipocytes or because male mice are resistant to the effect.

The transcriptional co-regulator LDB1 is required for brown adipose function

ObjectiveBrown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. Exploiting the energy uncoupling capacity of BAT may reveal targets for obesity therapies. This exploitation requires a greater understanding of the transcriptional mechanisms underlying BAT function. One potential regulator of BAT is the transcriptional co-regulator LIM domain-binding protein 1 (LDB1), which acts as a dimerized scaffold, allowing for the assembly of transcriptional complexes. Utilizing a global LDB1 heterozygous mouse model, we recently reported that LDB1 might have novel roles in regulating BAT function. However, direct evidence for the LDB1 regulation of BAT thermogenesis and substrate utilization has not been elucidated. We hypothesize that brown adipocyte-expressed LDB1 is required for BAT function. MethodsLDB1-deficient primary cells and brown adipocyte cell lines were assessed via qRT-PCR and western blotting for altered mRNA and protein levels to define the brown adipose-specific roles. We conducted chromatin immunoprecipitation with primary BAT tissue and immortalized cell lines. Potential transcriptional partners of LDB1 were revealed by conducting LIM factor surveys via qRT-PCR in mouse and human brown adipocytes. We developed a Ucp1-Cre-driven LDB1-deficiency mouse model, termed Ldb1ΔBAT, to test LDB1 function in vivo. Glucose tolerance and uptake were assessed at thermoneutrality via intraperitoneal glucose challenge and glucose tracer studies. Insulin tolerance was measured at thermoneutrality and after stimulation with cold or the administration of the β3-adrenergic receptor (β3-AR) agonist CL316,243. Additionally, we analyzed plasma insulin via ELISA and insulin signaling via western blotting. Lipid metabolism was evaluated via BAT weight, histology, lipid droplet morphometry, and the examination of lipid-associated mRNA. Finally, energy expenditure and cold tolerance were evaluated via indirect calorimetry and cold challenges. ResultsReducing Ldb1 in vitro and in vivo resulted in altered BAT-selective mRNA, including Ucp1, Elovl3, and Dio2. In addition, there was reduced Ucp1 induction in vitro. Impacts on gene expression may be due, in part, to LDB1 occupying Ucp1 upstream regulatory domains. We also identified BAT-expressed LIM-domain factors Lmo2, Lmo4, and Lhx8, which may partner with LDB1 to mediate activity in brown adipocytes. Additionally, we observed LDB1 enrichment in human brown adipose. In vivo analysis revealed LDB1 is required for whole-body glucose and insulin tolerance, in part through reduced glucose uptake into BAT. In Ldb1ΔBAT tissue, we found significant alterations in insulin-signaling effectors. An assessment of brown adipocyte morphology and lipid droplet size revealed larger and more unilocular brown adipocytes in Ldb1ΔBAT mice, particularly after a cold challenge. Alterations in lipid handling were further supported by reductions in mRNA associated with fatty acid oxidation and mitochondrial respiration. Finally, LDB1 is required for energy expenditure and cold tolerance in both male and female mice. ConclusionsOur findings support LDB1 as a regulator of BAT function. Furthermore, given LDB1 enrichment in human brown adipose, this co-regulator may have conserved roles in human BAT.

The Them1 START Domain Regulates Nuclear Trafficking in Brown Adipocytes

Thioesterase superfamily member 1 (Them1) is highly expressed in brown adipose tissue, where it limits energy expenditure. Them1 is a fatty acyl‐CoA thioesterase with tandem hotdog fold thioesterase domains, an N‐terminal set of phosphorylation sites at S15, 18, and 25, and a C‐terminal lipid‐binding steroidogenic acute regulatory protein‐related lipid transfer (START) domain, which modulates thioesterase activity. Cell fractionation studies have demonstrated that Them1 thioesterase activity is localized in microsomes and nuclei, suggesting that it is targeted to specific intracellular compartments. Our aim was to determine the mechanism by which Them1 traffics to the nucleus and its function therein.MethodsIdentification of a putative nuclear localization signal was determined using cNLS mapper ( http://nls‐mapper.iab.keio.ac.jp/cgi‐bin/NLS_Mapper_form.cgi). Full‐length, mutant, or truncated THEM1 constructs were cloned and linked to EGFP. Plasmids or adenoviral vectors were transfected into a differentiated brown adipocyte cell line (MUCAR cells) that does not express Them1. The localization of Them1 was visualized by confocal microscopy via EFGP and mRNA expression was determined using qPCR. Confocal z‐stacks were used to produce 3‐D images with Volocity software. Them1 nuclear localization was evaluated in the presence or absence of the nuclear export inhibitor leptomycin B. Cyclohexamide was used to block new protein synthesis.ResultsPhosphomimic mutations that simulated Them1 phosphorylation at S15 resulted in a diffuse Them1 localization. Stimulation of cells with norepinephrine resulted in Them1 phosphorylation and its reorganization from discrete puncta to a diffuse localization. In the diffuse state, Them1 was transported into the nucleus. Treatment with cyclohexamide suggested that the nuclear Them1 pool was not newly synthesized. We identified a nuclear localization signal in a hinge region located between the 2nd thioesterase domain and the START domain. Deletion of either the nuclear localization signal or the START domain in full length Them 1 blocked nuclear targeting. Conversely, mutant Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus, as evidenced by exit blockade using leptomycin B. Nuclear Them1 regulated mRNA expression levels for a number of genes that regulate lipid metabolism in brown adipocytes.ConclusionsOur results demonstrate that trafficking of Them1 to the nucleus occurs via a nuclear localization signal within the START domain. We speculate that nuclear Them1 regulates the transcription of lipid metabolic enzymes by a mechanism that is distinct from its acyl‐CoA thioesterase activity.Support or Funding InformationThis work was supported by NIH DK103046 to DEC, SJH and EAO.

SUN-108 Dose-Dependent Effect of Progesterone on T37i Brown Adipocyte Differentiation

Brown adipose tissue (BAT) is a specialized organ that dissipates energy as heat by a unique mitochondrial protein, uncoupling protein 1 (UCP1). Sex differences on BAT function and abundance have been studied in many species: BAT of female mice has a greater thermogenic function due to a higher mitochondrial capacity than in males, and metabolically active BAT in the PET/CT imaging is more prevalent in women than in men. These sex-specific features are likely driven by sex hormones: estrogen and testosterone. However, the effect of progesterone, a major circulating female sex hormone during luteal phase and pregnancy, has been limitedly studied and available data are conflicting. We aimed to investigate the direct effects of progesterone on BAT differentiation and function using T37i, a female brown adipocyte cell line. T37i cells were differentiated with a standard regimen: 20 nM insulin and 2 nM triiodothyronine, with or without progesterone from 100 nM (a physiological circulating level) to 10 µM. After 9 days of differentiation, cells were collected for analysis or stimulated for 24 hours with 1 µM norepinephrine (NE). Progesterone dose-responsively reduced T37i differentiation. This is reflected by a 30% reduced lipid accumulation (quantified by Oil Red O staining), 37% decreased basal lipolytic activity (determined by glycerol release into the culture media), and decreased expression of adipogenic marker genes: Cd36 65%, Adipoq 68%, and Lep 93% reductions at the highest progesterone concentration. Concerning the thermogenic capacity of T37i cells, progesterone inhibited basal and NE-stimulated expressions of thermogenic genes (baseline Ucp1 98% and Ppargc1a 63% reductions; NE-stimulated Ucp1 97% and Ppargc1a 76% reductions) and reduced the NE-stimulated lipolysis by 50%. Moreover, short-term treatment with progesterone on differentiated T37i cells also reduced Ucp1, Adipoq, and Lep gene expressions. Interestingly, we could not detect Pgr mRNA expression in T37i cells. In conclusion, we demonstrated that progesterone inhibits the T37i brown adipocyte differentiation and thermogenic function, likely independently from the classical progesterone receptor, in a dose-dependent manner. Further studies are required to elucidate the mechanisms how progesterone inhibits BAT differentiation and functions.

Them1 Regulates the Subcellular Localization of Glycogen in Brown Adipocytes: Implications for Energy Homeostasis

Molecular mechanisms that integrate lipid and glycogen metabolism are not well understood. In brown adipose tissue (BAT), cold exposure increases energy expenditure by increasing mitochondrial β‐oxidation of lipid droplet‐derived fatty acids. This process is suppressed by thioesterase superfamily member 1 (Them1), a fatty acyl‐CoA thioesterase that is also upregulated by cold ambient temperatures. Them1 limits thermogenesis by reducing the availability of fatty acid for β‐oxidation in mitochondria. Previous studies suggested that the effects of Them1 can be blocked by PKCβ‐mediated phosphorylation at S15, but the cellular mechanism remains unclear. Here, we demonstrate that Them1 associates with glycogen and controls its 3‐D physical state in a phosphorylation‐dependent manner.MethodsThe full‐length cDNA for THEM1 and a series of mutants were cloned and linked to tandem dimer tomato (tdTomato) fluorescent protein or EGFP that included an engineered ascorbate peroxidase (APEX2) fusion protein at the C‐terminus. The plasmids were transfected into a differentiated brown adipocyte cell line that does not express Them1. The localization of Them1 was visualized by confocal microscopy (EFGP or tdTomato; living cells) or by electron microscopy (EM; APEX2). Volocity software was used to produce 3‐D images from confocal z‐stacks. Them1 localization was also evaluated in the presence or absence of inhibitors and activators of key effectors of the lipolysis pathway. Immunoprecipitation, using isolated lipid droplets from BAT and anti‐Them1, was used to evaluate potential binding partners by proteomics from Them1‐expressing vs deficient cold‐exposed mice.ResultsIn unstimulated cells, Them1 formed small (100–200 nm in diameter), punctate structures that were localized near lipid droplets and mitochondria and also circumferentially surrounded lipid droplets. EM studies confirmed that Them1 was associated with glycogen in both locations. In response to stimulation by norepinephrine (β3‐adrenergic receptor agonist), PKA, or adipose triglyceride lipase, Them1 was redistributed from a punctate to a diffuse localization. By contrast, inhibiting PKA or PKC retained Them1 in a punctate localization. The punctate form was also observed when the first 36 amino acids of Them1 were deleted or when the S15 phosphorylation site was mutated from S15 to A15. In the diffuse distribution, Them1 localized to individual β‐glycogen particles by EM. Proteomic analysis revealed glycogen phosphorylase as a likely binding partner for Them1.ConclusionsOur results show that Them1 is associated with glycogen in BAT cells, most likely via its interactions with glycogen phosphorylase. Them1 phosphorylation induces a major conformational rearrangement of glycogen, which in turn redistributes Them1 away from lipid droplets. This presumably prevents Them1 from suppressing lipolysis during a period of energy demand. We speculate that the Them1‐mediated glycogen rearrangement may also function to coordinate lipid and glucose metabolism within BAT.Support or Funding InformationThis work was supported by NIH DK103046 to DEC and SJH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vitro Models for Study of Brown Adipocyte Biology.

Brown adipocytes are the key cell type in brown adipose tissue (BAT) that express the genes required for heat production through the process of thermogenesis. Brown adipocyte cell culture models are important for researching the molecular pathways that control cell autonomous processes. In vitro tools for the study of brown adipocytes include BAT explant cultures and BAT primary cultures that are first proliferated and then differentiated. A number of stable brown preadipocyte cell lines have been generated by the expression transforming factors such as SV40 T antigen. The application of these cell lines reduces the requirement for animal tissue which is needed for primary culture and explants. Furthermore, brown adipocyte cell lines that effectively recapitulate the properties of brown adipocytes permit large-scale experimental procedures that are generally unfeasible with primary cultures that undergo a restricted number of cell divisions. Cell lines are valuable for applications such as large-scale endogenous protein expression, ChIP assay, and procedures requiring antibiotic selection over several cell divisions including stable exogenous gene expression and CRISR/Cas9 gene editing.

Carnitine Palmitoyltransferase 1 Increases Lipolysis, UCP1 Protein Expression and Mitochondrial Activity in Brown Adipocytes.

The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders.

Susceptibility of brown adipocytes to pro-inflammatory cytokine toxicity and reactive oxygen species.

Brown adipose tissue (BAT) cells have a very high oxidative capacity. On the other hand, in obesity and obesity-related diabetes, levels of pro-inflammatory cytokines are elevated, which might promote BAT dysfunction and consequently impair carbohydrate metabolism and thereby exacerbate cellular dysfunction and promote diabetes progression. Therefore, the antioxidative enzyme status of a brown adipocyte cell line and its susceptibility towards pro-inflammatory cytokines, which participate in the pathogenesis of diabetes, and reactive oxygen species (ROS) were analysed. Mature brown adipocytes exhibited significantly higher levels of expression of mitochondrially and peroxisomally located antioxidative enzymes compared with non-differentiated brown adipocytes. Pro-inflammatory cytokines induced a significant decrease in the viability of differentiated brown adipocytes, which was accompanied by a massive ROS production and down-regulation of BAT-specific markers, such as uncoupling protein 1 (UCP-1) and β-Klotho. Taken together, the results strongly indicate that pro-inflammatory cytokines cause brown adipocyte dysfunction and death through suppression of BAT-specific proteins, especially of UCP-1 and β-Klotho, and consequently increased oxidative stress.

Histone deacetylase 1 plays an important role in brown adipogenesis (817.4)

Induction of brown adipocytes in white fat plays an important role in the regulation of energy homeostasis. Here, we have investigated the role of HDAC1 in brown adipogenesis. The RNA and protein levels of HDAC1 are lower in mice brown compared to white adipose tissue, in brown adipose tissue of mice exposed to cold compared to that of mice residing in room temperature, and reduced during brown adipocyte differentiation. Overexpression of HDAC1 inhibits, whereas genetic deletion of HDAC1 results in further enhancement of isoproterenol‐stimulated expression of uncoupling protein 1 (UCP1) and other BAT‐specific genes, including peroxisome proliferative activated receptor γ coactivator 1α (PGC1α), PGC1β, PR domain containing 16 (PRDM16), type 2 deiodinase (DIO2), fibroblast growth factor 21 (FGF21) and myelin protein zero‐like 2 (Eva1) in brown adipocyte cell lines BAT‐1 and HIB1B. Chromatin immunoprecipitation assay indicates that histone H3 and H4 acetylation is significantly increased at the UCP1 promoter in β‐adrenergic stimulated brown adipocytes, which is also associated with increased active histone markers H3 lysine 4 tri‐methylation, and H3 lysine 27 acetylation. Thus, our data suggest that epigenetics plays an important role in brown adipogenesis and thermogenesis, and identify HDAC1 as an important regulator during this process.Grant Funding Source: NIHR01HL107500

Miglitol prevents diet-induced obesity by stimulating brown adipose tissue and energy expenditure independent of preventing the digestion of carbohydrates

Miglitol is an alpha-glucosidase inhibitor that improves post-prandial hyperglycemia, and it is the only drug in its class that enters the bloodstream. Anecdotally, miglitol lowers patient body weight more effectively than other alpha-glucosidase inhibitors, but the precise mechanism has not been addressed. Therefore, we analyzed the anti-obesity effects of miglitol in mice and in the HB2 brown adipocyte cell line. Miglitol prevented diet-induced obesity by stimulating energy expenditure without affecting food intake in mice. Long-term miglitol treatment dose-dependently prevented diet-induced obesity and induced mitochondrial gene expression in brown adipose tissue. The anti-obesity effect was independent of preventing carbohydrate digestion in the gastrointestinal tract. Miglitol effectively stimulated energy expenditure in mice fed a high-fat high-monocarbohydrate diet, and intraperitoneal injection of miglitol was sufficient to stimulate energy expenditure in mice. Acarbose, which is a non-absorbable alpha glucosidase inhibitor, also prevented diet-induced obesity, but through a different mechanism: it did not stimulate energy expenditure, but caused indigestion, leading to less energy absorption. Miglitol promoted adrenergic signaling in brown adipocytes in vitro. These data indicate that circulating miglitol stimulates brown adipose tissue and increases energy expenditure, thereby preventing diet-induced obesity. Further optimizing miglitol's effect on brown adipose tissue could lead to a novel anti-obesity drug.

PAZ6 Cells Constitute a Representative Model for Human Brown Pre-Adipocytes

The role of brown adipose tissue (BAT) in human metabolism and its potential as an anti-obesity target organ have recently received much renewed attention. Following radiological detection of substantial amounts of BAT in adults by several independent research groups, an increasing number of studies are now dedicated to uncover BAT’s genetic, developmental, and environmental determinants. In contrast to murine BAT, human BAT is not present as a single major fat depot in a well-defined location. The distribution of BAT in several areas in the body significantly limits its availability to research. A human brown adipocyte cell line is therefore critical in broadening the options available to researchers in the field. The human BAT-cell line PAZ6 was created to address such a need and has been well characterized by several research groups around the world. In the present review, we discuss their findings and propose potential applications of the PAZ6 cells in addressing the relevant questions in the BAT field, namely for future use in therapeutic applications.

Assay of the Multiple Energy-Producing Pathways of Mammalian Cells

BackgroundTo elucidate metabolic changes that occur in diabetes, obesity, and cancer, it is important to understand cellular energy metabolism pathways and their alterations in various cells.Methodology and Principal FindingsHere we describe a technology for simultaneous assessment of cellular energy metabolism pathways. The technology employs a redox dye chemistry specifically coupled to catabolic energy-producing pathways. Using this colorimetric assay, we show that human cancer cell lines from different organ tissues produce distinct profiles of metabolic activity. Further, we show that murine white and brown adipocyte cell lines produce profiles that are distinct from each other as well as from precursor cells undergoing differentiation.ConclusionsThis technology can be employed as a fundamental tool in genotype-phenotype studies to determine changes in cells from shared lineages due to differentiation or mutation.

Brown Adipose Tissue Responds to Cold and Adrenergic Stimulation by Induction of FGF21

Fibroblast growth factor-21 (FGF21) is a pleiotropic protein involved in glucose, lipid metabolism and energy homeostasis, with main tissues of expression being the liver and adipose tissue. Brown adipose tissue (BAT) is responsible for cold-induced thermogenesis in rodents. The role of FGF21 in BAT biology has not been investigated. In the present study, wild-type C57BL/6J mice as well as a brown adipocyte cell line were used to explore the potential role of cold exposure and β3-adrenergic stimulation in the expression of FGF21 in BAT. Our results demonstrate that short-term exposure to cold, as well as β3-adrenergic stimulation, causes a significant induction of FGF21 mRNA levels in BAT, without a concomitant increase in FGF21 plasma levels. This finding opens new routes for the potential use of pharmaceuticals that could induce FGF21 and, hence, activate BAT thermogenesis.

Differential expression of adipokines due to hypoxia in brown and white adipocytes

Background: Adipose tissue, consisting of white and brown adipocytes, comprises an important endocrine regulator, secreting hormones specified as adipokines. While recent studies demonstrated that adipose tissue responds to hypoxia, the different roles of regulation in brown and white adipocytes are still unclear. In this study the effect of hypoxia on murine brown and white adipocyte cell lines was analyzed. Methods: In cultured adipocytes hypoxia was induced chemically with desferoxamine and cobaldchlorid or physically in an atmosphere of 1% oxygen. Vitality and differentiation was tested by oil-red-staining. Western Blot analysis was performed for hypoxia inducible factor 1 alpha (HIF 1 alpha) in nuclear protein extracts. In corresponding cellular protein extracts HIF 1 alpha regulated proteins (leptin, adiponectin, vascular endothelial growth factor (VEGF) and angiotensinogen) were evaluated by western blot. Results: Chemical as well as physical hypoxia inhibits differentiation of the brown and white adipocyte cell lines. Hypoxia leads to increased HIF 1 alpha-activation in brown and white adipocytes. The expression of adiponectin increased in white adipocytes and decreased in the brown adipocytes under hypoxic conditions. In contrast the amount of leptin was increased in brown and white adipocytes. VEGF- and angiotensinogen-expression did not change in both cell types following hypoxic stimulation. Conclusions: Hypoxia induces similar activation of HIF 1 alpha but differential protein expression in brown and white adipocytes. Different expression of adipokines under hypoxic conditions in the two cell types may represent diverging roles in hypoxia and thus requires an approach distinguishing between these cells for further investigations.

Quantifying Reductive Carboxylation Flux of Glutamine to Lipid in a Brown Adipocyte Cell Line

We previously reported that glutamine was a major source of carbon for de novo fatty acid synthesis in a brown adipocyte cell line. The pathway for fatty acid synthesis from glutamine may follow either of two distinct pathways after it enters the citric acid cycle. The glutaminolysis pathway follows the citric acid cycle, whereas the reductive carboxylation pathway travels in reverse of the citric acid cycle from alpha-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-(13)C]glutamine or [5-(13)C]glutamine and analyzed the mass isotopomer distribution of key metabolites using models that fit the isotopomer distribution to fluxes. We also investigated inhibitors of NADP-dependent isocitrate dehydrogenase and mitochondrial citrate export. The results indicated that one third of glutamine entering the citric acid cycle travels to citrate via reductive carboxylation while the remainder is oxidized through succinate. The reductive carboxylation flux accounted for 90% of all flux of glutamine to lipid. The inhibitor studies were compatible with reductive carboxylation flux through mitochondrial isocitrate dehydrogenase. Total cell citrate and alpha-ketoglutarate were near isotopic equilibrium as expected if rapid cycling exists between these compounds involving the mitochondrial membrane NAD/NADP transhydrogenase. Taken together, these studies demonstrate a new role for glutamine as a lipogenic precursor and propose an alternative to the glutaminolysis pathway where flux of glutamine to lipogenic acetyl-CoA occurs via reductive carboxylation. These findings were enabled by a new modeling tool and software implementation (Metran) for global flux estimation.