Thermogenic Markers Research Articles

Parathyroid hormone receptor stimulation induces human adipocyte lipolysis and browning.

Activation of brown adipose tissue is a promising strategy to treat and prevent obesity and obesity-related disorders. Activation of uncoupling protein 1 (UCP1) leads to uncoupled respiration and dissipation of stored energy as heat. Induction of UCP1-rich adipocytes in white adipose tissue, a process known as 'browning', serves as an alternative strategy to increase whole body uncoupling capacity. Here, we aim to assess the association between parathyroid hormone (PTH) receptor expression and UCP1 expression in human adipose tissues and to study PTH effects on human white and brown adipocyte lipolysis and UCP1 expression. A descriptive study of human neck adipose tissue biopsies substantiated by an interventional study on human neck-derived adipose tissue cell models. Thermogenic markers and PTH receptor gene expression are assessed in human neck adipose tissue biopsies and are related to individual health records. PTH-initiated lipolysis and thermogenic gene induction are assessed in cultured human white and brown adipocyte cell models. PTH receptor involvement is investigated by PTH receptor silencing. PTH receptor gene expression correlates with UCP1 gene expression in the deep-neck adipose tissue in humans. In cell models, PTH receptor stimulation increases lipolysis and stimulates gene transcription of multiple thermogenic markers. Silencing of the PTH receptor attenuates the effects of PTH indicating a direct PTH effect via this receptor. PTH 1 receptor stimulation by PTH may play a role in human adipose tissue metabolism by affecting lipolysis and thermogenic capacity.

Unraveling the Developmental Roadmap toward Human Brown Adipose Tissue.

SummaryIncreasing brown adipose tissue (BAT) mass and activation is a therapeutic strategy to treat obesity and complications. Obese and diabetic patients possess low amounts of BAT, so an efficient way to expand their mass is necessary. There is limited knowledge about how human BAT develops, differentiates, and is optimally activated. Accessing human BAT is challenging, given its low volume and anatomical dispersion. These constraints make detailed BAT-related developmental and functional mechanistic studies in humans virtually impossible. We have developed and characterized functionally and molecularly a new chemically defined protocol for the differentiation of human pluripotent stem cells (hPSCs) into brown adipocytes (BAs) that overcomes current limitations. This protocol recapitulates step by step the physiological developmental path of human BAT. The BAs obtained express BA and thermogenic markers, are insulin sensitive, and responsive to β-adrenergic stimuli. This new protocol is scalable, enabling the study of human BAs at early stages of development.

The treatment with an extract from Calafate (Berberis microphylla) induces transcript and protein expression of molecules involved in thermogenesis and adipocyte browning in adipose tissue from obese mice

BACKGROUND: Polyphenols intake increases the function of brown adipose tissue (BAT), stimulating energy expenditure (EE). Calafate (Berberis microphylla) is a polyphenol-rich Chilean native fruit. OBJECTIVE: To analyse the effect of a treatment with a Calafate extract in the thermogenic activity of mice adipose tissues. METHODS: Forty adult C57BL/6J male mice were subdivided into four groups (n=10 each): control diet, control+Calafate (extract: 50mg total polyphenols/kg weight), high-fat diet (HF) and HF+Calafate. RESULTS: Calafate prevented the increase in body weight and the decrease EE induced by HF. In BAT, Ucp-1 transcript was influenced by the interaction between diet and Calafate (p<0.01), Pparα showed the same expression pattern as Ucp-1 and both, diet (p<0.01) and Calafate (p<0.05), induced significant effects in Sirt1. In inguinal adipose tissue, Pgc1α, Pparα, Prdm16, Sirt1, and Dio2 transcripts presented a decreased expression caused by HF, that was reversed by Calafate. In BAT, an effect of diet (p<0.05) and an interaction between diet and Calafate (p<0.01) was observed in UCP-1 protein levels. CONCLUSIONS: A treatment with Calafate drives less weight gain in mice fed with HF, and reverses the effects generated by it on the expression of thermogenic and browning markers.

SIRT3 (Sirtuin-3) Prevents Ang II (Angiotensin II)-Induced Macrophage Metabolic Switch Improving Perivascular Adipose Tissue Function.

Infiltrated macrophages actively promote perivascular adipose tissue remodeling and represent a dominant population in the perivascular adipose tissue microenvironment of hypertensive mice. However, the role of macrophages in initiating metabolic inflammation remains uncertain. SIRT3 (sirtuin-3), a NAD-dependent deacetylase, is sensitive to metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated metabolic shift in regulating NLRP3 (Nod-like receptor family pyrin domain-containing 3) inflammasome activation. Approach and Results: Here, we report that Ang II (angiotensin II) accelerates perivascular adipose tissue inflammation and fibrosis, accompanied by NLRP3 inflammasome activation and IL (interleukin)-1β secretion in myeloid SIRT3 knockout (SIRT3-/-) mice. This effect is associated with adipose tissue mitochondrial dysfunction. In vitro studies indicate that the deletion of SIRT3 in bone marrow-derived macrophages induces IL-1β production by shifting the metabolic phenotype from oxidative phosphorylation to glycolysis. Mechanistically, SIRT3 deacetylates and activates PDHA1 (pyruvate dehydrogenase E1 alpha) at lysine 83, and the loss of SIRT3 leads to PDH activity decrease and lactate accumulation. Knocking down LDHA (lactate dehydrogenase A) or using carnosine, a buffer against lactic acid, attenuates IL-1β secretion. Furthermore, the blockade of IL-1β from macrophages into brown adipocytes restores thermogenic markers and mitochondrial oxygen consumption. Moreover, NLRP3 knockout (NLRP3-/-) mice exhibited reduced IL-1β production while rescuing the mitochondrial function of brown adipocytes and alleviating perivascular adipose tissue fibrosis. SIRT3 represents a potential therapeutic target to attenuate NLRP3-related inflammation. Pharmacological targeting of glycolytic metabolism may represent an effective therapeutic approach.

White adipose remodeling during browning in mice involves YBX1 to drive thermogenic commitment

ObjectiveIncreasing adaptive thermogenesis by stimulating browning in white adipose tissue is a promising method of improving metabolic health. However, the molecular mechanisms underlying this transition remain elusive. Our study examined the molecular determinants driving the differentiation of precursor cells into thermogenic adipocytes. MethodsIn this study, we conducted temporal high-resolution proteomic analysis of subcutaneous white adipose tissue (scWAT) after cold exposure in mice. This was followed by loss- and gain-of-function experiments using siRNA-mediated knockdown and CRISPRa-mediated induction of gene expression, respectively, to evaluate the function of the transcriptional regulator Y box-binding protein 1 (YBX1) during adipogenesis of brown pre-adipocytes and mesenchymal stem cells. Transcriptomic analysis of mesenchymal stem cells following induction of endogenous Ybx1 expression was conducted to elucidate transcriptomic events controlled by YBX1 during adipogenesis. ResultsOur proteomics analysis uncovered 509 proteins differentially regulated by cold in a time-dependent manner. Overall, 44 transcriptional regulators were acutely upregulated following cold exposure, among which included the cold-shock domain containing protein YBX1, peaking after 24 h. Cold-induced upregulation of YBX1 also occurred in brown adipose tissue, but not in visceral white adipose tissue, suggesting a role of YBX1 in thermogenesis. This role was confirmed by Ybx1 knockdown in brown and brite preadipocytes, which significantly impaired their thermogenic potential. Conversely, inducing Ybx1 expression in mesenchymal stem cells during adipogenesis promoted browning concurrent with an increased expression of thermogenic markers and enhanced mitochondrial respiration. At a molecular level, our transcriptomic analysis showed that YBX1 regulates a subset of genes, including the histone H3K9 demethylase Jmjd1c, to promote thermogenic adipocyte differentiation. ConclusionOur study mapped the dynamic proteomic changes of murine scWAT during browning and identified YBX1 as a novel factor coordinating the genomic mechanisms by which preadipocytes commit to brite/beige lineage.

Dopamine receptor D1- and D2-agonists do not spark brown adipose tissue thermogenesis in mice

Brown adipose tissue (BAT) thermogenesis is considered a potential target for treatment of obesity and diabetes. In vitro data suggest dopamine receptor signaling as a promising approach; however, the biological relevance of dopamine receptors in the direct activation of BAT thermogenesis in vivo remains unclear. We investigated BAT thermogenesis in vivo in mice using peripheral administration of D1-agonist SKF38393 or D2-agonist Sumanirole, infrared thermography, and in-depth molecular analyses of potential target tissues; and ex vivo in BAT explants to identify direct effects on key thermogenic markers. Acute in vivo treatment with the D1- or D2-agonist caused a short spike or brief decrease in BAT temperature, respectively. However, repeated daily administration did not induce lasting effects on BAT thermogenesis. Likewise, neither agonist directly affected Ucp1 or Dio2 mRNA expression in BAT explants. Taken together, the investigated agonists do not seem to exert lasting and physiologically relevant effects on BAT thermogenesis after peripheral administration, demonstrating that D1- and D2-receptors in iBAT are unlikely to constitute targets for obesity treatment via BAT activation.

The Rosmarinus Bioactive Compound Carnosic Acid Is a Novel PPAR Antagonist That Inhibits the Browning of White Adipocytes.

Thermogenic brown and brite adipocytes convert chemical energy from nutrients into heat. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to control fat mass such as in obesity or cachexia. The peroxisome proliferator-activated receptor (PPAR) family plays key roles in the maintenance of adipose tissue and in the regulation of thermogenic activity. Activation of these receptors induce browning of white adipocyte. The purpose of this work was to characterize the role of carnosic acid (CA), a compound used in traditional medicine, in the control of brown/brite adipocyte formation and function. We used human multipotent adipose-derived stem (hMADS) cells differentiated into white or brite adipocytes. The expression of key marker genes was determined using RT-qPCR and western blotting. We show here that CA inhibits the browning of white adipocytes and favors decreased gene expression of thermogenic markers. CA treatment does not affect β-adrenergic response. Importantly, the effects of CA are fully reversible. We used transactivation assays to show that CA has a PPARα/γ antagonistic action. Our data pinpoint CA as a drug able to control PPAR activity through an antagonistic effect. These observations shed some light on the development of natural PPAR antagonists and their potential effects on thermogenic response.

Transferrin Receptor Functionally Marks Thermogenic Adipocytes.

BackgroundThermogenic adipocytes, including beige and brown adipocytes, are critical for thermogenesis and energy homeostasis. Identification of functional cell surface markers of thermogenic adipocytes is of significance for potential application in biological and clinical practices.MethodsWith a combination of RNA-sequencing of in vivo and in vitro models, we identified transferrin receptor (Tfr1), a receptor specialized for cellular iron uptake, as a previously unappreciated cell surface molecule for thermogenic adipocytes compared to white adipocytes. The alternation of Tfr1 levels under physiological and pathological stimuli was assessed, and the mitochondria functionality, browning capacity, and iron metabolism of mature adipocytes were examined with Tfr1 knockdown.ResultsTfr1 was expressed predominantly in thermogenic adipocytes versus white adipocyte, and its expression levels were tightly correlated with the activation or inhibition status of thermogenic adipocytes under external stimuli. Besides, Tfr1 gene deficiency in thermogenic adipocytes led to reduced thermogenic gene programs and mitochondrial integrity.ConclusionTfr1 functionally marks thermogenic adipocytes and could serve as a potential thermogenic adipocyte surface marker.

Lipocalin-2 deficiency may predispose to the progression of spontaneous age-related adiposity in mice

Lipocalin-2 (Lcn2) is an innate immune protein elevated by several orders of magnitude in various inflammatory conditions including aging and obesity. Recent studies have shown that Lcn2 is secreted by adipocytes in response to inflammation and is categorized as a new adipokine cross-linking innate immunity and metabolic disorders including obesity. However, the involvement of Lcn2 and its function during the progression of obesity is largely unknown. Recently, browning of white adipose tissue (WAT) has gained attention as a therapeutic strategy to combat obesity. Herein, we have shown that treatment of mature 3T3-L1 adipocytes with recombinant Lcn2 (rec-Lcn2) resulted in the up-regulation of thermogenic and beige/brown markers (UCP1, PRDM16, ZIC-1 and TBX1) and increased mitochondrial activity. Additionally, global Lcn2 genetic knockout (Lcn2KO) mice exhibited accelerated weight gain and visceral fat deposition with age, when compared to wild type (WT) mice. Taken together, both in vitro and in vivo studies suggest that Lcn2 is a naturally occurring adipokine, and may serve as an anti-obesity agent by upregulating the thermogenic markers resulting in the browning of WAT. Therefore, Lcn2 and its downstream signaling pathways could be a potential therapeutic target for obesity.

Decreased UCP-1 expression in beige adipocytes from adipose-derived stem cells of type 2 diabetes patients associates with mitochondrial ROS accumulation during obesity

ObjectiveAdipose derived stem cells (ADSC) are defective in metabolic disorders in various functionalities and properties including differentiation, multipotent state, metabolism and immunomodulation. However, the role of ADSC beiging potential in promoting of type 2 diabetes mellitus (T2DM) development remains unclear. Here we uncover association between potential of subcutaneous ADSC to beige differentiation and T2DM in patients with obesity. MethodsADSC were isolated from subcutaneous adipose tissue of patients with long morbid obesity (BMI > 35 kg/m2) and normal glucose tolerance (NGT) or T2DM. ADSC were differentiated into white or beige adipocytes and levels of thermogenic markers, lipid metabolism and electron transport chain (ETC) genes was analyzed by Western blotting and RT-PCR. ROS production was estimated by fluorescent microscopy. ResultsWe have shown decreased UCP-1 expression in beige adipocytes from T2DM patients. Nevertheless, signal and expression activities of lipolysis were equal in NGT and T2DM beige adipocytes. Expression analysis of ETC genes also has not shown any statistically significant differences. Interestingly, we revealed increased mitochondrial ROS production in T2DM beige adipocytes during beige differentiation. ConclusionsIn summary, compromised UCP1 expression in beige adipocytes of T2DM patients may cause increase of mitochondrial ROS. Elevated oxidative level is liable to act as damaging mechanism leading to insulin resistance or, alternatively, serve as compensatory mechanism for thermogenesis activation.

Biochanin A induces a brown-fat phenotype via improvement of mitochondrial biogenesis and activation of AMPK signaling in murine C3H10T1/2 mesenchymal stem cells.

In this study, we investigated the effect of Biochanin A (BioA), an O-methylated isoflavone on the brown-fat phenotype formation and on the associated thermogenic program including mitochondrial biogenesis and lipolysis in C3H10T1/2 MSCs. Our data demonstrates that Treatment with BioA in an adipogenic differentiation cocktail induced formation of brown-fat-like adipocytes from C3H10T1/2 MSCs without treatment with a known browning inducer (rosiglitazone or T3) at an early stage of differentiation. The formation of brown-fat-like adipocytes by BioA treatment was evidenced by upregulation of key thermogenic markers: Ucp1, Pgc1α, Prdm16, and Pparγ. BioA also increased the expression of beige (Cd137 and Fgf21) and brown (Elovl3 and Zic1)-specific markers. Additionally, BioA treatment promoted mitochondrial biogenesis, judging by the upregulation of genes; Cox8b, Cidea, Dio2, Sirt1, Opa1, and Fis1. BioA treatment increased the amount of mitochondrial DNA and its encoded proteins: oxidative phosphorylation complexes (I-V); this change was associated with high oxygen consumption by C3H10T1/2 MSCs. A small-interfering-RNA-induced gene knockdown and experiments with dorsomorphin-driven competitive inhibition revealed that BioA exerts the thermogenic action via activation of AMPK signaling. Our study shows the mechanism of BioA-induced promotion of a brown-fat phenotype. Nonetheless, clinical research is necessary to validate BioA as a brown-fat-like signature inducer.

The paradoxical lean phenotype of hypothyroid mice is marked by increased adaptive thermogenesis in the skeletal muscle

Obesity is a major health problem worldwide, given its growing incidence and its association with a variety of comorbidities. Weight gain results from an increase in energy intake without a concomitant increase in energy expenditure. To combat the obesity epidemic, many studies have focused on the pathways underlying satiety and hunger signaling, while other studies have concentrated on the mechanisms involved in energy expenditure, most notably adaptive thermogenesis. Hypothyroidism in humans is typically associated with a decreased basal metabolic rate, lower energy expenditure, and weight gain. However, hypothyroid mouse models have been reported to have a leaner phenotype than euthyroid controls. To elucidate the mechanism underlying this phenomenon, we used a drug-free mouse model of hypothyroidism: mice lacking the sodium/iodide symporter (NIS), the plasma membrane protein that mediates active iodide uptake in the thyroid. In addition to being leaner than euthyroid mice, owing in part to reduced food intake, these hypothyroid mice show signs of compensatory up-regulation of the skeletal-muscle adaptive thermogenic marker sarcolipin, with an associated increase in fatty acid oxidation (FAO). Neither catecholamines nor thyroid-stimulating hormone (TSH) are responsible for sarcolipin expression or FAO stimulation; rather, thyroid hormones are likely to negatively regulate both processes in skeletal muscle. Our findings indicate that hypothyroidism in mice results in a variety of metabolic changes, which collectively lead to a leaner phenotype. A deeper understanding of these changes may make it possible to develop new strategies against obesity.

The novel non-steroidal MR antagonist finerenone improves metabolic parameters in high-fat diet-fed mice and activates brown adipose tissue viaAMPK-ATGL pathway.

Mineralocorticoid receptor antagonists (MRAs) are recommended for the treatment of heart failure and hypertension, mainly due to their natriuretic and anti-fibrotic mode of action. Rodent studies have shown that MRAs can prevent adverse metabolic consequences of obesity but an elucidation of underlying molecular mechanisms is missing. Here, we investigated metabolic effects of the novel non-steroidal MRA finerenone (FIN) in a mouse model of high-fat diet (HFD)-induced obesity and the signaling pathways activated by MR antagonism at level of interscapular brown adipose tissue (iBAT). C57BL/6J male mice were fed a normal diet or a HFD (with60%kcalfrom fat) containing or not FIN for 3months. Metabolic parameters, adipose tissue morphology, gene and protein expression analysis were assessed. We also used brown adipocyte cultures (T37i cells) to investigate the effects of FIN-mediated MR antagonism upon lipid and mitochondrial metabolism. HFD+FIN-treated mice showed improved glucose tolerance together with increased multilocularity and higher expression of thermogenic markers at the level of iBAT, without differences in white adipose depots, suggesting an iBAT-specific effect of FIN. Mechanistically, FIN increased activation of AMP-activated protein kinase which, in turn, stimulated adipose triglyceride lipase activation, with subsequent increased expression of uncoupling protein-1 in brown adipocytes.

Lack of collagen XVIII leads to lipodystrophy and perturbs hepatic glucose and lipid homeostasis.

Lack of collagen XVIII leads to lipodystrophy and perturbs hepatic glucose and lipid homeostasis.

Sitagliptin mitigates oxidative stress and up-regulates mitochondrial biogenesis markers in Brown adipose tissues of high-fat diet fed obese mice through AMPK phosphorylation

AimsThe present study was designed to investigate the effects of Sitagliptin (SGN) on oxidative stress, 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, together with mitochondrial biogenesis and thermogenic markers in intercapsular brown adipose tissue (iBAT) of Swiss albino mice fed high-fat diet for sixteen weeks. MethodsMice were distributed into five groups with last eight weeks of treatment: Normal control (NC): control diet with regular drinking water; Obese control (OC): 60% high-fat diet with 20% fructose drinking water (HFFR); Treatment-1: (OC + MET): HFFR with metformin (MET) 100 mg/kg/day; Treatment-2 (OC + SGN30): HFFR with SGN 30 mg/kg/day; Treatment-3 (OC + SGN20): HFFR with SGN 20 mg/kg/day. Fasting blood glucose, serum free fatty acid and adiponectin levels were measured. Oxidative stress, P-AMPK protein expression and mRNA expression of PPARα, PGC1-α, NRF-1, TFAM, and UCP-1 in iBAT were studied. ResultsSGN treatment for eight weeks ameliorated oxidative stress, improved P-AMPK protein expression and enhanced mitochondrial biogenesis and thermogenic mediators in iBAT. ConclusionThe findings from the present study suggest the beneficial role of SGN in ameliorating obesity associated oxidative stress and up-regulated mitochondrial biogenesis and thermogenic markers in iBAT of obese mice, possibly through AMPK activation.

Omega-3 Fatty Acids and Adipose Tissue: Inflammation and Browning.

White adipose tissue (WAT) and brown adipose tissue (BAT) are involved in whole-body energy homeostasis and metabolic regulation. Changes to mass and function of these tissues impact glucose homeostasis and whole-body energy balance during development of obesity, weight loss, and subsequent weight regain. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), which have known hypotriglyceridemic and cardioprotective effects, can also impact WAT and BAT function. In rodent models, these fatty acids alleviate obesity-associated WAT inflammation, improve energy metabolism, and increase thermogenic markers in BAT. Emerging evidence suggests that ω-3 PUFAs can also modulate gut microbiota impacting WAT function and adiposity. This review discusses molecular mechanisms, implications of these findings, translation to humans, and future work, especially with reference to the potential of these fatty acids in weight loss maintenance.

1965-P: The Extracellular Matrix Modulates Adipocyte Thermogenic Capacity

Obesity is characterized by an increase in adipose mass and is the leading risk factor for type 2 diabetes. Increasing adipose thermogenic capacity, by activation of brown or beige fat, could be a treatment for metabolic diseases. Nevertheless, many approaches fail in maintaining transformed adipose tissue in vivo, in part, due to the limited understanding of how environmental factors control cell fate and maintenance. We have shown that mice with a null mutation in the laminin α4 exhibit resistance to obesity, enhanced expression of thermogenic fat markers (UCP1) in subcutaneous white adipose tissue (sWAT), increased energy expenditure and enhanced insulin sensitivity. The knockout (KO) of Lama4 results in complex changes in overall ECM composition. We used liquid chromatography/tandem mass spectrometry (LC/MS) to evaluate ECM composition in sWAT from wild type (WT) and KO mice. In addition to the expected absence of Lama4, collagen 1A1 (Col1A1) and collagen 3A1 (Col3A1) were significantly lower in KO mice. Also, integrins α7 (ITα7) and β1 (ITβ1) were dramatically reduced. Immunohistochemical staining confirmed lower levels of Itα7 in KO compared to WT mice (P<0.0001). Knocking down Itα7 with siRNA promoted UCP1 expression (P=0.003) in vitro. In addition, culture of ADSC on Lama4, Col3A1 and Col1A1 coated surfaces had lower UCP1 expression (P<0.0001). Finally, mRNA levels of ITα7 and Lama4 decreased following 12 days of beige differentiation in human ADSC (P<0.0001). These results demonstrate that ECM can regulate adipocyte thermogenic capacity. Specifically, Lama4, Itα7, Col1A1 and Col3A1 are involved in the modulation of metabolic function in adipocytes. A better understanding of the mechanisms underlying these interactions allows for the potential to specifically manipulate these cells to improve systemic energy metabolism and glucose homeostasis. Disclosure M.A. Gonzalez Porras: None. K. Stojkova: None. M.K. Vaicik: None. A.A. Goddi: None. R.N. Cohen: None. E. Brey: None.

STAT5 is required for lipid breakdown and beta-adrenergic responsiveness of brown adipose tissue

ObjectiveIncreasing energy expenditure through activation of brown adipose tissue (BAT) thermogenesis is an attractive approach to counteract obesity. It is therefore essential to understand the molecular mechanisms that control BAT functions. Until now several members of the Janus kinase (JAK) - signal transducer and activator of transcription (STAT) pathway have been implicated as being relevant for BAT physiology. However, whether the STAT family member STAT5 is important for the thermogenic property of adipose tissues is unknown. Therefore, we have investigated the role of STAT5 in thermogenic fat in this paper. MethodsWe performed metabolic and molecular analyses using mice that harbor an adipocyte-specific deletion of Stat5a/b alleles. ResultsWe found that STAT5 is necessary for acute cold-induced temperature maintenance and the induction of lipid mobilization in BAT following β3-adrenergic stimulation. Moreover, mitochondrial respiration of primary differentiated brown adipocytes lacking STAT5 was diminished. Increased sensitivity to cold stress upon STAT5 deficiency was associated with reduced expression of thermogenic markers including uncoupling protein 1 (UCP1), while decreased stimulated lipolysis was linked to decreased protein kinase A (PKA) activity. Additionally, brown remodeling of white adipose tissue was diminished following chronic β3-adrenergic stimulation, which was accompanied by a decrease in mitochondrial performance. ConclusionWe conclude that STAT5 is essential for the functionality and the β-adrenergic responsiveness of thermogenic adipose tissue.

The effect of TGFβ1 on thermogenic markers is dependent on the degree of adipocyte differentiation.

Transforming growth factor β (TGFβ) a multifunctional cytokine is known to regulate cell proliferation, differentiation, migration and survival. Although there is variable expression of modulators of TGFβ action during differentiation, a differential effect on fat cell metabolism at the different stages of adipocyte differentiation was unclear. In the present study, 3T3L1 cells were used as an in vitro model to study the effect of TGFβ on adipogenic and thermogenic markers at various stages of preadipocyte to mature adipocyte differentiation. As in our earlier studies on the effect of TGFβ on CEBP’s, we used a standard differentiation mix, and one with the addition of rosiglitazone. RhTGFβ1 was added to undifferentiated adipocytes (preadipocytes) and to adipocytes at day 0 (commitment stage) as well as day 10 (terminal differentiation). Cellular responses in terms of Pref1, PPARγ, TLE3, PGC1α, PRDM16, UCP1 and UCP2 mRNA levels and selected protein products, were determined. Increases in PPARγ, PRDM16, UCP1 and UCP2 mRNA and decreases in Pref1 are good indicators of successful differentiation. The early addition of rhTGFβ1 during commitment stage decreased PPARγ, PRDM16, TLE3, UCP1 and UCP2 mRNA and decreased PRDM16 protein consistent with our earlier report on the inhibition of CEBP’s by TGFβ and CCN2. The addition of rhTGFβ1 to mature adipocyte at day 10 increased UCP1 mRNA and increased PRDM16 and UCP1 proteins. In the present study, our results suggest that TGFβ1 added late enhances the thermogenic potential of mature cells and causes 3T3L1 cells to differentiate to resemble brown or beige rather than white adipose tissue.

SUN-015 Developmental Programming: Depot-Specific Inflammatory State and Distribution of Thermogenic Adipocytes Programmed by Gestational Testosterone Excess

Prenatal testosterone excess induces insulin resistance, dyslipidemia and cardiovascular defects in ewes. Considering adipose depots influence systemic metabolic status, disruptions in visceral (VAT), subcutaneous (SAT), pericardiac (PCAT) and perirenal (PRAT) adipose depots may contribute to these metabolic defects. While the changes in PCAT and PRAT in the prenatal T-treated sheep are unknown, we found an increase in the oxidative stress and inflammatory state in the VAT but not SAT, while insulin sensitivity is maintained in both depots (Biol Reprod 2016;94:113). Although all four depots are rich in white adipocytes that specialize in lipid storage, it is not clear what underlies the depot-specific differences. A potential contributor may relate to depot-specific differences in presence of brown/beige adipocytes. Brown/beige unlike white adipocytes are thermogenically active due to their expression of uncoupled protein 1 (UCP1), which uncouples mitochondrial oxidative phosphorylation to release stored energy as heat. Because they favor lipid utilization rather than storage they promote a metabolically healthy phenotype. We hypothesized that the relative distribution of brown/beige adipocytes may contribute to the depot-specific changes in inflammatory state. To test this, adipose depots from control (n=6) and prenatal T- (100mg T propionate twice a week from days 30-90 of gestation)-treated (n=5) female sheep were studied at 21 months of age. The changes in expression of inflammatory and thermogenic adipocyte markers were assessed by real time RT-PCR and data analyzed by Student’s t-test and Cohen’s effect size analysis. Prenatal T-treatment induced 1) significant increase in inflammatory cytokine interleukin 6 in VAT and PRAT while decreasing it in PCAT 2) significant increase in tumor necrosis factor in the VAT and a trend (evident by effect size analysis only) for an increase in SAT and PCAT and 3) significant increase in PRAT and a trend toward increase in the macrophage marker CD68 expression in VAT. Among the thermogenic gene markers, the expression of UCP1 was significantly increased in VAT and PCAT with a trend for an increase in PRAT. The expression of UCP2 and PPAR gamma co-activator 1 beta (PPARGC1B) were also significantly elevated in VAT with a trend for an increase in SAT. These findings are indicative of depot-specific differences in prenatal T-induced inflammatory status with effects being pronounced in VAT compared to other depots. The increases in thermogenic markers in adipose depots do not support our hypothesis but rather are reflective of a compensatory response to promote adipose depot insulin sensitivity and may have a bearing on function of organs in the proximity of respective depots. These findings are likely of translational significance in metabolic disorders associated with hyperandrogenic state.