Androgen Reduces Mitochondrial Respiration in Mouse Brown Adipocytes: A Model for Disordered Energy Balance in Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is a common endocrinopathy that is associated with an adverse metabolic profile including reduced postprandial thermogenesis. Although abnormalities in adipose tissue function have been widely reported in women with PCOS, less is known about direct effects of androgen on white and, particularly, brown adipocytes. The purpose of this study was to investigate the effect of the nonaromatizable androgen dihydrotestosterone (DHT) on (1) lipid accumulation and expression of adipogenic markers in immortalized mouse brown adipose cell lines (IMBATs), (2) mitochondrial respiration in IMBATs, (3) mitochondrial DNA content and gene expression, (4) expression of brown adipose tissue (BAT) markers and thermogenic activation. In addition, we profiled the relative levels of 38 adipokines secreted from BAT explants and looked at androgen effects on adipokine gene expression in both IMBATs and immortalized mouse white adipose (IMWATs) cell lines. Androgen treatment inhibited IMBAT differentiation in a dose-dependent manner, reduced markers of adipogenesis, and attenuated the β-adrenoceptor-stimulated increase in uncoupling protein-1 (UCP1) expression. In explants of mouse interscapular BAT, androgen reduced expression of UCP1, peroxisome proliferator-activated receptor-γ coactivator-1 (PCG-1) and Cidea. Significantly, as well as affecting genes involved in thermogenesis in BAT, androgen treatment reduced mitochondrial respiration in IMBATs, as measured by the Seahorse XF method. The results of this study suggest a role for excess androgen in inhibiting brown adipogenesis, attenuating the activation of thermogenesis and reducing mitochondrial respiration in BAT. Together, these data provide a plausible molecular mechanism that may contribute to reduced postprandial thermogenesis and the tendency to obesity in women with PCOS.     

Reduced Expression of Urokinase Plasminogen Activator in Brown Adipose Tissue of Obese Mouse Models

In recent decades, the obesity epidemic has resulted in morbidity and mortality rates increasing globally. In this study, using obese mouse models, we investigated the relationship among urokinase plasminogen activator (uPA), metabolic disorders, glomerular filtration rate, and adipose tissues. Two groups, each comprised of C57BL/6J and BALB/c male mice, were fed a chow diet (CD) and a high fat diet (HFD), respectively. Within the two HFD groups, half of each group were euthanized at 8 weeks (W8) or 16 weeks (W16). Blood, urine and adipose tissues were collected and harvested for evaluation of the effects of obesity. In both mouse models, triglyceride with insulin resistance and body weight increased with duration when fed a HFD in comparison to those in the groups on a CD. In both C57BL/6J and BALB/c HFD mice, levels of serum uPA initially increased significantly in the W8 group, and then the increment decreased in the W16 group. The glomerular filtration rate declined in both HFD groups. The expression of uPA significantly decreased in brown adipose tissue (BAT), but not in white adipose tissue, when compared with that in the CD group. The results suggest a decline in the expression of uPA in BAT in obese m models as the serum uPA increases. There is possibly an association with BAT fibrosis and dysfunction, which may need further study.     

DEPP Deficiency Contributes to Browning of White Adipose Tissue

Decidual protein induced by progesterone (DEPP) was originally identified as a modulator in the process of decidualization in the endometrium. Here, we define that DEPP is involved in adipose tissue thermogenesis, which contributes to metabolic regulation. Knockdown of DEPP suppressed adipocyte differentiation and lipid accumulation in 3T3-L1 cells, induced expression of brown adipose tissue (BAT) markers in primary brown adipocyte and induced mouse embryonic fibroblasts (MEFs) differentiation to brown adipocytes. Moreover, DEPP deficiency in mice induced white adipocyte browning and enhanced BAT activity. Cold exposure stimulated more browning of white adipose tissue (WAT) and maintained higher body temperature in DEPP knockout mice compared to that in wild-type control mice. DEPP deficiency also protected mice against high-fat-diet-induced insulin resistance. Mechanistic studies demonstrated that DEPP competitively binds SIRT1, inhibiting the interaction between peroxisome proliferator-activated receptor gamma (PPARγ) and Sirtuin 1 (SIRT1). Collectively, these findings suggest that DEPP plays a crucial role in orchestrating thermogenesis through regulating adipocyte programs and thus might be a potential target for the treatment of metabolic disorders.     

Overexpression of Adiponectin Receptor 1 Inhibits Brown and Beige Adipose Tissue Activity in Mice

Adult humans and mice possess significant classical brown adipose tissues (BAT) and, upon cold-induction, acquire brown-like adipocytes in certain depots of white adipose tissues (WAT), known as beige adipose tissues or WAT browning/beiging. Activating thermogenic classical BAT or WAT beiging to generate heat limits diet-induced obesity or type-2 diabetes in mice. Adiponectin is a beneficial adipokine resisting diabetes, and causing “healthy obese” by increasing WAT expansion to limit lipotoxicity in other metabolic tissues during high-fat feeding. However, the role of its receptors, especially adiponectin receptor 1 (AdipoR1), on cold-induced thermogenesis in vivo in BAT and in WAT beiging is still elusive. Here, we established a cold-induction procedure in transgenic mice over-expressing AdipoR1 and applied a live 3-D [¹⁸F] fluorodeoxyglucose-PET/CT (¹⁸F-FDG PET/CT) scanning to measure BAT activity by determining glucose uptake in cold-acclimated transgenic mice. Results showed that cold-acclimated mice over-expressing AdipoR1 had diminished cold-induced glucose uptake, enlarged adipocyte size in BAT and in browned WAT, and reduced surface BAT/body temperature in vivo. Furthermore, decreased gene expression, related to thermogenic Ucp1, BAT-specific markers, BAT-enriched mitochondrial markers, lipolysis and fatty acid oxidation, and increased expression of whitening genes in BAT or in browned subcutaneous inguinal WAT of AdipoR1 mice are congruent with results of PET/CT scanning and surface body temperature in vivo. Moreover, differentiated brown-like beige adipocytes isolated from pre-adipocytes in subcutaneous WAT of transgenic AdipoR1 mice also had similar effects of lowered expression of thermogenic Ucp1, BAT selective markers, and BAT mitochondrial markers. Therefore, this study combines in vitro and in vivo results with live 3-D scanning and reveals one of the many facets of the adiponectin receptors in regulating energy homeostasis, especially in the involvement of cold-induced thermogenesis.     

Brown Adipose Tissue and Its Role in Insulin and Glucose Homeostasis

The increased worldwide prevalence of obesity, insulin resistance, and their related metabolic complications have prompted the scientific world to search for new possibilities to combat obesity. Brown adipose tissue (BAT), due to its unique protein uncoupling protein 1 (UPC1) in the inner membrane of the mitochondria, has been acknowledged as a promising approach to increase energy expenditure. Activated brown adipocytes dissipate energy, resulting in heat production. In other words, BAT burns fat and increases the metabolic rate, promoting a negative energy balance. Moreover, BAT alleviates metabolic complications like dyslipidemia, impaired insulin secretion, and insulin resistance in type 2 diabetes. The aim of this review is to explore the role of BAT in total energy expenditure, as well as lipid and glucose homeostasis, and to discuss new possible activators of brown adipose tissue in humans to treat obesity and metabolic disorders.     

Cold-Induced Browning Dynamically Alters the Expression Profiles of Inflammatory Adipokines with Tissue Specificity in Mice

Cold exposure or β₃-adrenoceptor agonist treatment induces the adipose tissues remodeling, relevant for beige adipogenesis within white adipose tissue (WAT). It remains unclear whether this process influences inflammatory adipokines expression in adipose tissues. We determine the temporal profile of cold or β₃-adrenoceptor agonist (CL316,243)-induced changes in the expression of inflammatory adipokines in adipose tissues in mice or primary mice adipocytes. Male C57BL/6J mice at eight weeks old were exposed to 4 °C for 1–5 days. Interscapular brown adipose tissue (iBAT), inguinal subcutaneous WAT (sWAT) and epididymal WAT (eWAT) were harvested for gene and protein expression analysis. In addition, cultured primary mice brown adipocyte (BA) and white adipocyte (WA) treated with or without CL316,243 were harvested for gene expression analysis. The inflammatory adipokines expressed significantly higher in WAT than BAT at baseline. They were rapidly changed in iBAT, while down-regulated in sWAT and up-regulated in eWAT during the cold acclimation. Upon CL316,243 treatment, detected inflammatory adipokines except Leptin were transiently increased in both BA and WA. Our in vivo and in vitro data demonstrate that the browning process alters the inflammatory adipokines expression in adipose tissues, which is acutely responded to in iBAT, dynamically decreased in sWAT whilst increased in eWAT for compensation.     

Role of Brown and Beige Adipose Tissues in Seasonal Adaptation in the Raccoon Dog (Nyctereutes procyonoides)

Brown adipose tissue (BAT) expresses uncoupling protein-1 (UCP1), which enables energy to be exerted towards needed thermogenesis. Beige adipocytes are precursor cells interspersed among white adipose tissue (WAT) that possess similar UCP1 activity and capacity for thermogenesis. The raccoon dog (Nyctereutes procyonoides) is a canid species that utilizes seasonal obesity to survive periods of food shortage in climate zones with cold winters. The potential to recruit a part of the abundant WAT storages as beige adipocytes for UCP1-dependent thermogenesis was investigated in vitro by treating raccoon dog adipocytes with different browning inducing factors. In vivo positron emission tomography/computed tomography (PET/CT) imaging with the glucose analog ¹⁸F-FDG showed that BAT was not detected in the adult raccoon dog during the winter season. In addition, UCP1 expression was not changed in response to chronic treatments with browning inducing factors in adipocyte cultures. Our results demonstrated that most likely the raccoon dog endures cold weather without the induction of BAT or recruitment of beige adipocytes for heat production. Its thick fur coat, insulating fat, and muscle shivering seem to provide the adequate heat needed for surviving the winter.     

Cold Exposure Drives Weight Gain and Adiposity following Chronic Suppression of Brown Adipose Tissue

Therapeutic activation of thermogenic brown adipose tissue (BAT) may be feasible to prevent, or treat, cardiometabolic disease. However, rodents are commonly housed below thermoneutrality (~20 °C) which can modulate their metabolism and physiology including the hyperactivation of brown (BAT) and beige white adipose tissue. We housed animals at thermoneutrality from weaning to chronically supress BAT, mimic human physiology and explore the efficacy of chronic, mild cold exposure (20 °C) and β3-adrenoreceptor agonism (YM-178) under these conditions. Using metabolic phenotyping and exploratory proteomics we show that transfer from 28 °C to 20 °C drives weight gain and a 125% increase in subcutaneous fat mass, an effect not seen with YM-178 administration, thus suggesting a direct effect of a cool ambient temperature in promoting weight gain and further adiposity in obese rats. Following chronic suppression of BAT, uncoupling protein 1 mRNA was undetectable in the subcutaneous inguinal white adipose tissue (IWAT) in all groups. Using exploratory adipose tissue proteomics, we reveal novel gene ontology terms associated with cold-induced weight gain in BAT and IWAT whilst Reactome pathway analysis highlights the regulation of mitotic (i.e., G2/M transition) and metabolism of amino acids and derivatives pathways. Conversely, YM-178 had minimal metabolic-related effects but modified pathways involved in proteolysis (i.e., eukaryotic translation initiation) and RNA surveillance across both tissues. Taken together these findings are indicative of a novel mechanism whereby animals increase body weight and fat mass following chronic suppression of adaptive thermogenesis from weaning. In addition, treatment with a B3-adrenoreceptor agonist did not improve metabolic health in obese animals raised at thermoneutrality.     

Potential of Nutraceutical Supplementation in the Modulation of White and Brown Fat Tissues in Obesity-Associated Disorders: Role of Inflammatory Signalling

The high incidence of obesity is associated with an increasing risk of several chronic diseases such as cardiovascular disease, type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Sustained obesity is characterized by a chronic and unsolved inflammation of adipose tissue, which leads to a greater expression of proinflammatory adipokines, excessive lipid storage and adipogenesis. The purpose of this review is to clarify how inflammatory mediators act during adipose tissue dysfunction in the development of insulin resistance and all obesity-associated diseases. In particular, we focused our attention on the role of inflammatory signaling in brown adipose tissue (BAT) thermogenic activity and the browning of white adipose tissue (WAT), which represent a relevant component of adipose alterations during obesity. Furthermore, we reported the most recent evidence in the literature on nutraceutical supplementation in the management of the adipose inflammatory state, and in particular on their potential effect on common inflammatory mediators and pathways, responsible for WAT and BAT dysfunction. Although further research is needed to demonstrate that targeting pro-inflammatory mediators improves adipose tissue dysfunction and activates thermogenesis in BAT and WAT browning during obesity, polyphenols supplementation could represent an innovative therapeutic strategy to prevent progression of obesity and obesity-related metabolic diseases.     

Differential Effects of 17,18-EEQ and 19,20-EDP Combined with Soluble Epoxide Hydrolase Inhibitor t-TUCB on Diet-Induced Obesity in Mice

17,18-Epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) are bioactive epoxides produced from n-3 polyunsaturated fatty acid eicosapentaenoic acid and docosahexaenoic acid, respectively. However, these epoxides are quickly metabolized into less active diols by soluble epoxide hydrolase (sEH). We have previously demonstrated that an sEH inhibitor, t-TUCB, decreased serum triglycerides (TG) and increased lipid metabolic protein expression in the brown adipose tissue (BAT) of diet-induced obese mice. This study investigates the preventive effects of t-TUCB (T) alone or combined with 19,20-EDP (T + EDP) or 17,18-EEQ (T + EEQ) on BAT activation in the development of diet-induced obesity and metabolic disorders via osmotic minipump delivery in mice. Both T + EDP and T + EEQ groups showed significant improvement in fasting glucose, serum triglycerides, and higher core body temperature, whereas heat production was only significantly increased in the T + EEQ group. Moreover, both the T + EDP and T + EEQ groups showed less lipid accumulation in the BAT. Although UCP1 expression was not changed, PGC1α expression was increased in all three treated groups. In contrast, the expression of CPT1A and CPT1B, which are responsible for the rate-limiting step for fatty acid oxidation, was only increased in the T + EDP and T + EEQ groups. Interestingly, as a fatty acid transporter, CD36 expression was only increased in the T + EEQ group. Furthermore, both the T + EDP and T + EEQ groups showed decreased inflammatory NFκB signaling in the BAT. Our results suggest that 17,18-EEQ or 19,20-EDP combined with t-TUCB may prevent high-fat diet-induced metabolic disorders, in part through increased thermogenesis, upregulating lipid metabolic protein expression, and decreasing inflammation in the BAT.     

ZW290 Increases Cold Tolerance by Inducing Thermogenesis via the Upregulation of Uncoupling Protein 1 in Brown Adipose Tissue In Vitro and In Vivo

To provide molecular evidence on the thermogenic mechanism of primary brown adipocytes, western blot analysis was used to detect brown adipose tissue (BAT)-specific gene expressions. BAT protects the mammals from hypothermia injury with a large amount of mitochondria and high expression of uncoupling Protein 1 (UCP1), which is the vital protein to determine the heat production in BAT. In our previous study, the compound ZW290 (the structure shown in Fig. 1) was obtained by molecular docking with a UCP1 inducer. In the present study, ZW290 not only significantly upregulated the expression of UCP1 protein (p < 0.01) and its related signaling pathway in the primary brown adipocytes, but also remarkably decreased the mitochondrial membrane potential and the concentration of adenosine triphosphate (ATP) (p < 0.01). Kunming (KM) mice were kept under acute cold exposure (−20°C) to evaluate the preventive and protective effects of ZW290 on cold injury, and revealed its regulating mechanism in vitro. The rectal and body temperatures of ZW290-treated mice were significantly higher than those of the control (or model) group both at room temperature and at −20°C (p < 0.001). Hematoxylin–eosin (HE) staining and immunohistochemistry indicated that ZW290 notably decreased the size of lipid droplets in BAT and increased the content of mitochondria and the expression of UCP1 in BAT and white adipose tissue (WAT). Furthermore, the survival rate showed that ZW290 could prolong the overall survival of mice. Therefore, we obtained the conclusion that ZW290 might transform energy into heat by inhibiting ATP synthesis and increasing the expression of UCP1. Additionally, ZW290 may enhance cold tolerance by increasing heat production through increasing the content of mitochondria and the expression of UCP1 in BAT and WAT.     

Papain Ameliorates Lipid Accumulation and Inflammation in High-Fat Diet-Induced Obesity Mice and 3T3-L1 Adipocytes via AMPK Activation

Papain is a proteolytic enzyme present in the leaves, fruits, roots, and latex of the Carica papaya (papaya) plant. Although it exhibits a wide range of activities, there are no reports on the anti-obesity effects of papain. This study examined the anti-obesity effect and obesity-involved anti-inflammatory mechanism of papain in in vivo and in vitro models using high-fat diet (HFD)-induced obese mice and 3T3-L1 preadipocytes. Oral administration of papain reduced HFD-induced weight of the body, liver, and adipose tissues of mice. Papain also reduced hepatic lipid accumulation and adipocyte size. Moreover, serum total cholesterol and triglyceride levels were markedly reduced in papain-treated mice. In addition, papain inhibited the differentiation of preadipocytes and oil accumulation in 3T3-L1 preadipocytes and rat primary preadipocytes. Mechanistically, papain significantly downregulated the protein levels of key adipogenesis regulators and reversed the expression of pro-inflammatory cytokines and adipokines in HFD-induced obese mice and 3T3-L1 preadipocytes. Papain also markedly enhanced activation of the AMP-activated protein kinase pathway in both models. Collectively, these results suggest that papain exerts anti-obesity effects in HFD-induced mice and 3T3-L1 preadipocytes by regulating levels of adipogenic factors involved in lipid metabolism and inflammation; thus, it could be useful in the prevention and treatment of obesity.     

Insulin Modulates the Bioenergetic and Thermogenic Capacity of Rat Brown Adipocytes In Vivo by Modulating Mitochondrial Mosaicism

The effects of insulin on the bioenergetic and thermogenic capacity of brown adipocyte mitochondria were investigated by focusing on key mitochondrial proteins. Two-month-old male Wistar rats were treated acutely or chronically with a low or high dose of insulin. Acute low insulin dose increased expression of all electron transport chain complexes and complex IV activity, whereas high dose increased complex II expression. Chronic low insulin dose decreased complex I and cyt c expression while increasing complex II and IV expression and complex IV activity. Chronic high insulin dose decreased complex II, III, cyt c, and increased complex IV expression. Uncoupling protein (UCP) 1 expression was decreased after acute high insulin but increased following chronic insulin treatment. ATP synthase expression was increased after acute and decreased after chronic insulin treatment. Only a high dose of insulin increased ATP synthase activity in acute and decreased it in chronic treatment. ATPase inhibitory factor protein expression was increased in all treated groups. Confocal microscopy showed that key mitochondrial proteins colocalize differently in different mitochondria within a single brown adipocyte, indicating mitochondrial mosaicism. These results suggest that insulin modulates the bioenergetic and thermogenic capacity of rat brown adipocytes in vivo by modulating mitochondrial mosaicism.     

Brown adipose tissue triacylglycerol fatty acids of obese and lean mice: In situ and in transplants

The triacylglcyerols of white adipose tissue (WAT) from animals with high rates of lipogenesis, such as obese hyperglycemic mice or hypothalamically lesioned rats, contain high proportions of palmitoleic acid (16∶1) and low proportions of linoleic acid (18∶2). These differences appear to result from dilution of dietary 18∶2 by synthesized fatty acids, particularly 16∶1. To test this we have investigated the triacylglycerol fatty acid composition of brown and white adipose tissue of lean and obese mice, as brown adipose tissue (BAT) has a higher lipogenic rate than WAT and lipogenesis is faster in obese than in lean mice. Between three and eight weeks of age the proportions of fatty acids in the tissues changed, with a marked fall in milk-derived lauric and myristic acids. From 8 to 16 weeks they were more stable and the proportions of 16∶1 and 18∶2 in the different tissues were as expected, with the highest and lowest proportions, respectively, in BAT from obese mice. When BAT from obese mice was transplanted under the kidney capsule of lean mice, or vice versa, for one month, the fatty acid composition of the grafts changed toward that of the host BAT. The proportions of 18∶2 and, to a lesser extent, 16∶1 were slightly higher in the grafts than in the hosts but since this also occurred in lean-to-lean and obese-to-obese grafts it was probably a transplantation artifact. Overall, the results confirm than the physiological environment, rather than the source of the adipose tissue, is the major determinant of its fatty acid composition.     

Uncoupling Protein 1 Does Not Produce Heat without Activation

Mitochondrial uncoupling protein 1 (UCP1) is the crucial mechanistic component of heat production in classical brown fat and the newly identified beige or brite fat. Thermogenesis inevitably comes at a high energetic cost and brown fat, ultimately, is an energy-wasting organ. A constrained strategy that minimizes brown fat activity unless obligate will have been favored during natural selection to safeguard metabolic thriftiness. Accordingly, UCP1 is constitutively inhibited and is inherently not leaky without activation. It follows that increasing brown adipocyte number or UCP1 abundance genetically or pharmacologically does not lead to an automatic increase in thermogenesis or subsequent metabolic consequences in the absence of a plausible route of concomitant activation. Despite its apparent obviousness, this tenet is frequently ignored. Consequently, incorrect conclusions are often drawn from increased BAT or brite/beige depot mass, e.g., predicting or causally linking beneficial metabolic effects. Here, we highlight the inherently inactive nature of UCP1, with a particular emphasis on the molecular brakes and releases of UCP1 activation under physiological conditions. These controls of UCP1 activity represent potential targets of therapeutic interventions to unlock constraints and efficiently harness the energy-expending potential of brown fat to prevent and treat obesity and associated metabolic disorders.     

Dietary t10,c12-CLA but not c9,t11 CLA Reduces Adipocyte Size in the Absence of Changes in the Adipose Renin–Angiotensin System in <Emphasis Type="Italic">fa/fa</Emphasis> Zucker Rats

In obesity, increased activity of the local renin–angiotensin system (RAS) and enlarged adipocytes with altered adipokine production are linked to the development of obesity-related health problems and cardiovascular disease. Mixtures of conjugated linoleic acid (CLA) isomers have been shown to reduce adipocyte size and alter the production of adipokines. The objective of this study was to investigate the effects of feeding individual CLA isomers on adipocyte size and adipokines associated with the local adipose RAS. Male fa/fa Zucker rats received either (a) control, (b) cis(c)9,trans(t)11-CLA, or (c) t10,c12-CLA diet for 8 weeks. The t10,c12-CLA isomer reduced adipocyte size and increased cell number in epididymal adipose tissue. RT-PCR and Western blot analysis revealed that neither CLA isomer altered mRNA or protein levels of angiotensinogen or AngII receptors in adipose tissue. Likewise, levels of the pro-inflammatory cytokines TNF-α and IL-6 or the anti-inflammatory cytokine IL-10 were unchanged in adipose tissue. Similarly, neither CLA isomer had any effect on phosphorylation nor DNA binding of NF-κB. Our results suggest that although the t10,c12-CLA isomer had beneficial effects on reducing adipocyte size in obese rats, this did not translate into changes in the local adipose RAS or associated adipokines.     

Leptin Modulates the Response of Brown Adipose Tissue to Negative Energy Balance: Implication of the GH/IGF-I Axis

The growth hormone (GH)/insulin-like growth factor I (IGF-I) axis is involved in metabolic control. Malnutrition reduces IGF-I and modifies the thermogenic capacity of brown adipose tissue (BAT). Leptin has effects on the GH/IGF-I axis and the function of BAT, but its interaction with IGF-I and the mechanisms involved in the regulation of thermogenesis remains unknown. We studied the GH/IGF-I axis and activation of IGF-I-related signaling and metabolism related to BAT thermogenesis in chronic central leptin infused (L), pair-fed (PF), and control rats. Hypothalamic somatostatin mRNA levels were increased in PF and decreased in L, while pituitary GH mRNA was reduced in PF. Serum GH and IGF-I concentrations were decreased only in PF. In BAT, the association between suppressor of cytokine signaling 3 and the IGF-I receptor was reduced, and phosphorylation of the IGF-I receptor increased in the L group. Phosphorylation of Akt and cyclic AMP response element binding protein and glucose transporter 4 mRNA levels were increased in L and mRNA levels of uncoupling protein-1 (UCP-1) and enzymes involved in lipid anabolism reduced in PF. These results suggest that modifications in UCP-1 in BAT and changes in the GH/IGF-I axis induced by negative energy balance are dependent upon leptin levels.