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.     

SAMM50 Regulates Thermogenesis of Beige Adipocytes Differentiated from Human Adipose-Derived Stem Cells by Balancing Mitochondrial Dynamics

Brown/beige adipocyte thermogenesis is a process that is important for energy balance. The thermogenesis of brown/beige adipocytes occurs in the mitochondria, which is modulated by the dynamic balance between mitochondrial fusion and fission. Mitophagy is also involved in mitochondrial dynamics. The sorting and assembly machinery (SAM) complex protein, SAMM50, plays a key role in mitochondrial dynamics and quality control through regulating mitophagy. However, the roles of SAMM50 in the thermogenesis of beige adipocytes remain unknown. Thus, the objective of this study was to conduct functional analyses of SAMM50. The expression of mitochondrial fusion genes was repressed by SAMM50 knockdown but was not altered by SAMM50 overexpression. These results agreed with the distribution of the fluorescence-stained mitochondria and an mtDNA copy number. In contrast, the expression of mitochondrial fission genes showed an opposite outcome. As a result, suppression by the SAMM50 shRNA inhibited the expression of thermogenic genes (UCP1, PPARGC1A, DIO2, ELOVL3, CIDEA, and CIDEC) and mitochondrial-related genes (CYCS, COX7A1, TFAM, CPT1B, and CPT2). Conversely, SAMM50 overexpression promoted the expression of the thermogenic genes and mitochondrial genes. Thus, SAMM50 links the balance between the mitochondrial dynamics and thermogenesis of beige adipocytes.     

Therapeutic Perspectives of Thermogenic Adipocytes in Obesity and Related Complications

There is a rapidly increasing prevalence of obesity and related metabolic disorders such as type 2 diabetes worldwide. White adipose tissue (WAT) stores excess energy, whereas brown and beige adipose tissues consume energy to generate heat in the process of thermogenesis. Adaptive thermogenesis occurs in response to environmental cues as a means of generating heat by dissipating stored chemical energy. Due to its cumulative nature, very small differences in energy expenditure from adaptive thermogenesis can have a significant impact on systemic metabolism over time. Targeting brown adipose tissue (BAT) activation and converting WAT to beige fat as a method to increase energy expenditure is one of the promising strategies to combat obesity. In this review, we discuss the activation of the thermogenic process in response to physiological conditions. We highlight recent advances in harnessing the therapeutic potential of thermogenic adipocytes by genetic, pharmacological and cell-based approaches in the treatment of obesity and metabolic disorders in mice and the human.     

Divergent Response of Murine and Porcine Adipocytes to Stimulation of Browning Genes by 18‐Carbon Polyunsaturated Fatty Acids and Beta‐Receptor Agonists

Long‐chain fatty acids (LCFA) are known to activate brown and beige adipocytes. However, very little is known about the effects of the number and the position of double bonds in LCFA with the same length on brown fat‐specific gene expression. To determine the specificity of LCFA in the regulation of these genes in different adipocyte models, fully differentiated 10T1/2, 3T3‐L1, murine, or porcine primary adipocytes (obtained from the subcutaneous fat pad of C57BL/6 mice or Landrace × Yorkshire × Duroc crossbred piglets) were treated with 50 μM of the following 18‐carbon fatty acids: stearic acid (STA; 18:0), oleic acid (OLA; 18:1, Δ9), linoleic acid (LNA; 18:2, Δ9,12), α‐linolenic acid (ALA; 18:3, Δ9,12,15), γ‐linolenic acid (GLA; 18:3, Δ6,9,12), or pinolenic acid (PLA; 18:3, Δ5,9,12) for 24 h with or without 4‐h norepinephrine (NE) treatment. Expression levels of thermoregulatory markers were measured by quantitative real‐time PCR. LNA, ALA, GLA, and PLA upregulated Ucp1 expression and tended to upregulate Pgc1a expression in murine primary adipocytes, but not in 10T1/2, 3T3‐L1, and porcine primary adipocytes. In murine primary adipocytes, NE induced a higher expression of Ucp1 and Pgc1a than non‐NE‐treated cells, and PLA augmented the NE effect. In 10T1/2 cells, NE upregulated Ucp1 and Pgc1a expression, but there was no fatty acid effect. However, 3T3‐L1 cells were insensitive to both fatty acid and beta‐adrenergic agonist stimulation. These results indicate that different adipocyte cell types have different levels of sensitivity to both LCFA and beta agonists in regard to induction of brown fat‐specific gene expression.     

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.     

ApoA1 Deficiency Reshapes the Phenotypic and Molecular Characteristics of Bone Marrow Adipocytes in Mice

In the present study, we studied the effect of apolipoprotein A-1 (APOA1) on the spatial and molecular characteristics of bone marrow adipocytes, using well-characterized ApoA1 knockout mice. APOA1 is a central regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, and thus HDL; our recent work showed that deficiency of APOA1 increases bone marrow adiposity in mice. We found that ApoA1 deficient mice have greatly elevated adipocytes within their bone marrow compared to wild type counterparts. Morphologically, the increased adipocytes were similar to white adipocytes, and displayed proximal tibial-end localization. Marrow adipocytes from wild type mice were significantly fewer and did not display a bone-end distribution pattern. The mRNA levels of the brown/beige adipocyte-specific markers Ucp1, Dio2, Pat2, and Pgc1a; and the expression of leptin were greatly reduced in the ApoA1 knock-out in comparison to the wild-type mice. In the knock-out mice, adiponectin was remarkably elevated. In keeping with the close ties of hematopoietic stem cells and marrow adipocytes, using flow cytometry we found that the elevated adiposity in the ApoA1 knockout mice is associated with a significant reduction in the compartments of hematopoietic stem cells and common myeloid, but not of the common lymphoid, progenitors. Moreover, the ‘beiging’-related marker osteopontin and the angiogenic factor VEGF were also reduced in the ApoA1 knock-out mice, further supporting the notion that APOA1—and most probably HDL-C—regulate bone marrow microenvironment, favoring beige/brown adipocyte characteristics.     

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.     

Dietary Arachidonic Acid Has a Time-Dependent Differential Impact on Adipogenesis Modulated via COX and LOX Pathways in Grass Carp Ctenopharyngodon idellus

In this study, we explored the function of arachidonic acid (ARA) in adipogenesis in the grass carp (Ctenopharyngodon idellus) using in vivo and in vitro models. An 8‐week feeding trial was performed using three isonitrogenous and isoenergetic purified diets: ARA‐free, ARA, and ARA + acetylsalicylic acid [ASA, a cyclooxygenase (COX) inhibitor]. Fish were sampled after 4 and 8 weeks of feeding. Results showed that ARA‐fed fish had a significantly lower intraperitoneal fat index (IPFI) and smaller adipocytes; these decreases were reversed by ASA after 8 weeks of feeding. Nevertheless, at week 4, the IPFI and adipocyte size were higher in the ARA group, and they were comparable to those of fish fed ARA + ASA. To further investigate the influence of ARA on adipocyte differentiation, confluent pre‐adipocytes of grass carp were incubated with ARA for 3 days. This in vitro experiment demonstrated that ARA promoted adipogenesis in a dose‐dependent manner. Pre‐treatment with the lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid attenuated the pro‐adipogenic function of ARA. However, after treatment with ARA for 8 days, adipocytes had a lower lipid content than cells treated with oleic acid, and ASA could suppress this effect. We thus revealed the dual function of ARA in adipogenesis in grass carp. The LOX pathway may play a key role in pro‐adipogenesis after short‐term treatment with ARA, whereas the COX pathway is possibly responsible for the inhibition of adipogenesis after long‐term treatment.     

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.     

The Effect of a Sustained High-Fat Diet on the Metabolism of White and Brown Adipose Tissue and Its Impact on Insulin Resistance: A Selected Time Point Cross-Sectional Study

(1) Background: studies on the long-term dynamic changes in fat depot metabolism in response to a high-fat diet (HFD) on hepatic lipid deposition and insulin resistance are sparse. This study investigated the dynamic changes produced by HFD and the production of dysfunctional fat depots on insulin resistance and liver lipid metabolism. (2) Methods: mice fed a chow or HFD (45% kcal fat) diet had three fat depots, liver, and blood collected at 6, 10, 20, and 30 weeks. Anthropometric changes and gene markers for adipogenesis, thermogenesis, ECM remodeling, inflammation, and tissue insulin resistance were measured. (3) Results: early responses to the HFD were increased body weight, minor deposition of lipid in liver, increased adipocyte size, and adipogenesis. Later changes were dysfunctional adipose depots, increased liver fat, insulin resistance (shown by changes in ITT) accompanied by increased inflammatory markers, increased fibrosis (fibrosis > 2-fold, p < 0.05 from week 6), and the presence of crown cells in white fat depots. Later, changes did not increase thermogenic markers in response to the increased calories and decreased UCP1 and PRDM16 proteins in WAT. (4) Conclusions: HFD feeding initially increased adipocyte diameter and number, but later changes caused adipose depots to become dysfunctional, restricting adipose tissue expansion, changing the brown/beige ratios in adipose depots, and causing ectopic lipid deposition and insulin resistance.     

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.     

Toll-like Receptor 7 (TLR7) Is Expressed in Adipocytes and the Pharmacological TLR7 Agonist Imiquimod and Adipocyte-Derived Cell-Free Nucleic Acids (cfDNA) Regulate Adipocyte Function

Endosome-localized Toll-like receptors (TLRs) 3 and 9 are expressed and functionally active in adipocytes. The functionality and role of TLR7 in adipocyte biology and innate immunity of adipose tissue (AT) is poorly characterized. We analyzed TLR7 mRNA and protein expression in murine 3T3-L1 and primary adipocytes, in co-cultures of 3T3-L1 adipocytes with murine J774A.1 monocytes and in human AT. The effects of TLR7 agonists imiquimod (IMQ) and cell-free nucleic acids (cfDNA) on adipokine concentration in cell-culture supernatants and gene expression profile were investigated. We found that TLR7 expression is strongly induced during adipocyte differentiation. TLR7 gene expression in adipocytes and AT stroma-vascular cells (SVC) seems to be independent of TLR9. IMQ downregulates resistin concentration in adipocyte cell-culture supernatants and modulates gene expression of glucose transporter Glut4. Adipocyte-derived cfDNA reduces adiponectin and resistin in cell-culture supernatants and potentially inhibits Glut4 gene expression. The responsiveness of 3T3-L1 adipocytes to imiquimod is preserved in co-culture with J774A.1 monocytes. Obesity-related, adipocyte-derived cfDNA engages adipocytic pattern recognition receptors (PRRs), modulating AT immune and metabolic homeostasis during adipose inflammation.     

Pleiotrophin Deficiency Induces Browning of Periovarian Adipose Tissue and Protects against High-Fat Diet-Induced Hepatic Steatosis

(1) Background: Pleiotrophin preserves insulin sensitivity, regulates adipose tissue lipid turnover and plasticity, energy metabolism and thermogenesis. The aim of this study was to determine the role of pleiotrophin in hepatic lipid metabolism and in the metabolic crosstalk between the liver and brown and white adipose tissue (AT) in a high-fat diet-induced (HFD) obesity mice model. (2) Methods: We analyzed circulating variables, lipid metabolism (hepatic lipid content and mRNA expression), brown AT thermogenesis (UCP-1 expression) and periovarian AT browning (brown adipocyte markers mRNA and immunodetection) in Ptn^−/− mice either fed with standard-chow diet or with HFD and in their corresponding Ptn^+/+ counterparts. (3) Results: HFD-Ptn^−/− mice are protected against the development of HFD-induced insulin resistance, had lower liver lipid content and lower expression of the key enzymes involved in triacylglycerides and fatty acid synthesis in liver. HFD-Ptn^−/− mice showed higher UCP-1 expression in brown AT. Moreover, Ptn deletion increased the expression of specific markers of brown/beige adipocytes and was associated with the immunodetection of UCP-1 enriched multilocular adipocytes in periovarian AT. (4) Conclusions: Ptn deletion protects against the development of HFD-induced insulin resistance and liver steatosis, by increasing UCP-1 expression in brown AT and promoting periovarian AT browning.     

LPS-Induced Inhibition of miR-143 Expression in Brown Adipocytes Promotes Thermogenesis and Fever

Fever is an important part of inflammatory response to infection. Although brown adipose tissue (BAT) thermogenesis is known to be potently influenced by systemic inflammation, the role of BAT during infection-induced fever remains largely unknown. Here, we injected mice with a low dose of LPS and found that low-dose LPS can directly induce thermogenesis of brown adipocytes. It is known that miR-143 is highly expressed in the BAT, and miR-143 knockout mice exhibited stronger thermogenesis under cold exposure. Interestingly, miR-143 was negatively correlated with an LPS-induced increase of TNFα and IL-6 mRNA levels, and the IL-6 pathway may mediate the inhibition of miR-143 expression. Moreover, miR-143 is down-regulated by LPS, and overexpression of miR-143 in brown adipocytes by lentivirus could rescue the enhancement of UCP1 protein expression caused by LPS, hinting miR-143 may be an important regulator of the thermogenesis in brown adipocytes. More importantly, the knockout of miR-143 further enhanced the LPS-induced increase of body temperature and BAT thermogenesis, and this result was further confirmed by in vitro experiments by using primary brown adipocytes. Mechanistically, adenylate cyclase 9 (AC9) is a new target gene of miR-143 and LPS increases BAT thermogenesis by a way of inhibiting miR-143 expression, a negative regulator for AC9. Our study considerably improves our collective understanding of the important function of miR-143 in inflammatory BAT thermogenesis.     

4-Hydroxyderricin,as a PPARγ Agonist,Promotes Adipogenesis,Adiponectin Secretion,and Glucose Uptake in 3T3-L1 Cells

Adipocyte differentiation plays a pivotal role in maintaining the production of small‐size adipocytes with insulin sensitivity, and impaired adipogenesis is implicated in insulin resistance. 4‐Hydroxyderricin (4‐HD), a phytochemical component of Angelica keiskei, possesses diverse biological properties such as anti‐inflammatory, antidiabetic, and antitumor. In the present study, we investigated the effects of 4‐HD on adipocyte differentiation. 4‐HD promoted lipid accumulation in 3T3‐L1 cells, upregulated both peroxisome proliferator‐activated receptor (PPAR)‐γ mRNA and protein expression, and acted as a ligand for PPARγ in the luciferase assay. Moreover, 4‐HD increased the mRNA and protein expression levels of adiponectin. Additionally, it promoted insulin‐dependent glucose uptake into 3T3‐L1 adipocytes and increased Akt phosphorylation and glucose transporter (GLUT) 4 mRNA expression. In summary, these findings suggest that 4‐HD, which promoted adipogenesis and insulin sensitivity in 3T3‐L1 cells, might be a phytochemical with potent insulin‐sensitizing effects.     

Physical Activity Attenuates the Obesity-Induced Dysregulated Expression of Brown Adipokines in Murine Interscapular Brown Adipose Tissue

In recent years, brown adipose tissue (BAT), which has a high heat-producing capacity, has been confirmed to exist even in adults, and it has become a focal point for the prevention and the improvement of obesity and lifestyle-related diseases. However, the influences of obesity and physical activity (PA) on the fluid factors secreted from BAT (brown adipokines) are not well understood. In this study, therefore, we focused on brown adipokines and investigated the effects of obesity and PA. The abnormal expressions of gene fluid factors such as galectin-3 (Lgals3) and Lgals3 binding protein (Lgals3bp), whose proteins are secreted from HB2 brown adipocytes, were observed in the interscapular BAT of obese mice fed a high-fat diet for 4 months. PA attenuated the abnormalities in the expressions of these genes. Furthermore, although the gene expressions of factors related to brown adipocyte differentiation such as peroxisome proliferator-activated receptor gamma coactivator 1-α were also down-regulated in the BAT of the obese mice, PA suppressed the down-regulation of these factors. On the other hand, lipogenesis was increased more in HB2 cells overexpressing Lgals3 compared with that in control cells, and the overexpression of Lgals3bp decreased the mitochondrial mass. These results indicate that PA attenuates the obesity-induced dysregulated expression of brown adipokines and suggests that Lgals3 and Lgals3bp are involved in brown adipocyte differentiation.     

Factors Associated with White Fat Browning: New Regulators of Lipid Metabolism

Mammalian adipose tissue can be divided into white and brown adipose tissue based on its colour, location, and cellular structure. Certain conditions, such as sympathetic nerve excitement, can induce the white adipose adipocytes into a new type of adipocytes, known as beige adipocytes. The process, leading to the conversion of white adipocytes into beige adipocytes, is called white fat browning. The dynamic balance between white and beige adipocytes is closely related to the body’s metabolic homeostasis. Studying the signal transduction pathways of the white fat browning might provide novel ideas for the treatment of obesity and alleviation of obesity-related glucose and lipid metabolism disorders. This article aimed to provide an overview of recent advances in understanding white fat browning and the role of BAT in lipid metabolism.