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.

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.

Novel imaging biomarkers: epicardial adipose tissue evaluation.

Epicardial adipose tissue (EAT) is a metabolically activated beige adipose tissue, non-homogeneously surrounding the myocardium. Physiologically, EAT regulates toxic fatty acids, protects the coronary arteries against mechanical strain, regulates proinflammatory cytokines, stimulates the production of nitric oxide, reduces oxidative stress, and works as a thermogenic source against hypothermia. Conversely, EAT has pathologic paracrine interactions with the surrounded vessels, and might favour the onset of atrial fibrillation. In addition, initial atherosclerotic lesions can promote inflammation and trigger the EAT production of cytokines increasing vascular inflammation, which, in turn, may help the development of collateral vessels but also of self-stimulating, dysregulated inflammatory process, increasing coronary artery disease severity. Variations in EAT were also linked to metabolic syndrome. Echocardiography first estimated EAT measuring its thickness on the free wall of the right ventricle but does not allow accurate volumetric EAT estimates. Cardiac CT (CCT) and cardiac MR (CMR) allow for three-dimensional EAT estimates, the former showing higher spatial resolution and reproducibility but being limited by radiation exposure and long segmentation times, the latter being radiation-free but limited by lower spatial resolution and reproducibility, higher cost, and difficulties for obese patients. EAT radiodensity at CCT could to be related to underlying metabolic processes. The correlation between EAT and response to certain pharmacological therapies has also been investigated, showing promising results. In the future, semi-automatic or fully automatic techniques, machine/deep-learning methods, if validated, will facilitate research for various EAT measures and may find a place in CCT/CMR reporting.

Imaging adipose tissue browning using the TSPO-18kDa tracer [18F]FEPPA.

OBJECTIVES: The browning of white adipose tissue (WAT) into beige has been proposed as a strategy to enhance energy expenditure to combat the growing epidemic of obesity. Research into browning strategies are hampered by the lack of sensitive, translatable, imaging tools capable of detecting beige fat mass non-invasively. [18F]FDG is able to detect activated beige fat but provides little information on unstimulated beige fat mass. We have assessed the use of [18F]FEPPA, a tracer for the TSPO-18KDa found on the outer mitochondrial membrane, as an alternative imaging agent capable of detecting unstimulated brown fat (BAT) and beige fat. METHODS: Female Balb/c mice (n = 5) were treated for 7 days with the ß3 adrenergic agonist CL-316,243 to induce the browning of inguinal WAT (beige fat). Animals were imaged longitudinally with [18F]FDG and [18F]FEPPA and uptake in interscapular BAT and inguinal WAT assessed. The browning of inguinal WAT was confirmed using H&E and immunohistochemical detection of UCP-1 and TSPO. RESULTS: Repeated dosing with ß3-adrenergic agonist CL-316,243 caused a significant increase in [18F]FDG uptake in both interscapular BAT and inguinal WAT associated with the increased metabolic activity of brown and beige adipocytes respectively. [18F]FEPPA uptake was likewise increased in inguinal WAT but showed no increase in BAT uptake due to stimulation over the same time course. Furthermore, inguinal WAT uptake was unaffected by pharmacological blockade, indicating that [18F]FEPPA uptake is associated with the expression of mitochondria in BAT and beige adipocytes and independent of activation. CONCLUSION: These data show that [18F]FEPPA can detect BAT and newly formed beige fat under non-stimulated, thermoneutral conditions and that uptake after stimulation is linked to mitochondrial expression as opposed to activation.

A dual Ucp1 reporter mouse model for imaging and quantitation of brown and brite fat recruitment.

OBJECTIVES: Brown adipose tissue (BAT) dissipates nutritional energy as heat through uncoupling protein 1 (UCP1). The discovery of functional BAT in healthy adult humans has promoted the search for pharmacological interventions to recruit and activate brown fat as a treatment of obesity and diabetes type II. These efforts require in vivo models to compare the efficacy of novel compounds in a relevant physiological context. METHODS: We generated a knock-in mouse line expressing firefly luciferase and near-infrared red florescent protein (iRFP713) driven by the regulatory elements of the endogenous Ucp1 gene. RESULTS: Our detailed characterization revealed that firefly luciferase activity faithfully reports endogenous Ucp1 gene expression in response to physiological and pharmacological stimuli. The iRFP713 fluorescence signal was detected in the interscapular BAT region of cold-exposed reporter mice in an allele-dosage dependent manner. Using this reporter mouse model, we detected a higher browning capacity in female peri-ovarian white adipose tissue compared to male epididymal WAT, which we further corroborated by molecular and morphological features. In situ imaging detected a strong luciferase activity signal in a previously unappreciated adipose tissue depot adjunct to the femoral muscle, now adopted as femoral brown adipose tissue. In addition, screening cultured adipocytes by bioluminescence imaging identified the selective Salt-Inducible Kinase inhibitor, HG-9-91-01, to increase Ucp1 gene expression and mitochondrial respiration in brown and brite adipocytes. CONCLUSIONS: In our mouse model, firefly luciferase activity serves as a bona fide reporter for dynamic regulation of Ucp1. In addition, by means of iRFP713 we are able to monitor Ucp1 expression in a non-invasive fashion.

Whole-tissue 3D imaging reveals intra-adipose sympathetic plasticity regulated by NGF-TrkA signal in cold-induced beiging.

Sympathetic arborizations act as the essential efferent signals in regulating the metabolism of peripheral organs including white adipose tissues (WAT). However, whether these local neural structures would be of plastic nature, and how such plasticity might participate in specific metabolic events of WAT, remains largely uncharacterized. In this study, we exploit the new volume fluorescence-imaging technique to observe the significant, and also reversible, plasticity of intra-adipose sympathetic arborizations in mouse inguinal WAT in response to cold challenge. We demonstrate that this sympathetic plasticity depends on the cold-elicited signal of nerve growth factor (NGF) and TrkA receptor. Blockage of NGF or TrkA signaling suppresses intra-adipose sympathetic plasticity, and moreover, the cold-induced beiging process of WAT. Furthermore, we show that NGF expression in WAT depends on the catecholamine signal in cold challenge. We therefore reveal the key physiological relevance, together with the regulatory mechanism, of intra-adipose sympathetic plasticity in the WAT metabolism.

In vivo metabolic imaging and monitoring of brown and beige fat.

Activation of the thermogenic brown and beige fat is an effective means to increasing whole-body energy expenditure. In this work, a unique label-free method was developed to quantitatively assess the metabolism and thermogenesis of mouse adipose tissues in vivo. Specifically, an optical redox ratio (ORR) based on the endogenous fluorescence of mitochondrial metabolic coenzymes (nicotinamide adenine dinucleotide and flavin adenine dinucleotide) was used to measure the metabolic state of adipocytes. Our findings demonstrate that the ORR provides a label-free and real-time biomarker to determine the thermogenic response of brown, beige and white adipose tissues to a variety of physiological stimulations. In addition, the redox ratio also can be used to evaluate the degree of browning in the white fat of cold-acclimated mice. This technique is important to understand the recruitment and activation of thermogenic adipocytes in mammals and thus can help to develop therapeutic strategies against obesity.

An Adipose Tissue Atlas: An Image-Guided Identification of Human-like BAT and Beige Depots in Rodents.

[18F]Fluorodeoxyglucose-PET/CT (18F-FDG-PET/CT) imaging has been invaluable for visualizing metabolically active adipose tissues in humans with potential anti-diabetic and anti-obesity effects. To explore whether mice display human-like fat depots in anatomically comparable regions, we mapped fat depots using glucose or fatty acid imaging tracers, such as 18F-FDG through PET/CT or [123/125I]-ß-methyl-p-iodophenyl-pentadecanoic acid with SPECT/CT imaging, to analogous depots in mice. Using this type of image analysis with both probes, we define a large number of additional areas of high metabolic activity corresponding to novel fat pads. Histological and gene expression analyses validate these regions as bona fide fat pads. Our findings indicate that fat depots of rodents show a high degree of topological similarity to those of humans. Studies involving both glucose and lipid tracers indicate differential preferences for these substrates in different depots and also suggest that fatty acid-based visualized approaches may reveal additional brown adipose tissue and beige depots in humans.

Metabolic activity of brown, "beige," and white adipose tissues in response to chronic adrenergic stimulation in male mice.

Classical brown adipocytes such as those found in interscapular brown adipose tissue (iBAT) represent energy-burning cells, which have been postulated to play a pivotal role in energy metabolism. Brown adipocytes can also be found in white adipose tissue (WAT) depots [e.g., inguinal WAT (iWAT)] following adrenergic stimulation, and they have been referred to as "beige" adipocytes. Whether the presence of these adipocytes, which gives iWAT a beige appearance, can confer a white depot with some thermogenic activity remains to be seen. In consequence, we designed the present study to investigate the metabolic activity of iBAT, iWAT, and epididymal white depots in mice. Mice were either 1) kept at thermoneutrality (30°C), 2) kept at 30°C and treated daily for 14 days with an adrenergic agonist [CL-316,243 (CL)], or 3) housed at 10°C for 14 days. Metabolic activity was assessed using positron emission tomography imaging with fluoro-[(18)F]deoxyglucose (glucose uptake), fluoro-[(18)F]thiaheptadecanoic acid (fatty acid uptake), and [(11)C]acetate (oxidative activity). In each group, substrate uptakes and oxidative activity were measured in anesthetized mice in response to acute CL. Our results revealed iBAT as a major site of metabolic activity, which exhibited enhanced glucose and nonesterified fatty acid uptakes and oxidative activity in response to chronic cold and CL. On the other hand, beige adipose tissue failed to exhibit appreciable increase in oxidative activity in response to chronic cold and CL. Altogether, our results suggest that the contribution of beige fat to acute-CL-induced metabolic activity is low compared with that of iBAT, even after sustained adrenergic stimulation.