Preclinical in vivo imaging for brown adipose tissue

Life Sciences 249 (2020) 117500

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Preclinical in vivo imaging for brown adipose tissue ⁎

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Jiaojiao Gu, Xinlu Wang , Hua Yang, He Li, Jie Wang Department of Ultrasound, Shengjing Hospital of China Medical University, Shengjing Hospital, No. 36, Sanhao Street, Heping District, Shenyang, Liaoning, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Brown adipose tissue Positron-emission tomography (PET) Magnetic resonance imaging (MRI) Ultrasound

Brown adipose tissue (BAT) has multiple functions in the human body, including the production of heat and increasing energy consumption. However, BAT is also related to many kinds of diseases, such as obesity and metabolic disorders. The progression of such diseases occurs at the cellular level, and thus, imaging techniques could prove greatly beneficial for determining optimal therapeutic regimens. Currently, positron-emission tomography (PET) is considered to be the gold standard for assessing the function of activated BAT. However, PET also has inherent disadvantages, and, thus, recent efforts have been focused on exploring, and potentially developing, new imaging techniques to better observe BAT and evaluate its metabolic function. Researchers have already achieved promising success with computed tomography, magnetic resonance approaches, ultrasound, new tracers for use in PET, and other imaging techniques through in vivo and in vitro animal experiments. Since, these studies have shown that BAT may serve as an effective therapeutic target for treatment of metabolic dysfunction diseases, the development of an efficient in vivo BAT imaging technique that is applicable to humans will prove to be of great clinical value. In this review, classical PET imaging technique is highlighted as well as the current status of preclinical imaging methods developed for BAT examination.

1. Introduction Adipose tissue is categorized as either white (WAT) or brown (BAT) based on their associated colours. These two types of tissue exhibit distinctly unique characteristics. Beige adipocytes primarily exists in subcutaneous WAT and is also referred to as ‘browning’ WAT [1].WAT is primarily distributed throughout the inguinal, retroperitoneal, gonadal, mesenteric, and subcutaneous regions [2], and its primary function is to restore energy in the form of unilocular triglyceride droplets [3]. BAT, alternatively, has been reported to be primarily distributed throughout the interscapular region and is associated with heat production by consuming energy. BAT has the capacity to convert a large amount of caloric energy into heat [4]. The uncoupling protein 1 (UCP1), which is uniquely expressed in BAT, functions to uncouple mitochondrial respiration from ATP synthesis, thereby contributing to heat production. Moreover, BAT can promote thermogenesis and energy consumption through UCP1 in infants and hibernating mammals [5]. The volume of BAT is highest in infants, after which point it gradually decreases with aging [6]. BAT takes part in energy metabolism and non-shivering heat production throughout the human body [7]. BAT also has a strong heat production function in mice, which assists in combating cold environments through body temperature regulation [8–11]. Furthermore, the

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activation of BAT can prevent or reverse obesity, diabetes, heart failure, and other complications caused by diet [12–14], leading researchers to believe that BAT may be an ideal target for preventing or treating obesity and reducing plasma glucose and lipid levels by negatively shifting the chronic heat [15–17]. BAT plays an important role in maintaining the energy homeostasis in the human body. An epidemiological study found that the volume of BAT in type 2 diabetics was greatly lower than in normal controls [18]. Similarly, cold exposure experiments found that glucose uptake in BAT decreased in patients who suffered from diabetes [19], indicating that BAT is closely associated with diabetes. Studies have also indicated a relationship between BAT and heart failure. A recent study assessed the effects of neuroendocrine activation on BAT in heart failure in vivo [20]. This study revealed that the volume of BAT in heart failure samples was significantly lower than that in the control group, along with a decrease in the lipid globules and a four-fold increase in UCP1 mRNA levels. Further, novel discoveries suggest that BAT may function in the immune system as well. Positron-emission tomography-computed tomography (PET-CT) further verified that programmed death-ligand 1 (PD-L1) is expressed in BAT and that the intensity of the signal does not change with cold exposure or β-adrenergic activation [21]. Alternatively, recent studies have also reported that adipose tissue dysfunction in obese patients is closely associated with the development and progression of

Corresponding author. E-mail address: [email protected] (X. Wang).

https://doi.org/10.1016/j.lfs.2020.117500 Received 22 November 2019; Received in revised form 23 February 2020; Accepted 2 March 2020 Available online 05 March 2020 0024-3205/ © 2020 Elsevier Inc. All rights reserved.

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3. Imaging techniques used to study BAT

various cancers, including endometrial, postmenopausal breast, ovarian, and esophageal cancers. Moreover, the browning of adipose tissue usually results in a high incidence of cachexia in patients with advanced cancer, contributing to decreased survival rates and poor treatment outcomes [22], although, the underlying mechanism remains unknown. Furthermore, fludeoxyglucose (18F-FDG) PET-CT studies have confirmed a possible role for BAT in cancer progression and associated metabolic disturbances [23]. Stijn A. Bos et, al. chose 62 patients who had active cancer and 80 controls without active cancer and operated 18F-FDG PET/CT. Results showed that patients who had active cancer on PET/CT had higher BAT volume compared to patients without active cancer. The findings suggest a possible role of BAT in cancer activity and associated metabolic disturbances [23]. Based on these findings, the adipose tissue is considered to be a promising target for cancer prevention and future therapeutics. Imaging studies are essential for us to understand the structure and function of BAT, and can help us understand the correlation between structural and morphological changes in BAT and impaired function. So far, some methods have been applied to human research, but many methods cannot be applied to humans. The main purpose of scientific research is to apply these findings to the clinic. Compared with human experiments, preclinical animal experiments can provide us with more variety and larger amounts of data and information, thereby further promoting clinical development.

3.1. Imaging of BAT via PET As a relatively new method of inspection, PET is being employed more frequently and widely. Its imaging principle involves chemical linking of a positron-emitting nucleus (11C, 15N, 15O, 18F or other nuclides) to a specific biological molecule to develop a radiopharmaceutical (often referred to as a radioactive tracer). After the agent is injected into the human body, the tracer participates in the corresponding biological activities, and emits a positron beam, which is accepted by the PET system after being quenched and further converted into an image visible to the naked eye. Nowadays, many tracers are used in PET to better understand active BAT, and many new technologies are also been developed.

3.1.1. 18F-FDG 18 F-FDG is transported in the cell by the same transporter used for glucose and becomes phosphorylated by hexokinase to form 18FDG-6phosphate. Metabolic activity as an indicator is difficult to quantify, however, information can be obtained by observing glucose uptake and oxygen content by PET-CT [38–39]. BAT is identified by PET through the uptake of 18F-FDG in BAT. Under cold stimulation, uptake increases significantly. Although 18F-FDG has many known limitations, such as high cost, low spatial resolution and sensitivity, its uptake into adipose tissue following cold exposure continues to be recognized as the gold standard for identifying BAT and is used to describe the role that BAT has in vivo [40]. Although 18F-FDG is currently widely used to identify BAT, there is no globally accepted definition for the use of 18F-FDG PET-CT. Recent studies have shown that 18F-FDG PET is not reliable in measuring heat production by BAT [19]. Researchers believe that 18FFDG uptake may indicate lower BAT lipid content and higher angiogenesis than oxidative metabolism itself [41]. Mohammed K et al. carried out infrared thermal imaging and 18F-FDG PET/MRI experiments and concluded that stimulation by adrenaline can enhance 18FFDG uptake of BAT, independent of the thermogenic function of UCP1 [42]. Moreover, PET-CT scan revealed that 18F-FDG was almost entirely taken up by BAT after 1 h of exposure to cold in wild-type mice. However, APCmin mice, which are commonly used as a preclinical model for tumorigenesis and harbour a mutant allele of the adenomatous polyposis coli (APC)gene, also develop adenomatous polyp formation in the intestine, mimicking the phenotype of human familial adenomatous polyposis. Further, 18F-FDG in these mice was primarily distributed in the area of heart and bladder with no BAT aggregation observed. Thus, it was considered that BAT is dysfunctional in APCmin mice [43]. Additionally, Kimberly N et al. used Zucker lean (ZL) and obese (ZF) rats to show that the β3-adrenoreceptor agonist may have significant effects on BAT activation and contributes to obesity treatment [44]. Jin Won Park carried out small-animal PET-CT experiments in mice, sacrificed them, extracted the interscapular BAT and WAT depots, and then weighed and measured the tissues for 18F-FDG uptake. PET-CT showed low 18F-FDG uptake in both BAT and inguinal WAT at the start of the study. However, following single stimulation with the β3 agonist CL316,243, BAT uptake was seen to gradually increase, while the inguinal WAT uptake only increased slightly following the first stimulation, although it gradually increased to the levels in BAT after prolonged stimulation. The immunostaining of BAT and WAT for UCP1 content also confirmed that the prolonged stimulation of CL316,243 can induce WAT browning. UCP1 content was low in baseline inguinal WAT, yet demonstrated a linear increase after 10 days of CL316,243 injection. These results are consistent with that of 18F-FDG PET-CT experiments. The authors concluded that 18F-FDG PET-CT has the capacity to monitor brown adipocyte recruitment into WAT depots in vivo and may, thus, be useful for screening the efficacy of strategies to promote WAT browning [45].

2. Distribution characteristics of BAT 2.1. Distribution characteristics of BAT in small animals Nearly 500 years ago, brown fat was first discovered in the marmot by anatomical inspection [24]. BAT is found in most mammals, particularly in those that hibernate, however, it has been primarily studied in rodents, such as rats, mice, and hamsters. In rodents, BAT is predominantly localized in distinct deposits of the thoracic region, with the largest areas found in the intrascapular and dorso-cervical region [25] or in the axillary and subscapular regions [26]. Occasionally, BAT has been found to localize in the abdomen, in the area surrounding the kidneys [27]. Based on these studies, it has been reported that BAT in rodents is primarily distributed throughout the interscapular region, extending to the back, neck muscle tissue, armpit, chest, thymus, the area parallel to the thoracic aorta, and the abdomen, and wraps around the adrenal gland and the renal blood vessels [28–29]. Further, mice studies have found that the predominate BAT deposits are located around the spinal cord in the paravertebral area as well as in the mediastinum, especially around the heart, at the apex, and in the paraaortic area [30].

2.2. Distribution characteristics of BAT in humans The classical interscapular brown adipose tissue (iBAT) in human infants is myogenic origin which is similar to the fat depots in mice and other small mammals [31]. Researchers believe that the tissue decreases quickly with aging [32]. However, the 18F-FDG PET scanning on adult humans revealed that the metabolically active fat tissue can be detected in the supraclavicular and cervical areas, in a distinct fascial plane in the ventral neck, superficial and lateral to the sternocleidomastoid muscles [32–34]. Several researches also indicate that the BAT decreases with age and obesity [18,35–36]. BAT amount and activity was reported to be 4-fold higher in lean individuals compared to overweight/obese individuals [37]. Cypess AM et al. concluded that functionally active brown adipose tissue were present in adult humans and were more frequent in women than in men through 18F-FDG PET–CT scanning on 1013 women and 959 men [33].

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3.1.2. 15O-labelled oxygen and 11C-labelled acetate As described above, 18F-FDG can only be used to reflect the glucose metabolism process in BAT. The oxidative metabolism of BAT can be measured using 15O-labelled oxygen or 11C-labelled acetate; while nonesterified fatty acid uptake is measured using the fatty acid analogue 14(R,S)-[18]F-fluoro-6-thia-heptadecanoic acid ([18F]-FTHA)[46–48]. The short-lived, positron-emitting radioisotope of oxygen, 15O (t1/ 2 = 123 s), was first used with an invasive, intracarotid technique to study cerebral blood flow and oxygen utilization in humans by TerPogossian and colleagues [49–50], and is now considered the gold standard measure for tissue oxygen consumption (VO2). The study in humans by Muzik et al. showed that during cold stress, higher 18F-FDG standardized uptake values correlate with higher VO2 in BAT [51]. This technique demonstrates the overall oxidative metabolic rate of BAT, which also includes oxidation of all known possible substrates in BAT, including glucose, fatty acids, ketone bodies and amino acids. Another study [52] described a strong positive correlation between BAT daily energy expenditure and blood flow in humans, indicating the interdependence of both processes, suggesting that either UCP-1 mediated heat produced in BAT is distributed via circulation, or that the perfusion increases to provide oxygen and substrates for active BAT. This study also proposed that BAT oxidizes substrates at room temperature (RT), albeit in modest quantities compared with that under cold conditions, which suggests that BAT may be involved in thermogenesis at RT. However, it is unclear how much of the UCP-1 protein in BAT mitochondria become uncoupled at RT. PET-CT utilizing 15O-labelled oxygen or 11C-labelled acetate are more reliable than that with 18F-FDG for evaluating the BAT volume. One of the advantages of measuring oxygen consumption in BAT using 15OeO2 PET imaging is that it is a direct and non-invasive technique, which is not influenced by substrate availability and utilization rate, as may be the case with 18F-FDG and 18 F-FTHA radiotracers [53]. It has been demonstrated that the kinetics of 11C-acetate reflect tricarboxylic acid cycle flux and thus, myocardial oxygen consumption (MVO2) under a wide range of haemodynamic conditions in animals and humans. Another potential advantage of 11Cacetate is the potential for measuring myocardial blood flow and MVO2 simultaneously [53]. Furthermore, it is readily used for the direct measurement of both oxidative metabolism and Krebs cycle kinetics. However, the two methods have their own inherent limitations. These tracers have a short radioactive half-life and are rapidly metabolised in tissues; thus, only real-time PET can be carried out. Further, the tracers cannot be applied for the measurement of human BAT volume. While the uptake of [18F]-FTHA in BAT is lower than that in other tissues, it is difficult to distinguish between BAT and WAT. Thus far, these imaging methods have not been used in humans. Instead other techniques have been employed, although they too have associated disadvantages; for example, single photon emission tomography/computed tomography (SPETCT) has low resolution and specificity; while MRI as well as magnetic resonance spectroscopy (MRS) are easily affected by motion artefacts and partial volume [41].

Siemens Medical Solutions, and scanned rats for 90 min each, followed by scanning with the Inveon MM CT. Whole body scans revealed that [11C]Dalene uptake was observed in the adipose tissue of the interscapular region of the rat. Further, iBAT uptake of [11C]Dalene was slower than that of 4-[(11) C]methylamino-4′-N,N-dimethylaminoazobenzene ([11C]TAZA). In addition, compared to [11C]Dalene, [11C]TAZA was shown to have a higher level of uptake in BAT tissue. Moreover, in addition to the BAT in the interscapular region, BAT was also observed in other regions, such as cervical BAT and periaortic BAT. However, the uptake of [11C]TAZA by iBAT was initially slow, yet continued to rise after 90 min of scanning. In contrast, [11C]PIB appears to be rapidly taken up and rapidly cleared in iBAT. Additionally, micro PET-CT results found that tamoxifen competitively affects the binding of BAT to [11C]TAZA in all rat BAT tissues. Thus, the use of [11C]TAZA may allow for better visualization of BAT, however, only in the absence of competitive binding effects of norepinephrine (NE) transporter inhibitors [57]. Lastly, Olof Eriksson et al. [56] sliced BAT tissue and subcutaneous WAT tissue in rats, and subjected them to immunofluorescence staining with CB1 and UCP-1, followed by PET scanning to evaluate the binding of radiolabelled CB1 antagonist 18F-FMPEP-d 2 in BAT in vitro and in vivo. Results revealed that CB1 and UCP-1 were colocalized in BAT, however, not in WAT tissue. Further, significant binding of BAT tissue to 18F-FMPEP-d 2 was observed in rats in vivo, indicating that CB1 density was high; however, no obvious binding of 18 F-FMPEP-d 2 was observed in WAT. In conclusion, 18F-FMPEP-d 2 PET can be used non-invasively to quantify CB1 density in vivo in rats [56]. Although CB1 and UCP-1 exhibit similar expression patterns in humans, this technique cannot be readily applied to humans as it is in rats as 18F-FMPEP-d 2 exhibits high defluorination. Furthermore, BAT must be active during PET-CT scan, indicating that it is also unsuitable for post-mortem studies of BAT [58]. 3.1.4. 4Cu-NOTA-PD-1 mAb and 18F-B3 Programmed death 1(PD-1) is a critical negative co-receptor expressed on activated lymphocytes [59]. The major PD-1 ligand expressed on cells from solid tumors is PD-L1 (B7-H1) [60]. The PD pathway [59–61] can be blocked with antagonistic antibodies to either PD-1 or PD-L1, resulting in greatly enhanced antitumor immune responses [62]. The chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) was used to form stable complexes with the radioisotope 64Cu (t1/2 = 12.7 h) [63–65]. These tracers permitted the non-invasive determination of PD-1 and PD-L1 expression in malignant tumors and the biodistribution monitoring of the checkpoint-blocking antibodies. A PET-CT study used anti-mouse PD-L1 tracer in immunocompetent mice and observed the tracer in tumors and spleens [66]. Studies have also observed tumors and spleens in immunocompetent mice using the 64CuDOTA-anti-murine PD-1 mAb tracer. In immunoPET studies, PD-1 and PD-L1 were observed in healthy mice, tumor-bearing mice, and mice exposed to pathophysiological cytokine interferon-gamma. PD-L1 is found to be more readily taken up in the upper dorsal region, showing a butterfly-like shape and is located between the scapulae [67]. In mice with antigenic blockade and PD-L1 deficiency, the uptake of markers in lymphoid tissues and BAT tissues was significantly reduced or completely disappeared, further confirming the specificity of tissue uptake of PD-L1 mAb. Using 64Cu-NOTA-conjugated surrogate checkpoint blocker as a PET tracer, PD-L1 immunoPET indicated that the expression of ligands in BAT is significantly downregulated. This imaging technique allows for high sensitivity and resolution [68]. As was shown following immunization of an alpaca with purified ectodomain of mouse PD-L1, leading to the isolation, by phage display, of two single domain antibodies (VHHs), designated B3 and A12, both of which bind specifically to PD-L1 [21]; PET-CT with 18FeB3 revealed PD-L1 in BAT. Labeling of B3 with 18F or 64Cu produced equivalent results in terms of tissue distribution, with nonspecific staining in the lumen of the gut and kidney, a trait common to all VHHs used in PET imaging. Since the B3 VHH was found to bind PD-L1 in vivo, BAT was determined to express

3.1.3. [11C]2 PSNCBAM-1 is a potential allosteric antagonist of cannabinoid type1 receptor (CB1), which reduces the appetite of rodents and reduces body weight after oral administration. PET studies with [11C]2 revealed that radioactive uptake rate in BAT was high, while in animals pretreated with AM281, a cannabinoid receptor antagonist, radioactivity decreased. Animal studies have shown that [11C]2 may be a potent PET ligand for peripheral CB1 in BAT [54]. [11C] can specifically bind to peripheral CB1 in BAT with high affinity. Moreover, [11C]acetate can reflect the oxidizing ability of BAT in humans[19]. In recent years, researchers have discovered through rodent studies, that [11C]-metahydroxyephedrine can be used as a PET-CT tracer for the conversion of white fat to brown fat [55]; while the radiolabelled CB1R antagonist [18F]FMPEP-d2 can be used as a biomarker for observing BAT volume and density [56]. Another study [57] employed instruments from 3

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FBnTP imaging and CT scans; the scanned rats were divided into two groups based on administration of propranolol. The results revealed that at RT, 18F-FBnTP demonstrated rapid uptake and prolonged steadystate retention in BAT, while cold and β3-adrenergic agonist CL316,243 stimulation immediately washed out the 18F-FBnTP from BAT. Further, the washout process could be blocked by propranolol treatment. Using 18F-FBnTP PET, the mitochondrial membrane potential (ΔΨm) could be monitored in vivo, providing insights into the kinetic physiology of BAT. 18F-FBnTP PET imaging is highly sensitive and rapidly responsive to BAT. As such, it can efficiently support the further study of BAT physiology in vivo and be used as a novel set of quantitative metrics [72].

PD-L1. The presence of PD-L1 was evident not only in the intrascapular region, but also covering all brown fat in the thorax, with small deposits observed below the scapulae and in elongated structures parallel to the spinal column, presumably equivalent to the paravertebral BAT deposits seen in humans [29], independent of strain or age. The presence of PD-L1 within BAT was evident at the RNA level as well [21]. Relying on metabolic activity as the only means of detecting BAT has obvious limitations, particularly when aiming for modification of its activity either genetically or pharmacologically. The above study found that PD-L1 expression was largely independent of metabolic activity and could be used to label BAT in naive mice as well as in animals undergoing cold stress or treatment with β-agonists [21]. In a SPETCT study, PD-L1 was specifically detected in tumors and spleens [66]. Alternatively, another study indicated that PD-L1 can neither be detected in lymph nodes nor in BAT [69]. These completely contradictory results may be due to the relatively low spatial resolution and sensitivity of SPETCT, which could lead to inaccurate results. PD-1/PD-L1 immunoPET imaging has several advantages over immunohistochemistry such as being non-invasive, allowing for assessment of receptor expression in all metastatic lesions, and having the capacity to access regions that are otherwise inaccessible by puncture or in large masses that cannot be covered [70]. In many chronic inflammatory diseases, it is often critical to assess the expression of non-invasive markers. Moreover, the immunoPET technique is highly reproducible. However, further research is required to determine if PD-1/PD-L1 PET can respond to PD-1/PD-L1 expression levels before and after treatment. Based on those studies, PD-1/PD-L1 PET can be used to obtain quantitative and dynamic information regarding the antibody distribution throughout the body, observe the halftime of blood flow, and the extent of aggregation at tumor sites [71]. Compared with previous studies based on low resolution imaging and ex vivo scintillation counting, the VHH-based anti-PD-L1 immuno-PET exhibits superior resolution [21].

3.2. BAT imaging using magnetic resonance (MR) The multilocular lipid content, dense mitochondrial packing, and relatively higher capillarity within BAT lead to significantly higher water content and increased magnetic susceptibility, which makes it possible to use MR to observe BAT [5]. Compared with PET, MR approaches have better spatial resolution at lower cost and are much safer as they do not involve injection of radioactive tracers [73]. Moreover, MR offers non-invasive methods to identify fat fraction, such as singlevoxel 1H-MRS and MRI [5,72–74]. MRI and MRS are rapidly developing imaging techniques and have the advantage of not requiring ionizing radiation. Current MRI techniques can be used to observe the reduction of triglyceride levels in BAT tissue caused by cold stimulation [75]. It was found that the increase in triglyceride content in BAT was associated with decreased insulin sensitivity in the body [76]. Panagia M, et al. carried out MRI on mice that were pre-treated with permanent ligation of the left coronary artery after six weeks. Researchers performed T2 weighted MRI of BAT volume and blood oxygen level dependent (BOLD) MRI of BAT function. The T2* maps of BAT were acquired at different time points before and after administration of the β3 adrenergic agonist CL-316,243. The flow alternating inversion recovery (FAIR) approach was used to estimate the blood flow to BAT after CL injection. Results found that BAT volume was significantly lower in heart failure. CL injection increased BAT T2* in healthy animals, but not in mice with heart failure, consistent with an increase in flow in control BAT. This was confirmed by a significant difference in the FAIR response in BAT in control and heart-failure mice. It can be concluded that heart failure results in a decrease in BAT volume, and suggests the increase of BAT activity. Notably, the study also highlights that MRI is suitable for imaging BAT volume and studying the functional activity of BAT using BOLD and FAIR [20]. However, additional studies combining PET and MR should be carried out to capture both the molecular readout of PET and the sophisticated tissue-level analysis achieved by MRI. Indeed, some researchers have suggested that the fat content of BAT can be better determined by combining PET-CT tracer with MRI or MRS [77].

3.1.5. [18F]-AQ28A Wagner et al. used small-animal PET-MRI imaging with phosphodiesterase (PDE) 10A-specific radioligand [18F]-AQ28A[70]. Results indicated that interscapular BAT demonstrated strong uptake of [18F]AQ28A, compared with that by the surrounding muscles. Further, dynamic PET data analysis further confirmed that BAT tissue shows higher PDE10A expression than skeletal muscles. Additional blocking experiments were performed by injecting MP10, an inhibitor of PDE10A, before mice received [18F]-AQ28A; PET imaging results found that the uptake of [18F]-AQ28A decreased in BAT. To explore the importance of PDE10A expression function in the heat-producing BAT, researchers also carried out pharmacological [18F]-FDG-PET experiments on normal weight mice and diet-induced obese mice. Results indicated that acute administration of MP-10 resulted in a significant increase in [18F]-FDG uptake by BAT [66]. The investigators reanalyzed the results obtained from previous PET scans of subjects using the specific PDE10A radioligand [18F]-MNI-659, and observed a significant increase in uptake of [18F]-MNI-659 in the supraclavicular region. However, the uptake by skeletal muscle was lower, and the study of each subject indicated that the higher the BMI, the higher the BAT uptake of [18F]MNI-659 in the supraclavicular region of the subject [70]. Therefore, it was considered that PDE10A inhibitors are appropriate alternative treatments for obese patients, due to their dual effects of appetite suppression and increased energy expenditure. The study also found that long-term treatment of hyperglycaemia with MP-10 can improve insulin sensitivity. However, due to small sample size and gender factors, a definitive conclusion could not be reached.

3.3. Imaging of BAT using ultrasound Ultrasound technique has the advantages of being inexpensive, free of ionizing radiation, useful for dynamically observing numerous anatomical areas, and it is already widely used for both clinical and preclinical imaging [78–80]. Baron DM et al. [80] used contrast ultrasound (CU) to assess the blood flow of interscapular BAT by measuring intensity of continuously infused contrast microbubbles. To evaluate whether intact BAT activation is required to increase BAT blood flow, CU was performed in UCP1-deficient mice with impaired BAT activation. The authors of the study further investigated whether nitric oxide synthase enzyme (NOS) has any function associated with acute NE-induced changes in BAT blood flow. CU was performed with a 14-MHz linear transducer (Sequoia C512, Siemens, Mountain View, CA); and high-energy ultrasound frames (mechanical index 1.80, frame rate 30 Hz) were used to destroy the contrast microbubbles. The

3.1.6. 18F-FBnTP PET Igal Madar et al. [72] reported a technology for quantitative monitoring of principle kinetic components of BAT adaptive thermogenesis in living animals, using the PET imaging voltage sensor 18F-fluorobenzyltriphenylphosphonium (18F-FBnTP). The study used rats for 18F4

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Table 1 The disadvantages and advantages of the imaging modalities mentioned. Imaging modality PET 18 F-FDG

15

O-labelled oxygen

11

C-labelled acetate

4

Cu-NOTA-PD-1 mAb

18 18

F-AQ28A F-FBnTP

MR

High-resolution whole body 3-D MRI

Ultrasound

Autoradiography Infrared thermography

Near-infrared fluorescence

Advantages

Disadvantages

Widely used Riched clinical experience Describe the activation of BAT

Ionizing radiation Insufficient anatomic information Results dependent on imaging conditions and BAT activation level Short half-time Limited availability Short half-time Limited availability Low spatial resolution and sensitivity Limited availability

Direct measurement of oxygen consumption The uptake increased with BAT activation Activation-dependent uptake Non-invasive Having the capacity to access regions Highly reproducible Could compare with surrounding muscles High accumulation in inactive BAT Highly sensitive and rapidly responsive to BAT High spatial resolution and specificity Lower cost Safe Widely available Distinguish intra-abdominal adipose tissue from subcutaneous adipose tissue High resolution Non-radiation Inexpensive Dynamic Widely available Distinguish BAT from surrounding WAT High sensitivity Detect cold-induced BAT activation

Non-invasive High quality of the images Higher feasibility

Limited availability Decreased signal after BAT activation Limited for mixed BAT-WAT voxels Easily affected by motion artefacts and partial volume

Expensive Time-consuming Limited to superficial BAT depots Difficult to dissect BAT from surrounding WAT

With radiation Low spatial resolution Cannot detect inactive BAT Applicability and specificity remained controversial May cause other health problems Limited availability

difficult to dissect BAT from the surrounding WAT in human studies [81].

replenishment time course of the contrast microbubbles in the BAT was recorded for 10 s in real-time mode. After acquiring the CU results at baseline, NE was infused intravenously, and the effect of NE was verified by an increase in systemic blood pressure and heart rate after 10 min. The study analysed a total of 424 contrast microbubble replenishment curves from 55 animals to assess the feasibility of using CU to measure BAT blood flow. Results found that in wide type mice, NE induced a 15-fold increase in BAT blood flow, while in UCP1−/− mice, NE infusion caused only a 5-fold increase in blood flow compared with that at baseline, indicating that absence of UCP1 markedly suppressed the BAT blood flow. In conclusion, this experiment verified that CU can be used as a non-invasive method to estimate BAT blood flow in vivo in mice, and can also examine changes in blood flow when BAT is activated. It was considered that CU could be used to monitor the effect of anti-obesity therapies that regulate BAT function, and could be performed serially to follow-up BAT-modulating therapies when translated into humans [79]. CU has also been used in mice studies after NE stimulation to activate BAT and estimate the BAT blood flow, volume and mass. Results found that BAT blood flow increased after NE stimulation in all mice, although the increase in db/db mice, which have a genetic mutation that inactivates the leptin receptor causing them to develop severe obesity and diabetes, was much lower than that in wide-type mice. Further, CU-derived BAT mass was correlated with BAT mass obtained at necropsy. BAT mass was higher in mice fed a high-fat diet compared to those fed a low-fat diet. Hence, CU can be used to estimate BAT mass in mice when its vascularisation is not significantly impaired. This non-invasive technique can be applied in serial evaluation of therapies designed to augment BAT mass [80]. However, some researchers have argued that the measurements of BAT volume may not be reliable when BAT vascularisation and blood flow are significantly impaired. Furthermore, CU often overestimates BAT mass, as it assumes that the BAT density is homogeneous, and it is

3.4. Imaging of BAT using other technologies Autoradiography can be used to identify BAT. Chio Okuyama and his partners found that 123 I-Metaiodobenzylguanidine (MIBG) accumulates in BAT in the adrenergic nervous system. Pre-administration of 6-hydroxydopamine or reserpine resulted in lower MIBG concentrations in BAT. Activation of the β3-adrenergic receptor accelerated the washout of MIBG in BAT and caused an increase in concentration in WAT. This method can also distinguish BAT from surrounding WAT [82]. High-resolution whole body 3-D MRI can distinguish intra-abdominal adipose tissue from subcutaneous adipose tissue. Hence, this technique has great applications, as the two kinds of adipose tissue have different metabolic activities. Infrared thermography is also used to measure BAT activity [83], however, its applicability and specificity have remained controversial, as other tissues can also produce heat and result in changes in overlying skin blood flow, making it difficult to distinguish whether the increase in temperature and skin blood flow is a result of active BAT [84]. Nonetheless, the result of infrared thermography is similar to that of PET-CT, and therefore, infrared thermography may be useful to detect cold-induced BAT activation [85]. Recent developments in near-infrared (NIR) fluorescent proteins have greatly facilitated imaging of biological processes in vivo in mice, and are now widely used for in vivo imaging [86]. Fukuda A, et al. [87] proposed that NIR fluorescent protein is relatively less affected by absorption of biological components, moreover, iRFP720 is the most redshifted NIR fluorescent protein, and probably best suited for in vivo tissue imaging in live mice. Near-infrared photoluminescent singlewalled carbon nanotubes (CNTs) are considered as effectual bio-imaging tools. Yudasaka M, et al. [88] reported that CNTs coated with an 5

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treatment target for metabolic dysfunctions, such as obesity, diabetes, cardiovascular diseases, and even cancer. However, global efforts are required to develop more useful and efficient imaging methods to observe BAT and assess its function, so that it can be better understood and taken advantage of for the development of improved therapeutics.

amphiphilic and biocompatible polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate; PMB), generate highquality images of brown fat for its deep penetration and low scattering. The image brightness of adipose tissue can reflect the capillary density directly, and the thermogenic capability and brownness indirectly. CNTs are safer when compared to quantum dots because the quantum dots contain heavy metals, which may lead to health issues [89–90] (Table 1). The list of the abbreviations and their complete definition.

Author contribution JJ, G wrote the text; HY, HL, JW helped search relative literature and give useful ideas. XL, W is corresponding author and helped modify article.

Abbreviations

Declaration of competing interest

BAT Brown adipose tissue PET Positron-emission tomography MRI Magnetic resonance imaging UCP1 Uncoupling protein 1 PD-L1 Programmed death-ligand 1 18 F-FDG Fludeoxyglucose iBAT Interscapular brown adipose tissue WAT White adipose tissue APC Adenomatous polyposis coli 18 F-FTHA 14(R,S)-[18]F-fluoro-6-thia-heptadecanoic acid VO2 Oxygen consumption RT Room temperature MVO2 Myocardial oxygen consumption MRS Magnetic resonance spectroscopy CB1 Cannabinoid type-1 NE Norepinephrine PD-1 Programmed death 1 PDE 10A Phosphodiesterase 10A 18 F-FBnTP 18F-fluorobenzyltriphenylphosphonium MR Magnetic Resonance BOLD Blood oxygen level dependent FAIR Flow alternating inversion recovery CU Contrast ultrasound NOS Nitric oxide synthase enzyme MIBG 123 I-Metaiodobenzylguanidine NIR Near-infrared

The authors declare no conflicts of interest. Acknowledgments This work was supported by National Natural Science Foundation of China(NSFC). Project approval number: 81501496. Project Name: The role of NRP-1 in the proliferation and migration of ovarian cancer. References [1] K. Shinoda, I.H. Luijten, Y. Hasegawa, et al., Genetic and functional characterization of clonally derived adult human brown adipocytes[J], Nat. Med. 21 (2015) 389–394, https://doi.org/10.1038/nm.3819. [2] S.C. Lin, P. Li, CIDE-A, a novel link between brown adipose tissue and obesity[J], Trends Mol. Med. 10 (2004) 434–439, https://doi.org/10.1016/j.molmed.2004.07. 005. [3] S. Gesta, Y.H. Tseng, C.R. Kahn, Developmental origin of fat: tracking obesity to its source[J], Cell 131 (2007) 242–256, https://doi.org/10.1016/j.cell.2007.10.004. [4] G.G. Power, Biology of temperature: the mammalian fetus[J], J. Dev. Physiol. 12 (6) (1989) 295–304, https://doi.org/10.1002/jcp.1041410329. [5] S.C. Sampath, M.A. Bredella, et al., Imaging of brown adipose tissue: state of the art [J], Radiology 280 (1) (2016) 4–19, https://doi.org/10.1148/radiol.2016150390. [6] J.M. Heaton, The distribution of brown adipose tissue in the human[J], J Anat 112 (Pt 1) (1972) 35–39, https://doi.org/10.2307/2371925. [7] P. Huttunen, J. Hirvonen, V. Kinnula, The occurrence of brown adipose tissue in outdoor workers[J], Eur. J. Appl. Physiol. Occup. Physiol. 46 (4) (1981) 339–345, https://doi.org/10.1007/bf00422121. [8] S. Enerback, A. Jacobsson, E.M. Simpson, et al., Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese[J], Nature 387 (6628) (1997) 90–94, https://doi.org/10.1038/387090a0. [9] V. Golozoubova, E. Hohtola, A. Matthias, et al., Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold[J], FASEB J. 15 (11) (2001) 2048–2050, https://doi.org/10.1096/fj.00-0536fje. [10] V. Golozoubova, B. Cannon, J. Nedergaard, UCP1 is essential for adaptive adrenergic nonshivering thermogenesis[J], Am. J. Physiol. Endocrinol. Metab. 291 (2) (2006) E350–E357, https://doi.org/10.1152/ajpendo.00387.2005. [11] S. Oufara, H. Barre, J.L. Rouanet, et al., Adaptation to extreme ambient temperatures in cold-acclimated gerbils and mice[J], Am J Physiol 253 (1Pt2) (1987) R39–R45, https://doi.org/10.1152/ajpregu.1987.253.1.R39. [12] H.M. Feldmann, V. Golozoubova, B. Cannon, et al., UCP1 ablation induces obesity and abolishes diet-induced thermogenesis in mice exempt from thermal stress by living at thermoneutrality[J], Cell Metab 9 (2) (2009) 203–209, https://doi.org/10. 1016/j.cmet.2008.12.014. [13] C. Guerra, P. Navarro, A.M. Valverde, et al., Brown adipose tissue-specific insulin receptor knockout shows diabetic phenotype without insulin resistance[J], J. Clin. Invest. 108 (8) (2001) 1205–1213, https://doi.org/10.1172/JCI13103. [14] J.F. Berbée, M.R. Boon, P.P. Khedoe, et al., Brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development[J], Nat. Commun. 6 (2015) 6356, https://doi.org/10.1038/ncomms7356. [15] K.I. Stanford, R.J. Middelbeek, K.L. Townsend, et al., Brown adipose tissue regulates glucose homeostasis and insulin sensitivity[J], J. Clin. Invest. 123 (1) (2013) 215–223, https://doi.org/10.1172/JCI62308. [16] P.P. Khedoe, G. Hoeke, S. Kooijman, et al., Brown adipose tissue takes up plasma triglycerides mostly after lipolysis[J], J. Lipid Res. 56 (1) (2015) 51–59, https:// doi.org/10.1194/jlr.M052746. [17] A. Bartelt, O.T. Bruns, R. Reimer, et al., Brown adipose tissue activity controls triglycerie clearance[J], Nat. Med. 17 (2) (2011) 200–205, https://doi.org/10. 1038/nm.2297. [18] V. Ouellet, A. Routhier-Labadie, W. Bellemare, et al., Outdoor temperature, age, sex, body mass index, and diabetic status determine the prevalence, mass, and glucose-uptake activity of 18F-FDG-Detected BAT in humans[J], J. Clin. Endocrinol. Metab. 96 (1) (2011) 192–199, https://doi.org/10.1210/jc.2010-0989. [19] D.P. Blondin, S.M. Labbe, C. Noll, et al., Selective impairment of glucose but not fatty acid or oxidative metabolism in brown adipose tissue of subjects with type 2

4. Prospects There have been many imaging methods investigated for examination of BAT. PET remains the gold standard for BAT imaging, although it has its shortcomings such as high cost, ionizing radiation, and relatively low spatial resolution and sensitivity. Many tracers have been used to better evaluate the volume and activation of BAT, the mostly widely used being 18F-FDG, which is readily available and the technology is relatively advanced for clinical applications. However, in order to study the energy dynamics in BAT, 11C-acetate PET and 15Olabelled oxygen PET is useful, as it can directly measure oxygen consumption, and their uptake increases with activation of BAT. However, these tracers also have their shortcomings, such as limited availability and short half-life. The rapidly developing new methods such as CT and MRI have already garnered increased attention, as they offer superior spatial resolution. Specifically, MR does not require radiation and has relatively higher spatial resolution. Furthermore, it can be applied for dynamic activation studies, although it is not entirely useful in the measurement of BAT mass and has not been validated using large cohorts. Few studies have introduced BAT imaging using CU, which can provide dynamic and continuous information regarding BAT. Moreover, this method is also cost-effective and non-invasive, and can be readily reproduced. It is also a relatively reliable technique to quantify BAT volume. However, this technology is limited to superficial BAT depots and has not yet been well established for use in human subjects. Since BAT has many physiological functions, it can be used as a new 6

Life Sciences 249 (2020) 117500

J. Gu, et al.

[20]

[21]

[22]

[23]

[24] [25]

[26] [27]

[28] [29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47] E.B.M. Nascimento, W.D.V.M. Lichtenbelt, In vivo detection of human Brown adipose tissue during cold and exercise by PET/CT[J], Handb. Exp. Pharmacol. 251 (2019) 283–298, https://doi.org/10.1007/164_2018_121. [48] P. Santhanam, L. Solnes, J.C. Hannukainen, et al., Adiposity-related cancer and functional image of brown adipose tissue[J], Endocr. Pract. 21 (11) (2015) 1282–1290, https://doi.org/10.4158/EP15870.RA. [49] M.M. Ter-Pogossian, J.O. Eichling, D.O. Davis, et al., The determination of regional cerebral blood flow by means of water labeled with radioactive oxygen 15[J], Radiology 93 (1) (1969) 31–40, https://doi.org/10.1148/93.1.31. [50] M.M. Ter-Pogossian, J.O. Eichling, D.O. Davis, et al., The measure in vivo of regional cerebral oxygen utilization by means of oxyhemoglobin labeled with radioactive oxygen-15[J], J Clin Invest 49 (2) (1970) 381–391, https://doi.org/10. 1172/JCI106247. [51] O. Muzik, T.J. Mangner, W.R. Leonard, et al., 15O PET measurement of blood flow and oxygen consumption in cold-activated human brown fat[J], J Nucl Med 54 (4) (2013) 523–531, https://doi.org/10.2967/jnumed.112.111336. [52] U. Din M, J. Raiko, T. Saari, et al., Human brown adipose tissue [15O]O2PET imaging in the presence and absence of cold stimulus[J], Eur J Nucl Med Mol Imaging 3 (10) (2016) 1878–1886, https://doi.org/10.1007/s00259-016-3364-y. [53] L.J. Klein, F.C. Visser, P. Knaapen, et al., Carbon-11 acetate as a tracer of myocardial oxygen consumption[J], Eur. J. Nucl. Med. 28 (5) (2001) 651–668, https:// doi.org/10.1007/s002590000472. [54] T. Yamasaki, M. Fujinaga, Y. Shimoda, et al., Radiosynthesis and evaluation of new PET ligands for peripheral cannabinoid receptor type 1 imaging[J], Bioorg Med Chem Lett 27 (17) (2017) 4114–4117, https://doi.org/10.1016/j.bmcl.2017.07. 040. [55] C. Quarta, F. Lodi, R. Mazza, et al., (11)C-meta-hydroxyephedrine PET/CT imaging allows in vivo study of adaptive thermogenesis and white-to-brown fat conversion [J], Mol Metab 2 (3) (2013) 153–160, https://doi.org/10.1016/j.molmet.2013.04. 002. [56] O. Eriksson, K. Mikkola, D. Espes, et al., The cannabinoid receptor-1 is an imaging biomarker of brown adipose tissue[J], J. Nucl. Med. 56 (12) (2015) 1937–1941, https://doi.org/10.2967/jnumed.115.156422. [57] M.L. Pan, M.T. Mukherjee, H.H. Patel, et al., Evaluation of [11C]TAZA for amyloid β plaque imaging in postmortem human Alzheimer’s disease brain region and whole body distribution in rodent PET/CT[J], Synapse 70 (4) (2016) 163–176, https:// doi.org/10.1002/syn.21893. [58] P. Lee, J.R. Greenfield, K.K. Ho, M.J. Fulham, A critical appraisal of the prevalence and metabolic significance of brown adipose tissue in adult humans[J], Am. J. Physiol. Endocrinol. Metab. 299 (4) (2010) 601–606, https://doi.org/10.1152/ ajpendo.00298.2010. [59] T. Okazaki, S. Chikuma, Y. Iwai, S. Fagarasan, T. Honjo, A rheostat for immune responses: the unique properties of PD-1 and their advantages for clinical application[J], Nat. Immunol. 14 (12) (2013) 1212–1218, https://doi.org/10.1038/ni. 2762. [60] H. Dong, S.E. Strome, D.R. Salomao, et al., Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion[J], Nat Med 8 (8) (2002) 793–800, https://doi.org/10.1038/nm730. [61] G.J. Freeman, Engagement of the PD-1 Immunoinhibitory Receptor by a Novel B7 Family Member Leads to Negative Regulation of Lymphocyte Activation[J], J Exp Med 192 (7) (2000) 1027–1034, https://doi.org/10.1084/jem.192.7.1027. [62] L. Chen, X. Han, Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future[J], J. Clin. Invest. 125 (9) (2015) 3384–3391, https://doi.org/10.1172/ JCI80011. [63] R. Ferdani, C.J. Anderson, Copper-64 radiopharmaceuticals for PET imaging of cancer: advances in preclinical and clinical research[J], Cancer Biother. Radiopharm. 24 (4) (2009) 379–393, https://doi.org/10.1089/cbr.2009.0674. [64] A.F. Prasanphanich, P.K. Nanda, T.L. Rold, et al., [64Cu-NOTA-8-Aoc-BBN(7-14) NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues[J], Proc. Natl. Acad. Sci. U. S. A. 104 (30) (2007) 12462–12467, https://doi.org/10.1073/pnas.0705347104. [65] Y. Zhang, H. Hong, J.W. Engle, et al., Positron emission tomography imaging of CD105 expression with a 64Cu-labeled monoclonal antibody: NOTA is superior to DOTA[J], PLoS One 6 (12) (2011) e28005, , https://doi.org/10.1371/journal.pone. 0028005. [66] A. Josefsson, J.R. Nedrow, S. Park, et al., Imaging, biodistribution, and dosimetry of radionuclide-labeled PD-L1 antibody in an immunocompetent mouse model of breast cancer[J], Cancer Res. 76 (2) (2016) 472–479, https://doi.org/10.1158/ 0008-5472.CAN-15-2141. [67] A. Bartelt, J. Heeren, Adipose tissue browning and metabolic health[J], Nat Rev Endocrinol 10 (1) (2014) 24–36, https://doi.org/10.1038/nrendo.2013.204. [68] M. Hettich, F. Braun, M.D. Bartholomä, et al., High-resolution PET imaging with therapeutic antibody-based PD-1/PD-L1 checkpoint tracers[J], Theranostics 6 (10) (2016) 1629–1640, https://doi.org/10.7150/thno.15253. [69] S. Chatterjee, W.G. Lesniak, M. Gabrielson, et al., A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors[J], Oncotarget 7 (9) (2016) 10215–10227, https://doi.org/10.18632/oncotarget.7143. [70] M.K. Hankir, M. Kranz, T. Gnad, et al., A novel thermoregulatory role for PDE10A in mouse and human adipocytes[J], EMBO Mol Med 8 (7) (2016) 796–812, https:// doi.org/10.15252/emmm.201506085. [71] N.J. Rothwell, M.J. Stock, A role for brown adipose tissue in diet-induced thermogenesis[J], Nature 281 (5726) (1979) 31–35, https://doi.org/10.1038/ 281031a0. [72] I. Madar, E. Naor, D. Holt, et al., Brown adipose tissue response dynamics: in vivo insights with the voltage sensor 18F-fluorobenzyl triphenyl phosphonium[J], PLoS One 10 (6) (2015) e0129627, , https://doi.org/10.1371/journal.pone.0129627.

diabetes[J], Diabetes 64 (7) (2015) 2388–2397, https://doi.org/10.2337/db141651. M. Panagia, Y.C.I. Chen, H.H. Chen, et al., Functional and anatomical characterization of brown adipose tissue in heart failure with blood oxygen level dependent magnetic resonance[J], NMR Biomed. 29 (7) (2016) 978–984, https://doi.org/10. 1002/nbm.3557. J.R. Ingram, M. Dougan, M. Rashidian, et al., PD-L1 is an activation-independent marker of brown adipocytes[J], Nat. Commun. 8 (1) (2017) 647, https://doi.org/ 10.1038/s41467-017-00799-8. Y.X. Wang, N. Zhu, C.J. Zhang, et al., Friend or foe: multiple roles of adipose tissue in cancer formation and progression[J], J. Cell. Physiol. 234 (12) (2019) 21436–21449, https://doi.org/10.1002/jcp.28776. S.A. Bos, C.M. Gill, E.L. Martinez-Salazar, et al., Preliminary investigation of brown adipose tissue assessed by PET/CT and cancer activity[J], Skelet. Radiol. 48 (3) (2019) 413–419, https://doi.org/10.1007/s00256-018-3046-x. Gesner C, Conradi Gesneri medici Tigurini Historiae Animalium: Lib 1—De Quadrupedibus Viviparis (Zürich), 1551:842. N.C. Bal, S.K. Maurya, D.H. Sopariwala, et al., Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals[J], Nat Med 18 (10) (2012) 1575–1579, https://doi.org/10.1038/nm.2897. G. Heldmaier, G. Neuweiler, W. Rössler, Vergleichende Tierphysiologie, 2nd edn, Springer Spektrum, Berlin, 2012. R.E. Smith, J.C. Roberts, Thermogenesis of brown adipose tissue in cold-acclimated rats[J], Am. J. Phys. 206 (1) (1964) 143–148, https://doi.org/10.1152/ajplegacy. 1964.206.1.143. S.M. Aronson, G. Shwartzman, The histopathology of brown fat in experimental poliomyelitis.[J], Am. J. Pathol. 32 (2) (1956) 315–333. B. Cannon, J. Nedergaard, Brown adipose tissue: function and physiological significance[J], Physiol. Rev. 84 (1) (2004) 277–359, https://doi.org/10.1152/ physrev.00015.2003. M.T. Truong, J.J. Erasmus, R.F. Munden, E.M. Marom, B.S. Sabloffff, G.W. Gladish, et al., Focal FDG uptake in mediastinal brown fat mimicking malignancy:a potential pitfall resolved on PET/CT[J], AJR Am J Roentgenol 83 (4) (2004) 1127–1132, https://doi.org/10.2214/ajr.183.4.1831127. M.E. Lidell, M.J. Betz, O.D. Leinhard, et al., Evidence for two types of brown adipose tissue in humans[J], Nat. Med. 19 (5) (2013) 631–634, https://doi.org/10. 1038/nm.3017. L. Bahler, J.W. Deelen, J.B. Hoekstra, et al., Seasonal influence on stimulated BAT activity in prospective trials-A retrospective analysis of BAT visualized on 18F-FDG PET-CTs and 123I-mIBG SPECT[J], J Appl Physiol 120 (12) (2016) 1418–1423, https://doi.org/10.1152/japplphysiol.00008.2016. A.M. Cypess, S. Lehman, G. Williams, et al., Identification and importance of brown adipose tissue in adult humans[J], N. Engl. J. Med. 360 (15) (2009) 1509–1517, https://doi.org/10.1056/NEJMoa0810780. A. Cypess, L. Weiner, C. Roberts-Toler, et al., Activation of human Brown adipose tissue by a β3-adrenergic receptor agonist[J], Cell Metab. 21 (1) (2015) 33–38, https://doi.org/10.1016/j.cmet.2014.12.009. C. Brendle, M.K. Werner, M. Schmadl, et al., Correlation of brown adipose tissue with other body fat compartments and patient characteristics: a retrospective analysis in a large patient cohort using PET/CT[J], Acad. Radiol. 25 (1) (2018) 102–110, https://doi.org/10.1016/j.acra.2017.09.007. H. Prodhomme, J. Ognard, P. Robin, et al., Imaging and identification of brown adipose tissue on CT scan[J], Clin. Physiol. Funct. Imaging 38 (2) (2018) 186–191, https://doi.org/10.1111/cpf.12373. W.D. van Marken Lichtenbelt, J.W. Vanhommerig, N.M. Smulders, et al., Cold-activated brown adipose tissue in healthy men[J], N Engl J Med 360 (15) (2009) 1500–1508, https://doi.org/10.1056/NEJMoa0808718. Y.I. Chen, A.M. Cypess, C.A. Sass, et al., Anatomical and functional assessment of brown adipose tissue by magnetic resonance imaging[J], Obesity 20 (7) (2012) 1519–1526, https://doi.org/10.1038/oby.2012.22. A. Khanna, R.T. Branca, Detecting brown adipose tissue activity with BOLD MRI in mice[J], Magn. Reson. Med. 68 (4) (2012) 1285–1290, https://doi.org/10.1002/ mrm.24118. A.M. Cypess, C.R. Haft, M.R. Laughlin, et al., Brown fat in humans: consensus points and experimental guidelines[J], Cell Metab. 20 (3) (2014) 408–415, https://doi. org/10.1016/j.cmet.2014.07.025. D.P. Blondin, André C. Carpentier, The role of BAT in cardiometabolic disorders and ageing[J], Best Pract. Res. Clin. Endocrinol. Metab. 30 (4) (2016) 497–513, https:// doi.org/10.1016/j.beem.2016.09.002. M.K. Hankir, M. Kranz, S. Keipert, et al., Dissociation between brown adipose tissue [18] F-FDG uptake and thermogenesis in uncoupling protein 1 deficient mice[J], J. Nucl. Med. 58 (7) (2017) 1100–1103, https://doi.org/10.2967/jnumed.116. 186460. Z. Liu, Y. Xiao, Z. Zhou, et al., Extensive metabolic disorders are present in APCmin tumorigenesis mice[J], Mol. Cell. Endocrinol. 427 (2016) 57–64, https://doi.org/ 10.1016/j.mce.2016.03.004. K.N. Schade, A. Baranwal, C. Liang, et al., Preliminary evaluation of β3-adrenoceptor agonist-induced 18F-FDG metabolic activity of brown adipose tissue in obese Zucker rat[J], Nucl. Med. Biol. 42 (8) (2015) 691–694, https://doi.org/10.1016/j. nucmedbio.2015.04.003. J.W. Park, K.H. Jung, J.H. Lee, et al., 18F-FDG PET/CT monitoring of β3 agoniststimulated brown adipocyte recruitment in white adipose tissue[J], J. Nucl. Med. 56 (1) (2015) 153–158, https://doi.org/10.2967/jnumed.114.147603. M. Din, J. Raiko, T. Saari, et al., Human brown adipose tissue [(15)O]O2 PET imaging in the presence and absence of cold stimulus[J], Eur. J. Nucl. Med. Mol. Imaging 43 (10) (2016) 1878–1886, https://doi.org/10.1007/s00259-016-3364-y.

7

Life Sciences 249 (2020) 117500

J. Gu, et al.

10.1016/j.diabet.2017.03.008. [82] C. Okuyama, N. Sakane, T. Yoshida, et al., 123I- or 125I-metaiodobenzylguanidine visualization of brown adipose tissue[J], J. Nucl. Med. 43 (9) (2002) 1234–1240. [83] B. Martinez-Tellez, A. Perez-Bey, G. Sanchez-Delgado, et al., Concurrent validity of supraclavicular skin temperature measured with iButtons and infrared thermography as a surrogate marker of brown adipose tissue[J], J Therm Biol 82 (2019) 186–196, https://doi.org/10.1016/j.jtherbio.2019.04.009. [84] J. Law, J. Chalmers, D.E. Morris, et al., The use of infrared thermography in the measurement and characterization of brown adipose tissue activation[J], Temperature (Austin) 5 (2) (2018) 147–161, https://doi.org/10.1080/23328940. 2017.1397085. [85] L. Sun, S. Verma, N. Michael, et al., Brown adipose tissue: multimodality evaluation by PET, MRI, infrared thermography, and whole-body calorimetry (TACTICAL-II) [J], Obesity (Silver Spring) 27 (9) (2019) 1434–1442, https://doi.org/10.1002/ oby.22560. [86] G.S. Filonov, K.D. Piatkevich, L.M. Ting, et al., Bright and stable near-infrared fluorescent protein for in vivo imaging[J], Nat Biotechnol 29 (8) (2011) 757–761, https://doi.org/10.1038/nbt.1918. [87] A. Fukuda, S. Honda, N. Fujioka, et al., Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720[J], PLoS One 14 (11) (2019) e0225213, , https://doi.org/10.1371/journal.pone.0225213. [88] M. Yudasaka, Y. Yomogida, M. Zhang, et al., Near-infrared photoluminescent carbon nanotubes for imaging of brown fat[J], Sci Rep 7 (2017) 44760, https://doi. org/10.1038/srep44760. [89] K. Soga, et al., NIR bioimaging: development of liposome-encapsulated, rare-earthdoped Y2O3 nanoparticles as fluorescent probes[J], Eur J Inorg Chem 2010 (18) (2010) 2673–2677, https://doi.org/10.1002/ejic.201000201. [90] G. Hong, J.T. Robinson, Y. Zhang, et al., In vivo fluorescence imaging with Ag2S quantum dots in the second near-infrared region[J], Angew Chem Int Ed Engl 51 (39) (2012) 9818–9821, https://doi.org/10.1002/anie.201206059.

[73] H.H. Hu, D.L.S. Jr, K.S. Nayak, et al., Identification of brown adipose tissue in mice with fat–water IDEAL-MRI[J], J. Magn. Reson. Imaging 31 (5) (2010) 1195–1202, https://doi.org/10.1002/jmri.22162. [74] X.G. Peng, S. Ju, F. Fang, et al., Comparison of brown and white adipose tissue fat fractions in ob, seipin, and Fsp27 gene knockout mice by chemical shift-selective imaging and (1)H-MR spectroscopy[J], Am. J. Physiol. Endocrinol. Metab. 304 (2) (2013) 160–167, https://doi.org/10.1152/ajpendo.00401.2012. [75] E. Lundstrom, R. Strand, L. Johansson, et al., Magnetic resonance imaging coolingreheating protocol indicates decreased fat fraction via lipid consumption in suspected brown adipose tissue[J], PLoS One 10 (4) (2015) e0126705, , https://doi. org/10.1371/journal.pone.0126705. [76] J. Raiko, M. Holstila, K.A. Virtanen, et al., Brown adipose tissue triglyceride content is associated with decreased insulin sensitivity, independently of age and obesity [J], Diabetes Obes. Metab. 17 (5) (2015) 516–519, https://doi.org/10.1111/dom. 12433. [77] H.H. Hu, T.G. Perkins, J.M. Chia, V. Gilsanz, Characterization of human brown adipose tissue by chemical-shift water-fat MRI[J], AJR Am. J. Roentgenol. 200 (1) (2013) 177–183, https://doi.org/10.2214/AJR.12.8996. [78] A. Flynn, Q. Li, M. Panagia, et al., Contrast-enhanced ultrasound: a novel noninvasive, nonionizing method for the detection of brown adipose tissue in humans [J], J Am Soc Echocardiogr 28 (10) (2015) 1247–1254, https://doi.org/10.1016/j. echo.2015.06.014. [79] D.M. Baron, M. Clerte, P. Brouckaert, et al., In vivo noninvasive characterization of Brown adipose tissue blood flow by contrast ultrasound in mice[J], Circ Cardiovasc Imaging 5 (5) (2012) 652–659, https://doi.org/10.1161/CIRCIMAGING.112. 975607. [80] M. Clerte, D.M. Baron, P. Brouckaert, et al., Brown adipose tissue blood flow and mass in obesity: a contrast ultrasound study in mice[J], J. Am. Soc. Echocardiogr. 26 (12) (2013) 1465–1473, https://doi.org/10.1016/j.echo.2013.07.015. [81] L. Sun, J. Yan, L. Sun, et al., A synopsis of brown adipose tissue imaging modalities for clinical research[J], Diabetes Metab. 43 (5) (2017) 401–410, https://doi.org/

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