Imaging Adipose Tissue Browning using Mitochondrial Complex-I Tracer [18F]BCPP-EF

Browning of white adipose tissue (WAT) into beige adipocytes has been proposed as a strategy to tackle the ongoing obesity epidemic. Thermogenic stimuli have been investigated with the aim of converting existing white adipose tissue, primarily used for energy storage, into beige adipocytes capable of dissipating energy; however, evaluation is complicated by the dearth of noninvasive methodologies to quantify de novo beige adipocytes in WAT. Imaging with [18F]FDG is commonly used to measure brown adipose tissue (BAT) and beige adipocytes but the relationship between beige adipocytes, thermogenesis and [18F]FDG uptake is unclear. [18F]BCPP-EF, a tracer for mitochondrial complex-I (MC-I), acts as a marker of oxidative metabolism and may be useful for the detection of newly formed beige adipocytes. Mice received doses of the β3-adrenergic agonist CL-316,243 subchronically for 7 days to induce formation of beige adipocytes in inguinal white fat. PET imaging was performed longitudinally with both [18F]FDG (a marker of glycolysis) and [18F]BCPP-EF (an MC-I marker) to assess the effect of thermogenic stimulation on uptake in browning inguinal WAT and interscapular BAT. Treatment with CL-316,243 led to significant increases in both [18F]FDG and [18F]BCPP-EF in inguinal WAT. The uptake of [18F]BCPP-EF in inguinal WAT was significantly increased above control levels after 3 days of stimulation, whereas [18F]FDG only showed a significant increase after 7 days. The uptake of [18F]BCPP-EF in newly formed beige adipocytes was blocked by pretreatment with an adrenoceptor antagonist suggesting that beige adipocyte formation may be associated with the activation of MC-I. However, in BAT, uptake of [18F]BCPP-EF was unaffected by β3-adrenergic stimulation, potentially due to the high expression of MC-I. [18F]BCPP-EF can detect newly formed beige adipocytes in WAT generated after subchronic treatment with the β3-adrenergic agonist CL-316,243 and displays both higher inguinal WAT uptake and earlier detection than [18F]FDG. The MC-I tracer may be a useful tool in the evaluation of new therapeutic strategies targeting metabolic adipose tissues to tackle obesity and metabolic diseases.


Introduction
Adipose tissue is complex and plays an important role in energy homeostasis; dysfunctions can lead to increased risk of metabolic disease and obesity [1,2]. Two functionally distinct types of adipose tissue exist, white adipose tissue (WAT) consists of white adipocytes used for energy storage (mainly fatty acids and lipids) and brown adipose tissue (BAT) consists of metabolically active, thermogenic brown adipocytes. A third type, thermogenic beige adipocytes, is inducible in WAT in response to cold, exercise or stimulation with β-adrenergic receptor (β3AR) agonists [3]. ermogenesis in brown and beige adipocytes occurs via the action of uncoupling protein-1 (UCP1) [4]. UCP1 is located on the inner mitochondrial membrane and is associated with adaptive thermogenesis, UCP1 translocates protons through the mitochondrial intermembrane space without generating ATP, uncoupling respiration from ATP generation, resulting in energy dissipation as heat and stimulating fatty acid oxidation [5]. Adults with higher BAT mass exhibit improvements in lipid metabolism and lower body mass index, highlighting the therapeutic potential of increasing the amount or activity of BAT in overweight individuals [6,7]. Numerous thermogenic stimuli have been investigated with the aim of converting existing WAT into beige adipocytes; however, treatment efficacy is complicated by the dearth of noninvasive methodologies to quantify de novo beige adipocyte biogenesis in WAT [8]. [ 18 F]FDG is typically used in PET imaging of BAT and beige adipocytes; however, the link between beige adipocyte biogenesis and [ 18 F]FDG uptake is unclear [9]. Accurate quantitation of beige adipocytes and thermogenic activity will be critical for the development of new therapeutic strategies for weight loss and lipid regulation. Beige adipocytes and brown adipocytes share similar phenotypes; both are characterized by multilocular lipid droplets and appear brown due to their abundance of mitochondria [1,2,10]. In contrast white adipocytes have one large lipid droplet and very few mitochondria [11,12]. Prior studies have shown that imaging agents able to quantify mitochondrial expression can aid in the detection of new beige fat deposits in WAT after β3AR agonist stimulation [8,13]. Mitochondria contain a respiratory electron transfer chain consisting of five components (complexes I-V) which are responsible for the generation of the majority of the ATP required by the cell. Mitochondrial complex I (MC-I) is the first and the largest complex in the pathway for oxidative phosphorylation [14]. In this study, we evaluated the radiopharmaceutical, 2-tert-butyl-4-chloro-5-(6-[2-(2- [18]F-fluoroethoxy)-ethoxy]pyridine-3-ylmethoxy)-2H-pyridazin-3-one ( 18 F-BCPP-EF) which targets active MC-I [16] for its ability to detect the generation of beige adipocytes in inguinal WAT after subchronic β3AR stimulation, comparing uptake to [ 18 F]FDG and confirming beige adipocyte generation using histology and immunohistochemical detection of UCP1.

Animal Procedures.
Animal procedures adhered to Institutional Animal Care and Use Committee Singapore requirements. BALB/c mice were purchased from InVivos Singapore at 6-8 weeks of age, kept in standard ABSL2 housing with food and water ad libitum. Animals (n � 10 per group) were dosed intraperitoneally into the left inguinal white fat pad daily for 7 days with 1 mg/kg β3AR agonist CL-316,243 in saline or saline alone as a control. e β3AR antagonist L-748,328 (1 mg/kg) was used to understand the stimulation dependency of radiotracer uptake and was administered 1 hour prior to imaging on day 7.

PET-CT and MR Imaging.
e animals were imaged longitudinally on days 1, 3, and 7 at 60 minutes after dosing as previously described [8]. Static PET acquisitions were acquired with either [ 18 F]BCPP-EF (∼10MBq per animal, 30-50 min postinjection) or [ 18 F]FDG (∼5MBq per animal, 60-80 min postinjection) based on prior dynamic imaging data. Calibrated images were reconstructed and analysed using the Amide software (version 10.3 SourceForge). Uptake of radioactivity was measured using a volume of interest (VOI) around the interscapular BAT and inguinal WAT regions with the quadriceps muscle used to provide reference tissue values.

Histology.
Inguinal fat tissues were dissected out on day 7 and fixed in 10% neutral buffered formalin, paraffin embedded and sectioned. e induction of beige adipocytes was assessed using hematoxylin and eosin (H&E) staining and immunohistochemical antibody staining for UCP1 (ab23841, Abcam). Representative images were acquired and the percentage of multilocular fat cells was assessed from the H&E stained sections and UCP1 binding was assessed from the IHC sections. Number of cells or UCP1 binding was analysed in a masked manner to treatment assignment manually, calculated as the percentage of field of view covered per 10 fields of view assessed per sample.

Histology.
Staining with H&E shows that the presence of multilocular fat cells has increased significantly after subchronic treatment with CL-316,243 compared to the control (25.8 ± 12.8 vs 3.6 ± 1.7, * * p < 0.01, Figure 4

Discussion
Many imaging techniques are able to reliably detect BAT; however, detection of beige adipocytes dispersed in existing WAT is a major challenge [17,18] [19,20]. Furthermore, [ 18 F]FDG uptake in response to β3AR agonists is unaffected in UCP1 knockout mice despite defective BAT thermogenesis [21]. ese findings suggest that [ 18 F]FDG uptake may not be always associated with UCP1-mediated thermogenesis. Studies estimate that glucose contributes only a small percentage of the energy required for nonshivering thermogenesis with the majority from triglycerides [15]. In contrast, to [ 18 F]FDG, which reflects glycolysis (especially anaerobic); [ 18 F]BCPP-EF is considered an indicator of mitochondrial activity, reflecting oxidative metabolism [22,23]. In non-shivering thermogenesis triglycerides are hydrolysed to fatty acids and glycerol, the resulting fatty acids undergo β-oxidation. As β-oxidation is the final step in non-shivering thermogenesis [24], [ (Figure 1(a)). While both radiopharmaceuticals are proposed to be markers of metabolic activity, they showed different responses to β3AR agonist stimulation in BAT (Figure 1(b) [8].
Overall, the data suggest that [ 18 F]BCPP-EF may be a useful tool for the development of new therapeutic strategies for weight loss and lipid regulation. However, care must be taken when interpreting tracer uptake as beige adipocytes are significantly more vascularized than WAT and beige adipocyte expression is transient, after withdrawal of β3-AR agonist stimulation they have been observed rapidly reverting to the white phenotype [25,26]. Further studies will be required to fully understand the relationship between [ 18 F]BCPP-EF uptake and the extent of beige adipocyte formation in WAT browning in humans. Critically while β3AR stimulation is able to potently generate beige fat preclinically, cold acclimation and exercise regimes used to generate beige fat clinically are far less potent. Whether [ 18 F] BCPP-EF is able to measure beige adipocyte activity induced by these methods remains to be seen.

Data Availability
All the data are available on request to the corresponding author.

Conflicts of Interest
e authors declare no conflicts of interest.