Application of PET probe targeting sphingomyelin S1PR1 receptor in monitoring lipodystrophy in tail-suspended mice

Abstract

The present invention discloses the use of a PET probe targeting sphingosine-1-phosphate receptor 1 (S1P1) in monitoring lipodystrophy in tail-suspended mice. The present invention uses the S1P1-targeted PET probe 18F-TZ4877 and finds that tail suspension leads to a lipodystrophy-like phenotype, accompanied by activation of brown fat and white fat sphingolipid metabolic pathways and thermogenic activation, thereby revealing that S1P / S1P1 axis activation is closely related to lipodystrophy, which has important guiding significance for understanding the potential mechanism of acquired lipid dystrophy and developing more effective treatment strategies.

Description

Technical Field

[0001] The invention belongs to the field of experimental nuclear medicine, and in particular relates to the application of a PET probe targeting S1P1 in monitoring fat nutrition of tail-suspended mice. Background Art

[0002] Adipose tissue, as the largest "energy depot" in the body, is an important tissue for storing and mobilizing triglycerides. It is also an extremely important endocrine system. Adipose tissue is mainly divided into two categories: white adipose tissue (WAT) and brown adipose tissue (BAT). Under cold stimulation and other conditions, white fat can turn brown and is called beige fat (Carpentier, Blondin, Virtanen, et al. Brown Adipose Tissue Energy Metabolism in Humans. Front Endocrinol (Lausanne), 2018, 9:447.). The main function of white fat is energy storage, while the main function of brown fat is energy consumption and heat production.

[0003] Adipose tissue plays a key role in maintaining systemic metabolic balance. Excessive adipose tissue in obesity is closely associated with insulin resistance, fatty liver, and dyslipidemia, while a lack of functional adipose tissue leads to a pathological state called lipodystrophy, which manifests similar metabolic disorders (Melvin, Stears and Savage. Recent developments in lipodystrophy. Curr Opin Lipidol, 2019, 30(4): 284-90.). Lipodystrophy is a rare, heterogeneous disease that affects the body primarily through adipose tissue loss and leptin deficiency. It can be caused by genetic or acquired factors and is characterized by complete or partial loss of adipose tissue in certain parts of the body and / or abnormal distribution of adipose tissue (Garg. Acquired and inherited lipodystrophies. N Engl J Med, 2004, 350(12): 1220-34.). Although hereditary lipodystrophy is mainly caused by gene mutations, the causes of acquired lipodystrophy are more diverse (Knebel, Müller-Wieland and Kotzka. Lipodystrophies-Disorders of the Fatty Tissue. Int J Mol Sci, 2020, 21(22):.). The most common acquired lipodystrophy occurs in HIV patients receiving highly active antiretroviral therapy (Fiorenza, Chou and Mantzoros. Lipodystrophy: pathophysiology and advances intreatment. Nat Rev Endocrinol, 2011, 7(3): 137-50.). Other forms of acquired lipodystrophy are difficult to diagnose or have been understudied. Due to the heterogeneity of the disease, current treatments mainly target the corresponding metabolic complications symptomatically, with varying therapeutic effects (Fiorenza, Chou and Mantzoros. Lipodystrophy: pathophysiology and advances in treatment. Nat Rev Endocrinol, 2011, 7(3):137-50.). Therefore, there is an urgent need to understand the underlying mechanisms of acquired lipid dystrophy in order to develop more effective treatment strategies.

[0004] Sphingosine-1-phosphate (S1P) is one of the most important sphingolipid metabolites and a key factor in the occurrence of metabolic diseases. S1P is produced by the phosphorylation of sphingosine (Sph) by sphingosine kinases (SPHKs). On the one hand, it directly acts on intracellular targets through intracellular secretion; on the other hand, it binds to five cell surface receptors sphingosine-1-phosphate receptor 1-5 (S1P1-5) through autocrine, paracrine and endocrine pathways, thereby regulating the pathophysiological processes of multiple organ systems (Grewe, Knapstein, Donat, et al. The role of sphingosine-1-phosphate in bone remodeling and osteoporosis. Bone Res, 2022, 10(1): 34.). Based on studies of constitutive mutants and conditional mutants, S1P1 is the most important for maintaining normal physiological functions (Chun, Hla, Lynch, et al. International Union of Basicand Clinical Pharmacology. LXXVIII. Lysophospholipid receptor nomenclature. Pharmacol Rev, 2010, 62(4): 579-87.). The S1P axis mainly includes SPHKs, S1P and S1PR, and plays an important role in metabolic diseases (Duan, Gao, Chen, et al. Sphingosine-1-phosphate inmitochondrial function and metabolic diseases. Obes Rev, 2022, 23(6): e13426.). S1P-mediated lipolysis in mature adipocytes reduces lipid burden and limits the expansion of adipose tissue. In contrast, inhibition of preadipocyte differentiation mediated by S1P1 or S1P2 reduces the adaptive expansion of adipose tissue in response to positive energy balance (Fang, Pyne and Pyne. Ceramide and sphingosine 1-phosphate in adiposedysfunction. Prog Lipid Res, 2019, 74: 145-59.). Therefore, S1P is an important lipid mediator that regulates fat distribution and total body fat mass by regulating lipolysis and adipogenesis, as well as the differentiation and proliferation of preadipocytes.In addition, S1P also activates the PI3K / AKT pathway, thereby regulating the insulin signaling pathway and participating in insulin resistance (Green, Maceyka, Cowart, et al. Sphingolipids in metabolic disease: The good, the bad, and the unknown. Cell Metab, 2021, 33(7): 1293-306.). Studies have also reported that adipocyte-specific defects in nascent sphingolipid biosynthesis lead to lipodystrophy and insulin resistance (Lee, Lee, Song, et al. Adipocyte-Specific Deficiency of De Novo Sphingolipid Biosynthesis Leadsto Lipodystrophy and Insulin Resistance. Diabetes, 2017, 66(10): 2596-609.). In summary, the S1P / S1P1 axis may be a potential biomarker for lipodystrophy. However, the relationship between the S1P / S1P1 axis and metabolic disorders associated with lipodystrophy is still unclear.

[0005] Based on the above, the present invention discloses the application of a PET probe targeting S1P1 in monitoring lipodystrophy in tail-suspended mice. By constructing a tail-suspended model, the lipodystrophy-like phenotype of the model mice was verified, and the association between lipodystrophy and the S1P / S1P1 sphingolipid metabolic pathway was visualized by PET imaging targeting S1P1. This has important guiding significance for understanding the potential mechanism of acquired lipid dystrophy and developing more effective treatment strategies. Summary of the invention

[0006] Since the relationship between the S1P / S1P1 axis and metabolic disorders associated with lipodystrophy is still unclear, the present invention aims to remedy the above shortcomings and discloses the use of a PET probe targeting S1P1 in visualizing lipodystrophy with sphingolipid metabolism activation in tail-suspended mice.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions: The present invention first provides the use of a PET probe targeting S1P1 in visualizing lipodystrophy accompanied by activation of sphingolipid metabolism in tail-suspended mice.

[0008] Furthermore, the lipodystrophy model is a tail-suspended mouse model.

[0009] Furthermore, a tail-suspension mouse animal model was constructed by hindlimb unloading (HU) to simulate the lipodystrophy-like phenotype, and a normal control group (control, CTR) was set up.

[0010] Furthermore, based on CT images, the thickness of inguinal subcutaneous white fat (iWAT) was quantified to assess whether the model mice had adipose tissue loss. At the same time, interscapular BAT, inguinal subcutaneous white fat (iWAT) and epididymal white fat (eWAT) were sampled in vitro to observe the gross morphological changes of adipose tissue.

[0011] Furthermore, blood was collected from model mice to detect serum triglyceride (TG), The levels of cholesterol (cholesterol, CHO), high-density lipoprotein (high-density lipoprotein, HDL), low-density lipoprotein (low-density lipoprotein, LDL), non-esterified fatty acid (non-esterified fatty acid, NEFA), glucose (glucose, GLU) and insulin were measured to evaluate the changes in metabolic indicators of model mice.

[0012] Furthermore, the PET probe is mainly used to visualize the association between lipodystrophy and sphingolipid metabolism.

[0013] Furthermore, the probe for visualizing sphingolipid metabolism is a PET probe that specifically targets S1P1.

[0014] Furthermore, the relationship between lipodystrophy and sphingolipid metabolism was visualized mainly by quantifying the expression of S1P1 in adipose tissue.

[0015] Furthermore, the S1P1 targeting PET probe is [ 18 F] TZ4877 probe, the structural formula of which is shown in Formula I:

[0016] Furthermore, the [ 18 The preparation method of the F]TZ4877 probe is a radioisotope 18 Compound F reacts with a precursor compound to obtain 18 The precursor compound labeled with F is then reacted under acidic conditions and neutralized with NaOH to obtain [ 18 F] TZ4877 probe.

[0017] Furthermore, the structural formula of the precursor compound is shown in Formula II:

[0018] Furthermore, the 18 The precursor compound labeled with F is 18 F is obtained by replacing the p-toluenesulfonate group (-TsO) of the precursor compound.

[0019] Furthermore, the radioisotope 18 F compound is K[ 18 F].

[0020] Furthermore, the 18 The precursor compound labeled with F was 222 (cryptand K 222 ) / K2CO3 solution elution to obtain 18 F ions are prepared by reacting with precursor compounds and acetonitrile.

[0021] Preferably, the reaction temperature of the nucleophilic substitution reaction is 110° C. and the reaction time is 15 min.

[0022] Further, the reaction under the acidic conditions is 18 The precursor compound labeled with F is mixed with HCl and acetonitrile and reacted.

[0023] Preferably, the HCl concentration is 6M HCl, and the NaOH concentration is 6M NaOH.

[0024] Preferably, the reaction time under the acidic condition is 7 minutes at 110°C.

[0025] Furthermore, positron emission computed tomography (PET / CT) scanning was used to evaluate the intravenous 18 F-TZ4877 mice were dynamically scanned and reconstructed using 3-dimensional ordered subsets expectation maximization (3D OSEM).

[0026] Furthermore, the reconstructed PET / CT images were analyzed to delineate the regions of interest of left ventricular blood, target tissues (WAT, BAT) and reference tissues, and the time activity curves (TAC) of the corresponding regions were obtained. The TAC of left ventricular blood was used as the input function and the TAC of the target tissue was used as the output function. The reversible receptor binding pharmacokinetic model Logan AIF model was used to fit the original TAC of the target tissue, and the TAC of the target tissue was obtained. 18The distribution volume (DV) of F-TZ4877 in the target tissue; the distribution volume ratio (DVR) and binding potential (BP) of the target tissue were obtained by taking the tissue with low distribution volume as the reference tissue. ND ), thereby quantifying the expression of S1P1 in adipose tissue.

[0027] Furthermore, adipose tissue RNA sequencing was used to evaluate changes in related metabolic pathways in model mice, especially sphingolipid metabolic pathways, and the expression of related pathway genes and proteins was verified by qPCR and WB.

[0028] Furthermore, adipose tissue phenotype and expression of key proteins were evaluated by hematoxylin-eosin staining and immunohistochemical staining.

[0029] In summary, the present invention discloses the application of PET probe targeting S1P1 in visualizing lipodystrophy with activation of sphingolipid metabolism in tail-suspended mice. Through PET imaging targeting S1P1, the association between lipodystrophy and the S1P / S1P1 sphingolipid metabolism pathway is visualized, and the potential mechanism of acquired lipid dystrophy is further understood to develop more effective treatment strategies.

[0030] Description of the drawings.

[0031] Figure 1 HU causes a lipodystrophy-like phenotype.

[0032] Figure 1 A is the body weight change (%) curve (n = 6 in each group).

[0033] Figure 1 B: The thickness of subcutaneous iWAT was measured based on CT imaging (indicated by the white double-headed arrow).

[0034] Figure 1 C, Quantitative comparison of iWAT thickness based on CT imaging (n = 6 in each group).

[0035] Figure 1 D Left: Anatomical locations of BAT, iWAT, and eWAT (indicated by *), Figure 1 D right: General view of BAT, iWAT, and eWAT. White arrows indicate areas where tissue color changes. Data are presented as mean ± standard error. * P <0.05, ** P <0.01,*** P <0.001, **** P <0.0001. Two-way repeated measures analysis of variance and Sidak's multiple comparison test (A and C) were used for analysis of differences.

[0036] Figure 2HU led to decreased serum TG, NEFA and insulin levels. Figure 2 AG is a comparison of serum TG, NEFA, CHO, HDL, LDL, GLU, and insulin levels. Data are expressed as mean ± standard error. * P <0.05, ** P <0.01,*** P <0.001, ns = not significant. Differences were analyzed using two-tailed unpaired Student's t-test (AG).

[0037] Figure 3 for 18 Radiochemical labeling roadmap and quality control of F-TZ4877.

[0038] Figure 3 A is 18 Radiochemical labeling route of F-TZ4877.

[0039] Figure 3 B is the target product 18 Semi-preparative HPLC separation of F-TZ4877 with retention time Rt = 11.2 min.

[0040] Figure 3 C is the analytical HPLC quality control of 18F-TZ4877.

[0041] Figure 3 D is 18 F-TZ4877 was co-injected with the standard.

[0042] Figure 4 Schematic diagram of the two-tissue compartment model.

[0043] Figure 5 BAT in the HU model 18 F-TZ4877 uptake was increased.

[0044] Figure 5 A is sagittal 18 F-TZ4877 PET / CT image, the dashed oval indicates the anatomical location of BAT.

[0045] Figure 5 B is for BAT 18 F-TZ4877 uptake of TAC.

[0046] Figure 5 C is the Logan AIF graphic analysis fitting of the original BAT TAC using left ventricular blood TAC as the input function, and the slope of the straight line represents the distribution volume DV.

[0047] Figure 5D is for BAT 18 Area under the curve (AUC) of F-TZ4877 TAC.

[0048] Figure 5 E is the distribution volume ratio, DVR (DV BAT With DV lung ratio, with lung as the reference tissue).

[0049] Figure 5 F is the binding potential (BP ND ), BP ND = DVR -1.

[0050] Two-tailed unpaired Student's t-test (DF) was used for difference analysis.

[0051] Figure 6 For BAT RNA sequencing, differentially expressed genes were mainly enriched in the glucose and lipid metabolism processes and pathways.

[0052] Figure 6 A is the volcano plot of DEGs (n = 4 per group).

[0053] Figure 6 B is the GO cellular component enrichment analysis of all DEGs.

[0054] Figure 6 C is the KEGG pathway enrichment bubble plot of all DEGs showing the top 15 major enriched pathways.

[0055] Figure 6 D is Figure 6 C Z-score heat map of 69 DEGs mainly enriched in glucose and lipid metabolism-related pathways.

[0056] Figure 7 Sphingolipid metabolism in BAT and iWAT ( S1P1, Sphk1, Sphk2 ) Relative mRNA expression of related genes (n = 6 per group). Data are presented as mean ± standard error. ** P <0.01, *** P <0.001, ns = not significant. Differences were analyzed using two-tailed unpaired Student's t test.

[0057] Figure 8 Glucose uptake and metabolism in BAT and iWAT ( Glut1, Glut4, Glut5, Fgf21 ) and gluconeogenesis ( Gk, Pck1、Fbp1 ) Relative mRNA expression of related genes (n = 6 per group). Data are presented as mean ± standard error. * P <0.05, ** P<0.01, ns = not significant. Differences were analyzed using two-tailed unpaired Student's t test.

[0058] Fig. 9 Fatty acid oxidation in BAT and iWAT ( Acsl1 ) and lipid storage regulation ( Plin5, Leptin ) Relative mRNA expression of related genes (n = 6 per group). Data are presented as mean ± standard error. * P <0.05, ** P <0.01, *** P <0.001, ns = not significant. Differences were analyzed using two-tailed unpaired Student's t test.

[0059] Fig.10 Heat production in BAT and iWAT ( Ucp1, Pgc1α, Pparα, Dio2 ) Relative mRNA expression of related genes (n = 6 per group). Data are presented as mean ± standard error. ** P <0.01, ns = not significant. Differences were analyzed using two-tailed unpaired Student's t test.

[0060] Fig.11 Western blot analysis was performed to analyze the expression of S1P1 and UCP1 proteins in BAT, iWAT, and eWAT (n = 2 per group).

[0061] Fig.12 HU leads to BAT activation-like phenotype and WAT beige-like phenotype.

[0062] Fig.12 A is HE staining of BAT, iWAT and eWAT.

[0063] Fig.12 BC is based on HE, and the average adipocyte (AC) size and the number of adipocytes (ACs) in each field were semi-quantified by Image J (n = 3, 2 fields were randomly selected for quantification from each mouse). Data are expressed as mean ± standard error. *** P <0.001,*** P <0.0001. Two-tailed unpaired Student's t test (BC) was used to analyze the differences.

[0064] Fig.13 HU led to increased expression of the classic brown fat activation marker uncoupling protein UCP1 in brown fat (BAT) and white fat (iWAT and eWAT).

[0065] Fig.13 A: UCP1 IHC staining of BAT, iWAT and eWAT.

[0066] Fig.13 B is the semi-quantification of IHC staining of UCP1 based on histochemistry score (H-score) by Image J (n = 3, 2 fields of view were randomly selected for quantification from each mouse). Data are expressed as mean ± standard error. ** P <0.01,*** P <0.001. Differences were analyzed using a two-tailed unpaired Student's t test (B).

[0067] Fig.14 HU resulted in increased S1P1 expression in brown adipose tissue (BAT) and white adipose tissue (iWAT and eWAT).

[0068] Fig.14 A: S1P1 IHC staining of BAT, iWAT and eWAT.

[0069] Fig.14 B is the semi-quantification of IHC staining of S1P1 based on histochemical score (H-score) by Image J (n = 3, 2 fields of view were randomly selected for quantification from each mouse). Data are expressed as mean ± standard error. * P <0.05, ** P <0.01. Differences were analyzed using a two-tailed unpaired Student's t test (B). DETAILED DESCRIPTION

[0071] The present invention discloses the application of a PET probe targeting S1P1 in monitoring lipodystrophy in tail-suspended mice. In order to describe the present invention more specifically, the technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment in the art.

[0072] (1) Construction of an acquired lipodystrophy mouse model

[0073] Considering that most of the current animal models of lipodystrophy are hereditary gene mouse models or HIV-related acquired lipodystrophy mouse models, which have difficulties such as high technical requirements and long modeling cycles, the present invention uses a more accessible hindlimb unloading (HU) model to simulate the acquired lipodystrophy-like phenotype. The HU model is established using the classic tail suspension method, so it is also called the tail suspension model. A large number of studies have reported that tail suspension induces metabolic disorders, including glucose intolerance and insulin resistance, dyslipidemia, impaired skeletal muscle glucose uptake and metabolism, increased hepatic gluconeogenesis, activation of lipotoxic pathways in mouse liver, and increased fat accumulation (Biensø, Ringholm, Kiilerich, et al. GLUT4 and glycogen synthase are key players in bed rest-inducedinsulin resistance. Diabetes, 2012, 61(5): 1090-9; Jonscher, Alfonso-Garcia, Suhalim, et al. Spaceflight Activates Lipotoxic Pathways in Mouse Liver. PLoS One, 2016, 11(4): e0152877; Stuart, Kidder, Pietrzyk, et al. Rat tail suspension causes a decline in insulin receptors. Exp Toxicol Pathol, 1993,45(5-6): 291-5.). Therefore, the tail suspension model may be a suitable animal model for studying acquired lipodystrophy. The tail suspension cage used for modeling was purchased from Suzhou Suhang Technology Equipment Co., Ltd. Gently grasp the mouse to be suspended, minimize the stress of the mouse, and let it drill into the homemade mouse fixer to limit its movement, exposing only the tail, which is convenient for subsequent operations. Cut a medical cotton-based tape about twice the length of the pasting area (covering at least two-thirds of the tail length from the base of the tail) as the longitudinal traction tape, and place a plastic straw with the same width as the tape at the midpoint as the suspension point. Start pasting the traction tape along the base of the tail, cover the pasting area, bypass the plastic straw and paste it on the surface of the opposite tail, and press the tape to stick to the tail. The width of the tape is selected according to the thickness of the base of the mouse tail (about 0.5 cm), and ensure that there is no overlap between the traction tapes on both sides of the mouse tail to prevent tail ischemia. Cut 3-4 short strips of tape and wrap them around the longitudinal traction tape in a C shape at even intervals to fix the longitudinal traction tape and prevent tail ischemia.Then wrap the traction tape and the mouse tail with medical gauze to prevent the traction tape from falling off. The plastic straw passes through the S hook at the end of the stainless steel suspension chain of the suspension cage. The height of the suspension chain is adjusted to make the mouse's hind limbs hang in the air to unload the load. The trunk is at an angle of about 30° to the bottom of the suspension cage, ensuring that the hind limbs cannot touch the bottom of the suspension cage when fully extended, and the mouse is always in a head-down state. The stainless steel suspension chain can rotate and slide 360° so that the direction of the mouse is not restricted when using the forelimbs. The control group (control, CTR) mice were housed in the same tail suspension cage, but without tail suspension. The other conditions were consistent with the HU group. Only one mouse was housed in each suspension cage, and the suspension time was 4 weeks. The overall health of the mice was observed daily, whether the tail cuff fell off, and whether the tail was ischemic and necrotic. All animals were housed in an environment of 22-24℃, 12 h / 12 ​​h light-dark alternating cycle, and free food and water. Body weight was measured once a week. The results showed that HU induced a lean phenotype in mice with reduced body weight (. Figure 1 A).

[0074] After the modeling, the mice were subjected to CT imaging, and the thickness of the inguinal subcutaneous white adipose tissue (iWAT) was quantified to evaluate whether the model mice had adipose tissue loss. The thickness of the subcutaneous white fat layer on the left and right sides of the inguinal region was measured and the average value of the bilateral measurements was taken as the thickness of iWAT ( Figure 1 B). The results showed that HU led to a significant reduction in visceral fat and subcutaneous fat ( Figure 1 B). According to CT image quantification, the thickness of iWAT was significantly reduced ( Figure 1 C).

[0075] To further investigate the gross appearance of different adipose tissues, subcutaneous adipose tissue (iWAT), visceral adipose tissue (eWAT), and interscapular BAT were dissected at the same time. The gross appearance of BAT, iWAT, and eWAT showed that the adipose tissue volume of HU mice was smaller ( Figure 1 D). The color of BAT in the HU group was darker. Compared with the CTR group, the iWAT and eWAT of the HU group mice had scattered tissue color changes in the macroscopic view ( Figure 1 D) HU indeed caused gross changes in brown and white adipose depots. These results suggest that HU leads to a reduction in subcutaneous fat depot storage in mice, manifesting a lipodystrophy-like phenotype.

[0076] Then, blood was collected from the mice's eyes, and serum triglyceride (TG), cholesterol (CHO), high-density lipoprotein (HDL), and glucose (GLU) were detected using commercial kits and an automatic biochemical analyzer. Serum non-esterified fatty acid (NEFA), low-density lipoprotein (LDL), and insulin were measured by an ELISA reader according to the manufacturer's standard experimental procedures using commercial kits to evaluate changes in serum metabolic indicators in model mice. The results showed that serum TG, NEFA, and insulin levels in HU mice were significantly reduced, while serum CHO, HDL, LDL, and GLU levels did not change significantly ( Figure 2 AG). This shows that HU does lead to changes in the body's glucose and lipid metabolism.

[0077] (2) 18 Radiochemical labeling of F-TZ4877

[0078] Pre-labeled precursors and standards were synthesized by a third-party service company based on published papers (Liu, Luo, Gu, et al. In vivo Characterization of Four(18)F-Labeled S1PR1 Tracers for Neuroinflammation. Mol Imaging Biol, 2020, 22(5): 1362-69; Luo, Han, Liu, et al. Syntheses and in vitro biologicalevaluation of S1PR1 ligands and PET studies of four F-18 labeled radiotracersin the brain of nonhuman primates. Org Biomol Chem, 2018, 16(47): 9171-84.). 18 F-TZ4877 Radiochemical marking route map Figure 3 A.

[0079] 18 F ions are ordered from qualified companies. 18 F ions were pre-adsorbed on a Sep-pak light QMA column. 18F ions were eluted into a vial with a mixed aqueous solution of potassium carbonate (K2CO3) and Kryptofix 2.2.2 (0.9 mL acetonitrile, 0.1 mL H2O, 15 mg K2,2,2, 5 mg K2CO3). The vial was dried by azeotropic evaporation with acetonitrile (3 × 1 mL) three times under a nitrogen flow at 110°C. The precursor (1 mg) dissolved in 700 μL anhydrous acetonitrile was then added to the vial, capped, and reacted at 110°C for 15 min. The vial was detected by TLC, and the developing solvent was acetonitrile. Then 125 μL 6 M HCl was added to the reaction vial, capped, and heated at 110°C for 7 min. After natural cooling, 125 μL 6 M NaOH was added at room temperature to terminate the reaction. The vial was detected by TLC, and the developing solvent was acetonitrile. The product was further purified on an HPLC column (C18, 9.4 × 250 mm, 5 µm; mobile phase: 60% acetonitrile, 40% H2O, 0.1% trifluoroacetic acid in H2O; flow rate 4.0 mL / min; UV = 254 nm). The target HPLC fractions were collected in a 50 mL centrifuge tube, diluted with 50 mL of water, and then captured on a Sep-Pak C18 column. The collected product was eluted with 1 mL of ethanol, filtered through a 0.2 μm filter, then partially dried under a stream of nitrogen, and finally diluted with an appropriate amount of saline for animal injection. The purified product was injected separately onto an analytical HPLC column (C18, 4.6 × 250 mm, 5 µm; mobile phase: 60% acetonitrile, 40% H2O, 0.1% trifluoroacetic acid in H2O; flow rate 1.0 mL / min; UV = 254 nm) to determine the purity of the tracer. At the same time, the product and the standard were co-injected into the same analytical HPLC column for identification.

[0080] After the reaction, the product was purified by semi-preparative HPLC column to obtain the target product. 18 The peak time of F-TZ4877 is about 11.2 min (i.e., retention time R t = 10 - 12 min) ( Figure 3 B). The purified product was injected into the analytical HPLC column alone, and the peak time was about 11.2 min (i.e., retention time R t = 10 - 12 min), and only a single peak appeared, indicating that the tracer product obtained was of high purity ( Figure 3 C). The product and the standard were co-injected into the same analytical HPLC column for identification. The peak times of the product and the standard were consistent at about 11.2 min (i.e., retention time R t = 10 - 12 min), proving that the obtained product is the target product 18F-TZ4877( Figure 3 D). The calculated corrected radiochemical yield was 48.11% and the molar activity was 56.40 GBq / μmol.

[0081] (3) HU mice 18 F-TZ4877 PET imaging and quantitative analysis

[0082] 18 F-TZ4877 microPET / CT imaging: PET / CT images were acquired by a small animal microPET / CT imaging system (nanoScan PET / CT 82s). First, the pre-imaging mice were weighed and anesthetized with isoflurane (2% induction, 1% maintenance); a homemade indwelling needle was implanted in the tail vein, and the mice were placed in a prone position on the scanning bed. Throughout the imaging process, the important physiological parameters of the animals were continuously monitored and recorded by a physiological monitoring system. The temperature of the scanning room was controlled at 22-24°C. After acquiring a CT positioning image to help plan the scanning area for PET / CT acquisition, the radioactive tracer was injected into the tail vein via the indwelling needle catheter. 18 F-TZ4877 (10-15 μCi / g) was administered, and dynamic PET / CT images were immediately acquired for 30 min. CT spiral scanning was then performed for image fusion and attenuation correction. Finally, the acquired PET raw data were reconstructed into 25 frames (10 × 3 s; 3 × 10 s; 4 × 60 s; 6 × 150 s; 2 × 300 s) using the 3-dimensional ordered subsets expectation maximization (3D OSEM) algorithm.

[0083] 18 Semi-quantitative analysis of F-TZ4877 dynamic PET data: Dynamic raw data are analyzed using the software provided by the PET imaging system or the recognized PET data analysis software in the field. The volume of interest (VOI) is delineated in the left ventricular blood, BAT, and reference tissue (lung tissue in this example), and the original time activity curve (TAC) and semi-quantitative standardized uptake value (SUV) of the corresponding VOI are obtained. SUV = radioactivity concentration of VOIs (KBq / mL) / (injected dose (KBq) / body weight (Kg)).

[0084] PET data Logan AIF model fitting: S1P1 is a G protein-coupled receptor expressed on the cell membrane. 18 The binding of F-TZ4877 to S1P1 is a reversible receptor binding. Therefore, it is appropriate to use the reversible binding Logan AIF non-compartmental model to fit the dynamic data (Logan, Fowler, Volkow, et al. Graphical analysis of reversible radioligand binding from time-activity measurements applied to [N-11C-methyl]-(-)-cocaine PET studies in human subjects. J Cereb Blood Flow Metab, 1990, 10(5): 740-7.). The Logan AIF model was fitted with left ventricular blood as the input function and BAT TAC as the output function. The model parameters can be explained by the parameters corresponding to the two-tissue compartment model. Figure 4 is a schematic diagram of the two-tissue compartment model, where C B represents the concentration of tracer in whole blood, C P represents the concentration of tracer in plasma, C T represents the total tracer concentration in the tissue, C ND represents the concentration of undisplaced tracer in the tissue, C S represents the concentration of the specifically bound tracer in the tissue, νB represents the capillary volume fraction; νT represents the tissue volume fraction, νT=1-νB, VT (also often expressed as DV) represents the total distribution volume in the tissue; the activity concentration of a given ROI measured on a PET image, C ROI (t), can be expressed as:

[0085] Since vB≪1, C B ≈C P , equation (1) can be simplified to:

[0086] Based on the reversible two-tissue compartment model equations (3) and (4):

[0087] Where K1 represents the uptake rate constant from plasma to tissue, k2 represents the clearance rate constant from tissue to plasma, k3 represents the binding rate of the tracer to the specific binding site, and k4 represents the dissociation rate of the tracer to the specific binding site.

[0088] Combining equations (2), (3) and (4), we can deduce:

[0089] By definition

[0090] Since vB≪1, equation (5) can be simplified to:

[0091] By definition

[0092] Substituting into equation (7), the Logan AIF plot can be expressed by the straight line equation (10): Given an input function C P And the output function C ROI , the x and y values ​​at different time points can be calculated; based on multiple paired x and y values, the slope V can be fitted by linear least squares method T , the regression line with intercept Int; since Logan AIF usually meets linearity after time t*, time points earlier than t* are usually omitted during fitting, and the maximum goodness of fit (t*) is selected; the slope of the fitting line V T (also often expressed as DV) represents the total distribution volume of the tracer in the tissue. In this embodiment, the data of the 15th to 25th frames (fitting time 3 to 30 minutes) were used for Logan AIF fitting analysis.

[0093] In this example, dynamic PET images of the tracer distribution over time were first obtained by dynamic scanning. Preliminary visual assessment and SUV semi-quantitative analysis showed that interscapular BAT in HU mice showed strong 18 F-TZ4877 uptake, while BAT in control mice 18 F-TZ4877 uptake was lower ( Figure 5 A). Combining BAT 18 F-TZ4877 TAC curve ( Figure 5 B) The AUC of BAT TAC also showed that the BAT of HU mice 18F-TZ4877 uptake increased ( Figure 5 D). Since SUV is a semiquantitative method, further use of reversible receptor binding (i.e. 18 F-TZ4877 imaging) fully quantitative non-ventricular model-Logan AIF model analyzes the raw TAC data. With left ventricular blood TAC as the input function, BAT raw 18 Logan AIF graphical analysis of the F-TZ4877TAC data shows that the goodness-of-fit of the fitted straight line is good ( Figure 5 C). The slope of the fitted line represents the distribution volume (DV) of the region of interest (ROI). The distribution volume of BAT in the HU group was higher than that in the CTR group ( Figure 5 C). Due to the overall low uptake in the lung, the lung was used as the reference tissue in this example. The distribution volume ratio (DVR) and binding potential (BP) of BAT in the HU group were ND ) was significantly higher than that in the CTR group, which further indicated that the expression of S1P1 in BAT of the HU model was increased ( Figure 5 EF). However, quantitative analysis showed that there was a significant difference in the expression of EF in iWAT and eWAT between HU mice and control mice. 18 The difference in F-TZ4877 uptake was not significant (not shown).

[0094] (4) BAT RNA sequencing

[0095] because 18 The uptake of F-TZ4877 in BAT was more different than that in WAT, so RNA sequencing analysis was further performed on BAT. RNA sequencing of BAT (n = 4 per group) was performed by BGI Genomics Co., Ltd.

[0096] Total RNA extraction and concentration determination: Total RNA from adipose tissue was extracted using the QIAGEN RNeasy Lipid Tissue Mini Kit. Before the experiment, 4 volumes of anhydrous ethanol were added to the RPE buffer, and the mixture was shaken upside down to be used as the working solution. Approximately no more than 100 mg of adipose tissue was weighed and quickly ground into powder in a high-pressure sterilized mortar under liquid nitrogen. At the same time, 1.0 mL of QIAzol Lysis Reagent was added to fully lyse the cells, and the lysate was transferred into 2 mL of sterile RNase-free EP. The lysate was allowed to stand at room temperature for 5 min. Centrifuged at 12000 × g at 4ºC for 10 min to remove cell debris and residues. 200 μL of chloroform was added, vortexed for 15 s to mix, left at room temperature for 2-3 min, and centrifuged at 12000 × g at 4ºC for 15 min. Transfer the upper aqueous phase to a new 1.5 mL RNase-free EP tube (about 500-600 μL), add an equal volume of 70% ethanol (diluted with DEPC water), vortex to mix, and do not centrifuge. Transfer 700 μL of the mixture to the RNeasy Mini spin column in batches, centrifuge at 12,000 × g for 20 s at room temperature, and discard the waste liquid. Repeat the experimental operation until all the mixture has been transferred to the column. Add 700 μL of Buffer RW1 to the RNeasy Mini spin column, centrifuge at 12,000 × g for 15 s at room temperature, and discard the waste liquid. Add 500 μL of Buffer RPE to the RNeasy Mini spin column, centrifuge at 12,000 × g for 15 s at room temperature, and discard the waste liquid. Add 500 μL of Buffer RPE to the RNeasy Mini spin column, centrifuge at 12,000 × g for 2 min at room temperature, and discard the waste liquid. Centrifuge at the highest speed for 1 min to completely dry the remaining liquid. Place the RNasy Mini spin column in a clean 1.5 mL RNase-free tube, add 50 μL RNase-free water to the center of the column, and let it stand for 5 min. Centrifuge at 12000 × g for 1 min at room temperature to collect RNA. Repeat the elution to increase the yield. Nanodrop detection of RNA concentration: Prepare DEPC water (or RNase-free water) and extracted RNA stock solution, and use NanoDrop OneC ultra-micro UV photometer to detect RNA concentration.

[0097] Library construction: Agilent 2100 Bioanalyzer was used to detect the concentration, RIN value, 28S / 18S and fragment size of total RNA. The total RNA sample was denatured at a suitable temperature to open its secondary structure, and oligo(dT) magnetic beads were used to enrich mRNA. The shearing reagent was added to the mRNA obtained in the previous step, and the mRNA was fragmented by reacting at a suitable temperature for a certain period of time. The first-strand synthesis reaction system prepared in advance was added to the sheared mRNA, and the first-strand cDNA was synthesized according to the corresponding program on the PCR instrument. The second-strand synthesis reaction system was prepared, and the second-strand cDNA was synthesized by reacting at a suitable temperature for a certain period of time. The reaction system was prepared, and the reaction was carried out at a suitable temperature for a certain period of time to repair the ends of the double-stranded cDNA, and the A base was added to the 3' end. The adapter connection reaction system was prepared, and the reaction was carried out at a suitable temperature for a certain period of time to connect the adapter to the cDNA. The PCR reaction system was prepared, and the reaction program was set to amplify the connection product. After the PCR product is denatured into a single strand, a circularization reaction system is prepared, mixed thoroughly and reacted at an appropriate temperature for a certain period of time to obtain a single-stranded circular product. After digesting the uncircularized linear DNA molecules, the final library is obtained.

[0098] Library detection: Agilent 2100 Bioanalyzer was used to detect the fragment size and concentration of the library.

[0099] On-machine sequencing: Single-stranded circular DNA molecules replicate through rolling circles to form a DNA nanoball (DNB) containing more than 200 copies. The obtained DNBs are added to the mesh holes on the chip using high-density DNA nanochip technology. Sequencing is performed using combined probe anchor polymerization technology (cPAS) to obtain sequencing read lengths of 50 bp / 100 bp / 150 bp. SOAPnuke (v1.5.2) is used to filter the sequencing data to obtain clean reads, and then HISAT2 (v2.0.4) is used to map the obtained clean reads to the reference genome. RSEM (v1.2.12) is used to calculate gene expression levels.

[0100] Sequencing data analysis: The sequencing data were analyzed using the Dr. Tom BGI multi-omics data mining system, including GO cellular component enrichment analysis and KEGG pathway enrichment analysis of differentially expressed genes (DEGs).

[0101] The sequencing results showed that there were 1656 differentially expressed genes (DEGs), of which 775 genes were significantly upregulated and 881 genes were significantly downregulated ( Figure 6A). GO cellular component enrichment analysis showed that most DEGs were enriched in lipid metabolism and fatty acid metabolism ( Figure 6 B). Through KEGG pathway enrichment analysis, the six main enriched pathways related to glucose and lipid metabolism are insulin resistance, PPAR signaling pathway, sphingolipid metabolism, unsaturated fatty acid biosynthesis, fat decomposition regulation in adipocytes, and AMPK signaling pathway ( Figure 6 C). A total of 69 DEGs were enriched in these 6 major pathways. The Z-score heat map showed that the gene expression profiles were highly reproducible in each group consisting of 4 independent mouse samples. Among these 69 DEGs, HU led to upregulation of most genes and downregulation of a few genes ( Figure 6 D).

[0102] (5) RT-qPCR of adipose tissue

[0103] Subsequently, the genes differentially expressed by adipose tissue sequencing were further verified by RT-qPCR. First, Total RNA extraction and concentration determination were performed with reference to the RNA sequencing section. Then, qPCR primers were designed and synthesized. The primers used in this example are shown in Table 1.

[0104] cDNA was obtained by reverse transcription using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. Specifically, the genomic DNA removal system was prepared in an RNase-free centrifuge tube, i.e., 4 μL 4 × gDNAwiper Mix, 1 μg template RNA, and RNase-free ddH2O was added to a total volume of 16 μL. Mix by gently pipetting. The BioRad PCR instrument was placed on a 42°C machine for 2 min. 4 μL 5 ×HiScript III qRT SuperMix was directly added to the reaction tube in the previous step to a total volume of 20 μL. Mix by gently pipetting. The cDNA product was obtained by reverse transcription reaction on a BioRad PCR instrument under the reaction conditions of 37°C for 15 min and 85°C for 5 s.

[0105] Use the ChamQ Universal SYBR qPCR Master Mix kit for RT-qPCR. Specifically, prepare the qPCR reaction system in a qPCR tube (2×ChamQ Universal SYBR qPCR Master Mix 10 μL, forward primer 0.1 μL, reverse primer 0.1 μL, cDNA template 1.0 μL, add ddH2O to a total volume of 20 μL). Mix before use, add samples to a 96-well PCR plate according to the PCR reaction system, set up a set of no-template controls, seal the PCR film, balance the sealed PCR plate, and centrifuge at 1500 rpm for 2 min to reduce bubbles in the reaction system. Perform qPCR reactions on the QuantStudio 7 Flex real-time fluorescence quantitative PCR instrument:

[0106] Then, perform experimental data analysis: observe the amplification curve, set appropriate baselines and thresholds, observe the melting curve, and check whether there is nonspecific amplification or primer dimers in the reaction system. -∆∆CT The relative expression of mRNA was quantitatively analyzed by the method. That is, the starting template concentration of each gene was calculated by the cycle threshold (Ct) value after the reaction, according to the following formula: The change fold of the target gene expression in the experimental group relative to the target gene expression in the control group, that is, the differential expression fold = 2 -∆∆CT , ∆∆Ct=(Ct 实验组目的基因 -Ct 实验组内参基因β-actin )-(Ct 对照组目的基因 -Ct 对照组内参基因β-actin )

[0107] HU leads to activation of sphingolipid metabolism pathway: Sequencing results showed that most genes related to sphingolipid metabolism were upregulated. RT-qPCR confirmed that HU mice BAT Sphk1 and Sphk2 The relative expression of gene mRNA was significantly upregulated ( Figure 7 ). Meanwhile, BAT in HU mice S1p1 The relative upregulation of mRNA expression is consistent with the upregulation of BAT S1P1 expression found in PET imaging ( Figure 7 ). However, in iWAT only Sphk2 The relative mRNA expression level of Figure 7 ), S1p1, Sphk1 The relative expression levels of mRNA in iWAT were not significantly different. These clues indicate that HU leads to the activation of sphingolipid metabolism pathways in BAT.

[0108] HU leads to increased gluconeogenesis: Glucose, NEFA, dietary fatty acids (including saturated and unsaturated fatty acids), and glutamate are the main energy substrates for BAT. BAT has been reported to have high basic constitutive FFA uptake and inducible glucose uptake. Most of the energy for BAT comes from stored TG. TG lipolysis in BAT produces FFA for oxidative metabolism. Glucose is transported into cells through glucose transporters (GLUTs). Sequencing data show that BAT mainly expresses Glut4 and Glut5 ,and Glut1 ( Slc2a1 ) were significantly upregulated and enriched in the insulin resistance pathway ( Figure 6 D). Therefore, the next step was to study the Glut1 , Glut4 , Glut5 The relative expression level of mRNA in BAT of HU mice Glut1 The relative expression of mRNA increased. Glut4 , Glut5 The relative expression of mRNA decreased ( Figure 8 A). Myokine FGF21 is a key regulator of glucose metabolism. Based on sequencing data and RT-qPCR, FGF21 is expressed in BAT of HU mice. Fgf21 The relative expression of mRNA was also significantly decreased ( Figure 8 A). However, in the inguinal fat depot iWAT, during HU Glut1 , Glut4 , Glut5 and Fgf21 There was no significant change in the relative mRNA expression level ( Figure 8 A). RNA sequencing ( Figure 6 D) and RT-qPCR ( Figure 8 B) Further verification showed that gluconeogenesis-related genes, including glycerol kinase, G ), phosphoenolpyruvate carboxykinase 1, Pck1 ), fructosebisphosphatase 1 Fbp1 ), the relative expression level of mRNA was significantly increased. RT-qPCR quantitative analysis did not find that G , Pck1 , Fbp1 The relative expression levels of mRNAs in Figure 8 B). These results suggest that gluconeogenesis is increased in BAT of HU mice.

[0109] HU leads to increased TG lipolysis, increased FFA uptake and oxidation, and feedback positive regulation of lipid storage: adipose triacylglyceride lipase (ATGL) is a key lipase for triglyceride mobilization. RNA sequencing showed that BAT of HU mice contains patatin-like phospholipase domain containing 2, Pnpla2 , also known as Atgl ) mRNA expression was significantly increased, indicating increased TG lipolysis. For fatty acid uptake and oxidation genes, RNA sequencing showed that in BAT of HU mice CD36 , solutecarrier family 27 Slc27 , also called fatty acid transporter), acyl-CoA synthetase long-chain family member 1 (acyl-CoA synthetase long-chain family member 1, Acsl1 ) mRNA expression level was significantly increased ( Figure 6 D). RT-qPCR showed that BAT in HU mice Acsl1 mRNA expression was upregulated, while in iWAT Acsl1 The relative expression of mRNA was not upregulated ( Fig. 9 At the same time, it is worth noting that after 4 weeks of HU, Perilipin 5 ( Plin5 ) gene expression increased significantly ( Figure 6 D). The results were further verified by RT-qPCR. Plin5 The relative expression of mRNA was significantly increased ( Fig. 9 ). However, in iWAT Plin5 There was no significant change in expression ( Fig. 9 In contrast, according to sequencing data and RT-qPCR, after 4 weeks of HU, the expression of Leptin The relative expression of mRNA was significantly downregulated ( Fig. 9 These data further suggest that HU induces TG lipolysis and increases in FFA uptake and oxidation, as well as a positive feedback regulation of lipid storage.

[0110] HU leads to upregulation of thermogenic genes in BAT and WAT: BAT is the main source of non-tremor thermogenesis. Notably, uncoupling protein 1 (UNC1) Ucp1) is the most representative BAT marker gene, and its expression level is higher in the BAT tissue of HU mice. Other genes involved in thermogenesis in the BAT of HU mice, such as peroxisome proliferative activated receptor gamma coactivator 1 alpha, Pgc1α ), peroxisome proliferator activated receptor alpha (PPAR Pparα ), peroxisome proliferator activator receptor delta ( Pparδ ), type II iodothyronine deiodinase ( Dio2 ) mRNA relative expression level was significantly increased ( Figure 6 D), in which some genes were verified by RT-qPCR ( Fig.10 ). Meanwhile, in the inguinal fat depot iWAT, HU also led to the expression of thermogenesis-related genes Ucp1 , Pgc1 α、 Ppar The expression level of α increased, and Dio2 There was no significant change in the relative expression level of mRNA ( Fig.10 ). In summary, HU led to thermogenesis activation of BAT and WAT.

[0111] (6) Adipose tissue protein imprint After weighing the tissue, put it into an autoclaved mortar, pour in an appropriate amount of liquid nitrogen, and grind the tissue continuously. According to the weight of the tissue, add RIPA lysis buffer (BAT: 10 μL / mg; iWAT: 5 μL / mg; epiWAT: 5 μL / mg), add protease inhibitors (i.e., diluted to 1×), phosphatase inhibitors (i.e., diluted to 1×), and after sufficient grinding, collect the tissue homogenate in an autoclaved centrifuge tube, centrifuge it twice at 4℃ 14000 rcf, 10 min each time, and collect the supernatant. Use the BCA protein concentration determination kit (enhanced) to determine the protein concentration. According to the volume of the sample stock solution, add 1 / 4 of the stock solution volume of 5×SDA-PAGE protein loading buffer to dilute it to 1× protein loading buffer. After dilution, the sample concentration is 0.8× the stock solution concentration. Metal bath, 100℃, boil the protein for 10 minutes, and immediately put it on ice. Use Tris-MOPS-SDS electrophoresis buffer powder to prepare electrophoresis buffer according to the instructions. Install the electrophoresis tank and add the sample. The voltage was 120 V and the time was 95-105 min. After the electrophoresis, the membrane was transferred. The constant current was used for transfer, the current was 350 mA, and the duration was 90-100 min. The PVDF membrane after the transfer was added with blocking solution and blocked for 30 min on a shaker at 80 rpm at room temperature. Incubate with primary antibodies, including β-tubulin, S1P1, and UCP1, overnight for 12-16 h on a shaker at 80 rpm at 4°C. After washing the membrane, incubate with secondary antibodies on a shaker at 80 rpm at room temperature for 1 h. Cover the membrane with ECL luminescent solution and then image it with a fluorescent chemiluminescence imaging system.

[0112] Western blot analysis confirmed that during HU, S1P1 protein expression increased in BAT and iWAT, but did not change significantly in eWAT ( Fig.11 Obviously, during HU, the expression of UCP1 protein in BAT, iWAT and eWAT increased significantly ( Fig.11 ).

[0113] (7) Hematoxylin-eosin staining of adipose tissue In order to explore the microhistological changes of adipose tissue, HE staining was performed on BAT, iWAT and eWAT.

[0114] 5 μm thick paraffin sections were prepared using a rotary microtome. After sectioning, the sections were bleached at 40 °C and spread at 55 °C. The sections were baked at 60 °C for 120 min to ensure adequate dewaxing and prevent delamination. The sections were placed on a slice rack and sequentially placed in xylene I (10 min), xylene II (10 min), xylene III (10 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), 95% alcohol (3 min), 75% alcohol (3 min), 50% alcohol (3 min), and tap water (3 min), and then immersed in dewaxing and gradient hydration. The sections were stained with hematoxylin solution for 3 min, rinsed with tap water, differentiated with 1% hydrochloric acid ethanol differentiation solution for a few seconds, rinsed with tap water, blued with 1% ammonia water for a few seconds, and rinsed with tap water. The sections were dehydrated in 85% alcohol and 95% alcohol for 5 min each, stained with eosin solution for 3 min, and rinsed with tap water. The sections were placed in 50% alcohol, 75% alcohol, 95% alcohol, anhydrous ethanol I, and anhydrous ethanol II for 3 min each, xylene I (5 min), and xylene II (5 min), and the sections were placed in a fume hood to dry naturally. The sections were sealed with neutral gum. A high-resolution digital pathology slide scanner (Pannoramic 250 Flash III) was used to scan and image analysis was performed using Image software (National Institutes of Health, USA).

[0115] Histological HE staining showed that HU caused microstructural changes in interscapular BAT, including an increase in the number of adipocytes and lipid droplets, an increase in cytoplasmic components, and a significant decrease in the average adipocyte diameter and lipid droplet size ( Fig.12 AC). At the same time, HE staining also showed that iWAT and eWAT of HU mice showed a BAT-like phenotype. The adipocytes in WAT tissue of HU mice were multilocular and contained denser cytoplasmic components, which may be due to the increase in the number of mitochondria. At the same time, quantification also showed that the average adipocyte diameter of iWAT and eWAT in the HU group decreased, while the number of adipocytes and lipid droplets in each field of view increased ( Fig.12 AC).

[0116] (8) Immunohistochemical staining of adipose tissue Bake the slices at 60℃ for 120 min to ensure adequate dewaxing and prevent detachment. Place the slices on a slice rack and sequentially place in xylene I (10 min), xylene II (10 min), xylene III (10 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), 95% alcohol (3 min), 75% alcohol (3 min), 50% alcohol (3 min), and tap water (3 min) for soaking and dewaxing and gradient hydration. Soak in PBST for 5 min.

[0117] The slice rack was placed in an immunohistochemical cylinder, and an appropriate amount of citrate antigen repair solution was added to cover the slice. The immunohistochemical cylinder was sealed and placed in a pressure cooker. Water was added to the pot to cover the height of the repair solution in the cylinder. After heating to boiling, the pressure was maintained for 10 minutes, and then the heating was stopped. The slices were naturally cooled to room temperature before the subsequent steps. Rinse with PBST 2 times × 3 min, remove the water on the slices, and draw a circle with an immunohistochemical oil pen 2 mm away from the tissue. About 50 µL of peroxidase blocking solution was added to each tissue slice, incubated at room temperature for 10 min, and rinsed with PBST 3 times × 3 min. Remove PBST, and add about 50 µL of freshly prepared 10% normal goat serum or donkey serum to each tissue for blocking (the source of the serum is consistent with the host of the secondary antibody, 1:10 times PBS dilution), and incubate at room temperature for 60 min to saturate nonspecific antigen binding sites. Remove serum, add about 50 µL of freshly prepared PBS-diluted primary antibody, including S1P1 and UCP1 (set isotype IgG primary antibody control or blank PBS control at the same time), and incubate at 4ºC overnight for 12 h. After taking out from 4 ℃, naturally warm up at room temperature for 45 min, and rinse with PBST 3 times × 5 min. Remove PBST, add about 50 µL of secondary antibody (selected according to the primary antibody host) to each tissue, incubate at room temperature for 60 min, and rinse with PBST 3 times × 5 min. Remove PBST, add about 50 µL of freshly prepared DAB color reagent to each tissue for color development, control the color development time according to the color development situation, and rinse with tap water to stop the color development in time. Soak in hematoxylin counterstain for 20 s and rinse with tap water. Differentiate with 1% hydrochloric acid ethanol for a few seconds until the slice fades to light red, and rinse with tap water. 1% ammonia water turns blue, and rinse with tap water until the cell nucleus staining is clearly observed under the microscope. The slices were placed in 50% alcohol, 75% alcohol, 95% alcohol, anhydrous ethanol I, anhydrous ethanol II for 3 min each, xylene I (5 min), xylene II (5 min), and the slices were placed in a fume hood to dry naturally. The slices were sealed with neutral gum, taking care not to produce bubbles. After the slices were dried, they were stored in a slice box at room temperature. A high-resolution digital pathology slice scanner (Pannoramic 250 Flash III) was used to scan, and image analysis was performed using Image software (National Institutes of Health, USA).

[0118] UCP1 is the most representative BAT marker and has been widely reported to be upregulated during BAT activation and WAT browning. IHC staining showed that mitochondrial UCP1 expression increased significantly in both BAT and WAT during HU ( Fig.13 At the same time, IHC staining showed that S1P1 protein expression increased significantly, and quantitative analysis showed that the total S1P1 positive signal increased significantly ( Fig.14). Therefore, the staining results further indicated that HU caused browning of BAT and beige of WAT, accompanied by upregulation of S1P1 expression.

[0119] The above evidence shows that tail-suspended mice exhibit a lipodystrophy-like phenotype, and the PET probe targeting S1P1 reveals at the in vivo level that S1P / S1P1 axis activation is closely related to the lipodystrophy-like phenotype, which is of great guiding significance for understanding the potential mechanism of acquired lipid dystrophy and developing more effective treatment strategies.

[0120] The above description of the embodiments is to facilitate the understanding and application of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to the above embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made to the present invention by those skilled in the art based on the disclosure of the present invention should be within the scope of protection of the present invention.

[0121] References Biensø, RS, S. Ringholm, K. Kiilerich, et al. GLUT4 and glycogensynthase are key players in bed rest-induced insulin resistance. Diabetes ,2012, 61(5): 1090-9. Carpentier, AC, DP Blondin, KA Virtanen, et al. BrownAdipose Tissue Energy Metabolism in Humans. Front Endocrinol (Lausanne) ,2018, 9: 447. Chun, J., T. Hla, KR Lynch, et al. International Union of Basicand Clinical Pharmacology. LXXVIII. Lysophospholipid receptor nomenclature. Pharmacol Rev , 2010, 62(4): 579-87. Duan, M., P. Gao, S. X. Chen, et al. Sphingosine-1-phosphate inmitochondrial function and metabolic diseases. Obes Rev , 2022, 23(6): e13426. Fang, Z., S. Pyne, and N. J. Pyne. Ceramide and sphingosine 1-phosphate in adipose dysfunction. Prog Lipid Res , 2019, 74: 145-59. Fiorenza, C. G., S. H. Chou, and C. S. Mantzoros. Lipodystrophy:pathophysiology and advances in treatment. Nat Rev Endocrinol , 2011, 7(3):137-50. Garg, A. Acquired and inherited lipodystrophies. N Engl J Med , 2004,350(12): 1220-34. Green, C. D., M. Maceyka, L. A. Cowart, et al. Sphingolipids inmetabolic disease: The good, the bad, and the unknown. Cell Metab , 2021, 33(7): 1293-306. Grewe, J. M., P. R. Knapstein, A. Donat, et al. The role ofsphingosine-1-phosphate in bone remodeling and osteoporosis. Bone Res , 2022,10(1): 34. Jonscher, K. R., A. Alfonso-Garcia, J. L. Suhalim, et al. SpaceflightActivates Lipotoxic Pathways in Mouse Liver. PLoS One, 2016, 11(4): e0152877. Knebel, B., D. Müller-Wieland, and J. Kotzka. Lipodystrophies-Disorders of the Fatty Tissue. Int J Mol Sci , 2020, 21(22). Lee, S. Y., H. Y. Lee, J. H. Song, et al. Adipocyte-SpecificDeficiency of De Novo Sphingolipid Biosynthesis Leads to Lipodystrophy andInsulin Resistance. Diabetes , 2017, 66(10): 2596-609. Liu, H., Z. Luo, J. Gu, et al. In vivo Characterization of Four (18)F-Labeled S1PR1 Tracers for Neuroinflammation. Mol Imaging Biol , 2020, 22(5):1362-69. Logan, J., J. S. Fowler, N. D. Volkow, et al. Graphical analysis ofreversible radioligand binding from time-activity measurements applied to [N-11C-methyl]-(-)-cocaine PET studies in human subjects. J Cereb Blood Flow Metab , 1990, 10(5): 740-7. Luo, Z., J. Han, H. Liu, et al. Syntheses and in vitro biologicalevaluation of S1PR1 ligands and PET studies of four F-18 labeled radiotracersin the brain of nonhuman primates. Org Biomol Chem, 2018, 16(47): 9171-84. Melvin, A., A. Stears, and D. B. Savage. Recent developments inlipodystrophy. Curr Opin Lipidol , 2019, 30(4): 284-90. Stuart, C. A., L. S. Kidder, R. A. Pietrzyk, et al. Rat tailsuspension causes a decline in insulin receptors. Exp Toxicol Pathol , 1993,45(5-6): 291-5。

Claims

1. Application of PET probe targeting sphingosine-1-phosphate receptor 1 (S1P1) in monitoring lipodystrophy in tail-suspended mice.

2. The use according to claim 1, characterized in that: The lipodystrophy model is a tail-suspension mouse model.

3. The application according to claim 1, characterized in that: The PET probe is mainly used for visualizing lipodystrophy disease monitoring.

4. The use according to claim 3, characterized in that: The probe for visualizing sphingolipid metabolism is a PET probe that specifically targets S1P1.

5. The application according to claim 3, characterized in that: The relationship between lipodystrophy and sphingolipid metabolism is mainly monitored by quantifying the expression of S1P1 in adipose tissue.

6. The use according to claim 4, characterized in that: The S1P1 targeting PET probe is [ 18 F] TZ4877 probe, the structural formula of which is shown in Formula I: .

7. The use according to claim 6, characterized in that: Said 18 The preparation method of the F]TZ4877 probe is a radioisotope 18 The F compound is heated to react with the precursor compound to obtain 18 The precursor compound labeled with F was further reacted under acidic conditions and neutralized with NaOH to obtain [ 18 F] TZ4877 probe, the precursor compound is shown in formula II: .

8. The use according to claim 7, characterized in that: Said 18 The precursor compound labeled with F is 18 F is obtained by replacing the p-toluenesulfonate group (-TsO) of the precursor compound.

9. The use according to claim 7, characterized in that: The radioisotope 18 F compound is K[ 18 F].

10. The use according to claim 7, characterized in that: The reaction under the acidic conditions is 18 The precursor compound labeled with F is mixed with HCl and acetonitrile to react, and then neutralized with NaOH to obtain a product.

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