Assessment of the aging of the brown adipose tissue by 18F-FDG PET/CT imaging in the progeria mouse model LmnaG609G/G609G

Brown adipose tissue (BAT) is an important energy metabolic organ that is closely related to obesity, type 2 diabetes, and atherosclerosis. Aging is one of the most important determinants of BAT activity. In this study, we used 18F-FDG PET/CT imaging to assess the aging of the BAT in LmnaG609G/G609G mice. To evaluate the BAT activity, LmnaG609G/G609G and wild-type (WT) mice were injected with 18F-FDG, and PET/CT imaging was performed. The maximum standardized uptake value (SUVMax) of the BAT was measured and the target/nontarget (T/NT) values of BAT were calculated. The transcription and the protein expression levels of the uncoupling protein 1 (UCP1), beta3-adrenergic receptor (β3-AR), and the PRdomain-containing16 (PRDM16), were measured by quantitative real-time polymerase chain reaction (RT-PCR) and Western blotting or immunohistochemical analysis. Apoptosis and cell senescence of the BAT, in WT and LmnaG609G/G609G mice, was detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and by CDKN2A/p16INK4a immunohistochemical staining, respectively. At 14 weeks of age, the BAT SUVMax and the expression levels of UCP1, β3-AR and PRDM16 in LmnaG609G/G609G mice was significantly lower than that in WT mice. At the same time, the number of p16INK4a and TUNEL positively stained cells (%) increased in LmnaG609G/G609G mice. LmnaG609G/G609G mice are an ideal model for studying BAT aging. The aging characteristics and the aging mechanism of BAT in LmnaG609G/G609G mice can mimic normal BAT aging.


Introduction
Aging has been defined as the age-related deterioration of physiological functions of an organism. The essence of aging is the process of gradual decline of the functions of the organ system. Brown adipose tissue (BAT) is an adipose organ which is maintaining core temperature in small mammals and in newborn humans (Cannon and Nedergaard 2004). The functional status of BAT is closely related to obesity, type 2 diabetes and atherosclerosis (Berbee et al. 2015;Koksharova et al. 2017). BAT quality and activity are gradually decrease with age (Pfannenberg et al. 2010).
Aging is one of the most important determinants of BAT activity (Lecoultre and Ravussin 2011). Therefore, it is important to study the relationship between BAT functional status and aging. Since 1996, the researchers conducted long-term study of the relationship between BAT function and aging by measuring BAT-related indicators, including cold-induced heat generation, cold tolerance, body temperature and other macro physiological parameters and in the level of microscopic molecular biomarker such as Uncoupling protein 1 (UCP1), PPAR-g coactivator 1a (PGC1a), PRdomain-containing16 (PRDM16) and beta3-adrenergic receptor (beta3-AR) (Kirov et al. 1996;Lin et al. 2016;Yamashita et al. 1999). However, there are still some problems that cannot be ignored in the above research. Firstly, the research model, as the above study is the normal mouse aging process, and the mouse life of up to 32 weeks, so the research cycle is long, the degree of aging is difficult to unified control (Graber et al. 2015); secondly, the research method, the macroscopic physiological indicators or microscopic molecular markers used in the above studies are indirect data on the metabolic activity of BAT, and often do not accurately reflect the functional status of BAT. Thus, a visualization of the method is necessary to detect BAT metabolic activity of the models of aging.
Since 2003, the presence of adult BAT was confirmed during 18 F-FDG PET scanning (Cohade et al. 2003) . 18 F-FDG PET/CT is considered to be the "gold standard" of the current BAT function measurement, and the functional status of BAT can be measured directly in vivo by showing glucose metabolism activity (Cypess et al. 2014), and has been widely used in the basics and clinical studies of BAT (Cohade et al. 2003;Yeung et al. 2003). Hutchinson-Gilford progeria syndrome(HGPS) is caused by a mutation in the Lmna gene (De Sandre-Giovannoli et al. 2003a).
According to this phenomenon, the HGPS mouse model (Lmna G609G/G609G mice) was constructed and as the models of aging have been widely used in aging studies (Chen et al. 2012;Zhang et al. 2014). However, like any premature aging syndrome, Lmna G609G/G609G is only a partial representation of the multifactorial process of normal aging. The aging of some key organs will not be appearing in Lmna G609G/G609G models such as nervous system (Jung et al. 2012). Whether the BAT function of the Lmna G609G/G609G mice is premature senility; whether the level of 18 F-FDG uptake is related to the levels of the molecular markers which are associated with the aging of BAT. The above questions directly determine whether the model is suitable for BAT-related aging studies.
In this study, 18 F-FDG PET/CT imaging technique was used to qualitatively and quantitatively analyze the relationship between 18 F-FDG PET/CT imaging and aging of BAT in Lmna G609G/G609G mice. The relationship between BAT-related molecular markers UCP1 and β 3-AR levels and 18 F-FDG uptake was examined. In addition, the mechanism of BAT dysfunction in mice was studied.

Effects of age on the metabolic activity of BAT in mice
To study the effect of age on Lmna G609G/G609G mice and WT mice, PET/CT imaging was performed from 4 weeks on Lmna G609G/G609G mice and WT mice, once every two weeks and for 16 weeks. As shown in figure 1A, from 4 weeks to 12 weeks, there was no significant difference in SUV Max of BAT between Lmna G609G/G609G mice and WT mice, indicating that FDG uptake in BAT of Lmna G609G/G609G mice was no different from that of WT mice at first 12 weeks. As shown in figure 1B

Effects of age on BAT in mice
To clarify the causes of BAT 18 F-FDG uptake decreased in Lmna G609G/G609G mice, the mRNA and expression of UCP1 and beta3-AR were performed. As shown in Figure 2B, UCP1 and beta3-AR translation in Lmna G609G/G609G mice were significantly lower than that in WT mice at 14 weeks of age (UCP1 0.0474±0.0089 vs. Likewise, the number of TUNEL positive cells of BAT in Lmna G609G/G609G mice were more than that in WT mice (4.0±0.45% vs. 1.36±0.1202 P=0.0049).

Effects of age on body weight in mice
As shown in figure 4, the body weight of Lmna G609G/G609G mice began to decline in the 10th week, while the weight of wild mice continued to increase, indicating that Lmna G609G/G609G mice began to decline in the state of the body.

Discussion
In this study, we studied the changes of BAT function with age and analyzed the relationship between 18 F-FDG PET/CT imaging and aging of BAT in Lmna G609G/G609G mice and the mechanism of BAT dysfunction was explored. Our study led to these major findings: Firstly, The BAT function of the Lmna G609G/G609G mice decreased significantly, and the uptake of 18 F-FDG decreased at the age of 14 weeks. Secondly, Lmna G609G/G609G mice brown adipocytes produce reduced, increased number of senescence and apoptosis. Thirdly, the Lmna G609G/G609G mice began to decline in the state of the body at 10 weeks of age earlier than 18 F-FDG uptake decrease.
Aging is an irreversible natural process. It is projected that the combined senior and geriatric population will reach 2.1 billion by 2050 (Mba 2010  mice, which acts as a transcription coregulator that controls the development of brown adipocytes in BAT (Seale et al. 2011). Secondly, the number of BAT cells changed (Sellayah and Sikder 2014). We also found that the increase in BAT cell apoptosis and senescent cells in Lmna G609G / G609G mice resulted in a decrease in the number of cells.
Aging is a systemic change, Lmna G609G/G609G mice weight began to decrease in the 10th week. HGPS model were lean (Sullivan et al. 1999), and myopathic disease (Cutler et al. 2002), which lead to body weight decreased before BAT 18 F-FDG uptake decrease, indicating that Lmna G609G/G609G mice began to decline in the state of the body before BAT aging.
We studied the aging of BAT in Lmna G609G/G609G mice from the perspective of glucose metabolism. 18 F-FDG is current 'gold-standard' for the identification and quantification of BAT metabolic activity, but it cannot discriminate between oxidative and non-oxidative BAT glucose metabolism (Blondin et al. 2015a;Orava et al. 2011).
In addition to 18 F-FDG, there are still other tracers used in aging of BAT metabolic activity studies (Blondin et al. 2015b;U et al. 2016), to more comprehensive assessment of BAT metabolic activity, the application of other tracers is also necessary. Ensure the mice are fasting but with access to water. 200-300uCi of 18 F-FDG in 150ul of saline were intraperitoneal injected into each mouse. The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. Once anesthesia is induced, the animals were moved onto mice bed with its head resting within a cone face mask that continuously delivers Isoflurane (2%) at a flow rate of 1.5 L/min. An electric heating pad is placed under the animal to help maintain the body temperature using a heating pad provided with the small-animal PET/CT system. PET/CT data were acquired for 600s for each mouse with continuous anesthesia. All PET/CT images were processed and analyzed using Nucline nanoScan software (Mediso). For semi-quantitative analysis, three-dimensional (3D) regions of interest were carefully drawn and adjusted manually according to CT images over the borders of the BAT on small-animal PET images of each mice. Three-dimensional round regions of interest were delineated on the lung as a nontarget (NT) reference. Tracer uptake by BAT and lung was quantified as standardized uptake values (SUV) using the formula: SUV=tissue activity concentration (Bq/mL)/injected dose (Bq)×body weight (g). The uptake ratio (T/NT) of the mean BAT and mean NT uptake was calculated and compared.

Quantitative Real Time Reverse Transcription-PCR
After the mice were sacrificed, total RNA was isolated from BAT using an Total

Western blot analysis
After the mice were sacrificed, protein was extracted, the total protein concentration in the samples was determined using the bicinchoninic acid (BCA; Boster, Hubei, China) method and adding 5×loading buffering heated 5 min at 100 . (TBST) for 5 min five times and incubated for 1 h at room temperature with secondary antibody. The membrane was washed in TBST for 5 min five times. Blots were detected using enhanced chemiluminescence (ECL) method. The images were captured and analyzed by ImageJ software.

Immunohistochemistry Analysis
The BAT was harvested from dead mice and fixed in 10% formalin.
Formalin-fixed, paraffinembedded tissue blocks were serially cut into 3-mm-thick sections, which were dewaxed in xylene and rehydrated through a graded series of ethanol solutions. After 3 washes in PBS, heat-induced antigen was retrieved in 0.01 M citric acid buffer (pH 6.0) and autoclaved for 5 min at 120 . Nonspecific binding sites were blocked through preincubation with normal bovine serum for 30 min.
Quantification of the immunostaining was performed by digital image analysis with the Image-Pro Plus 6.0 software (Media Cybernetics). The integrated optical density (IOD) of all the positive staining in each field and areas of interest (AOI) were measured. The IOD was used to evaluate the area and intensity of the positive staining.
The mean density (IOD/AOI) represented the concentration of specific protein per unit area.

Detection of Cell Apoptosis in Brown Adipose Tissue
Tissue apoptosis was assessed using sections for Tunel staining (Beyotime, Beijing, China) according to the manufacturer's protocol. Briefly, after dewaxing and 1 3 hydration, sections were incubated with TUNEL reaction mixture. Nuclei were stained using DAPI. Afterwards, slides were observed using a fluorescence microscope (400×; TE-2000U, Nikon, Japan). For each staining, totally 3 sections per group are observed.

Statistical Analysis
All values are expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism Software. The differences between two groups were determined by Student's t test. The probability value of P < 0.05 was considered significant.