Transgenic mice with brown adipocyte-specific overexpression of mitoNEET (mitoNEET-Tg) in a C57BL/6J background were generated to express human mitoNEET driven by the mouse Ucp-1 promoter. Littermate mice without the human mitoNEET transgene served as wild type control mice. For the atherosclerosis study, mitoNEET-Tg mice were crossed with ApoE knockout (ApoE KO) mice (Stock# 002052, Jackson Laboratory) to obtain ApoE knockout mitoNEET-Tg mice (ApoE/mitoNEET-Tg). The offspring were genotyped by PCR analysis of DNA obtained from tail-snip biopsies using transgene-specific oligonucleotide primers for human mitoNEET and ApoE knockout. We selected two groups of mice for this study: (1) ApoE homozygous and positive for human mitoNEET, and (2) littermate control mice with a genotype of ApoE homozygous and negative for human mitoNEET. All experiments were conducted in 8-week-old male mice. The study protocol was approved by the Animal Research Ethics Committee of the University of Michigan.

Mice were anesthetized by isoflurane inhalation and fixed face down on a surgical heating pad (37°C). The subscapular hair was removed, and a 1 cm long incision on the midline skin was made to expose the interscapular BAT. Next, the intact BAT was completely separated from the interscapular trigonal pyramidal region. The vessels supplying blood to BAT and the neighboring cells at the trigonal pyramidal bottom were cauterized using an electronic cauterizer to permanently block bleeding and BAT regeneration after the BAT removal procedure. The skin wound was closed using wound clips. The mice were allowed to recover for 1 week at room temperature (22°C) before initiating the temperature, energy expenditure and atherosclerosis studies at 16°C (Chang et al., 2012b).

The mice were anesthetized by isoflurane inhalation. The neck hair was removed, and the skin was opened. A temperature monitoring microchip (Bio Medic Data Systems, 12 mm long and 2 mm in diameter) was surgically implanted in the subcutaneous area. After 3 days' recovery from the surgery, the temperature was manually recorded by remotely scanning the animal using a handheld reader system (DAS-7007R, Bio Medic Data Systems) at 9 a.m., 12 p.m., and 4 p.m. daily.

Intravascular temperature was monitored using a T-type thermocouple probe (ADInstruments MLT1405) which was inserted into the thoracic aorta through the left carotid artery of mice under anesthesia induced by isoflurane inhalation (Chang et al., 2012b). The thermocouple probe was connected to a data acquisition system (AdInstruments Powerlab) to monitor the temperature inside of aortic lumen. During the procedure, the mice were lying on their backs on a 35°C warm pad with constant inhalation of isoflurane, and cold stimulation was performed by submerging the tail and hind feet in 4°C water with the researcher blinded to the genotype of mice.

Oxygen consumption (VO₂), carbon dioxide production (VCO₂), spontaneous motor activity and food intake were measured using the Comprehensive Laboratory Monitoring System (CLAMS, Columbus Instruments), an integrated open-circuit calorimeter equipped with an optical beam activity monitoring device. Mice were individually placed into the sealed chambers (7.9′′ × 4′′ × 5′′) with free access to food and water. To determine the energy expenditure in mice upon acute cold exposure, the study was carried out in an experimentation room set at 22 or 4°C with 12-12 h (6:00 p.m.~6:00 a.m.) dark-light cycles. The measurements were carried out continuously for 48 h at 22 or 4°C. Mice were provided food and water through the feeding and drinking devices located inside the chamber without nesting material due to the fact that it blocks the beams that track activity. The amount of food consumed by each animal was monitored through a precision balance attached below the chamber. The body composition data were measured using an NMR analyzer when conscious mice were placed individually into the measuring tube with a minimum restrain. Total energy expenditure was calculated based on the values of VO₂, VCO₂, and the protein breakdown (Riachi et al., 2004).

For atherosclerosis experiments, 8-week-old male ApoE KO and ApoE/mitoNEET-Tg mice were fed a high-cholesterol diet (Harlan, TD.88137) for 3 months in a cold-temperature chamber (16°C) with a 12-h:12-h light-dark cycle and free access to water and diet as in our previous study (Chang et al., 2012b). Afterwards, the animals were sacrificed with excess CO₂. After collection of plasma, the mice were perfused with 20 ml normal saline solution through the heart, followed by 20 ml 37% formalin. The mice were fixed with formalin and the whole aortic tree was dissected under a surgical microscope. Next, the aortic trees were stained with Oil Red O solution (0.2% Oil Red O (w/v) in 3.5:1 of methanol:1N NaOH) for 50 min, followed by 70% ethanol for 30 min. Afterwards, the aortic trees were kept in ddH₂O. The attached connective tissues around the aortic trees were cleaned and pinned on a plate containing paraffin wax, and then the aorta was longitudinally opened with a Vannas scissor to expose the atherosclerotic lesions. The pictures of whole aortic trees were obtained using a digital camera, and the atherosclerotic lesion areas were calculated by an Image software (Meta Imaging Series 7.0, Molecular Devices, LLC).

Adipose tissues were harvested from mice that were anesthetized and fixed overnight via transcardial perfusion with 4% paraformaldehyde (pH 8.0). After dehydration, the samples were embedded in paraffin wax according to standard laboratory procedures. Sections of 5 μm were stained with H&E for routine histopathological examination with light microscopy.

The mice were housed at 4°C for 24 h, 16°C for 1 week or 3 months. The tissues indicated in the figures were harvested and frozen in liquid nitrogen for mRNA and protein analysis. Total RNA was isolated from tissues using TRIzol reagent (Invitrogen). The mRNA levels were measured by QT-PCR using a Bio-Rad thermocycler and a SYBR green kit (Bio-Rad). The mouse primers used for each gene were, respectively, as follows:

Ucp-1: 5′-AAAAACAGAAGGATTGCCGAAACT-3′ and 5′-TAAGCATTGTAGGTCCCCGTGTAG-3′;

Cidea: 5′-CTGTCGCCAAGGTCGGGTCAAG-3′ and 5′-CGAAAAGGGCGAGCTGGATGTAT-3′;

Pgc-1α: 5′-CTCCTCCCACAACTCCTCCTCATA-3′ and 5′-GGGCCGTTTAGTCTTCCTTTCCTC-3′;

Pgc-1β: 5′-CTACCGCCTGGCCATACCTGTCA-3′ and 5′-CTCCTCATCTTCCTCCCGCTTTTG-3′;

IL-6: 5′-TTCCCTACTTCACAAGTC-3′ and 5′-GTACAAAGCTCATGGAGA-3′;

TNF-α: 5′-CTCAGATCATCTTCTCAA-3′ and 5′-GGTTTGCCGAGTAGATCT-3′;

Mcp-1: 5′-CACCAGCACCAGCCAACTCTCACT-3′ and 5′-CATTCCTTCTTGGGGTCAGCACAG-3′;

Ckb: 5′-CCTGCTTCGTCCGGCATC-3′ and 5′-GTCCAAAGTAAAGCCGCTCG-3′;

Macrod1: 5′-ATTGTCAACGCTGCCAACAG-3′ and 5′-TTCTGTAGGGTGCGGCATTC-3′;

Fabp3: 5′-CAGGTGGCTAGCATGACCAA-3′ and 5′-ATGAGTTTGCCTCCGTCCAG-3′;

Idh2: 5′-TGTATCCATGGCCTCAGCAA-3′ and 5′-TGCCATGTACAGAGTACCCAC-3′;

Klf2: 5′-TAAAGGCGCATCTGCGTACA-3′ and 5′-GTGGCACTGAAAGGGTCTGT-3′.

The proteins in tissues were extracted by T-PER tissue protein extraction reagent (Thermo Scientific 78510) as indicated in the instructions manual. Protein separation by electrophoresis using 10% SDS-PAGE gels for 1 h at 150V in Tris/Glycine/SDS electrophoresis buffer (25 mM Tris, 190 mM glycine and 0.1% SDS) was performed on 30 μg protein/well in loading buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue and 0.125M Tris-HCl) after boiling at 100°C for 10 min. Proteins were transferred to a nitrocellulose membrane in transfer buffer (25 mM Tris, 190 mM glycine and 20% methanol) at 40V overnight at 4°C. The membrane was rinsed in TBST buffer (20 mM Tris pH7.5, 150 mM NaCl, 0.1% Tween 20) 3 times at room temperature. MitoNEET protein levels were detected by overnight incubation at 4°C in 5% milk containing 1:1000 anti-mitoNEET antibody (ProteintechTM Cat#: 16006-1-AP). Then the membrane was rinsed in TBST buffer, and incubated with Goat anti-Rabbit IgG antibody (IRDye680LT) for 2 h at room temperature. The blot image was captured and analyzed by BioRad LI-COR system.

All data were evaluated with a 2-tailed, unpaired Student's t-test or compared by one-way ANOVA followed by Newman-Keuls and were expressed as mean ± SD. A value of p < 0.05 was considered statistically significant.

Prior studies indicated that the characteristics of PVAT in mice make it almost identical to interscapular BAT (Fitzgibbons et al., 2011; Chang et al., 2012b). Thus, we reasoned that thermogenesis would be one of the properties of PVAT. Our previous study demonstrated that mice lacking PVAT had lower intravascular temperature in response to cold stimuli (Chang et al., 2012b). Since Pgc1α and Pgc1β are well-known nuclear receptor coactivators critically involved in BAT thermogenesis (Spiegelman, 2007a,b), we performed RNA deep sequencing analysis in PVAT of Pgc1α and Pgc1β knockout (KO) mice to screen for factors related to thermogenesis in PVAT. Firstly, we confirmed that Pgc1α mRNA or Pgc1β mRNA is efficiently deleted in both PVAT and BAT of Pgc1α KO (Figure 1A) or Pgc1β KO mice (Figure 1B), respectively. To investigate whether Pgc1α or Pgc1β in PVAT contribute to PVAT thermogenesis, we measured the intravascular temperature of Pgc1α or Pgc1β KO mice upon cold stimulus for 90 s. As shown in Figure 1C, upon 4°C cold stimulus, the intravascular temperatures in all mice are gradually reduced. However, the intravascular temperature of both Pgc1α or Pgc1β KO mice drops faster than that of wild type mice. After just 30 s of cold stimulus, the average temperature in thoracic aorta of wild type mice dropped 0.05 ± 0.03°C, while it dropped 0.13 ± 0.04°C and 0.12 ± 0.05°C in the aorta of Pgc1α KO and Pgc1β KO mice, respectively. After 60 s of cold stimulus it further dropped to a differential of 0.28 ± 0.05°C and 0.30 ± 0.07°C in Pgc1α KO and Pgc1β KO mice respectively vs. 0.10 ± 0.03°C in wild type mice. At the endpoint of cold exposure, 90 s, the average temperature in thoracic aorta of wild type mice is reduced 0.19 ± 0.03°C while it is reduced 0.44 ± 0.11°C in Pgc1α KO mice and 0.48 ± 0.14°C in Pgc1β KO mice (Figure 1C). These data indicate that deleting Pgc1α or Pgc1β in PVAT might contribute to the hypothermic phenotype independent of BAT. To investigate the common thermogenic genes, which might represent a cross point between Pgc1α and Pgc1β in terms of their shared thermogenic mechanisms, we compared the genes in PVAT of Pgc1α and Pgc1β showing a 1.5-fold change when compared to PVAT of wild type mice. About 37 genes are reduced in PVAT of Pgc1α KO mice, while about 112 genes are reduced in PVAT of Pgc1β KO mice. Among them, only 8 genes [Ivns1abp, Ckb, Macrod1, Fabp3, Lmpdh1, Idh2, Klf2 and Cisd1 (mitoNEET)], which might be directly involved as effectors of thermogenic mechanisms, are reduced in PVAT of both Pgc1α and Pgc1β KO mice (Figure 1D), consistent with known functions of these genes in thermogenesis and lipid metabolism in adipose tissue (Banerjee et al., 2003; Chen et al., 2011; Vergnes et al., 2011; Van der Zee, 2013; Lee et al., 2016). Using real-time PCR, we confirmed that 6 of 8 genes were down-regulated in PVAT of both Pgc1α and Pgc1β KO mice (Suppl. Figure IA, Figure 1E). MitoNEET protein levels were also significantly reduced in PVAT of Pgc1α and Pgc1β KO mice (Figure 1F). Although all the common differentially regulated genes might be critical for thermogenesis, we focused on mitoNEET in this study because mitoNEET is located on the mitochondrial membrane, making it a likely direct effector, and it is involved in WAT browning (Kusminski et al., 2014). Also, compared with WAT, mitoNEET mRNA levels in PVAT and BAT are about 15-fold higher, respectively (Figure 2A). These data suggest that mitoNEET in brown-like PVAT might be directly involved in the regulation of PVAT-dependent thermogenesis.

Next, we investigated whether mitoNEET is associated with thermogenesis. Even though mitoNEET is also highly expressed in mitochondria-rich organs such as skeletal muscle, heart and brain, however, upon cold stimulus, mitoNEET expression is not increased in those three tissues (Suppl. Figure I) while it is significantly increased in PVAT and BAT (Figures 2B,C). Despite of subcutaneous WAT (sWAT) being recognized as an Ucp1-positive beige adipose tissue, the mitoNEET mRNA in sWAT is comparable with that in pure gonadal WAT (gWAT) (Figure 2A). Consistently, the increase in mitoNEET levels in sWAT is not as marked as in PVAT and BAT (Figures 2B,C) upon 24 h cold stimulus. These data suggested that mitoNEET in brown-like PVAT might be strongly associated with cold-induced thermogenesis.

To investigate whether mitoNEET in brown adipocytes regulates thermogenesis, we generated mice with brown adipocyte-specific overexpression of human mitoNEET (mitoNEET-Tg) driven by the Ucp-1 promoter using the strategy outlined in Figure 3A. Western blot confirms that mitoNEET is specifically overexpressed in PVAT and BAT (Figure 3B), but not in subcutaneous and gonadal WAT (Suppl. Figure IIA). Overexpression of mitoNEET in brown adipocytes does not affect the morphology of interscapular BAT, PVAT and aorta (Figure 3C), or of subcutaneous and gonadal WAT (Suppl. Figure IIB). Next, we investigated whether mitoNEET overexpression in brown adipocytes enhances thermogenesis in mice. As shown in Figure 3D, in a 22°C environment, the body temperatures are comparable between wild type and mitoNEET-Tg mice. However, when the mice are placed in a 4°C environment, the body temperature is significantly reduced in the wild type animals while the mitoNEET-Tg mice are able to maintain the body temperature, suggesting that mitoNEET in brown adipocytes is able to prevent the temperature reduction observed in the wild type animals upon transfer to the cold environment. To exclude the potential contribution of BAT to intravascular temperature, we surgically removed the interscapular BAT in both the wild type and mitoNEET-Tg mice and measured the intravascular temperature in mice under anesthesia. As shown in Figure 3E, by immersing the hind feet and tail of mice with 4°C cold water, the reduction rate of intravascular temperatures of mitoNEET-Tg mice is slower than that of wild type mice. After 90 s of cold stimulation, the intravascular temperature in wild type mice dropped by 0.70 ± 0.08°C, while it only dropped by 0.44 ± 0.04°C in mitoNEET-Tg mice. Consistently, in mice housed in a mild cold environment (16°C) as a challenge for 1-week, the mRNA levels related to thermogenesis-associated genes such as Ucp1, Cidea, Evolv3, Dio2, and Pgc1α are increased in the PVAT of mitoNEET-Tg mice when compared with those in PVAT of wild type mice (Figure 4) while the adipogenic aP2 remains unchanged. These genes, except for Evolv3 are not increased in gWAT (Suppl. Figure IIIA). The increased expression of thermogenesis-associated genes in PVAT was also confirmed in the mitoNEET-Tg mice line originated from founder #27 (Suppl. Figure IIIB), ruling out an insertional effect in the #8 founder. These data further confirm that mitoNEET in PVAT positively contributes to cold-induced thermogenesis independently of the presence of BAT.

Next, we studied whether overexpression of mitoNEET in PVAT from mice with BAT removed promotes energy expenditure. Our results indicate that overexpression of mitoNEET in brown adipocytes does not affect the food intake (Figure 5A), body composition (Figure 5B) and total locomotor activity (Figure 5C) when the mice were housed in a 22°C environment. Additionally, there are no statistical differences in energy expenditure at room temperature, between that of wild type and mitoNEET-Tg mice (Figure 5D). Consistently, O₂ consumption (Figure 5E) and CO₂ production (Figure 5F) are comparable between wild type and mitoNEET-Tg mice at that temperature. When mice were housed at 4°C, the cold stimulus increases comparably the food intake and reduces fluid percentage and total locomotor activity of both wild type and mitoNEET-Tg mice (Figures 5A–C), indicating that overexpression of mitoNEET in PVAT does not change the food intake, body composition and movement behaviors of mice. However, when challenged with 4°C cold stimulus, both O₂ consumption (Figure 5E) and CO₂ production (Figure 5F) are significantly increased in the mitoNEET-Tg mice. Consistently, the energy expenditure (Figure 5D) was also significantly increased at 4°C in mitoNEET-Tg mice when compared with wild type mice, suggesting that mitoNEET in PVAT is involved in cold-induced energy expenditure.

Since acute 4°C cold exposure enhanced thermogenesis and energy expenditure in mitoNEET-Tg mice, we investigated the effects of overexpression of mitoNEET in PVAT on atherosclerosis when the interscapular BAT was removed and the mice were housed in a mild cold 16°C environment for 3 months. As shown in Figure 6A, after 16°C exposure and high cholesterol diet feeding for 3 months, ApoE/mitoNEET-Tg mice show higher expression of thermogenesis-related genes such as Ucp-1, Cidea, Cox8b, Evolv3, and Pgc1a in PVAT than those in the PVAT of ApoE knockout mice. Consistently, en face staining of lipid-rich lesions from mouse aortas showed that the total lesion area was significantly lower in aortas from ApoE/mitoNEET-Tg than those from ApoE knockout mice (Figures 6B,C), indicating that overexpression of mitoNEET in PVAT inhibits atherosclerosis in mild cold conditions. However, the body weights (Figure 6D) are comparable between ApoE knockout mice with and without mitoNEET overexpression in PVAT after 16°C exposure for 3 months. Compared to ApoE knockout mice, the total plasma cholesterol and triglyceride levels in ApoE/mitoNEET-Tg mice are significantly reduced in mild cold conditions (Figure 6E), suggesting increased lipid clearance. Remarkably, overexpression of mitoNEET significantly reduced the expression of the pro-inflammatory genes IL-1β, IL-6, Mcp-1, and TNFα in PVAT of ApoE/mitoNEET-Tg mice (Figure 6F). Consistently, the macrophage infiltration is significantly reduced in PVAT of ApoE/mitoNEET-Tg mice (Figures 6G,H). Taken together, these data indicate that increased levels of mitoNEET in PVAT reduce the development of atherosclerosis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.