Enhanced longevity and metabolism by brown adipose tissue with disruption of the regulator of G protein signaling 14

Summary Disruption of the regulator for G protein signaling 14 (RGS14) knockout (KO) in mice extends their lifespan and has multiple beneficial effects related to healthful aging, that is, protection from obesity, as reflected by reduced white adipose tissue, protection against cold exposure, and improved metabolism. The observed beneficial effects were mediated by improved mitochondrial function. But most importantly, the main mechanism responsible for the salutary properties of the RGS14 KO involved an increase in brown adipose tissue (BAT), which was confirmed by surgical BAT removal and transplantation to wild‐type (WT) mice, a surgical simulation of a molecular knockout. This technique reversed the phenotype of the RGS14 KO and WT, resulting in loss of the improved metabolism and protection against cold exposure in RGS14 KO and conferring this protection to the WT BAT recipients. Another mechanism mediating the salutary features in the RGS14 KO was increased SIRT3. This mechanism was confirmed in the RGS14 X SIRT3 double KO, which no longer demonstrated improved metabolism and protection against cold exposure. Loss of function of the Caenorhabditis elegans RGS‐14 homolog confirmed the evolutionary conservation of this mechanism. Thus, disruption of RGS14 is a model of healthful aging, as it not only enhances lifespan, but also protects against obesity and cold exposure and improves metabolism with a key mechanism of increased BAT, which, when removed, eliminates the features of healthful aging.

mechanisms that not only extend lifespan but also correct some of the comorbidities of old age are of importance.
There are two distinctly different types of fat found in mammals: white adipose tissue (WAT), which is an essential site for triglyceride storage, and brown adipose tissue (BAT). The BAT is a protective mechanism of recent interest. BAT enhances energy metabolism and protects against cold exposure and obesity (Stanford et al., 2013). A novel model to investigate the role of BAT in healthful aging and lifespan is the mouse model of the disruption of the regulator for G protein signaling 14 (RGS14), which has increased BAT. RGS14 is a complex RGS family member that contains a canonical RGS domain, a tandem (R1 and R2) Ras/Rap binding domain Wohlgemuth et al., 2005), as well as a GoLoco/GPR motif. Most prior work on RGS14 focused on its effects on embryonic development and on the visual cortex and central nervous system (Evans, Lee, Smith & Hepler, 2014;Lee et al., 2010;Lopez-Aranda et al., 2009;Shu, Ramineni & Hepler, 2010;Vellano, Brown, Blumer & Hepler, 2013). The role of BAT in RGS14 KO and its ability to enhance lifespan and improve metabolism, the focus of the present investigation, have never been explored. Sirtuins, which have been shown to be involved in mediating healthful aging (Merksamer et al., 2013;Sack & Finkel, 2012) were also examined. To confirm the essential role of BAT in mediating the protection in the RGS14 KO, we transplanted BAT from RGS14 KO to WT mice, a technique that is equivalent to a BAT KO, as it disrupts the salutary phenotype in the RGS14 KO and transplants these features to their WT, receiving the BAT. Finally, the evolutionary conservation of the effects of   As BAT is known to protect animals from the cold (Stanford et al., 2013), we induced a cold challenge and found that body temperature was preserved better in RGS14 KO than in WT littermates ( Figure 2f,g), with basal body temperatures similar in both the dark and the light cycles. In addition, mock surgery of BAT transplant in RGS14 KO and its WT littermates, that is, conducting the surgical procedure, but not removing BAT from RGS14 KO, did not alter the pattern of thermogenic function protection in RGS14 KO (Figure 2f, g). These data, taken together with Figure 1, demonstrate an increased BAT mass, improved metabolism, and protection against obesity and cold exposure in RGS14 KO mice.

| RGS14 KO improves metabolism and mitochondrial function
Both the absolute levels of NAD + and ratio of NAD + /NADH were elevated in skeletal muscle in the RGS14 KO mice (n = 4) (Figure 3a). To determine the mechanism and signaling pathways accounting for the induction of NAD + /NADH levels and SIRT3 by RGS14 deficiency, we investigated the CD38 level in skeletal muscle, which is the primary NADase in mammals and regulates cellular   (Aksoy, White, Thompson & Chini, 2006;Camacho-Pereira et al., 2016;Young, Choleris, Lund & Kirkland, 2006).
Both the absolute level of NAD + and NAD + /NADH ratio were elevated in the RGS14 KO mice (n = 4/group). (b) CD38, a mechanism for changes in NAD + , was reduced in RGS14 KO skeletal muscle, further confirmed in the increased NAD + level (n = 6-7/group). (c) Increase in mitochondrial DNA content in skeletal muscle was measured by mitochondrial DNA/nuclear DNA ratio (n = 5/group). (d) Complex I activity in skeletal muscle was significantly increased in RGS14 KO animals (n = 7/group). (e) PGC-1a, a key regulator of mitochondrial biogenesis, was increased at the protein level in the BAT of the RGS14 KO mice (n = 3/group). Results are expressed as mean AE SEM.

| RGS-2 loss of function reduces oxidative stress and extends Caenorhabditis elegans lifespan
The nematode C. elegans possesses 12 RGS proteins (Sierra et al., 2002). According to the results of Basic Local Alignment Search Tool (BLAST), two members of the family, rgs-1 and rgs-2, are closely related to the mammalian RGS14 ( Figure 6a). Phylogenetic studies show that the RGS domain is evolutionarily conserved among different species, including nematode, fruit fly, zebra fish, frog, mouse, and human (Figure 6b,c).
We next explored the effects of rgs-2 loss of function (LOF) in C. elegans. By using reporter strains, expressing green fluorescent protein (GFP) in mitochondria of the body wall muscles and intestine, we were able to assess mitochondrial content in these tissues.

| DISCUSSION
The major finding of this investigation was that disruption of the regulator for G protein signaling 14 is a novel model of longevity.
Moreover, the RGS14 KO homolog in C. elegans also showed increased survival, confirming the importance of this mechanism and its evolutionary conservation. Another major finding was that the RGS14 KO is a model of healthful aging, as it also reduces WAT and  improves metabolism and thermogenic function. Potentially, the most important new information is that the mechanism of the healthful aging in the RGS14 KO involves BAT, which is increased in this model. In support of the importance of BAT in healthful aging, BAT mass and BAT function are known to decay with aging (McDonald & Horwitz, 1999), while WAT mass is known to increase (Goncalves et al., 2017). Indeed, we found that the signature improvements in metabolism observed in the skeletal muscle of the RGS14 KO were recapitulated in BAT from RGS14 KO. These include enhanced oxidative phosphorylation, PGC-1a, SIRT3, and mitochondrial DNA ( Figure 5).
Another major finding of the current investigation was that the RGS14 KO mice showed an extension in lifespan in both males and females and did not exhibit many of the adverse effects of aging normally observed in mice, such as decreased thymus weight, body atrophy, or loss of body hair (Gui, Mustachio, Su & Craig, 2012;Tyner et al., 2002). The current field of aging-related research is focused more on extending a healthful lifespan, rather than simply extending lifespan, as older individuals with complications of aging that impair the quality of life often do not feel that further extension of lifespan is desirable. It was therefore important to determine whether these mice were also protected from risk factors that impair healthful aging, for example, protection against obesity (American Diabetes, 2004;Brown, Fujioka, Wilson & Woodworth, 2009;Guh et al., 2009) and impaired metabolism (Finkel, 2015;Nguyen, Samson, Reddy, Gonzalez & Sekhar, 2013;Roberts & Rosenberg, 2006). Indeed, the RGS14 KO mice have a reduced WAT/body weight ratio, are protected against the cold, and have increased mitochondrial biogenesis.
To prove that the increase in BAT activity was key for the improved metabolism and healthful aging in the RGS14 KO mice, we utilized a BAT KO simulation, that is, surgically removing BAT from RGS14 KO mice and transplanting it into WT recipients. The BAT transplantation reversed the phenotype of the RGS14 KO and WT; that is, WT BAT recipients had improved oxygen consumption and protection from cold exposure, thus demonstrating a phenotype similar to that of RGS14 KO with intact BAT, whereas the RGS14 KO donors lost these salutary features and were no longer different from WT. Thus, the WT mice with RGS14 KO BAT transplantation did not show the adverse aging-related features observed in WT mice without the BAT transplants. All of the above point to BAT as a key mechanism mediating the enhanced metabolism and healthful aging in the RGS14 KO mouse model.   (i) Worm survival is preserved upon paraquat treatment (4 mM) after rgs-2 RNAi compared to worms fed with empty vector. (j) Worm mobility is improved during aging after rgs-2 RNAi compared to worms fed with empty vector. (k) Worms exposed to rgs-2 RNAi live longer compared to control worms under basal conditions. (l) The observed increase in lifespan upon rgs-2 RNAi treatment is more important when worms are exposed to rgs-2 RNAi during larval stages than during adult life. (i-l) n = 100. Statistical significance was determined by the use of the log-rank (Mantel-Cox) method. p < .05 is considered as significant only by increasing its expression levels, but also by increasing the availability of NAD + , an important cofactor required for sirtuin function. SIRT3 activation, in turn, leads to improved mitochondrial biogenesis, providing the molecular basis for healthful aging in the RGS14 KO animals.
In summary, increased BAT in the RGS14 KO constitutes a novel model of healthful longevity. The salutary healthful aging properties of the RGS14 KO are mediated by BAT, involving a SIRT3 mechanism and its ability to improve metabolism and mitochondrial function, also known to mediate healthful aging (Bratic & Trifunovic, 2010;Brewer, Gibbs & Smith, 2016;Herranz et al., 2010). The latter point was confirmed by experiments with BAT transplants, which reversed the phenotypes of the RGS14 KO and WT, such that the WT with the BAT transplant did not exhibit the adverse phenotype of aging and demonstrated improved cold exposure tolerance and oxygen consumption, whereas the RGS14 KO donors no longer possessed these features of healthful aging.
Thus, the results of this investigation support the concept that finding a mechanism to translate the RGS14 KO or BAT phenotype to patients would be a novel therapeutic approach to promote healthful longevity.

| Generation of RGS14 KO mice
C57Bl6/J background mice with systemic RGS14 gene deficiency were developed as previously described (Lee et al., 2010). RGS14 KO mice (RGS14tm1-lex) were generated in the Transgenic Core Facility at Rutgers University-New Jersey Medical School, through the National Institutes of Health-sponsored Mutant Mouse Regional Resource Center at http://www.informatics.jax.

| Sample and subject selection
Sample sizes for all experiments were predetermined using power analysis and on the basis of extensive laboratory experience with these end points. Animals that survived through to the endpoint of the successful experiments were included in the analysis. These criteria were pre-established. No animals were excluded from the results. For all molecular and physiological experiments, animals were subjected to the same surgery and grouped on the basis of their genotypes after data collection.

| Animal experimental procedures
Pups were weaned at 28 days of age and housed individually to allow for measurement of food intake on standard diet, in a pathogen-free facility under a 12:12 hr light/dark cycle with access to water and food ad libitum. Age-and sex-matched mice were also followed as controls. Investigators were blinded until the data were collected. No randomization was used during animal handling. Thymuses were collected and weighed at defined time points. For aging phenotype, old mice were compared at an age of 24 months old and also documented for lifespan on the day they died.

| Calculation of adiposity index
Three-to five-month-old RGS14 KO or WT mice were sacrificed and their gonadal, retroperitoneal, inguinal, and brown fat pads were isolated and weighed. The white adiposity index was calculated using total adipose depot (gonadal, retroperitoneal, inguinal) weight divided against live body weight then multiplied by 100. The brown adiposity index was calculated using brown adipose depot weight divided against live body weight then multiplied by 100.

| Indirect calorimetry
The mice were individually housed in separate metabolic chambers (Accuscan Instruments Inc., OH) with ad libitum access to food and water. The chambers were placed into a controlled environment with regulated temperature and 12-hr day/night cycles. Oxygen consumption and production were recorded every 10 min for 24 hr.

| Brown adipose tissue (BAT) removal and transplantation
Three-to five-month-old male RGS14 KO or WT mice were anesthetized with pentobarbital sodium. The backs of the mice were shaved, and the mice were placed in the prone position. A 2-cm midscapular transverse incision was made on the back of the mouse, and the BAT was freed from surrounding muscles and removed through the skin incision. Transplantation was performed by removing BAT from RGS14 KO mice. The removed BAT from donor mice was incubated in 10 ml saline at 37°C for 20-30 min. Three-to five-month-old WT recipient mice were anesthetized. 0.1 g BAT from RGS14 KO donor was transplanted into the visceral cavity of each recipient WT mouse. The graft was carefully lodged deep between folds within the endogenous epididymal fat of the recipient mice. The skin incision in the recipient mice was either closed with 6-0 nylon sutures or with stainless steel wound clips. The animal was allowed to recover in a warm Thermocare unit. The removed BAT was used for BAT transplantation. These mice were allowed to recover for 3 days prior to the BAT thermogenic function studies.
These mice were also studied older, at 24 months of age. The shamoperated mice underwent the same procedure, except for receiving donor tissues.

| BAT thermogenic function
RGS14 KO and WT mice that had been acclimatized to thermoneutrality (30°C) for 4-10 days were transferred to 4°C with full access to water and food (Jin et al., 2011;Vila-Bedmar et al., 2012) Bacterial feeding RNAi experiments were carried out as described (Kamath, Martinez-Campos, Zipperlen, Fraser & Ahringer, 2001), and the clone, which was used for the experiments, was rgs-2 (F16H9.1). It was purchased from GeneService, and its identity was confirmed by sequencing.

| Lifespan assays
Caenorhabditis elegans lifespan assays were carried out at 20°C as described (Mouchiroud et al., 2011). Briefly, each condition included 100 worms and the scoring was performed every 2 days until all worms were dead. The reasons for censoring were the «exploded vulva» phenotype or animals that crawled off the plate. Where indicated, paraquat was added on top of the seeded agar plates. L4 stage worms were added to these plates once the paraquat solution was completely dried. For the paraquat experiments, the scoring was performed every day for the first 3-4 days.

| Mobility assessment
The movement of worms was recorded for 90 s at l4 stage and at days 1, 2, 3, 4, and 5 of adulthood using a Nikon DS-L2/DS-Fi1 camera and controller setup, attached to both a computer and a standard bright-field microscope. One hundred worms per condition were used. The movement of worms during aging was calculated by taking an integral of the speed value, which was assessed by following the worm centroids with a modified version of the Parallel Worm Tracker for MATLAB (Mouchiroud et al., 2013).

| GFP quantification
Fluorescence intensity in worm reporter strains expressing green fluorescent protein (GFP) was quantified using Victor X4 plate reader (Perkin-Elmer). Eighty worms at the corresponding age were used per condition. Worms were placed into M9 medium in black-walled 96-well plate, with 20 worms per well. Each experiment was repeated at least twice.

| Respirometry on fresh BAT
Fresh BAT was isolated from RGS14 WT or KO male mice. Mitochondrial function in fresh BAT was evaluated using high-resolution respirometry (Oroboros Oxygraph-2k; Oroboros Instruments, Austria), as previously described (Pirinen et al., 2014), with minor modifications. Briefly, Complex I leak respiration was measured by adding pyruvate (9.8 mM), glutamate (20 mM), and malate (1.6 mM). This was followed by the addition of ADP (4.8 mM) to assess Complex I OXPHOS capacity. Adding succinate (9.6 mM) stimulated Complex I + Complex II-driven coupled respiration. Finally, Complex II OXPHOS capacity was assessed by sequential addition of rotenone (0.1 mM), which inhibited Complex I activity. O 2 flux obtained in each step of the protocol was normalized by the weight of the tissue used for the analysis. VATNER ET AL.
| 9 of 11 4.14 | Statistics Data were presented as mean AE SEM. Statistical comparisons among groups (n ≥ 3) were calculated using two-way ANOVA with a Bonferroni analysis. Comparisons between two groups were calculated using a two-tailed Student's t test. To examine statistical significance of the longevity data, we used three statistical tests: (i) The Kaplan-Meier analysis was used for the total survival curve, specifically the log-rank (Mantel-Cox) test, as previously described (Bland & Altman, 2004). (ii) For median lifespan analysis, a Mood's median test (Glover et al., 2016), was used to determine differences in median lifespan.
(iii) A Student's t test was used to test differences in maximum lifespan. p values of < .05 were considered significant.

| Study approval
Animals used in this study were maintained in accordance with the