In order to further our understanding of where and when neuronal mTORC2 might be important, we examined the phosphorylation of its substrate AKT S473 in the brains of both male and female mice across their lifespans. We determined that neuronal mTORC2 signaling increases with age in distinct brain regions including the hypothalamus. In order to elucidate the roles of hypothalamic mTORC2 in the metabolic health and aging of mice, we created a model ( Rictor Nkx2.1−/− ) in which Rictor is deleted in a wide range of hypothalamic neurons using Nkx2.1‐Cre . We find that Rictor Nkx2.1−/− mice of both sexes exhibit lifelong increases in adiposity starting at an early age and have reduced spontaneous locomotor activity. Rictor Nkx2.1−/− mice have decreased glucose tolerance, develop insulin resistance as they age, display increased frailty, and ultimately have a reduced lifespan. Finally, we find that Rictor Nkx2.1−/− mice have increased susceptibility to diet‐induced obesity. Our results demonstrate a key role for hypothalamic mTORC2 in the regulation of metabolism, fitness, and longevity, and suggest that inhibition of this complex by pharmaceuticals must be approached with caution.

Over the last decade, significant progress has been made in understanding the roles of both mTOR complexes in the regulation of key metabolic tissues (Kennedy & Lamming, 2016 ). Less well understood is the role of mTOR complex signaling in the brain. mTOR Complex 1 is clearly an important regulator of neuronal behavior; hypothalamic mTORC1 is a key sensor of nutrient sufficiency and acute activation of hypothalamic mTORC1 suppresses food intake, while chronic activation selectively in POMC neurons can drive overnutrition and obesity (Cota et al., 2006 ; Mori et al., 2009 ; Yang et al., 2012 ). Genetic reduction of S6K1 , a key downstream effector of mTORC1, or prophylactic treatment with rapamycin, which can cross the blood‐brain barrier (Cloughesy et al., 2008 ; Gottschalk et al., 2011 ) delays or prevents the progression of Alzheimer's disease in mouse models (Caccamo et al., 2015 ; Majumder et al., 2012 ; Spilman et al., 2010 ) and also blocks age‐associated cognitive decline in wild‐type mice (Halloran et al., 2012 ). In contrast, the role of brain mTORC2 signaling in the regulation of metabolism, health, and longevity has been less studied. This knowledge gap has recently begun to narrow, with recent work showing that deletion of Rictor in male mice using the neuron‐specific Nestin‐Cre recombinase decreases energy expenditure and increases adiposity without affecting food intake, lowers body temperature, and disrupts glucose homeostasis (Kocalis et al., 2014 ). Body weight was also affected in male mice by selective loss of Rictor in POMC neurons, but in this case, the primary effect was on food intake rather than energy expenditure (Kocalis et al., 2014 ). Thus, mTORC2 signaling in the brain plays important roles in whole body metabolism, but the specific neuronal populations mediating these effects and the long‐term implications for health and longevity remain to be elucidated.

While it has long been presumed that inhibition of mTORC1 by rapamycin mediates its beneficial effects on longevity, we and others have found that prolonged treatment with rapamycin also inhibits mTORC2, both in cell culture and in vivo in mice (Lamming et al., 2012 ; Sarbassov et al., 2004 ; Schreiber et al., 2015 ). However, inhibition of mTORC2 by rapamycin is limited to specific cell lines and tissues, most likely determined by the relative expression of FK506‐binding proteins (FKBPs), FKBP12 and FKBP51 (Schreiber et al., 2015 ). In the nematode Caenorhabditis elegans , mTORC2 regulates metabolic processes via several distinct signaling pathways and can have positive or negative effects on lifespan depending on the tissue that is targeted, the temperature, and the food source (Mizunuma, Neumann‐Haefelin, Moroz, Li, & Blackwell, 2014 ; Robida‐Stubbs et al., 2012 ; Soukas, Kane, Carr, Melo, & Ruvkun, 2009 ). In mice, disruption of mTORC2 signaling via deletion of Rictor , which encodes an essential protein component, in the liver, adipose tissue, or skeletal muscle leads to insulin resistance (Bentzinger et al., 2008 ; Kumar et al., 2008 , 2010 ; Lamming, Demirkan, et al., 2014 ; Lamming, Mihaylova, et al., 2014 ; Polak et al., 2008 ; Tang et al., 2016 ). We also recently showed that deletion of hepatic Rictor , or postdevelopmental depletion of RICTOR in the whole body of mice, significantly reduced male lifespan (Lamming, Mihaylova, et al., 2014 ).

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that plays critical roles in the regulation of growth, metabolism, and aging. The mTOR protein kinase is found in two distinct protein complexes; mTOR complex 1 (mTORC1) integrates numerous environmental and hormonal cues, including the availability of amino acids (Wolfson & Sabatini, 2017 ), to regulate key anabolic processes including ribosomal biogenesis, protein translation, and autophagy, while mTOR complex 2 (mTORC2) plays a role in cytoskeletal organization and is a key effector of insulin/PI3K signaling (Kennedy & Lamming, 2016 ; Zhou & Huang, 2011 ). The pharmaceutical rapamycin, which acutely and robustly inhibits mTORC1, extends the lifespan in organisms including yeast, worms, flies, and mice, even when begun late in life or when treatment is intermittent (Apelo, Pumper, Baar, Cummings, & Lamming, 2016 ; Arriola Apelo & Lamming, 2016 ; Bitto et al., 2016 ; Bjedov et al., 2010 ; Dumas & Lamming, 2019 ; Hansen et al., 2007 ; Harrison et al., 2009 ; Kapahi et al., 2004 ; Miller et al., 2011 ; Powers, Kaeberlein, Caldwell, Kennedy, & Fields, 2006 ; Robida‐Stubbs et al., 2012 ; Selman et al., 2009 ).

Because the body weights of Rictor Nkx2.1−/− mice remain stably elevated over those of controls across most of the lifespan, there is little opportunity to capture a substantial energetic imbalance. In order to gain insight into the mechanism that establishes this weight difference in Rictor Nkx2.1−/− mice, we placed mice in metabolic chambers during the first week of HFHS diet feeding, a period of rapid weight gain. As in the previous experiment, we observed that female Rictor Nkx2.1−/− mice gained significantly more weight than controls within one week of HFHS exposure (Figure 6 e). Female Rictor Nkx2.1−/− mice maintained their low level of spontaneous activity following the shift from chow to HFHS diet (Figure 6 f and Figure S5 a). During the period of body weight gain on HFHS diet, there was no decrease in total energy expenditure on a per mouse basis or when adjusted for total body mass, suggesting that it could not account for the preferential weight gain of the Rictor Nkx2.1−/− mice (Figure 6 g and Table S1 ). Female Rictor Nkx2.1−/− mice also exhibited a small but significant increase in RER (Figure 6 h), suggesting a shift toward use of carbohydrates as an energy source and/or increased synthesis of fatty acids. Food intake was increased for the first three days during the dark period in both female and male Rictor Nkx2.1−/− mice (Figure 6 i), and subsequently remained measurably higher in males but not in females during continued exposure to HFHS diet (Figure 6 j). Thus, a modest increase in food consumption, rather than a decrease in energy expenditure, may be the primary driver of weight gain in Rictor Nkx2.1−/− mice.

We found that fasting blood glucose was significantly increased in both Rictor Nkx2.1−/− males and females fed a HFHS diet (Figure 6 c). Fasting and refed plasma insulin levels were increased in female Rictor Nkx2.1−/− on HFHS diet (Figure 6 d). Consistently, Rictor Nkx2.1−/− mice fed a HFHS diet tended to have impaired glucose tolerance and insulin sensitivity as compared to littermate controls fed the same HFHS diet (Figures S9 c–e). Plasma and liver triglycerides trended to increase in female Rictor Nkx2.1−/− mice fed a chow diet (Figure S9 f). Although there was no change in triglyceride levels in the plasma or livers of Rictor Nkx2.1−/− mice of either sex on HFHS diet as compared to littermate controls, lipid content was elevated in the gastrocnemius muscles of female Rictor Nkx2.1−/− mice (Figure S9 g–i). Thus, the lack of mTORC2 signaling in Nkx2.1 neurons exacerbates body weight gain and the resulting impairment of glucose homeostasis when mice are exposed to an unhealthy diet.

Rictor Nkx2.1−/− mice have increased susceptibility to diet‐induced obesity. (a and b) The body weight of control and Rictor Nkx2.1−/− mice of both sexes was tracked on chow diet and following a switch to a high‐fat, high‐sucrose (HFHS) diet as indicated, and (a) weight and (b) percentage weight gain on HFHS diet were plotted ( n = 5‐12/group; Sidak test following two‐way ANOVA, * = p < .05, ** = p < .01, *** = p < .001). The overall effect of genotype (GT), time (T), and the interaction represents the p ‐value from (a) a two‐way ANOVA or a (b) RM ANOVA. (c and d) Mice fed a HFHS diet for 3 weeks were fasted overnight and then refed for 4 hr, with collection of blood for the determination of (c) blood glucose and (d) insulin ( n = 4–12 mice/group; Sidak's test following two‐way ANOVA, * = p < .05, ** p = < .01). (e–i) Metabolic chambers were used to interrogate the metabolic effects of 1 week of HFHS diet feeding. (e) Body weight (f) spontaneous activity (g), energy expenditure per mouse (h) RER, and (i) average food intake during days 1–3 of HFHS feeding ( n = 6 mice/group; Sidak's test following two‐way ANOVA, * = p < .05, ** = p < .01, *** = p < .001). (c–i) The overall effect of genotype (GT), sex, and the interaction represents the p ‐value from a two‐way ANOVA. (j) Food intake (left) and body weight (right) of control and Rictor Nkx2.1−/− mice of both sexes was tracked on a HFHS diet ( n = 6 mice/group; Sidak's test following RM two‐way ANOVA, * = p < .05, ** = p < .01, *** = p < .001, blue/pink stars indicate significant difference vs. male/female controls). The overall effect of genotype (GT), time on diet (T), and the interaction represents the p ‐value from a two‐way ANOVA. Error bars represent the SEM

Deletion of Rictor in the whole brain or POMC neurons (Kocalis et al., 2014 ), or in Nkx2.1‐expressing neurons (this report), increases adiposity in male mice under chow feeding. However, the interactions of these genotypes with high calorie diets that are more relevant to current eating habits have not been investigated and no previous studies have included females. We therefore challenged Rictor Nkx2.1−/− mice with a high‐fat, high‐sucrose (HFHS) diet. Both sexes lacking hypothalamic Rictor exhibited increased susceptibility to diet‐induced obesity, with significantly greater weight gain in females detectable even during the first week (Figure 6 a,b). Adipose mass was significantly increased in Rictor Nkx2.1−/− mice as compared to littermate controls fed the same HFHS diet (Figure S9 a,b).

Rictor Nkx2.1−/− mice have lifelong impairment of glucose tolerance and develop insulin resistance. Metabolic health was assessed by performing (a–c) a fasting glucose tolerance test (GTT) and (d–f) a fasting insulin tolerance test (ITT) on both sexes of control and Rictor Nkx2.1−/− mice at approximately (a and d) 3 months, (b and e) 6 months, and (c and f) 18 months of age. (a and d) n = 6–14 mice/group, 2–3 months of age; (b and e) n = 9–10 mice/group, 5–6 months of age; (c and f) n = 20–32 mice/group, 15–20 months of age. Area under the curve: the overall effect of genotype (GT), sex, and the interaction represents the p ‐value from a two‐way ANOVA; * = p < .05 from a Sidak's post‐test examining the effect of parameters identified as significant in the two‐way ANOVA. Error bars represent the SEM

Genetic disruption of mTORC2 signaling in several key metabolic tissues, including adipose tissue, liver, pancreas, and skeletal muscle, is associated with disruption of glucose intolerance and insulin resistance (Bentzinger et al., 2008 ; Blair, Archer, & Hand, 2013 ; Kumar et al., 2008 , 2010 ; Lamming, Demirkan, et al., 2014 ; Lamming, Mihaylova, et al., 2014 ; Lamming et al., 2012 ; Polak et al., 2008 ; Tang et al., 2016 ). We found that both male and female Rictor Nkx2.1−/− mice exhibit lifelong mild glucose intolerance (statistically significant in males at all ages tested and in females only at 6 months of age, Figure 5 a–c). Insulin sensitivity of young Rictor Nkx2.1−/− mice was similar to that of their littermate controls in both sexes (Figure 5 d,e). However, Rictor Nkx2.1−/− mice developed age‐related insulin resistance, an effect that was particularly prominent in males (Figure 5 f). Collectively, these results demonstrate a critical role for hypothalamic mTORC2 in maintaining physiological and metabolic health with age.

Hypothalamic mTORC2 signaling is essential for healthspan and lifespan. (a and b) Frailty was assessed longitudinally in (a) female and (b) male mice starting at 21 months of age ( n = numbers vary month by month; 2–24 mice/group at each time point). (c) Kaplan–Meier plot showing the lifespan of female and male control and Rictor Nkx2.1−/− mice. The overall effect of genotype ( Rictor Nkx2.1−/− ) and sex (M) was determined using a Cox proportional hazards test (HR, hazard ratio). The table shows the median lifespan for each group, the percentage decrease in median lifespan for each sex, and the two‐tailed stratified log‐rank p ‐value for the decrease in lifespan as a result of deletion of hypothalamic Rictor . Error bars represent the SEM

We next sought to determine the overall effect of hypothalamic Rictor deletion on health and longevity. Mice and humans become increasingly frail with age, and we applied a recently validated mouse frailty index that permits the quantification of the accumulating deficits that occur with age and predicts mortality risk (Kane et al., 2016 ; Rockwood et al., 2017 ; Whitehead et al., 2014 ). We observed that both female and male Rictor Nkx2.1−/− mice develop significantly greater frailty than their control littermates (Figure 4 a,b). As portended by this increased frailty, we find that loss of hypothalamic Rictor shortens the lifespan of both female and male mice ( p = .005, log‐rank test stratified by genotype) (Figure 4 c). Cox regression likewise indicated a significant negative effect of hypothalamic deletion of Rictor on survival (hazard rate (HR) = 1.69), and no interaction of genotype with sex was detected.

The fact that Rictor Nkx2.1−/− mice maintain increased adiposity despite high leptin is consistent with the possibility that they are leptin resistant. Since many leptin‐responsive neurons are located within the hypothalamus, one possibility is that mTORC2 is directly required downstream of leptin to suppress food intake. To test this possibility, we injected control and knockout mice with recombinant leptin and found that high‐dose leptin suppresses food intake and body weight in Rictor Nkx2.1−/− mice (Figure S8 a,b). We also observed similar levels of pSTAT3 in the hypothalamus of control and Rictor Nkx2.1−/− mice (Figure S8 c,d), suggesting that proximal leptin signaling is at normal levels. To further probe potential mechanisms underlying the dysregulated body weight in Rictor Nkx2.1−/− mice, we examined the expression of hypothalamic neuropeptides. The orexigenic neuropeptide NPY was significantly increased in females, whereas the anorexigenic POMC and CART were reduced by ~50% and 25%, respectively, in males (Figure S8 e,f). Thus, Rictor deletion in Nkx2.1‐expressing neurons altered the expression of several hypothalamic neuropeptides known to influence satiety and food intake.

As Nkx2.1‐Cre has been shown to be active not only in the hypothalamus, but also in cells within the thyroid and pituitary (Xu et al., 2008 ), we next assessed whether Rictor Nkx2.1−/− mice displayed changes in related pathways that might contribute to their growth and body weight phenotypes. Intriguingly, we found a significant increase in the circulating level of insulin‐like growth factor 1 (IGF‐1) in both sexes of Rictor Nkx2.1−/− mice relative to their wild‐type littermates (Figure S7 a). Consistent with increased growth hormone/IGF‐1 action, we observed a statistically significant increase in the femur lengths of female Rictor Nkx2.1−/− mice (Figure S7 b) as well as increases in the weights of lean tissues, including liver and skeletal muscle, in three‐month‐old mice (Figure S7 c,d). In contrast, we found no significant differences in T4 or corticosterone levels between Rictor Nkx2.1−/− knockouts and their littermate controls (Figure S7 e,f). Thus, we consider it unlikely that the metabolic phenotypes we observe are a result of altered mTORC2 activity in the thyroid or changes in the hypothalamus–pituitary–adrenal axis, but increased IGF‐1 signaling may play a role in the increased lean mass. Notably, growth hormone/IGF‐1 signaling is normally associated with decreased adiposity (Bengtsson et al., 1993 ; Berryman, Glad, List, & Johannsson, 2013 ) and thus cannot explain the expanded adipose tissue mass in Rictor Nkx2.1−/− mice.

To address the possibility of altered food seeking behavior in Rictor Nkx2.1−/− mice, we assessed their motivation to obtain food. Mice were trained to press a lever to obtain a food pellet, then subjected to a progressive ratio (PR) schedule of reinforcement, where the number of lever presses required to obtain each food pellet increased exponentially (Alhadeff & Grill, 2014 ; Betley et al., 2015 ). Break point analysis (number of pellets received prior to a 10‐min break in lever pressing) revealed that control and Rictor Nkx2.1−/− mice are equally motivated to obtain food under both fed and fasted conditions in this operant task, whether assessed for one hour during the light period or overnight during the dark period (Figure 3 f). Next, we determined the willingness of Rictor Nkx2.1−/− mice to press the lever in the absence of a food pellet reward (extinction) and observed no significant effects (Figure 3 g). Together, these data indicate that Rictor Nkx2.1−/− mice have no change in motivation to obtain food, which suggests that goal‐oriented activity is retained in Rictor Nkx2.1−/− mice despite altered basal activity (Krashes et al., 2011 ). We next placed running wheels in home cages to assess the intrinsic motivation of Rictor Nkx2.1−/− mice toward physical activity. Rictor Nkx2.1−/− mice exhibited a major decrease in voluntary wheel running when fed ad libitum (Figure 3 h). Upon food deprivation, running wheel activity was partly restored during the dark phase in Rictor Nkx2.1−/− mice (Figure 3 i), but the expected increase in activity during the light period relative to ad libitum fed mice was largely blunted (Krizo et al., 2018 ). These data support the conclusion that Rictor Nkx2.1−/− mice have a reduced intrinsic drive to be physically active.

In rodents, locomotor activity can be categorized as spontaneous (e.g., voluntary activity or exploration) or motivated (e.g., food seeking). To first establish that the decreased locomotor activity in Rictor Nkx2.1−/− mice was not due to motor deficits, we tested running using an accelerating treadmill protocol (Frederick et al., 2015 ). Both sexes of Rictor Nkx2.1−/− mice were able to run (Figure 3 e), exhibiting activity levels far in excess of spontaneous home cage movement (Majdak et al., 2016 ; Zombeck, Deyoung, Brzezinska, & Rhodes, 2011 ). We observed an overall effect of genotype on the total distance run at exhaustion, with a small but significant reduction for female Rictor Nkx2.1−/− mice (Figure 3 e). This small effect is not consistent with a major motor deficit, and may be attributable to the increased body weight of mice lacking hypothalamic Rictor , and/or their decreased habitual level of activity.

A neural circuit involving the preoptic area and dorsomedial hypothalamus was recently shown to influence both physical activity and core body temperature (Zhao et al., 2017 ), and mice lacking Rictor in the whole brain have decreased core body temperature (Kocalis et al., 2014 ). Using implanted telemetry probes, we observed a subtle but consistent reduction in core body temperature in ad libitum fed female Rictor Nkx2.1−/− mice during the middle of the dark period, with a similar tendency that did not reach statistical significance in males (Figure S6 a–d).

Voluntary home cage and running wheel activity but not goal‐oriented tasks is reduced in Rictor Nkx2.1−/− mice. (a and b) Traces of average home cage activity of 13‐ to 14‐wk‐old female (a) and male (b) mice under the conditions indicated, as determined by telemetry with counts binned into 10‐min blocks. The overall effect of genotype (GT), time, and the interaction represents the p ‐value from a two‐way RM ANOVA. (c and d) Quantification of the data in panels a and b; activity during the fed condition represents a two day average; during fasting and refeeding over ~24‐hr time period ( n = 4–5 mice/group; Sidak test following two‐way ANOVA, * = p < .05, ** = p < .01, *** = p < .001). (e) Average distance run on a treadmill at exhaustion for 11‐wk‐old male and female mice ( n = 4–10 mice/group, Sidak test following two‐way ANOVA, * = p < .05, ** = p < .01). (f) Eight‐month‐old control and Rictor Nkx2.1−/− mice of both sexes were trained to press a lever to obtain food pellets. Pellets received prior to a 10‐min gap without earning a pellet (the “break point”) in a progressive ratio operant task conducted for one hour during the light period under fed and fasted conditions (1h PR) or during an overnight progressive ratio operant task under fed and fasted conditions (overnight PR). ( n = 5–7 mice/group; Sidak test following two‐way ANOVA, * = p < .05). (g) Number of active lever presses during an overnight extinction paradigm where mice do not receive food pellets in response to lever presses ( n = 5–7 mice/group; Sidak test following two‐way ANOVA, * = p < .05). (e–g) The overall effect of genotype (GT), sex, and the interaction represents the p ‐value from a two‐way ANOVA. (h and i) Voluntary running wheel activity of 4‐month‐old male mice during (h) ad libitum feeding and (i) 24‐hr food deprivation. Data represented as revolutions per 10‐min bin. Inset, cumulative running wheel activity during the light and dark periods ( n = 5‐8/group; Sidak test following two‐way RM ANOVA, * = p < .05, *** = p < .001). (h and i) The overall effect of genotype (GT), time, and the interaction represents the p ‐value from a two‐way ANOVA. Error bars represent the SEM

To more definitively assess spontaneous locomotor activity in Rictor Nkx2.1−/− mice in their home cage environment, we employed telemetry. We found that activity was robustly decreased in both sexes, although the effect was most pronounced in females (Figure 3 a–d), owing in part to the fact that wild‐type females are considerably more active than their male counterparts. Fasting markedly increased locomotor activity, as expected (Yamanaka et al., 2003 ), but the decreased activity phenotype remained in Rictor Nkx2.1−/− mice. Body weight and locomotor activity were not correlated in this experiment, suggesting that the phenotype was not secondary to changes in body weight per se (Figure S5 d).

We did not detect significant differences in body weight, respiratory exchange ratio (RER), food intake, or locomotor activity in 4‐week‐old Rictor Nkx2.1−/− mice (Figure S2 f–i). Although Rictor Nkx2.1−/− mice displayed a slight decrease in energy expenditure on a per animal basis, the effect was not significant after correcting for the slightly lower body weight of the female Rictor Nkx2.1−/− mice at this age, either by dividing energy expenditure by mass or by using ANCOVA (Figure S2 j and Table S1 ). While no consistent change in food intake was detected during the period of body weight gain (Figure S2 k), we did note a trend toward increased hyperphagia in male Rictor Nkx2.1−/− mice upon refeeding (Figure S2 l), indicating some dysregulation of the mechanism controlling satiety. In adult (24–33 week‐old) mice, body weight was higher in Rictor Nkx2.1−/− mice of both sexes than in their wild‐type littermates (Figure S2 m), while average energy expenditure per mouse and food intake were not affected by genotype in either gender (Figure 2 h,i). Body mass adjusted energy expenditure (ANCOVA) was reduced in adult female Rictor Nkx2.1−/− mice relative to their wild‐type littermates, which is consistent with their increased adiposity since adipose tissue consumes less energy per unit mass (Table S1 ). At ten months of age, opposite trends were observed in the energy expenditure between genders; total energy expenditure per mouse was slightly increased in females and slightly reduced in male Rictor Nkx2.1−/− mice. The effect in females was absent after dividing energy expenditure by body weight, but remained marginally significant when adjusting by ANCOVA. Thus, energy expenditure is unchanged or increased in females, suggesting that food intake must explain the higher body weight. In contrast, the decreased energy expenditure in males occurs despite their larger size and unchanged food intake, suggesting that decreased calorie output might contribute to the maintenance of higher body mass in this sex (Figure S3 a–c and Table S1 ). However, neither food intake nor daily energy expenditure were significantly changed in subsequent measures (at 18 months of age) from the same Rictor Nkx2.1−/− mice, with or without adjustment for body weight by ANCOVA (Figure S4 a–c and Table S1 ). Intriguingly, beam break analysis revealed decreased locomotor activity in 6‐ and 10‐month‐old Rictor Nkx2.1−/− mice (Figure S5 a–c), a phenotype that has not been previously associated with the mTORC2 signaling pathway.

Leptin is an adipose‐derived hormone that decreases food intake and promotes energy expenditure (Campbell et al., 2017 ; Chua et al., 1996 ; Halaas et al., 1995 ; Pelleymounter et al., 1995 ). Loss of leptin signaling is sufficient to cause drastic weight gain, whereas weight gain due to other mechanisms is associated with hyperleptinemia as a compensatory response. As Rictor Nkx2.1−/− mice have increased adiposity, we determined leptin levels in knockouts and their wild‐type littermates. We observed that plasma leptin was significantly increased in Rictor Nkx2.1−/− mice, as was leptin mRNA expression in adipose tissue (Figure 2 f and Figure S2 c). Intriguingly, the increase in plasma leptin was observed in Rictor Nkx2.1−/− mice prior to a measurable increase in body weight or adipose mass (Figure 2 f,g), and without obvious differences in adipocyte size (Figure S2 d,e). Together, our results suggest that mTORC2 signaling in Nkx2.1 neurons may have primary effects on leptin expression independent of adipose mass.

Early onset of obesity in mice lackingin hypothalamic neurons. (a and b) The weights of (a) female and (b) male control andmice were tracked from 3 to 8 weeks of age (varies by time point and group,= 4–31; Holm–Sidak test following two‐way ANOVA, * =.05, ** =.01). (c and d) Lean and fat mass in (c) 10‐wk‐old female mice (= 5‐6/group; Holm–Sidak test following two‐way ANOVA, * =.05, ** =.01, *** =.001, solid lines indicate comparisons of fat mass and spotted lines indicate comparison of lean mass) and (d) 20‐ to 22‐week‐old male mice (= 5‐9/group;test, ** =.01). (e) H&E‐stained BAT and gonadal white adipose tissue from 24‐ to 26‐wk‐old chow fed male mice. (f) Plasma leptin levels of female and malemice (= 6–8 mice/group; Sidak test following two‐way ANOVA, * =.05, ** =.01, *** =.001, blue/pink stars indicate significant difference vs. male/female controls). (Corresponding body weight curve is represented in Figure 6 a, week four to nine on chow diet) (g) Fat mass (Left) and body weight (Right) of male control andmice (= 5–6 mice/group; Sidak test following two‐way ANOVA, * =.05, *** =.001). (h) Energy expenditure of 24‐ to 33‐wk‐old female and male mice; per mouse basis (= 6 mice/group; Sidak test following two‐way ANOVA, * =.05). (i) Twenty‐four hour food intake of 24‐ to 33‐wk‐old female and male mice on normal chow (= 6 mice/group; Sidak test following two‐way ANOVA, * =.05).The overall effect of either genotype (GT) and age (panels A, B, F, G), GT and feeding status (C) or GT and sex (panels H‐I), and the interaction represents the‐value from a two‐way ANOVA. Error bars represent the

To characterize the development of these body weight and composition phenotypes, we analyzed a second cohort of Rictor Nkx2.1−/− mice and their wild‐type littermates from the time of weaning. Rictor Nkx2.1−/− mice of both sexes tended to be lighter than littermate controls at weaning, an effect that was statistically significant in males, yet by 5–7 weeks of age Rictor Nkx2.1−/− mice of both sexes weighed more than littermate controls (Figure 2 a,b). Weight gain generally occurred over a distinct period with subsequent stabilization at a new set point relative to controls. Increased body weight in young Rictor Nkx2.1−/− mice reflected an increase in fat mass without any change in lean mass (Figure 2 c,d). Consistently, fat pads were heavier in Rictor Nkx2.1−/− mice (Figure S2 a,b), and larger lipid droplets and adipocyte hypertrophy were observed in brown and white adipose tissue, respectively (Figure 2 e).

We monitored the body weight and composition of Rictor Nkx2.1−/− mice and their wild‐type littermates. We observed that mice lacking hypothalamic Rictor weighed more than their wild‐type littermates throughout their lifespan, a difference that was statistically significant up to 22 months of age in females and 26 months of age in males (Figure 1 f). Periodic assessment of body composition demonstrated that in the aging cohort of Rictor Nkx2.1−/− mice, both sexes had increases in fat mass (Figure 1 g) and to a lesser extent, lean mass (Figure 1 h); the overall effect was a lifelong increase in adiposity (Figure S1 e,f).

Mice lacking mTORC2 signaling in hypothalamic neurons were generated by crossing mice conditionally expressing Rictor to mice expressing Cre recombinase under the control of the Nkx2.1 promoter ( Rictor Nkx2.1−/− ) (Shiota, Woo, Lindner, Shelton, & Magnuson, 2006 ; Xu, Tam, & Anderson, 2008 ). This promoter is active in most of the hypothalamic nuclei during early development, with the exception of the suprachiasmatic nucleus (Mieda, Hasegawa, Kessaris, & Sakurai, 2017 ; Ring & Zeltser, 2010 ; Xu et al., 2008 ). We verified deletion of Rictor in the hypothalamus by determining the expression of Rictor mRNA and RICTOR protein from both male and female mice (Figure 1 c–e and Figure S1 c). mTORC2 activity was assessed by determining phosphorylation of the mTORC2 substrate AKT S473, as well as phosphorylation of mTOR itself at S2481, an autophosphorylation site associated with incorporation into mTORC2 (Copp, Manning, & Hunter, 2009 ). As expected, the phosphorylation of AKT S473 and mTOR S2481 was reduced in Rictor Nkx2.1−/− mice. Brain size was normal in Rictor Nkx2.1−/− mice with no gross abnormalities apparent (Figure S1 d).

Hypothalamic mTORC2 signaling increases with age and regulates body weight homeostasis. (a) Quantification of phosphorylated AKT residues in whole brain lysate from fasted female and male C57BL.6J.Nia mice; young refers to 6‐month‐old males and females (10 males, 5 females), middle refers to 24‐month‐old males and 22‐month‐old females (10 males, 5 females), and old refers to 30‐month‐old males and 26‐month‐old females (8 males, 4 females). Quantification of phosphorylated proteins are relative to total protein (Dunnett's test following two‐way ANOVA, * = p < .05, ** = p < .01, *** = p < .001). (b) mTORC2 activity, as determined by IHC‐IF for phosphorylated Akt S473 (in red), is increased in the hypothalamus of overnight fasted 23‐month‐old female C57BL.6J.Nia mice relative to young 8‐month‐old mice. A neuronal nuclei marker is targeted by the NeuN antibody (in green), showing the mTORC2 signaling is increased in aged neurons in these regions. Shown are representative images of hypothalamic regions (total n examined = 4 mice/group). Scale bar = 100 µm. (c) Expression of Rictor mRNA in hypothalamic tissue of 3‐ to 6‐month‐old Rictor Nkx2.1−/− mice and controls ( n = 5‐8/group; *** = p < .001, Holm–Sidak test following two‐way ANOVA). (d) Hypothalamic protein lysates from 6‐month‐old male control and Rictor Nkx2.1−/− mice were immunoblotted for phosphorylated and total AKT, phosphorylated and total mTOR, RICTOR, and β‐ACTIN. (e) Quantification of RICTOR expression relative to β‐ACTIN and phosphorylated mTOR and AKT relative to total protein ( n = 5 control and 9 Rictor Nkx2.1−/− mice; left: ** = p < .01, t test; right: Holm–Sidak test following two‐way ANOVA, * = p < .05, *** = p < .001). (f) Longitudinal assessment of body weight of control and Rictor Nkx2.1−/− mice ( n = 5–35 per group; p < .05 indicates significant difference between genotypes at each time point within the indicated range, Holm–Sidak test following two‐way ANOVA). (g, h) Longitudinal assessment of (g) fat mass and (H) lean mass of control and Rictor Nkx2.1−/− mice ( n = 5–29 mice/group; Holm–Sidak test following two‐way ANOVA, * = p < .05, ** = p < .01, *** = p < .001). (f–h) The overall effect of genotype (GT), age, and the interaction represents the p ‐value from a two‐way ANOVA. Error bars represent the SEM

We studied brains from three different age‐groups of C57BL/6J.Nia mice obtained from the NIA Aged Rodent Colony: a “young” group, aged 6 months; a “middle” group of 22‐month‐old females and 24‐month‐old males (approximately 70% survival for each sex, based on published lifespan curves for C57BL/6J.Nia mice (Turturro et al., 1999 )); and an “old” group of 26‐month‐old females and 30‐month‐old males (approximately 30% survival). We observed increased phosphorylation of the mTORC2 target AKT S473 in whole brain lysates from 22‐ and 26‐month‐old females and 30‐month‐old males relative to young control mice (Figure 1 a and Figure S1 a). This effect was specific to mTORC2 and not representative of a generalized increase in insulin/IGF‐1 signaling, as phosphorylation of AKT T308, an mTORC2‐independent site downstream of insulin signaling, was not increased in aged mice of either sex. In order to identify the specific regions of the brain that contributed to the increased mTORC2 signaling, we performed immunohistochemistry with antibodies against phosphorylated AKT S473 and NeuN, a marker of neuronal nuclei. We found that phosphorylation of AKT S473 increased in specific regions of the aged mouse brain, including the neurons of the hypothalamus as well as cells within the cortex and thalamus (Figure 1 b, Figure S1 b).

3 DISCUSSION

The mTOR complexes are ancient sensors of nutrient status and metabolic state that have profound tissue‐specific effects on health and longevity. Inhibition of these complexes via rapamycin or genetic interventions that target mTORC1 signaling extends lifespan across species. Although the role of mTORC2 is comparatively less studied, targeting of this pathway in the liver is sufficient to shorten the lifespan of male mice, whereas disrupting mTORC2 in worms can alternately lead to increased or decreased longevity (Lamming, Mihaylova, et al., 2014; Mizunuma et al., 2014; Robida‐Stubbs et al., 2012; Soukas et al., 2009). Here, we report that hypothalamic mTORC2 activity increases with age in mice and that genetically ablating this complex in hypothalamic neurons is detrimental to metabolic health and longevity. RictorNkx2.1−/− mice are hypoactive, predisposed to adiposity and diet‐induced weight gain, become measurably more frail as they age, and have decreased overall survival.

The Nkx2.1 promoter drives expression of Cre recombinase in a wide range of hypothalamic nuclei (Ring & Zeltser, 2010; Xu et al., 2008). Reporter gene expression has also been mapped to scattered cells in the cerebral cortex, striatum, and globus pallidus, and in the thyroid, pituitary, and lung during development. Currently, there are no other genetic tools available that targets the majority of the neuronal subtypes that reside within the hypothalamus. The Nkx2.1‐Cre system has therefore been used widely to study the hypothalamus despite the limitations of its specificity (Burmeister et al., 2017; Chong, Greendyk, Greendyk, & Zeltser, 2015; Chong, Vogt, Vogt, Hill, Brüning, & Zeltser, 2015; Heinrich, Meece, Wardlaw, & Accili, 2014; Ring & Zeltser, 2010). We attempted to overcome this limitation using an inducible Nkx2.1.Cre model to delete Rictor during adulthood (Taniguchi et al., 2011). However, we did not observe a decrease in Rictor mRNA expression in the hypothalamus or difference in body weight and adiposity in this mouse model (data not shown). This is consistent with a prior report that Nkx2.1 expression is substantially reduced after birth (Magno, Catanzariti, Nitsch, Krude, & Naumann, 2009). Thus, with currently available systems, we are unable to completely the avoid the potential of off‐target effects resulting from constitutive expression of Nkx2.1‐Cre. However, we note that thyroid hormone levels are not affected in RictorNkx2.1−/− mice, suggesting that the phenotypes we observe here do not result from inactivation of mTORC2 in the thyroid.

Both obesity and physical inactivity are thought to accelerate age‐related decline, either independently or in combination, and reduce life expectancy. While obesity per se is consistently related to all‐cause mortality across studies (Anon, 2016; Flegal, Kit, Orpana, & Graubard, 2013), it is increasingly appreciated that rapid weight gain early in life can be especially detrimental (Wagener, Müller, & Brockmann, 2013). Restricted in utero growth and/or transient lower body weight postnatally can trigger rapid catch‐up growth that is associated with shorter lifespan independently from adiposity (Hou, Bolt, & Bergman, 2011; Jennings, Ozanne, Dorling, & Hales, 1999; Ozanne & Hales, 2004; Ricklefs, 2006; Rollo, 2002; Sayer et al., 1998). We find that disruption of Rictor in hypothalamic neurons leads to lower body weight at the time of weaning followed by a rapid, excessive gain in body weight during postnatal development. In general, changes in food intake and/or energy expenditure were too modest to detect in young mice on chow diets. We view food intake as the most likely explanation for weight gain, given that subtle changes in food consumption are sufficient to explain considerable changes in body weight (Tschop et al., 2011), and that males are hyperphagic during refeeding. Moreover, food intake was clearly the major contributing factor in the accelerated weight gain experienced by both genders after switching to HFHS diet. We did, however, detect a modest decrease in energy expenditure in chow fed males at a single time point (10 months of age). Thus, it remains possible that there is also a small and potentially sex‐specific contribution of altered energy expenditure to the weight gain or higher weight maintenance of RictorNkx2.1−/− mice.

Weight gain in RictorNkx2.1−/− mice primarily reflected an increase in adiposity, yet we also observed a modest increase in circulating IGF‐1, femur length, and lean mass in the aging cohorts. Growth hormone‐releasing hormone, a neuropeptide expressed in the hypothalamus, stimulates the pituitary gland to release growth hormone, a major regulator of IGF‐1 expression (Junnila, List, Berryman, Murrey, & Kopchick, 2013). Thus, the increased levels of IGF‐1 that we observe could be due to altered hypothalamic release of growth hormone‐releasing hormone. Alternatively, they could also be a direct consequence of altered pituitary function. Further research will be required to distinguish between these possibilities and to determine the role of IGF‐1 in the metabolic effects we observed. Reduced signaling through the growth hormone/IGF‐1 axis due to genetic mutations or caloric restriction is associated with increased healthspan and lifespan in model organisms (Mao et al., 2018; Milman, Huffman, & Barzilai, 2016). These results support the idea that the early‐onset obesity observed in RictorNkx2.1−/− mice and the higher circulating level of IGF‐1 could have a combined long‐term negative impact on health and lifespan.

In addition, the majority of studies have found positive correlations between physical activity and longevity in rodents (Bronikowski et al., 2003; Holloszy, 1993; Holloszy, Smith, Vining, & Adams, 1985; Lokkegaard, Larsen, & Christensen, 2016; Mlekusch et al., 1996; Vogel et al., 2009) as well as humans (Lokkegaard et al., 2016; Rizzuto & Fratiglioni, 2014; Vogel et al., 2009). In normal weight individuals, regular physical activity has been estimated to extend life by 7.2 years, and conversely, inactivity to decrease life by 3.1 years (Moore et al., 2012). RictorNkx2.1−/− mice have substantially reduced spontaneous activity, and understanding the pathways that control this intrinsic drive to move could lead to new approaches to target a key modifiable factor that imparts resistance to stress and injury in older adults and delays the onset of age‐associated diseases (Cabanas‐Sánchez et al., 2018; Huffman, Schafer, & LeBrasseur, 2016).

Physical activity is an important component of energy expenditure in humans that negatively correlates with body weight gain and can act independently from changes in food intake (Bamman et al., 2014; Johannsen & Ravussin, 2008; Ladabaum, Mannalithara, Myer, & Singh, 2014; Mozaffarian, Hao, Rimm, Willett, & Hu, 2011; Pontzer et al., 2016; Warburton, Nicol, & Bredin, 2006). Thus, our finding that locomotor activity decreased in the absence of a measurable change in energy expenditure in young adult RictorNkx2.1−/− mice may appear counterintuitive. However, several recent studies have indicated that the effect of locomotor activity on total energy expenditure is far less in mice than in humans, and may be negligible under the conditions used for most experiments (Abreu‐Vieira, Xiao, Gavrilova, & Reitman, 2015; Dauncey & Brown, 1987; Moruppa, 1990; O'Neal, Friend, Guo, Hall, & Kravitz, 2017; Virtue, Even, & Vidal‐Puig, 2012). While past estimates have placed the fraction of total energy expenditure devoted to physical activity in mice as high as 38% (Dauncey & Brown, 1987), Virtue et al. (2012) have suggested that these studies overestimated the contribution of physical activity per se because other energy consuming processes correlate with activity. They determined that the true energetic cost of physical activity is ~10% of total energy expenditure at thermoneutrality, and much less under standard housing conditions, consistent with a prior study that used similar methods to estimate ~5% (Moruppa, 1990). Thus, even the profound decrease in locomotor activity that we observe in RictorNkx2.1−/− mice is likely to account for only a very small change in energy expenditure or weight gain. However, the neural pathways controlling the set point for activity level are of significant interest, given the clear benefits of both deliberate exercise and spontaneous movement for human health (Bamman et al., 2014; Johannsen & Ravussin, 2008; Pontzer et al., 2016; Warburton et al., 2006). Elevated home cage activity early in the dark period and during food deprivation can be associated with food seeking behavior, even when total food intake is unchanged (Mistlberger, 1994; Yang et al., 2015). To clarify whether motivation to obtain food was altered in the RictorNkx2.1−/− mice, we measured lever pressing in a progressive ratio operant task. The results clearly indicate that RictorNkx2.1−/− mice are equally motivated to obtain food under both ad libitum feeding and fasting conditions. Collectively, our findings support a direct regulation of physical activity level by neuronal mTORC2, rather than a secondary effect of food seeking behavior.

Mice lacking Rictor in Nkx2.1‐expressing cells display markedly increased susceptibility to diet‐induced obesity, a phenotype that was not previously assessed in mice lacking neuronal mTORC2 activity (Kocalis et al., 2014). Total energy expenditure was unaffected by genotype in mice consuming a high‐fat, high‐sucrose diet, suggesting that food intake (or absorption) plays a major role in weight gain. Consistently, higher food intake was recorded in both sexes over the first few days of HFHS diet feeding, when the rate of weight gain was highest, and food intake remained high in males over the subsequent weeks. Obesity and adiposity are well known to be associated with impaired glucose homeostasis, and thus, a limitation of the present study is that we cannot directly assess the direct versus indirect regulation of glucose homeostasis by hypothalamic mTORC2. We note that on chow diet, glucose intolerance is more prevalent in male RictorNkx2.1−/− mice, whereas the increase in adiposity is more pronounced in females, suggesting that the two effects may be somewhat independent. As we observed changes in the levels of several hypothalamic neuropeptides (e.g., AgRP, NPY, POMC, CART) involved in satiety, food intake, and energy balance, we consider it likely that the metabolic effects of hypothalamic Rictor on distinct neuronal populations mediate growth, adiposity, and metabolic phenotypes. A direct effect of hypothalamic Rictor loss on glucose tolerance would be consistent with the previously proposed role for central and hypothalamic insulin resistance in the maintenance of systemic glucose homeostasis (Chen, Balland, & Cowley, 2017; Koch et al., 2010).

It will be of significant interest to elucidate the molecular events that lie upstream and downstream of mTORC2 activity in hypothalamic neurons. Insulin signaling is known to stimulate mTORC2‐dependent phosphorylation of Akt S473 in multiple cell types, and neuron‐specific disruption of the insulin receptor (IR) driven by Nestin‐Cre increases body weight and fat mass (Bruning et al., 2000; Kappeler et al., 2008). However, deletion of the IR in Nkx2.1‐expressing neurons does not have any effect on body weight or composition (Chong, Greendyk, et al., 2015), possibly due to the IGF‐1 receptor playing a more prominent role than the IR in the hypothalamus (Kleinridders, Ferris, Cai, & Kahn, 2014) or due to activation of PI3‐kinase downstream of the leptin receptor (Lamming, 2014). Intriguingly, disruption of the IR in the arcuate nucleus reduces physical activity in young mice (Lin et al., 2010; Taguchi, Wartschow, & White, 2007), and re‐establishment of IR expression specifically in POMC neurons is sufficient to restore physical activity (Lin et al., 2010). These findings support the notion that the hypothalamic IR/mTORC2/Akt signaling cascade plays an important role in determining body weight homeostasis and locomotor activity in vivo. It is interesting to speculate that the age‐dependent increase of mTORC2 activity we observed in the hypothalamus of wild‐type mice may help to preserve fitness and longevity by promoting physical activity.