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Experimental Gerontology 96 (2017) 155–161

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Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero

Life-extending dietary restriction and ovariectomy each increase leucine oxidation and alter leucine allocation in grasshoppers John D. Hatle a,⁎, Ayesha Awan a, Justin Nicholas a, Ryan Koch a, Julie R. Vokrri a, Marshall D. McCue b, Caroline M. Williams c,1, Goggy Davidowitz d, Daniel A. Hahn e a

Department of Biology, 1 UNF Drive, Univ. of North Florida, Jacksonville, FL 32224, USA Department of Biological Sciences, St. Mary’s University, San Antonio, TX 78228, USA Department of Integrative Biology, University of California Berkeley, Berkeley, CA 94720, USA d Department of Entomology, University of Arizona, Tucson, AZ 85721, USA e Department of Entomology & Nematology, University of Florida, Gainesville, FL 32611, USA b c

a r t i c l e

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Article history: Received 22 February 2017 Received in revised form 14 June 2017 Accepted 27 June 2017 Available online 29 June 2017 Section Editor: Holly M. Brown-Borg Keywords: Branched-chain amino acids Glucose Oleic acid Storage Target of rapamycin

a b s t r a c t Reduced reproduction and dietary restriction each extend lifespan in many animal models, but possible contributions of nutrient oxidation and allocation are largely unknown. Ovariectomy and eating 70% of ad libitum-feeding each extend lifespan in lubber grasshoppers. When feeding levels between the two groups are matched, ovariectomy increases fat and protein storage while dietary restriction reduces fat storage. Because of these disparities in nutrient investment, metabolism may differ between these two life-extending treatments. Therefore, we examined the allocation and organismal oxidation of one representative of each macronutrient class: leucine, oleic acid, and glucose. Ovariectomy and dietary restriction each increased oxidation of dietary leucine. Dietary leucine may play a special role in aging because amino acids stimulate cellular growth. Speeding oxidation of leucine may attenuate cellular growth. Allocation of leucine to muscle was the clearest difference between ovariectomy and dietary restriction in this study. Ovariectomy reduced allocation of leucine to femur muscle, whereas dietary restriction increased allocation of leucine to femur muscle. This allocation likely corresponds to muscle maintenance for locomotion, suggesting dietary restriction increases support for locomotion, perhaps to search for food. Last, ovariectomy decreased oxidation of dietary oleic acid and glucose, perhaps to save them for storage, but the site of storage is unclear. This study suggests that the oxidation of branched-chain amino acids may be an underappreciated mechanism underlying lifespan extension. © 2017 Published by Elsevier Inc.

1. Introduction Reduced reproduction and dietary restriction each extend lifespan in many animal models (Hansen et al., 2013; Nakagawa et al., 2012). Because the degree of dietary restriction required to extend lifespan almost always reduces reproduction as well, the traditional paradigm was that these two treatments act through the same mechanisms in the animal. A recent study first showed a mechanistic connection between the two, demonstrating how steroidal signaling upon dietary restriction inhibits reproduction (Thondamal et al., 2014). Despite this discovery, our understanding of how dietary restriction extends lifespan is greater than our understanding of how reduced reproduction extends lifespan. This disparity in knowledge has contributed to the persistent dogma that reduced reproduction and dietary restriction act through the same means. ⁎ Corresponding author. E-mail address: [email protected] (J.D. Hatle). 1 Formerly at Department of Entomology and Nematology, University of Florida.

http://dx.doi.org/10.1016/j.exger.2017.06.019 0531-5565/© 2017 Published by Elsevier Inc.

Lubber grasshoppers provide an excellent system for studying the organismal physiology of aging (Lee et al., 2015). Reduced reproduction by ovariectomy and dietary restriction each extend grasshopper lifespan by 16% (Drewry et al., 2011), and the combination of ovariectomy and dietary restriction extends lifespan 31%. Ovariectomy results in an approximately 40% reduction in feeding, strikingly similar to the feeding level typically used for life-extending dietary restriction. Indeed, when the feeding rate of ovariectomized females was measured, and this same amount was fed daily to a sham-operated group, the survivorship of the sham-operated group was increased (Hatle et al., 2013). This diet matching approach allows direct testing of the effects of reduced reproduction, without confounding effects of reduced feeding. Ovariectomy doubled fat body mass and hemolymph volume (the primary sites of lipid and protein storage respectively), while matched-fed sham-operated grasshoppers slightly reduced fat body mass (Hatle et al., 2013). These massive differences in energy storage reveal that the mechanisms underlying life-extension by reduced reproduction and dietary restriction are different, but the mechanistic details of how these treatments extend longevity are as yet not known. To address this in a

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resource (i.e., nutrient) investment context (O'Brien et al., 2008), we examine aspects of nutrient metabolism underlying reduced reproduction and dietary restriction. In particular, we test the effects of ovariectomy, dietary restriction, or both on organismal oxidation and allocation of representatives of the three classes of macronutrients (proteins, lipids, and carbohydrates). High-protein diets maximize reproduction and shorten lifespan, and circulating branched-chain amino acids in particular have been implicated in promoting aging (Lee et al., 2008; Solon-Biet et al., 2014), but see (Mansfeld et al., 2015). Branched-chain amino acids are especially potent at activating Target of Rapamycin (TOR) (Chotechuang et al., 2009), a central cellular pathway of protein sensing and growth. Leucine, the most prevalent of the three branched-chain amino acids, was recently shown to signal the TOR pathway via sestrin2 (Wolfson et al., 2016). Further, when leucine is oxidized to make ATP, carbon-1 is the first carbon released in CO2 (when NADH is produced). The CO2 is exhaled and is detected by our oxidation assay (see below). Because of its tight association with TOR and the clear interpretation of its oxidation, we chose to investigate oxidation and allocation of dietary 13C-1leucine. Lipid oxidation changes have been linked to longevity in several animal models. Since the demonstration that germline ablation extends lifespan (Hsin and Kenyon, 1999), work in C. elegans has characterized mechanisms of how reduced reproduction extends lifespan (Amrit et al., 2016). For the present study, we chose specifically to measure the oxidation of dietary oleic acid, in part because increased levels of oleic acid are associated with longevity in C. elegans (Goudeau et al. 2011). Finally, dietary carbohydrates are believed to promote aging via the accumulation of advanced glycation end-products (Semba et al., 2010). For grasshoppers in particular, both simple and complex carbohydrates are abundant in their plant-based diets, but these plant diets have relatively low levels of proteins and very low levels of lipids. Hence, we tested oxidation and allocation of dietary glucose as a representative sugar. Our working hypothesis is that ovariectomy and dietary restriction extend the lifespan of grasshoppers in different ways. We predict that these two different interventions would produce distinct patterns of organismal nutrient oxidation and allocation to tissues. Specifically, we predicted that dietary restriction would increase oxidation of all tested nutrients during the first few hours after a meal and tracer ingestion because animals on an energy deficit are likely to use ingested nutrients to fuel immediate metabolic demands. Finally, we predicted that when oxidation of any nutrient was increased, the allocation of that nutrient to non-digestive tissues would be decreased. Our hypothesis of distinct patterns underlying dietary restriction and ovariectomy was supported, but most the specifics of oxidation and allocation were different than we predicted. 2. Materials and methods 2.1. Gas exchange experiments Animal rearing and experimental design - Eastern lubber grasshoppers (Romalea microptera) were obtained as juveniles from Miami, FL, USA and reared to adulthood in the lab. Once individuals molted to adults they were isolated and reared in individual 500 ml containers at 35 °C (light), 27 °C (dark) with 14 h of light and 10 h of darkness and at 50% humidity. New adult females were serially assigned into four treatment groups: Sham-operated & full diet, Ovariectomized (OVX) & full diet, Sham-operated & dietary restricted, and OVX & dietary restricted. Surgeries were performed as previously described (Hatle et al., 2003; Hatle et al., 2013). The Sham-full diet and the OVXfull diet groups were fed fresh Romaine lettuce ad libitum daily, and their feeding rates were measured each week (Hatle et al., 2013). As we have shown previously, ovariectomy reduced feeding rate (Drewry et al., 2011; Tetlak et al., 2015). Therefore, we experimentally set the daily meal sizes for the Sham-dietary restricted group equal to the

average daily feeding rate of the OVX-full diet group (see Fig. S1). This removes feeding rate as a potential confounding variable when comparing these two groups. Instead, the Sham-dietary restricted group differed from the OVX-full diet group only in reproduction. After the animals were 40 d old, this dietary restriction treatment resulted in meals typically between 2.0 and 2.5 g daily. The OVX-dietary restricted group was fed 70% of the amount consumed by the OVX-full diet group. Rates of oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured in old adults, at a median adult age of 112 d (range 85–130 d). For context, the median lifespan in another recent experiment with similar rearing conditions was 147.5 d (Tetlak et al., 2015). The time of day at which the animals were fed was standardized two weeks prior to the day of respirometry testing, and the respirometry readings for all individuals in the study were taken at about the same time within the photophase, to control for any circadian rhythm in metabolic rates. Each individual was tested 1 h prior to feeding, and then 1, 5, and 8 h after feeding. Each measurement included a 20 min acclimation period in the test chamber, and then gas exchange was monitored for 40 min. A few weeks after respirometry, the animals were tested for feeding rates over a 1 h period. That same day, each individual was dissected and its hemolymph volume and fat body mass were measured. Similar to previous studies (Hatle et al., 2013), ovariectomy approximately tripled fat body mass and hemolymph volume (both P b 0.0001), while dietary restriction reduced fat body mass ~50% (P = 0.0167). 2.2. Respirometry procedures We first examined gas exchange, with a focus on whether there were clearly identifiable patterns relative to the time of daily feeding in metabolic rate, lipid oxidation, or carbohydrate oxidation. Openflow respirometry was performed using a FoxBox Respirometry System (Sable Systems International [SSI], North Las Vegas, NV, USA) consisting of an infrared CO2 analyzer (CA-10, SSI) and an O2 fuel cell (SSI). Instruments were zeroed and spanned using dry nitrogen and a calibrated span gas (500 ppm CO2, balance nitrogen; Prax-Air) or room air (20.95% O2) before beginning the experiments. Flow rates of 100 ml/min of dry, CO2-free air, generated by using a DrieriteAscarite-Drierite column, were maintained using the mass-flow valve in the FoxBox. Readings were baselined to a 10-min recording taken on an empty chamber at the beginning and end of each run, and data were acquired at 1 Hz frequency by a SSI UI-2 analog-digital interface driven by Expedata software (SSI). Temperature was controlled by keeping the grasshoppers in an environmental chamber set at 35 °C. Metabolic rates (in ml/min) were estimated as O2 consumption (ml O2/min) divided by femur length in mm to adjust for body size (McCue et al., 2016b). Respiratory Exchange Ratios (RERs) were calculated as the ratio of the rate of CO2 produced to the rate of O2 consumed. All repeated-measures data were analyzed using two-way MANOVA with time as a dependent variable. 2.3. Nutrient specific oxidation and allocation The RER provides an unambiguous indication of fuel substrate when only carbohydrates are metabolized (RER = 1.0) or when only lipids are metabolized (RER = 0.7). All intermediate values are either a combination of the two or indicate protein metabolism (RER = 0.8). Therefore, we measured oxidation of amino acid (leucine), one lipid (oleic acid), and one carbohydrate (glucose). 2.4. Animal rearing and experimental design Eastern lubber grasshoppers were collected as juveniles from Jacksonville, FL, USA and reared to adulthood in the lab. Adult rearing conditions, the assignment to treatment groups, surgeries, and feeding regimens were identical to the gas exchange experiments.

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2.5. Procedures for nutrient-specific oxidation Grasshoppers were tested for nutrient-specific oxidation at a median age of 60 d (range 50–64 d). This equates to middle age for lubber grasshoppers, and middle age has been shown to be a critical period for determining lifespan in humans (Levine et al., 2014). On test days, prior to any exposure to 13C-labeled tracers, a baseline breath sample was collected from each grasshopper. To collect the breath, the grasshopper was placed in a 50-ml gas tight syringe for ~ 30 min, then 20 ml of the gas in the syringe was removed and transferred to a 12 ml, evacuated Exetainer vial (Labco Limited, High Wycombe, Bucks, UK). This volume and duration consistently allowed accumulation of CO2 to at least 2% but b 5%. This range of concentrations permitted accurate measurement of 13C, but was not high enough to induce hypercapnia (Welch et al., 2016). Lubber grasshoppers are docile (Hatle and Faragher, 1998), and there were no problems with animals becoming hyperactive in the syringes. To apply the dose of 13C tracer, each grasshopper was fed one-half a dietary restriction meal, then force fed with a known amount of a specific nutrient labeled with 13C, and then fed the remainder of its daily meal. The amount of 13C-labeled CO2 in the breath of the grasshoppers was used to calculate the rates of nutrient oxidation. Force feeding was accomplished by drawing the sample into a 10 μl or gel loading pipette tip, gently pressing the pipette tip on the mandibles, and when the mandibles and labrum opened slowly ejecting the sample into the oral cavity. Visual inspection ensured that the entire sample was ingested. The three tracers were delivered as: 13C-1-leucine, 0.45 mg in 30 μl water (30% of the leucine in a typical DR meal); 13C-1-oleic acid, 1.0 mg as 1.1 μl liquid (11-fold of the oleic acid oil in a typical DR meal); 13C-1-glucose, 0.5 mg in 5 μl water (6% of the glucose in a typical DR meal). All isotopes were purchased from Isotec (Sigma-Aldrich, St. Louis, MO, USA). After tracer ingestion, breath samples were collected every hour for the subsequent 11 h, and then again at 24 h (with the 24 h samples omitted from the figures for greater resolution). The measurement of 13 C in the exhaled CO2 and the calculation of the rate of tracer oxidation was done as previously described (McCue et al., 2016a; McCue et al., 2015). All statistical tests of oxidation rates included the samples from 1 h after to 24 h after tracer ingestion, with the time zero baseline recordings dropped because they lacked any noticeable differences. All repeated-measures data were analyzed using two-way MANOVA with time as a dependent variable. 2.6. Allocation of nutrients The tissues from the same individuals tested for nutrient-specific oxidation were dissected out 24 h later to measure allocation of the isotope to specific tissues. The femur muscle, mandibular muscle, and the total visceral fat body were excised and stored frozen to measure nutrient allocation. In addition, the gut was cut open longitudinally, scraped clean, and the mass of the empty gut and the mass of the contents of the gut were measured (see Fig. S2). The δ13C of each sample was measured using a Picarro (Sunnyvale, CA, USA) G2121-i Cavity Ring-Down Spectroscopy δ13C stable isotope analyzer with an A0502 ambient CO2 interface paired with an A0201 Combustion Module (Picarro) and an A0301 gas interface (CM-CRDS). Measures of allocation using stable isotopes must account for discrimination, which is an underlying bias for a particular isotope in the biochemical reactions, leading to a biased accumulation of one isotope in the tissue (del Rio et al., 2009). A well-known example of discrimination is that 14N is preferentially excreted over 15N in many animals, so 15 N accumulates in animal tissues over time. To adjust for discrimination of 13C due to dietary restriction or ovariectomy in the present experiment, we used 13C levels in animals that had not been fed tracer (from previous projects). The mean 13C level in the tissue of the experimental group (e.g., on dietary restriction, or ovariectomized) was

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subtracted from the mean 13C level in the tissue of the control group (i.e., fully fed and not ovariectomized). Discrimination due to dietary restriction was accounted for using data from a previous project (Heck et al., 2017); we did not report 13C levels in that paper, but they were measured at the time of the study, and we now use those 13C measures for calculations of discrimination upon dietary restriction in the present paper. Discrimination due to ovariectomy was accounted for using data from (Judd et al., 2011). Both projects used middle-aged female lubber grasshoppers. For dietary restriction and for ovariectomy, muscle showed 13C discrimination b 0.1‰. For fat body, the discrimination was −0.54‰ for dietary restriction and +0.71‰ for ovariectomy (Table 1). That is, fat bodies from animals on dietary restriction had slightly higher levels of 13C than did animals on full diets, while fat bodies from ovariectomized animals had slightly lower levels of 13C than did unoperated animals. In all cases, the degree of discrimination was subtracted from the 13C value for the tissue before statistical analysis and reporting. Any tissues that showed a statistical difference due only to the discrimination factor were not considered significant. All allocation data were tested as separate ANOVAs for each tissue, due to incomplete sets of all samples for several individuals. 3. Results 3.1. Feeding rates To identify differences in consumption rates, we measured the amount of a 2.0 g meal that was eaten during the first 1 h period of daily feeding (n = 33). This experiment used the same animals as the gas exchange experiments, and it was conducted on the day of dissection for each individual. Overall, feeding rates were strongly affected by treatment (F3,34 = 11.33; P b 0.0001). Dietary restriction significantly increased the amount eaten (P = 0.0007), while ovariectomy significantly decreased the amount eaten (P = 0.0006). The interaction term was not quite significant (P = 0.0762). The amounts eaten were: Sham-operated & full diet group = 1.01 ± 0.14 g (mean ± SE); OVX & full diet group = 0.72 ± 0.16 g; Sham-operated & dietary restriction group = 1.81 ± 0.13 g; OVX & dietary restriction group = 1.00 ± 0.14 g. These results show that our dietary restriction treatment increased short-term consumption, and our ovariectomy treatment decreased consumption. 3.2. Gas exchange experiments Lubber grasshoppers showed little specific dynamic action response (Secor, 2009); that is, there were no increases in metabolic rate upon eating a meal. Relative to Sham-operated & full diet grasshoppers, metabolic rate was generally reduced by either ovariectomy or dietary restriction, both before and after the daily feeding. This trend was not consistently significant through the duration of the experimental day (Pillai's Trace F3,19 = 2.31; P = 0.1092; Fig. 1A). The strongest effect was 1 h after feeding (F3,24 = 7.52; Poverall = 0.0013), with a significant

Table 1 Discrimination was determined using control grasshoppers that were not fed tracers in previous studies (Heck et al. 2017 for dietary restriction, and Judd et al., 2011 for ovariectomy). The discrimination from the control was determined by first taking the average 13C level in the tissue of control animals (fully fed and not ovariectomized), and then taking the average of the 13C level in the same tissue for experimental animals (either dietary restricted or ovariectomized). The discrimination from control was equal to the 13C level for control animals minus the 13C level for the experimental animals. Dietary restriction

femur muscle mandibular muscle fat body

Discrimination from control (‰) −0.091 −0.010 −0.542

Ovariectomy n 30 32 29

Discrimination from control (‰) 0.037 N/A 0.710

n 14 13

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effect of diet (Pdiet = 0.0251) and a significant interaction of diet and surgery (Pinteraction = 0.0010). Respiratory exchange ratios (RER) can indicate the oxidative substrate used by the animal. Values close to 1.0 indicate exclusive carbohydrate metabolism, values close to 0.7 indicate exclusive lipid metabolism, and intermediate values may be either protein metabolism (RER = 0.83) or a mixture of substrates (Flatt, 1995). Respiratory exchange ratios did not differ among treatment groups immediately after feeding, but by 8 h post-feeding RER was lower in OVX compared to Sham-operated grasshoppers (F3,24 = 2.90; Poverall = 0.0589; Psurgery = 0.0190; Fig. 1B). Overall, RERs were surprisingly low a few hours after feeding on lettuce (which has two-thirds of its calories as carbohydrates), suggesting lipids are the primary fuel even a few hours after a high carbohydrate meal. 3.3. Leucine oxidation and allocation Alterations in leucine oxidation and allocation were the clearest and most important effects identified in this study. Oxidation was tested by force-feeding a single 13C–labeled nutrient at the daily meal time. Dietary restriction increased leucine oxidation at 2 h (F3,18 = 2.26; Poverall = 0.1237; Pdiet = 0.0218; Fig. 2) and 3 h (F3,18 = 2.55; Poverall = 0.0944; Pdiet = 0.0197) after tracer ingestion. Ovariectomy increased

leucine oxidation at 7 h (F3,18 = 4.51; Poverall = 0.0191; Psurgery = 0.0030), 8 h (F3,18 = 3.94; Poverall = 0.0294; Psurgery = 0.0084), and 9 h (F3,18 = 3.36; Poverall = 0.0472; Psurgery = 0.0111) after tracer ingestion. At 24 h after tracer ingestion (e.g., samples collected during the 30 min from 23.5 to 24 h), leucine oxidation was higher in ovariectomized grasshoppers (F3,18 = 3.36; Poverall = 0.0472; Psurgery = 0.0078). Neither diet nor surgery affected leucine oxidation at any other collection period. In general, for each individual, the leucine oxidation increased over the first few hours after feeding and then gradually returned toward pre-feeding values. This creates a distinct peak in oxidation for each individual. Hence, we compared the times of peak oxidation (i.e., the maximum for each individual) across groups, as well as the levels of these peaks. The trend was for dietary restriction to slightly increase the peak rate of leucine oxidation (F3,18 = 1.60; Poverall = 0.2312; Pdiet = 0.0462). No other treatment effects for the maximum rates of leucine oxidation were observed. Allocation of specific nutrients to the skeletal muscle or fat body was tested 24 h after tracer ingestion. All measures were corrected for any discrimination of 13C due to dietary restriction or ovariectomy (see Materials and methods and Table 1). Allocation of leucine to the femur muscle was increased by dietary restriction (F3,10 = 8.49; Poverall = 0.0099; Pdiet = 0.0058; Fig. 3) but decreased by ovariectomy (Psurgery = 0.0202). Somewhat similarly, leucine allocation to mandibular muscle (n = 18) tended to increase upon dietary restriction (F3,17 = 2.76; Poverall = 0.0816; Pdiet = 0.0360), but was not affected by ovariectomy (Psurgery = 0.6395). Allocation of leucine to fat body (n = 20) was not affected by any treatment (all P N 0.20). 3.4. Oleic acid oxidation Our experiments with oleic acid were hampered by the failure of several animals to absorb the large bolus of lipid. All individuals (n = 13) with high levels of 13C from oleic acid in the feces for the 24 h after force feeding were omitted from the study. We set the threshold for omission at 13C ≥ 0‰. For comparison, lettuce 13C is −28‰. Ovariectomy decreased oleic acid oxidation at 1 h (F3,11 = 16.24; Poverall = 0.0105; Psurgery = 0.0027), 4 h (F3,11 = 9.18; Poverall = 0.0288; Psurgery = 0.0147), and 8 h (F3,11 = 10.48; Poverall = 0.0230; Psurgery = 0.0075) after tracer ingestion (Fig. 4). Over the first 11 h of the experiment, rates of oleic acid oxidation remained relatively constant, in contrast to leucine. Because the maximum rate and time of oleic acid oxidation for each individual was not distinct, we did not test for statistical effects on the peak of oxidation. Data on allocation of oleic acid was preliminary (Fig. S3). The nonsignificant trend was for allocation to mandibular muscle to be reduced by ovariectomy.

Fig. 1. A) Metabolic rates for female lubber grasshoppers (total n = 25). Each individual was tested for 40 min four times in one day: once before feeding and three times after feeding. Metabolic rate was higher in sham-full diet grasshoppers only at 1 h postfeeding. B) Respiratory exchange ratios measured simultaneously to the metabolic rates. Use of b/= indicates a non-significant overall effect but a significant main effect of surgery. Respiratory exchange ratios of 1.0 can indicate exclusive carbohydrate metabolism, ratios of 0.7 can indicate exclusive lipid metabolism, and ratios between 0.7 and 1.0 indicate either protein metabolism or a mix of fuels. For both panels, data markers are staggered for clarity and show LSmeans ± SE.

Fig. 2. Oxidation of 13C-1-leucine, as measured by the appearance of 13C in exhaled CO2, in female lubber grasshoppers (total n = 19). Ovariectomy also increased oxidation of leucine at 24 h after tracer ingestion (data not shown). This was the strongest effect on nutrient-specific metabolism in our experiments (R2 = 0.47 at 7 h, 0.44 at 8 h, and 0.43 at 24 h). Data markers are staggered for clarity and show LSmeans ± SE. Use of N/= indicates a non-significant overall effect but a significant main effect of diet level.

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Fig. 3. Allocation of leucine to femur muscle (n = 11), mandibular muscle (n = 18), and fat body (n = 20), in female lubber grasshoppers. Each individual was fed 0.45 mg of 13 C-1-leucine, and then 24 h later was dissected to harvest tissues. All values are corrected for isotopic discrimination (see Table 1). Higher (i.e., less negative) values indicate greater nutrient allocation. Data sets are LSmeans ± SE; in some cases the error bars are smaller than data marker. Use of b/= indicates a non-significant overall effect but a significant main effect of diet. Dietary restriction increased allocation to muscle, while ovariectomy decreased allocation to femur muscle.

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Fig. 5. Oxidation of 13C-1-glucose, as measured by the appearance of 13C in exhaled CO2, in female lubber grasshoppers (total n = 31). Ovariectomy marginally decreased the oxidation of glucose at 1 h after tracer ingestion and increased the time until the maximum rate of oxidation. Data markers are staggered for clarity and show LSmeans ± SE.

glucose to fat body was similar to the magnitude of the discrimination correction, so the allocation was not considered significant. 4. Discussion

3.5. Glucose oxidation and allocation We previously determined that dietary restriction had little or no effect on glucose oxidation (Fig. S4). Therefore, we omitted the diet treatment for the main study, which facilitated increasing the sample sizes for studying the effects of ovariectomy. Ovariectomy tended to decrease the rate of glucose oxidation 1 h after tracer ingestion (F1,30 = 3.79; P = 0.0612; Fig. 5). No other time point showed a difference in glucose oxidation (all P N 0.10). For the maximum level (i.e., peak) of oxidation of glucose, there was no significant effect between treatments (t-test; P = 0.145). The data set on the time at the maximum rate of oxidation (i.e., peak time) of glucose had one significant outlier (Grubb's test; Z = 3.43, which is greater than the critical value of 2.59). After removing this outlier, the time at the maximum rate of oxidation of glucose was 1 h later in ovariectomized grasshoppers than in sham-operated controls (t-test; P = 0.0425); a similar result was obtained when the outlier was retained and the data was tested with the non-parametric MannWhitney U test (U-value = 64.5, which is less than the critical value of 70; P = 0.030). Finally, allocation of glucose to femur muscle was decreased by ovariectomy (t-test; P = 0.0241; Fig. 6). The increase in allocation of

Fig. 4. Oxidation of 13C-1-oleic acid, as measured by the appearance of 13C in exhaled CO2, in female lubber grasshoppers (total n = 8). Ovariectomy significantly decreased oxidation at 1, 4, and 8 h. Data markers are staggered for clarity and show LSmeans ± SE.

This study suggests that the regulation of oxidation of leucine may be an underappreciated mechanism underlying lifespan extension. To our knowledge, this study includes the first direct measures of organismal oxidation of specific nutrients upon life-extending dietary restriction or reduced reproduction. Ovariectomy and dietary restriction each increased organismal oxidation of leucine. While some work in C. elegans suggests reduced oxidation of branched-chain amino acids can increase lifespan (Mansfeld et al., 2015), our study found increased oxidation of leucine was associated with longevity. Our study is consistent with work showing that supplemental dietary branch-chain amino acids improve metabolic health in mice (Fontana et al., 2016). The increases in oxidation of leucine, upon dietary restriction and especially upon ovariectomy, should decrease the amount of leucine available to many tissues in the animal (but this was not true for muscle upon dietary restriction, see paragraph 5 of this Discussion). Provided there is less leucine available to other non-muscle tissues, this alteration of metabolism may contribute to longevity. Leucine is especially potent

Fig. 6. Allocation of dietary glucose to femur muscle, mandibular muscle, and fat body (n = 26 for all), in female lubber grasshoppers. Each individual was fed 0.45 mg of 13C-1glucose, and then 24 h later was dissected to harvest tissues. All values are corrected for isotopic discrimination (see Table 1). Data sets are LSmeans ± SE; in some cases the error bars are obscured by the data marker. Ovariectomy decreased allocation of glucose to femur muscle.

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at stimulating the TOR pathway (Edwards et al., 2015; Wolfson et al., 2016). Removing leucine quickly by oxidizing it could attenuate TOR signaling, leading to multiple salubrious effects (Edwards et al., 2015). Further studies on oxidation of branched-chain amino acids are warranted, especially if they link this oxidation to TOR activity, autophagy, or other beneficial mechanisms. Ovariectomy increased leucine oxidation, and this was the strongest effect on nutrient-specific metabolism in our experiments. Because the increase in leucine oxidation did not occur immediately after feeding, we interpret it as a steady-state response to ovariectomy that is obscured by other fuel use shortly after a meal. Our best guess is that increased oxidation at 7–8 h represents the period after which leucine has been absorbed into cells but before the leucine has been incorporated into cellular proteins. The long-term increase in oxidation (i.e., at 24 h) may be because leucine is not used for egg production in ovariectomized animals. Crickets also oxidize greater proportions of protein when not actively reproducing (Zera and Zhao, 2006). Dietary restriction does not increase short-term oxidation of all ingested nutrients. Specifically, while oxidation of leucine was increased, oxidation of oleic acid and glucose was unaffected. Our prediction was that there would be an energy deficit incurred upon dietary restriction that would result in increased oxidation of all nutrients. It may be that dietary restriction is not creating a great energy deficit, perhaps because reproduction is reduced. Last and most important, we observed that oxidation of only leucine and not glucose or oleic acid is altered by dietary restriction. This implies that the changes in leucine oxidation are regulated in animals as part of life-extending interventions, and that these changes in leucine oxidation are not simply stoichiometric. 4.1. Nutrient allocation Nutrient allocation upon dietary restriction is consistent with supporting muscle maintenance, perhaps for locomotion, while allocation upon ovariectomy does not. Dietary restriction generally increased allocation of leucine to skeletal muscle. One of the classic effects of dietary restriction is an increase in locomotor activity (Katewa et al., 2012), presumably to seek out new food sources. Increased locomotion would be facilitated by robust femur muscle. Further, taking advantage of some types of food once found, especially sub-optimal food sources, would require robust mandibular muscle (Ibanez et al., 2013). The increased allocation of leucine to femur and mandibular muscles seen in this study is consistent with this scenario of preparing to move and consume food during a prolonged dietary restriction. This may be particularly true for leucine because it promotes muscle maintenance (Pereira et al., 2015). Ovariectomy reduced allocation of all tested nutrients to femur muscle. Ovariectomy in grasshoppers results in massive increases in storage (Hatle et al., 2013), and also increased gut size (Fig. S2). These increases in storage and organ size previously had been attributed to the reduction in investment in reproduction (Hansen et al., 2013). The consistent decrease in allocation of ingested nutrients to the femur muscle suggests reduced reproduction may also cause a reduced investment in muscle maintenance, perhaps especially for locomotory muscle. This reduced investment in femur muscle is the opposite of the increased investment in muscle upon dietary restriction, and this difference creates the clearest contrast between ovariectomy and dietary restriction in this study. 4.2. Confidence in allocation measures The actual values underlying the allocation measures in this study (for both ovariectomy and dietary restriction) were low, given that the δ13C for Romaine lettuce is −28‰ (Judd et al., 2010). This suggests that little 13C accumulated in the tissues during the 24 h of the study. At first glance, the magnitude of the differences in δ13C initially may appear small (e.g., δ13C ~ 0.4 decrease in leucine allocation upon ovariectomy).

However, our experience with tissue 13C measures (Heck et al., 2017; Judd et al., 2011; Wessels et al., 2011) indicates they are typically small, highly precise, and very consistent across individuals. Indeed, our past studies showed clearly detectable differences with δ13C b 1.0. Most important, all 13C measures reported here are properly adjusted for discrimination, so they represent allocation, not just accumulation. Further, the change in 13C levels is at least 4-fold greater than the discrimination adjustment, establishing that these conclusions are not based only on discrimination. Taken together, these details increase our confidence in our estimates of nutrient allocation in this study overall. This study includes a comparison of matched-fed reproductive and non-reproductive grasshoppers (i.e., Sham-operated & dietary restricted vs. OVX & full diet). We have previously demonstrated that two different means of reducing reproduction (viz., ovariectomy or RNAi against vitellogenin) reduce feeding (Drewry et al., 2011; Tetlak et al., 2015). Studies in C. elegans on the mechanisms by which reduced reproduction increases lifespan have not included a control for any potential change in feeding. Our results indicate that life-extending reductions in reproduction, independent of daily feeding, can affect aspects of metabolism. Sham-operated & dietary restricted grasshoppers oxidized less leucine (at 7–8 h after ingestion) and allocated more to leucine to femur muscle, in comparison to OVX & full diet grasshoppers. 4.3. Differences from studies in other aging models We predicted, based on a study in mice (Bruss et al., 2010), that dietary restriction would result in temporal changes in fuel use relative to the timing of feeding. However, we saw no patterns in RERs relative to feeding time in grasshoppers on dietary restriction. This is despite the fact that we trained grasshoppers to feed at a specific time of day, and they completed their meals rapidly. Our nutrient-specific oxidation experiments also failed to show any temporal changes in fuel use. In retrospect, the lack of temporal pattern in RERs may be due in part to changes in protein oxidation after meal ingestion. Oxidation of pure protein results in an RER of ~ 0.83 (Flatt, 1995). The substantial and changing oxidation of leucine we observed after feeding would obscure clear patterns in carbohydrate or lipid use that could be observed via RER. Extensive work with germ-line ablated C. elegans implicates a faster turnover of lipids as a contributing factor to life-extension in non-reproductive worms (Amrit et al., 2016; Wang et al., 2008). Surprising to us, in grasshoppers ovariectomy reduced oxidation of recently ingested oleic acid. Because the force-fed bolus of lipid in our experiments was not absorbed satisfactorily by several individuals, our study on oleic acid suffers from a low sample size. It was recently shown that another fatty acid, palmitic acid, in nectar needs to be emulsified to enable uptake in hawkmoths (Levin et al., 2017). Nonetheless, our study suggests that lipid turnover could be an important difference across species. Near as we can tell, this indicates a need for further examination of the relationship between life-extending reduced reproduction and lipid turnover across additional model species. Acknowledgements We thank Andre Zaharchenya for collecting breath samples and members of the Hatle lab for advice. Thanks to Peter Magyari for helpful discussions about leucine. Supported by NIH awards 2R15AG02851202A1 and 1R15AG050218-01A1 to JDH, NSF awards IOS-1051890 and IOS 1257298 to DAH, NSF award IOS 1558159 to CMW, and NSF award IOS-1053318 to GD. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.exger.2017.06.019.

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