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Purpose: To evaluate the potential of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect.Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized [1-13C]-pyruvate MRSI after systemic delivery of [1-13C] pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 μg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed in vitro. LDH activity was assessed in tumor homogenates.Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized [1-13C]-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11 in vitro. LDH activity correlated to FX-11 sensitivity.Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect. Clin Cancer Res; 24(13); 3137–48. ©2018 AACR.

Mice bred in 2017 and entered into the C2017 cohort were tested for possible lifespan benefits of (R/S)-1,3-butanediol (BD), captopril (Capt), leucine (Leu), the Nrf2-activating botanical mixture PB125, sulindac, syringaresinol, or the combination of rapamycin and acarbose started at 9 or 16 months of age (RaAc9, RaAc16). In male mice, the combination of Rapa and Aca started at 9 months and led to a longer lifespan than in either of the two prior cohorts of mice treated with Rapa only, suggesting that this drug combination was more potent than either of its components used alone. In females, lifespan in mice receiving both drugs was neither higher nor lower than that seen previously in Rapa only, perhaps reflecting the limited survival benefits seen in prior cohorts of females receiving Aca alone. Capt led to a significant, though small (4% or 5%), increase in female lifespan. Capt also showed some possible benefits in male mice, but the interpretation was complicated by the unusually low survival of controls at one of the three test sites. BD seemed to produce a small (2%) increase in females, but only if the analysis included data from the site with unusually short-lived controls. None of the other 4 tested agents led to any lifespan benefit. The C2017 ITP dataset shows that combinations of anti-aging drugs may have effects that surpass the benefits produced by either drug used alone, and that additional studies of captopril, over a wider range of doses, are likely to be rewarding.

Dietary restriction (DR) extends lifespan in many animal models. In mice, a range of factors affects the magnitude of the pro-longevity effect of DR, including the strain, restriction level, sex, and age of treatment onset. Early onset DR dramatically prolongs lifespan, whereas DR started later in life has diminished or no significant effect. The lifespan responses of males and females to DR also differ. Whether there is a sex difference in the lifespan response to the age of onset of DR has not been examined. Probing the basis for the variable response to DR can help elucidate the underlying mechanisms that mediate lifespan extension. We diet restricted UM-HET3 mice of both sexes from either 5 months (early-onset) or 20 months (late-onset) of age and monitored their survival. Early onset DR significantly prolonged lifespan in both males and females, which increased median lifespans by 54% and 18.5%, respectively. By contrast, although late onset DR prolonged lifespans in both sexes, the magnitude of increase in males (1.4%) was significantly lower than in females (15.8%, p=0.0474). Moreover, the decrease in the magnitude of the response to late-onset DR compared to that of early-onset DR is more dramatic in males than females. Further analysis found a significant interaction (p=0.0001) between early and late-onset DR in males but not in females (p=0.3818). Together, these results indicate that the lifespan response to delayed onset of DR diminishes more in males than in females, providing another model to probe the underlying mechanisms of DR.

Canagliflozin (Cana) is an inhibitor of the sodium glucose transporter 2 (SGLT2), and is thought to act by blocking renal reuptake and intestinal absorption of glucose. Cana is FDA-approved for treatment of diabetes, and affords protection from cardiovascular and kidney diseases. In the context of the mouse Interventions Testing Program, genetically heterogeneous mice were given chow containing 180 ppm Cana at 7 months of age until their death. Cana extended median survival of male mice by 14%, with p < 0.001 by log-rank test. Cana also increased by 9% the age for 90th percentile survival (p < 0.001 by Wang/Allison test), with parallel effects seen at each of three test sites. Cana did not alter the distribution of inferred cause of death, nor of incidental pathology findings at end-of-life necropsies. No benefits were seen in female mice. The lifespan benefit of Cana is likely to reflect blunting of peak glucose levels, because similar longevity effects are seen in mice given acarbose, a diabetes drug that blocks glucose surges through a distinct mechanism, i.e. slowing breakdown of carbohydrate in the intestine. Interventions that control daily peak glucose levels deserve attention as possible preventive medicines to protect from a wide range of late-life neoplastic and degenerative diseases.

Purpose: To evaluate the potential of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect. Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized [1-13C]-pyruvate MRSI after systemic delivery of [1-13C] pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 μg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed in vitro. LDH activity was assessed in tumor homogenates. Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized [1-13C]-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11 in vitro. LDH activity correlated to FX-11 sensitivity. Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect. Clin Cancer Res; 24(13); 3137–48. ©2018 AACR.

Aging is associated with replacement of normal kidney parenchyma by fibrosis. Because hydrogen sulfide (H2S) ameliorates kidney fibrosis in disease models, we examined its status in the aging kidney. In the first study, we examined kidney cortical H2S metabolism and signaling pathways related to synthesis of proteins including matrix proteins in young and old male C57BL/6 mice. In old mice, increase in renal cortical content of matrix protein involved in fibrosis was associated with decreased H2S generation and AMPK activity, and activation of insulin receptor (IR)/IRS-2-Akt-mTORC1-mRNA translation signaling axis that can lead to increase in protein synthesis. In the second study, we randomized 18–19 month-old male C57BL/6 mice to receive 30 μmol/L sodium hydrosulfide (NaHS) in drinking water vs. water alone (control) for 5 months. Administration of NaHS increased plasma free sulfide levels. NaHS inhibited the increase in kidney cortical content of matrix proteins involved in fibrosis and ameliorated glomerulosclerosis. NaHS restored AMPK activity and inhibited activation of IR/IRS-2-Akt-mTORC1-mRNA translation axis. NaHS inhibited age-related increase in kidney cortical content of p21, IL-1β, and IL-6, components of the senescence-associated secretory phenotype. NaHS abolished increase in urinary albumin excretion seen in control mice and reduced serum cystatin C levels suggesting improved glomerular clearance function. We conclude that aging-induced changes in the kidney are associated with H2S deficiency. Administration of H2S ameliorates aging-induced kidney changes probably by inhibiting signaling pathways leading to matrix protein synthesis.

Summary Loss of SURF1, a Complex IV assembly protein, was reported to increase lifespan in mice despite dramatically lower cytochrome oxidase (COX) activity. Consistent with this, our previous studies found advantageous changes in metabolism (reduced adiposity, increased insulin sensitivity, and mitochondrial biogenesis) in Surf1 −/− mice. The lack of deleterious phenotypes in Surf1 −/− mice is contrary to the hypothesis that mitochondrial dysfunction contributes to aging. We found only a modest (nonsignificant) extension of lifespan (7% median, 16% maximum) and no change in healthspan indices in Surf1 −/− vs. Surf1 +/+ mice despite substantial decreases in COX activity (22%–87% across tissues). Dietary restriction (DR) increased median lifespan in both Surf1 +/+ and Surf1 −/− mice (36% and 19%, respectively). We measured gene expression, metabolites, and targeted expression of key metabolic proteins in adipose tissue, liver, and brain in Surf1 +/+ and Surf1 −/− mice. Gene expression was differentially regulated in a tissue‐specific manner. Many proteins and metabolites are downregulated in Surf1 −/− adipose tissue and reversed by DR, while in brain, most metabolites that changed were elevated in Surf1 −/− mice. Finally, mitochondrial unfolded protein response (UPRmt)‐associated proteins were not uniformly altered by age or genotype, suggesting the UPRmt is not a key player in aging or in response to reduced COX activity. While the changes in gene expression and metabolism may represent compensatory responses to mitochondrial stress, the important outcome of this study is that lifespan and healthspan are not compromised in Surf1 −/− mice, suggesting that not all mitochondrial deficiencies are a critical determinant of lifespan.

We examined the effect of rapamycin on the life span of a mouse model of type 2 diabetes, db/db mice. At 4 months of age, male and female C57BLKSJ-lepr (db/db) mice (db/db) were placed on either a control diet, lacking rapamycin or a diet containing rapamycin and maintained on these diets over their life span. Rapamycin was found to reduce the life span of the db/db mice. The median survival of male db/db mice fed the control and rapamycin diets was 349 and 302 days, respectively, and the median survival of female db/db mice fed the control and rapamycin diets was 487 and 411 days, respectively. Adjusting for gender differences, rapamycin increased the mortality risk 1.7-fold in both male and female db/db mice. End-of-life pathological data showed that suppurative inflammation was the main cause of death in the db/db mice, which is enhanced slightly by rapamycin treatment.

The mammalian target of rapamycin (mTOR) plays an important role in cardiac development and function. Inhibition of mTOR by rapamycin has been shown to attenuate pathological cardiac hypertrophy and improve the function of aging heart, accompanied by an inhibition of the cardiac proteasome activity. The current study aimed to determine the potential mechanism(s) by which mTOR inhibition modulates cardiac proteasome. Inhibition of mTOR by rapamycin was found to reduce primarily the immunoproteasome in both H9c2 cells in vitro and mouse heart in vivo, without significant effect on the constitutive proteasome and protein ubiquitination. Concurrent with the reduction of the immunoproteasome, rapamycin reduced two important inflammatory response pathways, the NF-κB and Stat3 signaling. In addition, rapamycin attenuated the induction of the immunoproteasome in H9c2 cells by inflammatory cytokines, including INFγ and TNFα, by suppressing NF-κB signaling. These data indicate that rapamycin indirectly modulated immunoproteasome through the suppression of inflammatory response pathways. Lastly, the role of the immunoproteasome during the development of cardiac hypertrophy was investigated. Administration of a specific inhibitor of the immunoproteasome ONX 0914 attenuated isoproterenol-induced cardiac hypertrophy, suggesting that the immunoproteasome may be involved in the development of cardiac hypertrophy and therefore could be a therapeutic target. In conclusion, rapamycin inhibits the immunoproteasome through its effect on the inflammatory signaling pathways and the immunoproteasome could be a potential therapeutic target for pathological cardiac hypertrophy.

The tuberous sclerosis complex 1/2 (TSC1/2) is an endogenous regulator of the mechanistic target of rapamycin (mTOR). While mTOR has been shown to play an important role in health and aging, the role of TSC1/2 in aging has not been fully investigated. In the current study, a constitutive TSC1 transgenic (Tsc1tg) mouse model was generated and characterized. mTORC1 signaling was reduced in majority of the tissues, except the brain. In contrast, mTORC2 signaling was enhanced in Tsc1tg mice. Tsc1tg mice are more tolerant to exhaustive exercises and less susceptible to isoproterenol-induced cardiac hypertrophy at both young and advanced ages. Tsc1tg mice have less fibrosis and inflammation in aged as well as isoproterenol-challenged heart than age-matched wild type mice. The female Tsc1tg mice exhibit a higher fat to lean mass ratio at advanced ages than age-matched wild type mice. More importantly, the lifespan increased significantly in female Tsc1tg mice, but not in male Tsc1tg mice. Collectively, our data demonstrated that moderate increase of TSC1 expression can enhance overall health, particularly cardiovascular health, and improve survival in a gender-specific manner., Scientific Reports, 7, ISSN:2045-2322

The inhibition of mTOR (mechanistic target of rapamycin) by the macrolide rapamycin has many beneficial effects in mice, including extension of lifespan and reduction or prevention of several age-related diseases. At the same time, chronic rapamycin treatment causes impairments in glucose metabolism including hyperglycemia, glucose intolerance and insulin resistance. It is unknown whether these metabolic effects of rapamycin are permanent or whether they can be alleviated. Here, we confirmed that rapamycin causes glucose intolerance and insulin resistance in both inbred and genetically heterogeneous mice fed either low fat or high fat diets, suggesting that these effects of rapamycin are independent of genetic background. Importantly, we also found that these effects were almost completely lost within a few weeks of cessation of treatment, showing that chronic rapamycin treatment does not induce permanent impairment of glucose metabolism. Somewhat surprisingly, chronic rapamycin also promoted increased accumulation of adipose tissue in high fat fed mice. However, this effect too was lost when rapamycin treatment was ended suggesting that this effect of rapamycin is also not permanent. The reversible nature of rapamycin's alterations of metabolic function suggests that these potentially detrimental side-effects might be managed through alternative dosing strategies or concurrent treatment options.

Atypical Chemokine Receptor 1 (ACKR1), previously known as Duffy Antigen Receptor for Chemokines, stands out among chemokine receptors for high selective expression on cerebellar Purkinje neurons. Although ACKR1 ligands activate Purkinje cells in vitro, evidence for ACKR1 regulation of brain function in vivo is lacking. Here we demonstrate that Ackr1 (-/-) mice have markedly impaired balance and ataxia on a rotating rod and increased tremor when injected with harmaline, which induces whole-body tremor by activating Purkinje cells. Ackr1 (-/-) mice also exhibited impaired exploratory behavior, increased anxiety-like behavior and frequent episodes of marked hypoactivity under low-stress conditions. Surprisingly, Ackr1 (+/-) had similar behavioral abnormalities, indicating pronounced haploinsufficiency. The behavioral phenotype of Ackr1 (-/-) mice was the opposite of mouse models of cerebellar degeneration, and the defects persisted when Ackr1 was deficient only on non-hematopoietic cells. Together, the results suggest that normal motor function and behavior may partly depend on negative regulation of Purkinje cell activity by Ackr1.

Calorie restriction (CR), which lengthens lifespan in many species, is associated with moderate hyperadrenocorticism and attenuated inflammation. Given the anti‐inflammatory action of glucocorticoids, we tested the hypothesis that the hyperadrenocorticism of CR contributes to its attenuated inflammatory response. We used a corticotropin‐releasing‐hormone knockout (CRHKO) mouse, which is glucocorticoid insufficient. There were four controls groups: CRHKO mice and wild‐type (WT) littermates fed either ad libitum (AL) or CR (60% of AL food intake), and three experimental groups: (a) AL‐fed CRHKO mice given corticosterone (CORT) in their drinking water titrated to match the integrated 24‐hr plasma CORT levels of AL‐fed WT mice, (b) CR‐fed CRHKO mice given CORT to match the 24‐hr CORT levels of AL‐fed WT mice, and (c) CR‐fed CHRKO mice given CORT to match the 24‐hr CORT levels of CR‐fed WT mice. Inflammation was measured volumetrically as footpad edema induced by carrageenan injection. As previously observed, CR attenuated footpad edema in WT mice. This attenuation was significantly blocked in CORT‐deficient CR‐fed CRHKO mice. Replacement of CORT in CR‐fed CRHKO mice to the elevated levels observed in CR‐fed WT mice, but not to the levels observed in AL‐fed WT mice, restored the anti‐inflammatory effect of CR. These results indicate that the hyperadrenocorticism of CR contributes to the anti‐inflammatory action of CR, which may in turn contribute to its life‐extending actions.

Interference in insulin and/or insulin-like growth factor 1 (IGF-1) signaling can extend invertebrate life span, and interference in IGF-1 signaling can extend murine life span. Whether interference with murine insulin signaling, which can be diabetogenic and pathological, is also life-extending is controversial. We therefore measured life span in 3 murine strains genetically modified to reduce or increase insulin sensitivity. Mice with reduced insulin sensitivity were hemizygous for a null mutation in the insulin receptor (insulin receptor knockout mice; IRKO(+/-)). Mice with increased insulin sensitivity either had a null mutation of protein tyrosine phosphatase 1B (PTP-1B(-/-)) or overexpressed Peroxisome proliferator-activated receptor-α coactivator (PGC)-1α (PGC-1α(TG)). Life span of insulin insensitive IRKO(+/) mice was increased (males) or unaffected (females). Life spans of mice with increased insulin sensitivity were shortened overall (PTP-1B(-/-) mice) or partially (PGC-1α(TG): survival at the 25th percentile was reduced). These results show that insulin sensitivity in some murine genotypes is inversely related to longevity and provide further evidence for evolutionary conservation of this pathway as a modulator of longevity.

Dietary restriction (DR), one of the most robust life-extending manipulations, is usually associated with reduced adiposity. This reduction is hypothesized to be important in the life-extending effect of DR, because excess adiposity is associated with metabolic and age-related disease. Previously, we described remarkable variation in the lifespan response of 41 recombinant inbred strains of mice to DR, ranging from life extension to life shortening. Here, we used this variation to determine the relationship of lifespan modulation under DR to fat loss. Across strains, DR life extension correlated inversely with fat reduction, measured at midlife (males, r= -0.41, P

Purpose: Non-invasive magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized (HP) 13C-labeled pyruvate and its metabolite lactate is being used to monitor the metabolic flux in solid tumors. In this study, we evaluate the potential of MRSI of HP [1-13C]-pyruvate and [1-13C]-lactate, in prostate cancer as a predictive biomarker for targeting lactate dehydrogenase. Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured, after intravenous delivery of hyperpolarized [1-13C] pyruvic acid, by MRSI. Steady state metabolomic analysis of xenograft tumors was performed by mass spectrometry and steady state lactate levels were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 µg/mouse/day for 5 days x 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates and oxygen consumption of the prostate cancer cell lines was analyzed in vitro. Protein levels of glycolysis regulators were assessed with immunoblotting. Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with HP [1-13C] MRSI relative to PC3 tumors (21% higher 13C-lactate/pyruvate p Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer xenografts predicted the efficacy of targeting LDH when steady state markers of glycolysis, in vivo and in vitro, did not. Citation Format: Bradley T. Scroggins, Masayuki Matsuo, Ayla O. White, Keita Saito, Jeeva P. Munasinghe, Carole Sourbier, Kazutoshi Yamamoto, Vivian Diaz, James B. Mitchell, Murali C. Krishna, Deborah E. Citrin. Hyperpolarized [1-13C]-pyruvate/lactate magnetic resonance spectroscopic imaging of prostate cancer in vivo predicts response to lactate dehydrogenase inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4106.

This study examined the effect of housing density on the longevity-extending and disease-delaying actions of calorie restriction (CR). Singly or multiply housed (four per cage) mice were either fed ad libitum (AL) or were on CR beginning at 2 months. All CR mice were fed 40% less food than were multiply housed AL mice. CR increased median longevity by 19%, and housing density had no effect on this increase. CR also reduced neoplastic lesions in both housing groups, but lymphoma, the most common neoplasm, was reduced more in singly than in multiply housed mice. Singly housed AL mice ate 40% more food than did multiply housed AL mice, but weighed the same and lived as long as multiply housed AL mice. These results indicate that CR can extend life span as effectively in multiply as in singly housed mice, even though housing density can differentially affect the cancer-reducing effect of CR.

Rapamycin was found to increase (11% to 16%) the lifespan of male and female C57BL/6J mice most likely by reducing the increase in the hazard for mortality (i.e., the rate of aging) term in the Gompertz mortality analysis. To identify the pathways that could be responsible for rapamycin's longevity effect, we analyzed the transcriptome of liver from 25-month-old male and female mice fed rapamycin starting at 4 months of age. Few changes (4,500) changed significantly in females. Using multidimensional scaling and heatmap analyses, the male mice fed rapamycin were found to segregate into two groups: one group that is almost identical to control males (Rapa-1) and a second group (Rapa-2) that shows a change in gene expression (>4,000 transcripts) with more than 60% of the genes shared with female mice fed Rapa. Using ingenuity pathway analysis, 13 pathways were significantly altered in both Rapa-2 males and rapamycin-fed females with mitochondrial function as the most significantly changed pathway. Our findings show that rapamycin has a major effect on the transcriptome and point to several pathways that would likely impact the longevity.

Department of Integrative Physiology, University of Colorado,Boulder, CO 80309, USAOur recent report (Liao et al., 2010) that dietary restriction(DR) shortened lifespan in many strains of recombinant inbred(RI) mice was unexpected in the context of a long history ofresearch on the life-extending action of DR (Weindruch W Masoro, 2005). It is important to recognize,however,thatourreportisnotthe first(see Liaoet al.,2010,fora number of earlier reports in which DR had no effect onlifespan, or even led to shorter lifespan). Although we hadhypothesized that there would be strain variation in the extentof lifespan extension by DR, we too had overlooked dissonantdata, which in retrospect would have led us to expand ourhypothesis to include possible negative effects of DR. Thegenetic variation we have found in the response to DR, particu-larly given its magnitude, provides a valuable tool for elucidatingunderlying genes and pathways that mediate the lifespan-mod-ulatingactionofDR–theultimategoalofresearch inthisarea.We are pleased to respond to the commentary by Mattson(2010) and the opportunity it offers to provide additional infor-mation bearing on mechanisms by which DR can shorten as wellas lengthen lifespan. The commentary offers some interestingsuggestions about these mechanisms. Here, we provide datathat largely refute these suggestions. We also respond to othercomments and speculations raised in the commentary thatdeserveclarification.The author’s primary explanation for the lifespan shorteningby DR of some strains is based on postulated effects of multiplehousing. The author assumes that multiple housing results incompetition for limited food, thereby leading to dominancebehaviour and unequal distribution of food among the individu-als. The commentary speculates that the dominant mousewouldeat adisproportionate shareof the food,andthus receiveless than the normal 40% restriction, and that subordinate micein the cage would be restricted more than 40% leading to earlydeath, i.e., shortened lifespan in relation to ad libitum (AL) fedcontrols of the same strain. Our published data, as well as newdata presented here, do not support this idea. First, exclusionfrom the analysis of early deaths, which in Mattson’s modelwould represent subordinate mice with exceptionally low foodintake, has little impact on the frequency or distribution of RIstrains with shortened lifespan (supporting Fig. S3, Liao et al.,2010). Second, taking body weight as an indicator of feedingdominance, there is no evidence that such dominance led toshortened life of subordinates. Inmales, arguedby theauthor tobe more prone to feeding dominance, mean lifespan of themost subordinate (i.e. smallest) mouse was no shorter than thatof the most dominant (i.e., largest) mouse, and the same is truefor females, both for the strains that had significantly shortenedlifespans under DR (Fig. 1), as well as for strains with shortenedlifespans that did not reach statistical significance (data notshown). Finally, although we were unable to measure the foodconsumption of individual mice housed multiply, using bodyweight as a measure of food consumption provides little evi-dence formarkedinequality offood intakeamongcagemates.Iffood were unequally distributed among cagemates because ofcompetition, the range and variance of body weight in DR cageswould be greater than that in AL fed cages. Neither range (datanot shown) nor coefficient of variation of body weight was sig-nificantly greater in the DR than in the AL cages (Table 1). Thesefindings are consistent with our behavioural observations of themice during feeding in this study as well as previously (Ikenoet al., 2005). Although we are in agreement with the commen-tary that DR mice consume virtually all of their allotment within1–2 h of feeding, we see no fighting during this period. Whenfood is available, each DR mouse spends its time obtainingpellets of food from the hopper and consuming them. In somecases, a mouse will take the pellet from another mouse butthat mouse will obtain another pellet from the hopper andboth will have food to eat (C.-Y. Liao, V. Diaz, J. F. Nelson;unpublished observations). The food hoppers are large enoughtoallowallmicetoaccessfoodsimultaneously.The most compelling evidence that competition for food doesnot underlie the DR shortening of lifespan is the finding that DR

Target of rapamycin inhibition by rapamycin feeding has previously been shown to extend life in genetically heterogeneous mice. To examine whether it similarly affected mouse health, we fed encapsulated rapamycin or a control diet to C57BL/6Nia mice of both sexes starting at 19 months of age. We performed a range of health assessments 6 and 12 months later. Rapamycin feeding significantly reduced mTOR activity in most but not all tissues. It also reduced total and resting metabolic rate during the light (inactive) phase of the light:dark cycle in females only but had no effect on spontaneous activity or metabolism during the dark (active) phase of either sex. Males only had less fragmented sleep when fed rapamycin, whereas stride length and rotarod performance were improved in both sexes. Survival was also improved by this late-life rapamycin feeding, and some pathological lesions were delayed. We found no adverse health consequences associated with rapamycin treatment.

The study was undertaken to explore the effect of rapamycin, an anti-inflammatory agent, on the metabolic profile of type 2 diabetic mice. Seven-month-old diabetic db/db mice and their lean littermate non-diabetic controls (db/m) were randomized to receive control chow or chow mixed with rapamycin (2.24 mg/kg/day) (each group n=20, males and females) for 4 months and sacrificed. Serum samples were analyzed for the measurement of glucose, creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT), total cholesterol, total triglyceride, and total protein, using the automated dry chemistry analysis. Rapamycin elevated serum glucose in female diabetic mice. Serum creatinine tended to be higher in diabetic mice but was not affected by rapamycin; there was no difference in BUN levels among the groups. Serum ALP was elevated in diabetic mice and rapamycin lowered it only in female diabetic mice; serum ALT levels were increased in female diabetic mice, unaffected by rapamycin. Serum total protein was elevated in diabetic mice of both genders but was not affected by rapamycin. Diabetic mice from both genders had elevated serum cholesterol and triglycerides; rapamycin did not affect serum cholesterol but decreased serum total triglycerides in male diabetic mice. We conclude that rapamycin elicits complex metabolic responses in aging diabetic mice, worsening hyperglycemia in females but improving ALP in female diabetic and total triglycerides in male diabetic mice, respectively. The metabolic effects of rapamycin should be considered while performing studies with rapamycin in mice. Keywords: alkaline phosphatase; alanine aminotransferase; cholesterol; triglycerides (Published: 23 April 2012) Citation: Pathobiology of Aging & Age-related Diseases 2012, 2 : 15896 - http://dx.doi.org/10.3402/pba.v2i0.15896

Dietary restriction (DR) and rapamycin (Rapa) have been shown to increase the lifespan of a variety of organisms leading to the speculation that these interventions increase lifespan through related mechanisms. However, both these interventions have a detrimental effect in the G93A mutant mouse model of amyotrophic lateral sclerosis (ALS). Our previous work indicated that different ALS SOD1 mutant mouse models differ in disease pathogenesis; therefore in this study we measured the effect of DR and Rapa in a second ALS mutant mouse model (the H46R/H48Q mutant). Interestingly, in mice expressing this mutant SOD1 protein, DR significantly delays disease onset and extends lifespan, while Rapa has no effect. These findings suggest that: (1) the effect of DR in ALS is not mediated through pathways common with Rapa, (2) the deleterious effect of DR and Rapa in the G93A ALS mouse model may not be universal to disease caused by all SOD1 mutations, and (3) the results reinforce our previous conclusions that the pathogenic mechanisms in G93A and H46R/H48Q mice are distinct.

A case of steroid-resistant nephrotic syndrome with focal segmental glomerulosclerosis leading to renal failure in a 4-year-old girl is described by her mother, with special emphasis on the problems resulting from recurrence of this disease, with graft loss in three successive kidney transplants. This report chronicles the gradual evolution from a family's initial heavy dependence upon medical solutions to their ultimate primary emphasis upon spiritual values, with medicine seen as the support toward achieving the child's psychological well-being and spiritual growth. The physician's role in balancing medical decision making, non-medical alternatives, and demands on limited and precious resources in such difficult cases is discussed.

In 1935 McCay et al. (1935) reported that underfed rats “attained extreme ages beyond those of either sex that grew normally.” Since then, chronic reduction of food intake (dietary restriction or DR) has become the most common environmental intervention used to extend lifespan and probe mechanisms specifying longevity. DR extends lifespan across a variety of taxa (Weindruch & Walford, 1988; Finch, 1990; Masoro, 2003) and is considered to be among the most robust life-extending interventions (Weindruch & Walford, 1988; Masoro, 2005). Clinical studies are underway to test the effect of DR on various mortality risk factors in humans (Holloszy & Fontana, 2007), and members of one organization, the Calorie Restriction Society, practice self-imposed DR in an effort to extend their lives (Fontana et al., 2008). However, life extension by DR may not be universal (Carey et al., 2002; Cooper et al., 2004). Several reports indicate that DR does not extend lifespan or has minimal effects in some rodent strains (Weindruch & Walford, 1988; Harper et al., 2006; Turturro et al., 1999). Others even report that DR shortens lifespan in some strains (Barrow & Roeder, 1965; Fernandes et al., 1976; Harrison & Archer, 1987; Forster et al., 2003), but these studies have not been conclusive given that other studies have shown lifespan extension under different conditions (Weindruch & Walford, 1988; Turturro et al., 1999). A systematic, unbiased screen to determine the efficacy of moderate DR across a range of genotypes is lacking. Here, we undertook such a study -- testing the hypothesis that the lifespan response to DR is subject to naturally-occurring genetic variation encompassing null or even negative effects. This study used 41 ILSXISS recombinant inbred (RI) mouse strains (Williams et al., 2004) (formerly called LXS) originally developed to analyze genetic variation in alcohol sensitivity (Bennett et al., 2006). Mice were typically maintained 5/cage (Supplementary Table S1) and started at 2–5 months of age fed ad libitum (AL) or DR diets (60% of strain-specific AL intake) in a specific-pathogen-free vivarium dedicated to murine aging research (Ikeno et al., 2005). The DR rations, which were not implemented gradually, were calculated on the basis of AL food intake measured weekly for each strain, adjusted for wastage (Ikeno et al., 2005), and the rations were given daily just before lights out. At 12 months of age, the DR rations were fixed to avoid tracking the reduction of food intake that occurs during aging. We have followed this DR protocol at 60% of AL intake for over 30 years (Ikeno et al., 2005; Yu et al., 1982; McCarter et al., 2007). This level of restriction is one of the most common (Turturro et al., 1999; de Cabo et al., 2005), although DR levels from 40% to 80% of AL intake have been used to achieve life extension (Weindruch & Walford, 1988). We found that the RI strains exhibited marked genetic variation in lifespan under both AL and DR conditions (Figs. 1 A, B; Supplementary Table S1). Mean lifespan under AL feeding ranged two- to three-fold: 504 to 1152 days in males and 407 to 1208 days in females. This variation in AL lifespan is comparable to that of 31 inbred strains selected for their genetic diversity (Yuan et al., 2009) (Supplementary Fig. S1). Strain variation of mean lifespan in mice under DR was even greater, ranging six- to ten-fold: 217 to 1215 days in males and 113 to 1225 days in females. Effect of strain on lifespan was significant for both sexes under both feeding conditions (p < 1×10−6, ANOVA). Heritability of lifespan under AL feeding was 28% (males) and 36% (females) and under DR was 55% (males) and 53% (females). Fig. 1 Strain variation in mean lifespan of ILSXISS recombinant inbred (RI) mice under ad libitum (AL) and dietary restriction (DR) diets. Lifespans were typically obtained from 10 AL and 10 DR mice from each strain (5 males & 5 females per treatment ... Strikingly, the majority of strains showed no extension of lifespan under the level of DR used in this study (Figs. 1C, D). Only 5% of the strains for males and 21% of the strains for females showed statistically significant life extension under DR, using single strain p values < 0.05. DR shortened lifespan in more strains (27% and 26%; males and females, respectively; p < 0.05 – 0.001). Although sample sizes were small, mean lifespans of males and females were significantly correlated under both AL (r = 0.50, p = 0.002) and DR (r = 0.42. p = 0.012) conditions. In addition, doubling sample size by combining the two sexes yielded a similar result: DR shortened life in more strains than showed lengthened life (Supplementary Fig. S2). Maximum lifespan (age at death of oldest mouse) was highly correlated with mean lifespan across strains under both AL and DR regimens (AL males, r = 0.81; AL females, r = 0.82; DR males, r = 0.92; DR females, r = 0.94; all p < 1×10−9), indicating that the strain variation in mean lifespan was not disproportionately affected by early deaths that can arise in DR mice. That early deaths in DR mice contributed to lifespan shortening is not supported by the finding that exclusion of deaths occurring before 12 months of age had negligible effect on the frequency of lifespan shortening (Supplementary Fig. S3). These results, using a large genetic screen, buttress previous but often overlooked results showing no extension or shortening of lifespan by DR (Weindruch & Walford, 1988; Harper et al., 2006; Turturro et al., 1999; Barrow & Roeder, 1965; Fernandes et al., 1976; Harrison & Archer, 1987; Forster et al., 2003). However, whether strains showing no increase in lifespan under 40% or other fixed level of DR show no increase in lifespan under less stringent level of DR remains to be determined. Of note, the longest lifespans achieved under DR did not exceed the longest achieved under AL feeding (Figs. 1A, B). The average of the mean lifespans of the five longest-lived strains under DR (1103±40 and 1108±32 days in males and females) did not exceed that of the five longest-lived, albeit different, strains under AL feeding (1098±20 and 1088±31 days). Future studies are needed to determine why DR cannot further extend the lifespan of long-lived strains in this RI panel. One testable hypothesis is that the lifespan extending biochemical pathways modulated by DR are already maximally modulated in strains that are long-lived under AL conditions. The biological basis for the strikingly different responses of lifespan to the commonly used level of DR, including life shortening, is important to determine. For example, some lines in this study may have unusual nutritional needs, and thus 40% DR could cause nutritional deficiencies that might outweigh the beneficial effects of DR. However, the possibility that some strains are vulnerable to a mineral or vitamin deficiency under DR is unlikely because, with the exception of selenium and choline, the diet used (Harlan-Teklad 7912) exceeded by several fold the minimum requirements established by the National Research Council (Nutrient Requirements of Laboratory Animals, 1995) (Supplementary Table S2). Also, even with diets supplemented with vitamins, the lifespan of male DBA/2J mice was either not extended (Forster et al., 2003) or minimally lengthened (Turturro et al., 1999). There also was no correlation between DR lifespan and the large strain variation in absolute food intake (Table 1), suggesting that the strains most likely to encounter deficiency were not more likely to have reduced survival under DR. Table 1 Absence of correlation between lifespan under dietary restriction (DR) and lifespan, food consumption, fertility and ethanol sensitivity under ad libitum (AL) feeding.f Considering the derivation of the ILSXISS strains, we tested whether the lifespan variation in response to DR might be related to the segregation of alleles for extreme differences in ethanol sensitivity, which could potentially reflect differences in vitality or stress resistance. However, there was no correlation between sensitivity to this stressor and lifespan in DR mice (Table 1). Another potential measure of vigor, female fertility, also showed no correlation with DR lifespan (Table 1). These results argue against the notion that strains in which DR shortened lifespan lacked overall vitality. Many other testable possibilities exist to explain life-shortening of some strains under DR. These include vulnerability a) to stresses requiring energy expenditure, such as cold stress; b) to inbreeding depression (recessive alleles) not reflected by the variation in AL lifespan or fertility; and c) to a 40% reduction in food intake that would not be present at a 30% or 20% reduction. Nevertheless, the variable response of these strains to DR provides a valuable tool for identifying quantitative trait loci (genes) that modulate DR’s mechanism of action. In addition, mechanistic traits hypothesized to underlie the lifespan modulating effect of DR should correlate positively with the variation in the lifespan response to DR. In summary, these findings, coupled with earlier reports, show that even though DR extends lifespan across a variety of taxa, a prolongevity effect may not be a foregone conclusion for many genotypes. The marked genetic variation among RI strains provides a tool for identifying genes and biochemical pathways that mediate lifespan modulation by DR. Finally, the results raise a cautionary note concerning the application of DR to humans and a critical need for predictors of efficacy.

Genetic instability has been implicated as a causal factor in cancer and aging. Caloric restriction (CR) and suppression of the somatotroph axis significantly increase life span in the mouse and reduce multiple symptoms of aging, including cancer. To test if in vivo spontaneous mutation frequency is reduced by such mechanisms, we crossed long-lived Ames dwarf mice with a C57BL/6J line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from tissues and organs into Escherichia coli to measure mutant frequencies. Four cohorts were studied: (1) ad lib wild-type; (2) CR wild-type; (3) ad lib dwarf; and (4) CR dwarf. While both CR wild-type and ad lib dwarf mice lived significantly longer than the ad lib wild-type mice, under CR conditions dwarf mice did not live any longer than ad lib wild-type mice. While this may be due to an as yet unknown adverse effect of the C57BL/6J background, it did not prevent an effect on spontaneous mutation frequencies at the lacZ locus, which were assessed in liver, kidney and small intestine of 7- and 15-month-old mice of all four cohorts. A lower mutant frequency in the ad lib dwarf background was observed in liver and kidney at 7 and 15 months of age and in small intestine at 15 months of age as compared to the ad lib wild-type. CR also significantly reduced spontaneous mutant frequency in kidney and small intestine, but not in liver. In a separate cohort of lacZ-C57BL/6J mice CR was also found to significantly reduce spontaneous mutant frequency in liver and small intestine, across three age levels. These results indicate that two major pro-longevity interventions in the mouse are associated with a reduced mutation frequency. This could be responsible, at least in part, for the enhanced longevity associated with Ames dwarfism and CR.

PROPÓSITO: Los tumores pulmonares con diferenciación neuroendocrina (DN) son un grupo heterogéneo de neoplasias que incluyen tumores carcinoides típicos, carcinoides atípicos, carcinomas neuroendocrinos de células grandes (CNCG) y carcinoma pulmonar de células pequeñas. Los CNCG constituyen menos del 5% de los carcinomas pulmonares no células pequeñas (CPNCP). En este trabajo se describe una serie de CNCG, tratados en un solo centro a lo largo de 10 años. MATERIAL Y MÉTODOS: Se analizan 11 pacientes diagnosticados de CNCG (5 con histologías mixtas). RESULTADOS: La edad media de los pacientes fue de 66 años, 5 fueron varones, 4 tuvieron enfermedad localizada, 5 localmente avanzada y 2 diseminada. Siete pacientes, con tumores localizados, fueron tratados con cirugía radical, 2 de ellos recibieron quimioterapia adyuvante; 1 quimioterapia y radioterapia y los otros 3 solo quimioterapia. La mediana de supervivencia de la serie es de 24 meses, y la supervivencia global a 2 y 5 años del 45% y 27% respectivamente. CONCLUSIÓN: Los datos de nuestra serie corroboran las recomendaciones de que el manejo de los CNCG debe hacerse de forma similar al del resto de los CPNCP. La cirugía radical es el tratamiento fundamental en los tumores localizados. No hay datos suficientes que indiquen una peor respuesta a los tratamientos de quimioterapia o radioterapia en este tipo de tumores.

Dietary restriction (DR) has been shown to inhibit almost all the age-related diseases, e.g. cardiomyopathy and cancers, in rodents. However, there is little information for the effect of DR on atherosclerosis. In the present study, we examined the effect of DR on the development of atherosclerosis in mice homozygous knockout for apolipoprotein E gene (ApoE(-/-)). The ApoE(-/-) mice were fed either ad libitum (AL) or 60% of the diet consumed by the mice fed AL. Atherosclerotic lesions in the proximal aorta of these mice were measured. Our results showed that ApoE(-/-) mice fed the calorie-restricted diet had smaller and relatively early stages of atherosclerotic lesions (e.g. foam cells and free lipids) when compared to ApoE(-/-) mice fed AL, who developed more advanced lesions (e.g. fibrous caps and acellular areas). In addition, ApoE(-/-) mice fed the calorie-restricted diet showed a significant decrease in the level of lipid hydroperoxides and the production of superoxide and hydrogen peroxide in the aorta as compared to ApoE(-/-) mice fed AL. These observations suggest that reduction of oxidative stress in the arterial wall may contribute to the anti-atherogenic effect of DR in ApoE(-/-) mice.