Weekly administration of rapamycin improves survival and biomarkers in obese male mice on high‐fat diet

Recent discoveries have revealed the key role of mTOR (target of rapamycin) in aging. Furthermore, rapamycin extends lifespan in mice, especially in female mice. Here, we treated obese male mice on high‐fat diet with rapamycin given intermittently: either weekly (once a week) or alternating bi‐weekly (three injections every other week). While only marginally reducing obesity, intermittent administration of rapamycin significantly extended lifespan. Significance was achieved for weekly treated group and for the three rapamycin‐received groups combined. In weekly treatment group, 100% mice were alive by the age of 2 years, whereas 60% of mice died in untreated group by this age. The effect of weekly treatment on survival was highly significant and cannot be fully explained by partial reduction in obesity. Alternating bi‐weekly treatments seem to be less effective than weekly treatment, although effects of additional factors (see 3 ) may not be excluded. After one year of treatment, all survived mice were sacrificed 8 days after the last administration of rapamycin to avoid its direct interference with parameters examined. Fasting levels of cardiac and hepatic p‐S6, a marker of mTORC1 activity, were lower in weekly treatment group compared with control mice. In contrast, levels of p‐Akt (S473), glucose, triglycerides and insulin were unchanged, whereas leptin and IGF‐1 tended to be lower. Thus, weekly treatment with rapamycin may slow down aging in obese male mice on high‐fat diet.     

Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects

Calorie restriction (CR) and rapamycin (RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin (mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10‐week RP or 40% CR. Protein abundance and turnover were measured in vivo using ²H₃–leucine heavy isotope labeling followed by LC‐MS/MS, and translation was assessed by polysome profiling. We observed 35–60% increased protein half‐lives after CR and 15% increased half‐lives after RP compared to age‐matched controls. Surprisingly, the effects of RP and CR on protein turnover and abundance differed greatly between canonical pathways, with opposite effects in mitochondrial (mt) dysfunction and eIF2 signaling pathways. CR most closely recapitulated the young phenotype in the top pathways. Polysome profiles indicated that CR reduced polysome loading while RP increased polysome loading in young and old mice, suggesting distinct mechanisms of reduced protein synthesis. CR and RP both attenuated protein oxidative damage. Our findings collectively suggest that CR and RP extend lifespan in part through the reduction of protein synthetic burden and damage and a concomitant increase in protein quality. However, these results challenge the notion that RP is a faithful CR mimetic and highlight mechanistic differences between the two interventions.     

Mechanistic target of rapamycin controls homeostasis of adipogenesis

Signaling mediated by the mechanistic target of rapamycin (mTOR) is believed to play a critical and positive role in adipogenesis, based on pharmacological evidence and genetic manipulation of mTOR regulators and targets. However, there is no direct genetic evidence for an autonomous role of mTOR itself in preadipocyte differentiation. To seek such evidence, we employed a conditional knockdown approach to deplete mTOR in preadipocytes. Surprisingly, while knockdown of S6K1, a target of mTOR, impairs 3T3-L1 preadipocyte differentiation, reduction of mTOR levels leads to increased differentiation. This enhanced adipogenesis requires the remaining mTOR activity, as mTOR inhibitors abolish differentiation in the mTOR knockdown cells. We also found that mTOR knockdown elevates the levels of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Furthermore, partial reduction of mTOR levels alleviates inhibition of Akt by mTORC1 via IRS1, while at the same time maintaining its positive input through mTORC1 into the adipogenic program. The greater sensitivity of the IRS1-Akt pathway to mTOR levels provides a mechanism that explains the net outcome of enhanced adipogenesis through PPARγ upon mTOR knockdown. Our observations reveal an unexpected role of mTOR in suppressing adipogenesis and suggest that mTOR governs the homeostasis of the adipogenic process by modulating multiple signaling pathways.     

Young and old genetically heterogeneous HET3 mice on a rapamycin diet are glucose intolerant but insulin sensitive

Rapamycin, an inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, extends the life span of yeast, worms, flies, and mice. Interventions that promote longevity are often correlated with increased insulin sensitivity, and it therefore is surprising that chronic rapamycin treatment of mice, rats, and humans is associated with insulin resistance (J Am Soc Nephrol., 19 , 2008, 1411; Diabetes, 00, 2010, 00; Science , 335, 2012, 1638). We examined the effect of dietary rapamycin treatment on glucose homeostasis and insulin resistance in the genetically heterogeneous HET3 mouse strain, a strain in which dietary rapamycin robustly extends mean and maximum life span. We find that rapamycin treatment leads to glucose intolerance in both young and old HET3 mice, but in contrast to the previously reported effect of injected rapamycin in C57BL/6 mice, HET3 mice treated with dietary rapamycin responded normally in an insulin tolerance test. To gauge the overall consequences of rapamycin treatment on average blood glucose levels, we measured HBA1c. Dietary rapamycin increased HBA1c over the first 3 weeks of treatment in young animals, but the effect was lost by 3 months, and no effect was detected in older animals. Our results demonstrate that the extended life span of HET3 mice on a rapamycin diet occurs in the absence of major changes in insulin sensitivity and highlight the importance of strain background and delivery method in testing effects of longevity interventions.     

Chronic mTOR inhibition in mice with rapamycin alters T,B, myeloid,and innate lymphoid cells and gut flora and prolongs life of immune‐deficient mice

The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age‐related debilities including increasing antigen‐specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long‐term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T‐ and B‐lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer‐ and infection‐prone mice supporting disease mitigation as a mechanism for mTOR suppression‐mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension.     

p53 induces miR-199a-3p to suppress mechanistic target of rapamycin activation in cisplatin-induced acute kidney injury

How p53 participates in acute kidney injury (AKI) progress and what are the underlying mechanisms remain illusive. For this issue, it is important to probe into the role of p53 in cisplatin-induced AKI. We find that p53 was upregulated in cisplatin-induced AKI, yet, pifithrin-α inhibites the p53 expression to attenuated renal injury and cell apoptosis both in vivo cisplatin-induced AKI mice and in vitro HK-2 human renal tubular epithelial cells. To knock down p53 by siRNA significantly decreased the miRNA, miR-199a-3p, expression in HK-2 cells. Blockade of miR-199a-3p significantly reduced cisplatin-induced cell apoptosis and inhibited caspase-3 activity. Mechanistically, we identified that miR-199a-3p directly bound to mechanistic target of rapamycin (mTOR) 3′-untranslated region and overexpressed miR-199a-3p reduce the expression and phosphorylation of mTOR. Furthermore, we demonstrated that p53 inhibited mTOR activation through activating miR-199a-3p. In conclusion, our findings reveal that p53, upregulating the expression of miR-199a-3p affects the progress of cisplatin-induced AKI, which might provide a promising therapeutic target of AKI.     

Short‐term rapamycin treatment increases ovarian lifespan in young and middle‐aged female mice

Although age‐related ovarian failure in female mammals cannot be reversed, recent strategies have focused on improving reproductive capacity with age, and rapamycin is one such intervention that has shown a potential for preserving the ovarian follicle pool and preventing premature ovarian failure. However, the application is limited because of its detrimental effects on follicular development and ovulation during long‐term treatment. Herein, we shortened the rapamycin administration to 2 weeks and applied the protocol to both young (8 weeks) and middle‐aged (8 months) mouse models. Results showed disturbances in ovarian function during and shortly after treatment; however, all the treated animals returned to normal fertility 2 months later. Following natural mating, we observed prolongation of ovarian lifespan in both mouse models, with the most prominent effect occurring in mice older than 12 months. The effects of transient rapamycin treatment on ovarian lifespan were reflected in the preservation of primordial follicles, increases in oocyte quality, and improvement in the ovarian microenvironment. These data indicate that short‐term rapamycin treatment exhibits persistent effects on prolonging ovarian lifespan no matter the age at initiation of treatment. In order not to disturb fertility in young adults, investigators should in the future consider applying the protocol later in life so as to delay menopause in women, and at the same time increase ovarian lifespan.     

Comparison of rapamycin schedules in mice on high-fat diet

At a wide range of doses, rapamycin extends life span in mice. It was shown that intraperitoneal injections (i.p.) of rapamycin prevent weight gain in mice on high-fat diet (HFD). We further investigated the effect of rapamycin on weight gain in female C57BL/6 mice on HFD started at the age of 7.5 months. By the age of 16 and 23 months, mice on HFD weighed significantly more (52 vs 33 g; p = 0.0001 and 70 vs 38 g; p < 0.0001, respectively) than mice on low fat diet (LFD). The i.p. administration of 1.5 mg/kg rapamycin, 3 times a week every other week, completely prevented weight gain, whereas administration of rapamycin by oral gavash did not. Rapamycin given in the drinking water slightly decreased weight gain by the age of 23 months. In addition, metabolic parameters were evaluated at the age of 16 and 23 months, 6 and 13 days after last rapamycin administration, respectively. Plasma leptin levels strongly correlated with body weight, (P < 0.0001, r=0.86), suggesting that the difference in weight was due to fat tissue mass. Levels of insulin, glucose, triglycerides and IGF1 were not statistically different in all groups, indicating that these courses of rapamycin treatment did not impair metabolic parameters at least after rapamycin discontinuation. Despite rapamycin discontinuation, cardiac levels of phospho-S6 and pAKT(S473) were low in the i.p.-treated group. This continuous effect of rapamycin can be explained by prevention of obesity in the i.p. group. We conclude that intermittent i.p. administration of rapamycin prevents weight gain without causing gross metabolic abnormalities. Intermittent gavash administration minimally affected weight gain. Potential clinical applications are discussed.     

Target of rapamycin activation predicts lifespan in fruit flies

Aging and age-related diseases are one of the most important health issues that the world will confront during the 21^st century. Only by understanding the proximal causes will we be able to find treatments to reduce or delay the onset of degenerative diseases associated with aging. Currently, the prevalent paradigm in the field is the accumulation of damage. However, a new theory that proposes an alternative explanation is gaining momentum. The hyperfunction theory proposes that aging is not a consequence of a wear and tear process, but a result of the continuation of developmental programs during adulthood. Here we use Drosophila melanogaster, where evidence supporting both paradigms has been reported, to identify which parameters that have been previously related with lifespan best predict the rate of aging in wild type flies cultured at different temperatures. We find that mitochondrial function and mitochondrial reactive oxygen species (mtROS) generation correlates with metabolic rate, but not with the rate of aging. Importantly, we find that activation of nutrient sensing pathways (i.e. insulin-PI3K/Target of rapamycin (Tor) pathway) correlates with lifespan, but not with metabolic rate. Our results, dissociate metabolic rate and lifespan in wild type flies and instead link nutrient sensing signaling with longevity as predicted by the hyperfunction theory.     

The anti-tumor effects of the combination of microwave hyperthermia and lobaplatin against breast cancer cells in vitro and in vivo

Background: Breast cancer is the main lethal disease among females. The combination of lobaplatin and microwave hyperthermia plays a crucial role in several kinds of cancer in the clinic, but its possible mechanism in breast cancer has remained indistinct.Methods: Mouse models were used to detect breast cancer progression. Cell growth was explored with MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphonyl)-2H-tetrazolium) and colony formation assays. Cell migration and invasion were investigated with a transwell assay. Cell apoptosis was probed with flow cytometry. The expression of apoptosis-associated proteins was examined with Western blots.Result: Combination treatment decreased breast cancer cell viability, colony formation, cell invasion and metastasis. In addition, the treatment-induced breast cancer cell apoptosis and autophagy, activated the c-Jun N-terminal kinase (JNK) signaling pathway, suppressed the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, and down-regulated IAP and Bcl-2 family protein expression.Conclusion: These results indicate that lobaplatin is an effective breast cancer anti-tumor agent. Microwave hyperthermia was a useful adjunctive treatment. Combination treatment was more efficient than any single therapy. The possible mechanism for this effect was mainly associated with activation of the JNK signaling pathway, inactivation of the AKT/mTOR signaling pathway and down-regulation of the Bcl-2 and IAP families.     

Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice

Early diabetic nephropathy is characterized by renal hypertrophy that is mainly due to proximal tubular hypertrophy. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, and its signaling has been reported to regulate protein synthesis and cellular growth, specifically, hypertrophy. Therefore, we examined the effect of mTOR signaling on diabetic renal hypertrophy by using the specific inhibitor for mTOR, rapamycin. Ten days after streptozotocin-induced diabetes, mice showed kidney hypertrophy with increases in the phosphorylation of p70S6kinase and the expression of cyclin kinase inhibitors, p21(Cip1) and p27(Kip1), in the kidneys. The intraperitoneal injection of rapamycin (2 mg/kg/day) markedly attenuated the enhanced phosphorylation of p70S6kinase, the increment of cyclin-dependent kinase inhibitors, and renal enlargement without any changes of clinical parameters, including blood glucose, blood pressure, and food intake. Overexpression of a constitutive active form of p70S6kinase resulted in increased cell size of cultured mouse proximal tubule cells; thus, activation of p70S6kinase causes hypertrophy of proximal tubular cells. Our findings suggest that activation of mTOR signaling causes renal hypertrophy at the early stage of diabetes.     

Chronic rapamycin treatment or lack of S6K1 does not reduce ribosome activity in vivo

Reducing activity of the mTORC1/S6K1 pathway has been shown to extend lifespan in both vertebrate and invertebrate models. For instance, both pharmacological inhibition of mTORC1 with the drug rapamycin or S6K1 knockout extends lifespan in mice. Since studies with invertebrate models suggest that reducing translational activity can increase lifespan, we reasoned that the benefits of decreased mTORC1 or S6K1 activity might be due, at least in part, to a reduction of general translational activity. Here, we report that mice given a single dose of rapamycin have reduced translational activity, while mice receiving multiple injections of rapamycin over 4 weeks show no difference in translational activity compared with vehicle-injected controls. Furthermore, mice lacking S6K1 have no difference in global translational activity compared with wild-type littermates as measured by the percentage of ribosomes that are active in multiple tissues. Translational activity is reduced in S6K1-knockout mice following single injection of rapamycin, demonstrating that rapamycin’s effects on translation can occur independently of S6K1. Taken together, these data suggest that benefits of chronic rapamycin treatment or lack of S6K1 are dissociable from potential benefits of reduced translational activity, instead pointing to a model whereby changes in translation of specific subsets of mRNAs and/or translation-independent effects of reduced mTOR signaling underlie the longevity benefits.