Intermittent rapamycin feeding recapitulates some effects of continuous treatment while maintaining lifespan extension.

OBJECTIVE: Rapamycin, a powerful geroprotective drug, can have detrimental effects when administered chronically. We determined whether intermittent treatment of mice can reduce negative effects while maintaining benefits of chronic treatment. METHODS: From 6 months of age, male and female C3B6F1 hybrid mice were either continuously fed with 42 mg/kg rapamycin, or intermittently fed by alternating weekly feeding of 42 mg/kg rapamycin food with weekly control feeding. Survival of these mice compared to control animals was measured. Furthermore, longitudinal phenotyping including metabolic (body composition, GTT, ITT, indirect calorimetry) and fitness phenotypes (treadmil, rotarod, electrocardiography and open field) was performed. Organ specific pathology was assessed at 24 months of age. RESULTS: Chronic rapamycin treatment induced glucose intolerance, which was partially ameliorated by intermittent treatment. Chronic and intermittent rapamycin treatments increased lifespan equally in males, while in females chronic treatment resulted in slightly higher survival. The two treatments had equivalent effects on testicular degeneration, heart fibrosis and liver lipidosis. In males, the two treatment regimes led to a similar increase in motor coordination, heart rate and Q-T interval, and reduction in spleen weight, while in females, they equally reduced BAT inflammation and spleen weight and maintained heart rate and Q-T interval. However, other health parameters, including age related pathologies, were better prevented by continuous treatment. CONCLUSIONS: Intermittent rapamycin treatment is effective in prolonging lifespan and reduces some side-effects of chronic treatment, but chronic treatment is more beneficial to healthspan.

Reduced insulin signaling in neurons induces sex-specific health benefits.

Reduced activity of insulin/insulin-like growth factor signaling (IIS) extends health and life span in mammals. Loss of the insulin receptor substrate 1 (Irs1) gene increases survival in mice and causes tissue-specific changes in gene expression. However, the tissues underlying IIS-mediated longevity are currently unknown. Here, we measured survival and health span in mice lacking IRS1 specifically in liver, muscle, fat, and brain. Tissue-specific loss of IRS1 did not increase survival, suggesting that lack of IRS1 in more than one tissue is required for life-span extension. Loss of IRS1 in liver, muscle, and fat did not improve health. In contrast, loss of neuronal IRS1 increased energy expenditure, locomotion, and insulin sensitivity, specifically in old males. Neuronal loss of IRS1 also caused male-specific mitochondrial dysfunction, activation of Atf4, and metabolic adaptations consistent with an activated integrated stress response at old age. Thus, we identified a male-specific brain signature of aging in response to reduced IIS associated with improved health at old age.

Long-lasting geroprotection from brief rapamycin treatment in early adulthood by persistently increased intestinal autophagy.

The licensed drug rapamycin has potential to be repurposed for geroprotection. A key challenge is to avoid adverse side effects from continuous dosing. Here we show that geroprotective effects of chronic rapamycin treatment can be obtained with a brief pulse of the drug in early adulthood in female Drosophila and mice. In Drosophila, a brief, early rapamycin treatment of adults extended lifespan and attenuated age-related decline in the intestine to the same degree as lifelong dosing. Lasting memory of earlier treatment was mediated by elevated autophagy in intestinal enterocytes, accompanied by increased levels of intestinal LManV and lysozyme. Brief elevation of autophagy in early adulthood itself induced a long-term increase in autophagy. In mice, a 3-month, early treatment also induced a memory effect, with maintenance similar to chronic treatment, of lysozyme distribution, Man2B1 level in intestinal crypts, Paneth cell architecture and gut barrier function, even 6 months after rapamycin was withdrawn.

Lithium can mildly increase health during ageing but not lifespan in mice.

Lithium is a nutritional trace element, used clinically as an anti-depressant. Preclinically, lithium has neuroprotective effects in invertebrates and mice, and it can also extend lifespan in fission yeast, C. elegans and Drosophila. An inverse correlation of human mortality with the concentration of lithium in tap water suggests a possible, evolutionarily conserved mechanism mediating longevity. Here, we assessed the effects of lithium treatment on lifespan and ageing parameters in mice. Lithium has a narrow therapeutic dose range, and overdosing can severely affect organ health. Within the tolerable dosing range, we saw some mildly positive effects of lithium on health span but not on lifespan.

FoxO1 Is a Novel Regulator of 20S Proteasome Subunits Expression and Activity.

Proteostasis collapses during aging resulting, among other things, in the accumulation of damaged and aggregated proteins. The proteasome is the main cellular proteolytic system and plays a fundamental role in the maintenance of protein homeostasis. Our previous work has demonstrated that senescence and aging are related to a decline in proteasome content and activities, while its activation extends lifespan in vitro and in vivo in various species. However, the mechanisms underlying this age-related decline of proteasome function and the down-regulation in expression of its subunits remain largely unclear. Here, we demonstrate that the Forkhead box-O1 (FoxO1) transcription factor directly regulates the expression of a 20S proteasome catalytic subunit and, hence, proteasome activity. Specifically, we demonstrate that knockout of FoxO1, but not of FoxO3, in mice severely impairs proteasome activity in several tissues, while depletion of IRS1 enhances proteasome function. Importantly, we show that FoxO1 directly binds on the promoter region of the rate-limiting catalytic ß5 proteasome subunit to regulate its expression. In summary, this study reveals the direct role of FoxO factors in the regulation of proteasome function and provides new insight into how FoxOs affect proteostasis and, in turn, longevity.

Longevity in response to lowered insulin signaling requires glycine N-methyltransferase-dependent spermidine production.

Reduced insulin/IGF signaling (IIS) extends lifespan in multiple organisms. Different processes in different tissues mediate this lifespan extension, with a set of interplays that remain unclear. We here show that, in Drosophila, reduced IIS activity modulates methionine metabolism, through tissue-specific regulation of glycine N-methyltransferase (Gnmt), and that this regulation is required for full IIS-mediated longevity. Furthermore, fat body-specific expression of Gnmt was sufficient to extend lifespan. Targeted metabolomics showed that reducing IIS activity led to a Gnmt-dependent increase in spermidine levels. We also show that both spermidine treatment and reduced IIS activity are sufficient to extend the lifespan of Drosophila, but only in the presence of Gnmt. This extension of lifespan was associated with increased levels of autophagy. Finally, we found that increased expression of Gnmt occurs in the liver of liver-specific IRS1 KO mice and is thus an evolutionarily conserved response to reduced IIS. The discovery of Gnmt and spermidine as tissue-specific modulators of IIS-mediated longevity may aid in developing future therapeutic treatments to ameliorate aging and prevent disease.

Growth Hormone Receptor Regulation in Cancer and Chronic Diseases.

The GHR signaling pathway plays important roles in growth, metabolism, cell cycle control, immunity, homeostatic processes, and chemoresistance via both the JAK/STAT and the SRC pathways. Dysregulation of GHR signaling is associated with various diseases and chronic conditions such as acromegaly, cancer, aging, metabolic disease, fibroses, inflammation and autoimmunity. Numerous studies entailing the GHR signaling pathway have been conducted for various cancers. Diverse factors mediate the up- or down-regulation of GHR signaling through post-translational modifications. Of the numerous modifications, ubiquitination and deubiquitination are prominent events. Ubiquitination by E3 ligase attaches ubiquitins to target proteins and induces proteasomal degradation or starts the sequence of events that leads to endocytosis and lysosomal degradation. In this review, we discuss the role of first line effectors that act directly on the GHR at the cell surface including ADAM17, JAK2, SRC family member Lyn, Ubc13/CHIP, proteasome, ßTrCP, CK2, STAT5b, and SOCS2. Activity of all, except JAK2, Lyn and STAT5b, counteract GHR signaling. Loss of their function increases the GH-induced signaling in favor of aging and certain chronic diseases, exemplified by increased lung cancer risk in case of a mutation in the SOCS2-GHR interaction site. Insight in their roles in GHR signaling can be applied for cancer and other therapeutic strategies.

Intestinal Fork Head Regulates Nutrient Absorption and Promotes Longevity.

Reduced activity of nutrient-sensing signaling networks can extend organismal lifespan, yet the underlying biology remains unclear. We show that the anti-aging effects of rapamycin and reduced intestinal insulin/insulin growth factor (IGF) signaling (IIS) require the Drosophila FoxA transcription factor homolog Fork Head (FKH). Intestinal FKH induction extends lifespan, highlighting a role for the gut. FKH binds to and is phosphorylated by AKT and Target of Rapamycin. Gut-specific FKH upregulation improves gut barrier function in aged flies. Additionally, it increases the expression of nutrient transporters, as does lowered IIS. Evolutionary conservation of this effect of lowered IIS is suggested by the upregulation of related nutrient transporters in insulin receptor substrate 1 knockout mouse intestine. Our study highlights a critical role played by FKH in the gut in mediating anti-aging effects of reduced IIS. Malnutrition caused by poor intestinal absorption is a major problem in the elderly, and a better understanding of the mechanisms involved will have important therapeutic implications for human aging.

Reduced insulin/insulin-like growth factor signaling decreases translation in Drosophila and mice.

Down-regulation of insulin/insulin-like growth factor signaling (IIS) can increase lifespan in C. elegans, Drosophila and mice. In C. elegans, reduced IIS results in down-regulation of translation, which itself can extend lifespan. However, the effect of reduced IIS on translation has yet to be determined in other multicellular organisms. Using two long-lived IIS models, namely Drosophila lacking three insulin-like peptides (dilp2-3,5(-/-)) and mice lacking insulin receptor substrate 1 (Irs1(-/-)), and two independent translation assays, polysome profiling and radiolabeled amino acid incorporation, we show that reduced IIS lowers translation in these organisms. In Drosophila, reduced IIS decreased polysome levels in fat body and gut, but reduced the rate of protein synthesis only in the fat body. Reduced IIS in mice decreased protein synthesis rate only in skeletal muscle, without reducing polysomes in any tissue. This lowered translation in muscle was independent of Irs1 loss in the muscle itself, but a secondary effect of Irs1 loss in the liver. In conclusion, down-regulation of translation is an evolutionarily conserved response to reduced IIS, but the tissues in which it occurs can vary between organisms. Furthermore, the mechanisms underlying lowered translation may differ in mice, possibly associated with the complexity of the regulatory processes.

Fos-Zippered GH Receptor Cytosolic Tails Act as Jak2 Substrates and Signal Transducers.

Members of the Janus kinase (Jak) family initiate the majority of downstream signaling events of the cytokine receptor family. The prevailing principle is that the receptors act in dimers: 2 Jak2 molecules bind to the cytosolic tails of a cytokine receptor family member and initiate Jak-signal transducer and activator of transcription signaling upon a conformational change in the receptor complex, induced by the cognate cytokine. Due to the complexity of signaling complexes, there is a strong need for in vitro model systems. To investigate the molecular details of the Jak2 interaction with the GH receptor (GHR), we used cytosolic tails provided with leucine zippers derived from c-Fos to mimic the dimerized state of GHR. Expressed together with Jak2, fos-zippered tails, but not unzippered tails, were stabilized. In addition, the Jak-signal transducer and activator of transcription signaling pathway was activated by the fos-zippered tails. The stabilization depended also on α-helix rotation of the zippers. Fos-zippered GHR tails and Jak2, both purified from baculovirus-infected insect cells, interacted via box1 with a binding affinity of approximately 40nM. As expected, the Jak kinase inhibitor Ruxolitinib inhibited the stabilization but did not affect the c-Fos-zippered GHR tail-Jak2 interaction. Analysis by blue-native gel electrophoresis revealed high molecular-weight complexes containing both Jak2 and nonphosphorylated GHR tails, whereas Jak2-dissociated tails were highly phosphorylated and monomeric, implying that Jak2 detaches from its substrate upon phosphorylation.

Lithium Promotes Longevity through GSK3/NRF2-Dependent Hormesis.

The quest to extend healthspan via pharmacological means is becoming increasingly urgent, both from a health and economic perspective. Here we show that lithium, a drug approved for human use, promotes longevity and healthspan. We demonstrate that lithium extends lifespan in female and male Drosophila, when administered throughout adulthood or only later in life. The life-extending mechanism involves the inhibition of glycogen synthase kinase-3 (GSK-3) and activation of the transcription factor nuclear factor erythroid 2-related factor (NRF-2). Combining genetic loss of the NRF-2 repressor Kelch-like ECH-associated protein 1 (Keap1) with lithium treatment revealed that high levels of NRF-2 activation conferred stress resistance, while low levels additionally promoted longevity. The discovery of GSK-3 as a therapeutic target for aging will likely lead to more effective treatments that can modulate mammalian aging and further improve health in later life.

The Jak/STAT signaling pathway is downregulated at febrile temperatures.

BACKGROUND: The Janus family of kinases (JAKs), Jak1, Jak2, Jak3, and Tyk2, constitute a subgroup of non-receptor protein tyrosine kinases. Upon cytokine binding, the receptor-associated kinases are activated and phosphorylate tyrosine residues in their cognate cytokine receptors. Their activities are controlled at several levels and include cellular concentration, auto-activation, and degradation. PRINCIPAL FINDINGS: Our findings show that elevated temperatures in the fever range irreversibly aggregate Jak2 and considerably reduce functional Jak2 protein levels. Jak2 synthesis remains unaltered. We observed that also the protein level of the signal transducer and activator of transcription, STAT5b, is transiently decreased at temperatures above 37°C. Consequently, the signaling response, e.g. via the growth hormone receptor, is reduced. CONCLUSIONS/SIGNIFICANCE: These findings predict that elevated body temperatures lower the responsiveness of cytokine receptors.