Fibroblasts derived from long‐lived insulin receptor substrate 1 null mice are not resistant to multiple forms of stress

Reduced signalling through the insulin/insulin-like growth factor-1 signalling (IIS) pathway is a highly conserved lifespan determinant in model organisms. The precise mechanism underlying the effects of the IIS on lifespan and health is currently unclear, although cellular stress resistance may be important. We have previously demonstrated that mice globally lacking insulin receptor substrate 1 (Irs1^−/−) are long-lived and enjoy a greater period of their life free from age-related pathology compared with wild-type (WT) controls. In this study, we show that primary dermal fibroblasts and primary myoblasts derived from Irs1^−/− mice are no more resistant to a range of oxidant and nonoxidant chemical stressors than cells derived from WT mice.     

Metformin treatment of diverse Caenorhabditis species reveals the importance of genetic background in longevity and healthspan extension outcomes

Metformin, the most commonly prescribed anti‐diabetes medication, has multiple reported health benefits, including lowering the risks of cardiovascular disease and cancer, improving cognitive function with age, extending survival in diabetic patients, and, in several animal models, promoting youthful physiology and lifespan. Due to its longevity and health effects, metformin is now the focus of the first proposed clinical trial of an anti‐aging drug—the Targeting Aging with Metformin (TAME) program. Genetic variation will likely influence outcomes when studying metformin health effects in human populations. To test for metformin impact in diverse genetic backgrounds, we measured lifespan and healthspan effects of metformin treatment in three Caenorhabditis species representing genetic variability greater than that between mice and humans. We show that metformin increases median survival in three C. elegans strains, but not in C. briggsae and C. tropicalis strains. In C. briggsae, metformin either has no impact on survival or decreases lifespan. In C. tropicalis, metformin decreases median survival in a dose‐dependent manner. We show that metformin prolongs the period of youthful vigor in all C. elegans strains and in two C. briggsae strains, but that metformin has a negative impact on the locomotion of C. tropicalis strains. Our data demonstrate that metformin can be a robust promoter of healthy aging across different genetic backgrounds, but that genetic variation can determine whether metformin has positive, neutral, or negative lifespan/healthspan impact. These results underscore the importance of tailoring treatment to individuals when testing for metformin health benefits in diverse human populations.     

SIRT1 but not its increased expression is essential for lifespan extension in caloric‐restricted mice

The SIRT1 deacetylase is one of the best-studied putative mediators of some of the anti-aging effects of calorie restriction (CR), but its role in CR-dependent lifespan extension has not been demonstrated. We previously found that mice lacking both copies of SIRT1 displayed a shorter median lifespan than wild-type mice on an ad libitum diet. Here, we report that median lifespan extension in CR heterozygote SIRT1^+/− mice was identical (51%) to that observed in wild-type mice, but SIRT1^+/− mice displayed a higher frequency of certain pathologies. Although larger studies in additional genetic backgrounds are needed, these results provide strong initial evidence for the requirement of SIRT1 for the lifespan extension effects of CR, but suggest that its high expression is not required for CR-induced lifespan extension.     

Lithium reduces apoptosis and autophagy after neonatal hypoxia�Cischemia

Lithium is used in the treatment of bipolar mood disorder. Reportedly, lithium can be neuroprotective in models of adult brain ischemia. The purpose of this study was to evaluate the effects of lithium in a model of neonatal hypoxic–ischemic brain injury. Nine-day-old male rats were subjected to unilateral hypoxia–ischemia (HI) and 2 mmol/kg lithium chloride was injected i.p. immediately after the insult. Additional lithium injections, 1 mmol/kg, were administered at 24-h intervals. Pups were killed 6, 24 or 72 h after HI. Lithium reduced the infarct volume from 24.7±2.9 to 13.8±3.3 mm³ (44.1%) and total tissue loss (degeneration + lack of growth) from 67.4±4.4 to 38.4±5.9 mm³ (43.1%) compared with vehicle at 72 h after HI. Injury was reduced in the cortex, hippocampus, thalamus and striatum. Lithium reduced the ischemia-induced dephosphorylation of glycogen synthase kinase-3β and extracellular signal-regulated kinase, the activation of calpain and caspase-3, the mitochondrial release of cytochrome c and apoptosis-inducing factor, as well as autophagy. We conclude that lithium could mitigate the brain injury after HI by inhibiting neuronal apoptosis. The lithium doses used were in the same range as those used in bipolar patients, suggesting that lithium might be safely used for the avoidance of neonatal brain injury.     

Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3β (GSK-3β), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3β, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3β, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.     

Possible Role of the Glycogen Synthase Kinase-3 Signaling Pathway in Trimethyltin-Induced Hippocampal Neurodegeneration in Mice

Trimethyltin (TMT) is an organotin compound with potent neurotoxic effects characterized by neuronal destruction in selective regions, including the hippocampus. Glycogen synthase kinase-3 (GSK-3) regulates many cellular processes, and is implicated in several neurodegenerative disorders. In this study, we evaluated the therapeutic effect of lithium, a selective GSK-3 inhibitor, on the hippocampus of adult C57BL/6 mice with TMT treatment (2.6 mg/kg, intraperitoneal [i.p.]) and on cultured hippocampal neurons (12 days in vitro) with TMT treatment (5 µM). Lithium (50 mg/kg, i.p., 0 and 24 h after TMT injection) significantly attenuated TMT-induced hippocampal cell degeneration, seizure, and memory deficits in mice. In cultured hippocampal neurons, lithium treatment (0–10 mM; 1 h before TMT application) significantly reduced TMT-induced cytotoxicity in a dose-dependent manner. Additionally, the dynamic changes in GSK-3/β-catenin signaling were observed in the mouse hippocampus and cultured hippocampal neurons after TMT treatment with or without lithium. Therefore, lithium inhibited the detrimental effects of TMT on the hippocampal neurons in vivo and in vitro, suggesting involvement of the GSK-3/β-catenin signaling pathway in TMT-induced hippocampal cell degeneration and dysfunction.     

How does the mood stabilizer lithium bind ATP,the energy currency of the cell: Insights from solid-state NMR

BackgroundLithium, in the form of a salt, is a mood stabilizer and a leading drug for the treatment of bipolar disorder. It has a very narrow therapeutic range and a variety of side effects. Lithium can replace magnesium and other cations in enzymes and small molecules, among them ATP, thereby affecting and inhibiting many biochemical pathways. The form of binding of lithium ions to ATP is not known.MethodsHere we extract the binding environment of lithium in solid ATP using a multi-nuclear multi-dimensional solid-state NMR approach.ResultsWe determine that the coordination sphere of lithium includes, at a distance of 3.0(±0.4) Å, three phosphates; the two phosphates closest to the ribose ring from one ATP molecule, and the middle phosphate from another ATP molecule. A water molecule most probably completes the fourth coordination. Despite the use of excess lithium in the preparations, sodium ions still remain bound to the sample, at distances of 4.3–5.5 Å from Li, and coordinate the first phosphate and two terminal phosphates.ConclusionsSolid-state NMR enables to unravel the exact coordination of lithium in ATP showing binding to three phosphates from two molecules, none of which are the terminal gamma phosphate.General significanceThe methods we use are applicable to study lithium bound to a variety of ATP-bound enzymes, or to other cellular targets of lithium, consequently suggesting a molecular basis for its mode of action.     

Health Effects of Long-Term Rapamycin Treatment: The Impact on Mouse Health of Enteric Rapamycin Treatment from Four Months of Age throughout Life

Rapamycin, an mTOR inhibitor, has been shown to extend lifespan in a range of model organisms. It has been reported to extend lifespan in multiple strains of mice, administered chronically or acutely early or late in life. The ability of rapamycin to extend health (healthspan) as opposed to life is less well documented. To assess the effects chronic rapamycin treatment on healthspan, enteric rapamycin was given to male and female C57BL/6J mice starting at 4 months of age and continued throughout life. Repeated, longitudinal assessments of health in individual animals were made starting at 16 months of age (=12 months of treatment) until death. A number of health parameters were improved (female grip strength, female body mass and reduced sleep fragmentation in both sexes), others showed no significant difference, while at least one (male rotarod performance) was negatively affected. Rapamycin treatment affected many measures of health in a highly sex-specific manner. While sex-specific phenotypic effects of rapamycin treatment have been widely reported, in this study we document sex differences in the direction of phenotypic change. Rapamycin-fed males and females were both significantly different from controls; however the differences were in the opposite direction in measures of body mass, percent fat and resting metabolic rate, a pattern not previously reported.     

Reevaluation of the effect of dietary restriction on different recombinant inbred lines of male and female mice

Dietary restriction (DR) was reported to either have no effect or reduce the lifespan of the majority of the 41‐recombinant inbred (RI) lines studied by Liao et al. (Aging Cell, 2010, 9, 92). In an appropriately power longevity study (n > 30 mice/group), we measured the lifespan of the four RI lines (115‐RI, 97‐RI, 98‐RI, and 107‐RI) that were reported to have the greatest decrease in lifespan when fed 40% DR. DR increased the median lifespan of female RI‐115, 97‐RI, and 107‐RI mice and male 115‐RI mice. DR had little effect (<4%) on the median lifespan of female and male 98‐RI mice and male 97‐RI mice and reduced the lifespan of male 107‐RI mice over 20%. While our study was unable to replicate the effect of DR on the lifespan of the RI mice (except male 107‐RI mice) reported by Liao et al. (Aging Cell, 2010, 9, 92), we found that the genotype of a mouse had a major impact on the effect of DR on lifespan, with the effect of DR ranging from a 50% increase to a 22% decrease in median lifespan. No correlation was observed between the changes in either body composition or glucose tolerance induced by DR and the changes observed in lifespan of the four RI lines of male and female mice. These four RI lines of mice give the research community a unique resource where investigators for the first time can study the anti‐aging mechanism of DR by comparing mice in which DR increases lifespan to mice where DR has either no effect or reduces lifespan.     

Role of CBP and SATB-1 in Aging,Dietary Restriction,and Insulin-Like Signaling

How dietary restriction (DR) increases lifespan and decreases disease burden are questions of major interest in biomedical research. Here we report that hypothalamic expression of CREB-binding protein (CBP) and CBP-binding partner Special AT-rich sequence binding protein 1 (SATB-1) is highly correlated with lifespan across five strains of mice, and expression of these genes decreases with age and diabetes in mice. Furthermore, in Caenorhabditis elegans, cbp-1 is induced by bacterial dilution DR (bDR) and the daf-2 mutation, and cbp-1 RNAi specifically in adults completely blocks lifespan extension by three distinct protocols of DR, partially blocks lifespan extension by the daf-2 mutation but not of cold, and blocks delay of other age-related pathologies by bDR. Inhibiting the C. elegans ortholog of SATB-1 and CBP-binding partners daf-16 and hsf-1 also attenuates lifespan extension by bDR, but not other protocols of DR. In a transgenic Aβ42 model of Alzheimer''s disease, cbp-1 RNAi prevents protective effects of bDR and accelerates Aβ42-related pathology. Furthermore, consistent with the function of CBP as a histone acetyltransferase, drugs that enhance histone acetylation increase lifespan and reduce Aβ42-related pathology, protective effects completely blocked by cbp-1 RNAi. Other factors implicated in lifespan extension are also CBP-binding partners, suggesting that CBP constitutes a common factor in the modulation of lifespan and disease burden by DR and the insulin/IGF1 signaling pathway.     

Pharmacometabolomic Signature of Ataxia SCA1 Mouse Model and Lithium Effects

We have shown that lithium treatment improves motor coordination in a spinocerebellar ataxia type 1 (SCA1) disease mouse model (Sca1^154Q/+). To learn more about disease pathogenesis and molecular contributions to the neuroprotective effects of lithium, we investigated metabolomic profiles of cerebellar tissue and plasma from SCA1-model treated and untreated mice. Metabolomic analyses of wild-type and Sca1^154Q/+ mice, with and without lithium treatment, were performed using gas chromatography time-of-flight mass spectrometry and BinBase mass spectral annotations. We detected 416 metabolites, of which 130 were identified. We observed specific metabolic perturbations in Sca1^154Q/+ mice and major effects of lithium on metabolism, centrally and peripherally. Compared to wild-type, Sca1^154Q/+ cerebella metabolic profile revealed changes in glucose, lipids, and metabolites of the tricarboxylic acid cycle and purines. Fewer metabolic differences were noted in Sca1^154Q/+ mouse plasma versus wild-type. In both genotypes, the major lithium responses in cerebellum involved energy metabolism, purines, unsaturated free fatty acids, and aromatic and sulphur-containing amino acids. The largest metabolic difference with lithium was a 10-fold increase in ascorbate levels in wild-type cerebella (p<0.002), with lower threonate levels, a major ascorbate catabolite. In contrast, Sca1^154Q/+ mice that received lithium showed no elevated cerebellar ascorbate levels. Our data emphasize that lithium regulates a variety of metabolic pathways, including purine, oxidative stress and energy production pathways. The purine metabolite level, reduced in the Sca1^154Q/+ mice and restored upon lithium treatment, might relate to lithium neuroprotective properties.     

Essential role of p38 MAPK for activation of skeletal muscle glucose transport by lithium

Lithium increases glucose transport and glycogen synthesis in insulin-sensitive cell lines and rat skeletal muscle, and has been used as a non-selective inhibitor of glycogen synthase kinase-3 (GSK-3). However, the molecular mechanisms underlying lithium action on glucose transport in mammalian skeletal muscle are unknown. Therefore, we examined the effects of lithium on glucose transport activity, glycogen synthesis, insulin signaling elements (insulin receptor (IR), Akt, and GSK-3beta), and the stress-activated p38 mitogen-activated protein kinase (p38 MAPK) in the absence or presence of insulin in isolated soleus muscle from lean Zucker rats. Lithium (10 mM LiCl) enhanced basal glucose transport by 62% (p < 0.05) and augmented net glycogen synthesis by 112% (p < 0.05). Whereas lithium did not affect basal IR tyrosine phosphorylation or Akt ser(473) phosphorylation, it did enhance (41%, p < 0.05) basal GSK-3beta ser(9) phosphorylation. Lithium further enhanced (p < 0.05) the stimulatory effects of insulin on glucose transport (43%), glycogen synthesis (44%), and GSK-3beta ser(9) phosphorylation (13%). Lithium increased (p < 0.05) p38 MAPK phosphorylation both in the absence (37%) and presence (41%) of insulin. Importantly, selective inhibition of p38 MAPK (using 10 microM A304000) completely prevented the basal activation of glucose transport by lithium, and also significantly reduced (52%, p < 0.05) the lithium-induced enhancement of insulin-stimulated glucose transport. Theses results demonstrate that lithium enhances basal and insulin-stimulated glucose transport activity and glycogen synthesis in insulin-sensitive rat skeletal muscle, and that these effects are associated with a significant enhancement of GSK-3beta phosphorylation. Importantly, we have documented an essential role of p38 MAPK phosphorylation in the action lithium on the glucose transport system in isolated mammalian skeletal muscle.     

Lithium suppresses signaling and induces rapid sequestration of beta2-adrenergic receptors

Lithium is a monovalent cation used therapeutically to treat a range of affective disorders (1), although the cellular mechanisms of lithium regulation that might contribute to its therapeutic effects at the level of neurotransmitter receptors are not known. Herein we report the ability of lithium to stimulate the internalization of beta2-adrenergic receptors. Lithium treatment of A431 human epidermoid carcinoma cells resulted in a rapid, prominent desensitization and internalization of beta2-adrenergic receptors. The ability of these receptors to generate a cyclic AMP response was strongly inhibited by lithium, at concentrations therapeutic in humans. Receptors for the serotonin (5HT1c) and for opiates (mu-opioid), in sharp contrast, resisted the effects of lithium on internalization. These data provide the first receptor-based mechanism to be described for lithium that could explain, in part, the therapeutic effects of lithium on affective disorders.     

Detrimental Effects of RNAi: A Cautionary Note on Its Use in Drosophila Ageing Studies

RNA interference (RNAi) provides an important tool for gene function discovery. It has been widely exploited in Caenorhabditis elegans ageing research because it does not appear to have any non-specific effects on ageing-related traits in that model organism. We show here that ubiquitous, adult-onset activation of the RNAi machinery, achieved by expressing a double stranded RNA targeting GFP or lacZ for degradation, or by increasing expression of Dicer substantially reduces lifespan in Drosophila melanogaster. Induction of GFPRNAi construct also alters the response of lifespan to nutrition, exacerbating the lifespan-shortening effects of food containing a high quantity of yeast. Our study indicates that activation of the RNAi machinery may have sequence-independent side-effects on lifespan, and that caution needs to be exercised when employing ubiquitous RNAi in Drosophila ageing studies. However, we also show that RNAi restricted to certain tissues may not be detrimental to lifespan.