Caloric restriction (CR) protects against aging and disease but the mechanisms by which this affects mammalian lifespan are unclear. We show in mice that deletion of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway component ribosomal S6 protein kinase 1 (S6K1) led to increased lifespan and resistance to age-related pathologies such as bone, immune and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian lifespan, and suggest therapeutic manipulation of S6K1 and AMPK might mimic CR and provide broad protection against diseases of aging. Genetic studies in S. cerevisiae, C. elegans and D. melanogaster implicate several mechanisms in the regulation of lifespan. These include the insulin and insulin-like growth factor 1 (IGF-1) signaling (IIS) and mammalian target of rapamycin (mTOR) pathways which both activate the downstream effector ribosomal protein S6 kinase 1 (S6K1) (1, 2). Although the role of these pathways in mammalian aging is less clear, there is mounting evidence that IIS regulates lifespan in mice (1). Global deletion of one allele of the IGF1 receptor (Igf1r), adipose-specific deletion of the insulin receptor (Insr), global deletion of insulin receptor substrate protein 1 (Irs1) or neuron-specific deletion of Irs2 all increase mouse lifespan (1). Lifespan-extending mutations in the somatotropic axis also appear to work through attenuated IIS (3). Igf1r has also been implicated as a modulator of human longevity (4). However, the action of downstream effectors of IIS or mTOR signaling in mammalian longevity is not fully understood.S6K1 transduces anabolic signals that indicate nutritional status to regulate cell size and growth and metabolism through various mechanisms (5). These include effects on the translational machinery and on cellular energy levels through the activity of adenosine monophosphate (AMP)-activated protein kinase (AMPK) (6, 7). Furthermore, S6K1 serine phosphorylates IRS1 and IRS2 thereby decreasing insulin signaling (5). Given the key role of S6K1 in IIS and mTOR signaling, and the regulation of aging in lower organisms by mTOR, S6K, and their downstream effectors (2) we used log rank testing to evaluate differences in lifespan of wild-type (WT) and S6K1 -/-littermate mice on a C57BL/6 background (8). Data for both sexes combined showed median lifespan in S6K1 -/-mice increased by 80 days (from 862 to 942 days) or 9% relative to that of WT mice (X 2 = 10.52, p < 0.001) ( Fig. 1A and Table 1). Maximum lifespan (mean lifespan of the oldest 10% within a cohort) was also increased (1077±16 and 1175±24 days, p < 0.01 for WT and S6K1 -/-mice, respectively). Analysis of each sex separately showed that median lifespan in female S6K1 -/-mice was increased, by 153 d...

Hypothalamic AMP-activated protein kinase (AMPK) has been suggested to act as a key sensing mechanism, responding to hormones and nutrients in the regulation of energy homeostasis. However, the precise neuronal populations and cellular mechanisms involved are unclear. The effects of long-term manipulation of hypothalamic AMPK on energy balance are also unknown. To directly address such issues, we generated POMCα2KO and AgRPα2KO mice lacking AMPKα2 in proopiomelanocortin-(POMC-) and agouti-related protein-expressing (AgRP-expressing) neurons, key regulators of energy homeostasis. POMCα2KO mice developed obesity due to reduced energy expenditure and dysregulated food intake but remained sensitive to leptin. In contrast, AgRPα2KO mice developed an age-dependent lean phenotype with increased sensitivity to a melanocortin agonist. Electrophysiological studies in AMPKα2-deficient POMC or AgRP neurons revealed normal leptin or insulin action but absent responses to alterations in extracellular glucose levels, showing that glucose-sensing signaling mechanisms in these neurons are distinct from those pathways utilized by leptin or insulin. Taken together with the divergent phenotypes of POMCα2KO and AgRPα2KO mice, our findings suggest that while AMPK plays a key role in hypothalamic function, it does not act as a general sensor and integrator of energy homeostasis in the mediobasal hypothalamus.

Recent evidence suggests that alterations in insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) can increase mammalian life span. For example, in several mouse mutants, impairment of the growth hormone (GH)/IGF1 axis increases life span and also insulin sensitivity. However, the intracellular signaling route to altered mammalian aging remains unclear. We therefore measured the life span of mice lacking either insulin receptor substrate (IRS) 1 or 2, the major intracellular effectors of the IIS receptors. Our provisional results indicate that female Irs1-/- mice are long-lived. Furthermore, they displayed resistance to a range of age-sensitive markers of aging including skin, bone, immune, and motor dysfunction. These improvements in health were seen despite mild, lifelong insulin resistance. Thus, enhanced insulin sensitivity is not a prerequisite for IIS mutant longevity. Irs1-/- female mice also displayed normal anterior pituitary function, distinguishing them from long-lived somatotrophic axis mutants. In contrast, Irs2-/- mice were short-lived, whereas Irs1+/- and Irs2+/- mice of both sexes showed normal life spans. Our results therefore suggest that IRS1 signaling is an evolutionarily conserved pathway regulating mammalian life span and may be a point of intervention for therapies with the potential to delay age-related processes.

Polymorphisms in the fat mass and obesity-associated gene (FTO) are associated with human obesity and obesity-prone behaviors, including increased food intake and a preference for energy-dense foods. FTO demethylates N 6 -methyladenosine, a potential regulatory RNA modification, but the mechanisms by which FTO predisposes humans to obesity remain unclear. In adiposity-matched, normal-weight humans, we showed that subjects homozygous for the FTO "obesity-risk" rs9939609 A allele have dysregulated circulating levels of the orexigenic hormone acyl-ghrelin and attenuated postprandial appetite reduction. Using functional MRI (fMRI) in normal-weight AA and TT humans, we found that the FTO genotype modulates the neural responses to food images in homeostatic and brain reward regions. Furthermore, AA and TT subjects exhibited divergent neural responsiveness to circulating acyl-ghrelin within brain regions that regulate appetite, reward processing, and incentive motivation. In cell models, FTO overexpression reduced ghrelin mRNA N 6 -methyladenosine methylation, concomitantly increasing ghrelin mRNA and peptide levels. Furthermore, peripheral blood cells from AA human subjects exhibited increased FTO mRNA, reduced ghrelin mRNA N 6 -methyladenosine methylation, and increased ghrelin mRNA abundance compared with TT subjects. Our findings show that FTO regulates ghrelin, a key mediator of ingestive behavior, and offer insight into how FTO obesity-risk alleles predispose to increased energy intake and obesity in humans.Introduction FTO is an AlkB-like 2-oxoglutarate-dependent nucleic acid demethylase of uncertain cellular function (1). Genome-wide association studies (GWAS) have reliably established that SNPs within the first intron of FTO are robustly associated with increased BMI and adiposity across different ages and populations (2-6). Subjects homozygous for the "obesity-risk" A allele of FTO rs9939609 have a 1.7-fold increased risk for obesity compared with subjects homozygous for the low-risk T allele (2). Evidence to date suggests that the association between SNPs in FTO and BMI is predominantly driven by increased energy intake. Subjects homozygous for the obesity-risk A allele of rs9939609 exhibit overall increased ad libitum food-intake (7-9), particularly fat consumption (7, 9-11), and impaired satiety (12, 13). Furthermore, preschool AA children (AA denotes homozygosity for the A obesity-risk allele in the rs9939609 FTO variant) exhibit obesity-prone eating behaviors, including increased food responsiveness and a tendency to eat in

Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in β cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and β cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced β cell mass. Overt diabetes did not ensue, because β cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced β cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in β cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis. IntroductionInsulin regulates peripheral energy homeostasis by acting on multiple tissues to control carbohydrate, lipid, and protein metabolism (1). Gene targeting in mice has shown that β cell deletion of the insulin receptor causes reduced first-phase insulin release, reduced β cell insulin content, and progressive deterioration in glucose tolerance (2). Early studies of the effects of insulin in the CNS demonstrated a role for intracerebroventricularly administered insulin in the control of food intake and body weight (3). Mouse brain insulin receptor deletion causes mild hyperphagia and adiposity in female mice, diet-sensitive obesity, and defects in reproductive function (4). Results from studies in which insulinomimetics and insulin receptor antisense were centrally administered also support a role for CNS insulin signaling in energy homeostasis regulation (5, 6). Insulin signaling mechanisms therefore regulate β cell and CNS function, but it is unclear which postreceptor compo-

SummaryPI3K signaling is thought to mediate leptin and insulin action in hypothalamic pro-opiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, key regulators of energy homeostasis, through largely unknown mechanisms. We inactivated either p110α or p110β PI3K catalytic subunits in these neurons and demonstrate a dominant role for the latter in energy homeostasis regulation. In POMC neurons, p110β inactivation prevented insulin- and leptin-stimulated electrophysiological responses. POMCp110β null mice exhibited central leptin resistance, increased adiposity, and diet-induced obesity. In contrast, the response to leptin was not blocked in p110α-deficient POMC neurons. Accordingly, POMCp110α null mice displayed minimal energy homeostasis abnormalities. Similarly, in AgRP neurons, p110β had a more important role than p110α. AgRPp110α null mice displayed normal energy homeostasis regulation, whereas AgRPp110β null mice were lean, with increased leptin sensitivity and resistance to diet-induced obesity. These results demonstrate distinct metabolic roles for the p110α and p110β isoforms of PI3K in hypothalamic energy regulation.

As impaired insulin signalling (IIS) is a risk factor for Alzheimer’s disease we crossed mice (Tg2576) over-expressing human amyloid precursor protein (APP), with insulin receptor substrate 2 null (Irs2−/−) mice which develop insulin resistance. The resulting Tg2576/Irs2−/− animals had increased tau phosphorylation but a paradoxical amelioration of Aβ pathology. An increase of the Aβ binding protein transthyretin suggests that increased clearance of Aβ underlies the reduction in plaques. Increased tau phosphorylation correlated with reduced tau-phosphatase PP2A, despite an inhibition of the tau-kinase glycogen synthase kinase-3. Our findings demonstrate that disruption of IIS in Tg2576 mice has divergent effects on pathological processes—a reduction in aggregated Aβ but an increase in tau phosphorylation. However, as these effects are accompanied by improvement in behavioural deficits, our findings suggest a novel protective effect of disrupting IRS2 signalling in AD which may be a useful therapeutic strategy for this condition.

OBJECTIVEBariatric surgery causes durable weight loss. Gut hormones are implicated in obesity pathogenesis, dietary failure, and mediating gastrointestinal bypass (GIBP) surgery weight loss. In mice, we determined the effects of diet-induced obesity (DIO), subsequent dieting, and GIBP surgery on ghrelin, peptide YY (PYY), and glucagon-like peptide-1 (GLP-1). To evaluate PYY’s role in mediating weight loss post-GIBP, we undertook GIBP surgery in PyyKO mice.RESEARCH DESIGN AND METHODSMale C57BL/6 mice randomized to a high-fat diet or control diet were killed at 4-week intervals. DIO mice underwent switch to ad libitum low-fat diet (DIO-switch) or caloric restriction (CR) for 4 weeks before being killed. PyyKO mice and their DIO wild-type (WT) littermates underwent GIBP or sham surgery and were culled 10 days postoperatively. Fasting acyl-ghrelin, total PYY, active GLP-1 concentrations, stomach ghrelin expression, and colonic Pyy and glucagon expression were determined. Fasting and postprandial PYY and GLP-1 concentrations were assessed 30 days postsurgery in GIBP and sham pair-fed (sham.PF) groups.RESULTSDIO progressively reduced circulating fasting acyl-ghrelin, PYY, and GLP-1 levels. CR and DIO-switch caused weight loss but failed to restore circulating PYY to weight-appropriate levels. After GIBP, WT mice lost weight and exhibited increased circulating fasting PYY and colonic Pyy and glucagon expression. In contrast, the acute effects of GIBP on body weight were lost in PyyKO mice. Fasting PYY and postprandial PYY and GLP-1 levels were increased in GIBP mice compared with sham.PF mice.CONCLUSIONSPYY plays a key role in mediating the early weight loss observed post-GIBP, whereas relative PYY deficiency during dieting may compromise weight-loss attempts.