Mario G. Mirisola

Low Protein Intake Is Associated with a Major Reduction in IGF-1, Cancer, and Overall Mortality in the 65 and Younger but Not Older Population

Mice and humans with growth hormone receptor/IGF-1 deficiencies display major reductions in age-related diseases. Because protein restriction reduces GHR-IGF-1 activity, we examined links between protein intake and mortality. Respondents aged 50-65 reporting high protein intake had a 75% increase in overall mortality and a 4-fold increase in cancer death risk during the following 18 years. These associations were either abolished or attenuated if the proteins were plant derived. Conversely, high protein intake was associated with reduced cancer and overall mortality in respondents over 65, but a 5-fold increase in diabetes mortality across all ages. Mouse studies confirmed the effect of high protein intake and GHR-IGF-1 signaling on the incidence and progression of breast and melanoma tumors, but also the detrimental effects of a low protein diet in the very old. These results suggest that low protein intake during middle age followed by moderate to high protein consumption in old adults may optimize healthspan and longevity.

Interventions to Slow Aging in Humans: Are We Ready?

The workshop entitled ‘Interventions to Slow Aging in Humans: Are We Ready?’ was held in Erice, Italy, on October 8–13, 2013, to bring together leading experts in the biology and genetics of aging and obtain a consensus related to the discovery and development of safe interventions to slow aging and increase healthy lifespan in humans. There was consensus that there is sufficient evidence that aging interventions will delay and prevent disease onset for many chronic conditions of adult and old age. Essential pathways have been identified, and behavioral, dietary, and pharmacologic approaches have emerged. Although many gene targets and drugs were discussed and there was not complete consensus about all interventions, the participants selected a subset of the most promising strategies that could be tested in humans for their effects on healthspan. These were: (i) dietary interventions mimicking chronic dietary restriction (periodic fasting mimicking diets, protein restriction, etc.); (ii) drugs that inhibit the growth hormone/IGF‐I axis; (iii) drugs that inhibit the mTOR–S6K pathway; or (iv) drugs that activate AMPK or specific sirtuins. These choices were based in part on consistent evidence for the pro‐longevity effects and ability of these interventions to prevent or delay multiple age‐related diseases and improve healthspan in simple model organisms and rodents and their potential to be safe and effective in extending human healthspan. The authors of this manuscript were speakers and discussants invited to the workshop. The following summary highlights the major points addressed and the conclusions of the meeting.

A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan

Prolonged fasting (PF) promotes stress resistance, but its effects on longevity are poorly understood. We show that alternating PF and nutrient-rich medium extended yeast lifespan independently of established pro-longevity genes. In mice, 4 days of a diet that mimics fasting (FMD), developed to minimize the burden of PF, decreased the size of multiple organs/systems, an effect followed upon re-feeding by an elevated number of progenitor and stem cells and regeneration. Bi-monthly FMD cycles started at middle age extended longevity, lowered visceral fat, reduced cancer incidence and skin lesions, rejuvenated the immune system, and retarded bone mineral density loss. In old mice, FMD cycles promoted hippocampal neurogenesis, lowered IGF-1 levels and PKA activity, elevated NeuroD1, and improved cognitive performance. In a pilot clinical trial, three FMD cycles decreased risk factors/biomarkers for aging, diabetes, cardiovascular disease, and cancer without major adverse effects, providing support for the use of FMDs to promote healthspan.

Prolonged Fasting Reduces IGF-1/PKA to Promote Hematopoietic-Stem-Cell-Based Regeneration and Reverse Immunosuppression

Immune system defects are at the center of aging and a range of diseases. Here, we show that prolonged fasting reduces circulating IGF-1 levels and PKA activity in various cell populations, leading to signal transduction changes in long-term hematopoietic stem cells (LT-HSCs) and niche cells that promote stress resistance, self-renewal, and lineage-balanced regeneration. Multiple cycles of fasting abated the immunosuppression and mortality caused by chemotherapy and reversed age-dependent myeloid-bias in mice, in agreement with preliminary data on the protection of lymphocytes from chemotoxicity in fasting patients. The proregenerative effects of fasting on stem cells were recapitulated by deficiencies in either IGF-1 or PKA and blunted by exogenous IGF-1. These findings link the reduced levels of IGF-1 caused by fasting to PKA signaling and establish their crucial role in regulating hematopoietic stem cell protection, self-renewal, and regeneration.

cDNA cloning, sequence analysis and allergological characterization of Par j 2.0101, a new major allergen of the Parietaria judaica pollen

A clone (P2) coding for an allergen of Parietaria judaica (Pj) pollen has been isolated and sequenced from a cDNA library in lambda ZAP using a pool of 23 sera from Pj‐allergic patients. The clone contained an insert of 622 nucleotides with an open reading frame of 133 amino acids (aa) and a putative signal peptide of 31 aa giving a deduced mature processed protein of 102 aa with a molecular mass of 11 344 Da. The expressed recombinant protein, named rPar j 2.0101, was a major allergen since it reacted with IgE of 82% (23/28) of the sera of Pj‐allergic subjects analyzed. It was shown to be a new allergen since (i) the amino acid sequence homology with the already reported recombinant allergen Par j 1.0101 was 45% and (ii) there was no cross‐inhibition between rPar j 2.0101 and rPar j 1.0101. In addition, rPar j 2.0101 inhibited 35% of the specific IgE for 10–14 kDa native allergens and preincubation of sera from Pj‐allergic patients with both rPar j 2.0101 and rPar j 1.0101 fully abolished the IgE recognition of the 10–14 kDa native allergen region, suggesting that these two allergens contributed to the region.

Serine- and threonine/valine-dependent activation of PDK and Tor orthologs converge on Sch9 to promote aging

Dietary restriction extends longevity in organisms ranging from bacteria to mice and protects primates from a variety of diseases, but the contribution of each dietary component to aging is poorly understood. Here we demonstrate that glucose and specific amino acids promote stress sensitization and aging through the differential activation of the Ras/cAMP/PKA, PKH1/2 and Tor/S6K pathways. Whereas glucose sensitized cells through a Ras-dependent mechanism, threonine and valine promoted cellular sensitization and aging primarily by activating the Tor/S6K pathway and serine promoted sensitization via PDK1 orthologs Pkh1/2. Serine, threonine and valine activated a signaling network in which Sch9 integrates TORC1 and Pkh signaling via phosphorylation of threonines 570 and 737 and promoted intracellular relocalization and transcriptional inhibition of the stress resistance protein kinase Rim15. Because of the conserved pro-aging role of nutrient and growth signaling pathways in higher eukaryotes, these results raise the possibility that similar mechanisms contribute to aging in mammals.

Continental and subcontinental distributions of mtDNA control region types

When the mtDNA profile of a crime scene matches that of a suspect, it is necessary to determine the probability of a chance match by consulting the frequencies of the identified allele in a “reference population”. The ceiling principle suggests that that population should be chosen in which the allele of the suspect is found at the highest frequency, in order to give the suspect the maximum benefit of doubt. Recently, we advocated the use of a worldwide mitochondrial database combined with a geographical information system to identify the regions of the world with the highest frequencies of matching mtDNA types. Here, we demonstrate that the alternative approach of defining a ceiling reference population on the basis of continent or phenotype (race) is too coarse for a non-negligible percentage of mtDNA control region types.

RAS residues that are distant from the GDP binding site play a critical role in dissociation factor-stimulated release of GDP.

We have previously shown that a conserved glycine at position 82 of the yeast RAS2 protein is involved in the conversion of RAS proteins from the GDP‐ to the GTP‐bound form. We have now investigated the role of glycine 82 and neighbouring amino acids of the distal switch II region in the physiological mechanism of activation of RAS. We have introduced single and double amino acid substitutions at positions 80–83 of the RAS2 gene, and we have investigated the interaction of the corresponding proteins with a yeast GDP dissociation stimulator (SDC25 C‐domain). Using purified RAS proteins, we have found that the SDC25‐stimulated conversion of RAS from the GDP‐bound inactive state to the GTP‐bound active state was severely impaired by amino acid substitutions at positions 80–81. However, the rate and the extent of conversion from the GDP‐ to the GTP‐bound form in the absence of dissociation factor was unaffected. The insensitivity of the mutated proteins to the dissociation factor in vitro was paralleled by an inhibitory effect on growth in vivo. The mutations did not significantly affect the interaction of RAS with adenylyl cyclase. These findings point to residues 80–82 as important determinants of the response of RAS to GDP dissociation factors. This suggests a molecular model for the enhancement of nucleotide release from RAS by such factors.

Calorie Restriction in Mammals and Simple Model Organisms

Calorie restriction (CR), which usually refers to a 20–40% reduction in calorie intake, can effectively prolong lifespan preventing most age-associated diseases in several species. However, recent data from both human and nonhumans point to the ratio of macronutrients rather than the caloric intake as a major regulator of both lifespan and health-span. In addition, specific components of the diet have recently been identified as regulators of some age-associated intracellular signaling pathways in simple model systems. The comprehension of the mechanisms underpinning these findings is crucial since it may increase the beneficial effects of calorie restriction making it accessible to a broader population as well.

Tor-Sch9 deficiency activates catabolism of the ketone body-like acetic acid to promote trehalose accumulation and longevity

In mammals, extended periods of fasting leads to the accumulation of blood ketone bodies including acetoacetate. Here we show that similar to the conversion of leucine to acetoacetate in fasting mammals, starvation conditions induced ketone body‐like acetic acid generation from leucine in S. cerevisiae. Whereas wild‐type and ras2Δ cells accumulated acetic acid, long‐lived tor1Δ and sch9Δ mutants rapidly depleted it through a mitochondrial acetate CoA transferase‐dependent mechanism, which was essential for lifespan extension. The sch9Δ‐dependent utilization of acetic acid also required coenzyme Q biosynthetic genes and promoted the accumulation of intracellular trehalose. These results indicate that Tor‐Sch9 deficiency extends longevity by switching cells to an alternative metabolic mode, in which acetic acid can be utilized for the storage of stress resistance carbon sources. These effects are reminiscent of those described for ketone bodies in fasting mammals and raise the possibility that the lifespan extension caused by Tor‐S6K inhibition may also involve analogous metabolic changes in higher eukaryotes.