Aging research has been very successful at identifying signaling pathways and evolutionarily conserved genes that extend lifespan with the assumption that an increase in lifespan will also increase healthspan. However, it is largely unknown whether we are extending the healthy time of life or simply prolonging a period of frailty with increased incidence of age-associated diseases. Here we use Caenorhabditis elegans, one of the premiere systems for lifespan studies, to determine whether lifespan and healthspan are intrinsically correlated. We conducted multiple cellular and organismal assays on wild type as well as four long-lived mutants (insulin/insulin-like growth factor-1, dietary restriction, protein translation, mitochondrial signaling) in a longitudinal manner to determine the health of the animals as they age. We find that some long-lived mutants performed better than wild type when measured chronologically (number of days). However, all long-lived mutants increased the proportion of time spent in a frail state. Together, these data suggest that lifespan can no longer be the sole parameter of interest and reveal the importance of evaluating multiple healthspan parameters for future studies on antiaging interventions.

The insulin/IGF-1 signalling (IIS) pathway has diverse roles from metabolism to longevity1–5. In Caenorhabditis elegans, the single forkhead box O (FOXO) homologue, DAF-16, functions as the major target of the IIS pathway2,3,6,7. One of two isoforms4,5,8, DAF-16a, is known to regulate longevity, stress response and dauer diapause8–11. However, it remains unclear how DAF-16 achieves its specificity in regulating these various biological processes. Here we identify a new isoform, DAF-16d/f, as an important isoform regulating longevity. We show that DAF-16 isoforms functionally cooperate to modulate IIS-mediated processes through differential tissue enrichment, preferential modulation by upstream kinases, and regulating distinct and overlapping target genes. Promoter-swapping experiments show both the promoter and the coding region of DAF-16 are important for its function. Importantly, in mammals, four FOXO genes have overlapping and different functions6,12, and in C. elegans, a single FOXO/DAF-16 uses distinct isoforms to fine-tune the IIS-mediated processes in the context of a whole organism.

Mitochondria are key players in aging and in the pathogenesis of age-related diseases. Recent mitochondrial transcriptome analyses revealed the existence of multiple small mRNAs transcribed from mitochondrial DNA (mtDNA). Humanin (HN), a peptide encoded in the mtDNA 16S ribosomal RNA region, is a neuroprotective factor. An in silico search revealed six additional peptides in the same region of mtDNA as humanin; we named these peptides small humanin-like peptides (SHLPs). We identified the functional roles for these peptides and the potential mechanisms of action. The SHLPs differed in their ability to regulate cell viability in vitro. We focused on SHLP2 and SHLP3 because they shared similar protective effects with HN. Specifically, they significantly reduced apoptosis and the generation of reactive oxygen species, and improved mitochondrial metabolism in vitro. SHLP2 and SHLP3 also enhanced 3T3-L1 pre-adipocyte differentiation. Systemic hyperinsulinemic-euglycemic clamp studies showed that intracerebrally infused SHLP2 increased glucose uptake and suppressed hepatic glucose production, suggesting that it functions as an insulin sensitizer both peripherally and centrally. Similar to HN, the levels of circulating SHLP2 were found to decrease with age. These results suggest that mitochondria play critical roles in metabolism and survival through the synthesis of mitochondrial peptides, and provide new insights into mitochondrial biology with relevance to aging and human biology.

The nematode Caenorhabditis elegans has been employed as a model organism to study human obesity due to the conservation of the pathways that regulate energy metabolism. To assay for fat storage in C. elegans, a number of fat-soluble dyes have been employed including BODIPY, Nile Red, Oil Red O, and Sudan Black. However, dye-labeled assays produce results that often do not correlate with fat stores in C. elegans. An alternative label-free approach to analyze fat storage in C. elegans has recently been described with coherent anti-Stokes Raman scattering (CARS) microscopy. Here, we compare the performance of CARS microscopy with standard dye-labeled techniques and biochemical quantification to analyze fat storage in wild type C. elegans and with genetic mutations in the insulin/IGF-1 signaling pathway including the genes daf-2 (insulin/IGF-1 receptor), rict-1 (rictor) and sgk-1 (serum glucocorticoid kinase). CARS imaging provides a direct measure of fat storage with unprecedented details including total fat stores as well as the size, number, and lipid-chain unsaturation of individual lipid droplets. In addition, CARS/TPEF imaging reveals a neutral lipid species that resides in both the hypodermis and the intestinal cells and an autofluorescent organelle that resides exclusively in the intestinal cells. Importantly, coherent addition of the CARS fields from the C-H abundant neutral lipid permits selective CARS imaging of the fat store, and further coupling of spontaneous Raman analysis provides unprecedented details including lipid-chain unsaturation of individual lipid droplets. We observe that although daf-2, rict-1, and sgk-1 mutants affect insulin/IGF-1 signaling, they exhibit vastly different phenotypes in terms of neutral lipid and autofluorescent species. We find that CARS imaging gives quantification similar to standard biochemical triglyceride quantification. Further, we independently confirm that feeding worms with vital dyes does not lead to the staining of fat stores, but rather the sequestration of dyes in lysosome-related organelles. In contrast, fixative staining methods provide reproducible data but are prone to errors due to the interference of autofluorescent species and the non-specific staining of cellular structures other than fat stores. Importantly, both growth conditions and developmental stage should be considered when comparing methods of C. elegans lipid storage. Taken together, we confirm that CARS microscopy provides a direct, non-invasive, and label-free means to quantitatively analyze fat storage in living C. elegans.

Mitochondria have been largely considered as ‘end-function’ organelles, servicing the cell by producing energy and regulating cell death in response to complex signals. Being cellular entities with vital roles, mitochondria communicate back to the cell and actively engage in determining major cellular policies. These signals, collectively referred to as retrograde signals, are encoded in the nuclear genome, or are secondary products of mitochondrial metabolism. Here, we discuss humanin, the first small peptide of a putative set of mitochondrial-derived peptides, which exhibits strong cytoprotective actions against various stress and disease models. The study of humanin and other mitochondrial-derived retrograde signal peptides will aid in the identification of genes and peptides with therapeutic and diagnostic potential in treating human diseases.

PurposeTo investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress–induced cell death, mitochondrial bioenergetics, and senescence.MethodsHumanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5–10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress–induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment.ResultsA prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress–induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress–induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death.ConclusionsHumanin protected RPE cells against oxidative stress–induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD.

The discovery of humanin, a novel, mitochondrial-derived peptide, has created a potentially new category of biologically active peptide. As more research unravels the endogenous role of humanin as well as its potential pharmacological use, its role in stress resistance has become clearer. Humanin protects cells from oxidative stress, serum starvation, hypoxia, and other insults in vitro and also improves cardiovascular disease as well as Alzheimer's disease in vivo. In this review, we discuss the emerging role of humanin in stress resistance and its proposed mechanism of action.

Increased transcriptional complex activity, or pharmacological mimics of increased complex activity, predict lifespan in mice and mediate the protective effects of dietary restriction during aging.