Heritability of in vitro phenotypes exhibited by murine adipose-derived stromal cells.

Adipose-derived stromal cells (ADSCs) exhibit significant potential as therapeutic agents to promote tissue regeneration. Success of ADSC-based therapies is dependent upon efficient cell expansion in vitro as well as postinjection survival in the caustic milieu of damaged tissue. Genetic background regulates ADSC proliferative capacity and stress resistance, but the extent of the genetic effect size is not completely defined. The present study aimed to quantify phenotypic ranges and heritability of in vitro ADSC characteristics. ADSCs were isolated from mice representing 16 genetically diverse inbred mouse strains, including 12 classical inbred strains and four wild-derived strains. Cells were grown in vitro, and proliferative capacity and oxidative stress resistance were assessed. The fold change for ADSC growth ranged from 0.87 (BALB/cByJ) to 23.60 (POHN/DehJ), relative to original seeding density. The heritability of proliferative capacity was estimated to be 0.6462 (p = 9.967 × 10(-15)), and this phenotype was not associated with other ADSC traits. Cell viability following H2O2 treatment ranged from 39.81 % (CAST/EiJ) to 91.60 % (DBA/2 J), and the heritability of this phenotype was calculated as 0.6146 (p = 1.22 × 10(-12)). Relationships between cell viability and weight of the donor fat pad were also discovered. Donor genetic background is a major determinant of in vitro ADSC phenotypes. This study supports the development of forward genetics strategies to identify genes that underlie ADSC phenotypic diversity, which will inform efforts to improve cell-based therapies.

Rapamycin fed late in life extends lifespan in genetically heterogeneous mice.

Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in a mammalian species was unknown. Here we report that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. On the basis of age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. To our knowledge, these are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases.

Genetic coregulation of age of female sexual maturation and lifespan through circulating IGF1 among inbred mouse strains.

We previously reported that mouse strains with lower circulating insulin-like growth factor 1 (IGF1) level at 6 mo have significantly extended longevity. Here we report that strains with lower IGF1 have significantly delayed age of female sexual maturation, measured by vaginal patency (VP). Among strains with normal lifespans (mean lifespan >600 d), delayed age of VP associated with greater longevity (P = 0.015), suggesting a genetically regulated tradeoff at least partly mediated by IGF1. Supporting this hypothesis, C57BL/6J females had 9% lower IGF1, 6% delayed age of VP, and 24% extended lifespan compared with C57BL/6J.C3H/HeJ-Igf1, which carries a C3H/HeJ allele on chromosome (Chr) 10 that increases IGF1. To identify genetic loci/genes that regulate female sexual maturation, including loci that mediate lifespan tradeoffs, we performed haplotype association mapping for age of VP and identified significant loci on Chrs 4 (Vpq1) and 16 (Vpq2 and 3). At each locus, wild-derived strains share a unique haplotype that associates with delayed VP. Substitution of Chr 16 of C57BL/6J with Chr 16 from a wild-derived strain significantly reduced IGF1 and delayed VP. Strains with a wild-derived allele at Vpq3 have significantly extended longevity compared with strains with other alleles. Bioinformatic analysis identified Nrip1 at Vpq3 as a candidate gene. Nrip1(-/-) females have significantly reduced IGF1 and delayed age of VP compared with Nrip1(+/+) females. We conclude that IGF1 may coregulate female sexual maturation and longevity; wild-derived strains carry specific alleles that delay sexual maturation; and Nrip1 is involved in regulating sexual maturation and may affect longevity by regulating IGF1 level.

Genetically diverse mice are novel and valuable models of age-associated susceptibility to Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis, the disease due to Mycobacterium tuberculosis, is an important cause of morbidity and mortality in the elderly. Use of mouse models may accelerate insight into the disease and tests of therapies since mice age thirty times faster than humans. However, the majority of TB research relies on inbred mouse strains, and these results might not extrapolate well to the genetically diverse human population. We report here the first tests of M. tuberculosis infection in genetically heterogeneous aging mice, testing if old mice benefit from rapamycin. FINDINGS: We find that genetically diverse aging mice are much more susceptible than young mice to M. tuberculosis, as are aging human beings. We also find that rapamycin boosts immune responses during primary infection but fails to increase survival. CONCLUSIONS: Genetically diverse mouse models provide a valuable resource to study how age influences responses and susceptibility to pathogens and to test interventions. Additionally, surrogate markers such as immune measures may not predict whether interventions improve survival.

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

This study investigated the prevalence and progression of primary osteoarthritis (OA) in aged UM-HET3 mice. Using the Osteoarthritis Research Society International (OARSI) scoring system, we assessed articular cartilage (AC) integrity in 182 knee joints of 22-25 months old mice. Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), and the NLR family pyrin domain containing-3 (NLRP3) inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. Using micro-CT, we examined the correlations between subchondral bone (SCB) morphology traits and AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Finally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. In conclusion, our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

BACKGROUND: Primary osteoarthritis (OA) occurs without identifiable underlying causes such as previous injuries or specific medical conditions. Age is a major contributing factor to OA, and as one ages, various joint tissues undergo gradual change, including degeneration of the articular cartilage, alterations in subchondral bone (SCB) morphology, and inflammation of the synovium. METHODS: We investigated the prevalence of primary OA in aged, genetically diverse UM-HET3 mice. Articular cartilage (AC) integrity and SCB morphology were assessed in 182 knee joints of 22-25 months old mice using the Osteoarthritis Research Society International (OARSI) scoring system and micro-CT, respectively. Additionally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. RESULTS: Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13, inducible nitric oxide synthase, and the NLR family pyrin domain containing-3 inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. CONCLUSIONS: Our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Development of primary osteoarthritis during aging in genetically diverse UM-HET3 mice.

Background: Primary osteoarthritis (OA) occurs without identifiable underlying causes such as previous injuries or specific medical conditions. Age is a major contributing factor to OA, and as one ages, various joint tissues undergo gradual change, including degeneration of the articular cartilage, alterations in subchondral bone (SCB) morphology, and inflammation of the synovium. Methods: We investigated the prevalence of primary OA in aged, genetically diverse UM-HET3 mice. Articular cartilage (AC) integrity and SCB morphology were assessed in 182 knee joints of 22-25 months old mice using the Osteoarthritis Research Society International (OARSI) scoring system and micro-CT, respectively. Additionally, we explored the effects of methylene blue (MB) and mitoquinone (MitoQ), two agents that affect mitochondrial function, on the prevalence and progression of OA during aging. Results: Aged UM-HET3 mice showed a high prevalence of primary OA in both sexes. Significant positive correlations were found between cumulative AC (cAC) scores and synovitis in both sexes, and osteophyte formation in female mice. Ectopic chondrogenesis did not show significant correlations with cAC scores. Significant direct correlations were found between AC scores and inflammatory markers in chondrocytes, including matrix metalloproteinase-13, inducible nitric oxide synthase, and the NLR family pyrin domain containing-3 inflammasome in both sexes, indicating a link between OA severity and inflammation. Additionally, markers of cell cycle arrest, such as p16 and ß-galactosidase, also correlated with AC scores. In male mice, no significant correlations were found between SCB morphology traits and cAC scores, while in female mice, significant correlations were found between cAC scores and tibial SCB plate bone mineral density. Notably, MB and MitoQ treatments influenced the disease's progression in a sex-specific manner. MB treatment significantly reduced cAC scores at the medial knee joint, while MitoQ treatment reduced cAC scores, but these did not reach significance. Conclusions: Our study provides comprehensive insights into the prevalence and progression of primary OA in aged UM-HET3 mice, highlighting the sex-specific effects of MB and MitoQ treatments. The correlations between AC scores and various pathological factors underscore the multifaceted nature of OA and its association with inflammation and subchondral bone changes.

Targeting mitochondrial dysfunction using methylene blue or mitoquinone to improve skeletal aging.

Methylene blue (MB) is a well-established antioxidant that has been shown to improve mitochondrial function in both in vitro and in vivo settings. Mitoquinone (MitoQ) is a selective antioxidant that specifically targets mitochondria and effectively reduces the accumulation of reactive oxygen species. To investigate the effect of long-term administration of MB on skeletal morphology, we administered MB to aged (18 months old) female C57BL/J6 mice, as well as to adult male and female mice with a genetically diverse background (UM-HET3). Additionally, we used MitoQ as an alternative approach to target mitochondrial oxidative stress during aging in adult female and male UM-HET3 mice. Although we observed some beneficial effects of MB and MitoQ in vitro, the administration of these compounds in vivo did not alter the progression of age-induced bone loss. Specifically, treating 18-month-old female mice with MB for 6 or 12 months did not have an effect on age-related bone loss. Similarly, long-term treatment with MB from 7 to 22 months or with MitoQ from 4 to 22 months of age did not affect the morphology of cortical bone at the mid-diaphysis of the femur, trabecular bone at the distal-metaphysis of the femur, or trabecular bone at the lumbar vertebra-5 in UM-HET3 mice. Based on our findings, it appears that long-term treatment with MB or MitoQ alone, as a means to reduce skeletal oxidative stress, is insufficient to inhibit age-associated bone loss. This supports the notion that interventions solely with antioxidants may not provide adequate protection against skeletal aging.

Contributions of mouse genetic strain background to age-related phenotypes in physically active HET3 mice.

We assessed aging hallmarks in skin, muscle, and adipose in the genetically diverse HET3 mouse, and generated a broad dataset comparing these to individual animal diagnostic SNPs from the 4 founding inbred strains of the HET3 line. For middle- and old-aged HET3 mice, we provided running wheel exercise to ensure our observations were not purely representative of sedentary animals, but age-related phenotypes were not improved with running wheel activity. Adipose tissue fibrosis, peripheral neuropathy, and loss of neuromuscular junction integrity were consistent phenotypes in older-aged HET3 mice regardless of physical activity, but aspects of these phenotypes were moderated by the SNP% contributions of the founding strains for the HET3 line. Taken together, the genetic contribution of founder strain SNPs moderated age-related phenotypes in skin and muscle innervation and were dependent on biological sex and chronological age. However, there was not a single founder strain (BALB/cJ, C57BL/6J, C3H/HeJ, DBA/2J) that appeared to drive more protection or disease-risk across aging in this mouse line, but genetic diversity in general was more protective.

Lifespan effects in male UM-HET3 mice treated with sodium thiosulfate, 16-hydroxyestriol, and late-start canagliflozin.

Genetically heterogeneous UM-HET3 mice born in 2020 were used to test possible lifespan effects of alpha-ketoglutarate (AKG), 2,4-dinitrophenol (DNP), hydralazine (HYD), nebivolol (NEBI), 16α-hydroxyestriol (OH_Est), and sodium thiosulfate (THIO), and to evaluate the effects of canagliflozin (Cana) when started at 16 months of age. OH_Est produced a 15% increase (p = 0.0001) in median lifespan in males but led to a significant (7%) decline in female lifespan. Cana, started at 16 months, also led to a significant increase (14%, p = 0.004) in males and a significant decline (6%, p = 0.03) in females. Cana given to mice at 6 months led, as in our previous study, to an increase in male lifespan without any change in female lifespan, suggesting that this agent may lead to female-specific late-life harm. We found that blood levels of Cana were approximately 20-fold higher in aged females than in young males, suggesting a possible mechanism for the sex-specific disparities in its effects. NEBI was also found to produce a female-specific decline (4%, p = 0.03) in lifespan. None of the other tested drugs provided a lifespan benefit in either sex. These data bring to 7 the list of ITP-tested drugs that induce at least a 10% lifespan increase in one or both sexes, add a fourth drug with demonstrated mid-life benefits on lifespan, and provide a testable hypothesis that might explain the sexual dimorphism in lifespan effects of the SGLT2 inhibitor Cana.

Astaxanthin and meclizine extend lifespan in UM-HET3 male mice; fisetin, SG1002 (hydrogen sulfide donor), dimethyl fumarate, mycophenolic acid, and 4-phenylbutyrate do not significantly affect lifespan in either sex at the doses and schedules used.

In genetically heterogeneous (UM-HET3) mice produced by the CByB6F1 × C3D2F1 cross, the Nrf2 activator astaxanthin (Asta) extended the median male lifespan by 12% (p = 0.003, log-rank test), while meclizine (Mec), an mTORC1 inhibitor, extended the male lifespan by 8% (p = 0.03). Asta was fed at 1840 ± 520 (9) ppm and Mec at 544 ± 48 (9) ppm, stated as mean ± SE (n) of independent diet preparations. Both were started at 12 months of age. The 90th percentile lifespan for both treatments was extended in absolute value by 6% in males, but neither was significant by the Wang-Allison test. Five other new agents were also tested as follows: fisetin, SG1002 (hydrogen sulfide donor), dimethyl fumarate, mycophenolic acid, and 4-phenylbutyrate. None of these increased lifespan significantly at the dose and method of administration tested in either sex. Amounts of dimethyl fumarate in the diet averaged 35% of the target dose, which may explain the absence of lifespan effects. Body weight was not significantly affected in males by any of the test agents. Late life weights were lower in females fed Asta and Mec, but lifespan was not significantly affected in these females. The male-specific lifespan benefits from Asta and Mec may provide insights into sex-specific aspects of aging.

Canagliflozin retards age-related lesions in heart, kidney, liver, and adrenal gland in genetically heterogenous male mice.

Canagliflozin (Cana), a clinically important anti-diabetes drug, leads to a 14% increase in median lifespan and a 9% increase in the 90th percentile age when given to genetically heterogeneous male mice from 7 months of age, but does not increase lifespan in female mice. A histopathological study was conducted on 22-month-old mice to see if Cana retarded diverse forms of age-dependent pathology. This agent was found to diminish incidence or severity, in male mice only, of cardiomyopathy, glomerulonephropathy, arteriosclerosis, hepatic microvesicular cytoplasmic vacuolation (lipidosis), and adrenal cortical neoplasms. Protection against atrophy of the exocrine pancreas was seen in both males and females. Thus, the extension of lifespan in Cana-treated male mice, which is likely to reflect host- or tumor-mediated delay in lethal neoplasms, is accompanied by parallel retardation of lesions, in multiple tissues, that seldom if ever lead to death in these mice. Canagliflozin thus can be considered a drug that acts to slow the aging process and should be evaluated for potential protective effects against many other late-life conditions.

Age-related changes to adipose tissue and peripheral neuropathy in genetically diverse HET3 mice differ by sex and are not mitigated by rapamycin longevity treatment.

Neural communication between the brain and adipose tissues regulates energy expenditure and metabolism through modulation of adipose tissue functions. We have recently demonstrated that under pathophysiological conditions (obesity, diabetes, and aging), total subcutaneous white adipose tissue (scWAT) innervation is decreased ('adipose neuropathy'). With advanced age in the C57BL/6J mouse, small fiber peripheral nerve endings in adipose tissue die back, resulting in reduced contact with adipose-resident blood vessels and other cells. This vascular neuropathy and parenchymal neuropathy together likely pose a physiological challenge for tissue function. In the current work, we used the genetically diverse HET3 mouse model to investigate the incidence of peripheral neuropathy and adipose tissue dysregulation across several ages in both male and female mice. We also investigated the anti-aging treatment rapamycin, an mTOR inhibitor, as a means to prevent or reduce adipose neuropathy. We found that HET3 mice displayed a reduced neuropathy phenotype compared to inbred C56BL/6 J mice, indicating genetic contributions to this aging phenotype. Compared to female HET3 mice, male HET3 mice had worse neuropathic phenotypes by 62 weeks of age. Female HET3 mice appeared to have increased protection from neuropathy until advanced age (126 weeks), after reproductive senescence. We found that rapamycin overall had little impact on neuropathy measures, and actually worsened adipose tissue inflammation and fibrosis. Despite its success as a longevity treatment in mice, higher doses and longer delivery paradigms for rapamycin may lead to a disconnect between life span and beneficial health outcomes.

Long-term effects of canagliflozin treatment on the skeleton of aged UM-HET3 mice.

Sodium glucose cotransporter-2 inhibitors (SGLT2is) promote urinary glucose excretion and decrease plasma glucose levels independent of insulin. Canagliflozin (CANA) is an SGLT2i, which is widely prescribed, to reduce cardiovascular complications, and as a second-line therapy after metformin in the treatment of type 2 diabetes mellitus. Despite the robust metabolic benefits, reductions in bone mineral density (BMD) and cortical fractures were reported for CANA-treated subjects. In collaboration with the National Institute on Aging (NIA)-sponsored Interventions Testing Program (ITP), we tested skeletal integrity of UM-HET3 mice fed control (137 mice) or CANA-containing diet (180 ppm, 156 mice) from 7 to 22 months of age. Micro-computed tomography (micro-CT) revealed that CANA treatment caused significant thinning of the femur mid-diaphyseal cortex in both male and female mice, did not affect trabecular bone architecture in the distal femur or the lumbar vertebra-5 in male mice, but was associated with thinning of the trabeculae at the distal femur in CANA-treated female mice. In male mice, CANA treatment is associated with significant reductions in cortical bone volumetric BMD by micro-CT, and by quantitative backscattered scanning electron microscopy. Raman microspectroscopy, taken at the femur mid-diaphyseal posterior cortex, showed significant reductions in the mineral/matrix ratio and an increased carbonate/phosphate ratio in CANA-treated male mice. These data were supported by thermogravimetric assay (TGA) showing significantly decreased mineral and increased carbonate content in CANA-treated male mice. Finally, the sintered remains of TGA were subjected to X-ray diffraction and showed significantly higher fraction of whitlockite, a calcium orthophosphate mineral, which has higher resorbability than hydroxyapatite. Overall, long-term CANA treatment compromised bone morphology and mineral composition of bones, which likely contribute to increased fracture risk seen with this drug.

Rapamycin/metformin co-treatment normalizes insulin sensitivity and reduces complications of metabolic syndrome in type 2 diabetic mice.

Rapamycin treatment has positive and negative effects on progression of type 2 diabetes (T2D) in a recombinant inbred polygenic mouse model, male NONcNZO10/LtJ (NcZ10). Here, we show that combination treatment with metformin ameliorates negative effects of rapamycin while maintaining its benefits. From 12 to 30 weeks of age, NcZ10 males were fed a control diet or diets supplemented with rapamycin, metformin, or a combination of both. Rapamycin alone reduced weight gain, adiposity, HOMA-IR, and inflammation, and prevented hyperinsulinemia and pre-steatotic hepatic lipidosis, but exacerbated hyperglycemia, hypertriglyceridemia, and pancreatic islet degranulation. Metformin alone reduced hyperinsulinemia and circulating c-reactive protein, but exacerbated nephropathy. Combination treatment retained the benefits of both while preventing many of the deleterious effects. Importantly, the combination treatment reversed effects of rapamycin on markers of hepatic insulin resistance and normalized systemic insulin sensitivity in this inherently insulin-resistant model. In adipose tissue, rapamycin attenuated the expression of genes associated with adipose tissue expansion (Mest, Gpam), inflammation (Itgam, Itgax, Hmox1, Lbp), and cell senescence (Serpine1). In liver, the addition of metformin counteracted rapamycin-induced alterations of G6pc, Ppara, and Ldlr expressions that promote hyperglycemia and hypertriglyceridemia. Both rapamycin and metformin treatment reduced hepatic Fasn expression, potentially preventing lipidosis. These results delineate a state of "insulin signaling restriction" that withdraws endocrine support for further adipogenesis, progression of the metabolic syndrome, and the development of its comorbidities. Our results are relevant for the treatment of T2D, the optimization of current rapamycin-based treatments for posttransplant rejection and various cancers, and for the development of treatments for healthy aging.

Sex- and age-dependent genetics of longevity in a heterogeneous mouse population.

DNA variants that modulate life span provide insight into determinants of health, disease, and aging. Through analyses in the UM-HET3 mice of the Interventions Testing Program (ITP), we detected a sex-independent quantitative trait locus (QTL) on chromosome 12 and identified sex-specific QTLs, some of which we detected only in older mice. Similar relations between life history and longevity were uncovered in mice and humans, underscoring the importance of early access to nutrients and early growth. We identified common age- and sex-specific genetic effects on gene expression that we integrated with model organism and human data to create a hypothesis-building interactive resource of prioritized longevity and body weight genes. Finally, we validated Hipk1, Ddost, Hspg2, Fgd6, and Pdk1 as conserved longevity genes using Caenorhabditis elegans life-span experiments.

Lifespan benefits for the combination of rapamycin plus acarbose and for captopril in genetically heterogeneous mice.

Mice bred in 2017 and entered into the C2017 cohort were tested for possible lifespan benefits of (R/S)-1,3-butanediol (BD), captopril (Capt), leucine (Leu), the Nrf2-activating botanical mixture PB125, sulindac, syringaresinol, or the combination of rapamycin and acarbose started at 9 or 16 months of age (RaAc9, RaAc16). In male mice, the combination of Rapa and Aca started at 9 months and led to a longer lifespan than in either of the two prior cohorts of mice treated with Rapa only, suggesting that this drug combination was more potent than either of its components used alone. In females, lifespan in mice receiving both drugs was neither higher nor lower than that seen previously in Rapa only, perhaps reflecting the limited survival benefits seen in prior cohorts of females receiving Aca alone. Capt led to a significant, though small (4% or 5%), increase in female lifespan. Capt also showed some possible benefits in male mice, but the interpretation was complicated by the unusually low survival of controls at one of the three test sites. BD seemed to produce a small (2%) increase in females, but only if the analysis included data from the site with unusually short-lived controls. None of the other 4 tested agents led to any lifespan benefit. The C2017 ITP dataset shows that combinations of anti-aging drugs may have effects that surpass the benefits produced by either drug used alone, and that additional studies of captopril, over a wider range of doses, are likely to be rewarding.

Evaluation of matrix effects in analysis of estrogen using liquid chromatography-tandem mass spectrometry.

Matrix effects of different biological samples, including phosphate-buffered saline-bovine serum albumin (PBS-BSA), gelded horse serum, mouse serum, and mouse brain, were investigated for the determination of 17α- and ß-estradiol using derivatization with dansyl chloride prior to LC-MS/MS. Matrix effects were evaluated based on the slopes of regression lines plotted from results obtained in biological matrices versus pure standard solutions. Such plots indicate the enhancement or suppression of signal based on the presence of a particular biological fluid for a particular method. The matrix effects from PBS-BSA were similar to those of mouse serum. In contrast, analyses performed from horse serum and mouse brain yielded significant ion suppression, especially for 17ß-estradiol. Precipitation during derivatization was observed when pre-concentrated samples were processed with ethyl acetate as an extraction solvent. This was overcome with the use of methyl tert-butyl ether; however, matrix effects from this preparation were still present, evidenced by signal suppression and poor linearity in the standard curve. This work affirms that caution should be taken in the transfer of methods for use with different biological matrices, especially in the case where surrogate matrices are necessary for calibration purposes.

17-a-estradiol late in life extends lifespan in aging UM-HET3 male mice; nicotinamide riboside and three other drugs do not affect lifespan in either sex.

In genetically heterogeneous mice produced by the CByB6F1 x C3D2F1 cross, the "non-feminizing" estrogen, 17-α-estradiol (17aE2), extended median male lifespan by 19% (p < 0.0001, log-rank test) and 11% (p = 0.007) when fed at 14.4 ppm starting at 16 and 20 months, respectively. 90th percentile lifespans were extended 7% (p = 0.004, Wang-Allison test) and 5% (p = 0.17). Body weights were reduced about 20% after starting the 17aE2 diets. Four other interventions were tested in males and females: nicotinamide riboside, candesartan cilexetil, geranylgeranylacetone, and MIF098. Despite some data suggesting that nicotinamide riboside would be effective, neither it nor the other three increased lifespans significantly at the doses tested. The 17aE2 results confirm and extend our original reports, with very similar results when started at 16 months compared with mice started at 10 months of age in a prior study. The consistently large lifespan benefit in males, even when treatment is started late in life, may provide information on sex-specific aspects of aging.