Heterogeneous Mice Research Articles

Hypothalamic sex-specific metabolic shift by canagliflozin during aging

The hypothalamus undergoes significant changes with aging and plays crucial roles in age-related metabolic alterations. Sodium-glucose co-transporter 2 inhibitors (SGLT2i) are anti-diabetic agents that promote glucose excretion, and metabolic homeostasis. Recent studies have shown that a SGLT2i, Canagliflozin (Cana), can extend the median survival of genetically heterogeneous UM-HET3 male mice and improve central metabolic control via increases in hypothalamic insulin responsiveness in aged males, as well as reduced age-associated hypothalamic inflammation. We studied the long- and short-term effects of Cana on hypothalamic metabolic control in UM-HET3 mice. Starting the treatment from 7 months of age, we show that 4 weeks of Cana treatment significantly reduced body weight and fat mass in male but not female mice that was associated with enhanced glucose tolerance and insulin sensitivity observed by 12 months. Indirect calorimetry showed that Cana treatment increased energy expenditure in male, but not female mice, at 12 months of age. Long-term Cana treatment increased metabolic rates in both sexes, and markedly increasing formation of both orexigenic and anorexigenic projections to the paraventricular nucleus of the hypothalamus (PVH) mostly in females by 25 months. Hypothalamic RNA-sequencing analysis revealed increased sex-specific genes and signaling pathways related to insulin signaling, glycogen catabolic pathway, neuropeptide signaling, and mitochondrial function upregulated by Cana, with males showing a more pronounced and sustained effect on metabolic pathways at both age groups. Overall, our data provide critical evidence for sex-specific mechanisms that are affected by Cana during aging suggesting key targets of hypothalamic Cana-induced neuroprotection for metabolic control.

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.

The Gehan test identifies life-extending compounds overlooked by the log-rank test in the NIA Interventions Testing Program: Metformin, Enalapril, caffeic acid phenethyl ester, green tea extract, and 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride

The National Institute on Aging Interventions Testing Program (ITP) has so far identified 12 compounds that extend the lifespan of genetically heterogeneous mice using the log-rank test. However, the log-rank test is relatively insensitive to any compound that does not uniformly reduce mortality across the lifespan. This test may thus miss compounds that only reduce mortality before midlife, for example, a plausible outcome if a compound only mitigates risk factors before midlife or if its efficacy is reduced at later ages. We therefore reanalyzed all data collected by the ITP from 2004–2022 using the Gehan test, which is more sensitive to mortality differences earlier in the life course and does not assume a uniformly reduced mortality hazard across the lifespan. The Gehan test identified 5 additional compounds, metformin, enalapril, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), caffeic acid phenethyl ester (CAPE), and green tea extract (GTE), which significantly increased survival but were previously missed by the log-rank test. Three (metformin, enalapril, and 17-DMAG) were only effective in males and two (CAPE and GTE) were only effective in females. In addition, 1,3-butanediol, which by log-rank analysis increased survival in females but not males, increased survival in males by the Gehan test. These results suggest that statistical tests sensitive to non-uniformity of drug efficacy across the lifespan should be included in the standard statistical testing protocol to minimize overlooking geroprotective interventions.

Association of tameness and sociability but no sign of domestication syndrome in mice selectively bred for active tameness.

Domesticated animals have been developed by selecting desirable traits following the initial unconscious selection stage, and now exhibit phenotypes desired by humans. Tameness is a common behavioural trait found in all domesticated animals. At the same time, these domesticated animals exhibit a variety of morphological, behavioural, and physiological traits that differ from their wild counterparts of their ancestral species. These traits are collectively referred to as domestication syndrome. However, whether this phenomenon exists is debatable. Previously, selective breeding has been used to enhance active tameness, a motivation to interact with humans, in wild heterogeneous stock mice derived from eight wild inbred strains. In the current study, we used tame mice to study how selective breeding for active tameness affects behavioural and morphological traits. A series of behavioural and morphological analyses on mice showed an increased preference for social stimuli and a longer duration of engagement in non-aggressive behaviour. However, no differences were observed in exploratory or anxiety-related behaviours. Similarly, selection for tameness did not affect ultrasonic vocalisations in mice, and no changes were observed in known morphological traits associated with domestication syndrome. These results suggest that there may be a link between active tameness and sociability and provide insights into the relationship between tameness and other behaviours in the context of domestication.

Metformin treatment of juvenile mice alters aging-related developmental and metabolic phenotypes in sex-dependent and sex-independent manners.

Metformin has attracted increasing interest for its potential benefits in extending healthspan and longevity. This study examined the effects of early-life metformin treatment on the development and metabolism of C57BL/6J (B6) mice, with metformin administered to juvenile mice from 15 to 56days of age. Metformin treatment led to decreased body weight in both sexes (P < 0.05, t-test). At 9weeks of age, mice were euthanized and organ weights were recorded. The relative weight of retroperitoneal fat was decreased in females, while relative weights of perigonadal and retroperitoneal fat were decreased, and relative liver weight was increased in males (P < 0.05, t-test). Glucose and insulin tolerance tests (GTT and ITT) were conducted at the age of 7weeks. ANOVA revealed a significant impairment in insulin sensitivity by the treatment, and a significantly interactive effect on glucose tolerance between sex and treatment, underscoring a disparity in GTT between sexes in response to the treatment. Metformin treatment reduced circulating insulin levels in fasting and non-fasting conditions for male mice, with no significant alterations observed in female mice. qRT-PCR analysis of glucose metabolism-related genes (Akt2, Glut2, Glut4, Irs1, Nrip1, Pi3k, Pi3kca, Pkca) in the liver and skeletal muscle reveals metformin-induced sex- and organ-specific effects on gene expression. Comparison with previous studies in heterogeneous UM-HET3 mice receiving the same treatment suggests that genetic differences may contribute to variability in the effects of metformin treatment on development and metabolism. These findings indicate that early-life metformin treatment affects development and metabolism in both sex- and genetics-dependent manners.

DIETARY RESTRICTION OF ISOLEUCINE INCREASES THE HEALTHSPAN AND LIFESPAN OF GENETICALLY HETEROGENEOUS MICE

Abstract Low protein (LP) diets promote health and longevity in diverse species, andreducing dietary levels of the branched-chain amino acids (BCAAs) leucine, isoleucine and valine recapitulates these effects in C57BL/6J mice, promoting metabolic health in both sexes, and increases lifespan while reducing frailty in males. Each BCAA has unique metabolic effects, and we recently showed that restriction of isoleucine is both sufficient to promote metabolic health and required for the metabolic benefits of an LP diet in C57BL/6J males. Here, we tested the hypothesis that specifically restricting dietary isoleucine could promote healthy aging in adult genetically heterogenous UM-HET3 mice. We find that isoleucine restriction (IleR) improves the metabolic health of both young and old HET3 mice, promoting leanness and glycemic control in both sexes, and reprograms hepatic metabolism, blunting age-related molecular changes in males and, to a lesser extent, in females. Finally, we find that IleR reduces frailty and extends both the median and maximum lifespan of both male and female HET3 mice, but to a much greater degree in males. Our results demonstrate that restricting dietary isoleucine can increase health span and longevity in a genetically diverse population of mice and suggest that reducing dietary levels of isoleucine, or drug regimens that mimic this effect, may be a novel intervention to promote healthy aging.

THE ROLE OF CASTRATION BEFORE OR AFTER PUBERTY ON SEX DIFFERENCES IN GROWTH AND LONGEVITY IN UM-HET3 MICE

Abstract Sex differences in human aging and longevity are well known. Mortality rates of males are higher throughout life with no definitive mechanisms identified. Previously, we reported that genetically heterogeneous mice have sex differences in longevity like those of humans. Here, we report that prepubertal castration significantly reduced early adulthood mortality and increased median lifespan by 12.6% (n=236/group) to match female levels. It also shifted growth trajectories toward the female pattern, including a reduced rate of body weight gain and a prolonged growth period. Our findings suggest that testicular hormones are critical for these sex differences. We next tested whether a) exposure to gonadal hormones from puberty throughout life is required or b) only hormone exposure during the short period of pubertal development is sufficient to cause these sex differences. We castrated or sham-operated mice (n = 60/group) prepubertally at 20 days, or after puberty at 60 or 240 days. Prepubertal castration prolonged the period of growth as before, with dramatically increased fat mass and reduced lean mass, which reduced the lean/body mass ratio to a level indistinguishable from that of females. Although post-pubertal castrated groups also had reduced lean/body mass ratios, they were significantly higher than prepubertally castrated mice, but were unchanged between 60d and 240d groups. These results suggest that the narrow window of testicular hormone exposure during puberty is necessary for the full expression of sex differences in body composition trajectories. Its effect on lifespan will be presented at the meeting when mice are old enough for assessment.

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.

Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice

Low-protein diets promote health and longevity in diverse species. Restriction of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine recapitulates many of these benefits in young C57BL/6J mice. Restriction of dietary isoleucine (IleR) is sufficient to promote metabolic health and is required for many benefits of a low-protein diet in C57BL/6J males. Here, we test the hypothesis that IleR will promote healthy aging in genetically heterogeneous adult UM-HET3 mice. We find that IleR improves metabolic health in young and old HET3 mice, promoting leanness and glycemic control in both sexes, and reprograms hepatic metabolism in a sex-specific manner. IleR reduces frailty and extends the lifespan of male and female mice, but to a greater degree in males. Our results demonstrate that IleR increases healthspan and longevity in genetically diverse mice and suggests that IleR, or pharmaceuticals that mimic this effect, may have potential as a geroprotective intervention.

THU297 Metformin Treatment Of Juvenile Mice Alters Aging-Related Developmental And Metabolic Phenotypes In Sex-Dependent And -Independent Manners

Abstract Disclosure: Y. Zhu: None. M. Engmann: None. D. Medina: None. A. Bartke: None. B.S. Ellsworth: None. R. Yuan: None. Metformin is a widely used drug for the treatment of type 2 diabetes that also has a strong potential for long-term effects which may extend healthspan and longevity. In the current study, C57BL/6J (B6) juvenile female and male mice were treated with metformin between the ages of 15 and 56 days. Saline was used as the control. The results show that early-life treatment with metformin has profound effects on developmental and metabolic traits. Body weight was significantly reduced by metformin treatment in both sexes (P&amp;lt;0.05, t test). At 10 weeks of age, mice were euthanized, and organ weights were recorded. In treated males, weights of epididymal, perirenal, and brown fat depots were significantly reduced (P&amp;lt;0.05, t test). Pancreas weight was significantly reduced in both sexes (P&amp;lt;0.05, t test). Hypothalamus weight was significantly reduced in the females, and whole brain weight was significantly reduced in the males. Tail length as well as age of sexual maturation were not significantly altered in either sex. No significant difference was found in circulating insulin-like growth factor 1 (IGF1) or adiponectin. Circulating insulin was significantly reduced by the treatment under fasting and non-fasting conditions in the male mice, while in the females no significant difference was detected. Insulin sensitivity, calculated by the quantitative insulin-sensitivity check index (QUICKI), was suggestively increased in the male mice (P=0.062, t test). Glucose tolerance test (GTT) did not show a significant difference in treated female mice; however, comparing the area under curve, metformin treatment significantly improved the glucose tolerance of male mice (P=0.014, t test). Insulin tolerance test (ITT) showed no significant difference in the male mice; but, unexpectedly, in female mice, metformin treatment significantly reduced insulin sensitivity. This study revealed that early-life metformin treatment alters development and metabolism of mice in both sex-specific and non-specific manners, which may have long-term impacts on metabolism in aging. Interestingly, comparing the results of this study with previous studies of metformin-treated heterogenous UM-HET3 mice, further analyses revealed that metformin effects on body weight, age of sexual maturation, body size, IGF1, adiponectin, insulin, GTT, and ITT, are genetically dependent. Funding: SIU collaborative grant to Rong Yuan and Buffy S. Ellsworth. SIU Geriatric foundation by Andrzej Bartke Presentation: Thursday, June 15, 2023

Radiation cataract in Heterogeneous Stock mice after γ-ray or HZE ion exposure

Health effects of space radiation are a serious concern for astronauts on long-duration missions. The lens of the eye is one of the most radiosensitive tissues in the body and, therefore, ocular health risks for astronauts is a significant concern. Studies in humans and animals indicate that ionizing radiation exposure to the eye produces characteristic lens changes, termed “radiation cataract,” that can affect visual function. Animal models of radiation cataractogenesis have previously utilized inbred mouse or rat strains. These studies were essential for determining morphological changes and dose-response relationships between radiation exposure and cataract. However, the relevance of these studies to human radiosensitivity is limited by the narrow phenotypic range of genetically homogeneous animal models. To model radiation cataract in genetically diverse populations, longitudinal cataract phenotyping was nested within a lifetime carcinogenesis study in male and female heterogeneous stock (HS/Npt) mice exposed to 0.4 Gy HZE ions (n = 609) or 3.0 Gy γ-rays (n = 602) and in unirradiated controls (n = 603). Cataractous change was quantified in each eye for up to 2 years using Merriam-Focht grading criteria by dilated slit lamp examination. Virtual Optomotry™ measurement of visual acuity and contrast sensitivity was utilized to assess visual function in a subgroup of mice. Prevalence and severity of posterior lens opacifications were 2.6-fold higher in HZE ion and 2.3-fold higher in γ-ray irradiated mice compared to unirradiated controls. Male mice were at greater risk for spontaneous and radiation associated cataracts. Risk for cataractogenesis was associated with family structure, demonstrating that HS/Npt mice are well-suited to evaluate genetic determinants of ocular radiosensitivity. Last, mice were extensively evaluated for cataract and tumor formation, which revealed an overlap between individual susceptibility to both cancer and cataract.

Physical Resilience as a Predictor of Lifespan and Late-Life Health in Genetically Heterogeneous Mice.

Dynamic measures of resilience-the ability to resist and recover from a challenge-may be informative of the rate of aging before overt manifestations such as chronic disease, disability, and frailty. From this perspective mid-life resilience may predict longevity and late-life health. To test this hypothesis, we developed simple, reproducible, clinically relevant challenges, and outcome measures of physical resilience that revealed differences between and within age groups of genetically heterogeneous mice, and then examined associations between mid-life resilience and both lifespan and late-life measures of physiological function. We demonstrate that time to recovery from isoflurane anesthesia and weight change following a regimen of chemotherapy significantly differed among young, middle-aged, and older mice, and were more variable in older mice. Females that recovered faster than the median time from anesthesia (more resilient) at 12 months of age lived 8% longer than their counterparts, while more resilient males in mid-life exhibited better cardiac (fractional shortening and left ventricular volumes) and metabolic (glucose tolerance) function at 24 months of age. Moreover, female mice with less than the median weight loss at Day 3 of the cisplatin challenge lived 8% longer than those that lost more weight. In contrast, females who had more weight loss between Days 15 and 20 were relatively protected against early death. These data suggest that measures of physical resilience in mid-life may provide information about individual differences in aging, lifespan, and key parameters of late-life health.

LAMP2A, and other chaperone-mediated autophagy related proteins, do not decline with age in genetically heterogeneous UM-HET3 mice.

Chaperone-mediated autophagy (CMA) selectively degrades proteins that are crucial for glycolysis, fatty acid metabolism, and the progression of several age-associated diseases. Several previous studies, each of which evaluated males of a single inbred mouse or rat strain, have reported that CMA declines with age in many tissues, attributed to an age-related loss of LAMP2A, the primary and indispensable component of the CMA translocation complex. This has led to a paradigm in the field of CMA research, stating that the age-associated decline in LAMP2A in turn decreases CMA, contributing to the pathogenesis of late-life disease. We assessed LAMP2A levels and CMA substrate uptake in both sexes of the genetically heterogeneous UM-HET3 mouse stock, which is the current global standard for the evaluation of anti-aging interventions. We found no evidence for age-related changes in LAMP2A levels, CMA substrate uptake, or whole liver levels of CMA degradation targets, despite identifying sex differences in CMA.

Prepubertal castration eliminates sex differences in lifespan and growth trajectories in genetically heterogeneous mice.

Sex differences in aging and longevity have been widely observed, with females consistently outliving males across human populations. However, the mechanisms driving these disparities remain poorly understood. In this study, we explored the influence of post-pubertal testicular effects on sex differences in aging by prepubertally castrating genetically heterogeneous (UM-HET3) mice, a unique mouse model that emulates human sex differences in age-related mortality. Prepubertal castration eliminated the longevity disparity between sexes by reducing the elevated early- to mid-life mortality rate observed in males and extending their median lifespan to match that of females. Additionally, castration extended the duration of body weight growth and attenuated the inverse correlation between early-age body weight and lifespan in males, aligning their growth trajectories with those of females. Our findings suggest that post-pubertal testicular actions in genetically diverse mice are primarily responsible for sex differences in longevity as well as growth trajectories. These findings offer a foundation for further investigation into the fundamental mechanisms driving sex-specific aging patterns and the development of potential pro-longevity interventions.

Behavioral and neural network characterizations of the reinforcement of voluntary physical activity in heterogenous mice

Maintaining healthy and consistent levels of physical activity (PA) is a cost-effective means of reducing the onset of &gt;40-chronic diseases; however, these benefits go largely unrealized by the ~97% of adult Americans who fail to meet activity guidelines. Our prior work suggests that acute activity engages aversion-related brain regions, which may act as a barrier to PA adherence. Chronic PA is reinforcing and creates neural remodeling a across a network of brain regions belonging to the mesocorticolimbic system - comprising of stress and aversion-related brain regions [such as the basolateral amygdala (BLA)] and important reinforcement brain regions [i.e., the ventral tegmental area (VTA)]. A primary focus of this study is to determine how neural inputs to the nucleus accumbens (NAc) -the central point of convergence for this system– are engaged during acute and chronic PA. Objectives: Here we tested whether sex differences in the reinforcing value of wheel-running exist in a genetically diverse population of mice, the Heterogeneous Stock/Northport (HS/Npt). Moreover, we sought to identify the neural circuits engaged during acute and chronic voluntary wheel-running in these mice. Hypothesis: Given established findings that female rodents run more than males, we hypothesized that female HS/Npt mice would find wheel-running less aversive than males. We further hypothesized that 1-week of voluntary wheel-running would increase cFos expression (compared to mice with 1-week of wheel-lock) in brain regions associated with stress and aversion, whereas 4-weeks of wheel-running would increase neural engagement in brain regions associated with reinforcement, including the NAc, insular cortex, VTA and the medial preoptic area (a sexually dimorphic brain region critical for the estrogenic control of wheel-running in female rodents). Methods: As a proxy for their hedonic state, mice were provided a two-bottle choice saccharin preference test following acute or chronic PA. To better define the mesocorticolimbic system, we used an established retrograde tracer (rAAV-retro) to label the neural inputs to the NAc, which helped to identify important brain region involvement at two distinct stages of PA reinforcement. Results: There were no differences in running distance between female and male mice at 1- or 4-weeks and no differences in saccharin preference at 1-week. In contrast, male HS/Npt showed lower saccharin preference compared to females at 4-weeks of voluntary wheel running, suggesting a potential sex difference in the reinforcement value of chronic PA. Whole brain cFOS imaging is currently underway. Conclusions: It appears that chronic voluntary activity may confer lower hedonic states in male mice. Moreover, an improved understanding of how neural circuits across the mesocorticolimbic system are engaged during crtical stages of PA (in males and females) may prove important for determining neural and behavioral barriers to exercise adherence in humans. American College of Sports Medicine This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Early or Late-Life Treatment With Acarbose or Rapamycin Improves Physical Performance and Affects Cardiac Structure in Aging Mice.

Pharmacological treatments can extend the life span of mice. For optimal translation in humans, treatments should improve health during aging, and demonstrate efficacy when started later in life. Acarbose (ACA) and rapamycin (RAP) extend life span in mice when treatment is started early or later in life. Both drugs can also improve some indices of healthy aging, although there has been little systematic study of whether health benefits accrue differently depending on the age at which treatment is started. Here we compare the effects of early (4 months) versus late (16 months) onset ACA or RAP treatment on physical function and cardiac structure in genetically heterogeneous aged mice. ACA or RAP treatment improve rotarod acceleration and endurance capacity compared to controls, with effects that are largely similar in mice starting treatment from early or late in life. Compared to controls, cardiac hypertrophy is reduced by ACA or RAP in both sexes regardless of age at treatment onset. ACA has a greater effect on the cardiac lipidome than RAP, and the effects of early-life treatment are recapitulated by late-life treatment. These results indicate that late-life treatment with these drugs provide at least some of the benefits of life long treatment, although some of the benefits occur only in males, which could lead to sex differences in health outcomes later in life.

STAT-3 signaling role in an experimental model of nephropathy induced by doxorubicin.

The focal segmental glomerulosclerosis (FSGS) is one of the most frequent glomerulopathy in the world, being considered a significative public health problem worldwide. The disease is characterized by glomerular loss mainly due to inflammation process and collagen fibers deposition. STAT-3 is a transcription factor associated with cell differentiation, migration and proliferation and in renal cells it has been related with fibrosis, acting on the progression of the lesion. Considering this perspective, the present study evaluated the involvement of STAT-3 molecule in an experimental model of FSGS induced by Doxorubicin (DOX). DOX mimics primary FSGS by causing both glomerular and tubular lesions and the inhibition of the STAT3 pathway leads to a decrease in fibrosis and attenuation of kidney damage. We described here a novel FSGS experimental model in a strain of genetically heterogeneous mice which resembles the reality of FSGS patients. DOX-injected mice presented elevated indices of albuminuria and glycosuria, that were significantly reduced in animals treated with a STAT-3 inhibitor (STATTIC), in addition with a decrease of some inflammatory molecules. Moreover, we detected that SOCS-3 (a regulator of STAT family) was up-regulated only in STATTIC-treated mice. Finally, histopathological analyzes showed that DOX-treated group had a significant increase in a tubulointerstitial fibrosis and tubular necrosis, which were not identified in both control and STATTIC groups. Thus, our results indicate that STAT-3 pathway possess an important role in experimental FSGS induced by DOX and may be an important molecule to be further investigated.

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 9months 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.

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.

Abstract LB065: Genetically heterogeneous mouse models identify IFNg signaling as a shared signature of acquired resistance to immune checkpoint inhibitors

Abstract Immune checkpoint inhibitors (ICI) for cancer therapy have improved outcomes for a variety of malignancies, however many patients fail to benefit. To date, most pre-clinical studies investigating the tumor microenvironment (TME) have utilized different spontaneous or implanted tumor lines to separately investigate immunologically cold or hot TMEs. These models are often in different mouse strains, leading to confounding variables and a disconnect as to whether the tumor or the host is contributing to the observed immunological phenotype. To account for host diversity and reduce variation on the part of the tumor, we have developed immunotherapy models using genetically heterogeneous Diversity Outbred (DO) and recombinant inbred Collaborative Cross (CC) mice. We crossed DO mice with C57BL/6 (B6) to generate DOB6F1 mice that reliably accept B6-syngeneic B16F0 tumors after subcutaneous inoculation. DOB6F1 mice (n=142) treated with combined anti-PD1/anti-CTLA-4 ICI on days 3, 6, and 10 after inoculation exhibited a wide variation in tumor latency, up to a maximum of 65 days, with 19 mice never developing tumor. Quantitative Trait Locus analysis revealed multiple loci influencing response to ICI. We utilized this data to challenge 12 CCB6F1 strains selected based on predicted response where ICI outcomes range from non-responsive to near complete response. In addition, we show evidence that the DOB6F1 model recreates acquired resistance to ICI, with 9 mice having an extremely delayed tumor latency (&amp;gt;40 days). Melanin-free regions were observed in 3/9 of these tumors, suggesting tumor editing. Acquired resistance was also noted 3/10 ICI-treated CC051B6F1 mice. Whole transcriptomic analysis compared tumors from non-responder versus delayed latency DOB6F1 mice. Despite implantation within genetically heterogeneous mice, transcriptomic profiles from late-onset tumors cluster together. Gene Set Enrichment Analysis identified immune processes, with antigen processing and presentation as the most significantly dysregulated gene set. Many of the upregulated genes in late-onset tumors are driven by IFNγ, suggesting IFNγ signaling may contribute to immune escape. We tested this directly by culturing B16 cells with IFNγ prior to inoculation. Short exposure of B16 to IFNγ results in aggressive growth regardless of treatment, but long exposure increases tumor immunogenicity and responsiveness to ICI in inbred B6 mice. Experiments in ICI-responsive CT26-bearing BALB/c mice indicate IFNγ signaling contributes to ICI response, as demonstrated by increased tumor burden after a single 250 µg dose of neutralizing anti-IFNγ antibody concurrent with ICI treatment. Collectively, our DO and CC F1 models allow for reduced tumor variation with a focus on the host and associated TME, and we show differential roles of IFNγ in response to ICI based on the timing of IFNγ exposure. Citation Format: Justin B. Hackett, James Glassbrook, Nasrin Movahhedin, Madeline Bross, Alicia Kevelin, Maria Múniz, Heather Gibson. Genetically heterogeneous mouse models identify IFNg signaling as a shared signature of acquired resistance to immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB065.