Middle-aged Mice Research Articles

Abstract A003: The role of aging in promoting immune mediated reactivation from metastatic melanoma dormancy

Abstract Metastasis is the primary cause of melanoma death. A major problem with the vast majority of previous studies is that young mice (8-weeks old, equivalent to 20 human years) are used to investigate the mechanisms of metastatic outgrowth, whereas the median age of melanoma diagnosis in humans is ∼65 years. Advanced age also correlates with metastasis, mortality, and immune dysfunction. Moreover, most studies often only focus on lung metastases, despite melanoma commonly metastasizing to sites such as the liver in patients, which are significantly more resistant to checkpoint inhibitors (ICIs) compared to patients with lung metastases. We have begun addressing these core limitations by developing novel syngeneic mouse models of melanoma metastasis and colonization in young (8-week), middle-aged (12-month), and geriatric (18-22 month) mice, in which metastatic colonization/dissemination can be targeted to either the lung or liver alone, or to both simultaneously. Our data now show that aging promotes reactivation of dormant tumor cells and subsequent outgrowth of lung and liver metastases. We have found that frontline innate lymphocytes such as NK-cells and γδ T-cells are decreased in the pre-metastatic lung and livers of aged mice relative to young mice and remain decreased during early and late-stage metastasis. Using inducible KO mouse models and antibody depletion methods in young growth restrictive mice, we find that depletion of either NK-cells or γδ T-cells is sufficient to promote reactivation from dormancy and resistance to anti-PD1 therapy. Secondly, we further delineated changes in the post-metastatic lung and liver niche. We find that PROS1 secretion by reactivated melanoma cells expressing high levels of the MER tyrosine kinase receptor (MER) in the aged metastatic lung and liver increases CD11b myeloid cells expressing the immunosuppressive factor ARG1. Within this CD11b subset, we see an increase in polymorphonuclear myeloid derived suppressor cells (PMN MDSCs). Treatment with recombinant ARG1 promotes reactivation from dormancy in young mice whilst depletion of PMN MDSCs inhibits age induced outgrowth in the lung and liver. Overall, our study highlights common immune-mediated age-related changes in the pre and post metastatic lung and liver which contribute to aggressive metastatic initiation, reactivation from dormancy, progression, and resistance to anti-PD1 Citation Format: Kelly Coutant, Jhon Passamonte, Christopher Price, Pulkit Datt, Mitchell Fane. The role of aging in promoting immune mediated reactivation from metastatic melanoma dormancy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A003.

OxLDL/LOX-1 mediated sex, age, stiffness, and endothelial dependent alterations in mouse thoracic aortic vascular reactivity

Elevated plasma levels of oxidized low-density lipoprotein (oxLDL) are a risk factor and key component that accelerates and worsens cardiovascular disease fueling inflammation, plaque buildup and vascular damage. OxLDL can elicit its detrimental action via lectin-like oxLDL receptor 1 (LOX-1). In this study, we determined whether oxLDL, via LOX-1, alters aortic vascular reactivity and determined if sex and age differences exist. Thoracic aortic endothelium-intact or -denuded ring segments were isolated from 7 to 12 months old intact C57BL/6J female and male mice and pre-incubated with oxLDL ex vivo (50ug/dL; 2 h). Using wire myography, cumulative concentration-response curves to phenylephrine (PE) were generated to determine contractile responses. From these curves, the EC50 was determined and used to contract rings to assess acetylcholine (ACh) dependent relaxation. Calculated aortic stiffness and remodeling were also assessed. BI-0115 (10 μM; selective LOX-1 inhibitor) was used to determine LOX-1 dependence. We observed differential sex, age, endothelial cell, and LOX-1 dependent alterations to the efficacy of PE-induced contractile responses and ACh-mediated vasorelaxation in thoracic aortic rings following oxLDL exposure. Additionally, we observed a distinct sex and age effect on thoracic aortic stiffness following exposure to oxLDL. There was also a sex effect on calculated vessel diameter, as well as an age effect on oxLDL-mediated aortic remodeling that was LOX-1 dependent. Thus, LOX-1 inhibition and the resulting attenuation of oxLDL/endothelial-mediated alterations in aortic function suggests that there are differential sex differences in the role of oxLDL/LOX-1 in the thoracic aorta of middle-aged male and female mice. NEW and NOTEWORTHY. We investigated the effects of oxLDL via the LOX-1 receptor on murine thoracic aortic vasoreactivity, stiffness, and remodeling across age and sex. Acute exposure to oxLDL led to altered vasoreactivity, endothelial dysfunction, and changes in aortic stiffness and remodeling. These effects were in-part age, sex, endothelial, and LOX-1 dependent. This study reveals potential complex interactions in oxLDL/LOX-1-mediated vascular responses that could serve as potential therapeutic intervention for vascular diseases such as atherosclerosis and stroke.

Sex Influences on Hippocampal Kindling-Induced Seizures in Middle-Aged Mice

Sex Influences on Hippocampal Kindling-Induced Seizures in Middle-Aged Mice

Long-term administration of atorvastatin had no effect on the occurrence and progression of spontaneous hypertensive intracerebral hemorrhage in mice

Abstract Background Intracerebral hemorrhage (ICH) is a catastrophic disease with limited treatment options. Statins and low low-density lipoprotein cholesterol (LDL-C) levels have been reported to be associated with increased risk of ICH. Safety concerns of statins were raised as statins become the cornerstone of anti-atherosclerosis therapy. To date, the effect of statins on the incidence of ICH still remains a controversial topic. Purpose The aim of this study was to investigate the effect of atorvastatin on the occurrence and progression of hypertensive ICH in mice. Methods Male wild-type C57BL/6J mice, 13 months old, were randomly divided into atorvastatin (Ato, n=20) group and vehicle (Veh, n=20) group. The atorvastatin group and vehicle group were given atorvastatin (20 mg/kg) and same volume of 0.5% sodium carboxymethylcellulose by oral gavage for 8 weeks, separately. Angiotensin II (Ang II) and L-NAME were then used to induce hypertensive ICH. The time of stroke symptom onset was documented. Hemorrhage volumes were measured by spectrophotometric hemoglobin assay. Results After 8 weeks of atorvastatin or vehicle administration, serum LDL-C levels were significantly lower in the mice treated with atorvastatin than vehicle controls (0.705 vs. 1.008 mmol/L, P<0.001). The administration of atorvastatin did not show significant effects on the systolic blood pressure (SBP) levels when compared with the vehicle group (164 vs. 166 mmHg, P=0.497) after the induction of Ang II and L-NAME. Hemorrhage was observed in brain morphology, and was further verified by hematoxylin and eosin staining or MRI scans in mice that showed signs of stroke. During the 28-day experimental period, 75% (15/20) of mice in the atorvastatin group developed signs of ICH, and 70% (14/20) of mice in the vehicle group developed in the similar time window. The incidence of hypertensive ICH had no significant difference between atorvastatin and vehicle groups (Log-rank P=0.761). Hemorrhage volumes were also comparable between the two groups (1.462 vs. 1.392 uL, P=0.788). Conclusion Reducing serum LDL-C levels by long-term administration of atorvastatin had no effect on the occurrence and progression of spontaneous hypertensive ICH in middle-aged mice.

Blood pressure variability compromises vascular function in middle-aged mice.

Blood pressure variability (BPV) has emerged as a novel risk factor for cognitive decline and dementia, independent of alterations in average blood pressure (BP). However, the underlying consequences of large BP fluctuations on the neurovascular complex are unknown. We developed a novel mouse model of BPV in middle-aged mice based on intermittent Angiotensin II infusions. Using radio telemetry, we demonstrated that the 24-hr BP averages of these mice were similar to controls, indicating BPV without hypertension. Chronic (20-25 days) BPV led to a blunted bradycardic response and cognitive deficits. Two-photon imaging of parenchymal arterioles showed enhanced pressure-evoked constrictions (myogenic response) in BPV mice. Sensory stimulus-evoked dilations (neurovascular coupling) were greater at higher BP levels in control mice, but this pressure-dependence was lost in BPV mice. Our findings support the notion that large BP variations impair vascular function at the neurovascular complex and contribute to cognitive decline.

Glibenclamide Prevents Inflammation by Targeting NLRP3 Inflammasome Activation In Vitro

The NLRP3 inflammasome is known to play a significant role in the development of neurodegeneration and physiological aging, as well as the development of metabolic inflammation, which has generated significant interest in the scientific community in finding effective inhibitors of the NLRP3 inflammasome and assessing their effects. The purpose of this study was to evaluate the effect of pharmacological modulation of NLRP3 activity using an indirect NLRP3 inflammasome inhibitor, glibenclamide, on the expression of metaflammasome components in in vitro brain cells obtained from middle-aged mice. The study revealed that glibenclamide reduces the expression of pro-inflammatory markers NLRP3 and IL18 in cell culture, which in turn leads to the prevention of phosphorylation of protein kinases of the metaflammasome complex – PKR and IKKβ. However, we did not observe changes in the expression of pathologically phosphorylated IRS, as well as in the number of senescent cells in cultures after the exposure to glibenclamide.

Aged Gut Microbiome Induces Metabolic Impairment and Hallmarks of Vascular and Intestinal Aging in Young Mice.

Aging, an independent risk factor for cardiometabolic diseases, refers to a progressive deterioration in physiological function, characterized by 12 established hallmarks. Vascular aging is driven by endothelial dysfunction, telomere dysfunction, oxidative stress, and vascular inflammation. This study investigated whether aged gut microbiome promotes vascular aging and metabolic impairment. Fecal microbiome transfer (FMT) was conducted from aged (>75 weeks old) to young C57BL/6 mice (8 weeks old) for 6 weeks. Wire myography was used to evaluate endothelial function in aortas and mesenteric arteries. ROS levels were measured by dihydroethidium (DHE) staining and lucigenin-enhanced chemiluminescence. Vascular and intestinal telomere function, in terms of relative telomere length, telomerase reverse transcriptase expression and telomerase activity, were measured. Systemic inflammation, endotoxemia and intestinal integrity of mice were assessed. Gut microbiome profiles were studied by 16S rRNA sequencing. Some middle-aged mice (40-42 weeks old) were subjected to chronic metformin treatment and exercise training for 4 weeks to evaluate their anti-aging benefits. Six-week FMT impaired glucose homeostasis and caused vascular dysfunction in aortas and mesenteric arteries in young mice. FMT triggered vascular inflammation and oxidative stress, along with declined telomerase activity and shorter telomere length in aortas. Additionally, FMT impaired intestinal integrity, and triggered AMPK inactivation and telomere dysfunction in intestines, potentially attributed to the altered gut microbial profiles. Metformin treatment and moderate exercise improved integrity, AMPK activation and telomere function in mouse intestines. Our data highlight aged microbiome as a mechanism that accelerates intestinal and vascular aging, suggesting the gut-vascular connection as a potential intervention target against cardiovascular aging and complications.

Effects of aging on otolith morphology and functions in mice.

Increased fall risk caused by vestibular system impairment is a significant problem associated with aging. A vestibule is composed of linear acceleration-sensing otoliths and rotation-sensing semicircular canals. Otoliths, composed of utricle and saccule, detect linear accelerations. Otolithic organs partially play a role in falls due to aging. Aging possibly changes the morphology and functions of otoliths. However, the specific associations between aging and otolith changes remain unknown. Therefore, this study aimed to clarify these associations in mice. Young C56BL/6 N (8 week old) and old (108-117 weeks old) mice were used in a micro-computed tomography (μCT) experiment for morphological analysis and a linear acceleration experiment for functional analysis. Young C56BL/6 N (8 week old) and middle-aged (50 week old) mice were used in electron microscopy experiments for morphological analysis. μCT revealed no significant differences in the otolith volume (p = 0.11) but significant differences in the otolith density (p = 0.001) between young and old mice. μCT and electron microscopy revealed significant differences in the structure of striola at the center of the otolith (μCT; p = 0.029, electron microscopy; p = 0.017). Significant differences were also observed in the amplitude of the eye movement during the vestibulo-ocular reflex induced by linear acceleration (maximum amplitude of stimulation = 1.3G [p = 0.014]; maximum amplitude of stimulation = 0.7G [p = 0.015]), indicating that the otolith function was worse in old mice than in young mice. This study demonstrated the decline in otolith function with age caused by age-related morphological changes. Specifically, when otolith density decreased, inertial force acting on the hair cells decreased, and when the structure of striola collapsed, the function of cross-striolar inhibition decreased, thereby causing a decline in the overall otolith function.

Impacts of time-restricted feeding on middle-aged and old mice with obesity.

Time-restricted feeding is known to ameliorate obesity in young mice. However, evaluation of its effect in old age is still lacking. The current work aims to investigate the effects of time-restricted feeding on treating pre-existing obesity in old animals. The study utilized middle-aged and old high fat diet-induced obese mice and subjected them to 8h daily time-restricted feeding. Aged obese mice did not lose fat mass but lost lean mass after 8 weeks of treatment. In addition, time-restricted feeding reduced adiposity in brown adipose tissue, reversed excessive hepatic lipid accumulation, and improved glucose homeostasis in middle-aged and old obese mice. Mechanistic studies show that these metabolic benefits were mediated by transcriptional downregulation of essential genes responsible for hepatic adipogenesis and adipose tissue chronic inflammation. These results demonstrate that time-restricted feeding improves metabolic health and has beneficial effects in combating diet-induced obesity in aged obese mice. KEY POINTS: Contrary to in young obese mice, in old obese mice time-restricted feeding did not significantly reduce body fat but decreased lean mass. Time-restricted feeding reduced adipose tissue inflammation, reversed fatty liver, and improved glucose homeostasis in aged mice with diet-induced obesity. Time-restricted feeding is effective in improving metabolic homeostasis in aged mice, but less effective in terms of reducing obesity. Future studies should investigate the underlying mechanism of how ageing impaired intermittent fasting induced fat loss.

Hyperactive mTORC1/4EBP1 signaling dysregulates proteostasis and accelerates cardiac aging.

The mechanistic target of rapamycin complex 1 (mTORC1) has a major impact on aging by regulation of proteostasis. It is well established that mTORC1 signaling is hyperactivated with aging and age-related diseases. Previous studies have shown that partial inhibition of mTOR signaling by rapamycin reverses age-related deteriorations in cardiac function and structure in old mice. However, the downstream signaling pathways involved in this protection against cardiac aging have not been established. mTORC1 phosphorylates 4E-binding protein 1 (4EBP1) to promote the initiation of cap-dependent translation. The objective of this project is to examine the role of the mTORC1/4EBP1 axis in age-related cardiac dysfunction. We used a whole-body 4EBP1 KO mouse model, which mimics a hyperactive mTORC1/4EBP1/eIF4E axis, to investigate the effects of hyperactive mTORC1/4EBP1 axis in cardiac aging. Echocardiographic measurements of middle-aged 4EBP1 KO mice show impaired diastolic function and myocardial performance compared to age-matched WT mice and these parameters are at similar levels as old WT mice, suggesting that 4EBP1 KO mice experience accelerated cardiac aging. Old 4EBP1 KO mice show further decline in systolic and diastolic function compared to middle-aged counterparts and have worse systolic and diastolic function than age-matched WT mice. Gene expression levels of heart failure markers are not different between 4EBP1 KO and WT hearts. However, ribosomal biogenesis and protein ubiquitination are significantly increased in 4EBP1 KO hearts when compared to WT controls, suggesting dysregulated proteostasis in 4EBP1 KO hearts. Together, these results show that a hyperactive mTORC1/4EBP1 axis accelerates cardiac aging, potentially by dysregulating proteostasis.

8599 Development of an androgen receptor transgenic mouse that displays the complete attributes of Metabolic Syndrome

Abstract Disclosure: C. Alvarado: None. L.K. Beitel: None. M. Paliouras: None. M.A. Trifiro: None. The mouse androgen receptor (AR) shares over 90% homology with the human ortholog, but it lacks the CAG encoded poly-Q tract; instead, it contains a mixed glutamine/histidine tract. To study AR functionality in a more structured fashion, we created a knock-in mouse model that has only the equivalent of human poly-Q tract while maintaining the remaining mouse receptor sequence intact. Our humanized androgen receptor mouse (AR-19Q) displays a phenotype that is best described as Metabolic Syndrome (MetS). The mice gain excess weight, have hypertension, hyperglycemia, pancreatic islet cell abnormalities, and progressive non-alcoholic fatty liver disease (NAFLD). Features of MetS begin at approximately 4 months and progress till 12-14 months. Obesity: AR-19Q mice are significantly heavier, with a 35% gain of weight vs C57BL/6 mice. However, these mice are not hyperphagic at any time and eat identical quantities of chow. To assess obesity status, and found AR-19Q mice to have a significantly higher depositions of total white adipose visceral, lingual and subcutaneous vs age-matched controls. NAFLD: The progression of NAFLD is observed in AR-19Q mice. AR-19Q Histological analysis and immunohistochemistry reveal microvesicular steatosis, superseded by macrovesicular steatosis with hepatocyte balloon degeneration, scattered lobular inflammation (macrophages and lymphocytes) (6-9 months) and further superseded by perisinusoidal fibrosis (12 months). Biochemical blood analysis confirms. that there is a significant increase in alanine aminotransferase (ALT) levels, indicative of liver damage. With time classical NASH hepatitis and subsequent NASH fibrosis ensues; with several mice developing hepatocellular carcinoma (overall 20% of mice). Gene expression profiling AR-19Q mice for fatty liver genes, we observe an upregulation of inflammation genes Il-6, Il-10 and Tnf. In addition, increased Lpl (lipoprotein lipase) is indicative of increased blood triglyceride levels. On the other hand, down-regulation of G6pc (glucose-6-phosphatase) could be linked to glycogen storage disease. Down-regulation of Slc2a2 and Slc2a4, that encode GLUT2 and GLUT4, should have an influence on glucose levels, glucose homeostasis and insulin sensitivity, as insulin promotes glucose uptake through GLUT4. Type 2 Diabetes: Glucose intolerance is observed by 3-4 months followed by fasting hyperglycemia. Pancreases show advanced islet hyperplasia, concurrent with insulin resistance and type 2 diabetes. Pancreases of middle-age AR-19Q mice present with a significant islet hyperplasia, suggesting peripheral insulin resistance. We propose the use of the AR-19Q mice as a model to study the metabolic dysfunction/ deficiency leading to the MetS. The model will allow us to a better understanding into the mechanisms responsible for T2D and NAFLD-related disorders. Presentation: 6/2/2024

9285 Development of an androgen receptor transgenic mouse that displays the complete attributes of Metabolic Syndrome

Abstract Disclosure: C. Alvarado: None. L.K. Beitel: None. M. Paliouras: None. M.A. Trifiro: None. The mouse androgen receptor (AR) shares over 90% homology with the human ortholog, but it lacks the CAG encoded poly-Q tract; instead, it contains a mixed glutamine/histidine tract. To study AR functionality in a more structured fashion, we created a knock-in mouse model that has only the equivalent of human poly-Q tract while maintaining the remaining mouse receptor sequence intact. Our humanized androgen receptor mouse (AR-19Q) displays a phenotype that is best described as Metabolic Syndrome (MetS). The mice gain excess weight, have hypertension, hyperglycemia, pancreatic islet cell abnormalities, and progressive non-alcoholic fatty liver disease (NAFLD). Features of MetS begin at approximately 4 months and progress till 12-14 months. Obesity: AR-19Q mice are significantly heavier, with a 35% gain of weight vs C57BL/6 mice. However, these mice are not hyperphagic at any time and eat identical quantities of chow. To assess obesity status, and found AR-19Q mice to have a significantly higher depositions of total white adipose visceral, lingual and subcutaneous vs age-matched controls. NAFLD: The progression of NAFLD is observed in AR-19Q mice. AR-19Q Histological analysis and immunohistochemistry reveal microvesicular steatosis, superseded by macrovesicular steatosis with hepatocyte balloon degeneration, scattered lobular inflammation (macrophages and lymphocytes) (6-9 months) and further superseded by perisinusoidal fibrosis (12 months). Biochemical blood analysis confirms. that there is a significant increase in alanine aminotransferase (ALT) levels, indicative of liver damage. With time classical NASH hepatitis and subsequent NASH fibrosis ensues; with several mice developing hepatocellular carcinoma (overall 20% of mice). Gene expression profiling AR-19Q mice for fatty liver genes, we observe an upregulation of inflammation genes Il-6, Il-10 and Tnf. In addition, increased Lpl (lipoprotein lipase) is indicative of increased blood triglyceride levels. On the other hand, down-regulation of G6pc (glucose-6-phosphatase) could be linked to glycogen storage disease. Down-regulation of Slc2a2 and Slc2a4, that encode GLUT2 and GLUT4, should have an influence on glucose levels, glucose homeostasis and insulin sensitivity, as insulin promotes glucose uptake through GLUT4. Type 2 Diabetes: Glucose intolerance is observed by 3-4 months followed by fasting hyperglycemia. Pancreases show advanced islet hyperplasia, concurrent with insulin resistance and type 2 diabetes. Pancreases of middle-age AR-19Q mice present with a significant islet hyperplasia, suggesting peripheral insulin resistance. We propose the use of the AR-19Q mice as a model to study the metabolic dysfunction/ deficiency leading to the MetS. The model will allow us to a better understanding into the mechanisms responsible for T2D and NAFLD-related disorders. Presentation: 6/2/2024

An atlas of the aging mouse proteome reveals the features of age-related post-transcriptional dysregulation

To what extent and how post-transcriptional dysregulation affects aging proteome remains unclear. Here, we provide proteomic data of whole-tissue lysates (WTL) and low-solubility protein-enriched fractions (LSF) of major tissues collected from mice of 6, 15, 24, and 30 months of age. Low-solubility proteins are preferentially affected by age and the analysis of LSF doubles the number of proteins identified to be differentially expressed with age. Simultaneous analysis of proteome and transcriptome using the same tissue homogenates reveals the features of age-related post-transcriptional dysregulation. Post-transcriptional dysregulation becomes evident especially after 24 months of age and age-related post-transcriptional dysregulation leads to accumulation of core matrisome proteins and reduction of mitochondrial membrane proteins in multiple tissues. Based on our in-depth proteomic data and sample-matched transcriptome data of adult, middle-aged, old, and geriatric mice, we construct the Mouse aging proteomic atlas (https://aging-proteomics.info/), which provides a thorough and integrative view of age-related gene expression changes.

Longitudinal Study of Changes in Ammonia, Carbon Dioxide, Humidity and Temperature in Individually Ventilated Cages Housing Female and Male C57BL/6N Mice during Consecutive Cycles of Weekly and Bi-Weekly Cage Changes.

Housing conditions are essential for ensuring animal welfare and high-quality research outcomes. In this study, we continuously monitored air quality-specifically ammonia, carbon dioxide, relative humidity, and temperature-in Individually Ventilated Cages (IVCs) housing five female or male C57BL/6N mice. The cages were cleaned either weekly or bi-weekly, and the data were collected as the mice aged from 100 to 348 days. The survival rate remained above 96%, with body weight increasing by 35-52% during the study period. The ammonia levels rose throughout the cleaning cycle, but averaged below 25 ppm. However, in the older, heavier mice with bi-weekly cage cleaning, the ammonia levels reached between 25 and 75 ppm, particularly in the males. While circadian rhythms influenced the ammonia concentration only to a small extent, the carbon dioxide levels varied between 800 and 3000 ppm, increasing by 30-50% at night and by 1000 ppm with body weight. Humidity also correlated primarily with the circadian rhythms (10% higher at night) and, to a lesser extent, with body weight, reaching ≥70% in the middle-aged mice. The temperature variations remained minimal, within a 1 °C range. We conclude that air quality assessments in IVCs should be conducted during animals' active periods, and both housing density and biomass must be considered to optimise welfare.

Melatonin protects against sarcopenia in middle-aged mice.

Sarcopenia is a common age-related disease. Melatonin (MEL) is an age-related endocrine hormone, which displays a crucial role in resisting oxidative stress during aging. Importantly, the antioxidant properties of MEL can be mediated by mitochondria. Therefore, we wondered whether MEL could mitigate oxidative stress caused by mitochondria in sarcopenia. The middle-aged mice were administered 5 mg/kg/d and 10 mg/kg/d of MEL for 2 months. Young mice were used as the control group. After treatment with MEL, the grip strength of the fore/hind limbs, running time, and distance were elevated, and the weights of the gastrocnemius (GA), tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL) were enhanced in middle-aged mice. Additionally, MEL was observed to alleviate histological damage and increase the cross-sectional area of muscle fibers in GA tissues of middle-aged mice. Furthermore, following MEL treatment, there was an increase in the percentage and size of normal mitochondria as well as mtDNA copy number but a reduction in the levels of malondialdehyde (MDA), protein carbonyl, and reactive oxygen species (ROS) in the GA tissues of middle-aged mice. At the molecular level, MEL repressed the levels of ATROGIN-1, muscle RING-finger protein-1 (MURF-1), and the ratio of p-P38/P38, but elevated the expression of cytochrome c oxidase subunit 4 (COX4), cystatin C (CYTC), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in the GA tissues of middle-aged mice. Importantly, 10 mg/kg MEL was more efficacious in the treatment of sarcopenia than 5 mg/kg MEL. MEL attenuates sarcopenia in middle-aged mice, and the mechanism may relate to mitochondria-induced oxidative stress and the PGC-1α/TFAM pathway.

Age-dependent sex differences in cofilin1 pathway (LIMK1/SSH1) and its association with AD biomarkers after chronic systemic inflammation in mice

Chronic systemic inflammation (CSI) results in neuroinflammation and neurodegeneration. Cofilin1 is a stress protein that activates microglia and induces neuroinflammation, but its role in CSI at different aging stages remains unidentified. Therefore, the study aims to identify cofilin1 and its upstream regulators LIMK1 and SSH1 after CSI in young-, middle-, and advanced-aged mice. CSI was induced by injecting the male and female mice with a sub-lethal dose of Lipopolysaccharide weekly for six weeks. The results showed that normal male mice did not show cofilin pathway dysregulation, but a significant dysregulation was observed in CSI advanced-aged mice. In females, cofilin1 dysregulation was observed in healthy and CSI advanced-aged mice, while significant cofilin1 dysregulation was observed in middle-aged mice during CSI. Furthermore, cofilin1 pathway dysregulations correlated with Alzheimer's disease (AD) biomarkers in the brain and saliva, astrocyte activation, synaptic degeneration, neurobehavioral impairments, gut-microbiota abnormalities, and circulatory inflammation. These results provide new insights into cofilin1 sex and age-dependent mechanistic differences that might help identify targets for modulating neuroinflammation and early onset of AD.

Challenging Sarcopenia: Exploring AdipoRon in Aging Skeletal Muscle as a Healthspan-Extending Shield.

Sarcopenia, characterized by loss of muscle mass, quality, and function, poses significant risks in aging. We previously demonstrated that long-term treatment with AdipoRon (AR), an adiponectin receptor agonist, alleviated myosteatosis and muscle degeneration in middle-aged obese mice. This study aimed to determine if a shorter AR treatment could effectively offset sarcopenia in older mice. Two groups of old mice (20-23 months) were studied, one untreated (O) and one orally-treated with AR (O-AR) at 50 mg/kg/day for three months, compared with control 3-month-old young mice (Y) or 10-month-old young-adult mice (C-10). Results showed that AR remarkably inversed the loss of muscle mass by restoring the sarcopenia index and fiber count, which were greatly diminished with age. Additionally, AR successfully saved muscle quality of O mice by halving the accumulation of tubular aggregates and aberrant mitochondria, through AMPK pathway activation and enhanced autophagy. AR also bolstered muscle function by rescuing mitochondrial activity and improving exercise endurance. Finally, AR markedly curbed muscle fibrosis and mitigated local/systemic inflammation. Thus, a late three-month AR treatment successfully opposed sarcopenia and counteracted various hallmarks of aging, suggesting AR as a promising anti-aging therapy for skeletal muscles, potentially extending healthspan.

Effect of aging and exercise on hTERT expression in thymus tissue of hTERT transgenic bacterial artificial chromosome mice.

Telomere shortening occurs with aging in immune cells and may be related to immunosenescence. Exercise can upregulate telomerase activity and attenuate telomere shortening in immune cells, but it is unknown if exercise impacts other immune tissues such as the thymus. This study aimed to examine human telomerase reverse transcriptase (hTERT) alternative splicing (AS) in response to aging and exercise in thymus tissue. Transgenic mice with a human TERT bacterial artificial chromosome integrated into its genome (hTERT-BAC) were utilized in two different exercise models. Mice of different ages were assigned to an exercise cage (running wheel) or not for 3weeks prior to thymus tissue excision. Middle-aged mice (16months) were exposed or not to treadmill running (30min at 60% maximum speed) prior to thymus collection. hTERT transcript variants were measured by RT-PCR. hTERT transcripts decreased with aging (r = - 0.7511, p < 0.0001) and 3weeks of wheel running did not counteract this reduction. The ratio of exons 7/8 containing hTERT to total hTERT transcripts increased with aging (r = 0.3669, p = 0.0423) but 3weeks of voluntary wheel running attenuated this aging-driven effect (r = 0.2013, p = 0.4719). Aging increased the expression of senescence marker p16 with no impact of wheel running. Thymus regeneration transcription factor, Foxn1, went down with age with no impact of wheel running exercise. Acute treadmill exercise did not induce any significant changes in thymus hTERT expression or AS variant ratio (p > 0.05). In summary, thymic hTERT expression is reduced with aging. Exercise counteracted a shift in hTERT AS ratio with age. Our data demonstrate that aging impacts telomerase expression and that exercise impacts dysregulated splicing that occurs with aging.

Improvement of glucose metabolism in middle-aged mice on a high-fat diet by whole-grain highland barley is related to low methionine levels

Methionine restriction (MR) is an effective dietary strategy to regulate energy metabolism and alleviate oxidative stress and inflammation in the body, especially in the middle-aged and elderly population. However, the high methionine content of meat products makes this dietary strategy impossible to combine with protein supplementation and MR. Highland barley (HB), a low-methionine cereal, not only provides the body with protein but also has improved glucose metabolism and antioxidant and anti-inflammatory properties. Therefore, this study evaluated the feasibility of HB as a source of methionine-restricted dietary protein and the potential mechanisms. Middle-aged C57BL/6J mice were fed a control diet (CON), a high-fat diet (HFD), a whole-grain HB high-fat diet (HBHF), or a HBHF + methionine diet (HBHFmet) for 25 weeks. The results showed that the HBHF could keep the body weight, fasting glucose, insulin, homeostasis model assessment of insulin resistance (HOMA-IR), blood lipids, inflammation, and oxidative stress of HFD mice at normal levels. Compared with the HFD groups, HBHF inhibited pancreatic cell apoptosis and improved insulin secretion while improving hepatic and skeletal muscle glucose metabolism. However, these efficacies were attenuated in HBHFmet group mice. These findings suggest that HBHF has an MR strategy.

Abstract P228: Kdm6a Regulates Extracellular Matrix Genes During Arterial Stiffening and High Blood Pressure in Female Mice

Arterial stiffness measured by pulse wave velocity (PWV) increases steeply during menopause due to estradiol decline. Longitudinal studies show higher PWV in postmenopausal women compared to age-matched men. Previously, we have demonstrated increased PWV in ovariectomized (OVX) and middle-aged female mice. However, the impact of sex chromosomes on PWV is unknown in female mice. We hypothesize that a decline in estradiol unleashes the female sex chromosomes effect that promotes arterial stiffening. This study used four core genotype mice (n=6-10/group) of XX or XY sex chromosomes left intact or OVX for 8 weeks. Arterial stiffening was assessed with pulse wave Doppler and 24-hour blood pressure by radiotelemetry. Carotid artery passive myography and vascular reactivity of 2 nd order mesenteric artery were assessed by pressure myography. To induce hypertension, XX and XY mice were treated with deoxycorticosterone acetate (DOCA; 50 mg/pellet) for 21 days and 1% saline. In vitro, isolated aortic vascular smooth muscle cells (VSMC) were stretched on collagen matrix or treated with Kdm6a inhibitor GSKJ-1. PWV was significantly increased in OVX than in gonadal intact XX and XY mice (P&lt;0.001). However, an interaction effect was indicated with higher PWV in XX than XY OVX mice (4.9±0.1 vs. 4.2±0.2 m/s; P&lt;0.001), indicating a sex chromosome effect. Carotid artery stress-strain curves showed a leftward shift of XX than XY mice, suggesting increased structural stiffness. Masson’s trichrome staining of the aorta showed increased collagen in the adventitia of XX than XY (30% vs. 26%; P=0.005) mice. Higher systolic blood pressure (SBP) was indicated in XX than in XY mice (Baseline; 123±3 vs. 116±4 mmHg; P=0.02). Aortic RNAseq showed increased X-linked genes, including Kdm6a, in XX compared to XY mice. Stretched aortic VSMC showed increased Col1a1 and decreased Itga4 and Lama2 genes reversed by GSKJ-1. DOCA-induced hypertension blunted the PWV difference between XX and XY mice; however, SBP was higher in XX than in XY mice (Week 1: 135±13 vs.127±3 mmHg; P=0.03), but not weeks 2 and 3. We observed impaired acetylcholine-mediated relaxation in hypertensive XX than XY mice (47% vs. 24%; P=0.03). Our data suggested that OVX mice with XX sex chromosomes promote arterial stiffening and high blood pressure, and Kdm6a regulates extracellular matrix genes in XX female VSMC. DOCA-induced hypertension promotes endothelial dysfunction and blunts PWV in ovariectomized XX and XY mice.