Background: The burden of metabolic dysfunction-associated steatotic liver disease (MASLD) is of immediate concern, as its prevalence is increasing worldwide. MASLD often progresses to liver fibrosis, posing significant health risks. Age-independent non-invasive tools to evaluate fibrosis are needed to improve diagnostic accuracy across all age groups.
Methods: 84 inflammatory, hematological, and metabolic variables were quantified in the blood of n=63 individuals with MASLD with different degrees of fibrosis and n=22 age-matched controls. Linear regression models were employed to identify markers strongly correlated with liver fibrosis but not influenced by age. Logistic regression models were used to evaluate the ability of various indexes to discriminate between no/mild and severe liver fibrosis.
Results:. Levels of glutamine and propionate were identified as strongly correlated to fibrosis but not age and combined to form the GP index. The GP index demonstrated superior predictive power for liver fibrosis compared to existing scores, like circulating creatinine. It showed higher discriminatory ability (AUC = 0.872) and better model fit, indicating its robustness and reliability across all age groups.
Conclusions: The study introduces the GP index, an age-independent tool for diagnosing and monitoring liver fibrosis in MASLD patients. By excluding age-dependent markers, the GP index can potentially reduce false positives and improve diagnostic accuracy, particularly in older populations. The combination of glutamine and propionate in this index reflects a novel approach, capturing both intrinsic hepatic metabolic changes and extrinsic influences from gut microbiota, offering a simple yet effective solution for liver fibrosis staging.

On behalf of Working Package 1 Spoke 3 AGE-IT

Accumulation of lipids in the liver characterizes metabolic dysfunction-associated steatotic liver disease (MASLD), the most prevalent chronic liver disease worldwide. Here, the first knockout mouse model for mitogen-activated protein kinase 15 (MAPK15) was characterized, revealing a critical role for this protein in controlling lipid homeostasis in the liver. Indeed, Mapk15^-/- mice exhibited liver steatosis in the context of a MASLD-like phenotype while hepatocellular in vitro models allowed us to demonstrate that dysregulated accumulation of lipids was due to increased expression and membrane localization of the CD36 fatty acid translocase. Consistently, Mapk15^-/- mice exhibited elevated hepatic levels of CD36 and feeding them with a western-type high-fat diet significantly accelerated their progression to a steatohepatitis-like phenotype. Importantly, transcriptomic analysis of human cohorts revealed increased liver expression of MAPK15 in MASLD patients, suggesting a compensatory role in disease progression and opening to the possibility of counteracting hepatic steatosis in humans by pharmacologically or genetically activating this MAP kinase. Overall, our data highlight a critical role for MAPK15 in liver physiopathology, by contributing to maintaining physiological intracellular levels of lipids in this tissue. Importantly, as preclinical studies have demonstrated that reverting hepatic steatosis is also able to resolve liver inflammation, liver fibrosis, and diabetes, our data provide a new actionable therapeutic target with potential for preventing or reverting MASH and, possibly, contribute to reduce its frequent debilitating and deadly consequences, i.e. cirrhosis and HCC.

Age-related changes in mitochondrial DNA integrity and copy number (mtDNA-CN) are strongly associated with coronary artery disease (CAD) and adverse cardiovascular events. Mitochondrial dysfunction—particularly when aggravated by environmental stressors—may play a key role in accelerating vascular aging and increasing susceptibility to atherosclerosis and early-onset coronary artery disease (EOCAD), which is rising at an alarming rate.

To investigate this hypothesis, we examined the relationship between leukocyte mtDNA-CN and the mtDNA⁴⁹⁷⁷ deletion and the risk of EOCAD in 118 patients (≤60 years) compared to 150 healthy controls. Quantitative RT-PCR was employed to measure mtDNA-CN and the frequency of the mtDNA⁴⁹⁷⁷ deletion.

In parallel, we assessed mtDNA copy number and mitochondrial gene expression profiles in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) under two senescence models: replicative senescence (RS) and stress-induced premature senescence (SIPS).

Our study revealed that EOCAD is associated with a high prevalence of modifiable cardiovascular risk factors, as well as increased mitochondrial damage and dysfunction. In vitro, our preliminary findings suggest that both RS and SIPS elicit comparable mitochondrial changes in HUVECs and VSMCs. These results highlight the potential of mitochondrial-targeted therapies to mitigate vascular aging and damage stemming from mitochondrial dysfunction.

Background
Copper and zinc are essential for hematopoiesis and oxidative balance. Their ratio (Cu/Zn) has been implicated in immune dysfunction, anemia, and cardiovascular risk. This study examined the association between the Cu/Zn ratio and anemia in older adults with reduced kidney function using data from NHANES 2011–2012.
Methods
A cross-sectional analysis was performed on 5,972 participants (mean age: 60 ± 7 years) with available data on serum copper, zinc, hemoglobin, kidney function (eGFR via CKD-EPI 2021), and chronic diseases. Anemia was defined using WHO criteria. Participants were divided into Cu/Zn tertiles. Linear regression assessed the relationship between Cu/Zn and hemoglobin levels; logistic regression examined the risk of anemia across tertiles. Models were adjusted for age, sex, race, eGFR, and comorbidities.
Results
Higher Cu/Zn ratios were inversely associated with hemoglobin levels, with stronger effects in those with eGFR < 90 ml/min/1.73 m². Compared to the lowest tertile (Cu/Zn = 0.353–1.22), the second tertile was associated with higher anemia risk (OR: 1.03, 95% CI: 1.01–1.07), which increased further in the highest tertile (OR: 1.10, 95% CI: 1.07–1.18).
Conclusions
An elevated Cu/Zn ratio is independently associated with increased anemia risk in older adults, especially those with reduced kidney function. These findings underscore the relevance of trace metal homeostasis in hematologic health and suggest the Cu/Zn ratio may serve as a useful marker in assessing anemia risk in CKD populations.

Biological ageing is no longer an abstract concept—it is a measurable, reversible process with profound implications for public and occupational health. Over the past three years, the BioAgingLab (Occupational Medicine Unit, University of Padua) has played a leading role in AGE-IT (Spoke 2 – WP4_Task 4.11), developing translational strategies to identify and mitigate environmental, occupational, and lifestyle-driven accelerators of ageing.

Through a multi-omics platform integrating digital PCR and Next Generation Sequencing, we assessed epigenetic age (DNAmAge), telomere length (TL), and mitochondrial DNA copy number (mtDNAcn) in ageing workers, patients with IPF and mild cognitive impairment, and participants in space analogue confinement models. Key ageing accelerators were identified, including low birth weight, oxidative stress linked to inactivity and circadian disruption, and even mild or asymptomatic SARS-CoV-2 infections.

In parallel, we tested two non-pharmacological interventions: Monarda didyma L. extract, which stabilized biological age markers and improved inflammation and quality of life, and Cognitive Activation Therapy (CAT), which enhanced telomere length and cognitive function, especially in men with mild neurocognitive disorders.

Our results lay the groundwork for scalable, biomarker-guided screening tools and prevention frameworks. Beyond research, BioAgingLab’s impact has reached public audiences and institutions through national outreach events (Via Salutis, Science4All, ASI Roma, INNOFARM). Looking ahead, we are exploring the launch of a spin-off that integrates biotech, digital health, and personalized prevention—bringing science to life for individuals, employers, and health systems.

Introduction
Sarcopenia, frailty, and malnutrition are prevalent geriatric syndromes with significant implications for morbidity, mortality, and quality of life in older adults. These conditions often overlap and share complex pathophysiological mechanisms—including mitochondrial dysfunction, chronic inflammation, and neurodegeneration. We aimed at identifying reliable blood-based biomarkers that could improve early diagnosis and guide personalized interventions for these common geriatric syndromes.

Methods

This investigation was conducted within the framework of the Age-It project, a national initiative funded by Italy’s National Recovery and Resilience Plan (PNRR), focused on advancing the science of aging.

Through a Delphi consensus involving biologists and physicians from various Italian universities, we identified a panel of key biomarkers: FGF21 and GDF15, both mitokines released in response to mitochondrial stress; sRAGE, a soluble form of the receptor for advanced glycation end products (RAGE), generated through the shedding of membrane-bound RAGE, which acts as a decoy receptor by competitively inhibiting RAGE-ligand interactions and attenuating downstream inflammatory responses; and NfL, a marker of axonal damage. Participants were recruited from the FRASNET cohort which enrolled healthy community-dwelling volunteers in 2017-2020 with the aim of investigating renal aging, frailty, and sarcopenia. Participants underwent multidimensional assessments, including anthropometric evaluations, bioelectrical impedance analysis, frailty and malnutrition measurements, and blood sampling. Collected blood samples were subsequently bio-banked for future analyses. Follow-up assessments were conducted between 2023 and 2024. During follow-up, participants who attended underwent repeated multidimensional evaluations, and additional blood samples were collected and bio-banked. Blood biomarker determination was conducted using the ELLA method. Age- sex and comorbidity adjusted linear regression analyses and Spearman correlations were performed to assess the association between biomarkers and frailty, malnutrition and sarcopenia.

Results

Selected biomarkers were measured in samples of 52 individuals. The median age of this subgroup was 76 years, with 38.5% being male. The median Body Mass Index was 26.7, and the prevalence of frailty was 21%, with a median frailty index of 0.13. Cognitive performance in this group was generally high, with a median Mini-Mental State Examination score of 29, while nutritional status was good, indicated by a median Mini Nutritional Assessment Short Form score of 14.

NfL levels in 2017-2020 were found to be cross-sectionally associated with higher scores on the Fatigue Severity Scale (B = 0.05, 95% C.I. 0.02 - 0.87, p=0.005) prospectively associated with both the risk of sarcopenia (B = 0.074; 95% C.I. 0.034 - 0.11, p = 0.001), the decline in physical performance (B = - 0.55; 95% C.I. -0.10 - -0.01, p=0.02) the risk of malnutrition (B – 0.4, 95% C.I. -0.07 - -0.004, p = 0.03), physical frailty (r = 0.29, p = 0.046) and ability in performing activities of daily living (r=-0.34, p = 0.02). Plasma Nfl in 2023-2024 correlated with cognitive performance (r= -0.27, r = 0.003), physical performance battery score (r= -0.29, p = 0.002), muscle thickness (r = -0.21, p = 0.02), gait sped (r= -0.27, p = 0.003) and ability in performing instrumental activity of daily living (r = -0.27, r = 0.003).

FGF21 levels were found to be both cross-sectionally (B = -0.06, 95% C.I. -0.12 - 0.001, p= 0.05) and longitudinally associated with lower physical activity levels (B = - 0.06; 95% C.I. -0.11 - -0.005, p=0.03), malnutrition (B – 0.002, 95% C.I. -0.005 – 0.00005, p = 0.05) and reduced muscle mass (B = - 0.012; 95% C.I. -0.02 - -0.004, p=0.004). sRAGE levels were found to be longitudinally associated with reduced calf circumference (B = - 0.002; 95% C.I. -0.003 - -0.00008, p=0.04) and risk of malnutrition (B – 0.001, 95% C.I. -0.002 - -0.0002, p =0.01). GDF15 levels were found to be cross-sectionally associated with an increased risk of physical frailty (B = 0.0004, 95% C.I. 0.000001 - 0.001, p = 0.049), greater fatigue (B = 0.008, 95% C.I. 0.002 - 0.01, p=0.009), and poorer physical performance (B = -0.001, 95% C.I. -0.001 - -0.00009, p = 0.03) at baseline. Additionally, during follow-up visits, GDF15 levels were cross-sectionally associated with poorer balance (B = -0.001, 95% C.I. -0.001 - -0.0009, p = 0.009) and lower physical activity levels (B = -0.03, 95% C.I. -0.04 - -0.009, p= 0.005) in older adults. Longitudinally, elevated GDF15 levels were linked to an increased risk of sarcopenia (B = 0.002; 95% C.I. 0.001 - 0.002, p < 0.001), lower physical activity levels (B = - 0.03; 95% C.I. -0.04 - -0.01, p=0.003), poorer physical performance (B = - 0.001; 95% C.I. -0.002 - -0.00005, p=0.04) and increased risk of malnutrition (B -0.001, 95% C.I. -0.002 - -0.0004, p = 0.006).

Conclusions

In a population of community-dwelling older Italian adults, NfL, GDF15, FGF21, and sRAGE emerged as valuable biomarkers capturing different facets of age-related vulnerability, particularly sarcopenia, malnutrition, and frailty. Each biomarker reflected distinct underlying biological processes—mitochondrial stress, chronic inflammation, and neurodegeneration—suggesting that the heterogeneity in their associations mirrors the complex, multifactorial nature of these geriatric syndromes.