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Intermittent Fasting Drives Metabolic Reprogramming Through Tissue-Specific and Shared Molecular Adaptations

ABSTRACT: Intermittent fasting (IF) has emerged as a powerful dietary intervention with significant metabolic benefits, yet the tissue-specific molecular mechanisms underlying these effects remain poorly understood. In this study, we employed a multi-omics approach, integrating proteomics and transcriptomics, to investigate the systemic and organ-specific adaptations to IF in male C57BL/6 mice. Following a 16-hour daily fasting regimen (IF16) over four months, IF significantly reduced blood glucose, HbA1c, and cholesterol levels while increasing ketone bodies, indicative of enhanced metabolic flexibility. Proteomic profiling of the liver, muscle, and cortex revealed tissue-specific responses, with the liver exhibiting the most pronounced changes, including upregulation of pathways involved in fatty acid oxidation, ketogenesis, and glycan degradation, alongside downregulation of steroid hormone biosynthesis and cholesterol metabolism. In muscle, IF’s proteomic profile indicates enhanced pyruvate metabolism, fatty acid biosynthesis, and AMPK signaling, while suppressing oxidative phosphorylation and thermogenesis. The cortex displayed unique adaptations, with upregulation of autophagy, PPAR signaling, and metabolic pathways, coupled with downregulation of TGF-beta and p53 signaling, suggesting a shift toward energy conservation and stress resilience. Notably, Serpina1C emerged as the only protein commonly upregulated across all three tissues, highlighting its potential role in systemic adaptation to IF. Integrative transcriptomic and proteomic analyses revealed partial concordance between mRNA and protein expression, underscoring the complexity of post-transcriptional regulation. Shared biological processes, such as synaptic vesicle localization, were identified across tissues, suggesting a unifying mechanism linking metabolic changes to cellular communication. These findings provide a comprehensive map of the molecular adaptations to IF, revealing both conserved and tissue-specific responses that optimize energy utilization, enhance metabolic flexibility, and promote cellular resilience. This study advances our understanding of the physiological benefits of IF and highlights potential therapeutic targets for metabolic and neurodegenerative disorders.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skeletal Muscle Cell, Liver, Cerebral Cortex, Leg Muscle

SUBMITTER: Rohan Lowe

LAB HEAD: Thiruma V. Arumugam

PROVIDER: PXD063124 | Pride | 2026-03-18

REPOSITORIES: Pride

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Project description:Cholesterol has attracted significant attention as a possible lifespan regulator. It has been reported that serum cholesterol levels have an impact on mortality due to age-related disorders such as cardiovascular disease. Diet is also known to be an important lifespan regulator. Dietary restriction retards the onset of age-related diseases and extends lifespan in various organisms. Although cholesterol and dietary restriction are known to be lifespan regulators, it remains to be established whether cholesterol is involved in dietary restriction-induced longevity. Here, we show that cholesterol deprivation suppresses longevity induced by intermittent fasting, which is one of the dietary restriction regimens that effectively extend lifespan. We also found that cholesterol is required for the fasting-induced upregulation of transcriptional target genes such as the insulin/IGF-1 pathway effector DAF-16 and that cholesterol deprivation suppresses the long lifespan of the insulin/IGF-1 receptor daf-2 mutant. Remarkably, we found that cholesterol plays an important role in the fasting-induced nuclear accumulation of DAF-16. Moreover, knockdown of the cholesterol-binding protein NSBP-1, which has been shown to bind to DAF-16 in a cholesterol-dependent manner and to regulate DAF-16 activity, suppresses both fasting-induced longevity and DAF-16 nuclear accumulation. Furthermore, this suppression was not additive to the cholesterol deprivation-induced suppression, which suggests that NSBP-1 mediates, at least in part, the action of cholesterol to promote fasting-induced longevity and DAF-16 nuclear accumulation. These findings identify a novel role for cholesterol in the regulation of lifespan. Two independent replicates were performed. Total RNA was extracted with TRIzol (Invitrogen). The extracted RNA was purified with PureLink RNA Micro Kit (Invitrogen) and analyzed with Agilent 2100 Bioanalyzer to assess the RNA integrity. The microarray procedures were performed according to Affymetrix protocols. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.

Project description:Intermittent fasting (IF) increases lifespan, decreases metabolic disease phenotypes, and cancer risk in model organisms, but the mechanisms mediating these effects are not fully characterized. In particular, the altered transcriptional programming has yet to be defined in key fasting responsive tissues such as liver from animals undergoing intermittent fasting. In this study, we employed every-other-day-fasting (EODF) in mice and high-resolution proteome analysis of liver and blood plasma as a screening tool to identify key regulated pathways with comparison to ad libitum fed animals. We observed many changes in the liver proteome abundance profile that were distinct from those observed after a single bout of fasting. Key among these were the induction by EODF of de novo lipogenesis (DNL) and cholesterol biosynthesis pathway enzymes, which were mirrored by related metabolite changes such as increased triacylglycerides and HMG-CoA in EODF liver of fed mice. Paradoxically, we also observed the up-regulation of mitochondrial proteins associated with fatty-acid beta oxidation including ACOT2, which is known to accelerate this pathway in vivo. The most surprising observation was the EODF-mediated 16-fold down-regulation of alpha-1-antitrypsin (SERPINA1E) in liver, which is an abundant plasma protein made exclusively in this tissue. Plasma proteome analysis confirmed a 3-fold decrease in SERPINA1E after 2 weeks of EODF among other significant changes such as increased levels of APOA4, a finding in common with previous human EODF intervention studies. We determined that in liver the SREBP1c and HNF4A transcription factors were playing a major role in the up-regulation of lipid/cholesterol synthesis and down-regulation of AAT, respectively. Further characterization of HNF4A function suggested a global inhibition of its ability to bind promoters of target genes in livers from EODF animals, which we hypothesize is mediated by either increased linoleic acid binding, or post translational modifications of HNF4A protein in EODF animal liver tissue. Together these data provide a comprehensive Omics resource highlighting the key changes observed during the intermittent fasting response in a model animal.

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Intermittent Fasting Drives Metabolic Reprogramming Through Tissue-Specific and Shared Molecular Adaptations

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