Differential protein expression and functional analysis in response to intermittent fasting across multiple tissues.

(A) Heatmap of proteins significantly differentially expressed in IF group and AL group in liver (A), in muscle (C) and in cerebral cortex (E). The color scale denotes z-score normalized protein abundances. Red denotes higher protein abundance, and blue lower protein abundance. (B) Volcan plot of all protein expression in IF groups in comparison to the AL group in liver (B), in muscle (D) and in cerebral cortex (F). Each dot represents a protein. Red dots showing protein with p<0.05 and |log2FC|>1. Bule dots showing proteins with p <0.05 and 0<|log2FC|<1. Green dots showing proteins with p>0.05 and |log2FC>1|. (G) Enrichment analysis of modulated processes and pathways in IF groups in comparison to the AL group in both upregulated and downregulated pathways in liver (G,H), muscle (I, J) and cerebral cortex (K, L). The FDR was controlled at 0.05. Each bar represents a functional process or pathway. Red denotes higher protein abundance, and blue lower protein abundance. n = 7 mice in each group.

Differential expression patterns of proteins, functional analysis, and associated protein networks across multiple tissues in response to intermittent fasting across multiple tissues.

(A) Heatmap of significantly different proteins involved in metabolic process in IF group in comparison to AL group in liver (A), muscle (B) and cerebral cortex (C). (D) Heatmap of significantly different proteins involved in signaling pathways in IF group in comparison to AL group in liver (D), muscle (E) and cerebral cortex (F). (G) Functional transition network that shows metabolic processes (G) and fatty acid metabolism (H, I) in response to IF in liver and metabolic processes (K, L) in response to IF in muscle. (J) Heatmap of fatty acid metabolism related protein expression in response to IF in liver. (M) Heatmap of metabolic pathways related protein expression in response to IF in muscle. The FDR was controlled at 0.05 for GO biological processes and pathway databases. The functional transition network was based one gene set similarity as p value<0.01. n = 7 mice in each group.

Integrative analysis of protein expression and functional enrichment across multiple tissues.

(A) Venn diagram illustrating common proteins shared among the liver, muscle, and cerebral cortex. (B) Venn diagram depicting common differentially expressed proteins across the liver, muscle, and cortex. (C) Expression levels of Serpin A1c in the liver, muscle, and cerebral cortex. (D) alpha1-antitrypsin staining in green in the liver of both AL and IF mice. n = 3 mice in each group. (E) Heatmap of overlapping differentially expressed proteins between the liver and muscle. (F) Heatmap of overlapping differentially expressed proteins between the liver and cerebral cortex. (G) Heatmap of overlapping differentially expressed proteins between the cerebral cortex and muscle. (H) Venn diagram highlighting common biological processes shared among the liver, muscle, and cerebral cortex. data was presented as mean ± SEM with p<0.05 was defined as significantly different. n = 7 mice in each group.

Multi-omics integration reveals co-regulated transcriptomic and proteomic networks in response to intermittent fasting across multiple tissues.

Integrated heatmaps displaying the expression profiles of the robust molecular signatures selected by DIABLO in liver (A), muscle (C), and cerebral cortex (E). Relevance interaction networks visualizing the functional connectivity and high-confidence correlations between transcripts and proteins in liver (B), muscle (D), and cerebral cortex (F). The integrated signatures were identified using supervised N-integration block sparse PLS-DA (DIABLO) with 10-repeated 7-fold cross-validation to minimize the balanced error rate. n = 7 mice in each group.

Experimental design and metabolic analysis.

Male mice were divided into two groups: Ad Libitum (AL) and 16:8 fasting (IF) (A). Metabolic parameters measured at termination included: fasting blood glucose (B), HbA1c (C), ketone bodies (D), and total cholesterol (E) (mean ± SEM; two-tailed paired t-test: **p<0.01, ***p<0.001, ****p<0.0001 vs AL). Panels F and G show weekly body weight trajectories and net weight change (mean ± SEM; two-way ANOVA with Tukey’s post hoc: *p<0.05, **p<0.01 vs AL). (n=8 per group). Panel A created using BioRender.com.

3D Principal component analysis (PCA) of proteomic profiles.

3D PCA plots visualizing the separation of proteomic profiles between the AL group and IF group in liver (A), muscle (B), and cerebral cortex (C). Combined 3D PCA plot illustrating the global separation of all samples across the three tissues (Liver, Muscle, Cortex) and dietary conditions (AL vs. IF) (D). Each dot represents an individual biological replicate (n = 7 mice per group).

Network mapping of IF-responsive proteins in liver

Protein-protein interaction networks generated using STRING analysis highlight multiple functional pathways associated with proteins differentially expressed in liver in response to intermittent fasting. Red denotes increased expression, and blue denotes decreased expression. n= 7 each group.

Network mapping of IF-responsive proteins in muscle

Protein-protein interaction networks generated using STRING analysis highlight multiple functional pathways associated with proteins differentially expressed in muscle in response to intermittent fasting. Red denotes increased expression, and blue denotes decreased expression. n= 7 each group.

Network mapping of IF-responsive proteins in cortex

Protein-protein interaction networks generated using STRING analysis highlight multiple functional pathways associated with proteins differentially expressed in cortex in response to intermittent fasting. Red denotes increased expression, and blue denotes decreased expression. n= 7 each group.

Shared differentially expressed proteins across organs in response to intermittent fasting.

Heatmaps depict differentially expressed proteins common to (A) liver and muscle, (B) liver and cortex, and (C) cortex and muscle. Protein expression levels are represented by colours, corresponding to normalized protein intensities. Red denotes increased expression, and blue denotes decreased expression. n= 7 each group.

3D Principal component analysis (PCA) of transcriptomic profiles.

3D PCA plots visualizing the separation of transcriptomic proteomic profiles between the AL group and IF group in liver (A), muscle (B), and cerebral cortex (C). Combined 3D PCA plot illustrating the global separation of all samples across the three tissues (Liver, Muscle, Cortex) and dietary conditions (AL vs. IF) (D). Each dot represents an individual biological replicate (n = 7 mice per group).

Correlation between transcriptomic and proteomic profiles in the integrated DIABLO model.

Scatter plots illustrating the correlation between the first latent component of the transcriptomic and proteomic datasets for liver (A) , muscle (B), and cerebral cortex (C). Each dot represents an individual biological replicate, colored by dietary group (Blue: AL; Red: IF). n = 7 mice per group.

Gene ontology (GO) enrichment analysis of the integrated multi-omics signatures.

Dot plots displaying the top 10 significantly enriched GO Biological Processes associated with the robust integrated signatures identified by DIABLO in liver (A), muscle (B), and cerebral cortex (C). p.adj < 0.05 was defined as significantly difference using Benjamini-Hochberg correction for GO Biological Processes analysis. n = 7 mice in each group.