Dietary total, animal, and plant protein–energy ratio and risk of mortality: results from the NHANES III and a lifelong animal experiment

<i>Background</i>: Protein is essential for basic physiological functions of the body, but the relationship between excessive protein intake and health is controversial. The association between dietary protein and mortality may depend on protein intake and source. This study examined the links between the total/animal/plant protein-energy ratios and mortality from all causes, cardiovascular disease (CVD) and cancer. <i>Methods</i>: The population study included 13 490 participants with a median follow-up of 26.5 years from NHANES III. The study examined the relationship between total, animal, and plant protein-energy ratios, and their combinations, with mortality through Cox proportional hazards models, restricted cubic splines (RCS), and isocaloric (2.5%, 5%, and 10%) substitution analysis. In an animal experiment, 60 male Wistar rats were randomly assigned to normal protein diets (NC = 30) or isocaloric high-protein (HP) diets (HP = 30) for 101 weeks. Subsequently, renal histologic staining was performed using hematoxylin and eosin, and serum amino acid levels were measured through targeted metabolomics. Changes in hepatic gene expression profiles were assessed <i>via</i> principal component analysis (PCA), analyzed using protein interaction networks and modules with the STRING online database, and validated through quantitative real-time PCR (q-PCR). <i>Results</i>: Total protein-energy ratio was positively associated with all-cause mortality (<i>p</i>-trend = 0.003), and animal-protein-energy ratio was also positively associated with mortality (all-cause: <i>p</i>-trend = 0.007, cancer: <i>p</i>-trend = 0.020). RCS showed increased risks of all-cause and cancer mortality when total protein-energy ratio exceeded 14.8%. The risk of all-cause and cancer mortality showed a downward trend when isocaloric plant-protein-energy ratios replaced animal-protein-energy ratios. Rats fed high-animal-protein diets showed higher risk for tumorigenesis. HP diet may alter the transcriptome profiles of rat liver, with upregulated ACSM5, AMACR and TM7SF2, and downregulated HAO2. <i>Conclusions</i>: Keeping the total protein-energy ratio below 14.8% may be beneficial to reducing all-cause and cancer mortality. Substitution of animal protein with plant protein (by 2.5%, 5%, and 10% of energy) was associated with a decreasing trend in all-cause and cancer mortality. Animal experiments confirmed that the risk of tumorigenesis was associated with a high animal-protein-energy ratio, highlighting the effects of different sources of protein on health and the mechanisms of cancer development.