**Background:** Over 33% of Americans are obese, a major public health problem linked to metabolic syndrome, cardiovascular disease, and Type 2 diabetes. Prolonged higher-fat diet consumption alters the gut microbiome, contributing to obesity-related diseases. Probiotics, such as *Lacticaseibacillus paracasei*, may offer health benefits, but their effects on the host metabolome are poorly understood. This study aimed to determine if probiotic supplementation can modulate tissue metabolism in pigs fed a higher-fat diet.
**Methods:** Twenty Ossabaw pigs were randomized into four groups (n=5 each): (1) basal diet (18.5% kcal from protein, 71.0% carbohydrates, 10.5% fat) + placebo; (2) higher-fat diet (13% kcal from protein, 57% carbohydrates, 30% fat, supplemented with hydrogenated soybean oil, cholesterol, and cholic acid) + placebo; (3) basal diet + probiotic (*L. paracasei* subsp. *paracasei* L. casei W8, 1 × 10^10 cfu/day); (4) higher-fat diet + probiotic. Pigs were fed for 24 weeks with bi-weekly caloric adjustments. At sacrifice, tissue samples (brain cortex, heart, kidney, liver, skeletal muscle, pancreas) were collected, snap-frozen, and extracted with methanol:water (1:1). Metabolomic profiling was performed using LC-QToF in ESI positive and negative modes with HILIC and C18 columns. Data were processed with MassHunter and Mass Profiler Professional; features present in ≥60% of any cohort were analyzed. Metabolites were identified by MS/MS fragmentation matching to Metlin, HMDB, MoNA, NIST17, or MS-FINDER. Statistical analyses included fold-change, two-sample t-tests, Benjamini–Hochberg FDR correction, PCA, and correlation networks.
**Key Results:** PCA revealed clear metabolic differences among tissues, with heart and skeletal muscle clustering closely. The brain cortex and kidney were most sensitive to the higher-fat diet, with their metabolomes segregating from basal, basal+probiotic, and higher-fat+probiotic groups. In the brain cortex, higher-fat diet suppressed glutathione (below detection) and increased S-adenosylhomocysteine (SAH). In the heart and pancreas, betaine and trimethylamine N-oxide (TMAO) were lower; arabitol was lower in the heart. In the kidney, indoxyl sulfate was undetectable. The liver showed increased glycocholic acid and reduced PC/PE ratio. Skeletal muscle had elevated 7α-hydroxy-3-oxo-4-cholestenoic acid, UDP-GlcA, and inosine. Probiotic supplementation to the higher-fat diet reverted several metabolites to basal-like levels: in the brain cortex, SAH was suppressed and glutathione reappeared; in the kidney, indoxyl sulfate was detected in 3 of 5 pigs; in the heart, LysoPE (18:2) was reduced. Additionally, probiotic supplementation reduced UDP-GlcNAc in the kidney (undetected in all probiotic-fed pigs) and decreased saccharopine in the liver of basal+probiotic pigs. Correlation network analysis showed that the higher-fat metabotype was visually distinct from basal, but probiotic supplementation shifted the higher-fat+probiotic metabotype toward the basal pattern, especially in brain cortex, heart, and kidney.
**Clinical Implications:** This study demonstrates that a higher-fat diet induces widespread metabolic alterations in multiple tissues, with the brain cortex and kidney being particularly vulnerable. The reversion of aberrant metabolites (SAH, glutathione, indoxyl sulfate, LysoPE (18:2)) to healthy levels with probiotic supplementation suggests that *L. paracasei* may help counteract obesity-related metabolic dysfunction. These findings support the potential use of probiotics as a prophylactic strategy to mitigate neurocognitive decline, cardiovascular disease, and kidney toxicity associated with obesity. However, the mechanisms remain speculative, and further research is needed to confirm these effects in humans.