**Background:** The prevalence of type 2 diabetes (T2D) in China has reached epidemic proportions, with Asian Chinese showing greater susceptibility than European Caucasians, even at lower BMI. This has been linked to the "Thin on the Outside, Fat on the Inside" (TOFI) phenotype, characterized by preferential accumulation of visceral adipose tissue and ectopic organ fat, including intra-pancreatic fat deposition (IPFD). Whey protein isolate (WPI) is known to decrease postprandial hyperglycemia and promote insulin secretion. This study aimed to determine whether differences in postprandial metabolomic responses to WPI could distinguish between ethnic groups and between individuals with varying IPFD levels.
**Methods:** This was an acute, randomized, three-treatment crossover study involving 12 Asian Chinese and 12 European Caucasian women (aged 20–69 years, BMI 19.6–36.8 kg/m²) with prediabetes (FPG 5.6–6.9 mmol/L). Each participant attended three study visits with a minimum seven-day washout period. At each visit, after a fasted baseline sample (T=0), participants consumed 280 mL test drinks containing 0 g (water control), 12.5 g (low protein), or 50 g (high protein) WPI. Postprandial plasma samples were collected at 30, 60, 120, and 240 minutes. MRI-assessed IPFD was available for 20 participants (range 2.13–12.7%), with low and high IPFD defined as < and ≥ the cohort median of 4.66%, respectively. Polar metabolites were analyzed using HILIC-MS. A feature selection pipeline using linear mixed-effect modeling (with Meal, Time, Meal*Time, Age, and BMI as fixed effects), incremental AUC fold-change analysis, and support vector machine-recursive feature elimination (SVM-RFE) with 10-fold cross-validation was used to classify ethnicity and IPFD models. Metabolic network analysis and pathway enrichment were performed using MetScape and MetaboAnalyst.
**Key Results:** From 524 detected features, 125 were identified as relevant to the WPI response after filtering. The ethnicity SVM-RFE model achieved an optimal generalized error estimate of 0.042 with just four top-ranking features (imidazolelactic acid, uric acid, N(ϵ)-methyl-lysine, and L-cystine). The IPFD model required 19 features for an error estimate of 0.047. PLS-DA confirmed strong separation for ethnicity (R2Y 0.788, Q2 0.75) and moderate separation for IPFD (R2Y 0.501, Q2 0.354). The Chinese cohort had significantly lower mean age (42.0 ± 10.8 vs. 54.7 ± 15.6 years, p=0.038) and BMI (26.9 ± 3.8 vs. 31.4 ± 4.2 kg/m², p=0.014) but similar FPG and IPFD compared to Caucasians. The high IPFD cohort had significantly higher mean age (53.9 ± 8.1 vs. 35.9 ± 12.5 years, p=0.002) but similar BMI and FPG to the low IPFD cohort. Network analysis identified glycine as a key hub for both models. For ethnicity, enriched pathways included arginine and proline metabolism (5 hits, adjusted p=0.00377, impact 0.2905), alanine, aspartate, and glutamate metabolism (4 hits, adjusted p=0.018, impact 0.2837), and arginine biosynthesis (4 hits, adjusted p=0.000987, impact 0.2538). For IPFD, enriched pathways included glycine, serine, and threonine metabolism (5 hits, adjusted p=0.000971, impact 0.487), glyoxylate and dicarboxylate metabolism (5 hits, adjusted p=0.000839, impact 0.2593), and aminoacyl-tRNA biosynthesis (7 hits, adjusted p=1.36E-05, impact 0.1667). Glycine depletion was more prominent in the Chinese cohort and in the high IPFD cohort, despite comparable BMI between IPFD groups. Branched-chain amino acids (valine, leucine, isoleucine) were top-ranking only in the ethnicity model. Uric acid was the top-ranking metabolite for IPFD classification.
**Clinical Implications:** This study demonstrates that ethnicity has a stronger influence on the postprandial metabolomic response to WPI than IPFD in overweight women with prediabetes. The findings highlight that metabolic differences between Asian Chinese and European Caucasian individuals extend beyond simple anthropometric measures, involving distinct pathways in amino acid metabolism, particularly the urea cycle and glycine metabolism. The prominent glycine depletion observed in Chinese participants and those with high IPFD, despite comparable or lower BMI, suggests that the TOFI phenotype and ethnicity contribute to metabolic dysregulation independently of overall adiposity. The identification of uric acid as a key discriminator for IPFD supports emerging evidence linking purine metabolism to ectopic fat accumulation and insulin resistance. These results may inform personalized nutritional strategies for T2D prevention, suggesting that ethnicity-specific metabolic profiles should be considered when designing dietary interventions. The study also identified novel metabolites (imidazolelactic acid and N(ϵ)-methyl-lysine) that may reflect gut microbiota differences between ethnic groups, warranting further investigation.