This study characterized changes in the human metabolome following an acute glucose challenge and investigated variations in these responses between individuals consuming almonds versus graham crackers for 8 weeks. As shown previously [9,13–16], a progressive temporal shift in response to a glucose challenge was observed with alterations across amino acid, carbohydrate, and lipid metabolism pathways. Findings also exhibit a notable biphasic lipid response distinguished by higher levels of certain lipids such as unsaturated triglycerides in the earlier periods of a glucose challenge followed by lower levels in the latter period in the almond versus the cracker group. At the individual metabolite level, almond consumption was associated with distinct shifts in total AUC_{120 min} of specific amino acids and lipid mediators involved in metabolic and physiological processes.

The dynamic response of the human metabolome to a glucose challenge as depicted by an oGTT can help reveal metabolic alterations associated with health and disease [17]. The overall lack of significant metabolic changes over the first 15 minutes likely reflects a latent period in which glucose continues to reach peak absorption levels, to saturate perfused tissues, and the delay in the cell’s capacity to respond to an acute increase in extracellular glucose. This is in clear contrast to the latter oGTT time periods, which are characterized by more dynamic shifts in metabolic pathways. These shifts reveal the effects chronic almond consumption have on the cellular adaption to an acute glucose load and activation of specific metabolic pathways over longer durations [17]. The most dynamic metabolic changes have been reported over the first 90 minutes with no difference between the 90 and 120 minute oGTT time points [1]. While most studies have examined changes in metabolomic profiles in the fasted state, studies that have captured multivariate shifts in profiles during an oGTT demonstrate alterations in metabolites of dietary or microbial origin [1], those associated with mitochondrial efficiency and glucose oxidation [1], or markers of proteolysis and lipolysis associated with insulin sensitivity [18].

In the present study, the clear demarcation between the almond and cracker groups in the PLS-DA plots suggests that despite the isocaloric nature of the interventions, the inherent differences in the composition of the two foods, acutely and dynamically influence an array of metabolic pathways. As previously reported, at the end of the 8-week intervention, the almond group’s diet consisted of 42.7% carbohydrates and 41.7% fats, whereas the cracker group consumed 53.7% carbohydrates and 33.5% fats on average. These nutritional differences likely influenced the lipid responses during the glucose challenge, as further illustrated by the MESH class enrichment analysis. Since unsaturated triglycerides were enriched in the almond group in the end-of-study sample following an overnight fast [5], it may be reasonable to assume that the greater levels over the first 30 minutes of the glucose challenge reflect the already elevated circulating levels secreted to help support the short-term fasting metabolism [5]. These differences persist despite adjustment for time 0 values, providing some validation that the changes were induced by chronic almond consumption and reflect the corresponding metabolic adaptations. In contrast, saturated triglycerides were only enriched in the latter periods of the oGTT with lower levels observed in the almond group. The pattern mirrors the lower levels of unsaturated fats in the latter time periods suggesting that the glucose stimulated insulin may have facilitated the clearance of fats. Insulin released in response to the glucose bolus during the oGTT can enhance lipoprotein lipase activity. This increase in lipoprotein lipase activity and that of remnant receptors is essential for the efficient clearance of triglycerides from the bloodstream [19–21]. In this same study, we observed greater insulin sensitivity in the almond group compared to the cracker group [4] suggesting that this improvement in insulin sensitivity may facilitate the observed metabolism of TGs. These interactions suggest that chronic almond consumption selectively impacted the metabolism of saturated triglycerides differentially from unsaturated fats during the early stages of the glucose challenge particularly when comparing the effects to cracker consumption.

At the individual metabolite level, the AUC for isoheptadecanoic acid, a type of saturated fatty acid, was differentially lower in the almond group compared to the cracker group. Heptadecanoic acid, often used as a biomarker for dairy fat intake and linked to a decreased risk of type 2 diabetes, is a related compound [22,23]. High-fiber diets are found to reduce urine levels of this metabolite [24]. Its branched-chain variant, isoheptadecanoic acid, constitutes about 2% of dairy fat and can also be produced from branched chain amino acids through de novo synthesis [22,25]. Research involving children has shown an inverse relationship between plasma levels of isoheptadecanoic acid and hepatic steatosis [22]. The reduced AUC for isoheptadecanoic acid in the almond group suggests that chronic almond consumption shifts lipid metabolism from de novo lipogenesis to enhanced utilization of fatty acids, which may ameliorate the risk of T2D. The improvement in insulin sensitivity observed in the almond group may facilitate more efficient lipid metabolism, potentially reducing hepatic de novo lipogenesis [26] and the risk of hepatic steatosis.

In contrast, almond consumption resulted in a greater AUC for: a) AA, an omega-6 fatty acid precursor to eicosanoids; b) cyclooxygenase/lipoxygenase-derived hydroxy fatty acids (i.e., 11-HETE, 15-HETE and 12-HETE (p-value=0.092)), and c) cytochrome P450/soluble epoxide hydrolase-derived epoxy and dihydroxy fatty acids (i.e. 9,10-e-DiHO; 9,10-DiHODE, 12,13-EpOME and 12,13-DiHOME (p-value=0.089)). The effects of oxylipins on metabolic and physiological processes are nuanced and structurally dependent [27,28]. While 11-HETE and 15- HETE are associated with COX metabolism [29], 15-HETE can also exhibit anti-inflammatory properties [27]. Additionally, while 12-HETE decreases insulin secretion in vitro [30], it is also decreased in obesity and inversely associated with arterial stiffness even after BMI adjustment [31]. Little is known about the metabolic impacts of oleic acid and alpha-linolenic acid-derived epoxides and diols, however, the analogous linoleate metabolites have received some attention. For example, previous case studies have documented postprandial changes in 12,13 EpOME following high-carbohydrate challenges (such as consuming a banana) [32]. These epoxides are metabolized by soluble epoxide hydrolase (sEH) into 12,13-DiHOME and its counterpart, 9,10- DiHOME [33,34]. Evidence suggests that the 12,13-DiHOME may contribute to fatty acid uptake and oxidation in adipose tissue and skeletal muscle [33,35]. In addition, a negative correlation of circulating 12,13-DiHOME with insulin resistance has previously been demonstrated [35]. Adding to this evidence base, our study demonstrates a positive correlation of 12,13-DiHOME AUC with postprandial insulin sensitivity in response to chronic almond consumption, suggesting that the improved insulin sensitivity with almond consumption [37] could potentially be mediated via this lipokine. However, it’s important to note that the concentration of circulating 12,13-DiHOME can also be affected by several factors such as exercise and temperature [33].

Correlations between other omega-3 derived metabolites and MI provide additional insights. The negative correlation between MI and CE (22:6) AUC in the almond group suggests that individuals with higher insulin sensitivity may have a suppressed response in CE (22:6) production or release after glucose intake and implicates that those with better insulin sensitivity were more effective at utilizing or clearing it from the bloodstream during the glucose challenge. CE 22:6 is a cholesteryl ester containing DHA, an omega-3 fatty acid [38] and although not statistically significant, the almond group exhibited a trend toward higher CE 22:6 AUC compared to the cracker group (P =0.051). CE 22:6 has not been widely studied, and those that have, reported this metabolite in the pathogenesis of osteoarthritis, Parkinson’s disease, and bladder cancer [39–41]. However, it appears that in healthy individuals the incorporation of DHA in LDL cholesterol esters does not significantly promote LDL oxidation – a factor implicated in atherosclerosis [42]. Moreover, a systematic review reported that DHA intake can lower oxidative stress [43]. It’s essential to juxtapose this with the observation of reduced omega-3 total fatty acids (TFAs) in the almond group in the fasting state [5], which contrasts with the more dynamic lipid profile seen during the oGTT. Their reduced levels following an overnight fast in the almond group may indicate potential interactions between different unsaturated fatty acids, the intricate metabolism involving a series of steps [44], or possibly the overriding influence of other bioactive compounds in almonds.

Almond consumption also induced lower AUC for L-cystine compared to the cracker group. L-cystine is an oxidized dimer of L-cysteine which is a precursor for the synthesis of glutathione, a potent, non-enzymatic antioxidant [45]. Given that there were no significant differences in cysteine or L-cysteine-glutathione disulfide AUC between groups, and considering glutathione’s role in enhancing cellular redox balance and mitigating oxidative stress, the lower L-cystine AUC could instead indicate a more regulated or less stressed oxidative state in the almond-consuming individuals [46,47]. Since plasma glutathione levels are usually relatively low in normal, healthy humans [48] and glutathione synthesis is activated by acute increases in free radicals [49], the lower AUC for L-cystine without a change in cysteine, could indicate that chronic almond consumption helps maintains a balanced redox state through the efficient use of cysteine reserves during post-prandial, hyperglycemic excursions [50,51]. The lower L-cystine AUC may also reflect an environment where there’s less necessity to combat oxidative stress, possibly due to the inherent antioxidant properties of almonds [52,53]. This state could contribute to better cardiovascular health by maintaining efficient glutathione induced regulation of nitric oxide [46,47], aligning with the cardiovascular benefits associated with almond intake [54].

Almond consumption also resulted in lower AUC for N-acetylmannosamine, which is an important precursor in the biosynthesis of sialic acids [55]. Sialic acids are important components of glycoproteins and glycolipids and critically important in cellular communication and signaling [56,57]. The lower AUC of N-acetylmannosamine might reflect reduced synthesis and/or more efficient utilization of sialic acid-containing glycans. Notably, our previous analyses of fasting data revealed a positive correlation between fasting serum N-acetylmannosamine levels and the predicted microbial community potential to produce this metabolite in the almond group [5]. Given that the gut microbiome has been shown to predict postprandial glucose metabolism [58,59], it is plausible that a chronic alteration in gut microbiota as demonstrated in our study [5,60], could impact the synthesis and degradation of glycan-related compounds during metabolic challenges such as an oGTT.