br Materials and methods br

Materials and methods
Results Table 1 presents the metabolic phenotype of obese minipigs. After 60 d of HFHS feeding, minipigs not only showed an increased body weight (+45%), but also a perturbed Omaveloxolone sale homeostasis, with higher insulin levels (about +2-fold), HOMA-IR (+5.5-fold), and HOMA-%B (2-fold) indices and a reduced HOMA-%S index (−2-fold). Other metabolisms also were affected, as shown by increased TC (+1.4-fold) and HDL-C (+1.5-fold) levels, and reduced urea levels (−1.3-fold). BCAA and KA levels in plasma are shown in Figure 1. We observed that BCAA levels increased in two steps, with similar levels between days 0 and 7, and then increased at day 14, which remained steadily elevated to the end of the trial. The plasma KA profile followed a similar trend. However, we did not observe the two steps in the kinetics; KA levels increased progressively up to day 60. The PP BCAA profile also showed that BCAA handling was different after 60 d of HFHS feeding. Not only was the basal level higher in the HFHS-fed minipigs, but the entire profile after the meal was higher than after a regular meal, especially during the first 3 h. Liver BCAA metabolism is represented in Figure 2. The BCAA metabolism was differentially regulated by HFHS feeding according to the different steps of their catabolism. The transamination potential was reduced by the HFHS diet, with a twofold reduction in bcat2 mRNA levels and 21-fold diminution in BCAT activity. Concerning the oxidative deamination, although no changes were observed in the pBCKDH complex, the mRNA levels of some of its subunits were significantly reduced (30 to 40%), including bckdha, dbt, dld. The BCAA metabolism in the AT is shown in Figure 3. In VAT, despite the absence of changes in the pBCKDH status, BCAT2 activity was enhanced by the HFHS feeding by 15-fold. A smaller (fivefold) but significant increase in BCAT2 activity also was observed in SCAT, accompanied by a 50% increase in the pBCKDH complex and the mRNA levels of dld (P = 0.092). Finally, we explored the skeletal muscle BCAA metabolism (Fig. 4). We noticed only a few changes in BCAA metabolism induced by the HFHS diet, including a 50% increase in the mRNA levels of bckdk and a 40% increase in BCAT activity and mRNA levels. Spearman correlations between fasting and PP (0 → 510 min after the meal) BCAA levels and variables related to glucose and lipid homeostasis are shown in Table 2. Two clear and different correlation patterns were observed. First, fasting BCAA levels significantly correlated with variables related to glucose homeostasis, including HOMA-IR (r = 0.46), HOMA2-%B (r = 0.46), HOMA2-%S (r = −0.46), fasting plasma glucose (r = −0.65), and insulin (r = 0.54) levels, although only fasting glucose (r = 0.74) correlated with the PP BCAA levels. In contrast to the fasting BCAA, PP BCAA area under the curve (AUC) correlated with biochemical variables related to dyslipidemia, including fasting TGs (r = 0.69), TC (r = 0.95), and HDL-C (r = 0.93). Finally, we found a strong correlation of body weight with both fasting (r = 0.71) and PP (r = 0.91) BCAA levels. We also performed correlations between the major variable related to BCAA catabolism altered in the present study (pBCKDH in SCAT) and glucose and lipid homeostasis biomarkers (Table 3). SCAT pBCKDH correlated positively with both fasting (r = 0.77) and PP (r = 0.76) plasma BCAA levels, and also with body weight (r = 0.77) and two markers of dyslipidemia: fasting TC (r = 0.73) and HDL-C (r = 0.71). Finally, we found a correlation between PP BCAA and urea-level excursions after the meal (Fig. 5). A good positive correlation was found between both parameters but only after the first HFHS meal at day 0 (r = 0.615; P = 0.001). In contrast, after 2 mo of HFHS feeding, the variables were no longer correlated (r = −0.048; P = 0.836). Furthermore, no correlation was found between fasting BCAA and urea (data not shown).