Metabolic flux analysis of branched-chain amino and keto acids (BCAA, BCKA) and ß-hydroxy ß-methylbutyric acid across multiple organs in the pig.

Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and ß-hydroxy ß-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. However, their (inter)organ kinetics remain unclear. Therefore, branched-chain amino acids (BCAA) [leucine (Leu), valine (Val), isoleucine (Ile)], BCKA [α-ketoisocaproic acid (KIC), 3-methyl-2-oxovaleric acid (KMV), 2-oxoisovalerate (KIV)], and HMB across organ net fluxes were measured. In multi-catheterized pigs (n = 12, ±25 kg), net fluxes across liver, portal drained viscera (PDV), kidney, and hindquarter (HQ, muscle compartment) were measured before and 4 h after bolus feeding of a complete meal (30% daily intake) in conscious state. Arterial and venous plasma were collected and concentrations were measured by LC- or GC-MS/MS. Data are expressed as mean [95% CI] and significance (P < 0.05) from zero by the Wilcoxon Signed Rank Test. In the postabsorptive state (in nmol/kg body wt/min), the kidney takes up HMB (3.2[1.3,5.0]) . BCKA is taken up by PDV (144[13,216]) but no release by other organs. In the postprandial state, the total net fluxes over 4 h (in µmol/kg body wt/4 h) showed a release of all BCKA by HQ (46.2[34.2,58.2]), KIC by the PDV (12.3[7.0,17.6]), and KIV by the kidney (10.0[2.3,178]). HMB was released by the liver (0.76[0.49,1.0]). All BCKA were taken up by the liver (200[133,268]). Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.NEW & NOTEWORTHY Branched-chain amino acids (BCAA) and their metabolites the branched-chain keto acids (BCKA) and ß-hydroxy ß-methylbutyric acid (HMB) are involved in the regulation of key signaling pathways in the anabolic response to a meal. Substantial differences are present in (inter)organ metabolism and transport among the BCAA and its metabolites BCKA and HMB. The presented data in a translation animal model are relevant for the future development of optimized clinical nutrition.

Glucagon regulation of carbohydrate metabolism in rainbow trout: in vivo glucose fluxes and gene expression.

Glucagon increases fish glycaemia, but how it affects glucose fluxes in vivo has never been characterized. The goal of this study was to test the hypothesis that glucagon stimulates hepatic glucose production (rate of appearance, Ra) and inhibits disposal (rate of disposal, Rd) in rainbow trout. Changes in the mRNA abundance of key proteins involved in glycolysis, gluconeogenesis and glycogen breakdown were also monitored. The results show that glucagon increases glycaemia (+38%) by causing a temporary mismatch between Ra and Rd before the two fluxes converge below baseline (-17%). A novel aspect of the regulation of trout gluconeogenesis is also demonstrated: the completely different effects of glucagon on the expression of three Pepck isoforms (stimulation of pck1, inhibition of pck2a and no response of pck2b). Glycogen phosphorylase was modulated differently among tissues, and muscle upregulated pygb and downregulated pygm Glucagon failed to activate the cAMP-dependent protein kinase or FoxO1 signalling cascades. We conclude that trout hyperglycaemia results from the combination of two responses: (i) an increase in Ra glucose induced by the stimulation of gluconeogenesis through transcriptional activation of pck1 (and possibly glycogen phosphorylase), and (ii) a decrease in Rd glucose via inhibition of glycogen synthase and glycolysis. The observed decrease in glucose fluxes after 4 h of glucagon administration may be caused by a counter-regulatory response of insulin, potentially linked to the decrease in pygm transcript abundance. Overall, however, these integrated effects of glucagon only lead to modest changes in glucose fluxes that partly explain why trout seem to be unable to control glycaemia very tightly.

Cell bioenergetics in Leghorn male hepatoma cells and immortalized chicken liver cells in response to 4-hydroxy 2-nonenal-induced oxidative stress.

The major objectives of this study were to compare cell bioenergetics in 2 avian liver cell lines under control conditions and in response to oxidative stress imposed by 4-hydroxy 2-nonenal (4-HNE). Cells in this study were from a chemically immortalized Leghorn male hepatoma (LMH) cell line and a spontaneously immortalized chicken liver (CELi) cell line. Oxygen consumption rate (OCR) was monitored in specialized microtiter plates using an XF24 Flux Analyzer (Seahorse Bioscience, Billerica, MA). Cell bioenergetics was assessed by sequential additions of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and antimycin-A that enables the determination of a) OCR linked to adenosine triphosphate (ATP) synthase activity, b) mitochondrial oxygen reserve capacity, c) proton leak, and d) nonmitochondrial cytochrome c oxidase activity. Under control (unchallenged) conditions, LMH cells exhibited higher basal OCR and higher OCR attributed to each of the bioenergetic components listed above compared with CELi cells. When expressed as a percentage of maximal OCR (following uncoupling with FCCP), LMH cells exhibited higher OCR due to ATP synthase and proton leak activity, but lower mitochondrial oxygen reserve capacity compared with CELi cells; there were no differences in OCR associated with nonmitochondrial cytochrome c oxidase activity. Whereas the LMH cells exhibited robust ATP synthase activity up to 50 µM 4-HNE, CELi cells exhibited a progressive decline in ATP synthase activity with 10, 20, and 30 µM 4-HNE. The CELi cells exhibited higher mitochondrial oxygen reserve capacity compared with LMH cells with 0 and 20 µM 4-HNE but not with 30 µM 4-HNE. Both cell lines exhibited inducible proton leak in response to increasing levels of 4-HNE that was evident with 30 µM 4-HNE for CELi cells and with 40 and 50 µM 4-HNE in LMH cells. The results of these studies demonstrate fundamental differences in cell bioenergetics in 2 avian liver-derived cell lines under control conditions and in response to oxidative challenge due to 4-HNE.