The Effects of Long-Term High Fat and/or High Sugar Feeding on Sources of Postprandial Hepatic Glycogen and Triglyceride Synthesis in Mice.

BACKGROUND: In MASLD (formerly called NAFLD) mouse models, oversupply of dietary fat and sugar is more lipogenic than either nutrient alone. Fatty acids suppress de novo lipogenesis (DNL) from sugars, while DNL inhibits fatty acid oxidation. How such factors interact to impact hepatic triglyceride levels are incompletely understood. METHODS: Using deuterated water, we measured DNL in mice fed 18-weeks with standard chow (SC), SC supplemented with 55/45-fructose/glucose in the drinking water at 30% (w/v) (HS), high-fat chow (HF), and HF with HS supplementation (HFHS). Liver glycogen levels and its sources were also measured. For HS and HFHS mice, pentose phosphate (PP) fluxes and fructose contributions to DNL and glycogen were measured using [U-13C]fructose. RESULTS: The lipogenic diets caused significantly higher liver triglyceride levels compared to SC. DNL rates were suppressed in HF compared to SC and were partially restored in HFHS but supplied a minority of the additional triglyceride in HFHS compared to HF. Fructose contributed a significantly greater fraction of newly synthesized saturated fatty acids compared to oleic acid in both HS and HFHS. Glycogen levels were not different between diets, but significant differences in Direct and Indirect pathway contributions to glycogen synthesis were found. PP fluxes were similar in HS and HFHS mice and were insufficient to account for DNL reducing equivalents. CONCLUSIONS: Despite amplifying the lipogenic effects of fat, the fact that sugar-activated DNL per se barely contributes suggests that its role is likely more relevant in the inhibition of fatty acid oxidation. Fructose promotes lipogenesis of saturated over unsaturated fatty acids and contributes to maintenance of glycogen levels. PP fluxes associated with sugar conversion to fat account for a minor fraction of DNL reducing equivalents.

Mitochondria dysfunction induced by decyl-TPP mitochondriotropic antioxidant based on caffeic acid AntiOxCIN6 sensitizes cisplatin lung anticancer therapy due to a remodeling of energy metabolism.

The pharmacological interest in mitochondria is very relevant since these crucial organelles are involved in the pathogenesis of multiple diseases, such as cancer. In order to modulate cellular redox/oxidative balance and enhance mitochondrial function, numerous polyphenolic derivatives targeting mitochondria have been developed. Still, due to the drug resistance emergence in several cancer therapies, significant efforts are being made to develop drugs that combine the induction of mitochondrial metabolic reprogramming with the ability to generate reactive oxygen species, taking into consideration the varying metabolic profiles of different cell types. We previously developed a mitochondria-targeted antioxidant (AntiOxCIN6) by linking caffeic acid to lipophilic triphenylphosphonium cation through a 10-carbon aliphatic chain. The antioxidant activity of AntiOxCIN6 has been documented but how the mitochondriotropic compound impact energy metabolism of both normal and cancer cells remains unknown. We demonstrated that AntiOxCIN6 increased antioxidant defense system in HepG2 cells, although ROS clearance was ineffective. Consequently, AntiOxCIN6 significantly decreased mitochondrial function and morphology, culminating in a decreased capacity in complex I-driven ATP production without affecting cell viability. These alterations were accompanied by an increase in glycolytic fluxes. Additionally, we demonstrate that AntiOxCIN6 sensitized A549 adenocarcinoma cells for CIS-induced apoptotic cell death, while AntiOxCIN6 appears to cause metabolic changes or a redox pre-conditioning on lung MRC-5 fibroblasts, conferring protection against cisplatin. We propose that length and hydrophobicity of the C10-TPP+ alkyl linker play a significant role in inducing mitochondrial and cellular toxicity, while the presence of the antioxidant caffeic acid appears to be responsible for activating cytoprotective pathways.

Characterizing Early Cardiac Metabolic Programming via 30% Maternal Nutrient Reduction during Fetal Development in a Non-Human Primate Model.

Intra-uterine growth restriction (IUGR) is a common cause of fetal/neonatal morbidity and mortality and is associated with increased offspring predisposition for cardiovascular disease (CVD) development. Mitochondria are essential organelles in maintaining cardiac function, and thus, fetal cardiac mitochondria could be responsive to the IUGR environment. In this study, we investigated whether in utero fetal cardiac mitochondrial programming can be detectable in an early stage of IUGR pregnancy. Using a well-established nonhuman IUGR primate model, we induced IUGR by reducing by 30% the maternal diet (MNR), both in males (MNR-M) and in female (MNR-F) fetuses. Fetal cardiac left ventricle (LV) tissue and blood were collected at 90 days of gestation (0.5 gestation, 0.5 G). Blood biochemical parameters were determined and heart LV mitochondrial biology assessed. MNR fetus biochemical blood parameters confirm an early fetal response to MNR. In addition, we show that in utero cardiac mitochondrial MNR adaptations are already detectable at this early stage, in a sex-divergent way. MNR induced alterations in the cardiac gene expression of oxidative phosphorylation (OXPHOS) subunits (mostly for complex-I, III, and ATP synthase), along with increased protein content for complex-I, -III, and -IV subunits only for MNR-M in comparison with male controls, highlight the fetal cardiac sex-divergent response to MNR. At this fetal stage, no major alterations were detected in mitochondrial DNA copy number nor markers for oxidative stress. This study shows that in 90-day nonhuman primate fetuses, a 30% decrease in maternal nutrition generated early in utero adaptations in fetal blood biochemical parameters and sex-specific alterations in cardiac left ventricle gene and protein expression profiles, affecting predominantly OXPHOS subunits. Since the OXPHOS system is determinant for energy production in mitochondria, our findings suggest that these early IUGR-induced mitochondrial adaptations play a role in offspring's mitochondrial dysfunction and can increase predisposition to CVD in a sex-specific way.

Positional and compositional analysis of saturated, monounsaturated, and polyunsaturated fatty acids in human adipose tissue triglyceride by 13 C nuclear magnetic resonance.

The synthesis and turnover of triglyceride in adipose tissue involves enzymes with preferences for specific fatty acid classes and/or regioselectivity regarding the fatty acid position within the glycerol moiety. The focus of the current study was to characterize both the composition of fatty acids and their positional distribution in triglycerides of biopsied human subcutaneous adipose tissue, from subjects with wide ranges of body mass index (BMI) and insulin sensitivity, using 13 C nuclear magnetic resonance (NMR) spectroscopy. The triglyceride sn2 position was significantly more enriched with monounsaturated fatty acids compared with that of sn1,3, while the abundance of saturated fatty acids was significantly lower in the sn2 position compared with that of sn1,3. Furthermore, the analysis revealed significant positive correlations between the total fraction of palmitoleic acid with both BMI and insulin sensitivity scores (homeostatic model assessment of insulin resistance index). Additionally, we established that 13 C NMR chemical shifts for ω-3 signals, centered at 31.9 ppm, provided superior resolution of the most abundant fatty acid species, including palmitoleate, compared with the ω-2 signals that were used previously. 13 C NMR spectroscopy reveals for the first time a highly nonhomogenous distribution of fatty acids in the glycerol sites of human adipose tissue triglyceride, and that these distributions are correlated with different phenotypes, such as BMI and insulin sensitivity.

Integration of Liver Glycogen and Triglyceride NMR Isotopomer Analyses Provides a Comprehensive Coverage of Hepatic Glucose and Fructose Metabolism.

Dietary glucose and fructose are both efficiently assimilated by the liver but a comprehensive measurement of this process starting from their conversion to sugar phosphates, involvement of the pentose phosphate pathway (PPP), and conversion to glycogen and lipid storage products, remains incomplete. Mice were fed a chow diet supplemented with 35 g/100 mL drinking water of a 55/45 fructose/glucose mixture for 18 weeks. On the final night, the sugar mixture was enriched with either [U-13C]glucose or [U-13C]fructose, and deuterated water (2H2O) was also administered. 13C-isotopomers representing newly synthesized hepatic glucose-6-phosphate (glucose-6-P), glycerol-3-phosphate, and lipogenic acetyl-CoA were quantified by 2H and 13C NMR analysis of post-mortem liver glycogen and triglyceride. These data were applied to a metabolic model covering glucose-6-P, PPP, triose-P, and de novo lipogenesis (DNL) fluxes. The glucose supplement was converted to glucose-6-P via the direct pathway, while the fructose supplement was metabolized by the liver to gluconeogenic triose-P via fructokinase-aldolase-triokinase. Glucose-6-P from all carbohydrate sources accounted for 40-60% of lipogenic acetyl-CoA and 10-12% was oxidized by the pentose phosphate pathway (PPP). The yield of NADPH from PPP flux accounted for a minority (~30%) of the total DNL requirement. In conclusion, this approach integrates measurements of glucose-6-P, PPP, and DNL fluxes to provide a holistic and informative assessment of hepatic glucose and fructose metabolism.

Mitochondria-targeted anti-oxidant AntiOxCIN4 improved liver steatosis in Western diet-fed mice by preventing lipid accumulation due to upregulation of fatty acid oxidation, quality control mechanism and antioxidant defense systems.

Non-alcoholic fatty liver disease (NAFLD) is a health concern affecting 24% of the population worldwide. Although the pathophysiologic mechanisms underlying disease are not fully clarified, mitochondrial dysfunction and oxidative stress are key players in disease progression. Consequently, efforts to develop more efficient pharmacologic strategies targeting mitochondria for NAFLD prevention/treatment are underway. The conjugation of caffeic acid anti-oxidant moiety with an alkyl linker and a triphenylphosphonium cation (TPP+), guided by structure-activity relationships, led to the development of a mitochondria-targeted anti-oxidant (AntiOxCIN4) with remarkable anti-oxidant properties. Recently, we described that AntiOxCIN4 improved mitochondrial function, upregulated anti-oxidant defense systems, and cellular quality control mechanisms (mitophagy/autophagy) via activation of the Nrf2/Keap1 pathway, preventing fatty acid-induced cell damage. Despite the data obtained, AntiOxCIN4 effects on cellular and mitochondrial energy metabolism in vivo were not studied. In the present work, we proposed that AntiOxCIN4 (2.5 mg/day/animal) may prevent non-alcoholic fatty liver (NAFL) phenotype development in a C57BL/6J mice fed with 30% high-fat, 30% high-sucrose diet for 16 weeks. HepG2 cells treated with AntiOxCIN4 (100 µM, 48 h) before the exposure to supraphysiologic free fatty acids (FFAs) (250 µM, 24 h) were used for complementary studies. AntiOxCIN4 decreased body (by 43%), liver weight (by 39%), and plasma hepatocyte damage markers in WD-fed mice. Hepatic-related parameters associated with a reduction of fat liver accumulation (by 600%) and the remodeling of fatty acyl chain composition compared with the WD-fed group were improved. Data from human HepG2 cells confirmed that a reduction of lipid droplets size and number can be a result from AntiOxCIN4-induced stimulation of fatty acid oxidation and mitochondrial OXPHOS remodeling. In WD-fed mice, AntiOxCIN4 also induced a hepatic metabolism remodeling by upregulating mitochondrial OXPHOS, anti-oxidant defense system and phospholipid membrane composition, which is mediated by the PGC-1α-SIRT3 axis. AntiOxCIN4 prevented lipid accumulation-driven autophagic flux impairment, by increasing lysosomal proteolytic capacity. AntiOxCIN4 improved NAFL phenotype of WD-fed mice, via three main mechanisms: a) increase mitochondrial function (fatty acid oxidation); b) stimulation anti-oxidant defense system (enzymatic and non-enzymatic) and; c) prevent the impairment in autophagy. Together, the findings support the potential use of AntiOxCIN4 in the prevention/treatment of NAFLD.

Carbohydrate tolerance in Amazon tambaqui (Colossoma macropomum) revealed by NMR-metabolomics - Are glucose and fructose different sugars for fruit-eating fish?

In the present study, two approaches were followed to evaluate the metabolic responses of tambaqui (Colossoma macropomum), a frugivorous species, to intraperitoneal (IP) administration of glucose (GLU) and fructose (FRU) in fed (FED) and 10-day fasted (FAST) fish. Glucose and fructose tolerance tests were performed to assess the carbohydrate utilization and complementary NMR-metabolomics analyses were done to elucidate the impacts of sugar mobilization on the metabolic profile of plasma, liver and muscle. Blood was sampled from FED groups at 0, 3, 6 and 24 h; and at 0 and 24 h from FAST groups. Significant differences were observed in the hyperglycaemic peak between sugars at 3 h (GLU - 13.7 ± 2.0 mM vs. FRU - 8.7 ± 1.1 mM; saline 6.3 ± 0.6 mM) and on the return to normoglycaemia (GLU - 8.5 ± 2.2 mM vs. FRU - 5.2 ± 0.9 mM; saline 4.9 ± 0.6 mM) 6 h after IP on the FRU fish. The NMR-metabolomics approach allowed to conclude that tambaqui seems to be more responsive to the feeding regime (FED vs. FAST) than to the injected sugar (FRU vs. GLU). From the studied tissues, plasma showed no significant variations between feeding regimes at 24 h after IP, while muscle and liver revealed some variations on the final metabolome profile between FED and FAST groups. The metabolome variations between feeding regimes are indicative of changes on the amino acid utilization. Fish from FAST group seem to utilize amino acids as energy source rather than for protein synthesis and muscle growth. Variations on glucose concentration in muscle can also indicate different utilization of the sugars depending on the feeding regime.

Mitochondriotropic antioxidant based on caffeic acid AntiOxCIN4 activates Nrf2-dependent antioxidant defenses and quality control mechanisms to antagonize oxidative stress-induced cell damage.

Mitochondria are key organelles involved in cellular survival, differentiation, and death induction. In this regard, mitochondrial morphology and/or function alterations are involved in stress-induced adaptive pathways, priming mitochondria for mitophagy or apoptosis induction. We have previously shown that the mitochondriotropic antioxidant AntiOxCIN4 (100 µM; 48 h) presented significant cytoprotective effect without affecting the viability of human hepatoma-derived (HepG2) cells. Moreover, AntiOxCIN4 (12.5 µM; 72 h) caused a mild increase of reactive oxygen species (ROS) levels without toxicity to primary human skin fibroblasts (PHSF). As Nrf2 is a master regulator of the oxidative stress response inducing antioxidant-encoding gene expression, we hypothesized that AntiOxCIN4 could increase the resistance of human hepatoma-derived HepG2 to oxidative stress by Nrf2-dependent mechanisms, in a process mediated by mitochondrial ROS (mtROS). Here we showed that after an initial decrease in oxygen consumption paralleled by a moderate increase in superoxide anion levels, AntiOxCIN4 led to a time-dependent Nrf2 translocation to the nucleus. This was followed later by a 1.5-fold increase in basal respiration and a 1.2-fold increase in extracellular acidification. AntiOxCIN4 treatment enhanced mitochondrial quality by triggering the clearance of defective organelles by autophagy and/or mitophagy, coupled with increased mitochondrial biogenesis. AntiOxCIN4 also up-regulated the cellular antioxidant defense system. AntiOxCIN4 seems to have the ability to maintain hepatocyte redox homeostasis, regulating the electrophilic/nucleophilic tone, and preserve cellular physiological functions. The obtained data open a new avenue to explore the effects of AntiOxCIN4 in the context of preserving hepatic mitochondrial function in disorders, such as NASH/NAFLD and type II diabetes.

Digesta and Plasma Metabolomics of Rainbow Trout Strains with Varied Tolerance of Plant-Based Diets Highlights Potential for Non-Lethal Assessments of Enteritis Development.

The replacement of fishmeal in aquafeeds is essential to the sustainability of aquaculture. Besides the procurement of alternative protein sources, fish can also be selected for better performance on plant-based alternative diets. Rainbow trout (Oncorhynchus mykiss) is one such species in which the strain ARS-Sel has been selected for higher growth and enhanced utilization when fed soy-based diets. The aim of this study was to compare fish growth and plasma and digesta metabolomes between ARS-Sel and two commercial strains (CS-1 and CS-2), when fed plant-protein (PM) and fishmeal-based (FM) diets, and to correlate them with the onset of enteritis. An NMR-metabolomics approach was taken to assess plasma and digesta metabolite profiles. Diet and strain showed significant effects on fish growth, with the ARS-Sel fish receiving the PM diet reaching the highest final weight at sampling. Multivariate analysis revealed differences between plasma and digesta metabolite profiles of ARS-Sel and CS (CS-1 considered together with CS-2) PM-fed groups in the early stages of enteritis development, which was confirmed by intestinal histology. As reported in previous studies, the ARS-Sel strain performed better than the commercial strains when fed the PM diet. Our findings also suggest that metabolomic profiles of plasma and digesta, samples of which can be obtained through non-lethal methods, offer valuable insight in monitoring the occurrence of enteritis in carnivorous aquaculture species due to plant-based diets.

Sex-dependent vulnerability of fetal nonhuman primate cardiac mitochondria to moderate maternal nutrient reduction.

Poor maternal nutrition in pregnancy affects fetal development, predisposing offspring to cardiometabolic diseases. The role of mitochondria during fetal development on later-life cardiac dysfunction caused by maternal nutrient reduction (MNR) remains unexplored. We hypothesized that MNR during gestation causes fetal cardiac bioenergetic deficits, compromising cardiac mitochondrial metabolism and reserve capacity. To enable human translation, we developed a primate baboon model (Papio spp.) of moderate MNR in which mothers receive 70% of control nutrition during pregnancy, resulting in intrauterine growth restriction (IUGR) offspring and later exhibiting myocardial remodeling and heart failure at human equivalent ∼25 years. Term control and MNR baboon offspring were necropsied following cesarean-section, and left ventricle (LV) samples were collected. MNR adversely impacted fetal cardiac LV mitochondria in a sex-dependent fashion. Increased maternal plasma aspartate aminotransferase, creatine phosphokinase (CPK), and elevated cortisol levels in MNR concomitant with decreased blood insulin in male fetal MNR were measured. MNR resulted in a two-fold increase in fetal LV mitochondrial DNA (mtDNA). MNR resulted in increased transcripts for several respiratory chain (NDUFB8, UQCRC1, and cytochrome c) and adenosine triphosphate (ATP) synthase proteins. However, MNR fetal LV mitochondrial complex I and complex II/III activities were significantly decreased, possibly contributing to the 73% decreased ATP content and increased lipid peroxidation. MNR fetal LV showed mitochondria with sparse and disarranged cristae dysmorphology. Conclusion: MNR disruption of fetal cardiac mitochondrial fitness likely contributes to the documented developmental programming of adult cardiac dysfunction, indicating a programmed mitochondrial inability to deliver sufficient energy to cardiac tissues as a chronic mechanism for later-life heart failure.

Determining the contribution of a high-fructose corn syrup formulation to hepatic glycogen synthesis during ad-libitum feeding in mice.

Excessive sugar intake including high-fructose corn syrup (HFCS) is implicated in the rise of obesity, insulin resistance and non-alcoholic fatty liver disease. Liver glycogen synthesis is influenced by both fructose and insulin signaling. Therefore, the effect of HFCS on hepatic glycogenesis was evaluated in mice feeding ad-libitum. Using deuterated water: the fraction of glycogen derived from triose-P sources, Krebs cycle substrates, and direct pathway + cycling, was measured in 9 normal-chow fed mice (NC) and 12 mice fed normal chow plus a 55% fructose/45% glucose mix in the drinking water at 30% w/v (HFCS-55). This was enriched with [U-13C]fructose or [U-13C]glucose to determine the contribution of each to glycogenesis. For NC, direct pathway + cycling, Krebs cycle, and triose-P sources accounted for 66 ± 0.7%, 23 ± 0.8% and 11 ± 0.4% of glycogen synthesis, respectively. HFCS-55 mice had similar direct pathway + cycling (64 ± 1%) but lower Krebs cycle (12 ± 1%, p < 0.001) and higher triose-P contributions (24 ± 1%, p < 0.001). HFCS-55-fructose contributed 17 ± 1% via triose-P and 2 ± 0% via Krebs cycle. HFCS-55-glucose contributed 16 ± 3% via direct pathway and 1 ± 0% via Krebs cycle. In conclusion, HFCS-55 supplementation resulted in similar hepatic glycogen deposition rates. Indirect pathway contributions shifted from Krebs cycle to Triose-P sources reflecting HFCS-55-fructose utilization, while HFCS-55-glucose was incorporated almost exclusively by the direct pathway.

Metabolic incorporation of H218 O into specific glucose-6-phosphate oxygens by red-blood-cell lysates as observed by 13 C isotope-shifted NMR signals.

Water enriched with oxygen-18 (H218 O) is a potential tracer for evaluating the sources of glucose and glycogen synthesis since it is incorporated into specific sites of glucose-6-phosphate via specific enzyme-mediated exchange/addition mechanisms. Unlike 2 H, 18 O does not experience significant isotope effects for any of these processes. Therefore, H218 O might provide more precise estimates of endogenous carbohydrate synthesis compared with deuterated water provided that positional 18 O enrichments of glucose can be measured. As a proof of concept, H218 O was incorporated into a well characterized hemolysate model of sugar phosphate metabolism and 13 C NMR was applied to quantify positional 18 O enrichment of glucose-6-phosphate oxygens. Human erythrocyte hemolysate preparations were incubated overnight at 37 °C with a buffer containing sugar phosphate precursors and 20% (n = 5) and 80% (n = 1) H218 O. Enrichment of glucose-6-phosphate was analyzed by 13 C NMR analysis of 18 O-shifted versus unshifted signals following derivatization to monoacetone glucose (MAG). 13 C NMR MAG spectra from hemolysate revealed resolved 18 O-shifted signals in Positions 1-5. Mean 18 O enrichments were 16.4 ± 1.6% (Position 1), 13.3 ± 1.3% (Position 2), 4.1 ± 1.1% (Position 3), 12.6 ± 0.8% (Position 4), 10.7 ± 1.4% (Position 5), and no detectable enrichment of Position 6. No 18 O-shifted glucose-6-phosphate signals were detected in preparations containing sugar phosphate precursors only. H218 O is incorporated into Positions 1-5 of glucose-6-phosphate in accordance with spontaneous aldose hydration and specific enzymatic reaction mechanisms. This provides a basis for its deployment as a tracer for glucose and glycogen biosynthesis.

Estimating pentose phosphate pathway activity from the analysis of hepatic glycogen 13 C-isotopomers derived from [U-13 C]fructose and [U-13 C]glucose.

PURPOSE: The pentose phosphate pathway (PPP) is an important component of hepatic intermediary metabolism. Jin et al developed an elegant 13 C-NMR method for measuring hepatic PPP flux by quantifying the distribution of glucose 13 C-isotopomers formed from [U-13 C]glycerol. We demonstrate that this approach can be extended to exogenous [U-13 C]fructose and [U-13 C]glucose precursors by 13 C-NMR analysis of glycogen. METHODS: Twelve male C57BL/6 mice fed standard chow were provided a 55/45 mixture of fructose and glucose at 30% w/v in the drinking water for 18 wk. On the evening before sacrifice, the fructose component was enriched with 20% [U-13 C]fructose for 6 mice, while the glucose component was enriched with 20% [U-13 C]glucose for the remaining 6 mice. Mice were allowed to feed and drink naturally overnight, and then, euthanized. Livers were freeze-clamped and glycogen was extracted and derivatized for 13 C NMR spectroscopy. Flux of each sugar into the PPP relative to its incorporation into glycogen was quantified from selected 13 C glycogen isotopomer ratios. RESULTS: Both [U-13 C]fructose and [U-13 C]glucose precursors yielded glycogen 13 C-isotopomer distributions that were characteristic of PPP activity. The fraction of [U-13 C]glucose utilized by the PPP relative to its conversion to glycogen via the direct pathway was 14 ± 1%, while that from [U-13 C]fructose relative to its conversion to glycogen via the indirect pathway was significantly lower (10 ± 1%, P = .00032). CONCLUSIONS: Hepatic PPP fluxes from both [U-13 C]glucose and [U-13 C]fructose precursors were assessed by 13 C NMR analysis of glycogen 13 C-isotopomers. Glucose-6-phosphate generated via glucokinase and the direct pathway is preferentially utilized by the PPP.

Limitations to Starch Utilization in Barramundi (Lates calcarifer) as Revealed by NMR-Based Metabolomics.

Practical diets for commercial barramundi production rarely contain greater than 10% starch, used mainly as a binding agent during extrusion. Alternative ingredients such as digestible starch have shown some capacity to spare dietary protein catabolism to generate glucose. In the present study, a carnivorous fish species, the Asian seabass (Lates calcarifer) was subjected to two diets with the same digestible energy: Protein (P) - with high protein content (no digestible starch); and Starch (S) - with high digestible (pregelatinized) starch content. The effects of a high starch content diet on hepatic glycogen synthesis as well as the muscle and liver metabolome were studied using a complementary approach of 1H and 2H NMR. The hepatosomatic index was lower for fish fed high starch content diet while the concentration of hepatic glycogen was similar between groups. However, increased glycogen synthesis via the direct pathway was observed in the fish fed high starch content diet which is indicative of increased carbohydrate utilization. Multivariate analysis also showed differences between groups in the metabolome of both tissues. Univariate analysis revealed more variations in liver than in muscle of fish fed high starch content diet. Variations in metabolome were generally in agreement with the increase in the glycogen synthesis through direct pathway, however, this metabolic shift seemed to be insufficient to keep the growth rate as ensured by the diet with high protein content. Although liver glycogen does not make up a substantial quantity of total stored dietary energy in carnivorous fish, it is a key regulatory intermediate in dietary energy utilization.

Transfer of glucose hydrogens via acetyl-CoA, malonyl-CoA, and NADPH to fatty acids during de novo lipogenesis.

Deuterated water (2H2O) is widely used for measuring de novo lipogenesis (DNL). 2H is incorporated into fatty acids via exchange between body water and the hydrogens of acetyl-CoA, malonyl-CoA, and NADPH. Previous studies concluded that these exchanges are incomplete; therefore, fatty acid 2H enrichment requires correcting. In mice, we measured the 2H enrichment of fatty acid positions 2 and 3 and methyl hydrogens from [U-2H7]glucose to determine 2H transfer from glucose to fatty acid via malonyl-CoA, NADPH, and acetyl-CoA, respectively. Positional fatty acid 2H enrichments were compared with 13C enrichment of the same sites from an equivalent amount of [U-13C6]glucose provided alongside the [U-2H7]glucose tracer. Transfer of glucose 2H to fatty acid position 2 and methyl sites was low (2H enrichment of 0.06 ± 0.01 and 0.14 ± 0.01 relative to 13C) indicating extensive exchange at both malonyl- and acetyl-CoA, respectively. Transfer of glucose 2H into fatty acid position 3 was more extensive (0.46 ± 0.04 relative to 13C, P < 10-5 vs. position 2), indicating a more limited exchange of those glucose hydrogens that were transferred via NADPH. However, mice provided with [U-13C6]glucose and 2H2O had equivalent 2H enrichments of fatty acid positions 2 and 3, suggesting that in this setting, NADPH and body water 2H had exchanged extensively. This is explained by contributions of substrates other than exogenous glucose to DNL coupled with their extensive 2H enrichment from 2H2O prior to DNL. Under such conditions, 2H enrichment of fatty acids from 2H2O does not need correction.

Metabolic Effects of Dietary Glycerol Supplementation in Muscle and Liver of European Seabass and Rainbow Trout by 1H NMR Metabolomics.

The sustainable growth of fish aquaculture will require the procurement of non-marine feed sources. Glycerol is a potential feed supplement whose metabolism may spare the catabolism of dietary amino acids, thereby extending the use of the feed protein to other physiological functions such as growth. In the present study, the effects of dietary glycerol supplementation on the muscle and liver metabolomes of rainbow trout (Oncorhynchus mykiss) and European seabass (Dicentrarchus labrax) were evaluated. Fish juveniles were fed diets with 0%, 2.5%, and 5% glycerol. Muscle and liver aqueous fractions were extracted and 1H NMR spectra were acquired. Metabolite profiles derived from the 1H NMR signals were assessed using univariate and multivariate statistical analyses. The adenylate energy charge was determined in the muscle. For both species, the muscle metabolite profile showed more variability compared to that of the liver and was most perturbed by the 5.0% glycerol diet. For the liver metabolite profile, rainbow trout showed fewer differences compared to European seabass. No differences were observed in energy charge between experimental groups for either species. Thus, rainbow trout appeared to be less susceptible to tissue metabolite perturbations, compared to seabass, when the diet was supplemented with up to 5% glycerol.

Determining contributions of exogenous glucose and fructose to de novo fatty acid and glycerol synthesis in liver and adipose tissue.

The de novo synthesis of triglyceride (TG) fatty acids (FA) and glycerol can be measured with stable isotope tracers. However, these methods typically do not inform the contribution of a given substrate to specific pathways on these synthetic processes. We integrated deuterated water (2H2O) measurement of de novo lipogenesis (DNL) and glycerol-3-phosphate (GLY) synthesis from all substrates with a 13C nuclear magnetic resonance (NMR) method that quantifies TG FA and glycerol enrichment from a specific [U-13C]precursor. This allowed the [U-13C]precursor contribution to DNL and GLY to be estimated. We applied this method in mice to determine the contributions of fructose and glucose supplemented in the drinking water to DNL and GLY in liver, mesenteric adipose tissue (MAT) and subcutaneous adipose tissue (SCAT). In liver, fructose contributed significantly more to DNL of saturated fatty acids (SFA) and oleate as well as to GLY compared to glucose. Moreover, its contribution to SFA synthesis was significantly higher compared to that of oleate. MAT and SCAT had lower fractional rates of total DNL and GLY compared to liver and glucose was utilized more predominantly than fructose for TG synthesis in these tissues. This novel 2H2O/13C integrated method revealed for the first time, tissue specific selection of substrates for DNL, particularly fructose in regard to glucose in liver. Also, this approach was able to resolve the distribution of specific FAs into the TG sn2 and sn1,3 sites. This stable isotope integrated approach yielded information so far uncovered by other lipidomic tools and should powerfully assist in other nutritional, pathological or environmental contexts.

Impact of dietary starch on extrahepatic tissue lipid metabolism in farmed European (Dicentrarchus labrax) and Asian seabass (Lates calcarifer).

In aquaculture, there is high interest in substituting marine-derived with vegetable-based ingredients as energy source. Farmed carnivorous fish under high carbohydrate diets tend to increase adiposity but it remains unclear if this happens by increased lipid retention/accumulation, promotion of lipogenic pathways, or both. In order to determine the response of extrahepatic tissue to dietary starch, European (Dicentrarchus labrax) and Asian (Lates calcarifer) seabass were fed a control (low starch; LS) or experimental (high starch; HS) diet, for at least 21 days and then transferred for 6 days to saltwater enriched with deuterated water 2H2O. Incorporation of 2H-labelling follows well-defined metabolic steps, and analysis of triacylglycerols (TAG) 2H-enrichment by 2HNMR allowed evaluation of de novo lipogenesis (DNL) in muscle and visceral adipose tissue (VAT). Fractional synthetic rates for TAG-bound fatty acids and glycerol were quantified separately providing a detailed lipogenic profile. The FA profile differed substantially between muscle and VAT in both species, but their lipogenic fluxes revealed even greater differences. In European seabass, HS promoted DNL of TAG-bound FA, in muscle and VAT. High 2H-enrichment also found in muscle TAG-bound glycerol was indicative of its role on lipid cycling. In Asian seabass, HS had no effect on muscle FA composition and lipogenic flux, with no 2H-enriched TAG being detected. VAT on the other hand revealed a strong enhancement of DNL in HS-fed fish along with high TAG-bound glycerol cycling. This study consolidated the use of 2H2O as tracer for fish lipid metabolism in different tissues, under different dietary conditions and suitable to use in different fish models.

Reactive Oxygen Species Deficiency Due to Ncf1-Mutation Leads to Development of Adenocarcinoma and Metabolomic and Lipidomic Remodeling in a New Mouse Model of Dextran Sulfate Sodium-Induced Colitis.

Inflammatory bowel disease is characterized by chronic relapsing idiopathic inflammation of the gastrointestinal tract and persistent inflammation. Studies focusing on the immune-regulatory function of reactive oxygen species (ROS) are still largely missing. In this study, we analyzed an ROS-deficient mouse model leading to colon adenocarcinoma. Colitis was induced with dextran sulfate sodium (DSS) supplied via the drinking water in wild-type (WT) and Ncf1-mutant (Ncf1) B10.Q mice using two different protocols, one mimicking recovery after acute colitis and another simulating chronic colitis. Disease progression was monitored by evaluation of clinical parameters, histopathological analysis, and the blood serum metabolome using 1H nuclear magnetic resonance spectroscopy. At each experimental time point, colons and spleens from some mice were removed for histopathological analysis and internal clinical parameters. Clinical scores for weight variation, stool consistency, colorectal bleeding, colon length, and spleen weight were significantly worse for Ncf1 than for WT mice. Ncf1 mice with only a 7-day exposure to DSS followed by a 14-day resting period developed colonic distal high-grade dysplasia in contrast to the low-grade dysplasia found in the colon of WT mice. After a 21-day resting period, there was still ß-catenin-rich inflammatory infiltration in the Ncf1 mice together with high-grade dysplasia and invasive well-differentiated adenocarcinoma, while in the WT mice, high-grade dysplasia was prominent without malignant invasion and only low inflammation. Although exposure to DSS generated less severe histopathological changes in the WT group, the blood serum metabolome revealed an increased fatty acid content with moderate-to-strong correlations to inflammation score, weight variation, colon length, and spleen weight. Ncf1 mice also displayed a similar pattern but with lower coefficients and showed consistently lower glucose and/or higher lactate levels which correlated with inflammation score, weight variation, and spleen weight. In our novel, DSS-induced colitis animal model, the lack of an oxidative burst ROS was sufficient to develop adenocarcinoma, and display altered blood plasma metabolic and lipid profiles. Thus, oxidative burst seems to be necessary to prevent evolution toward cancer and may confer a protective role in a ROS-mediated self-control mechanism.

Response to dietary carbohydrates in European seabass (Dicentrarchus labrax) muscle tissue as revealed by NMR-based metabolomics.

INTRODUCTION: Feed optimization is a key step to the environmental and economic sustainability of aquaculture, especially for carnivorous species. Plant-derived ingredients can contribute to reduce costs and nitrogenous effluents while sparing wild fish stocks. However, the metabolic use of carbohydrates from vegetable sources by carnivorous fish is still not completely understood. OBJECTIVES: We aimed to study the effects of diets with carbohydrates of different digestibilities, gelatinized starch (DS) and raw starch (RS), in the muscle metabolome of European seabass (Dicentrarchus labrax). METHODS: We followed an NMR-metabolomics approach, using two sample preparation procedures, the intact muscle (HRMAS) and the aqueous muscle extracts (1H NMR), to compare the variations in muscle metabolome between the two diets. RESULTS: In muscle, multivariate analysis revealed similar metabolome shifts for DS and RS diets, when compared with the control diet. HRMAS of intact muscle, which included both hydrophobic and hydrophilic metabolites, showed increased lipid in DS-fed fish by univariate analysis. Regardless of the nature of the starch, increased glycine and phenylalanine, and decreased proline were observed when compared to the Ctr diet. Combined univariate analysis of intact muscle and aqueous extracts indicated specific diet related changes in lipid and amino acid metabolism, consistent with increased dietary carbohydrate supplementation. CONCLUSIONS: Due to differential sample processing, outputs differ in detail but provide complementary information. After tracing nutritional alterations by profiling fillet components, DS seems to be the most promising alternative to fishmeal-based diets in aquaculture. This approach should be reproducible for other farmed fish species and provide valuable information on nutritional and organoleptic properties of the final product.