Energy Metabolism In Liver Research Articles

Tissue Oxygenation and Mitochondrial Respiration under Different Modes of Intermittent Hypoxia

We compared the results of five modes of intermittent hypoxia training (IHT) on gastrocnemius muscle Po2 and heart and liver mitochondrial respiration in rats. Minutes of hypoxia, %O2, and recovery minutes on air in each mode were: 1) 5, 12%, 5; 2) 15, 12%, 15; 3) 5, 12%, 15; 4) 5, 7%, 5; and 5) 5, 7%, 15. Mode 1 proved best in that Pmo2 dropped minimally at the end of every hypoxic bout and recovered quickly after each bout. One, 2, and 3 week IHT in mode 1 each increased tissue PO2 in both normoxic and 30 min severe hypoxic (7% O2) tests. Adaptation to IHT in Mode 1 caused the substrate-dependent reorganization of liver and heart mitochondrial energy metabolism favoring NADH-dependent oxidation and improving the efficiency of oxidative phosphorylation. Mitochondrial adaptation occurred after 14 days of IHT in liver tissue, but after 21 days in myocardium, and was preserved during the 3 months following IHT termination. When using Mode 2, positive changes were also registered, but were less pronounced. Other IHT modes provoked negative effects on Pmo2 levels, both during hypoxic periods and reoxygenation. In conclusion, the most effective IHT regimen is 5 min 12% O2 with 5 min breaks, five cycles per day during 2 or 3 weeks depending on the task of IHT.

Effect of moderate dietary restriction on visceral organ weight, hepatic oxygen consumption, and metabolic proteins associated with energy balance in mature pregnant beef cows1,2

Twenty-two nonlactating multiparous pregnant beef cows (639 ± 68 kg) were used to investigate the effect of dietary restriction on the abundance of selected proteins regulating cellular energy metabolism. Cows were fed at either 85% (n = 11; LOW) or 140% (n = 11; HIGH) of total NE requirements. The diet consisted of a haylage-based total mixed ration containing 20% wheat straw. Cows were slaughtered by block (predicted date of parturition), beginning 83 d after the initiation of dietary treatments and every week thereafter for 6 wk, such that each block was slaughtered at approximately 250 d of gestation. Tissue samples from liver, kidney, sternomandibularis muscle, ruminal papilli (ventral sac), pancreas, and small intestinal muscosa were collected at slaughter and snap frozen in liquid N2. Western blots were conducted to quantify abundance of proliferating cell nuclear antigen (PCNA), ATP synthase, ubiquitin, and Na/K+ ATPase for all tissues; PPARγ, PPARγ coactivator 1 α (PGC-1α), and 5´-adenosine monophosphate-activated protein kinase (AMPK) and the activated form phosphorylated-AMPK (pAMPK) for liver, muscle, and rumen; phosphoenolpyruvate carboxykinase (PEPCK) for liver and kidney; and uncoupling protein 2 (UCP2) for liver. Statistical analysis was conducted using Proc Mixed in SAS and included the fixed effects of dietary treatment, cow age, block, and the random effect of pen. Dietary treatments resulted in cows fed HIGH having greater (P ≤ 0.04) ADG and final BW than cows fed LOW. Abundance of ubiquitin in muscle was greater (P = 0.009) in cows fed LOW, and PCG-1 α in liver was greater (P = 0.03) in cows fed HIGH. Hepatic O2 consumption was greater in HIGH (P ≤ 0.04). Feed intake can influence the abundance of important metabolic proteins and suggest that protein degradation may increase in muscle from moderately nutrient restricted cows and that energy metabolism in liver increases in cows fed above NE requirements.

Maternal obesity and post‐natal high fat diet disrupt hepatic circadian rhythm in rat offspring

Offspring of obese (Ob) rat dams gain greater body wt and fat mass when fed high fat diet (HFD) as compared to controls. Alterations of diurnal circadian rhythms are known to detrimentally impact metabolically active tissues such as liver. We sought to determine if maternal obesity (MOb) leads to perturbations of circadian rhythm and energy metabolism in offspring liver following a short (2 wk) high fat (HFD) challenge. mRNA expression on PND35 of putative circadian (CLOCK, Reverbα, Bmal1, Cry, Per) and metabolic (PPARα, SIRT1) genes were suppressed following HFD in lean and Ob groups. Further suppression was evident in Ob‐offspring indicating circadian disruption. Using a mathematical model for the circadian clock, analyses of PPARα and SIRT1 temporal changes in relative mRNA abundance revealed exposure to MOb and/or HFD disrupts the circadian rhythm by (i) decreasing mRNA synthesis via a rate limiting activation constant of expression of nuclear Bmal1 and (ii) increasing non‐specific mRNA degradation of both PPARα and SIRT1. HDAC3 a known regulator of circadian rhythmicity and hepatic lipid metabolism, was decreased at 5/6 time points (4 hr intervals) by exposure to HFD and MOb. HFD disrupts core hepatic clock machinery and key regulators of energy metabolism and is further exacerbated by MOb which may increase the risk for harmful adiposity gains in adulthood. Support NIHR01‐DK084225

Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase

Vitamin B12 (cobalamin, cbl) is a cofactor of methionine synthase (MTR) in the synthesis of methionine, the precursor of the universal methyl donor S-Adenosylmethionine (SAM), which is involved in epigenomic regulatory mechanisms. We have established a neuronal cell model with stable expression of a transcobalamin-oleosin chimer and subsequent decreased cellular availability of vitamin B12, which produces reduced proliferation, increased apoptosis and accelerated differentiation through PP2A, NGF and TACE pathways. Anti-transcobalamin antibody or impaired transcobalamin receptor expression produce also impaired proliferation in other cells. Consistently, the transcription, protein expression and activity of MTR are increased in proliferating cells of skin and intestinal epitheliums, in rat intestine crypts and in proliferating CaCo2 cells, while MTR activity correlates with DNA methylation in rat intestine villi. Exposure to nitrous oxide in animal models identified impairment of MTR reaction as the most important metabolic cause of neurological manifestations of B12 deficiency. Early vitamin B12 and folate deprivation during gestation and lactation of a 'dam-progeny' rat model developed in our laboratory is associated with long-lasting disabilities of behavior and memory capacities, with persisting hallmarks related to increased apoptosis, impaired neurogenesis and altered plasticity. We found also an epigenomic deregulation of energy metabolism and fatty acids beta-oxidation in myocardium and liver, through imbalanced methylation/acetylation of PGC-1alpha and decreased expression of SIRT1. These nutrigenomic effects display similarities with the molecular mechanisms of fetal programming. Beside deficiency, B12 loading increases the expression of MTR through internal ribosome entry sites (IRES) and down-regulates MDR-1 gene expression. In conclusion, vitamin B12 influences cell proliferation, differentiation and apoptosis in brain. Vitamin B12 and folate combined deficiency impairs fatty acid oxidation and energy metabolism in liver and heart through epigenomic mechanisms related to imbalanced acetylation/methylation. Some but not all of these effects reflect the upstream role of vitamin B12 in SAM synthesis.

Interactions between hypoxia tolerance and food deprivation in Amazonian oscars, Astronotus ocellatus (Agassiz)

Oscars are often subjected to a combination of low levels of oxygen and fasting during nest-guarding on Amazonian floodplains. We questioned whether this anorexia would aggravate the osmo-respiratory compromise. We compared fed and fasted oscars (10-14 days) in both normoxia and hypoxia (10-20 Torr, 4 h). Routine oxygen consumption rates (O2) were increased by 75% in fasted fish, reflecting behavioural differences, whereas fasting improved hypoxia resistance and critical oxygen tensions (Pcrit) lowered from 54 Torr in fed fish to 34 Torr when fasting. In fed fish, hypoxia reduced liver lipid stores by approximately 50% and total liver energy content by 30%. Fasted fish had a 50% lower hepatosomatic index, resulting in lower total liver protein, glycogen and lipid energy stores under normoxia. Compared with hypoxic fed fish, hypoxic fasted fish only showed reduced liver protein levels and even gained glycogen (+50%) on a per gram basis. This confirms the hypothesis that hypoxia-tolerant fish protect their glycogen stores as much as possible as a safeguard for more prolonged hypoxic events. In general, fasted fish showed lower hydroxyacylCoA dehydrogenase activities compared with fed fish, although this effect was only significant in hypoxic fasted fish. Energy stores and activities of enzymes related to energy metabolism in muscle or gills were not affected. Branchial Na(+) uptake rates were more than two times lower in fed fish, whereas Na(+) efflux was similar. Fed and fasted fish quickly reduced Na(+) uptake and efflux during hypoxia, with fasting fish responding more rapidly. Ammonia excretion and K(+) efflux were reduced under hypoxia, indicating decreased transcellular permeability. Fasted fish had more mitochondria-rich cells (MRC), with larger crypts, indicating the increased importance of the branchial uptake route when feeding is limited. Gill MRC density and surface area were greatly reduced under hypoxia, possibly to reduce ion uptake and efflux rates. Density of mucous cells of normoxic fasted fish was approximately fourfold of that in fed fish. Overall, a 10-14 day fasting period had no negative effects on hypoxia tolerance in oscars, as fasted fish were able to respond more quickly to lower oxygen levels, and reduced branchial permeability effectively.

Analysis of Liver Transcriptome in Broilers with Ascites and Regulation by L-Carnitine

Two experiments were conducted using male Ross-308 broilers. Exp. 1 was to determine the differences in liver transcriptomic profile of healthy broilers at 39 days (D39) vs. 21 days of age (D21), and of ascites vs. healthy broilers at D39 by GeneChips, under a low-temperature environment. Differentially expressed genes related to liver antioxidative capacity and energy metabolism were screened. Exp. 2 was to analyze the regulatory roles of L-carnitine on genes and biochemical parameters involved in liver oxidoreduction and energy metabolism. The results showed that the expression of 790 genes in D39 vs D21 healthy broilers (386 up- and 404 down-regulated) and expression of 178 genes in ascites vs. healthy broilers on D39 (128 up- and 50 down-regulated) was changed. The analysis of pathway and GO indicated that genes related to amino acid metabolism, fatty acid metabolism, Krebs cycle, carbohydrate metabolism, vitamin metabolism, cell apoptosis, immune response, oxidoreduction, angiogenesis, nitric oxide formation and erythrocyte differentiation were involved in the development of ascites. Liver MDA level of ascites broilers was significantly increased, but the activity of T-SOD was significantly decreased than healthy broilers. The activity of liver HK, MDH, PK and Na+-K+-ATPase was significantly lower in ascites broilers. The expression of liver HKDC1 and PCK1 was significantly down-regulated, and that of HNF-4α and AKR1B10 expression was significantly up-regulated. L-carnitine supplementation significantly increased the activity of T-SOD and PK, and significantly up-regulated HKDC1 and HNF-4α expression. It can be concluded that genes involved in many biological processes are differentially expressed due to ascites. Liver oxidation damage and energy generation obstruction were found in broilers with ascites. L-carnitine can alleviate the liver oxidative damage and promote the generation and utilization of energy. The present results demonstrated that L-carnitine could serve as a potential regulatory agent to reduce ascites susceptibility and mortality.

Liver-Specific PGC-1beta Deficiency Leads to Impaired Mitochondrial Function and Lipogenic Response to Fasting-Refeeding

PGC-1β plays pleiotropic roles in regulating intermediary metabolism and has been shown to regulate both catabolic and anabolic processes in liver. We sought to evaluate the effects of PGC-1β on liver energy metabolism by generating mice with postnatal, liver-specific deletion of PGC-1β (LS-PGC-1β−/− mice). LS-PGC-1β−/− mice were outwardly normal, but exhibited a significant increase in hepatic triglyceride content at 6 weeks of age. Hepatic steatosis was due, at least in part, to impaired capacity for fatty acid oxidation and marked mitochondrial dysfunction. Mitochondrial DNA content and the expression of genes encoding multiple steps in mitochondrial fatty acid oxidation and oxidative phosphorylation pathways were significantly diminished in LS-PGC-1β−/− mice. Liquid chromatography mass spectrometry-based analyses also revealed that acetylcarnitine and butyrylcarnitine levels were depleted whereas palmitoylcarnitine content was increased in LS-PGC-1β−/− liver, which is consistent with attenuated rates of fatty acid oxidation. Interestingly, loss of PGC-1β also significantly impaired inducible expression of glycolytic and lipogenic enzymes that occurs with high carbohydrate diet refeeding after a prolonged fast. These results suggest that PGC-1β plays dual roles in regulating hepatic fatty acid metabolism by controlling the expression of programs of genes involved in both fatty acid oxidation and de novo fatty acid synthesis.

Effect of rumen-protected choline supplementation on liver and adipose gene expression during the transition period in dairy cattle

We previously reported that supplementation of rumen-protected choline (RPC) reduces the hepatic triacylglycerol concentration in periparturient dairy cows during early lactation. Here, we investigated the effect of RPC on the transcript levels of lipid metabolism-related genes in liver and adipose tissue biopsies, taken at wk −3, 1, 3, and 6 after calving, to elucidate the mechanisms underlying this RPC-induced reduction of hepatic lipidosis. Sixteen multiparous cows were blocked into 8 pairs and randomly allocated to either 1 of 2 treatments, with or without RPC. Treatments were applied from 3wk before to 6wk after calving. Both groups received a basal diet and concentrate mixture. One group received RPC supplementation, resulting in an intake of 14.4g of choline per day, whereas controls received an isoenergetic mixture of palm oil and additional soybean meal. Liver and adipose tissue biopsies were taken at wk −3, 1, 3, and 6 to determine the mRNA abundance of 18 key genes involved in liver and adipose tissue lipid and energy metabolism. Milk samples were collected in wk 1, 2, 3, and 6 postpartum for analysis of milk fatty acid (FA) composition. The RPC-induced reduction in hepatic lipidosis could not be attributed to altered lipolysis in adipose tissue, as no treatment effect was observed on the expression of peroxisome proliferator-activated receptor γ, lipoprotein lipase, or FA synthase in adipose tissue, or on the milk FA composition. Rumen-protected choline supplementation increased the expression of FA transport protein 5 and carnitine transporter SLC22A5 in the liver, suggesting an increase in the capacity of FA uptake and intracellular transport, but no treatment effect was observed on carnitine palmitoyl transferase 1A, transporting long-chain FA into mitochondria. In the same organ, RPC appeared to promote apolipoprotein B-containing lipoprotein assembly, as shown by elevated microsomal triglyceride transfer protein expression and apolipoprotein B100 expression. Cows supplemented with RPC displayed elevated levels of glucose transporter 2 mRNA and a reduced peak in pyruvate carboxylase mRNA immediately after calving, showing that supplementation also resulted in improved carbohydrate metabolism. The results from this study suggest that RPC supplementation reduces liver triacylglycerol by improved FA processing and very-low-density lipoprotein synthesis, and RPC also benefits hepatic carbohydrate metabolism.

Effects of ischemic postconditioning on liver regeneration and energy metabolism in rats

To explore the effects of ischemic postconditioning on the remnant liver regeneration under ischemic reperfusion after subtotal hepatectomy. A total of 90 male SD rats weighting 230-280 g were randomly divided into 3 groups of simple subtotal hepatectomy (SH), ischemia reperfusion (IR) and ischemic postconditioning (IPO). The left and middle liver lobes were resected. And the remnant liver was treated as follows: blood flow was non-blocked in SH group, but blocked for 30 min in IR group, then reperfused. The IPO group was established by 30-30 s intermittent interruptions of blood flow thrice during the early phase of reperfusion. At 1, 6, 12, 24, 48 h respectively, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatocyte growth factor (HGF) and the positive ratio of proliferating cell nuclear antigen (PCNA) were detected. Also the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were measured and the regenerated liver weight was calculated. The activities of ALT and AST in IPO group from 1 - 48 h were (138 ± 20), (340 ± 22), (770 ± 55), (389 ± 60), (113 ± 25) U/L and (247 ± 31), (509 ± 51), (972 ± 77), (955 ± 159), (222 ± 44) U/L respectively at each time point and they were significantly lower than those (294 ± 37), (560 ± 69), (1222 ± 162), (540 ± 40), (161 ± 15) U/L and (439 ± 34), (669 ± 94), (1347 ± 118), (1274 ± 223), (403 ± 68) U/L in IR group (P < 0.05); the regenerated liver weight (18.8 ± 3.2, 34.0 ± 3.5%) and positive cell ratio of PCNA (43.9 ± 2.7, 56.2 ± 4.0%) in IPO group at 24 and 48 h were respectively higher than those in IR group ((11.3 ± 2.9), (26.8 ± 2.5)% and (34.5 ± 2.8), (48.8 ± 1.8)%)(P < 0.05); The HGF levels ((261 ± 54) and (252 ± 103) pg/ml) at 24 and 48 h in IPO group were significantly higher than those ((99 ± 47) and (70 ± 19) pg/ml) in IR group (P < 0.05); the contents of ATP in hepatic tissue at 24 and 48 h in IPO group were (22.20 ± 3.6) and (12.87 ± 2.49) µg/g respectively. And they were significantly higher than those in IR group (P < 0.05), but lower than those in SH group (P < 0.05). Ischemic postconditioning improves the remnant liver regeneration under ischemic reperfusion after subtotal hepatectomy through its actions of stimulating HGF production and maintaining hepatic energy metabolism.

Acute effects of deltamethrin on swimming velocity and biomarkers of the common prawn Palaemon serratus

The main purpose of the present study was to investigate the effects of deltamethrin on biomarkers and behavior of Palaemon serratus (common prawn), since this attempt to link different levels of biological organization will allow determining which biomarkers might be ecologically relevant and will be useful to complement the information about the effects of pesticides by using behavioral parameters. Therefore, parameters of liver antioxidant status, energy metabolism and neurotransmission were determined in different tissues of the common prawn and used to assess the effects at sub-individual level, whereas swimming velocity was used to assess the effects at the individual level. It was also investigated if the swimming velocity can be used as an endpoint in ecotoxicology bioassays and if it can be as sensitive as biomarker endpoints.Swimming velocity was significantly reduced in prawns exposed to deltamethrin, showing a lowest observed effect (LOEC) of 0.6ngL−1. Eye acetylcholinesterase (AChE) activity was significantly increased in prawns exposed to 0.6, 1.2 and 2.4ngL−1 deltamethrin, whereas muscle cholinesterase (ChE) activity was significantly increased in prawns exposed to 19 and 39ngL−1. On the other hand, lactate dehydrogenase (LDH) activity was significantly increased in muscle of prawns exposed to 0.6, 1.2, 2.4, 4.9ngL−1 deltamethrin, showing that organisms were requiring additional energy, but probably using it for detoxification processes rather than locomotion, since swimming velocity was inhibited. Glutathione S-transferase (GST) activity was significantly increased in the digestive gland of common prawn exposed to 19 and 39ngL−1 deltamethrin. Catalase (CAT) activity was significantly increased in digestive gland of prawn exposed to 19ngL−1 deltamethrin. However, CAT activity decreased in digestive gland of prawn exposed to 39ngL−1, suggesting an antioxidant defense system failure concomitant with high levels of lipid peroxidation. Thus, global results showed that decreased swimming velocity was not associated with cholinesterase inhibition. In fact, the impairment of swimming velocity may be due to allocation of energy for detoxification and antioxidant protection instead of swimming activity. The present study showed that swimming velocity could be used as an ecologically relevant tool and a sensitive endpoint to assess and complement the study of pesticide effects on marine organisms.

Altered feeding differentially regulates circadian rhythms and energy metabolism in liver and muscle of rats

Energy metabolism follows a diurnal pattern responding to the light/dark cycle and food availability. This study investigated the impact of restricting feeding to the daylight hours and feeding a high fat diet on circadian clock (bmal1, dbp, tef and e4bp4) and metabolic (pepck, fas, ucp3, pdk4) gene expression and markers of energy metabolism in muscle and liver of rats. The results show that in chow-fed rats switched to daylight feeding, the peak diurnal expression of genes in liver was shifted by 6–12h while expression of these genes in muscle remained in a similar phase to rats feeding ad libitum. High fat feeding during the daylight hours had limited effect on clock gene expression in liver or muscle but shifted the peak expression of metabolic genes (pepck, fas) in liver by 6–12h. The differential effects of daylight feeding on gene and protein expression in muscle and liver were accompanied by an 8% reduction in whole body energy expenditure, a 20–30% increased glycogen content during the light phase in muscle of day-fed rats and increased adipose tissue deposition per gram food consumed. These data demonstrate that a mismatch of feeding and light/dark cycle disrupts tissue metabolism in muscle with significant consequences for whole body energy homeostasis.

The European whitefish, Coregonus lavaretus (L.), under conditions of technogenic pollution in the Kostomuksha tailing pond

The population structure and biological parameters of the European whitefish, Coregonus lavaretus (L.), have been analyzed in a technogenically polluted water body. The biochemical state of the whitefish and the fauna and morphology of helminths parasitizing them have been assessed. It is shown that the species composition of the fish, plankton, benthos, and parasite faunas of this water body are depleted and the morphological parameters of common fish parasites are altered. It is noted that, in addition to adaptive rearrangements in biochemical metabolism, the whitefish from the transformed water body are characterized by a reduction in the level of protein synthesis in muscles and the rate of aerobic energy metabolism in the liver and muscles.

Short-term, Mild Hypothermia Can Increase the Beneficial Effect of Permissive Hypotension on Uncontrolled Hemorrhagic Shock in Rats

Our previous and other studies have shown that hypotensive or hypothermic resuscitation have beneficial effects on uncontrolled hemorrhagic shock. Whether hypothermia can increase the beneficial effect of hypotensive resuscitation on hemorrhagic shock is not known. Two-hundred and twenty Sprague-Dawley rats were used to make uncontrolled hemorrhagic shock. Before bleeding was controlled, rats received normotensive or hypotensive resuscitation (target mean arterial pressure at 80 or 50 mmHg) in combination with normal (37°C) or mild hypothermia (34°C) (phase II). After bleeding was controlled, rats received whole blood and lactated Ringer's solution resuscitation for 2 h (phase III). The animal survival, blood loss, fluid requirement, cardiac output, and coagulation functions, as well as vital organ function, mitochondrial function, and energy metabolism of liver, kidney and intestines, were noted. Short-term, mild hypothermia before bleeding was controlled increased the beneficial effect of hypotensive resuscitation. Hypothermia further decreased blood loss, oxygen consumption, and functional damage to the liver, kidney, and intestines during hypotensive resuscitation, protected mitochondrial function and energy metabolism (activity of Na(+)-K(+)-ATPase), and further improved survival time and survival rate (hypothermic/hypotensive combined group: survival rate, 9/10; survival time, 616 min; normothermic/normotensive group: 1/10, 256 min; hypothermic/normotensive group: 4/10, 293 min). Hypothermia slightly inhibited coagulation function. Mild hypothermia before bleeding is controlled can increase the beneficial effect of hypotensive resuscitation on uncontrolled hemorrhagic shock. The mechanism underlying the benefits of short-term hypothermia may be related to the decrease in oxygen consumption and metabolism, and protection of mitochondrial and organ functions.

Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression

Choline is an essential nutrient and the liver is a central organ responsible for choline metabolism. Hepatosteatosis and liver cell death occur when humans are deprived of choline. In the last few years, there have been significant advances in our understanding of the mechanisms that influence choline requirements in humans and in our understanding of choline's effects on liver function. These advances are useful in elucidating why nonalcoholic fatty liver disease (NAFLD) occurs and progresses sometimes to hepatocarcinogenesis. Humans eating low-choline diets develop fatty liver and liver damage. This dietary requirement for choline is modulated by estrogen and by single-nucleotide polymorphisms in specific genes of choline and folate metabolism. The spectrum of choline's effects on liver range from steatosis to development of hepatocarcinomas, and several mechanisms for these effects have been identified. They include abnormal phospholipid synthesis, defects in lipoprotein secretion, oxidative damage caused by mitochondrial dysfunction, and endoplasmic reticulum stress. Furthermore, the hepatic steatosis phenotype can be characterized more fully via metabolomic signatures and is influenced by the gut microbiome. Importantly, the intricate connection between liver function, one-carbon metabolism, and energy metabolism is just beginning to be elucidated. Choline influences liver function, and the dietary requirement for this nutrient varies depending on an individual's genotype and estrogen status. Understanding these individual differences is important for gastroenterologists seeking to understand why some individuals develop NAFLD and others do not, and why some patients tolerate total parenteral nutrition and others develop liver dysfunction.

Soy isoflavone Genistein affects energy metabolism in a steatotic liver model

Non-alcoholic fatty liver disease (NAFLD) is already the most common chronic liver disease in western countries. The complex metabolic disorder is strongly associated with insulin resistance. Epidemiological studies suggest beneficial effects of the natural soy isoflavone Genistein (GEN) on hepatic energy metabolism. Our aim was to establish a steatotic liver in vitro model to investigate the impact of GEN on energy metabolism focused on insulin signaling. Primary human hepatocytes (PHH) were isolated from resected liver tissues via a two-step collagenase perfusion technique. Steatosis was induced by adding oleic and palmitic acid for 24h to the culture medium. The lipid content was determined photometrically by Oil Red O and SRB staining. Cytotoxicity was evaluated by assays of XTT, AST/LDH and UREA. Phosphorylation levels of ERK½, AKT, FOXO1 and GSK3α/β were measured by Western blot analysis in PHH with and without insulin as well as after incubation with GEN for 24h. PHH treated with free fatty acids showed a significant increase of fat accumulation and a moderate lipotoxicity. Furthermore, in steatotic PHH we observed decreased insulin induced phosphorylation levels of ERK½, AKT, FOXO1 and GSK3α/β. Additional treatment with GEN showed in control PHH an increase of insulin induced phoshorylation levels of ERK½, AKT and GSK3α/β whereas steatotic PHH revealed decreased levels of ERK½ and AKT in a dose dependent manner. Our data show that the steatotic liver in vitro model is a reliable reproduction of in vivo conditions regarding fat accumulation, insulin resistance and lipotoxicity. Insulin resistance was observed by an impaired phosphorylation of ERK½, AKT, FOXO1 and GSK3α/β. GEN leads only in control PHH to an improvement of insulin signaling by increasing phosphorylation levels. However, in steatotic PHH a beneficial effect was not observed. Therefore we recommend further studies to investigate the impact of GEN on energy metabolism in steatotic liver.

Environmental Contaminant Mixtures at Ambient Concentrations Invoke a Metabolic Stress Response in Goldfish Not Predicted from Exposure to Individual Compounds Alone

Environmental contaminants from wastewater and industrial or agricultural areas are known to have adverse effects on development, reproduction, and metabolism. However, reliable assessment of environmental contaminant impact at low (i.e., ambient) concentrations using genomics and transcriptomics approaches has proven challenging. A goldfish model was used to investigate the effects of aquatic pollutant exposure in vivo by means of quantitative nuclear magnetic resonance metabolomics in multiple organs to elucidate a system-wide response. Animals were exposed to 4,4'-isopropylidenediphenol (Bisphenol-A, BPA), di-(2-ethylhexyl)-phthalate (DEHP), and nonylphenol (NP). Metabolite-specific spectral analysis combined with pathway-driven bioinformatics indicated changes in energy and lipid metabolism in liver following exposure to individual contaminants and a tertiary mixture. A dissimilar response in testis exposed to DEHP and mixture indicates disrupted AMPK and cAMP signaling. Uniquely, our observations (1) suggest that exposure to a contaminant mixture is characterized by a stress response not predicted from exposure to individual contaminants, even in the absence of other phenotypic features and (2) demonstrate the sensitivity of metabolomics in risk-assessment of environmental toxicant mixtures at ambient concentrations by detecting early stage metabolic dysregulation. These findings have general applicability in the assessment of "benign" compound mixtures in environmental and pharmaceutical development.

Resveratrol plus ethanol counteract the ethanol-induced impairment of energy metabolism: 31P NMR study of ATP and sn-glycerol-3-phosphate on isolated and perfused rat liver

The effects of trans-resveratrol (RSV) combined with ethanol (EtOH) were evaluated by 31P NMR on total ATP and sn-glycerol-3-phosphate (sn-G3P) contents measured in real time in isolated and perfused whole liver of the rat. Mitochondrial ATP turnover was assessed by using specific inhibitors of glycolytic and mitochondrial ATP supply (iodacetate and KCN, respectively). In RSV alone, the slight decrease in ATP content (−14±5% of the initial content), sn-G3P content and ATP turnover were similar to those in the Krebs-Henseleit buffer control. Compared to control, EtOH alone (14 or 70mmol/L) induced a decrease in ATP content (−24.95±2.95% of initial content, p<0.05) and an increase in sn-G3P (+158±22%), whereas ATP turnover tended to be increased. RSV (20μmol/L) combined with EtOH, (i) maintained ATP content near 100%, (ii) induced a 1.6-fold increase in mitochondrial ATP turnover (p=0.049 and p=0.004 vs EtOH 14 and 70mmol/L alone, respectively) and (iii) led to an increase in sn-G3P (+49±9% and +81±6% for 14 and 70mmol/L EtOH, respectively). These improvements were obtained only when glycolysis was efficient at the time of addition of EtOH+RSV. Glycolysis inhibition by iodacetate (IAA) evidenced an almost 21% contribution of this pathway to ATP content. RSV alone or RSV+EtOH prevented the ATP decrease induced by IAA addition (p<0.05 vs control). This is the first demonstration of the combined effects of RSV and EtOH on liver energy metabolism. RSV increased (i) the flux of substrates through ATP producing pathways (glycolysis and phosphorylative oxidation) probably via the activation of AMPkinase, and (ii) maintained the glycolysis deviation to sn-G3P linked to NADH+H+ re-oxidation occurring during EtOH detoxication, thus reducing the energy cost due to the latter.

Gut Microbiota Modulate the Metabolism of Brown Adipose Tissue in Mice

A two by two experimental study has been designed to determine the effect of gut microbiota on energy metabolism in mouse models. The metabolic phenotype of germ-free (GF, n = 20) and conventional (n = 20) mice was characterized using a NMR spectroscopy-based metabolic profiling approach, with a focus on sexual dimorphism (20 males, 20 females) and energy metabolism in urine, plasma, liver, and brown adipose tissue (BAT). Physiological data of age-matched GF and conventional mice showed that male animals had a higher weight than females in both groups. In addition, conventional males had a significantly higher total body fat content (TBFC) compared to conventional females, whereas this sexual dimorphism disappeared in GF animals (i.e., male GF mice had a TBFC similar to those of conventional and GF females). Profiling of BAT hydrophilic extracts revealed that sexual dimorphism in normal mice was absent in GF animals, which also displayed lower BAT lactate levels and higher levels of (D)-3-hydroxybutyrate in liver, plasma, and BAT, together with lower circulating levels of VLDL. These data indicate that the gut microbiota modulate the lipid metabolism in BAT, as the absence of gut microbiota stimulated both hepatic and BAT lipolysis while inhibiting lipogenesis. We also demonstrated that (1)H NMR metabolic profiles of BAT were excellent predictors of BW and TBFC, indicating the potential of BAT to fight against obesity.

Cloning and characterization of chicken fat mass and obesity associated (Fto) gene: fasting affects Fto expression

Fat mass and obesity associated gene (Fto), also known as Fatso, is a member of the Fe–II and 2-oxoglutarate-dependent dioxygenase superfamily. Recent studies in humans and rodents suggest that Fto is involved in food intake regulation and lipid metabolism, whereas single nucleotide mutations in the Fto gene are associated with obesity and type 2 diabetes. The Fto gene is highly conserved from green algae to humans, but little is known about the avian Fto gene or protein. The objectives of the current study were to clone full-length chicken Fto cDNA and to determine the effect of age or feeding status on Fto expression. With the use of rapid amplification of cDNA ends, the full-length chicken Fto cDNA was cloned and found to share 63% to 66% homology with the mammalian Fto nucleotide sequence. Several regions of the chicken Fto protein, including the substrate (2-oxoglutarate) binding domains, were found to be identical to mammalian Fto protein. Western blotting with anti-human Fto antibody and reverse transcription PCR studies showed that Fto protein and gene were ubiquitously expressed in various tissues of the chicken. With the use of quantitative PCR, Fto mRNA levels were found to be higher in liver and skeletal muscle of 8-wk-old chickens than in 4-wk-old chickens. In addition, alterations in feeding status resulted in significant changes in Fto mRNA and Fto protein expression in the liver but not in skeletal muscle and adipose tissue of broiler chickens. Taken together, our data suggest that Fto probably plays a significant role in liver function and energy metabolism in the chicken.

Metabolic effects of dietary sugar beet pulp or wheat bran in growing female pigs1

An experiment was conducted to determine the effects of feeding a moderate level of 2 different fiber sources on energy metabolites; mitochondrial biogenesis in the intestine, liver, and muscle; and the expression of some genes that regulate energy metabolism in intestine, liver, muscle, and adipose tissue. Female pigs (n = 36; BW = 15.0 ± 0.7 kg) were fed diets containing no added fiber, 12.5% sugar beet pulp (SBP), or 12.5% wheat bran (WB) for 24 d. Blood samples were collected on d 7 and 24 for cholesterol, glucose, NEFA, and triglyceride analyses. At completion of the experiment, ileum, colon, subcutaneous adipose, and LM samples were obtained from a subset (n = 6) of pigs fed each diet for analysis of tissue mitochondrial DNA (mtDNA) content and mRNA abundance by quantitative real-time reverse-transcription PCR. Glycogen and triglyceride content of liver and LM were determined, and colon content VFA was also determined. The addition of SBP or WB to the diet had no effect (P > 0.55) on ADG, ADFI, or G:F. Serum NEFA and triglycerides were increased (P < 0.05) in pigs fed SBP compared with pigs fed the control diet or WB on d 7, and NEFA remained increased (P < 0.05) on d 24 in pigs fed SBP. Dietary fiber had no effect (P > 0.24) on glycogen and triglyceride content of liver or LM, but colonic acetate concentrations were increased (P < 0.05) in pigs fed either SBP or WB. Pigs fed WB had an increased (P < 0.05) mtDNA content in ileum tissue and increased (P < 0.05) citrate synthase mRNA in colon tissue. In the liver, feeding either SBP or WB led to a decrease (P < 0.05) in mtDNA content, whereas feeding WB decreased (P < 0.05) mtDNA abundance in the LM, and feeding either SBP or WB decreased (P < 0.05) expression of citrate synthase mRNA. Quantitative reverse-transcription PCR revealed that feeding WB increased (P < 0.05) proliferating cell nuclear antigen mRNA abundance in the ileum and colon. Feeding WB increased (P < 0.05) mRNA abundance of a regulator of mitochondrial biogenesis, PPAR coactivator 1 α, in ileum tissue, and increased (P < 0.05) mRNA abundance of another mediator of mitochondrial biogensis, sirtuin 1, in colon tissue. Colonic mRNA expression of fasting-induced adipose factor was increased (P < 0.05) in pigs fed either SBP or WB, and adipose triglyceride lipase mRNA abundance was increased (P < 0.05) in adipose tissue of pigs fed SBP. These data indicate that increasing dietary fiber can increase the capacity of the intestine for oxidative metabolism and induce a repartitioning of energy metabolites depending on fiber source.