Mitochondrial-derived peptides and exercise

Acute exercise, and in particular aerobic exercise, increases skeletal muscle energy demand causing mitochondrial stress, and mitochondrial-related adaptations which are a hallmark of exercise training. Given that mitochondria are central players in the exercise response, it is imperative that they have networks that can communicate their status both intra- and inter-cellularly. Peptides encoded by short open-reading frames within mitochondrial DNA, mitochondrial-derived peptides (MDPs), have been suggested to form a newly recognised branch of this retrograde signalling cascade that contribute to coordinating the adaptive response to regular exercise. Here we summarise the recent evidence that acute high intensity exercise in humans can increase concentrations of the MDPs humanin and MOTS-c in skeletal muscle and plasma, and speculate on the mechanisms controlling MDP responses to exercise stress. Evidence that exercise training results in chronic changes in MDP expression within tissues and the circulation is conflicting and may depend on the mode, duration, intensity of training plan and participant characteristics. Further research is required to define the effect of these variables on MDPs and to determine whether MDPs other than MOTS-c have exercise mimetic properties. MOTS-c treatment of young and aged mice improves exercise capacity/performance and leads to adaptions that are similar to that of being physically active (weight loss, increased antioxidant capacity and improved insulin sensitivity), however, studies utilising a MOTS-c inactivating genetic variant or combination of exercise + MOTS-c treatment in mice suggest that there are distinct and overlapping pathways through which exercise and MOTS-c evoke metabolic benefits. Overall, MOTS-c, and potentially other MDPs, may be exercise-sensitive myokines and further work is required to define inter- and intra-tissue targets in an exercise context.     

Cardio-protective role of Humanin in myocardial ischemia-reperfusion

Mitochondria-derived peptides (MDPs) are bioactive peptides encoded by and secreted from the mitochondria. To date, a few MDPs including humanin, MOTS-c and SHLP1–6, and their diverse biological functions have been identified. The first and most studied MDP is humanin, a 24-amino-acid poly peptide. It was first identified in 2001 in the surviving neurons of patient with Alzheimer's disease, and since then has been well characterized for its neuro-protective effect through inhibition of apoptosis. Over the past two decades, humanin has been reported to play critical roles in aging as well as multiple diseases including metabolic disorders, cardiovascular diseases, and autoimmune disease. Humanin has been shown to modulate multiple biological processes including autophagy, ER stress, cellular metabolism, oxidative stress, and inflammation. A role for humanin has been shown in a wide range of cardiovascular diseases, such as coronary heart disease, atherosclerosis, and myocardial fibrosis. In this minireview, we will summarize the literature demonstrating a role for humanin in cardio-protection following myocardial ischemia-reperfusion induced injury and the potential mechanisms that mediate it.     

The emerging role of mitochondrial derived peptide humanin in the testis

The discovery of mitochondrial derive peptides (MDPs) has spotlighted mitochondria as central hubs in control and regulation of cell viability and metabolism in the testis in response to intracellular and extracellular stresses. MDPs (Humanin, MOTS-c and SHLP-2) are present in testes. Humanin, the first MDP, is predominantly expressed in Leydig cells, and moderately in germ cells and seminal plasma. The administration of synthetic humanin peptide agonist HNG protects male germ cells against apoptosis induced by intratesticular hormonal deprivation, testicular hyperthermia, and chemotherapeutic agents in rodent testes. Humanin interacting with IGFBP-3 and/or Bax (pro-apoptotic proteins) prevents the activation of germ cell apoptosis. Humanin participates in the network of IL-12/IL-27 family of cytokines to exert the immune-modulation of the testicular environment. Humanin and other MDPs may be important in the amelioration of testicular stress and prevention of cell injury with possible implications for male infertility, fertility preservation and contraceptive development.     

Quercetin in vesicular delivery systems: Evaluation in combating arsenic-induced acute liver toxicity associated gene expression in rat model

Arsenic, the environmental toxicant causes oxidative damage to liver and produces hepatic fibrosis. The theme of our study was to evaluate the therapeutic efficacy of liposomal and nanocapsulated herbal polyphenolic antioxidant Quercetin (QC) in combating arsenic induced hepatic oxidative stress, fibrosis associated upregulation of its gene expression and plasma TGF ß (transforming growth factor ß) in rat model.A single dose of Arsenic (sodium arsenite-NaAsO₂, 13 mg/kg b.wt) in oral route causes the generation of reactive oxygen species (ROS), arsenic accumulation in liver, hepatotoxicity and decrease in hepatic plasma membrane microviscosity and antioxidant enzyme levels in liver. Arsenic causes fibrosis associated elevation of its gene expression in liver, plasma TGF ß (from normal value 75.2 ± 8.67 ng/ml to 196.2 ± 12.07 ng/ml) and release of cytochrome c in cytoplasm. Among the two vesicular delivery systems formulated with QC, polylactide nanocapsules showed a promising result compared to liposomal delivery system in controlling arsenic induced alteration of those parameters. A single dose of 0.5 ml of nanocapsulated QC suspension (QC 2.71 mg/kg b.wt) when injected to rats 1 h after arsenic administration orally protects liver from arsenic induced deterioration of antioxidant levels as well as oxidative stress associated gene expression of liver. Histopathological examination also confirmed the pathological improvement in liver. Nanocapsulated plant origin flavonoidal compound may be a potent formulation in combating arsenic induced upregulation of gene expression of liver fibrosis through a complete protection against oxidative attack in hepatic cells of rat liver.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

Positive correlation between plasma nitrite and performance during high-intensive exercise but not oxidative stress in healthy men

Several studies suggest that exercise is associated with elevated oxidative stress which diminishes NO bioavailability. The aim of the present study was to investigate a potential link between NO synthesis and bioavailability and oxidative stress in the circulation of subjects performing high-intensive endurance exercise. Twenty-two male healthy subjects cycled at 80% of their maximal workload. Cubital venous blood was taken before, during and after exercise, and heparinized plasma was generated. Plasma concentrations of nitrite and nitrate were quantified by GC–MS and of the oxidative stress biomarker 15(S)-8-iso-PGF_2α by GC–MS/MS. pH and pCO₂ fell and HbO₂ increased upon exercise. The duration of the 80% phase (d80) was 740 ± 210 s. Subjects cycled at 89.2 ± 3.3% of their peak oxygen uptake. Plasma concentration of nitrite (P < 0.01) and 15(S)-8-iso-PGF_2α (P < 0.05) decreased significantly during exercise. At the end of exercise, plasma nitrite concentration correlated positively with d80 and performed work (w80) (each P < 0.05). Changes in nitrate concentration also correlated positively with d80 (P < 0.05) and w80/kg (P < 0.01). These findings provide evidence of a favorable effect of nitrite on high-intensive endurance exercise. The lack of association between 15(S)-8-iso-PGF_2α and NO bioavailability (nitrite concentration) and NO biosynthesis (nitrate concentration) suggest that oxidative stress, notably lipid peroxidation, is not linked to the l-arginine/NO pathway in healthy male subjects being on endurance exercise.     

Effects of excess dietary iron and fat on glucose and lipid metabolism

PurposeDiets rich in fat and energy are associated with metabolic syndrome (MS). Increased body iron stores have been recognized as a feature of MS. High-fat diets (HFs), excess iron loading and MS are closely associated, but the mechanism linking them has not been clearly defined. We investigated the interaction between dietary fat and dietary Fe in the context of glucose and lipid metabolism in the body.MethodsC57BL6/J mice were divided into four groups and fed the modified AIN-93G low-fat diet (LF) and HF with adequate or excess Fe for 7 weeks. The Fe contents were increased by adding carbonyl iron (2% of diet weight) (LF+Fe and HF+Fe).ResultsHigh iron levels increased blood glucose levels but decreased high-density lipoprotein cholesterol levels. The HF group showed increases in plasma levels of glucose and insulin and insulin resistance. HF+Fe mice showed greater changes. Representative indices of iron status, such hepatic and plasma Fe levels, were not altered further by the HF. However, both the HF and excess iron loading changed the hepatic expression of hepcidin and ferroportin. The LF+Fe, HF and HF+Fe groups showed greater hepatic fat accumulation compared with the LF group. These changes were paralleled by alterations in the levels of enzymes related to hepatic gluconeogenesis and lipid synthesis, which could be due to increases in mitochondrial dysfunction and oxidative stress.ConclusionsHigh-fat diets and iron overload are associated with insulin resistance, modified hepatic lipid and iron metabolism and increased mitochondrial dysfunction and oxidative stress.     

Isocaloric manipulation of macronutrients within a high-carbohydrate/moderate-fat diet induces unique effects on hepatic lipogenesis,steatosis and liver injury

Diets containing excess carbohydrate and fat promote hepatic steatosis and steatohepatitis in mice. Little is known, however, about the impact of specific carbohydrate/fat combinations on liver outcome. This study was designed to determine whether high-energy diets with identical caloric density but different carbohydrate and fat composition have unique effects on the liver. Four experimental diets were formulated with 60% kcal carbohydrate and 20% kcal fat, each in nearly pure form from a single source: starch-oleate, starch-palmitate, sucrose-oleate and sucrose-palmitate. The diets were fed to mice for 3 or 12 weeks for analysis of lipid metabolism and liver injury. All mice developed hepatic steatosis over 12 weeks, but mice fed the sucrose-palmitate diet accumulated more hepatic lipid than those in the other three experimental groups. The exaggerated lipid accumulation in sucrose-palmitate-fed mice was attributable to a disproportionate rise in hepatic de novo lipogenesis. These mice accrued more hepatic palmitate and exhibited more evidence of liver injury than any of the other experimental groups. Interestingly, lipogenic gene expression in mice fed the custom diets did not correlate with actual de novo lipogenesis. In addition, de novo lipogenesis rose in all mice between 3 and 12 weeks, without feedback inhibition from hepatic steatosis. The pairing of simple sugar (sucrose) and saturated fat (palmitate) in a high-carbohydrate/moderate-fat diet induces more de novo lipogenesis and liver injury than other carbohydrate/fat combinations. Diet-induced liver injury correlates positively with hepatic de novo lipogenesis and is not predictable by isolated analysis of lipogenic gene expression.     

Efficacy of azelaic acid on hepatic key enzymes of carbohydrate metabolism in high fat diet induced type 2 diabetic mice

Azelaic acid (AzA), a C9 linear α,ω-dicarboxylic acid, is found in whole grains namely wheat, rye, barley, oat seeds and sorghum. The study was performed to investigate whether AzA exerts beneficial effect on hepatic key enzymes of carbohydrate metabolism in high fat diet (HFD) induced type 2 diabetic C57BL/6J mice. C57BL/6J mice were fed high fat diet for 10 weeks and subjected to intragastric administration of various doses (20 mg, 40 mg and 80 mg/kg BW) of AzA daily for the subsequent 5 weeks. Rosiglitazone (RSG) was used as reference drug. Body weight, food intake, plasma glucose, plasma insulin, blood haemoglobin (Hb), blood glycosylated haemoglobin (HbA1c), liver glycolytic enzyme (hexokinase), hepatic shunt enzyme (glucose-6-phosphate dehydrogenase), gluconeogenic enzymes(glucose-6-phosphatase and fructose-1,6-bisphosphatase), liver glycogen, plasma and liver triglycerides were examined in mice fed with normal standard diet (NC), high fat diet (HFD), HFD with AzA (HFD + AzA) and HFD with rosiglitazone (HFD + RSG). Among the three doses, 80 mg/kg BW of AzA was able to positively regulate plasma glucose, insulin, blood HbA1c and haemoglobin levels by significantly increasing the activity of hexokinase and glucose-6-phosphate dehydrogenase and significantly decreasing the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase thereby increasing the glycogen content in the liver. From this study, we put forward that AzA could significantly restore the levels of plasma glucose, insulin, HbA1c, Hb, liver glycogen and carbohydrate metabolic key enzymes to near normal in diabetic mice and hence, AzA may be useful as a biomaterial in the development of therapeutic agents against high fat diet induced T2DM.     

C-C Chemokine Receptor 2 Inhibitor Ameliorates Hepatic Steatosis by Improving ER Stress and Inflammation in a Type 2 Diabetic Mouse Model

Hepatic steatosis is the accumulation of excess fat in the liver. Recently, hepatic steatosis has become more important because it occurs in the patients with obesity, type 2 diabetes, and hyperlipidemia and is associated with endoplasmic reticulum (ER) stress and insulin resistance. C-C chemokine receptor 2 (CCR2) inhibitor has been reported to improve inflammation and glucose intolerance in diabetes, but its mechanisms remained unknown in hepatic steatosis. We examined whether CCR2 inhibitor improves ER stress-induced hepatic steatosis in type 2 diabetic mice. In this study, db/db and db/m (n = 9) mice were fed CCR2 inhibitor (2 mg/kg/day) for 9 weeks. In diabetic mice, CCR2 inhibitor decreased plasma and hepatic triglycerides levels and improved insulin sensitivity. Moreover, CCR2 inhibitor treatment decreased ER stress markers (e.g., BiP, ATF4, CHOP, and XBP-1) and inflammatory cytokines (e.g., TNFα, IL-6, and MCP-1) while increasing markers of mitochondrial biogenesis (e.g., PGC-1α, Tfam, and COX1) in the liver. We suggest that CCR2 inhibitor may ameliorate hepatic steatosis by reducing ER stress and inflammation in type 2 diabetes mellitus.     

Disruption of hepatic one-carbon metabolism impairs mitochondrial function and enhances macrophage activity in methionine–choline-deficient mice

One-carbon metabolism is a collection of metabolic cycles that supports methylation and provides one-carbon bound folates for the de novo synthesis of purine and thymidine nucleotides. The methylation of phosphatidylethanolamine to form choline has been extensively studied in the context of fatty liver disease. However, the role of one-carbon metabolism in supporting nucleotide synthesis during liver damage has not been addressed. The objective of this study is to determine how the disruption of one-carbon metabolism influences nucleotide metabolism in the liver after dietary methionine and choline restriction. Mice (n=8) were fed a methionine–choline-deficient or control diet for 3 weeks. We treated mice with the compound alloxazine (0.5 mg/kg), a known adenosine receptor antagonist, every second day during the final week of feeding to probe the function of adenosine signaling during liver damage. We found that concentrations of several hepatic nucleotides were significantly lower in methionine- and choline-deficient mice vs. controls (adenine: 13.9±0.7 vs. 10.1±0.6, guanine: 1.8±0.1 vs. 1.4±0.1, thymidine: 0.0122±0.0027 vs. 0.0059±0.0027 nmol/mg dry tissue). Treatment of alloxazine caused a specific decrease in thymidine nucleotides, decrease in mitochondrial content in the liver and exacerbation of steatohepatitis as shown by the increased hepatic lipid content and altered macrophage morphology. This study demonstrates a role for one-carbon metabolism in supporting de novo nucleotide synthesis and mitochondrial function during liver damage.     

Hepatic metabolic adaptation and adipose tissue expansion are altered in mice with steatohepatitis induced by high-fat high sucrose diet

Background: Obesity is a chronic progressive disease with several metabolic alterations. Nonalcoholic fatty liver disease (NAFLD) is an important comorbidity of obesity that can progress to nonalcoholic steatohepatitis (NASH), cirrhosis or hepatocarcinoma. This study aimed at clarifying the molecular mechanisms underlying the metabolic alterations in hepatic and adipose tissue during high-fat high-sucrose diet-induced NAFLD development in mice. Methods: Twenty-four male mice (C57BL/6J) were randomly allocated into 3 groups (n = 8 mice per group) to receive a chow diet, a high-fat diet (HFD), or a high-fat high-sucrose diet (HF-HSD) for 20 weeks. At sacrifice, liver and adipose tissue were obtained for histopathological, metabolomic, and protein expression analyses. Results: HF-HSD (but not HFD) was associated with NASH and increased oxidative stress. These animals presented an inhibition of hepatic autophagy and alterations in AMP-activated protein kinase/mammalian target of rapamycin activity. We also observed that the ability of metabolic adaptation was adversely affected by the increase of damaged mitochondria. NASH development was associated with changes in adipose tissue dynamics and increased amounts of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids in visceral adipose tissue. Conclusion: HF-HSD led to a metabolic blockage and impaired hepatic mitochondria turnover. In addition, the continuous accumulation of fatty acids produced adipose tissue dysfunction and hepatic fat accumulation that favored the progression to NASH.     

Effects of dietary nitrate supplementation on the oxygen cost of exercise and walking performance in individuals with type 2 diabetes: a randomized,double-blind,placebo-controlled crossover trial

Dietary nitrate supplementation has been shown to reduce the oxygen (O₂) cost of exercise and enhance exercise tolerance in healthy individuals. This study assessed whether similar effects could be observed in individuals with type 2 diabetes (T2DM). In a randomized, double-blind, placebo-controlled crossover study, 48 participants with T2DM supplemented their diet for 4 days with either nitrate-rich beetroot juice (70 ml/day, 6.43 mmol nitrate/day) or nitrate-depleted beetroot juice as placebo (70 ml/day, 0.07 mmol nitrate/day). After each intervention period, resting plasma nitrate and nitrite concentrations were measured subsequent to participants completing moderate-paced walking. Pulmonary gas exchange was measured to assess the O₂ cost of walking. After a rest period, participants performed the 6-min walk test (6MWT). Relative to placebo, beetroot juice resulted in a significant increase in plasma nitrate (placebo, 57±66 vs beetroot, 319±110 µM; P < 0.001) and plasma nitrite concentration (placebo, 680±256 vs beetroot, 1065±607 nM; P < 0.001). There were no differences between placebo juice and beetroot juice for the O₂ cost of walking (946±221 vs 939±223 ml/min, respectively; P = 0.59) and distance covered in the 6MWT (550±83 vs 554±90 m, respectively; P = 0.17). Nitrate supplementation did not affect the O₂ cost of moderate-paced walking or improve performance in the 6MWT. These findings indicate that dietary nitrate supplementation does not modulate the response to exercise in individuals with T2DM.     

Arjunolic acid,a triterpenoid saponin,prevents acetaminophen (APAP)-induced liver and hepatocyte injury via the inhibition of APAP bioactivation and JNK-mediated mitochondrial protection

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug and is safe at therapeutic doses but its overdose frequently causes liver injury. In earlier studies, we demonstrated that arjunolic acid (AA) has a protective effect against chemically induced hepatotoxicity. The purpose of this study was to explore whether AA plays any protective role against APAP-induced acute hepatotoxicity and, if so, what molecular pathways it utilizes for the mechanism of its protective action. Exposure of rats to a hepatotoxic dose of acetaminophen (700 mg/kg, ip) altered a number of biomarkers (related to hepatic oxidative stress), increased reactive oxygen species production, reduced cellular adenosine triphosphate level, and induced necrotic cell death. Arjunolic acid pretreatment (80 mg/kg, orally), on the other hand, afforded significant protection against liver injury. Arjunolic acid also prevented acetaminophen-induced hepatic glutathione depletion and APAP metabolite formation although arjunolic acid itself did not affect hepatic glutathione levels. The results suggest that this preventive action of arjunolic acid is due to the metabolic inhibition of specific forms of cytochrome P450 that activate acetaminophen to N-acetyl-p-benzoquinone imine. In addition, administration of arjunolic acid 4 h after acetaminophen intoxication reduced acetaminophen-induced JNK and downstream Bcl-2 and Bcl-xL phosphorylation, thus protecting against mitochondrial permeabilization, loss of mitochondrial membrane potential, and cytochrome c release. In conclusion, the data suggest that arjunolic acid affords protection against acetaminophen-induced hepatotoxicity through inhibition of P450-mediated APAP bioactivation and inhibition of JNK-mediated activation of mitochondrial permeabilization.     

Vascular and Hepatic Impact of Short-Term Intermittent Hypoxia in a Mouse Model of Metabolic Syndrome

BackgroundExperimental models of intermittent hypoxia (IH) have been developed during the last decade to investigate the consequences of obstructive sleep apnea. IH is usually associated with detrimental metabolic and vascular outcomes. However, paradoxical protective effects have also been described depending of IH patterns and durations applied in studies. We evaluated the impact of short-term IH on vascular and metabolic function in a diet-induced model of metabolic syndrome (MS).MethodsMice were fed either a standard diet or a high fat diet (HFD) for 8 weeks. During the final 14 days of each diet, animals were exposed to either IH (1 min cycle, FiO₂ 5% for 30s, FiO₂ 21% for 30s; 8 h/day) or intermittent air (FiO₂ 21%). Ex-vivo vascular reactivity in response to acetylcholine was assessed in aorta rings by myography. Glucose, insulin and leptin levels were assessed, as well as serum lipid profile, hepatic mitochondrial activity and tissue nitric oxide (NO) release.ResultsMice fed with HFD developed moderate markers of dysmetabolism mimicking MS, including increased epididymal fat, dyslipidemia, hepatic steatosis and endothelial dysfunction. HFD decreased mitochondrial complex I, II and IV activities and increased lactate dehydrogenase (LDH) activity in liver. IH applied to HFD mice induced a major increase in insulin and leptin levels and prevented endothelial dysfunction by restoring NO production. IH also restored mitochondrial complex I and IV activities, moderated the increase in LDH activity and liver triglyceride accumulation in HFD mice.ConclusionIn a mouse model of MS, short-term IH increases insulin and leptin levels, restores endothelial function and mitochondrial activity and limits liver lipid accumulation.     

Radiosynthesis and biological evaluation of an fluorine-18 labeled galactose derivative [18F]FPGal for imaging the hepatic asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the surface of hepatocytes where it recognizes and endocytoses glycoproteins with galactosyl and N-acetylgalactosamine groups. Given its hepatic distribution, the asialoglycoprotein receptor can be targeted by positron imaging agents to study liver function using PET imaging. In this study, the positron imaging agent [¹⁸F]FPGal was designed to specifically target hepatic asialoglycoprotein receptor and its effectiveness was assessed in in vitro and in vivo models. The radiosynthesis of [¹⁸F]FPGal required 50 min with total radiochemical yields of [¹⁸F]FPGal from [¹⁸F]fluoride as 10% (corrected radiochemical yield). The K_d of [¹⁸F]FPGal to ASGPR in HepG2 cells was 1.99 ± 0.05 mM. Uptake values of 0.55% were observed within 30 min of incubation with HepG2 cells, which could be blocked by 200 mM d(+)-galactose (<0.1%). In vivo biodistribution analysis showed that the liver accumulation of [¹⁸F]FPGal at 30 min was 4.47 ± 0.96% ID/g in normal mice compared to 1.33 ± 0.07% ID/g in hepatic fibrotic mice (P < 0.01). Reduced uptake in the hepatic fibrosis mouse models was confirmed through PET/CT images at 30 min. Compared to normal mice, the standard uptake value (SUV) in the hepatic fibrosis mice was significantly lower when assessed through dynamic data collection for 1 h. Therefore, [¹⁸F]FPGal is a feasible PET probe that provide insight into ASGPR related liver disease.     

A Combination of Mitochondrial Oxidative Stress and Excess Fat/Calorie Intake Accelerates Steatohepatitis by Enhancing Hepatic CC Chemokine Production in Mice

Mitochondrial oxidative stress is considered as a key accelerator of fibrosis in various organs including the liver. However, the production of oxidative stress and progression of liver fibrosis may merely represent the independent consequences of hepatocellular injury caused by the primary disease. Because of a lack of appropriate experimental models to evaluate the sole effects of oxidative stress, it is virtually unknown whether this stress is causatively linked to the progression of liver fibrosis. Here, we examined the direct effects of mitochondrial reactive oxygen species (ROS) on the progression of high fat/calorie diet-induced steatohepatitis using Tet-mev-1 mice, in which a mutated succinate dehydrogenase transgene impairs the mitochondrial electron transport and generates an excess amount of ROS in response to doxycycline administration. Wild type and Tet-mev-1 mice that had been continuously given doxycycline-containing water were subsequently fed either normal chow or a cholesterol-free high-fat/high-sucrose diet for 4 months at approximately 1 or 2 years of age. Histopathological examinations indicated that neither the mitochondrial ROS induced in Tet-mev-1 mice nor the feeding of wild type animals with high-fat/high-sucrose diet alone caused significant liver fibrosis. Only when the Tet-mev-1 mice were fed a high-fat/high-sucrose diet, it induced lipid peroxidation in hepatocytes and enhanced hepatic CC chemokine expression. These events were accompanied by increased infiltration of CCR5-positive cells and activation of myofibroblasts, resulting in extensive liver fibrosis. Interestingly, this combinatorial effect of mitochondrial ROS and excess fat/calorie intake on liver fibrosis was observed only in 2-year-old Tet-mev-1 mice, not in the 1-year-old animals. Collectively, these results indicate that mitochondrial ROS in combination with excess fat/calorie intake accelerates liver fibrosis by enhancing CC chemokine production in aged animals. We have provided a good experimental model to explore how high fat/calorie intake increases the susceptibility to nonalcoholic steatohepatitis in aged individuals who have impaired mitochondrial adaptation.     

Lowering of oxidative stress improves endothelial function in healthy subjects with habitually low intake of fruit and vegetables: A randomized controlled trial of antioxidant- and polyphenol-rich blackcurrant juice

Inadequate intake of the recommended five-a-day fruit and vegetable portions might contribute to increased cardiovascular disease risk. We assessed the effects of dietary intake of a blackcurrant juice drink, rich in vitamin C and polyphenols, on oxidative stress and vascular function. This was a double-blind, placebo-controlled, parallel group study of 66 healthy adults who habitually consume <2 portions of fruit and vegetables per day. Participants were randomly allocated to consume 250 ml of placebo (flavored water) or low or high blackcurrant juice drink four times a day for 6 weeks. Flow-mediated dilation (FMD) and plasma concentrations of F₂-isoprostanes and vitamin C were measured. In the high blackcurrant juice drink group FMD increased significantly (5.8±3.1 to 6.9±3.1%, P=0.022) compared with the placebo group (6.0±2.2 to 5.1±2.4%). Plasma vitamin C concentration increased significantly in the low (38.6±17.6 to 49.4±21.0 µmol/L, P<0.001) and high (34.6±20.4 to 73.8±23.3 µmol/L, P<0.001) blackcurrant juice drink groups compared with the placebo group (38.1±21.0 to 29.0±17.6 µmol/L). F₂-isoprostane concentrations were significantly lower in the high blackcurrant juice drink group (225±64 pg/ml) compared with the low blackcurrant juice drink (257±69 pg/ml, P=0.002) and placebo group (254±59 pg/ml, P=0.003). At follow-up, changes in plasma vitamin C correlated significantly with changes in FMD (r=0.308, P=0.044). Consumption of blackcurrant juice drink high in vitamin C and polyphenols can decrease oxidative stress and improve vascular health in individuals with habitually low dietary fruit and vegetable intake.     

Antioxidant properties of tea blunt ROS-dependent lipogenesis: beneficial effect on hepatic steatosis in a high fat-high sucrose diet NAFLD obese rat model

Oxidative stress could trigger lipid accumulation in liver and thus hepatic steatosis. Tea is able to prevent liver disorders, but a direct link between antioxidant capacities and prevention of steatosis has not been reported yet. We aimed to investigate such relationship in a rat model of high fat-high sucrose diet (HFS)-induced obesity and to explore more deeply the mechanisms in isolated hepatocytes. Wistar rats were divided into a control group (standard diet), an HFS group (high fat-sucrose diet) and an HFS + tea group (HFS diet with ad-libitum access to tea drink). Body weight, fat mass, glycemic parameters in blood, lipid and oxidative stress parameters in blood and liver were measured in each group after 14 weeks. Isolated hepatocytes were treated with the reactive oxygen species (ROS) inducer t-BHP in the presence or not of antioxidants (tempol or tea), and superoxide anion production and lipid accumulation were measured using specific fluorescent probes. We reported that the HFS diet highly increased hepatic lipids content, while tea consumption attenuated steatosis and improved the oxidative status (decrease in hepatic oxidative stress, increase in plasma total antioxidant capacity). The role of antioxidant properties of tea in such phenomenon was confirmed in primary cultured rat hepatocytes. Indeed, the increase of mitochondrial ROS production with t-BHP resulted in lipid accumulation in hepatocytes (positive linear regression), and antioxidants (tempol or tea) normalized both. We reported that the antioxidant properties of tea protect rats from an obesogenic HFS diet-induced hepatic steatosis by counteracting the ROS-dependent lipogenesis.