Ambivalent effects of diazoxide on mitochondrial ROS production at respiratory chain complexes I and III

Background

Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H₂DCF) but had no direct impact on the H₂O₂ production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).

Methods

H₂O₂ generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.

Results

We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Q_o site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na⁺ or K⁺ excluding a role for putative mitochondrial K_ATP-channels.

General significance

A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion).     

Programming of Obesity and Comorbidities in the Progeny: Lessons from a Model of Diet-Induced Obese Parents

Aim

To determine the impact of paternal obesity, maternal obesity or the combination of two obese parents on markers of adult offspring metabolism, with a focus on body mass (BM), lipid and carbohydrate, components of lipogenesis and beta-oxidation in the liver, sex dimorphism in the offspring that received a SC diet during the postnatal period.

Materials and Methods

Male and female C57BL/6 mice were fed a high-fat diet (HF; 49% lipids) or standard chow (SC; 17% lipids) for 8 weeks before mating until lactation. The offspring were labeled according to sex, maternal diet (first letters), paternal diet (second letters), and received a SCdiet until 12-weeks of age when they were sacrificed. BM, eating behavior, glucose tolerance, plasma analysis, gene and protein expression of the components of lipogenesis and beta-oxidation in the liver of offspring were evaluated.

Results

HF diet-fed mothers and fathers were overweight, hyperglycemic and glucose intolerant and had a deteriorating lipid profile. The adult male and female offspring of HF-mothers were overweight, with an increased adiposity index, hyperphagic, had an impaired glucose metabolism, increased total cholesterol and triacylglycerol levels, increased lipogenesis concomitant with decreased beta-oxidation resulting in liver steatosis. The male and female offspring of HF-father had impaired glucose metabolism, exacerbated lipogenesis without influencing beta-oxidation and enhanced hepatic steatosis. These findings are independent of BM. Male and female offspring of a mother and father that received a HF diet demonstrated these effects most prominently in adult life.

Conclusion

Paternal obesity leads to alterations in glucose metabolism, increase in components of lipogenesis and liver steatosis. In contrast, maternal obesity leads to overweight and changes in the metabolic profile and liver resulting from activation of hepatic lipogenesis with impaired beta-oxidation. When both parents are obese, the effects observed in the male and female offspring are exacerbated.     

Exercise,but not quercetin,ameliorates inflammation,mitochondrial biogenesis,and lipid metabolism in skeletal muscle after strenuous exercise by high-fat diet mice

[Purpose]

The purpose of this study was to investigate whether moderate exercise and quercetin intake with a low fat diet contribute to inflammatory cytokine production, mitochondrial biogenesis, and lipid metabolism in skeletal muscle after strenuous exercise by high-fat diet mice.

[Methods]

Male C57BL/6 mice were randomly divided into four groups: (1) High-fat for 12 weeks and low-fat diet control (C; n = 6); (2) high-fat diet for 12 weeks and low-fat diet with quercetin (Q; n = 4); (3) high-fat diet for 12 weeks and low-fat diet with exercise (E; n = 4); or (4) high-fat diet for 12 weeks and low-fat diet with exercise and quercetin (EQ; n = 5). Quercetin (10 mg/kg) was administered once per day, 5 day/week for 8 weeks. Exercise training was performed at moderate intensity for 8 weeks, 5 days/week for 30–60 min/day. Mice were subjected to a strenuous exercise bout of 60 min at a speed of 25 m/min (VO₂ max 85%) conducted as an exercise-induced fatigue just before sacrifice.

[Results]

As results, body weights were significantly different among the groups. Exercise training significantly reduced inflammatory cytokines after strenuous exercise in skeletal muscle of high-fat diet mice. Exercise training increased Tfam mRNA in the soleus muscle after strenuous exercise. Exercise training significantly decreased lipogenesis markers in skeletal muscle of obese mice after strenuous exercise. Moderate exercise significantly increased lipolysis markers in the tibialis anterior muscle.

[Conclusion]

These findings suggest that exercise training reduced inflammatory cytokine levels and improved mitochondrial biogenesis and lipid metabolism. However quercetin supplementation did not affect these parameters. Thus, long-term moderate exercise training has positive effects on obesity.     

Propionyl-L-carnitine corrects metabolic and cardiovascular alterations in diet-induced obese mice and improves liver respiratory chain activity

Aims

Obesity is a primary contributor to acquired insulin resistance leading to the development of type 2 diabetes and cardiovascular alterations. The carnitine derivate, propionyl-L-carnitine (PLC), plays a key role in energy control. Our aim was to evaluate metabolic and cardiovascular effects of PLC in diet-induced obese mice.

Methods

C57BL/6 mice were fed a high-fat diet for 9 weeks and then divided into two groups, receiving either free- (vehicle-HF) or PLC-supplemented water (200 mg/kg/day) during 4 additional weeks. Standard diet-fed animals were used as lean controls (vehicle-ST). Body weight and food intake were monitored. Glucose and insulin tolerance tests were assessed, as well as the HOMA_IR, the serum lipid profile, the hepatic and muscular mitochondrial activity and the tissue nitric oxide (NO) liberation. Systolic blood pressure, cardiac and endothelial functions were also evaluated.

Results

Vehicle-HF displayed a greater increase of body weight compared to vehicle-ST that was completely reversed by PLC treatment without affecting food intake. PLC improved the insulin-resistant state and reversed the increased total cholesterol but not the increase in free fatty acid, triglyceride and HDL/LDL ratio induced by high-fat diet. Vehicle-HF exhibited a reduced cardiac output/body weight ratio, endothelial dysfunction and tissue decrease of NO production, all of them being improved by PLC treatment. Finally, the decrease of hepatic mitochondrial activity by high-fat diet was reversed by PLC.

Conclusions

Oral administration of PLC improves the insulin-resistant state developed by obese animals and decreases the cardiovascular risk associated to this metabolic alteration probably via correction of mitochondrial function.     

Methyl pyruvate rescues mitochondrial damage caused by SIGMAR1 mutation related to amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a disease caused by motor neuron degeneration. Recently, a novel SIGMAR1 gene variant (p.E102Q) was discovered in some familial ALS patients.

Methods

We address mechanisms underlying neurodegeneration caused by the mutation using Neuro2A cells overexpressing σ₁R^E102Q, a protein of a SIGMAR1 gene variant (p.E102Q) and evaluate potential amelioration by ATP production via methyl pyruvate (MP) treatment.

Results

σ₁R^E102Q overexpression promoted dissociation of the protein from the endoplasmic reticulum (ER) membrane and cytoplasmic aggregation, which in turn impaired mitochondrial ATP production and proteasome activity. Under ER stress conditions, overexpression of wild-type σ₁R suppressed ER stress-induced mitochondrial injury, whereas σ₁R^E102Q overexpression aggravated mitochondrial damage and induced autophagic cell death. Moreover, σ₁R^E102Q-overexpressing cells showed aberrant extra-nuclear localization of the TAR DNA-binding protein (TDP-43), a condition exacerbated by ER stress. Treatment of cells with the mitochondrial Ca^2 + transporter inhibitor Ru360 mimicked the effects of σ₁R^E102Q overexpression, indicating that aberrant σ₁R-mediated mitochondrial Ca^2 + transport likely underlies TDP-43 extra-nuclear localization, segregation in inclusion bodies, and ubiquitination. Finally, enhanced ATP production promoted by methyl pyruvate (MP) treatment rescued proteasome impairment and TDP-43 extra-nuclear localization caused by σ₁R^E102Q overexpression.

Conclusions

Our observations suggest that neurodegeneration seen in some forms of ALS are due in part to aberrant mitochondrial ATP production and proteasome activity as well as TDP-43 mislocalization resulting from the SIGMAR1 mutation.

General significance

ATP supplementation by MP represents a potential therapeutic strategy to treat ALS caused by SIGMAR1 mutation.     

Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance

Purpose

Evidence suggests an association between aberrant mitochondrial dynamics and cardiac diseases. Because myocardial metabolic deficiency caused by insulin resistance plays a crucial role in heart disease, we investigated the role of dynamin-related protein-1 (DRP1; a mitochondrial fission protein) in the pathogenesis of myocardial insulin resistance.

Methods and Results

DRP1-expressing H9c2 myocytes, which had fragmented mitochondria with mitochondrial membrane potential (ΔΨ_m) depolarization, exhibited attenuated insulin signaling and 2-deoxy-d-glucose (2-DG) uptake, indicating insulin resistance. Treatment of the DRP1-expressing myocytes with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP) significantly improved insulin resistance and mitochondrial dysfunction. When myocytes were exposed to hydrogen peroxide (H₂O₂), they increased DRP1 expression and mitochondrial fragmentation, resulting in ΔΨ_m depolarization and insulin resistance. When DRP1 was suppressed by siRNA, H₂O₂-induced mitochondrial dysfunction and insulin resistance were restored. Our results suggest that a mutual enhancement between DRP1 and reactive oxygen species could induce mitochondrial dysfunction and myocardial insulin resistance. In palmitate-induced insulin-resistant myocytes, neither DRP1-suppression nor TMPyP restored the ΔΨ_m depolarization and impaired 2-DG uptake, however they improved insulin signaling.

Conclusions

A mutual enhancement between DRP1 and ROS could promote mitochondrial dysfunction and inhibition of insulin signal transduction. However, other mechanisms, including lipid metabolite-induced mitochondrial dysfunction, may be involved in palmitate-induced insulin resistance.     

Disruption of oxidative phosphorylation and synaptic Na,K-ATPase activity by pristanic acid in cerebellum of young rats

Aims

Peroxisomal biogenesis disorders (PBD) are inherited disorders clinically manifested by neurological symptoms and brain abnormalities, in which the cerebellum is usually involved. Biochemically, patients affected by these neurodegenerative diseases accumulate branched-chain fatty acids, including pristanic acid (Prist) in the brain and other tissues.

Main methods

In the present investigation we studied the in vitro influence of Prist, at doses found in PBD, on oxidative phosphorylation, by measuring the activities of the respiratory chain complexes I–IV and ATP production, as well as on creatine kinase and synaptic Na⁺, K⁺-ATPase activities in rat cerebellum.

Key findings

Prist significantly decreased complexes I–III (65%), II (40%) and especially II–III (90%) activities, without altering the activities of complex IV of the respiratory chain and creatine kinase. Furthermore, ATP formation and synaptic Na⁺, K⁺-ATPase activity were markedly inhibited (80–90%) by Prist. We also observed that this fatty acid altered mitochondrial and synaptic membrane fluidity that may have contributed to its inhibitory effects on the activities of the respiratory chain complexes and Na⁺, K⁺-ATPase.

Significance

Considering the importance of oxidative phosphorylation for mitochondrial homeostasis and of Na⁺, K⁺-ATPase for the maintenance of cell membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission in cerebellum. We postulate that these pathomechanisms may contribute to the cerebellar alterations observed in patients affected by PBD in which Prist is accumulated.     

Chlorella modulates insulin signaling pathway and prevents high-fat diet-induced insulin resistance in mice

Aims

The search for natural agents that minimize obesity-associated disorders is receiving special attention. In this regard, the present study aimed to evaluate the prophylactic effect of Chlorella vulgaris (CV) on body weight, lipid profile, blood glucose and insulin signaling in liver, skeletal muscle and adipose tissue of diet-induced obese mice.

Main methods

Balb/C mice were fed either with standard rodent chow diet or high-fat diet (HFD) and received concomitant treatment with CV for 12 consecutive weeks. Triglyceride, free fatty acid, total cholesterol and fractions of cholesterol were measured using commercial assay. Insulin and leptin levels were determined by enzyme-linked immunosorbent assay (ELISA). Insulin and glucose tolerance tests were performed. The expression and phosphorylation of IRβ, IRS-1 and Akt were determined by Western blot analyses.

Key findings

Herein we demonstrate for the first time in the literature that prevention by CV of high-fat diet-induced insulin resistance in obese mice, as shown by increased glucose and insulin tolerance, is in part due to the improvement in the insulin signaling pathway at its main target tissues, by increasing the phosphorylation levels of proteins such as IR, IRS-1 and Akt. In parallel, the lower phosphorylation levels of IRS-1^ser307 were observed in obese mice. We also found that CV administration prevents high-fat diet-induced dyslipidemia by reducing triglyceride, cholesterol and free fatty acid levels.

Significance

We propose that the modulatory effect of CV treatment preventing the deleterious effects induced by high-fat diet is a good indicator for its use as a prophylactic–therapeutic agent against obesity-related complications.     

The role of mitochondrial electron transport in tumorigenesis and metastasis

Background

Tumor formation and spread via the circulatory and lymphatic drainage systems is associated with metabolic reprogramming that often includes increased glycolytic metabolism relative to mitochondrial energy production. However, cells within a tumor are not identical due to genetic change, clonal evolution and layers of epigenetic reprogramming. In addition, cell hierarchy impinges on metabolic status while tumor cell phenotype and metabolic status will be influenced by the local microenvironment including stromal cells, developing blood and lymphatic vessels and innate and adaptive immune cells. Mitochondrial mutations and changes in mitochondrial electron transport contribute to metabolic remodeling in cancer in ways that are poorly understood.

Scope of Review

This review concerns the role of mitochondria, mitochondrial mutations and mitochondrial electron transport function in tumorigenesis and metastasis.

Major Conclusions

It is concluded that mitochondrial electron transport is required for tumor initiation, growth and metastasis. Nevertheless, defects in mitochondrial electron transport that compromise mitochondrial energy metabolism can contribute to tumor formation and spread. These apparently contradictory phenomena can be reconciled by cells in individual tumors in a particular environment adapting dynamically to optimally balance mitochondrial genome changes and bioenergetic status.

General Significance

Tumors are complex evolving biological systems characterized by genetic and adaptive epigenetic changes. Understanding the complexity of these changes in terms of bioenergetics and metabolic changes will permit the development of better combination anticancer therapies. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.