Mitochondrial energy state controls AMPK-mediated foraging behavior in C. elegans.

Organisms surveil and respond to their environment using behaviors entrained by metabolic cues that reflect food availability. Mitochondria act as metabolic hubs and at the center of mitochondrial energy production is the protonmotive force (PMF), an electrochemical gradient generated by metabolite consumption. The PMF serves as a central integrator of mitochondrial status, but its role in governing metabolic signaling is poorly understood. We used optogenetics to dissipate the PMF in Caenorhabditis elegans tissues to test its role in food-related behaviors. Our data demonstrate that PMF reduction in the intestine is sufficient to initiate locomotor responses to acute food deprivation. This behavioral adaptation requires the cellular energy regulator AMP-activated protein kinase (AMPK) in neurons, not in the intestine, and relies on mitochondrial dynamics and axonal trafficking. Our results highlight a role for intestinal PMF as an internal metabolic cue, and we identify a bottom-up signaling axis through which changes in the PMF trigger AMPK activity in neurons to promote foraging behavior.

Oocyte mitochondria link maternal environment to offspring phenotype.

During maturation oocytes undergo a recently discovered mitochondrial proteome remodeling event in flies1, frogs1, and humans2. This oocyte mitochondrial remodeling, which includes substantial changes in electron transport chain (ETC) subunit abundance1,2, is regulated by maternal insulin signaling1. Why oocytes undergo mitochondrial remodeling is unknown, with some speculating that it might be an evolutionarily conserved mechanism to protect oocytes from genotoxic damage by reactive oxygen species (ROS)2. In Caenorhabditis elegans, we previously found that maternal exposure to osmotic stress drives a 50-fold increase in offspring survival in response to future osmotic stress3. Like mitochondrial remodeling, we found that this intergenerational adaptation is also regulated by insulin signaling to oocytes3. Here, we used proteomics and genetic manipulations to show that insulin signaling to oocytes regulates offspring's ability to adapt to future stress via a mechanism that depends on ETC composition in maternal oocytes. Specifically, we found that maternally expressed mutant alleles of nduf-7 (complex I subunit) or isp-1 (complex III subunit) altered offspring's response to osmotic stress at hatching independently of offspring genotype. Furthermore, we found that expressing wild-type isp-1 in germ cells (oocytes) was sufficient to restore offspring's normal response to osmotic stress. Chemical mutagenesis screens revealed that maternal ETC composition regulates offspring's response to stress by altering AMP kinase function in offspring which in turn regulates both ATP and glycerol metabolism in response to continued osmotic stress. To our knowledge, these data are the first to show that proper oocyte ETC composition is required to link a mother's environment to adaptive changes in offspring metabolism. The data also raise the possibility that the reason diverse animals exhibit insulin regulated remodeling of oocyte mitochondria is to tailor offspring metabolism to best match the environment of their mother.

Effect of skeletal muscle mitochondrial phenotype on H2O2 emission.

Reactive oxygen species (ROS) are a key output of the skeletal muscle mitochondrial information processing system both at rest and during exercise. In skeletal muscle, mitochondrial ROS release depends on multiple factors; however, fiber-type specific differences remain ambiguous in part owing to the use of mitochondria from mammalian muscle that consist of mixed fibers. To elucidate fiber-type specific differences, we used mitochondria isolated from rainbow trout (Oncorhynchus mykiss) red and white skeletal muscles that consist of spatially distinct essentially pure red and white fibers. We first characterized the assay conditions for measuring ROS production (as H2O2) in isolated fish red and white skeletal muscle mitochondria (RMM and WMM) and thereafter compared the rates of emission during oxidation of different substrates and the responses to mitochondrial electron transport system (ETS) pharmacological modulators. Our results showed that H2O2 emission rates by RMM and WMM can be quantified using the same protein concentration and composition of the Amplex UltraRed-horseradish peroxidase (AUR-HRP) detection system. For both RMM and WMM, protein normalized H2O2 emission rates were highest at the lowest protein concentration tested and decreased exponentially thereafter. However, the absolute values of H2O2 emission rates depended on the calibration curves used to convert fluorescent signals to H2O2 while the trends depended on the normalization strategy. We found substantial qualitative and quantitative differences between RMM and WMM in the H2O2 emission rates depending on the substrates being oxidized and their concentrations. Similarly, pharmacological modulators of the ETS altered the magnitudes and trends of the H2O2 emission differently in RMM and WMM. While comparable concentrations of substrates elicited maximal albeit quantitively different emission rates in RMM and WMM, different concentrations of pharmacological ETS modulators may be required for maximal H2O2 emission rates depending on muscle fiber-type. Taken together, our study suggests that biochemical differences exist in RMM compared with WMM that alter substrate oxidation and responses to ETS modulators resulting in fiber-type specific mitochondrial H2O2 emission rates.

Mitochondrial complex I ROS production and redox signaling in hypoxia.

Mitochondria are a main source of cellular energy. Oxidative phosphorylation (OXPHOS) is the major process of aerobic respiration. Enzyme complexes of the electron transport chain (ETC) pump protons to generate a protonmotive force (Δp) that drives OXPHOS. Complex I is an electron entry point into the ETC. Complex I oxidizes nicotinamide adenine dinucleotide (NADH) and transfers electrons to ubiquinone in a reaction coupled with proton pumping. Complex I also produces reactive oxygen species (ROS) under various conditions. The enzymatic activities of complex I can be regulated by metabolic conditions and serves as a regulatory node of the ETC. Complex I ROS plays diverse roles in cell metabolism ranging from physiologic to pathologic conditions. Progress in our understanding indicates that ROS release from complex I serves important signaling functions. Increasing evidence suggests that complex I ROS is important in signaling a mismatch in energy production and demand. In this article, we review the role of ROS from complex I in sensing acute hypoxia.

Exhaustive exercise alters native and site-specific H2O2 emission in red and white skeletal muscle mitochondria.

Mitochondrial reactive oxygen species (ROS) homeostasis is intricately linked to energy conversion reactions and entails regulation of the mechanisms of ROS production and removal. However, there is limited understanding of how energy demand modulates ROS balance. Skeletal muscle experiences a wide range of energy requirements depending on the intensity and duration of exercise and therefore is an excellent model to probe the effect of altered energy demand on mitochondrial ROS production. Because in most fish skeletal muscle exists essentially as pure spatially distinct slow-twitch red oxidative and fast-twitch white glycolytic fibers, it provides a natural system for investigating how functional specialization affects ROS homeostasis. We tested the hypothesis that acute increase in energy demand imposed by exhaustive exercise will increase mitochondrial H2O2 emission to a greater extent in red muscle mitochondria (RMM) compared with white muscle mitochondria (WMM). We found that native H2O2 emission rates varied by up to 6-fold depending on the substrate being oxidized and muscle fiber type, with RMM emitting at higher rates with glutamate-malate and palmitoylcarnitine while WMM emitted at higher rates with succinate and glyceral-3-phosphate. Exhaustive exercise increased the native and site-specific H2O2 emission rates; however, the maximal emission rates depended on the substrate, fiber type and redox site. The H2O2 consumption capacity and activities of individual antioxidant enzymes including the glutathione- and thioredoxin-dependent peroxidases as well as catalase were higher in RMM compared with WMM indicating that the activity of antioxidant defense system does not explain the differences in H2O2 emission rates in RMM and WMM. Overall, our study suggests that substrate selection and oxidation may be the key factors determining the rates of ROS production in RMM and WMM following exhaustive exercise.

Factors affecting liver mitochondrial hydrogen peroxide emission.

Mitochondria are key cellular sources of reactive oxygen species (ROS) and contain at least 12 known sites on multiple enzymes that convert molecular oxygen to superoxide and hydrogen peroxide (H2O2). Quantitation of site-specific ROS emission is critical to understand the relative contribution of different sites and the pathophysiologic importance of mitochondrial ROS. However, factors that affect mitochondrial ROS emission are not well understood. We characterized and optimized conditions for maximal total and site-specific H2O2 emission during oxidation of standard substrates and probed the source of the high H2O2 emission in unenergized rainbow trout liver mitochondria. We found that mitochondrial H2O2 emission capacity depended on the substrate being oxidized, mitochondrial protein concentration, and composition of the ROS detection system. Contrary to our expectation, addition of exogenous superoxide dismutase reduced H2O2 emission. Titration of conventional mitochondrial electron transfer system (ETS) inhibitors over a range of conditions revealed that one size does not fit all; inhibitor concentrations evoking maximal responses varied with substrate and were moderated by the presence of other inhibitors. Moreover, the efficacy of suppressors of electron leak (S1QEL1.1 and S3QEL2) was low and depended on the substrate being oxidized. We found that H2O2 emission in unenergized rainbow trout liver mitochondria was suppressed by GKT136901 suggesting that it is associated with NADPH oxidase activity. We conclude that optimization of assay conditions is critical for quantitation of maximal H2O2 emission and would facilitate more valid comparisons of mitochondrial total and site-specific H2O2 emission capacities between studies, tissues, and species.

Anoxia-reoxygenation modulates cadmium-induced liver mitochondrial reactive oxygen species emission during oxidation of glycerol 3-phosphate.

Aquatic organisms are frequently exposed to multiple stressors including low dissolved oxygen (O2) and metals such as cadmium (Cd). Reduced O2 concentration and Cd exposure alter cellular function in part by impairing energy metabolism and dysregulating reactive oxygen species (ROS) homeostasis. However, little is known about the role of mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) in ROS homeostasis in fish and its response to environmental stress. In this study, mGPDH activity and the effects of anoxia-reoxygenation (A-RO) and Cd on ROS (as hydrogen peroxide, H2O2) emission in rainbow trout liver mitochondria during oxidation of glycerol 3-phosphate (G3P) were probed. Trout liver mitochondria exhibited low mGPDH activity that supported a low respiratory rate but substantial H2O2 emission rate. Cd evoked a low concentration stimulatory-high concentration inhibitory H2O2 emission pattern that was blunted by A-RO. At specific redox centers, Cd suppressed H2O2 emission from site IQ, but stimulated emission from sites IIIQo and GQ. In contrast, A-RO stimulated H2O2 emission from site IQ following 15 min exposure and augmented Cd-stimulated emission from site IIF after 30 min exposure but did not alter the rate of H2O2 emission from sites IIIQo and GQ. Additionally, Cd neither altered the activities of catalase, glutathione peroxidase, or thioredoxin reductase nor the concentrations of total glutathione, reduced glutathione, or oxidized glutathione. Overall, this study indicates that oxidation of G3P drives ROS production from mGPDH and complexes I, II and III, whereas Cd directly modulates redox sites but not antioxidant defense systems to alter mitochondrial H2O2 emission.

Changes in the morphometry of the uterus, ovary, and foetus, and biochemistry of allantoic and amniotic membrane fluids of Yankasa ewes across the gestation period.

This study evaluated the uterine and fetal morphometric changes and fetal membrane fluids biochemistry across the gestation of Yankasa sheep. The amniotic and allantoic fluids are actively involved in the constant physiologic exchange between the fetus and maternal circulation. Hence, the knowledge regarding changes in the composition of fetal membrane fluids is important for understanding fetal metabolism, and the diagnosis of pathophysiological conditions during gestation. Gravid uteri from 37 ewes and their corresponding ovaries were sampled. The number and size of the placentomes in the second and third terms of gestation were significantly higher relative to the first term. The total protein, albumin, glucose, urea, creatinine, and calcium levels as well as alkaline phosphatase (ALP) and aspartate aminotransferase (AST) activities were higher in the allantoic fluid. In the allantoic fluid, the levels of total protein, globulin, and the activity levels of ALT and AST increased progressively with the advancement of gestation; contrarily, the levels of calcium, chloride, and the activity level of ALT decreased. For the amniotic fluid, the levels of total protein, globulin, urea, calcium, and the enzyme activities in the second and third terms did not differ but were higher than the level in the first term of gestation. In addition, the most significant increases in creatinine level and white blood cell count were observed in the third term of gestation. Therefore, notable differences in the levels of ALT, AST, total proteins, glucose, urea, creatinine, and WBC counts were observed in the two fetal membrane fluids.

Computational modeling and multi-objective optimization of engine performance of biodiesel made with castor oil.

In this research the engine performance of biodiesel made with castor oil through homogeneous alkali catalyzed transesterification was analyzed. The input variables for the performance analysis were biodiesel blend and engine speed while the response variables were break power (BP), basic specific fuel consumption (BSFC), break thermal efficiency (BTE), torque and unit cost. The engine performance was modeled using artificial neural network (ANN) and the ANN was subsequently used as the objective function for a non dominated sorting genetic algorithm (NSGA-II) for multi objective optimization of the engine performance. The ANN was equally coupled with a desirability function whose outputs were optimized using simulated annealing for multi objective optimization of the engine performance. Subsequent comparison of the two optimization models was done. The results show that biodiesel from castor oil could be a good replacement for biodiesels from fossil fuels. The ANN model predicted engine performance very well with the lowest value of the correlation coefficient between the experimental responses and ANN predictions being 0.9733. The multi objective optimization using desirability function performed excellently well with the optimum blend and speed being 78.7% and 1754.48 rpm respectively. The Pareto front from the NSGA-II algorithm generally has high desirability values. The Pareto front solution which is more flexible than the desirability function solution would serve as an excellent guide for engine designers. Finally, castor oil based biodiesel cost was for the first time integrated into engine performance optimization studies.

Assessment of age-related morphological changes in the testes of post-hatch light ecotype Nigerian indigenous chicken.

The age-related morphological changes of the testes in light ecotype Nigerian indigenous chicken were evaluated in this study using gross anatomical, histological and histomorphometric techniques. The results showed that the testes of 3- to 9-month-old birds were light pink while testes of sexually mature chicken were creamy white in colour. The left and right testicular weight, length, diameter, circumference and the organosomatic indices increased significantly (p < .05) with increasing age across the groups. Although the mean tubular diameter and epithelial height of the left and right seminiferous tubules increased significantly (p < .05) with age, the tubular diameter, epithelial height and luminal diameter did not vary significantly (p > .05) between the left and right testes of all the groups. The one-cell layer thick germinal epithelium of the left testes at 3 to 6 months old showed islands of cell proliferation that contained spermatogonia and spermatocytes. At 6 to 9 months, the left testes exhibited numerous early spermatids with occasional occurrence of late stage spermatids while the right testes showed scanty early stage spermatids. At 12 to 18 months, the germinal epithelia of both left and right testes were characterized by the presence of Sertoli cells, spermatogonia, primary spermatocytes, numerous early and late stage spermatids as well as spermatozoa. In conclusion, the morphological features highlighted in the present study show that at pre-pubertal periods, the left testes may develop faster than the right testes. However, both left and right testes may participate actively in the production of spermatozoa during the post-pubertal life.

Modulation of mitochondrial site-specific hydrogen peroxide efflux by exogenous stressors.

Oxygen (O2) deprivation and metals are common environmental stressors and their exposure to aquatic organisms can induce oxidative stress by disrupting cellular reactive oxygen species (ROS) homeostasis. Mitochondria are a major source of ROS in the cell wherein a dozen sites located on enzymes of the electron transport system (ETS) and substrate oxidation produce superoxide anion radicals (O2˙‾) or hydrogen peroxide (H2O2). Sites located on ETS enzymes can generate ROS by forward electron transfer (FET) and reverse electron transfer (RET) reactions; however, knowledge of how exogenous stressors modulate site-specific ROS production is limited. We investigated the effects of anoxia-reoxygenation and cadmium (Cd) on H2O2 emission in fish liver mitochondria oxidizing glutamate-malate, succinate or palmitoylcarnitine-malate. We find that anoxia-reoxygenation attenuates H2O2 emission while the effect of Cd depends on the substrate, with monotonic responses for glutamate-malate and palmitoylcarnitine-malate, and a biphasic response for succinate. Anoxia-reoxygenation exerts a substrate-dependent inhibition of mitochondrial respiration which is more severe with palmitoylcarnitine-malate compared with succinate or glutamate-malate. Additionally, specific mitochondrial ROS-emitting sites were sequestered using blockers of electron transfer and the effects of anoxia-reoxygenation and Cd on H2O2 emission were evaluated. Here, we find that site-specific H2O2 emission capacities depend on the substrate and the direction of electron flow. Moreover, anoxia-reoxygenation alters site-specific H2O2 emission rates during succinate and glutamate-malate oxidation whereas Cd imposes monotonic or biphasic H2O2 emission responses depending on the substrate and site. Contrary to our expectation, anoxia-reoxygenation blunts the effect of Cd. These results suggest that the effect of exogenous stressors on mitochondrial oxidant production is governed by their impact on energy conversion reactions and mitochondrial redox poise. Moreover, direct increased ROS production seemingly does not explain the increased adverse effects associated with combined exposure of aquatic organisms to Cd and low dissolved oxygen levels.

Effects of bioenergetics, temperature and cadmium on liver mitochondria reactive oxygen species production and consumption.

A by-product of mitochondrial substrate oxidation and electron transfer to generate cellular energy (ATP) is reactive oxygen species (ROS). Superoxide anion radical and hydrogen peroxide (H2O2) are the proximal ROS produced by the mitochondria. Because low levels of ROS serve critical regulatory roles in cell physiology while excessive levels or inappropriately localized ROS result in aberrant physiological states, mitochondrial ROS need to be tightly regulated. While it is known that regulation of mitochondrial ROS involves balancing the rates of production and removal, the effects of stressors on these processes remain largely unknown. To illuminate how stressors modulate mitochondrial ROS homeostasis, we investigated the effects of temperature and cadmium (Cd) on H2O2 emission and consumption in rainbow trout liver mitochondria. We show that H2O2 emission rates increase with temperature and Cd exposure. Energizing mitochondria with malate-glutamate or succinate increased the rate of H2O2 emission; however, Cd exposure imposed different patterns of H2O2 emission depending on the concentration and substrate. Specifically, mitochondria respiring on malate-glutamate exhibited a saturable graded concentration-response curve that plateaued at 5 µM while mitochondria respiring on succinate had a biphasic concentration-response curve characterized by a spike in the emission rate at 1 µM Cd followed by gradual diminution at higher Cd concentrations. To explain the observed substrate- and concentration-dependent effects of Cd, we sequestered specific mitochondrial ROS-emitting sites using blockers of electron transfer and then tested the effect of the metal. The results indicate that the biphasic H2O2 emission response imposed by succinate is due to site IIF but is further modified at sites IQ and IIIQo. Moreover, the saturable graded H2O2 emission response in mitochondria energized with malate-glutamate is consistent with effect of Cd on site IF. Additionally, Cd and temperature acted cooperatively to increase mitochondrial H2O2 emission suggesting that increased toxicity of Cd at high temperature may be due to increased oxidative insult. Surprisingly, despite their clear stimulatory effect on H2O2 emission, Cd, temperature and bioenergetic status did not affect the kinetics of mitochondrial H2O2 consumption; the rate constants and half-lives for all the conditions tested were similar. Overall, our study indicates that the production processes of rainbow trout liver mitochondrial H2O2 metabolism are highly responsive to stressors and bioenergetics while the consumption processes are recalcitrant. The latter denotes the presence of a robust H2O2 scavenging system in liver mitochondria that would maintain H2O2 homeostasis in the face of increased production and reduced scavenging capacity.

Effects of monosodium-L-glutamate administration on serum levels of reproductive hormones and cholesterol, epididymal sperm reserves and testicular histomorphology of male albino rats.

This study investigated the effects of administration of monosodium L-glutamate (MSG) on serum gonadotrophin-releasing hormone (GnRH), luteinising hormone (LH), testosterone and total cholesterol (TC), cauda epididymal sperm reserves (CESR) and testicular histomorphology of adult male albino rats. Eighty-four rats, randomly assigned to 7 groups of 12 rats each, were used for the study. Varying low doses (0.25, 0.50 or 1.00 g/kg body weight) of MSG were administered orally or subcutaneously at 48-h intervals for six weeks. Serum GnRH, LH, testosterone and TC, and CESR were evaluated on days 14, 28 and 42 of MSG administration. Testicular histomorphology was evaluated on day 42. The results showed that the mean serum GnRH, LH and testosterone levels, and the CESR of all the treated groups were significantly (P < 0.05) lower than those of the untreated control on days 14, 28 and 42 of MSG administration. The mean serum TC levels of all the treated groups were also significantly (P < 0.05) lower than those of the control group on days 14 and 28. No lesions were observed on sections of the testes. It was concluded that MSG administration for 14, 28 and 42 days led to significantly lower serum levels of GnRH, LH, testosterone and TC, and significantly lower CESR.