SIRT3 deficiency decreases oxidative metabolism capacity but increases lifespan in male mice under caloric restriction.

Mitochondrial NAD+ -dependent protein deacetylase Sirtuin3 (SIRT3) has been proposed to mediate calorie restriction (CR)-dependent metabolic regulation and lifespan extension. Here, we investigated the role of SIRT3 in CR-mediated longevity, mitochondrial function, and aerobic fitness. We report that SIRT3 is required for whole-body aerobic capacity but is dispensable for CR-dependent lifespan extension. Under CR, loss of SIRT3 (Sirt3-/- ) yielded a longer overall and maximum lifespan as compared to Sirt3+/+ mice. This unexpected lifespan extension was associated with altered mitochondrial protein acetylation in oxidative metabolic pathways, reduced mitochondrial respiration, and reduced aerobic exercise capacity. Also, Sirt3-/- CR mice exhibit lower spontaneous activity and a trend favoring fatty acid oxidation during the postprandial period. This study shows the uncoupling of lifespan and healthspan parameters (aerobic fitness and spontaneous activity) and provides new insights into SIRT3 function in CR adaptation, fuel utilization, and aging.

Obesity-dependent CDK1 signaling stimulates mitochondrial respiration at complex I in pancreatic ß-cells.

ß-Cell mitochondria play a central role in coupling glucose metabolism with insulin secretion. Here, we identified a metabolic function of cyclin-dependent kinase 1 (CDK1)/cyclin B1-the activation of mitochondrial respiratory complex I-that is active in quiescent adult ß-cells and hyperactive in ß-cells from obese (ob/ob) mice. In WT islets, respirometry revealed that 65% of complex I flux and 49% of state 3 respiration is sensitive to CDK1 inhibition. Islets from ob/ob mice expressed more cyclin B1 and exhibited a higher sensitivity to CDK1 blockade, which reduced complex I flux by 76% and state 3 respiration by 79%. The ensuing reduction in mitochondrial NADH utilization, measured with two-photon NAD(P)H fluorescence lifetime imaging (FLIM), was matched in the cytosol by a lag in citrate cycling, as shown with a FRET reporter targeted to ß-cells. Moreover, time-resolved measurements revealed that in ob/ob islets, where complex I flux dominates respiration, CDK1 inhibition is sufficient to restrict the duty cycle of ATP/ADP and calcium oscillations, the parameter that dynamically encodes ß-cell glucose sensing. Direct complex I inhibition with rotenone mimicked the restrictive effects of CDK1 inhibition on mitochondrial respiration, NADH turnover, ATP/ADP, and calcium influx. These findings identify complex I as a critical mediator of obesity-associated metabolic remodeling in ß-cells and implicate CDK1 as a regulator of complex I that enhances ß-cell glucose sensing.

Hypoxia induces selective modifications to the acetylome in the brain of zebrafish (Danio rerio).

Reversible protein acetylation is an important regulatory mechanism for modulating protein function. The cellular protein acetylome is in large part dictated by the cellular redox balance, and in particular [NAD+]. While the relationship between hypoxia, redox balance, energy charge and resulting mitochondrial dysfunction has been examined in the context of hypoxia-linked pathologies, little is known about the direct effects of decreases in environmental oxygen on reversible lysine acetylation, and the resulting modifications to mitochondrial metabolism. To address this knowledge gap, we exposed zebrafish (Danio rerio) to 16 h of hypoxia (2.21 kPa) and quantified acetylation levels of 1220 proteins using whole-cell proteomics in samples of brain taken from normoxic and hypoxic zebrafish. In addition, we examined the effects of hypoxia on cytoplasmic and mitochondrial redox status, whole-cell energetics, the activity of the mitochondrial NAD+-dependent deacetylase SIRT3, and electron transport chain complex activities to determine if there is an association between hypoxia-induced metabolic disturbances, protein acetylation, and mitochondrial function. Our results (1) reveal several key changes in the acetylation status of proteins in the brain, primarily within the mitochondria; (2) show significant fluctuations in cytoplasmic and mitochondrial redox status within the brain during hypoxia exposure; and (3) provide evidence that lysine acetylation may be related to large changes in electron transport and ATP-synthase complex activities and adenylate status in zebrafish exposed to hypoxic stress. Together, these data provide new insights into the role of protein modifications in mitochondrial metabolism during hypoxia.

Ethanol metabolism varies with hypoxia tolerance in ten cyprinid species.

During periods of severe hypoxia or anoxia, Carassius spp. are known for their ability to produce ethanol as their anaerobic end product, which diffuses into the environment thereby reducing the osmotic and acidotic load associated with "anaerobic" glycolysis. However, the relationship between alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities, key ethanol metabolizing enzymes, and hypoxia tolerance among Carassius spp. and their closely related non-ethanol-producing cyprinids remains unclear. To address this, we quantified the activity levels of key anaerobic enzymes in liver and muscle in species of cyprinids over 48 h of severe hypoxia exposure (0.7 kPa). As predicted, muscle ADH activity was highest in the two most hypoxia-tolerant species (Carassius spp.), with very low levels present in the other species examined. However, liver ADH activities showed an inverse relationship with hypoxia tolerance, with the most hypoxia-tolerant fish having the lowest ADH activity. There was no correlation between hypoxia tolerance and ALDH and LDH activities in muscle or liver. All species produced lactate, reaching their highest levels after 8 h, but returning to near-baseline levels by 48 h of sustained exposure to hypoxia, suggesting lactate oxidation or depressed ATP demand. Liver glycogen content was not affected by 48 h hypoxia exposure in the most hypoxia-tolerant species, whereas the least tolerant species consumed the majority of the liver glycogen stores, which is probably due to the greater relative hypoxia exposure experienced by these species. Our findings that liver ADH activities were inversely related to hypoxia tolerance suggests that in all but Carassius spp., the ethanol metabolizing pathways in cyprinids is largely similar to that observed in other vertebrates and plays a role in the detoxification of ethanol. Furthermore, conservation of glycogen stores may be the result of metabolic-depressing pathways in the more tolerant species, regardless of the ability to produce ethanol, or adaptations that improve oxygen uptake to reduce metabolic demands due to hypoxia.

Metabolic programming of the epigenome: host and gut microbial metabolite interactions with host chromatin.

The mammalian gut microbiota has been linked to host developmental, immunologic, and metabolic outcomes. This collection of trillions of microbes inhabits the gut and produces a myriad of metabolites, which are measurable in host circulation and contribute to the pathogenesis of human diseases. The link between endogenous metabolite availability and chromatin regulation is a well-established and active area of investigation; however, whether microbial metabolites can elicit similar effects is less understood. In this review, we focus on seminal and recent research that establishes chromatin regulatory roles for both endogenous and microbial metabolites. We also highlight key physiologic and disease settings where microbial metabolite-host chromatin interactions have been established and/or may be pertinent.

Using comparative biology to understand how aging affects mitochondrial metabolism.

Lifespan varies considerably among even closely related species, as exemplified by rodents and primates. Despite these disparities in lifespan, most studies have focused on intra-specific aging pathologies, primarily within a select few systems. While mice have provided much insight into aging biology, it is unclear if such a short-lived species lack defences against senescence that may have evolved in related longevous species. Many age-related diseases have been linked to mitochondrial dysfunction that are measured by decreased energy generation, structural damage to cellular components, and even cell death. Post translational modifications (PTMs) orchestrate many of the pathways associated with cellular metabolism, and are thought to be a key regulator in biological senescence. We propose hyperacylation as one such modification that may be implicated in numerous mitochondrial impairments affecting energy metabolism.

Steep, coincident, and concordant clines in mitochondrial and nuclear-encoded genes in a hybrid zone between subspecies of Atlantic killifish, Fundulus heteroclitus.

Steep genetic clines resulting from recent secondary contact between previously isolated taxa can either gradually erode over time or be stabilized by factors such as ecological selection or selection against hybrids. We used patterns of variation in 30 nuclear and two mitochondrial SNPs to examine the factors that could be involved in stabilizing clines across a hybrid zone between two subspecies of the Atlantic killifish, Fundulus heteroclitus. Increased heterozygote deficit and cytonuclear disequilibrium in populations near the center of the mtDNA cline suggest that some form of reproductive isolation such as assortative mating or selection against hybrids may be acting in this hybrid zone. However, only a small number of loci exhibited these signatures, suggesting locus-specific, rather than genomewide, factors. Fourteen of the 32 loci surveyed had cline widths inconsistent with neutral expectations, with two SNPs in the mitochondrial genome exhibiting the steepest clines. Seven of the 12 putatively non-neutral nuclear clines were for SNPs in genes related to oxidative metabolism. Among these putatively non-neutral nuclear clines, SNPs in two nuclear-encoded mitochondrial genes (SLC25A3 and HDDC2), as well as SNPs in the myoglobin, 40S ribosomal protein S17, and actin-binding LIM protein genes, had clines that were coincident and concordant with the mitochondrial clines. When hybrid index was calculated using this subset of loci, the frequency distribution of hybrid indices for a population located at the mtDNA cline center was non-unimodal, suggesting selection against advanced-generation hybrids, possibly due to effects on processes involved in oxidative metabolism.

Pancreatic ß-Cells From Mice Offset Age-Associated Mitochondrial Deficiency With Reduced KATP Channel Activity.

Aging is accompanied by impaired glucose homeostasis and an increased risk of type 2 diabetes, culminating in the failure of insulin secretion from pancreatic ß-cells. To investigate the effects of age on ß-cell metabolism, we established a novel assay to directly image islet metabolism with NAD(P)H fluorescence lifetime imaging (FLIM). We determined that impaired mitochondrial activity underlies an age-dependent loss of insulin secretion in human islets. NAD(P)H FLIM revealed a comparable decline in mitochondrial function in the pancreatic islets of aged mice (≥24 months), the result of 52% and 57% defects in flux through complex I and II, respectively, of the electron transport chain. However, insulin secretion and glucose tolerance are preserved in aged mouse islets by the heightened metabolic sensitivity of the ß-cell triggering pathway, an adaptation clearly encoded in the metabolic and Ca(2+) oscillations that trigger insulin release (Ca(2+) plateau fraction: young 0.211 ± 0.006, aged 0.380 ± 0.007, P < 0.0001). This enhanced sensitivity is driven by a reduction in KATP channel conductance (diazoxide: young 5.1 ± 0.2 nS; aged 3.5 ± 0.5 nS, P < 0.01), resulting in an ∼2.8 mmol/L left shift in the ß-cell glucose threshold. The results demonstrate how mice but not humans are able to successfully compensate for age-associated metabolic dysfunction by adjusting ß-cell glucose sensitivity and highlight an essential mechanism for ensuring the maintenance of insulin secretion.

Evidence for a bimodal distribution of hybrid indices in a hybrid zone with high admixture.

The genetic structure of a hybrid zone can provide insights into the relative roles of the various factors that maintain the zone. Here, we use a multilocus approach to characterize a hybrid zone between two subspecies of killifish (Fundulus heteroclitus, Walbaum 1792) found along the Atlantic coast of North America. We first analysed clinal variation along the Atlantic coast using a single-nucleotide polymorphism in the mitochondrial DNA (mtDNA) displacement loop (D-loop) and a panel of nine nuclear microsatellite markers. A model constraining all clines to the same width and centre was not significantly different from a model in which the clines were allowed to vary independently. Locus-by-locus analysis indicated that the majority of nuclear clines shared the same centre as the mtDNA cline, and the widths of these clines were also narrower than that predicted by a neutral model, suggesting that selection is operating to maintain the hybrid zone. However, two of the nuclear clines had widths greater than the neutral prediction and had centres that were displaced relative to the mtDNA cline centre. We also found that a marsh located near the centre of the mtDNA cline demonstrated a bimodal distribution of nuclear hybrid index values, suggesting a deficit of first-generation hybrids and backcrossed genotypes. Thus, selection against hybrid genotypes may be playing a role in maintaining this hybrid zone and the associated steep nuclear and mtDNA clines.

Interspecific differences in hypoxia-induced gill remodeling in carp.

The gills of many fish, but in particular those of crucian carp (Carassius carassius) and goldfish (Carassius auratus), are capable of extensive remodeling in response to changes in oxygen (O2), temperature, and exercise. In this study, we investigated the interspecific variation in hypoxia-induced gill modeling and hypoxia tolerance in 10 closely related groups of cyprinids (nine species, with two strains of Cyprinus carpio). There was significant variation in hypoxia tolerance, measured as the O2 tension (P(O2)) at which fish lost equilibrium (LOEcrit), among the 10 groups of carp. In normoxia, there was a significant, phylogenetically independent relationship between mass-specific gill surface area and LOEcrit, with the more hypoxia-tolerant carp having smaller gills than their less hypoxia-tolerant relatives. All groups of carp, except the Chinese bream (Megalobrama pellegrini), increased mass-specific gill surface area in response to 48 h of exposure to hypoxia (0.7 kPa) through reductions in the interlamellar cell mass (ILCM) volume. The magnitude of the hypoxia-induced reduction in the ILCM was negatively correlated with LOEcrit (and thus positively correlated with hypoxia tolerance), independent of phylogeny. The hypoxia-induced changes in gill morphology resulted in reduced variation in mass-specific gill surface area among species and eliminated the relationship between LOEcrit and mass-specific gill surface area. While behavioral responses to hypoxia differed among the carp groups, there were no significant relationships between hypoxia tolerance and the Po2 at which aquatic surface respiration (ASR) was initiated or the total number of ASR events observed during progressive hypoxia. Our results are the first to show that the extent of gill remodeling in cyprinids is associated with hypoxia tolerance in a phylogenetically independent fashion.

Variation in temperature tolerance among families of Atlantic salmon (Salmo salar) is associated with hypoxia tolerance, ventricle size and myoglobin level.

In fishes, performance failure at high temperature is thought to be due to a limitation on oxygen delivery (the theory of oxygen and capacity limited thermal tolerance, OCLTT), which suggests that thermal tolerance and hypoxia tolerance might be functionally associated. Here we examined variation in temperature and hypoxia tolerance among 41 families of Atlantic salmon (Salmo salar), which allowed us to evaluate the association between these two traits. Both temperature and hypoxia tolerance varied significantly among families and there was a significant positive correlation between critical maximum temperature (CTmax) and hypoxia tolerance, supporting the OCLTT concept. At the organ and cellular levels, we also discovered support for the OCLTT concept as relative ventricle mass (RVM) and cardiac myoglobin (Mb) levels both correlated positively with CTmax (R(2)=0.21, P<0.001 and R(2)=0.17, P=0.003, respectively). A large RVM has previously been shown to be associated with high cardiac output, which might facilitate tissue oxygen supply during elevated oxygen demand at high temperatures, while Mb facilitates the oxygen transfer from the blood to tissues, especially during hypoxia. The data presented here demonstrate for the first time that RVM and Mb are correlated with increased upper temperature tolerance in fish. High phenotypic variation between families and greater similarity among full- and half-siblings suggests that there is substantial standing genetic variation in thermal and hypoxia tolerance, which could respond to selection either in aquaculture or in response to anthropogenic stressors such as global climate change.

Intraspecific variation in the thermal plasticity of mitochondria in killifish.

Populations of the Atlantic killifish (Fundulus heteroclitus) inhabit salt marshes and estuaries along the eastern coast of North America from Newfoundland to northern Florida, and are thus exposed to a large range of temperatures. Previous studies have shown higher whole-organism metabolic rates in the northern subspecies (F. h. macrolepidotus) compared with the southern subspecies (F. h. heteroclitus) of these fish. Here, we examine phenotypic plasticity in the response to cold temperatures between the two subspecies by acclimating fish to 5, 15 and 25°C and comparing several mitochondrial and muscle properties. The relative area of oxidative muscle versus glycolytic muscle fibers was greater in the northern subspecies at the 5 and 15°C acclimation temperatures. However, there were no differences in capillary density between the two subspecies or at different temperatures. Mitochondrial volume and surface densities increased in response to cold temperature acclimation in red and white muscle, but only in the northern killifish. Citrate synthase activities also increased in the northern killifish at 5 and 15°C. The ratio of calculated [free ADP] to [ATP] increased in the 5°C acclimated southern killifish but not in the northern killifish at 5°C when compared with the 15°C acclimation group, suggesting that there are differences in adenylate signaling for mitochondrial respiration between subspecies at low temperature. Taken together, our data indicate that the northern subspecies have a greater ability to increase mitochondrial capacity at colder temperatures compared with the southern subspecies, providing one of the few examples of intraspecific variation in phenotypic plasticity in mitochondrial amount in response to cold temperatures.