Paul M. Craig

Temperature‐ and exercise‐induced gene expression and metabolic enzyme changes in skeletal muscle of adult zebrafish (Danio rerio)

Both exercise training and cold acclimatization induce muscle remodelling in vertebrates, producing a more aerobic phenotype. In ectothermic species exercise training and cold‐acclimatization represent distinct stimuli. It is currently unclear if these stimuli act through a common mechanism or if different mechanisms lead to a common phenotype. The goal of this study was to survey responses that represent potential mechanisms responsible for contraction‐ and temperature‐induced muscle remodelling, using an ectothermic vertebrate. Separate groups of adult zebrafish (Danio rerio) were either swim trained or cold acclimatized for 4 weeks. We found that the mitochondrial marker enzyme citrate synthase (CS) was increased by 1.5× in cold and by 1.3× with exercise (P < 0.05). Cytochrome c oxidase (COx) was increased by 1.2× following exercise training (P < 0.05) and 1.2× (P= 0.07) with cold acclimatization. However, only cold acclimatization increased β‐hydroxyacyl‐CoA dehydrogenase (HOAD) compared to exercise‐trained (by 1.3×) and pyruvate kinase (PK) relative to control zebrafish. We assessed the whole‐animal performance outcomes of these treatments. Maximum absolute sustained swimming speed (Ucrit) was increased in the exercise trained group but not in the cold acclimatized group. Real‐time PCR analysis indicated that increases in CS are primarily transcriptionally regulated with exercise but not with cold treatments. Both treatments showed increases in nuclear respiratory factor (NRF)‐1 mRNA which was increased by 2.3× in cold‐acclimatized and 4× in exercise‐trained zebrafish above controls. In contrast, peroxisome proliferator‐activated receptor (PPAR)‐α mRNA levels were decreased in both experimental groups while PPAR‐β1 declined in exercise training only. Moreover, PPAR‐γ coactivator (PGC)‐1α mRNA was not changed by either treatment. In zebrafish, both temperature and exercise produce a more aerobic phenotype, but there are stimulus‐dependent responses (i.e. HOAD and PK activities). While similar changes in NRF‐1 mRNA suggest that common responses might underlie aerobic muscle remodelling there are distinct changes (i.e. CS and PPAR‐β1 mRNA) that contribute to specific temperature‐ and exercise‐induced phenotypes.

The metabolic consequences of hepatic AMP-kinase phosphorylation in rainbow trout.

AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, is proposed to function as a “fuel gauge” to monitor cellular energy status in response to nutritional environmental variations. However, in fish, few studies have addressed the metabolic consequences related to the activation of this kinase. This study demonstrates that the rainbow trout (Oncorhynchus mykiss) possesses paralogs of the three known AMPK subunits that co-diversified, that the AMPK protein is present in the liver and in isolated hepatocytes, and it does change in response to physiological (fasting-re-feeding cycle) and pharmacological (AICAR and metformin administration and incubations) manipulations. Moreover, the phosphorylation of AMPK results in the phosphorylation of acetyl-CoA carboxylase, a main downstream target of AMPK in mammals. Other findings include changes in hepatic glycogen levels and several molecular actors involved in hepatic glucose and lipid metabolism, including mRNA transcript levels for glucokinase, glucose-6-phosphatase and fatty acid synthase both in vivo and in vitro. The fact that most results presented in this study are consistent with the recognized role of AMPK as a master regulator of energy homeostasis in living organisms supports the idea that these functions are conserved in this piscine model.

Fasted Zebrafish Mimic Genetic and Physiological Responses in Mammals: A Model for Obesity and Diabetes?

With worldwide rates of obesity and type-II diabetes increasing, it is essential to identify and understand the mechanisms involved during nutrient absorption and fuel allocation. Recent studies demonstrate that nutrients (e.g., lipids and carbohydrates) play a major regulatory role in gene transcription of glycolytic and lipogenic enzymes in addition to hormones, including insulin and glucagon. These nutrients generally exert their effects through key cellular nutrient/energy receptors. Fasting was used to identify these nutrient/energy receptors known from mammalian studies to ascertain if zebrafish (Danio rerio) are a suitable model for the study of metabolic disorders. Zebrafish were subjected to a fasting/re-feeding regime for 3 weeks, and gene expression of sterol responsive binding protein 1 and 2 (SREBP), the mammalian target of rapamycin (mTOR), cAMP response element binding protein 3-like 3 (CREB3l3), and AMP-activated protein kinase alpha (AMPKα) was assessed. Fasted zebrafish lost ∼10% of their body mass over the 3-week experiment, with an associated depression in oxygen consumption. Increases in liver AMPKα and CREB3l3 mRNA transcript level were noted, concurrent with increases in the activities of the β-oxidation and gluconeogenic markers β-hydroxyacyl CoA dehydrogenase and phosphoenolpyruvate carboxykinase, respectively. Conversely, a depression in liver mTOR and SREBP1 and 2 expression was noted, with a decrease in pyruvate kinase and alanine aminotransferase activities and decreases in liver lipid and glycogen contents. Twenty-four hours after re-feeding, zebrafish rapidly recover, and the majority of parameters return to control values. Taken together, these data suggest adult zebrafish are an appropriate model for the further study of human metabolic disorders.

Gene expression endpoints following chronic waterborne copper exposure in a genomic model organism, the zebrafish, Danio rerio

Although copper (Cu) is an essential micronutrient for all organisms, in excess, waterborne Cu poses a significant threat to fish from the cellular to population level. We examined the physiological and gene expression endpoints that chronic waterborne Cu exposure (21 d) imposes on soft-water acclimated zebrafish at two environmentally relevant concentrations: 8 microg/l (moderate) and 15 microg/l (high). Using a 16,730 65-mer oligonucleotide customized zebrafish microarray chip related to metal metabolism and toxicity to assess the transcriptomic response, we found that 573 genes in the liver responded significantly to Cu exposure. These clustered into three distinct patterns of expression. There was distinct upregulation of a majority of these genes under moderate Cu exposure and a significant downregulation under high Cu exposure. Microarray results were validated by qPCR of eight genes; two genes, metallothionein 2 (mt2) and Na(+)-K(+)-ATPase 1a1 (atp1a1), displayed increased expression under both Cu exposures, indicative of potential genetic endpoints of Cu toxicity, whereas the remaining six genes demonstrated opposing effects at each Cu exposure. Na(+)-K(+)-ATPase enzyme activity decreased during Cu exposure, which may be linked to Cus competitive effects with Na(+). Whole body cortisol levels were significantly increased in Cu-exposed fish, which prompted an analysis of the promoter region of all significantly regulated genes for glucocorticoid (GRE) and metal (MRE) response elements to dissociate metal- and stress-specific gene responses. Of the genes significantly regulated, 30% contained only a GRE sequence, whereas 2.5% contained only a consensus MRE. We conclude that the indirect effects of Cu exposure regulate gene expression to a much greater degree than the direct effects.

Methionine restriction affects the phenotypic and transcriptional response of rainbow trout (Oncorhynchus mykiss) to carbohydrate-enriched diets.

Mammalian studies report that methionine restriction (MR) as a dietary regimen extends life span, delays the onset of age-related diseases and enhances fat oxidation in obese subjects with metabolic syndromes. However, the underlying cellular signalling pathways are poorly understood. Rainbow trout (Oncorhynchus mykiss) is a glucose-intolerant species, providing an excellent model for the study of carbohydrate metabolism. MR diets in combination with 12 % (+/-) and 22 % (+/-) carbohydrate-rich meals were fed to rainbow trout for a period of 8 weeks and phenotypic and transcript expression changes in the liver and white muscle were assessed. Fish fed MR diets, irrespective of carbohydrate load, were shown to abolish the glucose-intolerant phenotype 6 h post-feeding. There was a distinct switch in glucose and glycogen content in the liver of fish fed MR diets, with a significantly higher concentration of glycogen, suggesting reduced glycolytic capacity. Transcriptional responses to MR demonstrated decreased expression of hepatic fatty acid synthase, sterol regulatory binding protein 1, PPARγ coactivator 1-α and PPARα, indicative of a reduction in the de novo synthesis of fatty acids and cholesterol, and a potential decrease in hepatic fat oxidative capacity. Muscle adenylate charge was depressed under MR, and increased expression of AMP-activated protein kinase α1 was detected, indicative of reduced energy availability. Total DNA methylation showed that carbohydrate load, rather than MR, dictated hypomethylation of genomic DNA. This is the first study which demonstrates that MR can abolish a glucose-intolerant phenotype in trout, and identifies trout as a suitable model for studying metabolic syndromes.

Dietary iron alters waterborne copper-induced gene expression in soft water acclimated zebrafish (Danio rerio)

Metals like iron (Fe) and copper (Cu) function as integral components in many biological reactions, and, in excess, these essential metals are toxic, and organisms must control metal acquisition and excretion. We examined the effects of chronic waterborne Cu exposure and the interactive effects of elevated dietary Fe on gene expression and tissue metal accumulation in zebrafish. Softwater acclimated zebrafish exposed to 8 microg/l Cu, with and without supplementation of a diet high in Fe (560 vs. 140 mg Fe/kg food) for 21 days demonstrated a significant reduction in liver and gut Cu load relative to waterborne Cu exposure alone. Gene expression levels for divalent metal transport (DMT)-1, copper transporter (CTR)-1, and the basolateral metal transporter ATP7A in the gills and gut increased when compared with controls, but the various combinations of Cu and high-Fe diet revealed altered levels of expression. Further examination of the basolateral Fe transporter, ferroportin, showed responses to waterborne Cu exposure in the gut and a significant increase with Fe treatment alone in the liver. Additionally, we examined metallothionein 1 and 2 (MT1 and MT2), which indicated that MT2 is more responsive to Cu. To explore the relationship between transcription and protein function, we examined both CTR-1 protein levels and gill apical uptake of radiolabeled Cu64, which demonstrated decreased Cu uptake and protein abundance in the elevated Cu treatments. This study shows that high dietary Fe can significantly alter the genetic expression pattern of Cu transporters at the level of the gill, liver, and gastrointestinal tract.

Profiling hepatic microRNAs in zebrafish: fluoxetine exposure mimics a fasting response that targets AMP-activated protein kinase (AMPK).

This study examined the similarities in microRNA profiles between fasted and fluoxetine (FLX) exposed zebrafish and downstream target transcripts and biological pathways. Using a custom designed microarray targeting 270 zebrafish miRNAs, we identified 9 differentially expressed miRNAs targeting transcripts in biological pathways associated with anabolic metabolism, such as adipogenesis, cholesterol biosynthesis, triacylglycerol synthesis, and insulin signaling. Exposure of female zebrafish to 540 ng/L FLX, an environmentally relevant concentration and a known metabolic repressor, increased specific miRNAs indicating greater inhibition of these pathways in spite of continued feeding. Further examination revealed two specific miRNAs, dre-let-7d and dre-miR-140-5p, were predicted in silico to bind to a primary regulator of metabolism, adenosine monophosphate-activated protein kinase (AMPK), and more specifically the two isoforms of the catalytic subunit, AMPKα1 and α2, respectively. Real-time analysis of the relative transcript abundance of the α1 and α2 mRNAs indicated a significant inverse relationship between specific miRNA and target transcript. This suggests that AMPK-related pathways may be compromised during FLX exposure as a result of increased miRNA abundance. The mechanism by which FLX regulates miRNA abundance is unknown but may be direct at the liver. The serotonin transporter, slc6a4, is the target of FLX and other selective serotonin reuptake inhibitors (SSRI) and it was found to be expressed in the liver, although treatment did not alter expression of this transporter. Exposure to FLX disrupts key hepatic metabolic pathways, which may be indicative of reduced overall fitness and these effects may be linked to specific miRNA abundance. This has important implications for the heath of fish because concentrations of SSRIs in aquatic ecosystems are continually increasing.

Water chemistry alters gene expression and physiological end points of chronic waterborne copper exposure in zebrafish, Danio rerio.

This is the first study to implement a genomic approach to ascertain both transcriptional and functional end points of chronic Cu toxicity in fish associated with experimentally manipulated water chemistries. Over 21 d, zebrafish acclimated to softwater (Na(+) = 0.06 mM, Ca(2+) = 0.08 mM, Mg(2+) = 0.03 mM) were exposed to the following: soft-water (Ctrl); 12 microg L(-1) Cu (Cu); 3.3 mM Na(+) (Na); 3.3 mM Na(+) + 12 microg L(-1) Cu (Na + Cu); 3.3 mM Ca(2+) (Ca); or 3.3 mM Ca(2+) + 12 microg L(-1) Cu (Ca + Cu). Although effective at reducing Cu load in all tissues, Na(+) in the presence of Cu did not decrease the degree of oxidative damage, particularly in the gill and gut. In contrast, Ca + Cu treatment decreased Cu accumulation in gill, but not liver or gut, with no reduction in oxidative damage. Transcriptional analysis of candidate genes (atp7a, ctr1, ECaC, esr1) showed principally a down regulation of transcripts with the Cu only treatment, while Ca + Cu treatment restored some of the genes to control levels. Conversely, the Na + Cu treatment had a strong, opposing affect when compared to that of Cu alone. Zebrafish Affymetrix GeneChips revealed significantly clustered patterns of expression. Changes in expression induced by Cu appeared to be opposite to the majority of the other treatments. Our data on the preventative or enhancing effects of Na(+) and Ca(2+) both alone and in the presence of Cu, may, in the future, facilitate the incorporation of gene expression end points into a biotic ligand model predicting chronic Cu toxicity in this tropical model species of genomic importance.

Sperm performance under hypoxic conditions in the intertidal fish Porichthys notatus

Hypoxia (low oxygen) exposure generally leads to decreased reproductive capacity, exhibited by reductions in testicular mass, reproductive hormones, and sperm swimming speed. However, in many fish species, reproduction occurs either periodically or exclusively under hypoxic conditions. In this study we assessed how hypoxia influences sperm performance in the plainfin midshipman (Porichthys notatus Girard, 1854), a species that lives in intertidal nests that become hypoxic during low tides. We exposed sperm from the same male to normoxic or hypoxic conditions and compared sperm characteristics and oxygen consumption between treatments. Sperm exposed to hypoxic water swam faster and consumed more oxygen than sperm swimming in normoxic conditions. Sperm swimming speed was positively related with oxygen consumption. For each male, the percentage of motile spermatozoa did not differ between treatments, suggesting that the same number of sperm were active but their performance was dependent on the dissolved oxy...

Understanding glucose uptake during methionine deprivation in incubated rainbow trout (Oncorhynchus mykiss) hepatocytes using a non-radioactive method

The role of methionine supplementation in fish metabolism remains largely unexplored. This study investigates the effects of methionine deprivation (MD) on glucose uptake in rainbow trout (Oncorhynchus mykiss) hepatocytes. To this end, primary hepatocytes were incubated in the presence (+M) or the absence (-M) of methionine for 48h and glucose uptake was assessed using a novel non-radioactive, fluorescent-linked enzymatic assay. Evidence indicated that glucose uptake increased under methionine deprivation, primarily due to the increased abundance of membrane bound sodium-glucose transporter 2 (SGLT2), which was likely facilitated by the cellular reduction in [ATP] resulting from increased mitochondrial uncoupling, as supported by elevated transcript levels of uncoupling protein 2a (UCP2a). This study is the first to suggest that the mechanisms responsible for the rapid glucose uptake associated with MR are facilitated by the greater abundance of SGLT2 glucose transporter and mitochondrial uncoupling.