Kenneth J. Rodnick

Contribution of impaired myocardial insulin signaling to mitochondrial dysfunction and oxidative stress in the heart

Background— Diabetes-associated cardiac dysfunction is associated with mitochondrial dysfunction and oxidative stress, which may contribute to left ventricular dysfunction. The contribution of altered myocardial insulin action, independent of associated changes in systemic metabolism, is incompletely understood. The present study tested the hypothesis that perinatal loss of insulin signaling in the heart impairs mitochondrial function. Methods and Results— In 8-week-old mice with cardiomyocyte deletion of insulin receptors (CIRKO), inotropic reserves were reduced, and mitochondria manifested respiratory defects for pyruvate that was associated with proportionate reductions in catalytic subunits of pyruvate dehydrogenase. Progressive age-dependent defects in oxygen consumption and ATP synthesis with the substrate glutamate and the fatty acid derivative palmitoyl-carnitine were observed. Mitochondria also were uncoupled when exposed to palmitoyl-carnitine, in part as a result of increased reactive oxygen species production and oxidative stress. Although proteomic and genomic approaches revealed a reduction in subsets of genes and proteins related to oxidative phosphorylation, no reductions in maximal activities of mitochondrial electron transport chain complexes were found. However, a disproportionate reduction in tricarboxylic acid cycle and fatty acid oxidation proteins in mitochondria suggests that defects in fatty acid and pyruvate metabolism and tricarboxylic acid flux may explain the mitochondrial dysfunction observed. Conclusions— Impaired myocardial insulin signaling promotes oxidative stress and mitochondrial uncoupling, which, together with reduced tricarboxylic acid and fatty acid oxidative capacity, impairs mitochondrial energetics. This study identifies specific contributions of impaired insulin action to mitochondrial dysfunction in the heart.

Effect of starvation on transcriptomes of brain and liver in adult female zebrafish (Danio rerio)

We used microarray and quantitative real-time PCR (qRT-PCR) analyses in adult female zebrafish (Danio rerio) to identify metabolic pathways regulated by starvation in the liver and brain. The transcriptome of whole zebrafish brain showed little response to 21 days of starvation. Only agouti-related protein 1 (agrp1) significantly responded, with increased expression in brains of starved fish. In contrast, a 21-day period of starvation significantly downregulated 466 and upregulated 108 transcripts in the liver, indicating an overall decrease in metabolic activity, reduced lipid metabolism, protein biosynthesis, proteolysis, and cellular respiration, and increased gluconeogenesis. Starvation also regulated expression of many components of the unfolded protein response, the first such report in a species other than yeast (Saccharomyces cerevisiae) and mice (Mus musculus). The response of the zebrafish hepatic transcriptome to starvation was strikingly similar to that of rainbow trout (Oncorhynchus mykiss) and less similar to mouse, while the response of common carp (Cyprinus carpio) differed considerably from the other three species.

Metabolism, Swimming Performance, and Tissue Biochemistry of High Desert Redband Trout (Oncorhynchus mykiss ssp.): Evidence for Phenotypic Differences in Physiological Function

Redband trout (Oncorhynchus mykiss ssp.) in southeastern Oregon inhabit high‐elevation streams that exhibit extreme variability in seasonal flow and diel water temperature. Given the strong influence and potential limitations exerted by temperature on fish physiology, we were interested in how acute temperature change and thermal history influenced the physiological capabilities and biochemical characteristics of these trout. To this end, we studied wild redband trout inhabiting two streams with different thermal profiles by measuring (1) critical swimming speed (Ucrit) and oxygen consumption in the field at 12° and 24°C; (2) biochemical indices of energy metabolism in the heart, axial white skeletal muscle, and blood; and (3) temperature preference in a laboratory thermal gradient. Further, we also examined genetic and morphological characteristics of fish from these two streams. At 12°C, maximum metabolic rate (Mo2 max) and metabolic power were greater in Little Blitzen redband trout as compared with those from Bridge Creek (by 37% and 32%, respectively). Conversely, Bridge Creek and Little Blitzen trout had similar values for Mo2 max and metabolic power at 24°C. The Ucrit of Little Blitzen trout was similar at the two temperatures (61 ± 3 vs. 57 ± 4 cm s−1). However, the Ucrit for Bridge Creek trout increased from 62 ± 3 cm s−1 to 75 ± 3 cm s−1 when water temperature was raised from 12° to 24°C, and the Ucrit value at 24°C was significantly greater than for Little Blitzen fish. Cost of transport was lower for Bridge Creek trout at both 12° and 24°C, indicating that these trout swim more efficiently than those from the Little Blitzen. Possible explanations for the greater metabolic power of Little Blitzen redband trout at 12°C include increased relative ventricular mass (27%) and an elevation in epaxial white muscle citrate synthase activity (by 72%). Bridge Creek trout had 50% higher lactate dehydrogenase activity in white muscle and presumably a greater potential for anaerobic metabolism. Both populations exhibited a preferred temperature of approximately 13°C and identical mitochondrial haplotypes and p53 gene allele frequencies. However, Bridge Creek trout had a more robust body form, with a relatively larger head and a deeper body and caudal peduncle. In summary, despite the short distance (∼10 km) and genotypic similarity between study streams, our results indicate that phenotypic reorganization of anatomical characteristics, swimming ability at environmentally pertinent temperatures and white axial muscle ATP‐producing pathways occurs in redband trout.

Differential Lowering by Manganese Treatment of Activities of Glycolytic and Tricarboxylic Acid (TCA) Cycle Enzymes Investigated in Neuroblastoma and Astrocytoma Cells Is Associated with Manganese-Induced Cell Death

Manganese (Mn) is a trace metal required for normal growth and development. Manganese neurotoxicity is rare and usually associated with occupational exposures. However, the cellular and molecular mechanisms underlying Mn toxicity are still elusive. In rats chronically exposed to Mn, their brain regional Mn levels increase in a dose-related manner. Brain Mn preferentially accumulates in mitochondria; this accumulation is further enhanced with Mn treatment in vivo. Exposure of mitochondria to Mn in vitro leads to uncoupling of oxidative phosphorylation. These observations prompted us to investigate the hypothesis that Mn induces alterations in energy metabolism in neural cells by interfering with the activities of various glycolytic and TCA cycle enzymes using human neuroblastoma (SK-N-SH) and astrocytoma (U87) cells. Treatments of SK-N-SH and U87 cells with MnCl2 induced cell death in these cells, in a concentration- and time-dependent manner, as determined by MTT assays. In parallel with the Mn-induced, dose-dependent decrease in cell survival, treatment of these cells with 0.01 to 4.0 mM MnCl2 for 48 h also induced dose-related decreases in their activities of hexokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, and malate dehydrogenase. Hexokinase in SK-N-SH cells was the most affected by Mn treatments, even at the lower range of concentrations. Mn treatment of SK-N-SH cells affected pyruvate kinase and citrate synthase to a lesser extent as compared to its effect on other enzymes investigated. However, citrate synthase and pyruvate kinase in U87 cells were more vulnerable than other enzymes investigated to the effects of Mn. The results suggest the two cell types exhibited differential susceptibility toward the Mn-induced effects. Additionally, the results may have significant implications in flux control because HK is the first and highly regulated enzyme in brain glycolysis. Thus these results are consistent with our hypothesis and may have pathophysiological implications in the mechanisms underlying Mn neurotoxicity.

Sexual dimorphism in hepatic gene expression and the response to dietary carbohydrate manipulation in the zebrafish (Danio rerio)

In this study, we tested for the presence of sexual dimorphism in the hepatic transcriptome of the adult zebrafish and examined the effect of long term manipulation of dietary carbohydrate on gene expression in both sexes. Zebrafish were fed diets comprised of 0%, 15%, 25%, or 35% carbohydrate from the larval stage through sexual maturity, then sampled for hepatic tissue, growth, proximate body composition, and retention efficiencies. Using Affymetrix microarrays and qRT-PCR, we observed substantial sexual dimorphism in the hepatic transcriptome. Males up-regulated genes associated with oxidative metabolism, carbohydrate metabolism, energy production, and amelioration of oxidative stress, while females had higher expression levels of genes associated with translation. Restriction of dietary carbohydrate (0% diet) significantly affected hepatic gene expression, growth performance, retention efficiencies of protein and energy, and percentages of moisture, lipid, and ash. The response of some genes to dietary manipulation varied by sex; with increased dietary carbohydrate, males up-regulated genes associated with oxidative metabolism (e.g. hadhbeta) while females up-regulated genes associated with glucose phosphorylation (e.g. glucokinase). Our data support the use of the zebrafish model for the study of fish nutritional genomics, but highlight the importance of accounting for sexual dimorphism in these studies.

The regulation and importance of glucose uptake in the isolated Atlantic cod heart: rate-limiting steps and effects of hypoxia

SUMMARY This study investigated the regulation of glucose uptake in Atlantic cod (Gadus morhua) hearts. Isolated hearts were perfused with or without glucose in the medium, under either normoxic or severely hypoxic conditions. Working at basal levels, hearts did not require extracellular glucose to maintain power under aerobic conditions. However, cardiac performance was significantly reduced without exogenous glucose under oxygen-limiting conditions. The addition of the glucose transporter inhibitor cytochalasin B caused hypoxic hearts to fail early, and hearts perfused with a glucose analogue, 2-deoxyglucose (2-DG), increased glucose uptake 3-fold under hypoxia. The uptake of 2-DG was only partially inhibited when cytochalasin B was added to the medium. Isolated ventricle strips were also incubated in the presence of 2-DG and the extracellular marker mannitol. Glucose uptake (glucose transport plus intracellular phosphorylation) was assessed by measuring the initial rate of 2-deoxyglucose-6-phosphate (2-DG-6-P) accumulation. At 1 mmol l-1 2-DG, the rate of 2-DG uptake remained linear for 60 min, and 2-DG-6-P, but not free 2-DG, accumulation was increased. The fact that intracellular 2-DG did not increase indicates that glucose transport is the rate-limiting step for glucose utilization in non-stimulated cardiac tissue. Replacement of Na+ by choline in the incubation medium did not affect 2-DG uptake, providing evidence that Na+-coupled glucose transport is absent in cod cardiac tissue. Similar to cytochalasin B, glucose uptake was also inhibited by phloridzin, suggesting that facilitated, carrier-mediated glucose transport occurs in cod hearts. Under the conditions employed in these experiments, it is clear that (1) activation of glucose transport is required to support hypoxic performance, (2) the rate-limiting step for glucose utilization is glucose transport rather than glucose phosphorylation, (3) 2-DG uptake accurately reflects glucose transport activity and (4) glucose uptake in cod hearts does not involve an Na+-dependent mechanism.

Pressure and volume overloads are associated with ventricular hypertrophy in male rainbow trout

We investigated whether ventricular hypertrophy in reproductively mature male trout ( Oncorhynchus mykiss) is associated with elevated hemodynamic loads. We measured ventral aortic blood pressure, pulse pressure dynamics, and blood volume in cannulated, unanesthetized trout with a wide range of relative ventricle masses (RVM, 0.076-0.199% of body wt). We also investigated in vitro pressure-volume dynamics in the bulbus arteriosus taken from trout with a wide range of RVMs. RVM was positively correlated with peak systolic pressure (SBP), mean blood pressure, and pulse pressure. Diastolic pressure and the absolute duration of arterial systole were similar among all animals, but a lower heart rate and a smaller relative duration of arterial systole were correlated with increasing RVM. Blood volume was expanded up to 34% as ventricles enlarged, and clearance of Evans blue dye was greater at higher SBP. Mass, maximal volume, and the pressure-volume dynamics of the bulbus were similar among all animals, suggesting that the bulbus did not compensate for ventricular enlargement. This conclusion was supported by the elevated maximal rates of arterial pressure development (+d P/d t) and decay (-d P/d t) observed as RVM increased. We conclude that 1) mature trout are hypertensive and hypervolemic, 2) the dynamics of the bulbus may contribute to increased afterload, and 3) these changes in hemodynamic load may promote ventricular hypertrophy.

Effects of Batrachochytrium dendrobatidis infection on ion concentrations in the boreal toad Anaxyrus (Bufo) boreas boreas.

Batrachochytrium dendrobatidis causes mortality in various amphibian species including the boreal toad Anaxyrus (Bufo) boreas boreas. The purpose of this study was to determine the physiological effects of this pathogen on experimentally infected boreal toads. Plasma osmolality, sodium, and potassium concentrations were analyzed to evaluate the differences between diseased and non-exposed animals. Infected animals with clinical signs of chytridiomycosis had significantly lower plasma osmolality, sodium, and potassium levels than non-infected animals (p < 0.06). On average, clinically infected animals housed in an aquatic environment had sodium and potassium levels of 60.1 (SE = 9.7) and 2.06 (SE = 0.32) mmol l(-1), respectively. These ion levels were significantly lower than the negative controls (sodium = 115.0 mmol l(-1), potassium = 3.7 mmol l(-1)) and consistent with the clinical signs observed in affected animals. We propose that infection with B. dendrobatidis results in an electrolyte disorder in boreal toads.

Dietary carbohydrate level affects transcription factor expression that regulates skeletal muscle myogenesis in rainbow trout.

Understanding the effects of dietary carbohydrates on transcription factors that regulate myogenesis provides insight into the role of nutrient sensing by satellite cells towards myocyte differentiation. We evaluated the influence of dietary carbohydrate level (0, 15, 25 or 35%) on the temporal mRNA expression patterns (4, 8 or 12 weeks) of transcription factors that regulate satellite cell myocyte addition (MA) in rainbow trout (Oncorhynchus mykiss), a vertebrate with indeterminate growth. Relative to the 0% carbohydrate (NC) diet, 15 (IC-15) and 25% (IC-25) carbohydrate containing diets significantly up-regulate MyoD and Myf5, but not Pax7, after 12 weeks of feeding. Simultaneously, the Pax7/MyoD mRNA expression ratio declined significantly with both the IC diets. Myogenin mRNA expression also increased in rainbow trout (RBT) fed the IC-15 diet. The high carbohydrate (HC) diet (35%) attenuated the increased mRNA expression of these transcription factors. It is of note that the 4 and 8 week samples lacked the promyogenic expression patterns. The myogenic gene expression in fish fed the IC-15 diet for 12 weeks indicate a transcriptional signature that reflects increased satellite cell myogenesis. Our results suggest a potential role for satellite cells in the nutrient sensing ability of a vertebrate with indeterminate skeletal muscle growth.

Metabolic, hemodynamic, and cardiac effects of captopril in young, spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) demonstrate elevated blood pressure, cardiac hypertrophy, glucose intolerance, and insulin resistance compared with age-matched Wistar-Kyoto rats (WKY). We investigated concurrent effects of captopril on blood pressure, cardiac mass, myocardial enzyme activities, glucose tolerance, and insulin action in young male SHR. At 10 weeks of age, SHR were randomized into two groups, one receiving distilled water, the other a captopril solution (50 mg/kg body weight/day). We also examined age-matched WKY receiving distilled water. Blood pressure was measured by tail-cuff during the 4-week treatment period and oral glucose tolerance was tested at the end of treatment. Hearts were weighed and ventricular tissue was assayed for activities of 3-hydroxyacyl-CoA dehydrogenase, citrate synthase, and hexokinase. Growth rates were similar between captopril-treated and control SHR, but less than those of WKY. Captopril reduced blood pressure (134 +/- 8 v 177 +/- 8 mm Hg, P < .05) and left ventricular mass (-18%, P < .05) in SHR. Cardiac enzyme activities also changed with captopril treatment, reflecting an increased capacity for beta-oxidation of fatty acids and reduced potential for glucose phosphorylation in the left ventricle of SHR. Serum concentrations of glucose, insulin, and free fatty acids after a brief fast and in response to oral glucose were not different after captopril treatment, suggesting no improvement in insulin action or glucose tolerance. In summary, treatment of young male SHR with captopril reduces blood pressure and cardiac mass, and promotes a small but significant increase in cardiac capacity for oxidation of fatty acids and reduction of glucose phosphorylation. In contrast, metabolic effects of captopril on oral glucose tolerance and insulin action were not evident.