Development of a TaqMan quantitative reverse transcription PCR assay to detect tilapia lake virus.

Tilapia lake virus disease (TiLVD) is an emerging viral disease associated with high morbidity and mortality in cultured tilapia worldwide. In this study, we have developed and validated a TaqMan quantitative reverse transcription PCR (RT-qPCR) assay for TiLV, targeting a conserved region within segment 10 of the genome. The RT-qPCR assay was efficient (mean ± SD: 96.71 ± 3.20%), sensitive with a limit of detection of 10 RNA viral copies per reaction, and detected TiLV strains from different geographic regions including North America, South America, Africa, and Asia. The intra- and inter-assay variability ranged over 0.18-1.41% and 0.21-2.21%, respectively. The TaqMan RT-qPCR assay did not cross-react with other RNA viruses of fish, including an orthomyxovirus, a betanodavirus, a picornavirus, and a rhabdovirus. Analysis of 91 proven-positive and 185 proven-negative samples yielded a diagnostic sensitivity of 96.7% and a diagnostic specificity of 100%. The TaqMan RT-qPCR assay also detected TiLV RNA in infected Nile tilapia liver tissue extracts following an experimental challenge study, and it successfully detected TiLV RNA in SSN-1 (E-11 clone) cell cultures displaying cytopathic effects following their inoculation with TiLV-infected tissue homogenates. Thus, the validated TaqMan RT-qPCR assay should be useful for both research and diagnostic purposes. Additionally, the TiLV qPCR assay returns the clinically relevant viral load of a sample which can assist health professionals in determining the role of TiLV during disease investigations. This RT-qPCR assay could be integrated into surveillance programs aimed at mitigating the effects of TiLVD on global tilapia production.

Farm-Level Cost Drivers of Salmonid Fish Health Inspections.

Regulatory costs on aquaculture farms have been shown to be of a magnitude that warrants additional analysis. The drivers of farm-level costs of fish health inspections were identified in this study from national survey data on U.S. salmonid farms. The greatest costs identified were related primarily to state fish health requirements for inspection and testing to certify that fish are free of specific pathogens prior to approval of necessary permits to sell and/or transport animals. Fish health inspection costs included laboratory testing, farm personnel time, veterinary fees, and shipping samples to laboratories, with laboratory testing and the value of farm personnel time being the most expensive components. Principal cost drivers were the number of tests and whether required sampling was farmwide or for each lot as identified by the collector. Farmers who primarily sold into recreational markets had greater fish health costs than farmers who primarily sold food fish because of the greater numbers of species and size-/age-classes of salmonids on their farms. Regulatory requirements to test all species and size-/age-classes on farms increased inspection costs by increasing the total number of tests, the total value of fish sacrificed, and shipping costs. Consequently, for farms with more than one species or more than one size-/age-class, annual farm-level testing was less costly than annual lot-based testing. Increased numbers of tests in a given year, although reported by only a few respondents, can increase costs dramatically and turn profitable farms unprofitable, even food fish farms. Smaller salmonid farms experienced disproportionately greater inspection cost burdens than did larger farms. The fish health inspection scenario of only one annual inspection of only the most susceptible species and size-/age-class showed a cost burden that did not generate economic distress, even on smaller salmonid farms. Other scenarios modeled (based on survey data) that included lot-based surveys of multiple species and size-/age-classes resulted in substantially greater fish health inspection costs that led to unprofitability for various farm sizes and business types. Study results suggest that implementing Comprehensive Aquaculture Health Program Standards might allow for risk- and pathogen-based reductions in the total number of inspections and fish sampled while maintaining equivalent or greater health status compared to current methods. American Fisheries Society-Fish Health Section Blue Book inspection methods are interpreted and applied inconsistently across states and generally yield lot- rather than farm-level health attestations because the history of testing results, risk assessment, and biosecurity practices are not typically taken into account. The cost effects of alternative fish health sampling and testing requirements should be considered in decisions and policy on fish health regulation.

Optimizing surveillance for early disease detection: Expert guidance for Ostreid herpesvirus surveillance design and system sensitivity calculation.

To keep pace with rising opportunities for disease emergence and spread, surveillance in aquaculture must enable the early detection of both known and new pathogens. Conventional surveillance systems (designed to provide proof of disease freedom) may not support detection outside of periodic sampling windows, leaving substantial blind spots to pathogens that emerge in other times and places. To address this problem, we organized an expert panel to envision optimal systems for early disease detection, focusing on Ostreid herpesvirus 1 (OsHV-1), a pathogen of panzootic consequence to oyster industries. The panel followed an integrative group process to identify and weight surveillance system traits perceived as critical to the early detection of OsHV-1. Results offer a road map with fourteen factors to consider when building surveillance systems geared to early detection; factor weights can be used by planners and analysts to compare the relative value of different designs or enhancements. The results were also used to build a simple, but replicable, model estimating the system sensitivity (SSe) of observational surveillance and, in turn, the confidence in disease freedom that negative reporting can provide. Findings suggest that optimally designed observational systems can contribute substantially to both early detection and disease freedom confidence. In contrast, active surveillance as a singular system is likely insufficient for early detection. The strongest systems combined active with observational surveillance and engaged joint industry and government involvement: results suggest that effective partnerships can generate highly sensitive systems, whereas ineffective partnerships may seriously erode early detection capability. Given the costs of routine testing, and the value (via averted losses) of early detection, we conclude that observational surveillance is an important and potentially very effective tool for health management and disease prevention on oyster farms, but one that demands careful planning and participation. This evaluation centered on OsHV-1 detection in farmed oyster populations. However, many of the features likely generalize to other pathogens and settings, with the important caveat that the pathogens need to manifest via morbidity or mortality events in the species, life stages and environments under observation.

Assessing the value of PCR assays in oral fluid samples for detecting African swine fever, classical swine fever, and foot-and-mouth disease in U.S. swine.

INTRODUCTION: Oral fluid sampling and testing offers a convenient, unobtrusive mechanism for evaluating the health status of swine, especially grower and finisher swine. This assessment evaluates the potential testing of oral fluid samples with real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) to detect African swine fever, classical swine fever, or foot-and-mouth disease for surveillance during a disease outbreak and early detection in a disease-free setting. METHODS: We used a series of logical arguments, informed assumptions, and a range of parameter values from literature and industry practices to examine the cost and value of information provided by oral fluid sampling and rRT-PCR testing for the swine foreign animal disease surveillance objectives outlined above. RESULTS: Based on the evaluation, oral fluid testing demonstrated value for both settings evaluated. The greatest value was in an outbreak scenario, where using oral fluids would minimize disruption of animal and farm activities, reduce sample sizes by 23%-40%, and decrease resource requirements relative to current individual animal sampling plans. For an early detection system, sampling every 3 days met the designed prevalence detection threshold with 0.95 probability, but was quite costly. LIMITATIONS: Implementation of oral fluid testing for African swine fever, classical swine fever, or foot-and-mouth disease surveillance is not yet possible due to several limitations and information gaps. The gaps include validation of PCR diagnostic protocols and kits for African swine fever, classical swine fever, or foot-and-mouth disease on swine oral fluid samples; minimal information on test performance in a field setting; detection windows with low virulence strains of some foreign animal disease viruses; and the need for confirmatory testing protocol development.

Mortality and Egg Production Patterns in the United States Prior to HP/LPAI H7N9 Detection.

In March 2017, two commercial broiler breeder operations were confirmed with H7N9 highly pathogenic avian influenza (HPAI), and an additional six commercial broiler breeder operations were found positive with an H7N9 low pathogenicity avian influenza virus (LPAIV) or an H7 LPAIV (N type not identified). To better understand conditions leading up to testing positive for AI, egg production and mortality data for the 6 mo before the outbreak were obtained from five case farms (two HPAIV-infected farms and three LPAIV-infected farms) and two control farms. Both HPAI farms experienced a sudden spike in mortality immediately before testing positive. Two LPAI farms experienced drops in egg production along with slight increases in mortality that occurred after a negative serologic test and before a positive PCR test. The third LPAI farm also had a notable drop in egg production with a coinciding increase in mortality before testing positive for AIV (last negative test date not available). Additionally, both HPAI farms and two LPAI farms reported mild respiratory illnesses in the weeks prior to testing positive for AI. Control farms did not experience similar drops in production or increase in mortality. Clinical signs on LPAI farms were mild and easily confused with background health patterns, suggesting the need for improved sensitivity to identify LPAI quickly. Applying a trigger of a 2% drop in egg production along with a mortality of 8 per 10 000 hens in individual barns showed that all case farms would be identified and uninfected farms would be falsely triggered on 1% of days monitored.

Mortalidad y patrones de producción de huevos en los Estados Unidos antes de la detección del virus de la influenza aviar H7N9 de baja y alta patogenicidad. En marzo del 2017, se confirmaron dos operaciones de reproductores pesados comerciales con influenza aviar altamente patógena H7N9 (HPAI) y adicionalmente se encontraron seis operaciones de reproductores pesados comerciales con un virus de influenza aviar de baja patogenicidad H7N9 (LPAIV) o con un virus de baja patogenicidad H7 con un subtipo de neuraminidasa no identificado. Para comprender mejor las condiciones que condujeron a un resultado positivo de influenza aviar, se recolectaron los datos de producción de huevo y mortalidad de cinco granjas que presentaron casos (dos granjas infectadas con el virus de alta patogenicidad y tres granjas infectadas con el virus de baja patogenicidad) y dos granjas control durante los seis meses anteriores al brote. Las dos grajas infectadas con el virus de alta patogenicidad experimentaron un aumento repentino en la mortalidad inmediatamente antes de que se determinaran como positivas. Dos granjas con el virus de baja patogenicidad experimentaron caídas en la producción de huevo junto con un ligero aumento en la mortalidad que se produjo después de que mostraran respuesta negativa a las pruebas serológicas y antes de resultar positivas mediante pruebas de RT-PCR. La tercera granja infectada con el virus de baja patogenicidad también tuvo una caída importante en la producción de huevo con un aumento coincidente en la mortalidad antes de que mostraran un resultado positivo para influenza aviar (la fecha de la última prueba negativa no se encontró disponible). Además, tanto las dos granjas con influenza aviar de alta patogenicidad como dos granjas con influenza aviar de baja patogenicidad reportaron enfermedades respiratorias leves en las semanas previas a la fecha cuando mostraron resultados positivos para la influenza aviar. Las granjas control no experimentaron caídas similares en la producción de huevo o aumento en la mortalidad. Los signos clínicos en las granjas de baja patogenicidad fueron leves y se confundieron fácilmente con los patrones de salud normales de la parvada, lo que sugiere la necesidad de mejorar la sensibilidad para identificar rápidamente la a la influenza aviar de baja patogenicidad. Con la aplicación de una alarma que se desencadene con una caída del 2% en la producción de huevo junto con una mortalidad de ocho por cada 10 mil gallinas en casetas individuales mostró que todas las granjas problema serían identificadas y que las granjas no infectadas podrían ser activadas falsamente en el 1% de los días monitoreados.

Is there an economic incentive for farmer participation in a uniform health standard for aquaculture farms? An empirical case study.

The growth of aquaculture, both in terms of the volume of production and the diversity of species and production systems, has created challenges for effective animal health policies. This paper presents results of a case study of the costs to a sector of U.S. aquaculture in which producers raising fish that are sold and shipped live contend with widely differing requirements for testing and certification of aquatic animal health. These are compared to related costs under a proposed uniform standard. The uniform standard scenario assumes adoption by the majority of the industry of a non-regulatory surveillance and biosecurity program with veterinary oversight, as an alternative to the current complex regulatory environment based on administrative political districts rather than on risk of disease transmission. Farm-level cost data were obtained through a survey that captured 74% of the national volume of baitfish and sportfish production in the U.S. Reflecting recent joint industry/federal efforts to develop a non-regulatory national U.S. program to set and verify a uniform standard for aquatic animal health, seven scenarios were modelled to determine the potential benefits and costs of such a program. Results showed that the net benefit of a uniform standard, if adopted nationally, could result in an estimated annual savings of $6.6 million to the U.S. baitfish and sportfish industry, and an average savings of $81,175/farm (with a range of $17,851/farm to $265,968/farm). Such cost savings provide an incentive for producers to support the program. Moreover, development of a uniform standard has potential to move aquatic animal health policies from the current framework of political administrative units to one based on epidemiological approaches and sound science.

Denman Island disease in Washington State, USA: distribution and prevalence in Pacific and Olympia oysters.

We sampled over 2400 wild, feral, and cultured Pacific oysters Crassostrea gigas and Olympia oysters Ostrea lurida in Washington State, USA, from 2002 to 2006 to estimate the prevalence of infection with Mikrocytos mackini, the causative agent of Denman Island disease. Both histology and qualitative PCR methods were used. Estimates of true prevalence of M. mackini infection in C. gigas, after accounting for imperfect test sensitivity, ranged from mean values of 0 to 10.0% by histology and 0 to 8.4% based on pooled PCR samples. M. mackini was not detected in any of the O. lurida samples. Results suggest a lower prevalence of the pathogen and severity of this oyster disease in Washington than that indicated in previous reports from British Columbia, Canada, potentially attributable to higher seawater temperatures in the Washington sample locations.

Establishing a definition of polar bear (Ursus maritimus) health: a guide to research and management activities.

The meaning of health for wildlife and perspectives on how to assess and measure health, are not well characterized. For wildlife at risk, such as some polar bear (Ursus maritimus) subpopulations, establishing comprehensive monitoring programs that include health status is an emerging need. Environmental changes, especially loss of sea ice habitat, have raised concern about polar bear health. Effective and consistent monitoring of polar bear health requires an unambiguous definition of health. We used the Delphi method of soliciting and interpreting expert knowledge to propose a working definition of polar bear health and to identify current concerns regarding health, challenges in measuring health, and important metrics for monitoring health. The expert opinion elicited through the exercise agreed that polar bear health is defined by characteristics and knowledge at the individual, population, and ecosystem level. The most important threats identified were in decreasing order: climate change, increased nutritional stress, chronic physiological stress, harvest management, increased exposure to contaminants, increased frequency of human interaction, diseases and parasites, and increased exposure to competitors. Fifteen metrics were identified to monitor polar bear health. Of these, indicators of body condition, disease and parasite exposure, contaminant exposure, and reproductive success were ranked as most important. We suggest that a cumulative effects approach to research and monitoring will improve the ability to assess the biological, ecological, and social determinants of polar bear health and provide measurable objectives for conservation goals and priorities and to evaluate progress.

Analysis of the diagnostic accuracy of the gamma interferon assay for detection of bovine tuberculosis in U.S. herds.

The goal of this study was to evaluate the test sensitivity (SE) and specificity (SP) of the gamma interferon (G-IFN) assay used for the detection of bovine tuberculosis (bTB) in U.S. cattle herds. In addition, the study assessed the association between G-IFN test results and bTB status of cattle, and explored different cut off values for classification of test results in adult cattle using receiver operating characteristics (ROC) curve analysis. Test SE was estimated using a population of 87 confirmed infected cattle from 14 herds distributed in 6 states. Test SP was estimated using a population of 4123 cattle representing 3000 premises in 3 states. These animals were from bTB free areas, accredited bTB free herds, or herds that were historically bTB free based on the absence of lesions found at slaughter and historical records of negative tests performed for bTB surveillance. The distribution of G-IFN results and its association with bTB infection status was also explored in a group of 914 exposed cattle in which infection was not confirmed. The results showed that the SE of the G-IFN for a cut-off value ≥0.1 was 83.9% (76.1, 91.6). The SP of the G-IFN was 90.7% (95% CI: 89.8, 91.6), 97% (95% CI: 96.5, 97.5), and 98.6%(95% CI: 98.2, 98.9), for cut off values of 0.1, 0.3, and 0.5, respectively. For a cut off value ≥0.1, the likelihood ratio of a positive G-IFN test was 9.03 (95% CI: 7.90, 10.31), and the likelihood ratio of a negative G-IFN test was 0.18 (95% CI: 0.11, 0.29). The area under the ROC curve was 0.976 (95% CI: 0.97, 0.98), characteristic of a highly accurate test. ROC analysis also showed that lower cut-off values, such as 0.1, have high SE with suitable SP for use in parallel testing, while cut-off values ranging between 0.3 and 0.6 provide the high SP desired in series-testing protocols with lower SE values. Findings from this study indicated that the G-IFN performs with high accuracy in the field, yielding SE and SP estimates comparable to those reported in previous evaluations (Ryan et al., 2000; Ameni et al., 2000; de la Rua-Domenech et al., 2006; Gormley et al., 2006).

Temporal and spatial variability in stable isotope compositions of a freshwater mussel: implications for biomonitoring and ecological studies.

Stable isotopes can be used to elucidate ecological relationships in community and trophic studies. Findings are calibrated against baselines, e.g. from a producer or primary consumer, assumed to act as a reference to the isotopic context created by spatio-temporal attributes such as geography, climate, nutrient, and energy sources. The ability of an organism to accurately represent a community base depends on how, and over what time-scale, it assimilates ambient materials. Freshwater mussels have served as references for trophic studies of freshwater communities and as indicators of change in nutrient pollution load or source. Their suitability as reference animals has not yet been fully explored, however. We conducted a series of studies examining the suitability of freshwater mussels as isotopic baselines, using their ability to reflect variation in ambient nutrient loads as a case scenario. (1) We analyzed bivalve foot tissue delta(15)N and delta(13)C from 22 stream reaches in the Piedmont region of North Carolina, USA to show that compositions varied substantially among locations. Site mean bivalve delta(13)C values correlated with site ambient particulate organic matter (POM) delta(13)C values, and site mean bivalve delta(15)N values correlated with site ambient water dissolved delta(15)N-NO(3) values. (2) Similarity of results among sample types demonstrated that the minimally invasive hemolymph sample is a suitable substitute for foot tissue in delta(15)N analyses, and that small sample sizes generate means representative of a larger population. Both findings can help minimize the impact of sampling on imperiled freshwater mussel populations. (3) In a bivalve transplantation study we showed that hemolymph delta(15)N compositions responded to a shift in ambient dissolved delta(15)N-NO(3), although slowly. The tissue turnover time for bivalve hemolymph was 113 days. We conclude that bivalves serve best as biomonitors of chronic, rather than acute, fluctuations in stream nutrient loads, and provide initial evidence of their suitability as time-integrated isotopic baselines for community studies.

Evaluation of a nonlethal technique for hemolymph collection in Elliptio complanata, a freshwater bivalve (Mollusca: Unionidae).

Hemolymph, the circulatory fluid of bivalves, transports nutrients, respiratory gases, enzymes, metabolic wastes, and toxicants throughout the body. Hemolymph can provide information pertinent to health assessment of animals or populations, but is not commonly used in freshwater bivalves partly because of the lack of tested, practical techniques for its nonlethal collection. The objective of this study was to evaluate the effect of hemolymph collection on the growth and survival of Elliptio complanata, a freshwater bivalve (Unionidae). We describe a simple technique for the collection of hemolymph from the anterior adductor muscle sinus of E. complanata. To evaluate the effect of hemolymph sampling on mussel survival and growth, 30 mussels sampled using the technique and 30 unsampled controls were followed for 3 mo post collection. Nine animals were sampled 3 times over 7 mo to monitor effects of repeated sampling. No negative impacts on survival or growth were observed in either the singly or repeatedly sampled animals. We also compared the composition of hemolymph collected from the adductor muscle sinus with that collected from the ventricle of the heart. Calcium levels and cell count of hemolymph obtained from the adductor sinus and ventricle were significantly different. There was no significant difference between collection sites for magnesium, phosphorus, ammonia, protein, sodium, potassium, or chloride. We conclude that collection of hemolymph from the adductor sinus is safe for sampled E. complanata and should be explored as a relatively non-invasive, and potentially useful, approach to the evaluation of freshwater mussel health.

Reference ranges for hemolymph chemistries from Elliptio complanata of North Carolina.

Hemolymph chemistries may be useful nonlethal measures of bivalve health. The prognostic value of hemolymph, however, depends on a comparison of chemistry results to reference ranges from healthy individuals. Currently, knowledge of expected hemolymph values in healthy and unhealthy freshwater mussels is extremely limited. The purpose of this study was to develop a set of reference ranges for clinical evaluation of hemolymph from a freshwater mussel species common to southeastern USA. We collected hemolymph from 380 Elliptio complanata from 19 apparently healthy populations from northwest of Raleigh, North Carolina, during May through July 2001. We present reference ranges for hemolymph parameters ammonia, glucose, calcium, magnesium, phosphorus, aspartate aminotransferase (AST), bicarbonate, protein and cell count, and for tissue glycogen. We compare the subpopulations of mussels from regions with an agricultural riparian buffer to those surrounded predominantly by forested lands. We further present correlations noted between hemolymph chemistries and physical or physiologic parameters. The only statistically significant differences between populations contiguous to agricultural and forested lands were in hemolymph calcium and glucose concentrations. Other statistically significant correlations identified were between gravidity and hemolymph protein concentration and tissue glycogen content, as well as between gravidity and parasite burden, and between shell length and hemolymph glucose, AST, calcium and bicarbonate concentrations. The results of this study will aid the interpretation of health measures from populations of E. complanata of similar geographic and seasonal origin.

Glycolytic buffering affects cardiac bioenergetic signaling and contractile reserve similar to creatine kinase.

Creatine kinase (CK) and glycolysis represent important energy-buffering processes in the cardiac myocyte. Although the role of compartmentalized CK in energy transfer has been investigated intensely, similar duties for intracellular glycolysis have not been demonstrated. By measuring the response time of mitochondrial oxygen consumption to dynamic workload jumps (tmito) in isolated rabbit hearts, we studied the effect of inhibiting energetic systems (CK and/or glycolysis) on transcytosolic signal transduction that couples cytosolic ATP hydrolysis to activation of oxidative phosphorylation. Tyrode-perfused hearts were exposed to 15 min of the following: 1) 0.4 mM iodoacetamide (IA; n = 6) to block CK (CK activity <3% vs. control), 2) 0.3 mM iodoacetic acid (IAA; n = 5) to inhibit glycolysis (GAPDH activity <3% vs. control), or 3) vehicle (control, n = 7) at 37 degrees C. Pretreatment tmito was similar across groups at 4.3 +/- 0.3 s (means +/- SE). No change in tmito was observed in control hearts; however, in IAA- and IA-treated hearts, tmito decreased by 15 +/- 3% and 40 +/- 5%, respectively (P < 0.05 vs. control), indicating quicker energy supply-demand signaling in the absence of ADP/ATP buffering by CK or glycolysis. The faster response times in IAA and IA groups were independent of the size of the workload jump, and the increase in myocardial oxygen consumption during workload steps was unaffected by CK or glycolysis blockade. Contractile function was compromised by IAA and IA treatment versus control, with contractile reserve (defined as increase in rate-pressure product during a standard heart rate jump) reduced to 80 +/- 8% and 80 +/- 10% of baseline, respectively (P < 0.05 vs. control), and significant elevations in end-diastolic pressure, suggesting raised ADP concentration. These results demonstrate that buffering of phosphate metabolites by glycolysis in the cytosol contributes appreciably to slower mitochondrial activation and may enhance contractile efficiency during increased cardiac workloads. Glycolysis may therefore play a role similar to CK in heart muscle.

Inhibition of autophagic proteolysis by inhibitors of phosphoinositide 3-kinase can interfere with the regulation of glycogen synthesis in isolated hepatocytes.

Amino acid-induced cell swelling stimulates conversion of glucose into glycogen in isolated hepatocytes. Activation of glycogen synthase (GS) phosphatase, caused by the fall in intracellular chloride accompanying regulatory volume decrease, and activation of phosphoinositide 3-kinase (PI 3-kinase), induced by cell swelling, have been proposed as underlying mechanisms. Because PI 3-kinase controls autophagic proteolysis, we examined the possibility that PI 3-kinase inhibitors interfere with glycogen production due to their anti-proteolytic action. The PI 3-kinase inhibitor wortmannin inhibited endogenous proteolysis, the production of glycogen from glucose and the activity of active (dephosphorylated) GS (GS a ) in the absence of added amino acids. The stimulation by amino acids of glycogen production and of GS a was only slightly affected by wortmannin. These effects of wortmannin could be mimicked by proteinase inhibitors. A combination of leucine, phenylalanine and tyrosine, which we showed previously to stimulate PI 3-kinase-dependent phosphorylation of ribosomal protein S6, did not stimulate glycogen production from glucose. In contrast with wortmannin, LY294002, another PI 3-kinase inhibitor, strongly inhibited both glycogen synthesis and GS a activity, irrespective of the presence of amino acids. Inhibition of glycogen synthesis by LY294002 could be ascribed in part to increased glycogenolysis and glycolysis. It is concluded that, in hepatocytes, activation of PI 3-kinase may not be responsible for the stimulation of glycogen synthesis by amino acids; LY294002 inhibits glycogen synthesis and stimulates glycogen breakdown by a mechanism that is unrelated to its action as an inhibitor of PI 3-kinase.

Clofibrate improves glucose tolerance in fat-fed rats but decreases hepatic glucose consumption capacity.

BACKGROUND/AIMS: High-fat (HF) diets cause glucose intolerance. Fibrates improve glucose tolerance. We have tried to obtain information on possible hepatic mechanisms contributing to this effect. METHODS: Rats were fed a HF diet, isocaloric with the control diet, for 3 weeks without or with clofibrate. Several parameters related to liver glucose and glycogen metabolism were measured. RESULTS: Clofibrate prevented the induction of glucose intolerance by 3 weeks HF feeding. Improved glucose tolerance by clofibrate was not due to increases in glucose phosphorylation or glycolysis in the liver, since both the HF diet and clofibrate suppressed glucokinase and pyruvate kinase activities with no effect on glucose 6-phosphatase. Clofibrate decreased glycogen storage in both control and HF rats. Clofibrate, with and without HF feeding, inhibited weight gain during the experimental period. Body temperature was significantly elevated by clofibrate, indicative of an increased basal metabolic rate. The capacity of liver mitochondria to oxidize long-chain fatty acids increased by clofibrate treatment. Mitochondria did not show uncoupling. CONCLUSIONS: Clofibrate does not improve glucose tolerance by improving hepatic glucose or glycogen metabolism. Peripheral glucose oxidation may be facilitated by increased energy dissipation.

Activation time of myocardial oxidative phosphorylation in creatine kinase and adenylate kinase knockout mice.

Our goal was to determine whether mice genetically altered to lack either creatine kinase (M/MtCK(-/-)) or adenylate kinase (AK(-/-)) show altered properties in the dynamic regulation of myocardial oxygen consumption (MVO(2)). We measured contractile function, oxygen consumption, and the mean response time of oxygen consumption to a step increase in heart rate [i.e., mitochondrial response time (t(mito))] in isolated Langendorff-perfused hearts from wild-type (n = 6), M/MtCK(-/-) (n = 6), and AK(-/-) (n = 4) mice. Left ventricular developed pressure was higher in M/MtCK(-/-) hearts (88.2 +/- 6.8 mmHg) and lower in AK(-/-) hearts (46.7 +/- 9.4 mmHg) compared with wild-type hearts (60.7 +/- 10.1 mmHg) at the basal pacing rate. Developed pressure fell slightly when heart rate was increased in all three groups. Basal MVO(2) at 300 beats/min was 19.1 +/- 2.4, 19.4 +/- 1.5, and 16.3 +/- 1.9 micromol x min(-1) x g dry wt(-1) for M/MtCK(-/-), AK(-/-), and wild type, respectively, which increased to 25.5 +/- 3.7, 25.4 +/- 2.6, and 22.0 +/- 2.6 micromol. min(-1) x g(-1), when heart rate was increased to 400 beats/min. The t(mito) was significantly faster in M/MtCK(-/-) hearts: 3.0 +/- 0.3 versus 7.3 +/- 0.6 and 8.0 +/- 0.4 s for M/MtCK(-/-), AK(-/-), and wild-type hearts, respectively. Our results demonstrate that MVO(2) of M/MtCK(-/-) hearts adapts more quickly to an increase in heart rate and thereby support the hypothesis that creatine kinase acts as an energy buffer in the cytosol, which delays the energy-related signal between sites of ATP hydrolysis and mitochondria.