Metabolic fuel kinetics in fish: swimming, hypoxia and muscle membranes.

Muscle performance depends on the supply of metabolic fuels and disposal of end-products. Using circulating metabolite concentrations to infer changes in fluxes is highly unreliable because the relationship between these parameters varies greatly with physiological state. Quantifying fuel kinetics directly is therefore crucial to the understanding of muscle metabolism. This review focuses on how carbohydrates, lipids and amino acids are provided to fish muscles during hypoxia and swimming. Both stresses force white muscle to produce lactate at higher rates than it can be processed by aerobic tissues. However, lactate accumulation is minimized because disposal is also strongly stimulated. Exogenous supply shows that trout have a much higher capacity to metabolize lactate than observed during hypoxia or intense swimming. The low density of monocarboxylate transporters and their lack of upregulation with exercise explain the phenomenon of white muscle lactate retention. This tissue operates as a quasi-closed system, where glycogen stores act as an 'energy spring' that alternates between explosive power release during swimming and slow recoil from lactate in situ during recovery. To cope with exogenous glucose, trout can completely suppress hepatic production and boost glucose disposal. Without these responses, glycemia would increase four times faster and reach dangerous levels. The capacity of salmonids for glucoregulation is therefore much better than presently described in the literature. Instead of albumin-bound fatty acids, fish use lipoproteins to shuttle energy from adipose tissue to working muscles during prolonged exercise. Proteins may play an important role in fueling muscle work in fish, but their exact contribution is yet to be established. The membrane pacemaker theory of metabolism accurately predicts general properties of muscle membranes such as unsaturation, but it does not explain allometric patterns of specific fatty acids. Investigations of metabolic fuel kinetics carried out in fish to date have demonstrated that these ectotherms use several unique strategies to orchestrate energy supply to working muscles and to survive hypoxia.

Exogenous lactate supply affects lactate kinetics of rainbow trout, not swimming performance.

Intense swimming causes circulatory lactate accumulation in rainbow trout because lactate disposal (Rd) is not stimulated as strongly as lactate appearance (Ra). This mismatch suggests that maximal Rd is limited by tissue capacity to metabolize lactate. This study uses exogenous lactate to investigate what constrains maximal Rd and minimal Ra. Our goals were to determine how exogenous lactate affects: 1) Ra and Rd of lactate under baseline conditions or during graded swimming, and 2) exercise performance (critical swimming speed, Ucrit) and energetics (cost of transport, COT). Results show that exogenous lactate allows swimming trout to boost maximal Rd lactate by 40% and reach impressive rates of 56 µmol·kg(-1)·min(-1). This shows that the metabolic capacity of tissues for lactate disposal is not responsible for setting the highest Rd normally observed after intense swimming. Baseline endogenous Ra (resting in normoxic water) is not significantly reduced by exogenous lactate supply. Therefore, trout have an obligatory need to produce lactate, either as a fuel for oxidative tissues and/or from organs relying on glycolysis. Exogenous lactate does not affect Ucrit or COT, probably because it acts as a substitute for glucose and lipids rather than extra fuel. We conclude that the observed 40% increase in Rd lactate is made possible by accelerating lactate entry into oxidative tissues via monocarboxylate transporters (MCTs). This observation together with the weak expression of MCTs and the phenomenon of white muscle lactate retention show that lactate metabolism of rainbow trout is significantly constrained by transmembrane transport.

Exhausting exercise and tissue-specific expression of monocarboxylate transporters in rainbow trout.

Transmembrane lactate movements are mediated by monocarboxylate transporters (MCTs), but these proteins have never been characterized in rainbow trout. Our goals were to clone potential trout MCTs, determine tissue distribution, and quantify the effects of exhausting exercise on MCT expression. Such information could prove important to understand the mechanisms underlying the classic "lactate retention" seen in trout white muscle after intense exercise. Four isoforms were identified and partially characterized in rainbow trout: MCT1a, MCT1b, MCT2, and MCT4. MCT1b was the most abundant in heart and red muscle but poorly expressed in the gill and brain where MCT1a and MCT2 were prevalent. MCT expression was strongly stimulated by exhausting exercise in brain (MCT2: +260%) and heart (MCT1a: +90% and MCT1b: +50%), possibly to increase capacity for lactate uptake in these highly oxidative tissues. By contrast, the MCTs of gill, liver, and muscle remained unaffected by exercise. This study provides a possible functional explanation for postexercise "lactate retention" in trout white muscle. Rainbow trout may be unable to release large lactate loads rapidly during recovery because: 1) they only poorly express MCT4, the main lactate exporter found in mammalian glycolytic muscles; 2) the combined expression of all trout MCTs is much lower in white muscle than in any other tissue; and 3) exhausting exercise fails to upregulate white muscle MCT expression. In this tissue, carbohydrates act as an "energy spring" that alternates between explosive power release during intense swimming (glycogen to lactate) and recoil during protracted recovery (slow glycogen resynthesis from local lactate).

Lactate kinetics of rainbow trout during graded exercise: do catheters affect the cost of transport?

Changes in lactate kinetics as a function of exercise intensity have never been measured in an ectotherm. Continuous infusion of a tracer is necessary to quantify rates of lactate appearance (Ra) and disposal (Rd), but it requires double catheterization, which could interfere with swimming. Using rainbow trout, our goals were to: (1) determine the potential effects of catheters and blood sampling on metabolic rate (O2), total cost of transport (TCOT), net cost of transport (NCOT) and critical swimming speed (Ucrit), and (2) monitor changes in lactate fluxes during prolonged, steady-state swimming or graded swimming from rest to Ucrit. This athletic species maintains high baseline lactate fluxes of 24 µmol kg(-1) min(-1) that are only increased at intensities >2.4 body lengths (BL) s(-1) or 85% Ucrit. As the fish reaches Ucrit, Ra is more strongly stimulated (+67% to 40.4 µmol kg(-1) min(-1)) than Rd (+41% to 34.7 µmol kg(-1) min(-1)), causing a fourfold increase in blood lactate concentration. Without this stimulation of Rd during intense swimming, lactate accumulation would double. By contrast, steady-state exercise at 1.7 BL s(-1) increases lactate fluxes to ~30 µmol kg(-1) min(-1), with a trivial mismatch between Ra and Rd that only affects blood concentration minimally. Results also show that the catheterizations and blood sampling needed to measure metabolite kinetics in exercising fish have no significant impact on O2 or TCOT. However, these experimental procedures affect locomotion energetics by increasing NCOT at high speeds and by decreasing Ucrit.

Hypoxia stimulates lactate disposal in rainbow trout.

Current understanding of lactate metabolism in fish is based almost entirely on the interpretation of concentration measurements that cannot be used to infer changes in flux. The goals of this investigation were: (1) to quantify baseline lactate fluxes in rainbow trout (Oncorhynchus mykiss) under normoxic conditions; (2) to establish how changes in rates of lactate appearance (R(a)) and disposal (R(d)) account for the increase in blood lactate elicited by hypoxia; and (3) to identify the tissues responsible for lactate production. R(a) and R(d) lactate of rainbow trout were measured in vivo by continuous infusion of [U-(14)C]lactate in trout exposed to 25% O(2) saturation or maintained in normoxia for 90 min. In normoxic fish, R(a) lactate decreased from 18.2 to 13.1 µmol kg(-1) min(-1) and R(d) lactate from 19.0 to 12.8. R(a) and R(d) were always matched, thereby maintaining a steady baseline blood lactate concentration of ∼0.8 mmol l(-1). By contrast, the hypoxic fish increased blood lactate to 8.9 mmol l(-1) and R(a) lactate from 18.4 to 36.5 µmol kg(-1) min(-1). This stimulation of anaerobic glycolysis was unexpectedly accompanied by a 52% increase in R(d) lactate from 19.9 to 30.3 µmol kg(-1) min(-1). White muscle was the main producer of lactate, which accumulated to 19.2 µmol g(-1) in this tissue. This first study of non-steady-state lactate kinetics in fish shows that the increase in lactate disposal elicited by hypoxia plays a strategic role in reducing the lactate load on the circulation. Without this crucial response, blood lactate accumulation would double.

Gemfibrozil disrupts the metabolism of circulating lipids in bobwhite quails.

The circulating lipids of birds play essential roles for egg production and as an energy source for flight and thermogenesis. How lipid-lowering pharmaceuticals geared to prevent heart disease in humans and that are routinely released in the environment affect their metabolism is unknown. This study assesses the impact of the popular drug gemfibrozil (GEM) on the plasma phospholipids (PL), neutral lipids (NL), and nonesterified fatty acids (NEFA) of bobwhite quails (Colinus virginianus). Results show that bird lipoproteins are rapidly altered by GEM, even at environmentally-relevant doses. After 4 days of exposure, pharmacological amounts cause an 83% increase in circulating PL levels, a major decrease in average lipoprotein size measured as a 56% drop in the NL/PL ratio, and important changes in the fatty acid composition of PL and NEFA (increases in fatty acid unsaturation). The levels of PL carrying all individual fatty acids except arachidonate are strongly stimulated. The large decrease in bird lipoprotein size may reflect the effects seen in humans: lowering of LDL that can cause atherosclerosis and stimulation of HDL that promote cholesterol disposal. Lower (environmental) doses of GEM cause a reduction of %palmitate in all the plasma lipid fractions of quails, but particularly in the core triacylglycerol of lipoproteins (NL). No changes in mRNA levels of bird peroxisome proliferator-activated receptor (PPAR) could be demonstrated. The disrupting effects of GEM on circulating lipids reported here suggest that the pervasive presence of this drug in the environment could jeopardize reproduction and migratory behaviours in wild birds.

Immunoglobulin mimicry by Hepatitis C Virus envelope protein E2.

Hepatitis C virus (HCV) establishes persistent infection in the majority of infected individuals. The currently accepted hypothesis of immune evasion by antigenic variation in hypervariable region 1 (HVR1) of glycoprotein E2 does not however, explain the lack of subsequent immune recognition. Here, we show that the N-terminal region of E2 is antigenically and structurally similar to human immunoglobulin (Ig) variable domains. E2 is recognized by anti-human IgG antibodies and also possesses common amino acid (aa) sequence features of the conserved v-gene framework regions of human Ig light chains in particular but also heavy chains and T cell receptors. Using a position specific scoring system, the degree of similarity of HVR1 to Ig types correlated with immune escape and persistence in humans and experimentally infected chimpanzees. We propose a unique role for threshold levels of Ig molecular mimicry in HCV biology that not only advances our concept of viral immune escape and persistent infection but also provides insight into host-dependent disease patterns.