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

Abstract

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.

Extracellular glucose supports lactate production but not aerobic metabolism in cardiomyocytes from both normoglycemic Atlantic cod (Gadus morhua) and low glycemic short-horned sculpin (Myoxocephalus scorpius)

“…Cytochalasin B significantly inhibited glucose metabolism in Atlantic cod myocytes at physiological levels of glucose concentration for this species. This finding is similar to that obtained with the inhibition of 2-deoxyglucose uptake in non-contracting ventricle strips from eel, Atlantic cod and rainbow trout under conditions of aerobic metabolism (Rodnick et al, 1997; Clow et al, 2004;Becker et al, 2013). Inhibition of glucose metabolism by cytochalasin B in short-horned sculpin myocytes was observed only under conditions of 10 mmol l −1 extracellular glucose, a level 20-fold higher than physiological concentrations.…”

“…At time 0, 30, 60, 120, 180 and 240 min, a 100 µl aliquot was taken and frozen in liquid nitrogen for later analysis of glucose and lactate. Glucose was measured as previously described by Clow et al (2004) and lactate as described above. Glucose disappearance and lactate appearance were determined from the linear regression of concentration versus time.…”

“…Hydrolysis was stopped after 2 h by adding 12% perchloric acid. Glucose from these samples was measured as described by Clow et al (2004). Free glucose from these samples without hydrolysis was also measured and subtracted from the glucose produced from glycogen.…”

“…Facilitated glucose diffusion is important in fish hearts as well, where the demand for ATP is much higher. Under aerobic conditions, the uptake of 2-deoxyglucose is inhibited 50-80% by cytochalasin B or phloridzin in ventricle strips from American eel (Anguilla rostrata) (Rodnick et al, 1997), Atlantic cod (Clow et al, 2004) and rainbow trout (Onchorynchus mykiss) (Becker et al, 2013). None of these species have exceptionally low levels of blood glucose.…”

Glucose uptake and metabolism by RBCs from fish with different extracellular glucose levels

“…Plasma was assayed as described by Clow et al (Clow et al, 2004) with the exception that absorbance was determined with a DTX 880 microplate reader (Beckman Coulter, Mississauga, ON, Canada). In glucose concentration experiments, RBCs were sampled immediately after the mixing of cells with incubation medium (considered to be time zero) and thereafter following 10min of incubation in the case of Atlantic salmon, and initially 30min of incubation for all other species.…”