3H2O Production Research Articles

The Role of PPARd in Alloreactive CD8 T cell Metabolism

Abstract Graft-versus-host disease (GVHD) is the most common cause of non-relapse mortality following allogeneic hematopoietic stem cell transplantation. Using a pre-clinical model of GVHD, previous work has found that CD8 T cells recovered on day 7 post-transplant upregulate the transcription factor, peroxisome proliferator activated receptor delta (PPARd). While PPARd has been shown to be a key regulator of fatty acid oxidation (FAO) in other tissues, it’s role in T cells is not well studied. We hypothesize that PPARd drives FAO in alloreactive CD8 T cells and that CD8 T cells lacking PPARd will be unable to oxidize fat and thus unable to cause GVHD. To investigate this hypothesis, ex vivo FAO was measured in day 7 WT versus PPARd KO CD8 T cells by quantitating conversion of 3H-palmitate to 3H2O. Production of 3H2O decreased by 50% in KO CD8 T cells, suggesting an inability to fully oxidize fat. To understand the mechanism underlying this decrease, we performed RNA sequencing and identified thirty genes that were differentially expressed in WT versus KO CD8s including 8 genes related to FAO. Strikingly, despite a decreased ability to oxidize fat, PPARd KO CD8 T cells were recovered in equal numbers on day 7 post transplantation, suggesting that CD8 T cells lacking PPARd adopt alternative metabolic pathways to generate sufficient energy for in vivo proliferation and activation. Ultimately, our analyses indicate that PPARd is required for FAO in alloreactive CD8 T cells, but this process is dispensable for short term proliferation and/or survival of CD8 T cells during GVHD. Future directions will identify compensatory metabolic pathways utilized by PPARd KO T cells through a combination of in vitro nutrient drop out experiments and metabolomic flux analyses.

Extracellular glucose supports lactate production but not aerobic metabolism in cardiomyocytes from both normoglycemic Atlantic cod and low glycemic short-horned sculpin.

Fish exhibit a wide range of species-specific blood glucose levels. How this relates to glucose utilization is yet to be fully realized. Here, we assessed glucose transport and metabolism in myocytes isolated from Atlantic cod (Gadus morhua) and short-horned sculpin (Myoxocephalus scorpius), species with blood glucose levels of 3.7 and 0.57 mmol l(-1), respectively. Glucose metabolism was assessed by the production of (3)H2O from [2-(3)H]glucose. Glucose metabolism was 3.5- to 6-fold higher by myocytes from Atlantic cod than by those from short-horned sculpin at the same level of extracellular glucose. In Atlantic cod myocytes, glucose metabolism displayed what appears to be a saturable component with respect to extracellular glucose, and cytochalasin B inhibited glucose metabolism. These features revealed a facilitated glucose diffusion mechanism that accounts for between 30% and 55% of glucose entry at physiological levels of extracellular glucose. Facilitated glucose diffusion appears to be minimal in myocytes for short-horned sculpin. Glucose entry by simple diffusion occurs in both cell types with the same linear relationship between glucose metabolism and extracellular glucose concentration, presumably due to similarities in membrane composition. Oxygen consumption by myocytes incubated in medium containing physiological levels of extracellular glucose (Atlantic cod 5 mmol l(-1), short-horned sculpin 0.5 mmol l(-1)) was similar in the two species and was not decreased by cytochalasin B, suggesting that these cells have the capability of oxidizing alternative on-board metabolic fuels. Cells produced lactate at low rates but glycogen levels did not change during the incubation period. In cells from both species, glucose utilization assessed by both simple chemical analysis of glucose disappearance from the medium and (3)H2O production was half the rate of lactate production and as such extracellular glucose was not available for oxidative metabolism. Overall, extracellular glucose makes only a minor contribution to ATP production but a sustained glycolysis may be necessary to support Ca(2+) transport mechanisms at either the sarcoplasmic reticulum or the sarcolemmal membrane.

High rates of glucose utilization in the gas gland of Atlantic cod (Gadus morhua) are supported by GLUT1 and HK1b.

The gas gland of physoclistous fish utilizes glucose to generate lactic acid that leads to the off-loading of oxygen from haemoglobin. This study addresses characteristics of the first two steps in glucose utilization in the gas gland of Atlantic cod (Gadus morhua). Glucose metabolism by isolated gas gland cells was 12- and 170-fold higher, respectively, than that in heart and red blood cells (RBCs) as determined by the production of (3)H2O from [2-(3)H]glucose. In the gas gland, essentially all of the glucose consumed was converted to lactate. Glucose uptake in the gas gland shows a very high dependence upon facilitated transport as evidenced by saturation of uptake of 2-deoxyglucose at a low extracellular concentration and a requirement for high levels of cytochalasin B for uptake inhibition despite the high efficacy of this treatment in heart and RBCs. Glucose transport is via glucose transporter1 (GLUT1), which is localized to the glandular cells. GLUT1 western blot analysis from whole-tissue lysates displayed a band with a relative molecular mass of 52 kDa, consistent with the deduced amino acid sequence. Levels of 52 kDa GLUT1 in the gas gland were 2.3- and 33-fold higher, respectively, than those in heart and RBCs, respectively. Glucose phosphorylation is catalysed by hexokinaseIb (HKIb), a paralogue that cannot bind to the outer mitochondrial membrane. Transcript levels of HKIb in the gas gland were 52- and 57-fold more abundant, respectively, than those in heart and RBCs. It appears that high levels of GLUT1 protein and an unusual isoform of HKI are both critical for the high rates of glycolysis in gas gland cells.

Regulation of Fatty Acid Oxidation in Mouse Cumulus-Oocyte Complexes during Maturation and Modulation by PPAR Agonists

Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of 3H2O from [3H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR) agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further decreased fatty acid oxidation during in vitro maturation and resulted in poor embryo development points to the developmental importance of fatty acid oxidation and the need for it to be optimized during in vitro maturation to improve this reproductive technology.

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

The aim of the present study was to assess whether mechanisms of glucose trafficking by red blood cells (RBCs) relate to species-specific extracellular glucose levels. Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar), cunner (Tautogolabrus adspersus) and short-horned sculpin (Myoxocephalus scorpius) had plasma glucose levels of 4, 4.1, 1.95 and 0.73 mmol l(-1), respectively. Glucose uptake by isolated RBCs was measured by the initial incorporation of [6-(14)C]-glucose and steady-state glucose metabolism was determined by the production of (3)H(2)O from [2-(3)H]-glucose. Saturation kinetics of glucose uptake and inhibition of both glucose uptake and metabolism by cytochalasin B and phloretin revealed that Atlantic cod, cunner and sculpin RBCs all had a facilitated transport component to glucose trafficking. RBCs from Atlantic salmon showed a linear relationship between glucose uptake and extracellular glucose level, but exhibited clear inhibition of glucose metabolism by cytochalasin B and phloretin, suggesting a component of facilitated glucose transport that is more elusive to detect. The production of (3)H(2)O was linear for at least 6 h and as such presents a rigorous approach to measuring glycolytic rate. Steady-state rates of glucose metabolism were achieved at extracellular levels of approximately 1 mmol l(-1) glucose for RBCs from all species, showing that within-species normal extracellular glucose level is not a primary determinant of the basal level of glycolysis. At physiological levels of extracellular glucose, the ratio of initial glucose uptake to glucose metabolism was 1.5 to 4 for all RBCs, suggesting that there is scope to increase metabolic rate without alteration of the basal glucose uptake capacity.

Western diet changes cardiac acyl-CoA composition in obese rats: a potential role for hepatic lipogenesis

The "lipotoxic footprint" of cardiac maladaptation in diet-induced obesity is poorly defined. We investigated how manipulation of dietary lipid and carbohydrate influenced potential lipotoxic species in the failing heart. In Wistar rats, contractile dysfunction develops at 48 weeks on a high-fat/high-carbohydrate "Western" diet, but not on low-fat/high-carbohydrate or high-fat diets. Cardiac content of the lipotoxic candidates--diacylglycerol, ceramide, lipid peroxide, and long-chain acyl-CoA species--was measured at different time points by high-performance liquid chromatography and biochemical assays, as was lipogenic capacity in the heart and liver by qRT-PCR and radiometric assays. Changes in membranes fluidity were also monitored using fluorescence polarization. We report that Western feeding induced a 40% decrease in myocardial palmitoleoyl-CoA content and a similar decrease in the unsaturated-to-saturated fatty acid ratio. These changes were associated with impaired cardiac mitochondrial membrane fluidity. At the same time, hepatic lipogenic capacity was increased in animals fed Western diet (+270% fatty acid elongase activity compared with high-fat diet), while fatty acid desaturase activity decreased over time. Our findings suggest that dysregulation of lipogenesis is a significant component of heart failure in diet-induced obesity.

Fenofibrate (FEN), a peroxisome proliferator activated receptor alpha (PPAR α) agonist, decreases dietary fat absorption and alters triglyceride (TG) metabolism in enterocytes of mice

FEN activates PPARα and lowers blood TG concentrations. The mechanism is complex and involves decreasing hepatic secretion of TG via increasing fatty acid oxidation (FAO) and increasing clearance of blood TG via lipolysis. We hypothesized that FEN may also lower blood TG conc. via altering dietary fat absorption and intestinal TG metabolism. We fed mice a high‐fat diet for 2 wks and then orally administered FEN (800 mg/kg bw/d) or vehicle (VEH) for 5 d and investigated TG metabolism in the small intestine. FEN effectively lowered fasting blood TG conc. compared to VEH (21±1 vs 27±2 mg/dL, P=0.02, n=7). Dietary fat absorption was lower in FEN compared to VEH mice as evidenced by fecal fat percent (10.7±0.6 vs 2.3±0.2% fat, P=0.003, n=4–5). Intestinal FAO was determined ex vivo by measuring the production of 3H2O from 3H‐oleate. FAO in FEN was 1.5 fold higher compared to VEH mice (P=0.03, n=11–12). TG storage was imaged by coherent anti‐Stokes Raman scattering microscopy. Very little to no TG storage was found in enterocytes of FEN compared to abundant TG storage in VEH mice. We determined TG secretion from enterocytes in chow fed mice orally administered FEN or VEH. We measured blood TG conc. before, 1, 2, and 3h post oil oral gavage. The AUC was interestingly, greater from FEN than VEH mice (P=0.045, n=7–10). These results confirm that FEN decreases dietary fat absorption and alters TG metabolism in enterocytes of mice.Grant Funding Source: American Heart Association

Islet adaptive changes to fructose-induced insulin resistance: β-cell mass, glucokinase, glucose metabolism, and insulin secretion

Beta-cell mass, hexokinase/glucokinase (HK/GK) activity, glucose metabolism and insulin secretion were studied in the islets of rats with fructose-induced insulin resistance (IR). Normal male Wistar rats were fed a standard commercial diet and water without (control, C) or with 10% fructose-rich diet (FRD) for 3 weeks. Blood glucose (strips), triglyceride (commercial kit), and insulin (RIA) levels were measured at the time of death. Glucose-induced insulin release, glucose metabolism ((14)CO(2) and (3)H(2)O production from D-[U-(14)C]- and D-[5-(3)H]-glucose) and HK/GK activity (G-6-P production), transcription (RT-PCR), protein expression (Western blot), and cellular compartmentalization were measured in isolated islets (collagenase digestion). FRD rats presented normoglycemia but impaired glucose tolerance, hypertriglyceridemia, hyperinsulinemia, and increased HOMA-IR index. In these rats, beta-cell mass decreased significantly by 33%, with a 44% increase in the percentage of apoptotic cells. Glucose-induced insulin release and islet glucose metabolism were higher in FRD rats. While GK activity (total and cytosolic fraction) and protein expression were significantly higher in FRD islets, HK showed no change in any of these parameters. Our results demonstrate that the changes induced by dietary-induced IR upon beta-cell function and mass are strongly conditional on the nutrient model used. In our model (intact animals with impaired glucose tolerance), GK activity increases through mechanisms previously shown only in vitro or under highly hyperglycemic conditions. Such an increase plays a pivotal role in the adaptive increased release of insulin in response to IR, even in the presence of marked beta-cell mass reduction.

Decreased islet sensitivity to insulin in hamsters with dietary-induced insulin resistance

In the present study, we evaluated the autocrine modulatory effect of insulin on glucose metabolism and glucose-induced insulin secretion in islets isolated from hamsters with insulin resistance (IR) induced by administration of a sucrose-rich diet (SRD) during 5 weeks. We used an approach of two metabolic pathways (glucose oxidation and utilization) based on the measurement of 14CO 2 and 3H 2O production from d-[U- 14C]-glucose and d-[5- 3H]-glucose, respectively, in isolated islets incubated with 3.3 and 16.7 mM glucose alone, or with 5 or 15 mU/ml insulin, anti-insulin guinea-pig serum (1:500), 25 μM nifedipine, or 150 nM wortmannin. Insulin release was measured by radioimmunoassay in islets incubated with 3.3 or 16.7 mM glucose, with or without 75, 150, and 300 nM wortmannin. Results showed that the stimulatory effect of insulin upon 14CO 2 and 3H 2O production in control islets was not observed in SRD islets. Addition of anti-insulin serum, nifedipine or wortmannin to the medium with 16.7 mM glucose decreased 14CO 2 and 3H 2O production in control but not in SRD islets. Whereas wortmannin did not decrease insulin release induced by 16.7mM glucose in SRD hamsters, it did in controls. We can conclude that the autocrine stimulatory effect of insulin upon glucose metabolism observed in normal islets is attenuated or even absent in islets from IR animals. Such decreased islet sensitivity to insulin did not prevent the compensatory secretion of insulin from maintaining glucose homeostasis, suggesting that, at least in this model, the islets can put forward alternative mechanisms to overcome such defect.

Meal Fatty Acid Uptake in Visceral Fat in Women

Differential meal fat uptake into adipose tissue depots may be a determinant of body fat distribution. We used the meal fat tracer/adipose tissue biopsy approach to compare the effects of meal fat content on the fat uptake into visceral and upper and lower body subcutaneous fat depots in 21 premenopausal women. [(3)H]triolein was used to trace the fate of fatty acids from a normal-fat or high-fat meal. The proportion of dietary fat uptake into the three depots did not differ between meals; visceral fat accounted for only approximately 5% of meal fat disposal irrespective of visceral fat mass. For the women consuming the normal-fat meal, the uptake of meal fatty acid into femoral fat (milligrams meal fat per gram lipid) increased as a function of leg fat mass (r = 0.68, P < 0.05), which we interpret as increased efficiency of uptake. The opposite pattern was seen in omental fat with the normal-fat meal and in all depots after the high-fat meal. For both meals, approximately 40% of meal fat was oxidized ((3)H(2)O production) after 24 h. We conclude that greater thigh adipose tissue in women is associated with greater efficiency of meal fat storage under conditions of energy balance, whereas the opposite is seen with visceral fat. These findings imply that different mechanisms may regulate fatty acid uptake in different depots, which may in turn impact on body fat distribution.

Metabolic Diapause in Pancreatic β-Cells Expressing a Gain-of-function Mutant of the Forkhead Protein Foxo1

Diabetes is associated with decreased pancreatic beta-cell function and mass. It is unclear whether diabetes treatment should aim at restoring beta-cell performance/mass or at inducing "beta-cell rest" to prevent further deterioration. The transcription factor Foxo1 protects beta-cells against oxidative stress induced by hyperglycemia and prevents beta-cell replication in insulin-resistant states. Here we show that these combined effects are associated with a concerted repression of genes involved in glycolysis, nitric-oxide synthesis, G protein-coupled receptor signaling, and ion transport. Conversely, Foxo1 increases expression of several neurotransmitter receptors and fails to regulate target genes predicted from Caenorhabditis elegans and Drosophila studies. Functional analyses show decreased glucose utilization and insulin secretion in beta-cells overexpressing Foxo1. We propose the definition of "metabolic diapause" for the changes induced by Foxo1 to protect beta-cells against oxidative stress. The data provide genetic underpinning for the concept of beta-cell rest as a treatment goal in diabetes.

Identification of Mouse Prp19p as a Lipid Droplet-associated Protein and Its Possible Involvement in the Biogenesis of Lipid Droplets

Prp19p is an integral component of the heteromeric protein complex (the NineTeen complex) in the nucleus, and it is essential for the structural integrity of NineTeen complex and its subsequent activation of the spliceosome. We identified Prp19p, which has never been reported in relation to any function outside of the nucleus, as a member of proteins associated with lipid droplets. Down-regulation of Prp19p expression with RNA interference in 3T3-L1 cells repressed lipid droplet formation with the reduction in the level of expression of perilipin and S3-12. The levels of expression of SCD1 (stearoyl-CoA desaturase-1), DGAT-1 (acyl-CoA diacylglycerol acyltransferase-1), and glycerol-3-phosphate acyltransferase were also reduced in Prp19p down-regulated cells, and a significant decrease in triglycerides was observed. Unlike perilipin, which is one of the most extensively studied lipid droplet-associated proteins, Prp19p is not essential for cAMP- and hormone-sensitive lipase-dependent lipolysis pathways, even though Prp19p is a component of the lipid droplet phospholipid monolayer, and down-regulation of Prp19p represses fat accretion significantly. These results suggest that Prp19p or Prp19-interacting proteins during lipid droplet biogenesis in adipocytes may be considered as another class of potential targets for attacking obesity and obesity-related problems.

Establishing a Reference Interval For Measurement of Flux through the Mitochondrial Fatty Acid Oxidation Pathway in Cultured Skin Fibroblasts

Mitochondrial fatty acid oxidation (FAO) represents a normal metabolic response to increased energy demands during periods of fasting, febrile illness, or muscular exertion. FAO is a complex pathway involving activation of free fatty acids to acyl-CoA species, transport into mitochondria, and cyclic oxidation to break the long-chain fatty acids into acetyl-CoA and a two-carbon chain-shortened acyl-CoA, which is then recycled. Acetyl-CoA is used in the Krebs cycle and in liver as a substrate for ketogenesis (1). Measurement of FAO flux in cultured cells provides valuable information about the integrity of the enzymes and transporters involved in FAO at all stages. Clinical indications for the measurement of FAO flux in skin fibroblasts include fatty acid oxidation enzyme defects, unexplained myopathies, cardiomyopathies, hypoglycemia, liver disease, sudden unexplained infant death, and hypoketotic hypoglycemia. FAO flux is typically measured by monitoring the conversion of radiolabeled fatty acids to carbon dioxide and water. Labeling can be with 14C with final analysis of the rate of production of 14CO2, or with 3H and analysis of production of 3H2O (2)(3). Palmitic acid (C16) is used to detect long-chain defects, including deficiencies in very long chain acyl-CoA dehydrogenase, long-chain l-3-hydroxy acyl-CoA dehydrogenase, long-chain 3-keto acyl-CoA thiolase, carnitine/acylcarnitine translocase, carnitine pamitoyltransferase 1, and carnitine pamitoyltransferase 2. Myristic acid (C14) is used to detect defects in medium-chain FAO, such as medium-chain acyl-CoA dehydrogenase deficiency. We use tritiated fatty acids labeled in the (9,10) position. The assay will not detect defects in carnitine palmitoyl transferase 1B; short-chain FAO defects, including short-chain acyl-CoA dehydrogenase and short-chain l-3-hydroxy acyl-CoA dehydrogenase; and short-chain-3-keto acyl-CoA thiolase deficiencies. Historically, patient samples have been compared with batch controls, and a reference interval was not established for this assay. In the present study we have assigned …

N-[3H]Benzoylglycylglycylglycine as a probe for hydroxyl radicals

N-[4-3H]Benzoylglycylglycylglycine ([3H]BzG3) was tested as a probe for detecting hydroxyl radicals (OH). Aerated solutions of l-ascorbate generated OH, which oxidized [3H]BzG3, yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. The 3H2O was separated from organic products and remaining [3H]BzG3 on Dowex-1. 3H2O production was much greater with OH than with other reactive oxygen species (ROS) (e.g., H2O2, superoxide). The slight 3H2O production in the presence of H2O2 or superoxide was blocked by OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H2O2 or superoxide. This indicates that 3H2O production was caused by OH and that other ROS only generated any 3H2O by forming traces of OH. Doses of OH that caused detectable nonenzymic polysaccharide scission also caused 3H2O production, indicating that [3H]BzG3 is a sensitive OH probe in studies of polymer scission. The ability of scavengers and chelators to protect against ascorbate-mediated polysaccharide scission paralleled their ability to inhibit concurrent 3H2O production, indicating that both processes were due to OH. Thus, [3H]BzG3 is a simple, specific, sensitive, and robust probe for detecting OH production in vitro. It may have applications for in vivo detection of extracellular OH in arthritic joints and of apoplastic OH in plant cell walls.

Beta-Hydroxybutyrate inhibits myocardial fatty acid oxidation in vivo independent of changes in malonyl-CoA content.

This study tested the hypothesis that an acute infusion of beta-hydroxybutyrate inhibits myocardial fatty acid uptake and oxidation in vivo. Anesthetized pigs were untreated (n = 6) or treated with an intravenous infusion of fat emulsion (n = 7) to elevate plasma free fatty acid levels. A third group received fat emulsion plus an intravenous infusion of beta-hydroxybutyrate (25 micromol.kg-1.min-1; n = 7) for 60 min. All animals received a continuous infusion of [3H]palmitate, and myocardial fatty acid oxidation was measured from the cardiac production of 3H2O. Plasma free fatty acid concentrations were elevated in the fat emulsion group (0.77 +/- 0.11 mM) compared with the untreated group (0.15 +/- 0.03 mM), which resulted in greater myocardial free fatty acid oxidation. In contrast, the group receiving beta-hydroxybutyrate in addition to fat emulsion had elevated beta-hydroxybutyrate concentration (0.87 +/- 0.11 vs. 0.04 +/- 0.01 mM), but suppressed fatty acid oxidation (0.053 +/- 0.013 micromol.g-1.min-1) (P < 0.05) compared with the fat emulsion group (0.116 +/- 0.029 micromol.g-1.min-1). There were no differences among the three groups in the tissue content for malonyl-CoA, acetyl-CoA, or free CoA or the activity of acetyl-CoA carboxylase; thus the inhibition of fatty acid oxidation by elevated beta-hydroxybutyrate did not appear to be due to malonyl-CoA inhibition of carnitine palmitoyl transferase-I or to an increase in the acetyl-CoA-to-free CoA ratio. In conclusion, fatty acid uptake and oxidation is blocked by an infusion of beta-hydroxybutyrate; this effect was not due to elevated myocardial malonyl-CoA content.

The role of calcium in the regulation of glucose uptake in isolated working rat heart.

Catecholamines or ischemia may increase myocardial glucose uptake by an increase in intracellular calcium. We tested the hypothesis that increasing or decreasing extracellular calcium supply would change glucose uptake. Hearts were perfused for 60 min at a physiological workload with Krebs-Henseleit buffer containing glucose (5 mM) and oleate (0.4 mM; bound to 1% BSA). Calcium concentration was 2.5 mM. In group A (control; n = 12), insulin (1 mU/ml) was added at 30 min. In Group B (n = 7), the calcium concentration was increased to 5.0 and 7.5 mM at 20 min and 40 min, respectively. In Group C (n = 7), verapamil was added at 20 min (0.25 microM) and 40 min (1.0 microM) to decrease calcium influx. In group D (n = 7), EDTA was added at 20 min (0.5 mM) and at 40 min (1.5 mM) to decrease the free extracellular calcium. Glucose uptake was measured by 3H2O production from [2-3H]glucose and cardiac work was measured simultaneously. Cardiac power in group B was 8.24 +/- 0.60 mW at 2.5 mM calcium, 9.45 +/- 0.50 mW at 5 mM calcium and 7.99 +/- 0.99 mW at 7.5 mM calcium (n.s.). The addition of verapamil decreased contractile function in a dose-dependent manner (8.50 +/- 0.74 vs. 3.11 +/- 0.84 vs. 1.48 +/- 0.39 mW, p < 0.01) suggesting that verapamil decreased cytosolic calcium concentration. A similar dose-dependent reduction in contractile performance was observed in the EDTA group (8.44 +/- 0.81 vs. 7.42 +/- 0.96 vs. 4.03 +/- 1.32 mW, p < 0.01). Glucose uptake was 1.35 +/- 0.11 micromol/min/g dry weight under control conditions. Glucose uptake increased threefold with the addition of insulin. Increasing extracellular [Ca2+] did not affect glucose uptake. Decreasing Ca2+ availability showed a trend towards a decrease in glucose uptake (n.s.), which was minor compared to the decrease in contractile function. We conclude that extracellular calcium does not regulate glucose uptake in the isolated working rat heart in the presence of glucose and fatty acids as substrates. The trend of decreased glucose uptake when calcium supply was limited may be due to dramatically reduced energy demand and not directly due to changes in calcium.

Differential effect of valproate and its Δ 2- and Δ 4-unsaturated metabolites, on the β-oxidation rate of long-chain and medium-chain fatty acids

Overall fatty acid oxidation rates were investigated in rat hepatocytes using [9,10- 3H]-palmitic, [9,10- 3H]-oleic, [9,10- 3H]-myristic and [2,3- 3H]-phenylpropionic acids. The effect of both valproate (VPA) (0–10 mM) and two of its unsaturated metabolites, Δ 2(E)-VPA and Δ 4-VPA (0–10 mM), on the overall 3H 2O production rate was studied. The results give evidence of a general inhibitory effect of VPA on the β-oxidation rate of all the tested substrates. Similar effects were observed with both VPA metabolites but these effects appeared to be dependent on the chain length of the substrate. When the effect on the oxidation of the medium-chain fatty acid 3-phenylpropionate (PPA) was studied, Δ 2(E)-VPA at 0.5 mM caused a 94% inhibition of the overall β-oxidation rate. However, with long-chain substrates, 0.5 mM Δ 4-VPA was a more potent inhibitor (20–30% of control activity) than 0.5 mM Δ 2(E)-VPA (60–80% of control activity). Our results suggest that VPA and/or its metabolites inhibit fatty acyl-CoA metabolism within the mitochondrion by two different mechanisms. The first mechanism involves CoASH sequestration, which affects the oxidation rate of all fatty acids with different chain length. The second mechanism is more specific in nature and involves selective inhibition of particular enzymes implicated in fatty acid β-oxidation.

Chylomicron metabolism by the isolated perfused mouse heart.

The catabolism of rat chylomicrons, labeled in their triacylglycerol (TG) component, was investigated using perfused working mouse hearts. Perfusion of mouse hearts with heparin increased lipoprotein lipase (LPL) activity in the perfusate. This heparin-releasable LPL pool remained constant over a variety of experimental conditions, including workload and fatty acid concentrations, making the mouse heart a suitable model to study chylomicron catabolism. Endothelium-bound LPL hydrolyzed radiolabeled (3)H-labeled chylomicrons (0.4 mM TG); the fate of LPL-derived (3)H-labeled fatty acids was split evenly between oxidation (production of (3)H(2)O) and esterification (incorporation into tissue lipids, mainly TG). In comparison, the oxidation of 0.4 mM [(3)H]palmitate complexed to albumin was fourfold greater than esterification into tissue lipids. Surprisingly, the addition of unlabeled palmitate (0.4 or 1.2 mM) to perfusions with (3)H-chylomicrons did not affect the fate (either oxidation or esterification) of LPL-derived (3)H-fatty acids. These results suggest that fatty acids produced from lipoprotein hydrolysis by the action of LPL and fatty acids from a fatty acid-albumin complex do not enter a common metabolic pool in the heart.

Kinetics of intramuscular triglyceride fatty acids in exercising humans.

A pulse ([(14)C]palmitate)-chase ([(3)H]palmitate) approach was used to study intramuscular triglyceride (imTG) fatty acid and plasma free fatty acid (FFA) kinetics during exercise at approximately 45% peak O(2) consumption in 12 adults. Vastus lateralis muscle was biopsied before and after 90 min of bicycle exercise; (3)H(2)O production, breath (14)CO(2) excretion and lipid oxidation (indirect calorimetry) rates were measured during exercise. during exercise, 8.2+/-1.2 and 8.4+/-0.7 micromol x kg(-1) x min(-1) of imTG fatty acids and plasma FFA, respectively, were oxidized according to isotopic measurements. The sum of these two values was not different (P = 0.6) from lipid oxidation by indirect calorimetry (15.4 +/-1.6 micromol x kg(-1) x min(-1)); the isotopic and indirect calorimetry values were correlated (r = 0.79, P<0.005). During exercise, imTG turnover rate was 0.32+/-0.07%/min (6.0+/-2.0 micromol of imTG x kg wet muscle(-1) x min(-1)) and plasma FFA were incorporated into imTG at a rate of 0.7+/-0.1 micromol x kg wet muscle(-1) x min(-1). The imTG pool size did not change during exercise. This pulse-chase, dual tracer appears to be a reasonable approach to measure oxidation and synthesis kinetics of imTG.

Alteration of glycogen and glucose metabolism in ischaemic and post-ischaemic working rat hearts by adenosine A1 receptor stimulation.

1. Cardioprotection by adenosine A1 receptor activation limits infarct size and improves post-ischaemic mechanical function. The mechanisms responsible are unclear but may involve alterations in myocardial glucose metabolism. 2. Since glycogen is an important source of glucose during ischaemia, we examined the effects of N6-cyclohexyladenosine (CHA), an A1 receptor agonist, on glycogen and glucose metabolism during ischaemia as well as reperfusion. 3. Isolated working rat hearts were perfused with Krebs-Henseleit solution containing dual-labelled 5-3H and 14C glucose and palmitate as energy substrates. Rates of glycolysis and glucose oxidation were measured directly from the production of 3H2O and 14CO2. Glycogen turnover was measured from the rate of change of [5-3H and 14C]glucosyl units in total myocardial glycogen. 4. Following low-flow (0.5 ml min-1) ischaemia (60 min) and reperfusion (30 min), left ventricular minute work (LV work) recovered to 22% of pre-ischaemic values. CHA (0.5 microM) improved the recovery of LV work 2 fold. 5. CHA altered glycogen turnover in post-ischaemic hearts by stimulating glycogen synthesis while having no effects on glycogen degradation. CHA also partially inhibited glycolysis. These changes accelerated the recovery of glycogen in CHA-treated hearts and reduced proton production. 6. During ischaemia, CHA had no measurable effect on glycogen turnover or glucose metabolism. Glycogen phosphorylase activity, which was elevated after ischaemia, was inhibited by CHA, possibly in response to CHA-induced inhibition of AMP-activated protein kinase activity. 7. These results indicate that CHA-induced cardioprotection is associated with alterations of glycogen turnover during reperfusion as well as improved metabolic coupling of glycolysis to glucose oxidation.