Cellular adaptations in fat tissue of exercise-trained miniature swine: role of excess energy intake.

This study examined the influence of energy expenditure and energy intake on cellular mechanisms regulating adipose tissue metabolism. Twenty-four swine were assigned to restricted-fed sedentary, restricted-fed exercise-trained, full-fed sedentary, or full-fed exercise-trained groups. After 3 mo of treatment, adipocytes were isolated and adipocyte size, adenosine A(1) receptor characteristics, and lipolytic sensitivity were measured. Swine were infused with epinephrine during which adipose tissue extracellular adenosine, plasma fatty acids, and plasma glycerol were measured. Results revealed that adipocytes isolated from restricted-fed exercised swine had a smaller diameter, a lower number of A(1) receptors, and a greater sensitivity to lipolytic stimulation, compared with adipocytes from full-fed exercised swine. Extracellular adenosine levels were transiently increased on infusion of epinephrine in adipose tissue of restricted-fed exercised but not full-fed exercised swine. These results suggest a role for adenosine in explaining the discrepancy between in vitro and in vivo lipolysis findings and underscore the notion that excess energy intake dampens the lipolytic sensitivity of adipocytes to beta-agonists and adenosine, even if accompanied by exercise training.     

Distribution of adenosine metabolising enzymes between adipocyte and stromal-vascular cells of adipose tissue

Rat adipose tissue was digested with collagenase and separated into adipocytes and stromal-vascular cells. The adipocytes accounted for 40% of the total adipose tissue adenosine deaminase activity, 32% of 5'-nucleotidase activity and 87% of adenosine kinase activity. This distribution suggests that adipocyte are the major cell type involved in adenosine utilization in adipose tissue. Furthermore, it suggests that the high sensitivity of isolated adipocytes to adenosine is representative of their sensitivity of isolated adipocytes to adenosine is representative of their sensitivity in vivo.     

Characterization of Cyclic AMP Efflux from Swine Adipocytes In Vitro

Objective : A variety of cell types transport cyclic AMP (cAMP) to the extracellular fluid; the purpose of this study was to determine if and how this process occurs in adipocytes. Research Methods and Procedures : Adipocytes were isolated from 3-month-old swine and incubated with stimulators of adenylate cyclase for 2 to 120 minutes to promote cAMP synthesis and efflux. Efflux was characterized in the presence of agents that inhibit ATP production, anion transport, intracellular cAMP metabolism, and extracellular cAMP metabolism. Extracellular cAMP was measured by enzyme immunoassay, then corrected for cell lysis by measuring lactate dehydrogenase release. Results : cAMP efflux averaged 24.7 fmol/min/cm² adipocyte surface area, was linear for 2 hours, and was proportional to adipocyte surface area (r = 0.94, p<0.05). Efflux was reduced by ∽35% in cells incubated with 1 4mUM antimycin, an inhibitor of ATP synthesis (p<0.05), and by ～55% in cells incubated with 2 mM probenecid, an anionpecific transport blocker (p<0.05). Extracellular cAMP levels more than doubled by the addition of 1 μM 1,3-dipropyl-8-p-sulfophenylxanthine, a purported inhibitor of extracellular phosphodiesterase. Discussion : Our data demonstrate that cAMP is transported from swine adipocytes by an energy-dependent anion transporter and can be metabolized extracellularly. Future studies will evaluate extracellular cAMP as a potential source of extracellular adenosine, a potent inhibitor of adipocyte lipolysis.     

Exercise training restores adenosine-induced relaxation in coronary arteries distal to chronic occlusion

We previously reported that canine collateral-dependent coronary arteries exhibit impaired relaxation to adenosine but not sodium nitroprusside. In contrast, exercise training enhances adenosine sensitivity of normal porcine coronary arteries. These results stimulated the hypothesis that chronic coronary occlusion and exercise training produce differential effects on cAMP- versus cGMP-mediated relaxation. To test this hypothesis, Ameroid occluders were surgically placed around the proximal left circumflex coronary artery (LCx) of female Yucatan miniature swine 8 wk before initiating sedentary or exercise training (treadmill run, 16 wk) protocols. Relaxation to the cAMP-dependent vasodilators adenosine (10(-7) to 10(-3) M) and isoproterenol (3 x 10(-8) to 3 x 10(-5) M) were impaired in collateral-dependent LCx versus nonoccluded left anterior descending (LAD) arterial rings isolated from sedentary but not exercise-trained pigs. Furthermore, adenosine-mediated reductions in simultaneous tension and myoplasmic free Ca(2+) were impaired in LCx versus LAD arteries isolated from sedentary but not exercise-trained pigs. In contrast, relaxation in response to the cAMP-dependent vasodilator forskolin (10(-9) to 10(-5) M) and the cGMP-dependent vasodilator sodium nitroprusside (10(-9) to 10(-4) M) was not different in LCx versus LAD arteries of sedentary or exercise-trained animals. These data suggest that chronic occlusion impairs receptor-dependent, cAMP-mediated relaxation; receptor-independent cAMP- and cGMP-mediated relaxation were unimpaired. Importantly, exercise training restores cAMP-mediated relaxation of collateral-dependent coronary arteries.     

Isomers of conjugated linoleic acid decrease plasma lipids and stimulate adipose tissue lipogenesis without changing adipose weight in post-prandial adult sedentary or trained Wistar rat

The respective effects and interactions of supplementation with two conjugated linoleic acid (CLA) isomers and exercise on plasma metabolic profile, activity of lipogenic enzymes and cellularity in two adipose tissue sites, those of the liver and heart, were examined in adult Wistar rats. Rats that were either sedentary or exercise-trained by treadmill running were fed one of four diets: a diet without CLA; a diet with either 1% cis 9, trans 11 CLA or 1% trans 10, cis 12 CLA; or a mixture of both isomers (1% of each) for 6 weeks. We observed that the exercise decreased lipogenic enzyme activities in epididymal and perirenal adipose tissue. Plasma cholesterol, insulin, and leptin concentrations were lower in exercise-trained rats than in sedentary rats. The ingestion of either CLA mixture or the trans 10, cis 12 CLA increased lipogenic enzyme activities in epididymal tissue and more markedly in perirenal adipose tissue, especially in sedentary rats, and without affecting adipose tissue weight or cellularity. A similar effect of trans 10, cis 12 CLA was observed in regard to malic enzyme activity in the liver. In addition, this isomer decreased plasma lipid and urea concentrations and increased plasma 3-hydroxybutyrate levels. The ingestion of cis 9, trans 11 CLA increased fatty acid synthase activity in perirenal adipose tissue in sedentary rats and decreased plasma cholesterol and leptin concentrations. These results show that isomers of CLA decrease plasma lipids and stimulate adipose tissue lipogenesis without changing adipose weight in adult sedentary or exercise-trained rat, thus suggesting a stimulation of adipose tissue turnover.     

Exercise training increases membrane bound form of tumor necrosis factor-alpha receptors with decreases in the secretion of soluble forms of receptors in rat adipocytes

We examined the effects of exercise training (treadmill running over 9 weeks) on the ability of isolated adipocytes to secrete tumor necrosis factor-alpha (TNF-alpha) and type 1 soluble TNF receptor (sTNFR1) in vitro in Wistar rats. We also examined the effects of exercise training on the expression of membrane bound forms of type 1 TNF receptor (mTNFR1) in adipocyte crude membranes of the same rat subjects. Exercise training significantly increased the secretions of TNF-alpha from isolated adipocytes. Treatment with a cyclooxygenase inhibitor, either indomethacin (100 microM) or eicosatetraynoic acid (100 microM), significantly blocked the release of TNF-alpha from adipocytes in both exercise-trained rat group and sedentary control rat group, suggesting that some cyclooxygenase metabolite(s) acts as a ligand in TNF-alpha synthesis. Decreased amounts of TNF-alpha were found to be significantly greater in both exercise-trained rat group than in sedentary control rat group after incubation with inhibitors. Thus, the inhibitory effect of both indomethacin and eicosatetraynoic acid was significantly greater in adipocytes from exercise-trained rats. Both plasma sTNFR1 levels and adipocytes-derived sTNFR1 were found to be significantly less in the exercise-trained rat group. Western blot analysis revealed that exercise training remarkably increased the expressions of mTNFR1 in adipocyte crude membrane. Thus, exercise training enhanced the ability of isolated adipocytes to secrete TNF-alpha with reduced secretion of sTNFR1, and provoked the greater expressions of mTNFR1 in adipocyte crude membrane. These alterations may induce enhanced the autocrine effects of TNF-alpha within adipocytes in exercise-trained rats.     

Isoproterenol increases active lipoprotein lipase in adipocyte medium and in rat plasma

White adipose tissue (WAT) lipoprotein lipase (LPL) activity channels diet fat towards storage in adipocytes. Adrenaline (ADR) is accepted to reduce WAT or adipocyte LPL activity (LPLa), but available data are not clear-cut regarding long exposure to ADR in vitro or in vivo. We studied the effects of long exposures to ADR or beta-adrenergic agonist on LPL: in isolated rat adipocytes (3 h) and in rats (>1 day). Isoproterenol (ISO) (1 microM) did not alter LPLmRNA nor LPLa in adipocytes, but increased LPLa in medium more than twofold (3.58 +/- 0.35 vs. 1.32 +/- 0.35 mU/10(6) adipocytes, P < 0.001). Effect was time (not present at 1 h, clear at 2 h) and concentration dependent (high sensitivity from 10 to 100 nM, max at 1 microM). Adenylate cyclase activator or cyclic AMP (cAMP) analogue produced a similar increase. Thus in adipocytes ISO produced an increase in LPLa release and/or a decrease in extracellular LPLa degradation. ADR or ISO treated rats had a two to fourfold decrease in WAT LPLa vs. unchanged LPLmRNA. This decrease was 10-fold in WAT heparin-releasable LPLa (5.7 +/- 0.6 vs. 57.3 +/- 10.2 mU/g, P < 0.001), which represents peri/extracellular LPLa. Plasma LPLa was increased 11-fold by ADR (3.30 +/- 0.58 vs. 0.32 +/- 0.08 mU/ml, P < 0.001) whereas only threefold by ISO (P > 0.01). We suggest that in vivo ADR increased release of active LPL to plasma from endothelial cells of LPL-rich tissue(s)-WAT was probably one of these tissues releasing LPL since it lost 90% of its peri/extracellular LPLa-and/or decreased degradation of plasma active LPL. Since liver LPLa was not increased, plasma active LPL might be kept away from hepatic degradation by binding to stabilising entities in plasma (fatty acids (FA), lipoproteins or soluble heparan sulphates (HS)). In conclusion, we believe this is the first report stating that: (a) ISO increases LPLa in isolated adipocyte medium, and (b) ADR administration to rats decreases WAT extracellular active LPL and increases preheparin plasma active LPL.     

Cyclic AMP metabolism by swine adipocyte microsomal and plasma membranes

Extracellular cyclic AMP is source of extracellular adenosine in brain and kidney. Whether this occurs in adipose tissue is unknown. The present study evaluated the capacity of swine adipocyte plasma membranes to metabolize cyclic AMP to AMP and adenosine, via phosphodiesterase (PDE) and 5'-nucleotidase (5'-NT), respectively. Plasma membranes (PM) and microsomal membranes (MM) were isolated from over-the-shoulder subcutaneous adipose tissue of 3 month-old male miniature swine. The purity of the membrane fractions was determined and PDE and 5'-NT activities in PM and MM fractions were corrected for cross-contamination. The maximal activity of MM-PDE was 7-fold greater than that of PM-PDE. MM-PDE was 100% inhibited by 5 microM cilostamide, while PM-PDE was unaffected by this PDE3B inhibitor. Inhibitors of PDE1, PDE2, PDE4 and PDE5 also failed to inhibit PM-PDE. However, 1 mM DPSPX inhibited PM-PDE activity by 72%. When PM were incubated with 0.8 microM cyclic AMP for 20 min, AMP accumulation was four times that of adenosine. These data demonstrate that cyclic AMP can be converted to AMP and adenosine by the PM-bound enzymes 5'-NT and PDE, and suggest that the PM-PDE responsible for extracellular cyclic AMP metabolism to AMP is distinct from the intracellular MM-PDE.     

Adenosine and the control of lipolysis in rat adipocytes during pregnancy and lactation.

The rate of noradrenaline-stimulated lipolysis is lower in fat-cells from lactating than from pregnant rats; this difference is eliminated by the addition of adenosine deaminase [Aitchison, Clegg & Vernon (1982) Biochem. J. 202, 243-247]. The activity of 5'-nucleotidase, and hence the capacity of the cells to synthesize adenosine, was the same in fat-cells and also stromal cells of adipose tissue from pregnant, lactating and male rats. The response and sensitivity of fat-cells to the anti-lipolytic effects of adenosine were measured by incubating cells in the presence of noradrenaline, adenosine deaminase (to remove endogenous adenosine) and various concentrations of the adenosine analogue N6-phenylisopropyladenosine (PIA). PIA caused a greater inhibition of the rate of noradrenaline-stimulated lipolysis in adipocytes from lactating than from pregnant rats. The concentration of PIA required to inhibit by 50% the rate of noradrenaline-stimulated lipolysis fell from over 100 nM for fat-cells from pregnant rats to 30 nM for fat-cells from lactating rats. The decreased rate of noradrenaline-stimulated lipolysis during lactation was not due to the smaller mean cell volume of adipocytes during this state.     

Extracellular Cyclic AMP—Adenosine Pathway in Isolated Adipocytes and Adipose Tissue

Objective: Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)–adenosine pathway in adipose tissue. Research Methods and Procedures: Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 μL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 μM isoproterenol, or 10 μM isoproterenol plus 1 mM α,β‐methylene adenosine 5′‐diphosphate (AMPCP), a 5′‐nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 μM isoproterenol, or 1 μM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Results: Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP‐provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. Discussion: These data suggest the existence of a cyclic AMP—adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.     

Endurance exercise training increases insulin responsiveness in isolated adipocytes through IRS/PI3-kinase/Akt pathway.

Endurance exercise training promotes important metabolic adaptations, and the adipose tissue is particularly affected. The aim of this study was to investigate how endurance exercise training modulates some aspects of insulin action in isolated adipocytes and in intact adipose tissue. Male Wistar rats were submitted to daily treadmill running (1 h/day) for 7 wk. Sedentary age-matched rats were used as controls. Final body weight, body weight gain, and epididymal fat pad weight did not show any statistical differences between groups. Adipocytes from trained rats were smaller than those from sedentary rats (205 +/- 16.8 vs. 286 +/- 26.4 pl; P < 0.05). Trained rats showed decreased plasma glucose (4.9 +/- 0.13 vs. 5.3 +/- 0.07 mM; P < 0.05) and insulin levels (0.24 +/- 0.012 vs. 0.41 +/- 0.049 mM; P < 0.05) and increased insulin-stimulated glucose uptake (23.1 +/- 3.1 vs. 12.1 +/- 2.9 pmol/cm(2); P < 0.05) compared with sedentary rats. The number of insulin receptors and the insulin-induced tyrosine phosphorylation of insulin receptor-beta subunit did not change between groups. Insulin-induced tyrosine phosphorylation insulin receptor substrates (IRS)-1 and -2 increased significantly (1.57- and 2.38-fold, respectively) in trained rats. Insulin-induced IRS-1/phosphatidylinositol 3 (PI3)-kinase (but not IRS-2/PI3-kinase) association and serine Akt phosphorylation also increased (2.06- and 3.15-fold, respectively) after training. The protein content of insulin receptor-beta subunit, IRS-1 and -2, did not differ between groups. Taken together, these data support the hypothesis that the increased adipocyte responsiveness to insulin observed after endurance exercise training is modulated by IRS/PI3-kinase/Akt pathway.     

Purine accumulation in human fat cell suspensions. Evidence that human adipocytes release inosine and hypoxanthine rather than adenosine

Human adipocytes are of limited viability (7 +/- 2% release of lactate dehydrogenase/h) and contain active ectophosphatases which are capable of sequentially degrading ATP to adenosine. At densities of 30,000-40,000 cells/ml, human fat cell suspensions accumulated adenosine, inosine, and hypoxanthine, and their concentrations were 38 +/- 8, 120 +/- 10, and 31 +/- 7 nmol/liter after 3 h of incubation. Dipyridamole (10 mumol/liter), an inhibitor of nucleoside transport, caused a 5-7-fold increase in adenosine accumulation which was reduced by 85% on inhibition of ectophosphatases by beta-glycerophosphate and antibodies against ecto-5'-nucleotidase or alpha, beta-methylene 5'-adenosine diphosphate (10 mumol/liter), respectively, indicating that most of the adenosine is produced in the extracellular compartment. Accordingly, the spontaneous accumulation of adenosine was reduced beyond 5 nmol/liter on inhibition of ectophosphatase activities or removal of extracellular AMP by AMP deaminase (4 units/ml). Added adenosine (30 nmol/liter) disappeared until its concentration approached 5 nmol/liter. Isoproterenol (1 mumol/liter) had no effect on adenosine accumulation regardless whether purine production from extracellular sources was minimized or not. In contrast to adenosine, the concentrations of inosine and hypoxanthine displayed only a modest decrease (30-50%) on inhibition of ectophosphatase activities. In addition, isoproterenol caused a 2-3-fold increase in inosine and hypoxanthine production which was concentration-dependent and could be inhibited by propranolol. It is concluded that the adenosine that accumulates in human adipocyte suspensions is almost exclusively derived from adenine nucleotides which are released by leaking cells. By contrast, inosine and hypoxanthine are produced inside the cells, and the release of these latter purines appears to be linked to ATP turnover via adenylate cyclase.     

ATP extracellular concentrations are increased in the rat striatum during in vivo ischemia

Interest is growing in the role of adenosine triphosphate (ATP) on P2 receptors during hypoxic/ischemic events in the brain. However, there is no direct evidence of an increase in extracellular ATP levels during cerebral ischemia in vivo. The aim of the present study was to evaluate ATP outflow from the rat striatum by the microdialysis technique associated with focal cerebral ischemia in vivo by intraluminal occlusion of the right middle cerebral artery (MCA). Between 1 and 4h after ischemia, rats showed a clear turning behavior contralateral to the ischemic side. Twenty-four hour after MCA occlusion, ischemic rats had definite neurological deficit and striatal and cortical damage. The ATP concentration (mean+/-S.E.M.) in the striatum of normoxic rats (n = 8) was 3.10+/-0.34 nM. During 220 min after MCA occlusion, the extracellular ATP levels significantly increased two-fold, being 5.90+/-0.61 nM (p < 0.01 versus normoxic level). ATP outflow showed a tendency to increase over time during the 220 min of ischemia. Since extracellular ATP is rapidly metabolized to adenosine, we also assessed ATP outflow in the presence of the ecto-5'-nucleotidase inhibitor, alpha,beta-methylene-adenosine diphosphate (AOPCP, 1 mM) directly perfused into the striatum. The ATP concentration in normoxic rats (n = 8) was increased three-fold in the presence of the ecto-5'-nucleotidase inhibitor (9.57+/-0.26 nM). During 220 min of ischemia, extracellular ATP levels significantly increased 1.3-fold in AOPCP-treated rats (12.62+/-0.65 nM, p < 0.01 versus normoxic level). The present study confirms that ATP is continuously released in the brain and demonstrates for the first time that ATP outflow increases during ischemia in vivo. These results confirm that ATP may be an important mediator in brain ischemia.     

Cyclic AMP efflux is regulated by occupancy of the adenosine receptor in pig aortic smooth muscle cells

Cultured pig aortic smooth muscle cells respond to extracellular adenosine by activating adenylate cyclase and by initiating the efflux of cAMP. In the presence of extracellular adenosine, efflux is first order with respect to intracellular cAMP concentration up to at least 125 pmol/10(6) cells. The apparent first-order rate constant for the efflux of cAMP increases in a dose-dependent manner in response to extracellular adenosine or 5-N-ethylcarboxamide adenosine. The EC50 for adenosine for promoting cAMP efflux is 12 microM. For cells stimulated with 5-N-ethylcarboxamide adenosine, the EC50 is 5 microM. When extracellular adenosine is removed, efflux stops abruptly. Cellular cAMP content falls but is still in a range that supports cAMP efflux when agonist is present. Efflux is not affected by H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP-dependent protein kinase. These data suggest that in pig aortic smooth muscle cells, the efficiency of cAMP efflux is regulated by A2 receptor occupancy.     

Exercise and the cAMP system in rat adipose tissue II. Nucleotide catabolism

In this study the effect of exercise training on the adenosine 3',5'-cyclic monophosphate (cAMP) system of rat adipose tissue has been investigated. The basal amount of cAMP for the exercising rats was 0.179 +/- 0.021 nmol/10(6) cells, the same value as for the controls. Phosphodiesterase activities (low and high Km) remained unaffected as a result of the program of treadmill running. Kinetic constants for the low- and high-Km phosphodiesterases revealed that the affinity of the enzymes for substrate (cAMP) was unaltered by physical training. Finally, ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, possibly through its effect on calmodulin, stimulated or inhibited (depending on concentration) phosphodiesterase activity in the same direction and to a similar extent in extracts of adipose tissue from runners and controls. Taken together, these data clearly demonstrate the exercise training has no effect on the cAMP system of adipose tissue in male rats.     

Regular exercise enhances insulin activation of IRS-1-associated PI3-kinase in human skeletal muscle.

Insulin action in skeletal muscle is enhanced by regular exercise. Whether insulin signaling in human skeletal muscle is affected by habitual exercise is not well understood. Phosphatidylinositol 3-kinase (PI3-kinase) activation is an important step in the insulin-signaling pathway and appears to regulate glucose metabolism via GLUT-4 translocation in skeletal muscle. To examine the effects of regular exercise on PI3-kinase activation, 2-h hyperinsulinemic (40 mU. m(-2). min(-1))-euglycemic (5.0 mM) clamps were performed on eight healthy exercise-trained [24 +/- 1 yr, 71.8 +/- 2.0 kg, maximal O(2) uptake (VO(2 max)) of 56.1 +/- 2.5 ml. kg(-1). min(-1)] and eight healthy sedentary men and women (24 +/- 1 yr, 64.7 +/- 4.4 kg, VO(2 max) of 44.4 +/- 2.7 ml. kg(-1). min(-1)). A [6, 6-(2)H]glucose tracer was used to measure hepatic glucose output. A muscle biopsy was obtained from the vastus lateralis muscle at basal and at 2 h of hyperinsulinemia to measure insulin receptor substrate-1(IRS-1)-associated PI3-kinase activation. Insulin concentrations during hyperinsulinemia were similar for both groups (293 +/- 22 and 311 +/- 22 pM for trained and sedentary, respectively). Insulin-mediated glucose disposal rates (GDR) were greater (P < 0.05) in the exercise-trained compared with the sedentary control group (9.22 +/- 0.95 vs. 6.36 +/- 0.57 mg. kg fat-free mass(-1). min(-1)). Insulin-stimulated PI3-kinase activation was also greater (P < 0.004) in the trained compared with the sedentary group (3.8 +/- 0.5- vs. 1.8 +/- 0.2-fold increase from basal). Endurance capacity (VO(2 max)) was positively correlated with PI3-kinase activation (r = 0.53, P < 0.04). There was no correlation between PI3-kinase and muscle morphology. However, increases in GDR were positively related to PI3-kinase activation (r = 0.60, P < 0.02). We conclude that regular exercise leads to greater insulin-stimulated IRS-1-associated PI3-kinase activation in human skeletal muscle, thus facilitating enhanced insulin-mediated glucose uptake.     

Hypoxia induces adenosine release from the rat carotid body

The effect of hypoxia on the release of adenosine was studied in vitro in the rat whole carotid body (CB) and compared with the effect of hypoxia (2%, 5% and 10% O(2)) on adenosine concentrations in superior cervical ganglia (SCG) and carotid arteries. Moderate hypoxia (10% O(2)) increased adenosine concentrations released from the CBs by 44%, but was not a strong enough stimulus to evoke adenosine release from SCG and arterial tissue. The extracellular pathways of adenosine production in rat CBs in normoxia and hypoxia were also investigated. S-(p-nitrobenzyl)-6-thioinosine (NBTI) and dipyridamole were used as pharmacological tools to inhibit adenosine equilibrative transporters (ENT) and alpha,beta-methylene ADP (AOPCP) to inhibit ecto-5'-nucleotidase. Approximately 40% of extracellular adenosine in the CB came from the extracellular catabolism of ATP, under both normoxic and hypoxic conditions. Low pO(2) triggers adenosine efflux through activation of NBTI-sensitive ENT. This effect was only apparent in hypoxia and when adenosine extracellular concentrations were reduced by the blockade of ecto-5'-nucleotidase. We concluded that CB chemoreceptor sensitivity could be related to its low threshold for the release of adenosine in response to hypoxia here quantified for the first time.     

Inhibition of lipolysis by palmitate, H2O2 and the sulfonylurea drug, glimepiride, in rat adipocytes depends on cAMP degradation by lipid droplets

The release of fatty acids and glycerol from lipid droplets (LD) of mammalian adipose cells is tightly regulated by a number of counterregulatory signals and negative feedback mechanisms. In humans unrestrained lipolysis contributes to the pathogenesis of obesity and type II diabetes. In order to identify novel targets for the pharmacological interference with lipolysis, the molecular mechanisms of four antilipolytic agents were compared in isolated rat adipocytes. Incubation of the adipocytes with insulin, palmitate, glucose oxidase (for the generation of H2O2) and the antidiabetic sulfonylurea drug, glimepiride, reduced adenylyl cyclase-dependent, but not dibutyryl-cAMP-induced lipolysis as well as the translocation of hormone-sensitive lipase and the LD-associated protein, perilipin-A, to and from LD, respectively. The antilipolytic activity of palmitate, H2O2 and glimepiride rather than that of insulin was dependent on rolipram-sensitive but cilostamide-insensitive phosphodiesterase (PDE) but was not associated with detectable downregulation of total cytosolic cAMP and insulin signaling via phosphatidylinositol-3 kinase and protein kinase B. LD from adipocytes treated with palmitate, H2O2 and glimepiride were capable of converting cAMP to adenosine in vitro, which was hardly observed with those from basal cells. Conversion of cAMP to adenosine was blocked by rolipram and the 5'-nucleotidase inhibitor, AMPCP. Immunoblotting analysis revealed a limited salt-sensitive association with LD of some of the PDE isoforms currently known to be expressed in rat adipocytes. In contrast, the cAMP-to-adenosine converting activity was stripped off the LD by bacterial phosphatidylinositol-specific phospholipase C. These findings emphasize the importance of the compartmentalization of cAMP signaling for the regulation of lipolysis in adipocytes, in general, and of the involvement of LD-associated proteins for cAMP degradation, in particular.     

Effects of chronic swimming training on cardiac sarcolemmal function and composition

Cardiac contractile function is dependent on the integrity and function of the sarcolemmal membrane. Swimming exercise training is known to increase cardiac contractile performance. The purpose of the present study was to examine whether a swimming exercise program would alter sarcolemmal enzyme activity, ion flux, and composition in rat hearts. After approximately 11 wk of exercise training, cardiac myosin and actomyosin Ca2+-adenosinetriphosphatase (ATPase) activity was significantly higher in exercised rat hearts than in sedentary control rat hearts. Glycogen content was increased in plantaris and gastrocnemius muscles from exercised animals as was succinic dehydrogenase activity in gastrocnemius muscle of exercised rats in comparison to sedentary rat preparations. Sarcolemmal vesicles were isolated from hearts of exercise-trained and control rats. Sarcolemmal Na+-K+-ATPase and K+-p-nitrophenylphosphatase activities, Na+-Ca2+ exchange, and passive Ca2+ binding did not differ between the two groups. ATP-dependent Ca2+ uptake and 5'-nucleotidase activity were elevated in the cardiac sarcolemmal vesicles isolated from exercised animals compared with sedentary control rats. Sarcolemmal phospholipid composition was not altered by the exercise training. Our results demonstrate that swimming training in rats does not affect most parameters of cardiac sarcolemmal function or composition. However, the elevated sarcolemmal Ca2+ pump activity in exercised rats may help to reduce intracellular Ca2+ and augment cardiac relaxation rates. The enhanced 5'-nucleotidase activity may stimulate adenosine production, which could affect myocardial blood flow. The present results further our knowledge on the subcellular response of the heart to swimming training in the rat.     

Effects of exercise on collateral development in myocardial ischemia in pigs

To study the effects of exercise on collateral development in myocardial ischemia, we induced coronary arterial stenosis of the left circumflex coronary artery (LCCA) in 18 of 30 pigs. During that surgery, we identified the coronary bed at risk. Nine of these pigs were then subjected to 5 mo of exercise training on a treadmill. After exercise training, we determined regional collateral and myocardial blood flow using radiolabeled microspheres. At autopsy, all animals had complete occlusion of the LCCA. Infarct size in the exercise-trained pigs was significantly less than in the sedentary pigs (5.9 +/- 1.0 vs. 11.7 +/- 1.0% of the left ventricle). The exercise-trained animals had a greater increase in collateral flow, 35.1 +/- 3.0 vs. 28.7 +/- 4.1 ml X min-1 X 100 g-1, in the noninfarcted jeopardized zone of the LCCA bed. The major findings of the study were the following: 1) chronic coronary artery stenosis progressing to occlusion stimulated development of the collateral circulation and salvaged tissue in the jeopardized myocardium of an animal model with sparse collaterals; 2) development of the collateral circulation and tissue salvage is increased by exercise training; 3) collaterals develop primarily in or near the ischemic zone; and 4) all collateral beds develop a circumferential flow gradient following occlusion.