Giant membrane vesicles as a model to study cellular substrate uptake dissected from metabolism.

In order to use giant vesicles for substrate uptake studies in metabolically important tissues, we characterized giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue. We investigated which cell types and which plasma membrane regions are involved in giant vesicle formation and we examined the presence of transporters for metabolic substrates. Analysis of giant vesicles with markers specific for distinct cell types and distinct domains of the plasma membrane reveals that the plasma membrane of parenchymal cells, but not endothelial cells, are the source of the vesicle membranes. In addition, plasma membrane regions enriched in caveolae and involved in docking of recycling vesicles from the endosomal compartment are retained in giant vesicles, indicating that KCl-induced alterations in recycling processes are involved in giant vesicle formation. Giant vesicles contain vesicular lumen consisting of the soluble constituents of the cytoplasm including, fatty-acid binding proteins. Furthermore, giant vesicles isolated from heart, liver, skeletal muscle and adipose tissue are similar in size (10-15 microm) and shape and do not contain subcellular organelles, providing the advantage that substrate fluxes in the different organs can be studied independently of the surface/volume ratio but most importantly in the absence of intracellular metabolism.

Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats.

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.

Skeletal muscle fatty acid transport and transporters.

While it has long been assumed that long chain fatty acids (LCFA) can freely diffuse across the plasma membrane, recent work has shown that LCFA uptake also involves a protein-mediated mechanism. Three putative LCFA transporters have been identified (FABPpm, FATP, and FAT/CD36), and all are expressed in rodent and human muscles. In a new model system (giant vesicles), we have demonstrated that (a) LCFA transport rates are scaled with the oxidative capacity of heart and muscle, (b) only FABPpm and FAT/CD36, but not FATP1, correlate with vesicular LCFA transport, and (c) LCFA transport can be increased by increasing (1) the FAT/CD36 protein of muscle (chronic adaptation) or (2) via the translocation of FAT/CD36 from an intracellular pool to the plasma membrane during muscle contraction (acute adaptation).

Acute regulation of fatty acid uptake involves the cellular redistribution of fatty acid translocase.

We used muscle contraction, which increases fatty acid oxidation, as a model to determine whether fatty acid transport is acutely regulated by fatty acid translocase (FAT/CD36). Palmitate uptake by giant vesicles, obtained from skeletal muscle, was increased by muscle contraction. Kinetic studies indicated that muscle contraction increased V(max), but K(m) remained unaltered. Sulfo-N-succinimidyl oleate, a specific inhibitor of FAT/CD36, fully blocked the contraction-induced increase in palmitate uptake. In giant vesicles from contracting muscles, plasma membrane FAT/CD36 was also increased in parallel with the increase in long chain fatty acid uptake. Further studies showed that like GLUT-4, FAT/CD36 is located in both the plasma membrane and intracellularly (endosomally). With muscle contraction, FAT/CD36 at the surface of the muscle was increased, while concomitantly, FAT/CD36 in the intracellular pool was reduced. Similar responses were observed for GLUT-4. We conclude that fatty acid uptake is subject to short term regulation by muscle contraction and involves the translocation of FAT/CD36 from intracellular stores to the sarcolemma, analogous to the regulation of glucose uptake by GLUT-4.

Exercise training and mouse mammary tumour metastasis.

The present study was designed to address whether exercise influences the experimental metastasis of a mouse mammary tumour; a related issue was the impact of timing of exercise onset relative to tumour exposure on NK and LAK mediated cytotoxicity. After 8 weeks of forced treadmill exercise or voluntary wheel running or remaining sedentary, female BALB/c mice received an intravenous (i.v.) injection of female BALB/c mice received an intravenous (i.v.) injection of MMT line 66 tumour cells. Mice were then randomized into continuation of activity (TT, WW), cessation of activity (TS, WS), initiation of activity (ST, SW) and maintenance of sedentary condition (SS) for three weeks. Tumour control (TC) mice, who were matched to the SS mice for age, received an i.v. injection of heat-killed MMT 66 tumour cells. In total there were 8 groups including the tumour control. The average number of lung tumours did not differ by activity condition; however, the mice in the continuous treadmill group (TT) tended to have a higher tumour multiplicity (162 +/- 22) and those in the treadmill x sedentary condition (TS) tended to have lower tumour multiplicity (109 +/- 16) compared with the other groups except the SW group (95 +/- 15). The lymphokine activated killer activity in the spleen was significantly elevated in the TS (49 +/- 3%) and WS (44 +/- 3%) mice compared with the sedentary animals (30 +/- 3%) (p < 0.003 and 0.05, respectively). NK activity was lower in the mice that had stopped exercising (TS and WS) after injection of tumour compared with sedentary animals. These data suggest that although exercise training influences natural immune cytotoxic mechanisms in vitro, this may not translate into clinically significant changes in tumour burden. The dissociation between natural immunity and tumour outcome may reflect the relative resistance of the tumour line to lysis by natural killer cells. It remains to be tested if infusion of IL-2 (to induce LAK activity) in exercise trained animals results in fewer tumour metastases.

Tissue distribution of radiolabelled tumor cells in wheel exercised and sedentary mice.

During the process of metastasis tumor cells must interact with vascular endothelial cells, basement membrane, and extracellular matrix. Specifically, tumor cells must bind to adhesion molecules to extravasate from the circulation into the tissue. Expression of adhesion molecules is altered by cytokines, such as tumor necrosis factor and interleukin-1. Exercise has been reported to alter circulatory levels of these cytokines. We have recently shown that exercise training reduces the number of experimental metastases in mice following intravenous (i.v.) injection of tumor cells. Thus, it was of interest to determine if one possible mechanism of reduced metastasis in exercised mice was a reduction in the initial retention of tumor cells in the pulmonary circulation. C3H/He and (C3H/H3XBALB/c)F1 mice were given access to running wheels or remained sedentary for nine weeks after which 51Cr labelled CIRAS 1 tumor cells (5 x 10(5) cells) were injected into a tail vein. Recovery of radioactivity in lungs, liver, spleen and kidney was assessed 5 min, 30 min, 90 min, 3 h and 24 h after injection. Wheel running mice displayed a lower retention of radioactivity in the lungs relative to sedentary mice 5 min (F1,34 = 11.91, p < 0.002; W = 59.1 +/- 3.6%, S = 75.0 +/- 2.9%; Mean +/- S.E.) and 30 min (F1,18 = 64.00, p < 0.001; W = 32.4 +/- 3.4%, S = 68.5 +/- 3.0%) post-injection. Radioactivity recovered in the lungs from wheel and sedentary mice was not significantly different at the other time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphokine activated killer cell activity following voluntary physical activity in mice.

The effect of eight weeks of voluntary wheel running exercise in female, outbred Swiss Webster mice on baseline splenic natural killer (NK) cell and interleukin-2 stimulated lymphokine activated killer (LAK) cell activity was studied. NK cell cytolytic activity against YAC-1 tumour targets was measured using a 51Cr release assay at the completion of a wheel running episode (end of dark cycle) or at 96 h after cessation of wheel running. LAK cell activity against tumour targets was generated from splenic NK cells by in vitro stimulation for 3 days with recombinant interleukin-2 (rIL-2). Wheel running was not associated with increases in baseline NK cytolytic activity either when sampled as a training effect (96 h after exercise) or immediately after exercise. Following an episode of wheel running exercise, LAK activity was significantly higher in the physically active compared to sedentary animals. These results support the concept that spontaneous wheel running activity enhances lymphokine activated killer cell activity following a cycle of active running; whether this greater LAK activity involves changes in IL-1, IL-2, or other cytokine concentrations or in the expression of IL-2 receptors on NK/LAK cells after exercise warrants further investigation. Given the clinical use of IL-2 to stimulate LAK cells in adoptive immunotherapy, it is possible (although untested) that exercise may have a potentially beneficial role as a treatment modality for some human cancers.

Effect of differential housing in mice on natural killer cell activity, tumor growth, and plasma corticosterone.

Various forms of stress have been shown to alter natural killer (NK) cell activity and tumorigenesis; however, few studies have measured these two variables simultaneously. Isolation of mice was utilized as a model of stress by which to study NK cell activity and pulmonary metastatic response following a tumor challenge. Male C3H mice were group or individually housed for 3 weeks, after which CIRAS 3 fibrosarcoma tumor cells or the tumor vehicle was injected intravenously (tail vein), NK cell activity, pulmonary metastasis, and plasma corticosterone were measured 1, 7, and 21 days following tumor cell inoculation. Individually housed mice, irrespective of tumor or vehicle condition, had a higher NK response on Day 1 relative to group-housed animals (P less than 0.001). By Day 21, tumor condition, rather than housing, was the major significant factor affecting NK activity (P less than 0.001). Nevertheless, individually housed, tumor-injected mice still had higher NK activity compared with the other treatment groups on Day 21. No effect of housing condition was present for the incidence of pulmonary metastases or frequency of metastases in affected animals. Plasma corticosterone levels generally increased over the study period, with no housing or injection effects at Days 1 and 7. Individually housed, vehicle-injected mice had higher corticosterone levels at Day 21 (P less than 0.01). These data suggest that in response to housing condition, NK cell activity differs in tumor-bearing mice and vehicle controls. Furthermore, CIRAS 3 pulmonary tumor formation is not affected by differences in NK activity consequent to housing condition. Plasma corticosterone does not appear to be a major in vivo regulator of NK activity in this experimental tumor system. Finally, the interpretation of housing effects on NK activity and plasma corticosterone levels depends on the temporal window in which sampling occurs.

Differential effects of exercise and housing condition on murine natural killer cell activity and tumor growth.

Acute exercise and exercise conditioning have been shown to affect the activity of natural killer (NK) cells as well as the growth of experimentally induced tumors in animals. Since psychosocial factors are also known to alter NK activity and tumor growth, isolation, a known psychosocial stressor of mice, was also investigated to see if housing condition could alter exercise-induced changes in NK cell activity and tumor growth. NK cell activity and concentration of asialo GM1 (ASGM1) positive splenocytes were measured in male C3H mice inoculated i.v. with CIRAS 3 tumor cells. Mice were housed individually or in groups of four and trained to run for eight weeks on a rodent treadmill; controls remained sedentary throughout the experimental period. At four weeks into the training protocol, mice were injected with the tumor cells and continued to run for four weeks after tumor exposure. There was a significant effect of physical activity (p less than 0.019) but not of housing on splenic NK cytotoxicity against tumor targets in vitro. When the data were analyzed by presence or absence of lung metastases, only those animals without visible lung tumors had significantly higher NK activity as a function of exercise relative to sedentary controls. There were no significant differences in the frequency of ASGM1+ splenocytes between trained and untrained animals, irrespective of presence or absence of lung tumor colonies. There was a significant effect of housing (p less than 0.02), but not of physical activity, in mice with successful tumor takes with greater numbers of group housed animals (29/59) with tumor relative to individually housed animals (13/60).(ABSTRACT TRUNCATED AT 250 WORDS)

Impact of changes in housing condition on mouse natural killer cell activity.

Various psychosocial stressors, such as housing condition and rotation, have been reported to influence tumour growth. This study assessed the influence of housing condition and change in housing condition on natural killer (NK) cell activity, an important component of natural immune defense against cancer. Mice which were individually housed for four weeks did not differ from group-housed mice in NK cell cytolytic activity against tumour targets in vitro or in the frequency of NK cells in the spleen. Switching of housing condition (group to individual, individual to group) for one week did not change the splenic NK cytolytic capacity relative to mice which were not switched. The two groups of mice which experienced a change in housing condition were, however, significantly different from each other. These data suggest that an acute change in housing condition, rather than the housing condition per se, has differential effects on the capacity to kill tumours by NK cells.

Lymphocyte proliferation responses after exercise in men: fitness, intensity, and duration effects.

This study investigated the effects of intensity and duration of exercise on lymphocyte proliferation as a measure of immunologic function in men of defined fitness. Three fitness groups--low [maximal O2 uptake (VO2max) = 44.9 +/- 1.5 ml O2.kg-1.min-1 and sedentary], moderate (VO2max = 55.2 +/- 1.6 ml O2.kg-1.min-1 and recreationally active), and high (VO2max = 63.3 +/- 1.8 ml O2.kg-1.min-1 and endurance trained)--and a mixed control group (VO2max = 52.4 +/- 2.3 ml O2.kg-1.min-1) participated in the study. Subjects completed four randomly ordered cycle ergometer rides: ride 1, 30 min at 65% VO2max; ride 2, 60 min at 30% VO2max; ride 3, 60 min at 75% VO2max; and ride 4, 120 min at 65% VO2max. Blood samples were obtained at various times before and after the exercise sessions. Lymphocyte responses to the T cell mitogen concanavalin A were determined at each sample time through the incorporation of radiolabeled thymidine [( 3H]TdR). Despite differences in resting levels of [3H]TdR uptake, a consistent depression in mitogenesis was present 2 h after an exercise bout in all fitness groups. The magnitude of the reduction in T cell mitogenesis was not affected by an increase in exercise duration. A trend toward greater reduction was present in the highly fit group when exercise intensity was increased. The reduction in lymphocyte proliferation to the concanavalin A mitogen after exercise was a short-term phenomenon with recovery to resting (preexercise) values 24 h after cessation of the work bout. These data suggest that single sessions of submaximal exercise transiently reduce lymphocyte function in men and that this effect occurs irrespective of subject fitness level.

Exercise and blood lymphocyte subset responses: intensity, duration, and subject fitness effects.

This study examined the effect of exercise intensity and duration on the percent blood lymphocytes in men of low [LF; maximal O2 uptake (VO2max) less than 50 ml.kg-1.min-1 and sedentary], moderate (MF; VO2max = 50-60 ml.kg-1.min-1 and recreationally active), and high (HF; VO2max greater than 60 ml.kg-1.min-1 and recent training history) fitness. Thirty healthy adult men (aged 20-31 yr) participated in four randomly ordered cycle ergometer rides: ride 1 (65% VO2max, 30 min), ride 2 (30% VO2max, 60 min), ride 3 (75% VO2max, 60 min), and ride 4 (65% VO2max, 120 min). Blood samples were drawn at various times before and after the exercise sessions. Lymphocyte subsets were determined by flow cytometry using monoclonal antibodies for total T (CD3+), T-helper (CD4+), and T-suppressor (CD8+) lymphocytes and for a subset of cells expressing a natural killer (NK) cell antigen (Leu7+). Plasma catecholamines were assayed to determine exercise stress. There were sharp reductions (P less than 0.01) in the percentage of pan-T and T-helper lymphocytes immediately after exercise across all fitness levels; the magnitude of this reduction was greatest after the highest intensity (ride 3) or longest duration (ride 4) work. In contrast, the absolute number of T and T-helper cells tended to increase after exercise and significantly so in the HF subjects (P less than 0.005). There was no significant effect of exercise or subject fitness category on the percentage of T-suppressor lymphocytes, although the absolute numbers of this subset increased significantly after exercise in LF subjects. Marked increases (P less than 0.01) in the percentage of NK cells occurred immediately after exercise at all intensities and durations tested; numerical increases in total NK cells were significant in all fitness groups after the highest intensity work (ride 3; P less than 0.005). Irrespective of whether the changes were expressed as percentage or total numbers, recovery to base line occurred at 30 min after exercise. The results suggest that the exercise effect on blood lymphocyte subset percentages in men is transient and occurs across all fitness levels. Concomitant changes in plasma catecholamine concentrations are only weakly associated with these lymphocyte subset percentage responses to exercise. Furthermore, this study shows that the exercise-induced changes in lymphocyte percentages do not consistently reflect changes in the absolute numbers of cells.

Lymphocyte subset responses to repeated submaximal exercise in men.

The effects of repeated bouts of submaximal cycle ergometry exercise on changes in the percentage of peripheral blood T-lymphocytes, the T-helper/inducer and T-cytotoxic/suppressor subsets, and natural killer (NK) cells were studied in 18 healthy young men who had no history of regular exercise training. Subjects were matched on the basis of maximal O2 uptake and assigned randomly to exercise or control groups, with controls resting quietly during the exercise sessions. The percentage of peripheral blood mononuclear leukocytes that reacted with monoclonal antibodies specific for T-lymphocytes (CD3+ cells), the helper/inducer subset (CD4+ cells) and cytotoxic/suppressor subset (CD8+ cells) of T-lymphocytes, and cells with NK activity (Leu7+ cells) were enumerated by fluorescence-activated flow cytometry for samples obtained immediately before and after exercise on days 1, 3, and 5 of a 5-day exercise regimen. The results of this study were mixed with decreases in the percentage of T-lymphocytes before vs. after exercise on days 1 and 3 (P less than 0.001), a decrease in the percentage of T-helper/inducer cells before vs. after exercise on day 3 (P less than 0.05), no effect of exercise on the percentage of T-cytotoxic/suppressor cells, and a marked increase in the percentage of NK cells after exercise on days 1 (P less than 0.05) and 3 (P less than 0.01). The total number of recovered NK cells in the mononuclear leukocyte fraction of blood also increased significantly after exercise on days 1 (P less than 0.05) and 3 (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Exercise stress alters the percentage of splenic lymphocyte subsets in response to mitogen but not in response to interleukin-1.

Results of previous work from this laboratory demonstrated that reduced murine splenic lymphocyte proliferation in response to Concanavalin A (Con A) is associated with acute exercise stress. The present study was conducted to determine whether the stress of physical work further affects the expression of splenic lymphocyte phenotypes following in vitro stimulation by the T-cell mitogen, Con A, and also by interleukin-1 (IL1). Mice in this study were assigned to one of five treatment conditions. Two groups of mice were exposed only to the noise and vibration of a treadmill for 8 weeks; one of these groups was given an acute exhaustive run. Three groups of mice were exposed to 8 weeks of treadmill training: one group was sacrificed immediately after training, a second group was sacrificed 72 h after training, and a third group was rested for 72 h and then given an acute exhaustive run. There was a significant effect of stimulation by Con A on the percentage of splenic lymphocytes positive for Thy1.2, Lyt-2, L3T4, and goat anti-mouse Ig, regardless of treatment condition. Acute exercise, however, affected the magnitude of the response. There was a significantly greater increase in the percentage of Thy1.2+ and, especially of, Lyt-2+ cells in stimulated splenic lymphocytes from untrained mice given an exhaustive exercise session compared with controls and with trained mice. There was no significant effect of the addition of IL1 to any culture, irrespective of treatment condition. These results suggest that reduced mitogenesis after acute exercise stress exposure may be related to the increased appearance of Lyt-2+ (T suppressor) cells.

Exercise stress alters murine lymphocyte subset distribution in spleen, lymph nodes and thymus.

Our previous work indicated that exercise stress in mice was associated with reduced splenic lymphocyte proliferation to T cell mitogens. The purpose of this study was to determine the effect of acute exercise stress and exercise training on the phenotype distribution of murine lymphocytes in the spleen, lymph nodes and thymus. In mice given an exhaustive bout of treadmill exercise, the percentage of Thy 1.2 and Lyt2 antigen bearing cells decreased in the thymus, but the percentage of L3T4-positive cells did not change significantly. Acute exercise did not alter the percentage of Thy1.2, L3T4 or Lyt2-positive cells in the secondary lymphoid compartments (nodes, spleen). By contrast, in mice given a chronic exercise training programme (8 weeks duration; 30 m/min, 8 degrees slope, 30 min/session, 5 daily sessions/week) followed by a 72 h rest period, the percentage of L3T4-positive cells increased by 53% in the spleen, 19% in the lymph nodes and 29% in the thymus compared with sedentary controls; no parallel increase in the percentage of Thy1.2 antigen bearing cells was observed. These results suggest that the effect of exercise on the frequencies of lymphocyte subpopulations in murine lymphoid compartments is dependent upon the chronicity of the stress and probably on the accompanying physiological adaptations to the stress.