INTRODUCTION

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THE INCREASE IN ENERGY expenditure elicited by acute exposure to a cold environment is met by several metabolic adaptations that provide oxidative tissues with carbohydrate and lipid substrates to maintain body temperature (reviewed in Ref. 15). One earlier report suggested that, in the rat, the metabolism of triglyceride (TG)-rich lipoproteins is also altered during an acute exposure to cold, as witnessed by a fall in circulating levels of TGs associated with very low-density lipoproteins (VLDL), the main blood TG carrier in the fasted state (27). A similar acute decrease in circulating TGs is observed in rats with other means of acutely increasing energy expenditure, such as physical activity (24-26). Along with nonesterified fatty acids (NEFA) released by adipose tissue, circulating TGs may contribute fatty acids to the pool of lipid substrates delivered to oxidizing tissues.

The mechanisms whereby an increase in energy expenditure is liable to acutely affect the metabolism of TG-rich lipoproteins include both synthetic and catabolic pathways. Although there is little information as to the adaptation of hepatic VLDL secretion to acute cold exposure, a previous study from our laboratory has shown that a single bout of treadmill running increased VLDL-TG secretion when assessed immediately after the running session (24). This contrasts with the reduction in the rate of TG secretion that has been reported after long-term physical training in rats, which was deemed responsible for the TG-lowering effect of training (20, 31, 36). In the latter case, however, TG secretion was assessed during periods of relative inactivity and therefore reflected longer-term metabolic adaptations to exercise rather than the acute consequences of increased energy expenditure. On the other hand, the contribution of the rate of intravascular TG hydrolysis to TG lowering after an acute increase in energy expenditure remains uncertain, as TG clearance has been reported to remain unaltered immediately after acute exposure to cold in humans (33) and to undergo a delayed increase after acute exercise (2). It appears that both ends of the triglyceridemia equation (secretion into the circulation and intravascular catabolism) have not yet been assessed concomitantly under identical conditions of increased energy expenditure.

Interventions that increase energy flux such as cold exposure and physical activity acutely alter several endocrine systems which themselves modulate lipoprotein metabolism. Specifically, the sympathoadrenal system is solicited, while insulin secretion is dampened (12). The involvement of these endocrine systems as mediators of the action of energy flux on lipoprotein metabolism and the pathways on which they exert their effects remain incompletely understood, particularly in the context of acute interventions.

This study was designed to assess the means by which an increase in energy expenditure through cold exposure acutely acts on TG-rich lipoprotein metabolism. To this end, the consequences of a short-term exposure to a cold environment on determinants of TG secretion and intravascular catabolism were evaluated. To gain insight into the mechanisms by which TG metabolism is affected by acute cold exposure, an assessment was made of changes in modulators of TG secretion and catabolism, namely plasma levels of insulin and NEFA, as well as postheparin plasma and tissue lipoprotein lipase (LPL) activity. Because the adrenergic system is strongly solicited by increased energy expenditure, the involvement of the -adrenergic system as an endocrine mediator of the cold-induced alterations in TG metabolism was also assessed.

	MATERIALS AND METHODS

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Animals and treatments. Two cohorts of 48 male Wistar rats each (Charles River, St. Constant, Québec, Canada) initially weighing 150-175 g were housed individually in stainless steel cages in a room maintained at 24 ± 1°C with a 12:12-h light/dark cycle (lights on at 0800). The animals were cared for and handled in compliance with the Canadian Guide for the Care and Use of Laboratory Animals, and the experimental procedures were approved by our institutional animal care committee. The animals had ad libitum access to tap water and a commercial nonpurified diet (Charles River rodent chow #5075) for two wk. All animals were then fitted with a permanent polyethylene cannula in the right jugular vein under isoflurane anesthesia. The goal of the study was to assess treatment effects on the metabolism of endogenous TG-rich lipoproteins in the postprandial state, at which time TG metabolism is most active. Therefore, after the cannulation procedure, the initial diet was replaced by a purified diet containing 65% of energy as carbohydrate (glucose-starch 1:1), 15% as lipid (corn oil), and 20% as protein (casein), and supplemented with vitamins, minerals, and fiber, with a gross energy content of 16.8 kJ/g (10). The diet was fed ad libitum for 3 days. During the last nighttime feeding period before the experiments, rats were fed the purified diet from which fat had been removed to avoid chylomicron formation, thereby allowing assessment of VLDL-TG kinetics. The animals were randomly assigned to 4 groups of 12 rats according to a 2 × 2 factorial design. The factors were environmental temperature with two levels, 24 and 10°C, and -adrenergic blockade with two levels, vehicle and propranolol. The experimental treatments consisted of acute exposure to 10°C for 3 h, which increases energy expenditure by ~60% (13), and of a single intraperitoneal administration of the nonselective -adrenergic antagonist DL-propranolol at a dose of 25 mg/kg body wt in saline. Propranolol at this dose was shown to completely prevent the increase in plasma NEFA brought by acute treadmill running in rats (26). In the morning after nighttime ad libitum access to the fat-free purified diet, food was removed. Thirty minutes later, two groups of animals were injected with saline and the other two with propranolol. Thirty minutes after the injection, one-half of the saline and propranolol groups were moved, inside their cages, to an adjacent cold room maintained at 10°C, whereas the other one-half was kept at 24°C. The first cohort of animals was used to sequentially determine the in vivo TG clearance and VLDL-TG secretion rates. The two procedures were separated by a 3-day period during in which the animals were returned to the 24°C environment. Postheparin plasma and tissue LPL activities as well as serum variables were determined in the second cohort. The postheparin plasma LPL procedure and tissue harvesting were separated by a 4-day period during which the animals were returned to 24°C.

In vivo TG clearance rate. Ten minutes before the end of the 3-h period of cold exposure, an initial blood sample (0.15 ml) was withdrawn through the venous catheter, and 0.75 ml/kg body wt of a 20% Intralipid emulsion (Vitrum, Stockholm, Sweden) was rapidly injected through the catheter. The latter was washed with saline, and blood samples (0.15 ml) were taken 1, 3, 6, and 9 min after Intralipid injection. The rate of clearance of TG from the circulation was calculated as the slope of the semilogarithmic plot of plasma TG versus time and was defined as k₂ (%/min) (7).

VLDL-TG secretion rate. Two hours into the period of cold exposure, an initial blood sample (0.15 ml) was withdrawn through the venous catheter, and rats were injected through the catheter with 300 mg/kg body wt of Triton WR1339 (Sigma, St. Louis, MO), a detergent that prevents intravascular TG catabolism (23). Blood samples (0.15 ml) were then taken 20, 40, and 60 min after the Triton injection. The rate of VLDL-TG secretion into the circulation was determined from regression analysis of TG accumulation in plasma versus time. Secretion rate was calculated by multiplying the slope of the regression line by plasma volume estimated from body weight and was expressed as micromoles per minute.

Postheparin plasma LPL. Approximately 0.5 ml of blood was drawn from the jugular catheter 10 min before and 10 min after the rapid intrajugular administration of 200 IU/kg body wt of sodium heparin (porcine intestinal mucosa, 1,000 USP/ml, Sigma Chemical) (14). Blood was centrifuged at 1,500 g, 4°C for 15 min, and plasma was stored at 70°C for later biochemical measurements.

Blood and tissue harvesting. Rats were killed by decapitation. Blood collected from the neck wound was centrifuged at 1,500 g, 4°C for 15 min. Serum was stored at 70°C for later biochemical measurements. Epididymal white adipose tissue (WAT), interscapular brown adipose tissue (BAT), red vastus lateralis muscle (VLM), and the heart were excised. BAT was cleaned of any adhering white fat and muscle tissues, and the heart was washed in saline. Tissues were weighed, ~50 mg were taken from WAT, BAT, the red portion of VLM, and the apex of the heart, and tissue samples were homogenized using all-glass tissue grinders (Kontes, Vineland, NJ). WAT and BAT were homogenized in 1 ml of a solution containing 0.25 M sucrose, 1 mM EDTA, 10 mM Tris · HCl, and 12 mM deoxycholate, pH 7.4. VLM and the heart were homogenized in 1 ml of a solution containing 1 M ethylene glycol, 50 mM Tris · HCl, 3 mM deoxycholate, 10 IU/ml heparin, and 5% (vol/vol) aprotinin (Trasylol, Miles Pharmaceuticals, Rexdale, Ontario, Canada), pH 7.4. These homogenizing media were found to yield optimal LPL activities in the individual tissues. Homogenates of VLM and the heart were quickly frozen and stored at 70°C until measurement of LPL activity. WAT and BAT homogenates were centrifuged at 12,000 g, 4°C for 20 min. The fraction between the upper fat layer and the bottom sediment was removed after tube slicing, diluted with four volumes of the homogenization solution without deoxycholate, and stored at 70°C until later measurement of LPL activity.

Serum variables. Insulin was quantitated by radioimmunoassay using a reagent kit from Incstar (Stillwater, MN) and rat insulin as standard. TG concentrations were assayed by an enzymatic method using a reagent kit from Boehringer Mannheim (Montréal, Québec, Canada), which allows correction for free glycerol. NEFA levels were measured enzymatically using reagents from Wako Chemicals (Richmond, VA).

Postheparin plasma and tissue LPL activities. LPL activity was measured in postheparin plasma and tissue homogenates as described earlier (25). Samples of 100 µl of postheparin plasma diluted 1:50 with saline and of WAT, BAT, VLM, and heart homogenates were incubated for 1 h at 28°C under gentle agitation with 100 µl of a substrate mixture consisting of a 0.2 M Tris · HCl buffer, pH 8.6, which contained 10 MBq/l [carboxyl-¹⁴C]triolein and 2.52 mM cold triolein emulsified in 5% gum arabic, as well as 2% fatty acid-free bovine serum albumin, 10% human serum as a source of apolipoprotein C-II, and either 0.1 or 2 M NaCl. Free oleate released by LPL was then separated from intact triolein and mixed with Universol (New England Nuclear, Montréal, Québec, Canada), and sample radioactivity was determined in an LKB Rackbeta liquid scintillation counter. LPL activity was calculated by subtracting non-LPL lipolytic activity determined in a final NaCl concentration of 1 M from total lipolytic activity determined in a final NaCl concentration of 0.05 M. In the present conditions, 1 M NaCl inhibited 82-91% of total lipolytic activity in all tissue homogenates and 30-40% of lipolytic activity (the remainder representing hepatic TG lipase) in postheparin plasma. Tissue LPL activity was expressed as microunits (1 µU = 1 µmol NEFAs released per hour of incubation at 28°C) and plasma LPL activity as microunits per milliliter plasma. The interassay coefficient of variation was 4.8% and was determined using bovine skim milk as a standard source of LPL. Protein content of the tissue extracts was determined by the method of Lowry et al. (18). Treatment effects were identical whether LPL activity was expressed per total tissue (total activity), per unit tissue weight, or per unit protein (specific activity), and only total activity is presented below.

Statistical analysis. Data are expressed as means ± SE. Main treatment effects and treatment interactions were analyzed using factorial analysis of variance. When significant interactions were revealed, individual between-group comparisons were performed using Fisher's protected least-squares difference post hoc test. Data were log transformed before analysis when group variances were not homogeneous (O'Brien's test), but untransformed values are presented below. Differences were considered statistically significant at P < 0.05.

	RESULTS

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Because single, acute interventions were studied, all groups were comparable in terms of final body and organ weights, as well as cumulative food intake (data not shown).

Figure 1 shows that the 3-h exposure to 10°C reduced serum TG levels to one-half of those observed in rats kept at 24°C. Propranolol did not alter TG levels in warm-exposed animals but blunted the cold-induced reduction in plasma TG, as witnessed by the significant treatment interaction. Post hoc analysis revealed that serum TG concentrations in cold-exposed rats injected with saline were lower than in the other three groups, which in turn were comparable to each other. Identical main and interactive treatment effects on plasma TG concentrations were also observed in blood obtained before measurement of TG clearance in the first cohort of animals (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Serum triglyceride (TG) concentrations in rats maintained in the warm (24°C) or exposed to cold (10°C) for 3 h after administration of saline (Sal) or 25 mg/kg body wt of propranolol (Prop). Bars represent means ± SE of 9 or 10 animals. Levels of significance from ANOVA of main effects of temperature (T), with 2 levels (24 and 10°C), and -blockade (B), with 2 levels (vehicle and propranolol), and those of treatment interactions (T × B): T, P = 0.009; B, P = not significant (NS); T × B, P = 0.05. Because a significant treatment interaction was revealed, individual between-group comparisons were made. Bars not sharing a common superscript are significantly different from each other at P < 0.05.

To assess which determinants of intravascular TG metabolism were affected by cold exposure and propranolol, the rate of TG secretion into the circulation during the last hour of exposure to cold was assessed. Figure 2A shows that cold exposure increased TG secretion by nearly 50% in saline-injected animals. Propranolol treatment per se did not alter TG secretion significantly, but interacted with temperature as it abolished the cold-induced stimulation of TG secretion (Fig. 2B). Post hoc analysis indicated that TG secretion rates in cold-exposed rats injected with saline were higher than in the other three groups, which in turn were comparable to each other. Serum levels of two major determinants of the rate of VLDL-TG secretion, namely NEFA and insulin, were assessed at the end of the period of cold exposure. Figure 2C shows that treatment effects and interaction on serum NEFA concentrations paralleled those on TG secretion. Cold exposure doubled NEFA levels, whereas propranolol abolished this effect while leaving NEFA unaltered in warm-exposed animals. In contrast, insulin concentrations (Fig. 2D) were slightly (22%) but significantly reduced by cold exposure and remained unaltered by - blockade at both environmental temperatures.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Serum TG concentrations and rates of TG secretion (above and below regression lines, in µmol/min) in rats maintained in the warm (24°C) or exposed to cold (10°C) for 3 h after administration of saline (A) or propranolol (B) and serum concentrations of nonesterified fatty acids (NEFA, C) and insulin (D). Circles and bars represent means ± SE of 9-12 animals. Levels of significance from ANOVA (as described in legend of Fig. 1): T, P = 0.02, 0.04, and 0.02; B, P = NS, NS and NS; and T × B, P = 0.02, 0.03 and NS for secretion rate, NEFA, and insulin, respectively. Means and bars not sharing a common superscript are significantly different from each other at P < 0.05.

In addition to their rate of secretion, the other major determinant of circulating TG levels is their rate of clearance from the circulation. Therefore, the rate of clearance of an Intralipid emulsion was assessed as an estimate of the global capacity for intravascular hydrolysis of TG. Both cold exposure and propranolol treatment exerted significant main effects on TG clearance without interacting with each other (Fig. 3, A and B). Cold exposure increased the rate of TG clearance by 3.6%/min (69% over control) in saline-injected rats and by 2.0%/min (61% over control) in propranolol-treated animals, whereas -blockade reduced TG clearance in both warm (1.9%/min, 37% below saline)- and cold-exposed (3.5%/min, 40% below saline) groups. Postheparin plasma LPL activity, one major determinant of the intravascular TG hydrolytic capacity, was not significantly affected by either treatment or their combination (Fig. 3C).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Serum TG concentrations and rates of TG clearance (above and below regression lines, in %/min) in rats maintained in the warm (24°C) or exposed to cold (10°C) for 3 h after administration of saline (A) or propranolol (B) and activity of lipoprotein lipase (LPL) in postheparin plasma (C). Circles and bars represent means ± SE of 9-12 animals. Levels of significance from ANOVA (as described in legend of Fig. 1): T, P = 0.002 and NS; B, P = 0.002 and NS; and T × B, P = NS and NS for clearance rate and LPL, respectively.

Despite the lack of change in LPL availability in the intravascular compartment, cold exposure and -blockade exerted major, tissue-specific actions on enzyme activity, as depicted in Fig. 4. LPL in epididymal WAT was decreased by one-half after 3 h of cold exposure, whereas -blockade remained without effect (Fig. 4A). In contrast, BAT LPL activity was more than doubled by cold exposure (Fig. 4B). Propranolol interacted significantly with the environmental temperature as it brought about a larger absolute decrease in brown adipose LPL activity in cold-exposed animals (27 µU) than in those maintained in the warm (9 µU). Figure 4C shows that vastus lateralis LPL was unaffected by treatments. Cold exposure resulted in a 50% increase in heart LPL activity, whereas propranolol treatment did not affect enzyme activity in this organ (Fig. 4D).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Total LPL activity in epididymal white adipose (A), interscapular brown adipose (B), vastus lateralis muscle (C), and heart (D) of rats maintained in the warm (24°C) or exposed to cold (10°C) for 3 h after administration of saline or propranolol. Bars represent means ± SE of 9-11 animals. Levels of significance from ANOVA (as described in legend of Fig. 1): T, P = 0.0005, 0.0001, NS, and 0.001; B, P = NS, 0.0001, NS, and NS; and T × B, P = NS, 0.009, NS, and NS for white adipose, brown adipose, vastus lateralis, and heart, respectively. Bars not sharing a common superscript are significantly different from each other at P < 0.05.

	DISCUSSION

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This study aimed to identify the metabolic steps involved in the acute hypotriglyceridemia brought about by increased energy expenditure through cold exposure and to assess the causative involvement of some determinants of TG metabolism as well as that of the -adrenergic pathway. The experimental approach had the advantage of evaluating both ends of the triglyceridemia equation (secretion and clearance) as well as several of their metabolic determinants within a single paradigm. The findings of this study point to intravascular TG hydrolysis as the cause of the acute cold-induced reduction in circulating TG, which occurred despite a concomitant elevation in the rate of TG secretion. Increased TG secretion appeared to be closely linked to NEFA availability. The results also show that TG clearance was increased in absence of a change in the availability of LPL in the intravascular compartment. Finally, the consequences of acute cold exposure on serum TG levels and their rates of secretion and clearance were greatly reduced by -adrenergic blockade.

The acute increase in VLDL-TG secretion after cold exposure is entirely consistent with the concomitant metabolic changes at the levels of adipocyte lipolysis and pancreatic function. Indeed, the increase in serum NEFA levels due to elevated adipose lipolysis, together with the lowering of insulinemia brought about by cold exposure (34), constitute two factors liable to stimulate hepatic TG synthesis and subsequent secretion. VLDL-TG secretion is sensitive to the amount of precursor NEFA delivered to the liver (16), and insulin tends to reduce VLDL-TG secretion by the insulin-sensitive liver both directly and indirectly through inhibition of adipose lipolysis (8, 16, 17). Therefore, the present observation of an acute increase in VLDL-TG secretion after cold exposure is in accordance with the effects of the intervention on two of its key modulators. It is noteworthy that a comparable increase in TG secretion has also been observed by our laboratory after acute treadmill exercise in rats (24). The acute stimulatory effect of increased energy expenditure, either by cold exposure or exercise, on VLDL-TG secretion must be contrasted with the lowering effect of chronic exercise training reported earlier (20, 31, 36). In at least some of these studies, however, TG secretion was measured in rats running spontaneously, but during periods of relative inactivity rather than immediately after exercise. At this time, the metabolic conditions liable to favor high rates of VLDL-TG secretion immediately after exercise may have been no longer present. Therefore, it can be suggested that repeated bouts of increased energy expenditure result in acute increases in VLDL-TG secretion followed by a reduction in TG secretion when metabolic conditions change some time after exposure.

The consequences of -adrenergic blockade in warm- and cold-exposed animals strongly suggest that circulating NEFA levels were a more determining factor than insulin concentrations in the response of TG secretion to cold exposure.  Blockade affected neither VLDL-TG secretion nor NEFA and insulin levels in warm-exposed animals, most likely because of a low level of sympathoadrenal activation in the 24°C environment. However, in rats exposed to cold, propranolol abolished both the cold-induced rise in VLDL-TG secretion and the increase in serum NEFA levels, without altering the cold-induced reduction in insulin levels. Blunting of the cold-induced rise in NEFA levels by propranolol confirms our earlier results with exercised rats (26) and is consistent with the fact that the sympathetic system is thought to be largely responsible for fat mobilization when energy demands increase (3, 11, 29). The combined actions of propranolol therefore strongly suggest that an increased availability of NEFA was responsible for the cold-induced increase in VLDL-TG secretion. These results do not rule out the direct involvement of insulin in the modulation of VLDL-TG secretion, which has been highlighted by recent in vivo and in vitro studies (4, 8, 16). The findings do suggest that, in the present conditions, changes in serum NEFA resulting from modulation of adipose lipolysis were more important determinants of the acute response of VLDL-TG secretion to short-term cold exposure than were the absolute circulating levels of insulin. This is further supported by the fact that, when VLDL-TG secretion has been found to be decreased some time after exercise in trained rats (20, 36), plasma NEFA levels were either unchanged or lower than in sedentary animals in the presence of lower insulin levels. Finally, the fact that epinephrine was shown in vitro to decrease, rather than increase, hepatocyte TG secretion (5) rules out a direct action of catecholamines on the liver and further supports an indirect mechanism involving NEFA release by adipose tissue and uptake from portal blood by the liver.

The concomitant assessment of TG secretion and intravascular clearance demonstrates that the acute cold-induced lowering of circulating TG was entirely due to an increase in their rate of intravascular hydrolysis. Indeed, cold exposure for 3 h reduced serum TG levels by one-half despite a concomitant 50% increase in VLDL-TG secretion. The intravascular hydrolytic capacity was not only able to compensate for the increase in VLDL-TG secretion, but its efficiency was such that the new steady state of serum VLDL-TG attained after cold exposure was one-half that measured in the warm. This increase in intravascular TG hydrolysis is not trivial, given that, after cold exposure, TG entry into the circulation within 1 h was equivalent to three times the plasma pool, as shown by the increase in plasma TG after Triton WR1339 administration (Fig. 3A). Furthermore, in the presence of such a large flux of endogenous TG, the hydrolytic system was still able to clear a load of exogenous TG (Intralipid) 70% faster than in warm conditions. This finding underlines the fact that the rate of intravascular TG hydrolysis is susceptible to remarkable positive modulation under conditions of increased energy expenditure. It should be noted that cold exposure of humans, which increased energy expenditure 2.5-fold, was found to be without effect on the rate of Intralipid clearance (33). Notwithstanding possible species differences, this discrepancy may be related to the fact that the subjects of the latter study were in the fasted state when the test was performed. In the present study, a high-carbohydrate diet was fed to the animals before cold exposure. Such a diet results in a significant postprandial increase in serum TG of endogenous origin (19), indicating a saturation of the capacity for intravascular TG hydrolysis. In the fasted state, however, TG hydrolysis is not likely to be saturated and may therefore be less susceptible to positive modulation. Alternatively, an increase in energy expenditure may alter TG clearance in fasting humans after some delay, as suggested by a study in which a 3-h exercise bout performed in the fasted state did not increase Intralipid clearance immediately after exercise, but did so 24 h later (2).

Another germane finding of the present study is the occurrence of a drastic lowering of serum TG due to enhanced clearance without any alteration in the intravascular availability of LPL. One earlier study suggested that postheparin plasma lipase activity was increased by a longer (24 h) exposure to more intense (4°C) cold (28), although lipase activity was measured in conditions that did not discriminate between LPL and hepatic lipase. Even if cold exposure may at some point result in an increase in endothelium-bound LPL, the present findings clearly show that TG clearance was greatly enhanced before such putative changes would have occurred. The lack of change in postheparin plasma LPL by short-term cold exposure therefore points to the existence of factors other than the absolute amount of endothelial LPL to account for this enhanced hydrolytic capacity. One likely factor is the increased blood flow that occurs in several vascular beds, such as BAT and skeletal muscle, during cold exposure of rats (1). An increase in the rate of delivery of TG-rich lipoproteins to the endothelium-bound LPL would be liable to increase the efficiency of interactions between TG-rich lipoproteins and the enzyme, resulting in a rapid lowering of circulating TG. An alternative mechanism is the possible existence of a "heparin-like" effect of cold exposure, as described by Oscai et al. (22) in the rat hindlimb after acute exercise, which consists of the detachment of LPL from the endothelial surface of capillary beds. Liberation of LPL results in a rapid lowering of circulating TG, as is the case with heparin administration. However, detachment of LPL from the endothelium after acute exercise reduces the amount of residual, heparin-releasable LPL (22, 25), which was not the case after cold exposure in the present study. Therefore, it is unlikely that this mechanism was operative. Be that as it may, the involvement of changes in blood flow as a determinant of the rate of TG hydrolysis represents a relevant possibility that remains to be fully characterized.

Despite a lack of change in the global availability of LPL in the intravascular compartment, LPL displayed the classic, tissue-specific modulation in response to cold exposure. The present results confirm other studies showing a reduction in WAT LPL and an increase in BAT LPL in response to cold exposure (9, 28, 32), which both involve changes in LPL mRNA levels (32). LPL also increased in the heart, but not in VLM, in which LPL may respond only after a delay of several hours after an elevation in energy expenditure, at least in the case of exercise (26), and participate in replenishing intramuscular TG stores. Tissue-specific modulation of LPL by cold exposure is therefore in accordance with the role of the enzyme in the routing of TG away from storage sites and toward oxidizing tissues. On the assumption that LPL activity measured in tissue homogenates was proportional to the fraction of the enzyme pool available at the endothelial surface (21), the divergent changes in LPL of specific tissues tended to cancel each other, because total endothelium-bound LPL measured in postheparin plasma remained unchanged by treatments. As to the effect of -blockade on tissue LPL, brown adipose was the sole site in which enzyme activity was altered, a finding that confirms the involvement of the -adrenergic pathway in the cold-induced stimulation of brown adipose LPL (6). The lack of effect of the blocker on white adipose and heart LPL is also in agreement with our earlier findings in rats subjected to acute treadmill running (26). It is worth noting that the changes in LPL activity in response to acute cold exposure differ from those observed after acute exercise, with the exception of WAT. We have shown that acute moderate treadmill running at least transiently reduces LPL activity in postheparin plasma and in all four tissues studied here when assessed immediately after the running session (25). As noted above, this might result from the heparin-like effect of exercise and subsequent rapid uptake of released LPL by the liver. Metabolic stimulation of BAT by cold, but not by exercise, as well as a larger solicitation of muscle by running than by shivering in the cold, constitutes other stimulus-related differences that may affect the tissue-specific response of LPL to increased energy expenditure.

The consequences of propranolol treatment before cold exposure clearly demonstrate the major involvement of the -adrenergic pathway in the cold-induced changes in TG-rich lipoprotein metabolism. In animals kept in the warm, propranolol did not alter TG secretion rate, but decreased the rate of Intralipid clearance by approximately one-third. This decrease in the global capacity for TG hydrolysis was apparently not large enough to constitute a limiting factor for clearance of endogenous TG, because serum TG remained unaffected. In cold-exposed animals, however, propranolol blunted the cold-induced increase in TG secretion. It also slowed TG clearance approximately twice as much, in absolute terms, as in rats kept in the warm. In fact, serum TG levels and their rates of secretion and clearance in propranolol-treated, cold-exposed animals were all indistinguishable from those of the saline-treated group kept in the warm. The mechanisms by which -adrenergic blockade influences determinants of intravascular TG metabolism remain to be fully identified. As discussed above, the parallelism between the effect of propranolol on NEFA levels and TG secretion strongly suggests that the blocker affected the latter indirectly through its inhibitory action on adipocyte lipolysis (11). As to TG clearance, it may be suggested that propranolol acted through dampening the cardiovascular adaptations to cold exposure that otherwise resulted in increased blood flow and efficiency of hydrolysis of circulating TG.

The acute effects of increased energy expenditure are to be distinguished to a certain extent from those of chronic adaptation to prolonged or repeated acute interventions. To the best of our knowledge, the consequences of long-term cold acclimation on TG kinetics have not yet been systematically addressed. However, as noted above, long-term physical training reduces the rate of VLDL-TG secretion measured in the resting state (20, 31, 36) and in humans increases high-density lipoprotein (HDL)-cholesterol (35) and skeletal muscle LPL activity (30). Therefore, long-term or repeated exposure to increased energy expenditure results in metabolic adaptations that are not all established immediately after a single exposure.

In summary, the lowering of serum TG after acute cold exposure in the postprandial state was entirely explained by an increase in their rate of disappearance from the circulation, as the rate of TG secretion not only did not decrease, as is the case late after chronic elevations in energy flux, but actually increased. This cold-induced increase in TG secretion appeared to result from an enhanced availability of lipogenic substrates (NEFA) and to be independent of the modest changes in insulinemia. The cold-induced enhancement in TG clearance occurred despite a lack of change in the absolute availability of LPL in the intravascular compartment. The integrated, acute response of TG metabolism to increased energy expenditure, such as that brought by exposure to cold, therefore involves an increased input of lipid substrates into the circulation in the form of NEFA, but also in the form of TGs, coupled with an increased capacity for hydrolysis of the latter. Tissue-specific alterations in LPL activity would favor the routing of fatty acids hydrolyzed from circulating TGs away from adipose stores and toward oxidizing tissues. This would constitute a means to increase the delivery of lipid substrates in a tissue-specific fashion, as opposed to the rather indiscriminate uptake of NEFAs released by adipose tissue. Finally, activation of the -adrenergic pathway appears to be largely responsible for the cold-induced effects on triglyceridemia as well as on TG secretion and clearance.

Perspectives