When present in excess, plasma NEFA are involved directly in the pathogenesis of metabolic disturbances leading to insulin resistance and the metabolic syndrome(Reference Stich and Berlan1). It has been suggested in animal as well as human studies that increased fat accumulation inside the adipocyte results in an increased lipolysis, thus resulting in increased plasma NEFA levels(Reference Scheen and Luyckx2). Regulation of lipolysis, especially in the visceral compartment(Reference Bosello and Zamboni3), is, therefore, important for the prevention of systemic metabolic disorders.
Since the typical Western diet is very high in fat, high-fat-diet regimens have been used frequently in animals as a model of obesity(Reference Kraegen, Storlien, Jenkins and James4, Reference Ghibaudi, Cook, Farley, van Heek and Hwa5) and more recently as a model to study the short-term development of obesity(Reference Gauthier, Couturier, Latour and Lavoie6, Reference Gauthier, Couturier, Charbonneau and Lavoie7). It is generally assumed that a high-fat-diet regimen leads to an increased basal and stimulated adipocyte lipolysis, thus contributing to increased plasma NEFA levels(Reference Berger and Barnard8, Reference Collin, Chapados, Dufresne, Corriveau, Imbeault and Lavoie9). On the other hand, exercise training has been reported to significantly reduce adipocyte fat accretion and plasma NEFA levels whether conducted concurrently or initiated during the course of a high-fat diet-induced obesity(Reference Gauthier, Couturier, Latour and Lavoie6, Reference Gauthier, Couturier, Charbonneau and Lavoie7). Stimulated lipolysis in adipocytes has been shown to be increased by exercise training in lean human (subcutaneous tissue) and animal adipose tissue(Reference Bukowiecki, Lupien, Follea, Paradis, Richard and LeBlanc10, Reference Crampes, Beauville, Riviere and Garrigues11) under normal feeding conditions. Whether basal and/or stimulated adipocyte lipolysis is also increased with exercise training under high-fat diet conditions is not known. It would be metabolically more appropriate if exercise training, under high-fat diet conditions, would result in a decrease instead of an increase in lipolysis. In this regard, it is revealing that in pinealectomised rats, in which adipocytes' lipolytic response to isoproterenol is increased, exercise training resulted in a decrease in this elevation in the epidymal fat pad(Reference Borges-Silva, Fonseca-Alaniz, Alonso-Vale, Takada, Andreotti, Peres, Cipolla-Neto, Pithon-Curi and Lima12). The main purpose of the present study was to test the hypothesis that exercise training in high-fat-fed animals is associated with a reduction in adipocyte lipolysis. This was tested in a visceral (mesenteric) and in a non-visceral (retroperitoneal) fat tissue. Visceral fat (i.e. omental, mesenteric) is usually associated more strongly with metabolic abnormalities such as dyslipidaemia, hypertension, insulin resistance and type 2 diabetes than is subcutaneous or total body fat(Reference Votruba and Jensen13). Catecholamine-induced rate of NEFA mobilisation from visceral fat cells is accelerated due to elevated rate of lipolysis in human obesity, mainly because of an increased β-3-adrenoceptor function, and a decreased α-2-adrenoceptor function. This promotes an increased release of NEFA to the portal system, which may contribute to the parallel metabolic disturbances observed in upper-body obesity(Reference Lönnqvist, Thöme, Nilsell, Hoffstedt and Arner14). A reduction in the elevated lipolysis, especially in the mesenteric tissue with exercise training in high-fat-fed rats would, therefore, constitute an asset in reducing the increased amount of NEFA reaching the portal system.
In recent years, a new component regulating the activity of hormone-sensitive lipase (HSL)-mediated lipolysis has been identified. Perilipins are phosphoproteins in adipocytes localised at the surface of the lipid droplet(Reference Greenberg, Egan, Wek, Garty, Blanchette-Mackie and Londos15). There is evidence that the access of HSL to the lipid droplets is determined by perilipin A, the main isoform of perilipin(Reference Londos, Brasaemle, Schultz, Segrest and Kimmel16, Reference Brasaemle, Rubin, Harten, Gruia-Gray, Kimmel and Londos17). Increased basal lipolysis in high-fat-fed rats may result from an increase in HSL mass or activity; alternatively, this increase may reflect the absence of a protective coat of perilipin on the lipid droplet(Reference Tansey, Sztalryd, Gruia-Gray, Roush, Zee, Gavrilova, Reitman, Deng, Li, Kimmel and Londos18). There is no information on the action of exercise training on perilipin in adipocytes. It has previously been suggested that exercise training might alter the amount of perilipin, thereby regulating the interaction of HSL with its substrate(Reference Nomura, Kawanami, Ueda, Kizaki, Ohno and Izawa19). In an effort to better understand how lipolysis may be affected by exercise training in high-fat-fed rats, we measured perilipin protein content and tried to associate it with changes in in vitro lipolysis in the mesenteric tissue.
Methods
Animal care
Female Sprague–Dawley strain rats (n 45) (Charles River, St-Constant, PQ, Canada), weighing 180–200 g upon their arrival were housed individually and had ad libitum access to food and tap water. Their environment was controlled in terms of light (12 h–12 h light–dark cycle starting at 06.00 hours), humidity and room temperature (20–23°C). All experiments described in this report were conducted according to the directives of the Canadian Council on Animal Care.
Diet and exercise protocol
A few days after their arrival, all animals were assigned to a high-fat diet while remaining sedentary (Sed) or being submitted to an exercise training programme (Tr) for 2 or 8 weeks (nine rats per group). Another group of rats were immediately killed to provide basal values (T = 0; n 6–9). The high-fat diet consisted of 42 % lipid (80 % lard, 20 % maize oil), 36 % carbohydrate, and 22 % protein (by energy content) and was provided in small pellets from ICN Pharmaceuticals (Plainview, NY, USA). Details of this diet have been presented elsewhere(Reference Gauthier, Couturier, Latour and Lavoie6). The exercise training programme was introduced concomitantly to the high-fat diet. Exercise training consisted of continuous running on a motor-driven rodent treadmill (Quinton Instruments, Seattle, WA, USA) five times per week for the duration of the experiment. During the first 2 weeks, rats were progressively run from 15 min/d at 15 m/min, 0 % slope up to 30 min/d at 26 m/min, 4 % slope. Thereafter, the training programme was progressively increased to reach 60 min at 26 m/min, 10 % slope for the last 4 weeks. All Tr animals were restrained from training 48 h before killing.
Blood and tissue sampling
At the end of their respective experimental condition, all animals were weighed and killed between 08.30 and 10.30 hours. Food was removed from the animals at least 2 h before killing. After complete anaesthesia (sodium pentobarbital, 50 mg/kg, intraperitoneal), the abdominal cavity was rapidly opened following the median line of the abdomen. Blood was rapidly ( < 45 s) drawn from the abdominal vena cava (about 4 ml) into syringes pre-treated with EDTA (15 %). Blood was centrifuged (4000 rpm for 10 min, 4°C) and the plasma kept for NEFA, glucose, insulin and leptin determinations. The mesenteric and retroperitoneal fat depots were, thereafter, rapidly excised and weighed. The mesenteric fat pad consisted of adipose tissue surrounding the gastrointestinal tract from the gastro-oesophageal sphincter to the end of the rectum with special care taken in distinguishing and removing pancreatic cells. The retroperitoneal fat pad was taken as that distinct deposit behind each kidney along the lumbar muscles. About 700 mg of the depots was immediately used for determination of lipolysis. The remaining portions of the mesenteric and retroperitoneal depots as well as plasma samples were stored at − 78°C until analyses.
Preparation of isolated adipocytes
Adipocytes were isolated according to the method of Rodbell(Reference Rodbell20). Tissue fragments (700 mg) from the mesenteric and the retroperitoneal tissue were minced in polyethylene vials containing Krebs–Ringer bicarbonate buffer in the presence of collagenase P (1 mg/ml) (Roche 1213865; Mannheim, Germany). Krebs–Ringer bicarbonate buffer also contained fatty acid-free bovine serum albumin (4 %) and glucose (0·5 g/l) (Krebs–Ringer bicarbonate buffer+albumin; KRBA). Thereafter, the atmosphere of the flasks was saturated with 95 % O2 and 5 % CO2. Adipocytes were then incubated in a shaking water-bath at 37°C for 30 min with a shaking frequency of 100 strokes/min. The digested pieces were filtered through a nylon mesh for separation of fat cells from the stroma. The filtrate was collected in a graduated polyethylene tube to avoid cell breakage. The aqueous phase was aspirated with polyethylene pipettes. The floating adipocytes were then re-suspended in 5 ml of fresh KRBA. This process was repeated three times to eliminate the remaining collagenase. The adipocytes were stored in the KRBA for 2 min between two washes.
Incubation of adipocytes and measurement of lipolysis
Basal and stimulated lipolysis was determined in isolated adipocytes (50 μl) incubated with a 10 ul solution of KRBA buffer and ascorbic acid (basal) and with isoproterenol adjusted to give concentrations of 10− 7, 10− 5 and 10− 4 m. Isoproterenol was used because it is a pure β-agonist and as such it elicits the greatest lipolytic response(Reference Berger and Barnard8). Isoproterenol produced a dose-dependent increase in lipolysis, with the maximal responses (plateau) reached at 10− 4 and 10− 5 m. In agreement with other groups, adenosine deaminase was not added to the incubation medium to block the anti-lipolytic action of adenosine(Reference Coe, Simpson and Bernlohr21, Reference Iglesias-Osma, Garcia-Barrado, Visentin, Pastor-Mansilla, Bour, Prevot, Valet, Moratinos and Carpene22). The incubation was performed at 37°C under a 95 % O2–5 % CO2 atmosphere with gentle shaking (eighty strokes per min) for 2 h. The reaction was stopped by adding 50 μl HCl (1 m) and then 50 μl NaOH (1 m) to neutralise the medium. The vials were vortexed and put on ice for 10 min. After a centrifugation of 5 s at 250 g, the aqueous phase was removed and stored at − 78°C until glycerol determination.
Lipolytic activity was measured by glycerol quantification with the use of commercially available kits from Roche Diagnostics (Mannheim, Germany). Glycerol quantification was expressed in millions of cells/h after counting the number of cells in each tested sample.
Cell count
The number of cells in each individual sample was determined by first diluting 100 μl of the isolated adipocyte preparation into 9·9 ml KRBA (100 μl) and then dropping 50 μl of this solution in 25 μl of trypan blue (0·4 % normal saline). The total number of cells in this final suspension was determined with the use of a Nikon-optiphot microscope (10 × ) and with Image-Pro plus version 4.5 (Media Cybernetics; Silver Spring, MD, USA).
Measurement of perilipin content
Mesenteric fat depots (about 100 mg) were homogenised in EDTA–sucrose buffer and centrifuged at 12 000 g, 4°C for 20 min. The infranatant was collected with a blunt-tipped Pasteur pipette and stored at − 80°C until perilipin determination. Perilipin A content in adipocytes was determined by Western blotting. All samples (25 μg of proteins) were separated on a 10 % SDS–polyacrylamide gel followed by protein transfer to a polyvinylidene difluoride membrane by electroblotting. The membrane was blocked in 5 % (w/v) skimmed milk in TBS (100 mm-2-amino-2-(hydroxymethyl)propane-1,3-diol, 100 mm-NaCl, pH 7·5) for 2 h at room temperature before overnight incubation with a rat perilipin A polyclonal antibody (ABR-Affinity Bio Reagents) with 0·05 % sodium azide. After three washes in TBS–Triton 0·5 %, the membrane was incubated for 1 h with an anti-rabbit IgG peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) diluted to 0·8 μg/ml in 0·1 % bovine serum albumin, 0·1 % Tween 20 and 1 % (w/v) skimmed milk in TBS. The membrane was washed three times for 10 min each time in TBS–Triton 0·5 % before a chemifluorescence substrate (enhanced chemiluminescence; Amersham, Baie D'Urfée, PQ, USA) was applied to the membrane. The resulting signal was detected on enhanced chemiluminescence film (Amersham), scanned with the use of Agfas Arcus II, and quantified with Imagepro 4.5 software (Media Cybernetics, Silver Spring, MD, USA) and expressed as arbitrary units.
Analytical procedures
Plasma glucose concentration was determined with the use of a glucose analyser (Yellow Springs Instruments 2300; Yellow Springs, OH, USA). Plasma insulin levels were measured with commercially available kits from MP Biomedicals, LLC (Orangeburg, NY, USA). Plasma NEFA levels were measured with commercially available kits from Roche Diagnostics (Mannheim, Germany). Leptin levels were measured with commercially available kits from Linco (St Charles, MO, USA).
Statistical analysis
Values are expressed as mean values with their standard errors. Statistical analyses were performed by a two-way ANOVA for non-repeated-measures design with exercise training and time as main effects at times 2 and 8 weeks, excluding time point 0 (since no diet treatment was given at that time). In a second step, the effect of time was analysed separately in Sed and Tr rats using a one-way ANOVA for non-repeated measures over all time points using the same values at time 0 for both groups. This second statistical analysis was performed to better characterise the effect of time in each condition including comparison with time 0. Fisher's post hoc test was used in the event of a significant (P < 0·05) F ratio.
Results
Changes in body composition and metabolic characteristics of Tr and Sed rats are presented in Table 1. The increase in body weight over time (P < 0·01) was similar in Tr and Sed rats. The increase in the weight of the mesenteric and the retroperitoneal fad pads, however, was higher (P < 0·05) in Sed than in Tr rats after 8 weeks of high-fat feeding. The same observation was made for plasma leptin levels, with higher (P < 0·05) values found in Sed than in Tr animals (Table 1). Plasma NEFA and glucose levels were also higher (P < 0·01) with time in both Tr and Sed rats, but to a significantly (P < 0·05) larger extent in Sed than in Tr animals. Plasma insulin levels were higher (P < 0·01) with time in both Tr and Sed rats (8 weeks).
Table 1 Body composition and metabolic characteristics of trained (Tr) and sedentary (Sed) rats at time week 0 (baseline values) and in response to 2 and 8 weeks of high-fat feeding (seven to nine rats/group)§
(Mean values with their standard errors)
Mean value was significantly different from that at baseline (time 0 weeks): *P < 0·05, **P < 0·01.
Mean value was significantly different from that at time 2 weeks: ††P < 0·01.
Mean value was significantly different from that of the Sed group: ‡P < 0·05, ‡‡P < 0·01.
§ All rats were fed a high-fat diet.
Basal as well as stimulated lipolysis levels were higher with time (P < 0·01) in the mesenteric tissue of Sed animals (Fig. 1). In Tr rats, basal lipolysis in the mesenteric tissue was not affected with time, while stimulated lipolysis was higher (P < 0·01) compared with time 0 but to a lesser extent than what was observed in Sed animals (Fig. 1). As a result, the elevation in stimulated lipolysis (10− 5–10− 4 m) in the mesenteric tissue was lower (P < 0·05) in Tr than in Sed animals (week 8; Fig. 1). In the retroperitoneal tissue, however, stimulated lipolysis (10− 5–10− 4 m) in Tr animals was increased to the same level as in Sed animals after 8 weeks, thus resulting in an absence of effects of training on lipolysis (Fig. 2).
Fig. 1 Adipocyte lipolysis in mesenteric adipose tissue of sedentary (■) and trained (□) rats at time 0 weeks (baseline values; 
) and in response to 2 and 8 weeks of high-fat feeding. Values are expressed as glycerol production per million cells per h in the basal state (A) and in response to isoproterenol (10− 7 m (B); 10− 5 m (C); 10− 4 m (D)) after 2 h of incubation. Values are means for seven to nine rats per group (n 6 at week 0), with standard errors represented by vertical bars. ** Mean value was significantly different from that at baseline (time 0 weeks) (P < 0·01). † Mean value was significantly different from that at 2 weeks (P < 0·05). ‡ Mean value was significantly different from that of the sedentary group (P < 0·05).
Fig. 2 Adipocyte lipolysis in retroperitoneal adipose tissue of sedentary (■) and trained (□) rats at time 0 weeks (baseline values; ) and in response to 2 and 8 weeks of high-fat feeding. Values are expressed as glycerol production per million cells per h in the basal state (A) and in response to isoproterenol (10− 7 M (B); 10− 5 M (C); 10− 4 M (D)) after 2 h of incubation. Values are means for seven to nine rats per group (n 6 at week 0), with standard errors represented by vertical bars. * Mean value was significantly different from that at baseline (time 0 weeks) (P < 0·05). † Mean value was significantly different from that at 2 weeks (P < 0·05).
Perilipin content in the mesenteric tissue expressed in arbitrary units was higher (P < 0·05) after 2 weeks in both Sed and Tr animals and decreased to initial values after 8 weeks (Fig. 3 (A)). This response was not significantly affected by the exercise training programme. When perilipin content was expressed per unit of fat pad weight, significantly lower (P < 0·01) levels were found after 8 weeks compared with basal levels in both Sed and Tr animals (Fig. 3 (B)).
Fig. 3 Perilipin content (A) and perilipin:fat ratio (B) measured in mesenteric tissue from sedentary (■) and trained (□) rats at time 0 weeks (baseline values; ) and in response to 2 and 8 weeks of high-fat feeding. Perilipin was measured by Western blotting and expressed in arbitrary units (AU). Perilipin:fat ratio was obtained by dividing the perilipin content by the absolute weight (g) of the mesenteric depot. Values are means for eight to nine rats per group (n 6 at week 0), with standard errors represented by vertical bars. Mean value was significantly different from that at baseline (time 0 weeks): *P < 0·05, **P < 0·01. Mean value was significantly different from that at 2 weeks: †P < 0·05, ††P < 0·01.
Discussion
In agreement with previous studies conducted in high-fat-fed rats(Reference Kraegen, Storlien, Jenkins and James4, Reference Gauthier, Couturier, Latour and Lavoie6, Reference Gauthier, Couturier, Charbonneau and Lavoie7), exercise training in the present study resulted in a significant reduction in fat accumulation in the mesenteric and the retroperitoneal tissues along with lower levels of plasma NEFA and leptin (week 8; Table 1). In line with these observations, the most novel finding of the present study is that the high-fat-induced increase in isoproterenol-stimulated lipolysis in the mesenteric tissue was reduced by 8 weeks of concurrent exercise training. This response is rather surprising considering that exercise training is generally associated with an increase, and not a decrease, in in vitro and in vivo stimulated adipose tissue lipolysis in normal dietary conditions(Reference Bukowiecki, Lupien, Follea, Paradis, Richard and LeBlanc10, Reference Crampes, Beauville, Riviere and Garrigues11, Reference Enevoldsen, Stallknecht, Fluckey and Galbo23). The fact that the elevation in stimulated lipolysis was reduced by training in high-fat-fed-rats might, at first glance, simply reflect a reduction in fat mass in the mesenteric tissue. As mentioned by other studies(Reference Enevoldsen, Stallknecht, Fluckey and Galbo23), this will not decrease the interest of the study since the reduction in fat mass is an effect of training. Most interestingly, however, stimulated lipolysis in the mesenteric tissue of 8-week Tr rats was lowered to the level measured in 2-week-old rats even though fat accumulation was still about 75 % higher in the former. This indicates that the large reduction in stimulated lipolysis in 8-week Tr rats cannot solely be attributed to the reduction in fat mass. Exercise training may act upon stimulated lipolysis in the mesenteric tissue of high-fat-fed rats so as to contribute to limit the amount of circulating NEFA resulting from daily sympathetic nerve activity. We previously reported that concurrent exercise conducted in high-fat-fed rats reduced plasma NEFA levels and completely prevented the high-fat diet-induced hepatic steatosis(Reference Gauthier, Couturier, Latour and Lavoie6). Taking into account that plasma NEFA levels reflect whole-body lipolysis, the present data extend these findings by being the first to indicate that under conditions of high-fat feeding exercise training reduced the elevation in isoproterenol-stimulated lipolytic activity of the mesenteric adipose tissue.
Although the present decrease in the elevation of isoproterenol-induced lipolysis with exercise training in the mesenteric tissue of high-fat-fed rats seems surprising, a similar finding has been recently reported in pinealectomised rats in which the absence of melatonin increased lipolysis(Reference Borges-Silva, Fonseca-Alaniz, Alonso-Vale, Takada, Andreotti, Peres, Cipolla-Neto, Pithon-Curi and Lima12). Interestingly, the authors suggest that melatonin is necessary for the balance between lipogenesis and lipolysis of the adipocytes in response to physical training in rats. Although the molecular mechanism remains to be clarified, it is possible that exercise training under a high-fat feeding condition increased the lipogenic capacity of the visceral adipocytes to the detriment of lipolysis.
The interest of the present finding that exercise training reduced the elevation in stimulated lipolysis in the mesenteric tissue of high-fat-fed rats is exacerbated by the fact that the same observation was not made in the retroperitoneal tissue. Stimulated lipolysis in the retroperitoneal tissue was increased by about 95 % (10− 5–10− 4 m) following 8 weeks of high-fat feeding. However, these increased lipolytic values were not affected by the exercise training programme (Fig. 2). In interpreting these data, it is important to recall that the retroperitoneal tissue, although considered an intra-abdominal tissue, is not a visceral tissue as is the intraperitoneal mesenteric depot(Reference Cinti24). Accordingly, the high-fat feeding protocol increased stimulated lipolytic activity to a greater extent in the mesenteric (about ten-fold) than in the retroperitoneal tissue (about two-fold; Figs. 1 and 2) although relative fat accumulation was higher in the retroperitoneal than in the mesenteric tissue (about seven- v. four-fold). The greater lipolytic response of the mesenteric tissue in response to the high-fat diet may be part of the explanation as to why exercise training specifically decreases lipolysis in this tissue in this dietary condition. In one of the rare studies that looked at the effects of exercise training on lipolysis in various adipose tissues in normally fed rats, exercise training did increase adrenaline-stimulated lipolysis in the retroperitoneal tissue but not in the mesenteric tissue, indicating that these two adipose tissues respond differently to exercise training under normal feeding conditions(Reference Enevoldsen, Stallknecht, Fluckey and Galbo23). Altogether, it seems that the effects of exercise training on reducing the elevation in lipolytic activity in high-fat-fed rats are particularly observed in the mesenteric tissue, suggesting that this effect might be specific to this visceral adipose tissue.
Basal lipolysis
Opposite of what was found in the Sed condition (week 8), exercise training did not result in higher levels of basal lipolysis in the mesenteric tissue compared with values at times 0 and 2 weeks (Fig. 1). Interpretation of basal lipolysis in the present study is complicated by the fact that the data were acquired in an in vitro situation, which may not reflect the normal basal in vivo conditions. This situation is not as important when lipolysis is stimulated by pharmacological agents since the stimulation overcomes the normal in vivo environment of the cells. Actually, several in vitro findings related to stimulated lipolysis have been confirmed by in vivo measurements(Reference Enevoldsen, Stallknecht, Fluckey and Galbo23). Notwithstanding the limitation of the in vitro measurements, the absence of a high rate of basal lipolysis in the mesenteric tissue of Tr rats fed a high-fat diet would be highly beneficial from a metabolic point of view by potentially decreasing the circulating levels of NEFA reaching the liver in the resting state and potentially reducing liver fat accumulation. It is not clear if exercise training affects basal lipolysis in normally fed human subjects and animals. Exercise training has been reported to be without effect or may reduce basal lipolysis in some fat depots in normally fed animals(Reference Nomura, Kawanami, Ueda, Kizaki, Ohno and Izawa19, Reference Enevoldsen, Stallknecht, Fluckey and Galbo23). The present data are, however, the first to indicate that exercise training limits the increase in basal in vitro lipolysis in the mesenteric fat depot of high-fat-fed rats.
Exercise and visceral adipose tissue in humans
It is well documented that visceral fat constitutes an important risk factor for the development of metabolic abnormalities and CVD in humans(Reference Votruba and Jensen13). Exercise training decreases visceral fat and this is associated with an improvement in glucose disposal and insulin sensitivity(Reference Ross, Dagnone, Jones, Smith, Paddags, Hudson and Janssen25). Recent evidence suggests that the improvement in insulin sensitivity with exercise training may be explained by a decreased availability of NEFA(Reference Shojaee-Moradie, Baynes and Pentecost26). A reduction in lipolytic activity of the visceral fat depot with exercise training could, therefore, contribute to the reduced availability of NEFA to the portal system and to the improvement in insulin sensitivity in obese individuals.
Lipolysis and perilipin
It is not readily apparent as to what mechanism might explain the exercise training-induced attenuation in the lipolytic activity of the mesenteric tissue in the present high-fat-fed rats. Plasma glucose and insulin were increased to a similar extent in Sed and Tr animals after 8 weeks of high-fat feeding. Nomura et al. (Reference Nomura, Kawanami, Ueda, Kizaki, Ohno and Izawa19) observing that basal activity of HSL was greater in Tr normally fed rats, but basal lipolysis was not, recalled the suggestion of a dual mechanism of lipolytic activation of lipolysis involving some factor on the surface of the lipid droplets (i.e. perilipin) that may be necessary for the hormonal stimulation of lipolysis(Reference Wise and Jungas27). In an attempt to explore this mechanistic avenue, perilipin content was measured in the mesenteric tissue of the present Tr and Sed animals. Interestingly, our data indicate a transient increase in perilipin content in absolute values after 2 weeks that decreased to initial values after 8 weeks (Fig. 3 (A)). This resulted in low perilipin content in relation to fat mass accretion after 8 weeks (Fig. 3 (B)). Since perilipin has been reported to be involved in the mechanism of stimulation of lipolysis(Reference Enevoldsen, Stallknecht, Fluckey and Galbo23, Reference Enevoldsen, Stallknecht, Langfort, Petersen, Holm, Ploug and Galbo28), the low level of perilipin expressed per unit of fat mass after 8 weeks may explain the increase in lipolysis in the present high-fat-fed rats kept in the Sed state. It is not clear what caused the decrease in perilipin content after 8 weeks of high-fat feeding. It could be related to the increased plasma leptin levels. Mice overexpressing leptin show a reduction in perilipin gene expression in white adipose tissue(Reference Ke, Qiu, Ogus, Shen, Kraemer and Chehab29). On the other hand, and most importantly, exercise training did not significantly affect perilipin content in the mesenteric tissue, whether reported in absolute or in relative values. The association between perilipin content and lipolysis is, however, complicated by the fact that other factors in addition to HSL and perilipin may contribute to the lipolytic response(Reference Sztalryd, Xu, Dorward, Tansey, Contreras, Kimmel and Londos30). The new enzyme adipose TAG lipase, that catalyses the initial step in TAG hydrolysis in mammalian adipose tissue, along with the adipocyte lipid-binding proteins that interact with HSL favouring the translocalisation from cytosol to lipid droplet, are potential candidates(Reference Zimmermann, Strauss and Haemmerle31, Reference Carmen and Victor32).
In summary, results of the present study indicate that exercise training lowers the elevation in stimulated in vitro lipolytic activity in the mesenteric tissue of rats concurrently fed with a high-fat diet. This effect was not observed in a non-visceral tissue (retroperitoneal) and seems to be independent of the perilipin content. Whether exercise training may decrease the lipolytic activity of the visceral adipose tissue of established obese individuals and thus constitute a health-promoting effect remains to be established.
Acknowledgements
The present study was supported by grants from the Natural Sciences and Engineering Research Council of Canada (J.-M. L.) and from the Canadian Institutes of Health Research (J.-M. L. and P. I.) of Canada.
There is no conflict of interest of any of the authors involved the present study. N. C. and P. Collins have been involved in all steps of the collection of the data, while P. I. and P. Corriveau have been involved in the technical aspects and the collection of the data. J.-M. L. and N. C. have been involved in the planning of the experiment and the writing of the manuscript. All authors have contributed to the correction of the manuscript.
