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Sequential changes in lipoprotein lipase activity and lipaemia induced by the Yoshida AH-130 ascites hepatoma in rats
Cancer Letters 116 (1997) 159–165
Sequential changes in lipoprotein lipase activity and lipaemia induced by the Yoshida AH-130 ascites hepatoma in rats Joaquı´n Lo´pez-Soriano, Josep M. Argile´s, Francisco J. Lo´pez-Soriano* Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071-Barcelona, Spain Received 14 January 1997; accepted 4 March 1997
Abstract The implantation of the Yoshida AH-130 ascites hepatoma to rats resulted in an exponential growth of the tumour cells followed by a late stationary phase. The tumour burden was accompanied by a dramatic decrease in body weight. Tumour growth was associated with a marked hypertriglyceridaemia during the period of exponential growth, while in the stationary phase the plasma triacylglycerol concentration was similar to that observed in the non-tumour-bearing animals. Similar increases were observed, following tumour inoculation, in the plasma concentrations of non-esterified fatty acids and glycerol, suggesting an intense lipolytic activity. These changes in lipaemia were associated with a marked decrease in LPL activity in white adipose tissue; in contrast, LPL activity was increased in the tumour-bearing animals in brown adipose tissue at day 6 following inoculation and in the heart during most of the period studied. Although the presence of the tumour did not induce any changes in blood lactate concentrations, it caused a decrease in circulating glucose; conversely, the tumour induced an important increase in the concentration of circulating ketone bodies, suggesting a metabolic adaptation of the tumour-bearing rats to glucose sparing and alternative fuel utilization. It may be suggested that the hyperlipidaemia present in the Yoshida AH-130 bearing rats is partly due to a decreased LPL activity in white adipose tissue which does not seem to be influenced by changes in insulin circulating concentrations. 1997 Elsevier Science Ireland Ltd. Keywords: Tumour growth; Circulating triglycerides; Lipoprotein lipase; Cancer cachexia; Rat
1. Introduction Hypertriglyceridaemia and depletion of fat stores have been observed both in patients [1,2] and experimental animals [3–5] during the growth of a wide variety of neoplasms. Understanding the mechanisms involved in the depletion of body fat may have clinical relevance because weight loss itself has a bad prognostic value [6] and the amount of remaining fat in * Corresponding author. Tel.: +34 3 4021525; fax: +34 3 4021559; e-mail: [email protected]
starved individuals closely correlates with the duration of survival [7]. Animal studies reveal that loss of fat is not caused by decreased food intake alone, since it precedes the onset of anorexia in mice [8] and it is more severe in tumour-bearing animals than in pair-fed controls [9]. Thus, increased catabolic events such as enhanced lipolytic rate [10,11] and a reduction in adipose tissue lipoprotein lipase (LPL) activity promote fat mobilization during tumour growth [4,11–13]. A decrease in LPL content or activity in a tissue usually correlates with a decreased uptake of triacylglycerol fatty acids
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by that tissue [14], which is also the case in tumourbearing animals [4]. In the latter, another contributing mechanism is an important decrease in the rate of lipogenesis in adipose tissue [11,15], which is consistent with a reduction in the activities of enzymes involved in this metabolic pathway [12]. Although a great deal of work concerning the metabolic effects caused by the presence of a malignant tumour has been carried out, the results concerning the effects of tumour burden on host lipid metabolism are not so clear, with contradictory results in both experimental animals and man. Lipid oxidation has been reported as either increased [8,16], unchanged [17] or decreased [18]. In addition, LPL activity decreases in adipose tissue [11,12], with a concomitant increase in plasma triacylglycerol levels [12]. Bearing all this in mind, the aim of the present investigation was to characterize the changes in LPL activity and lipaemia associated with tumour growth. In order to accomplish this objective, we have used the rat Yoshida AH-130 ascites hepatoma, a rapidly growing tumour.
2. Materials and methods 2.1. Animals and tumour implantation Female Wistar rats (12 weeks old) from our own colony were fed ad libitum on a chow diet (Panlab, Barcelona, Spain) consisting (by weight) of 54% carbohydrate, 17% protein and 5% fat (the residue was non-digestible material), with free access to drinking water, and were kept in individual polypropylene cages fitted with wire-mesh bottoms at an ambient temperature of 22 ± 2°C with a 12 h-light/12 h-dark cycle (lights on from 0800 h). Food intake and body weight were measured daily after tumour inoculation. A Yoshida AH-130 ascites hepatoma cell suspension (approx. 120 × 106 cells in 2 ml) was injected intraperitoneally, the control rats being injected with 2 ml of sterile 0.9% (w/v) NaCl solution. Total cell number was estimated using Trypan blue staining. 2.2. Biochemicals and radioactive compounds The biochemical and radioactive compounds were all reagent grade and obtained either from Boehringer
Mannheim S.A. (Barcelona, Spain) or from Sigma Chemical Co. (St. Louis, MO, USA). Radiochemicals were purchased from Amersham Int. (Amersham, Bucks., UK). 2.3. Lipoprotein lipase activity Perirenal white adipose tissue (WAT), heart and interscapular brown adipose tissue (BAT) LPL activities were measured by a modification of the technique of Nilsson-Ehle and Ekman [19]. Tissue samples were dried to a powder with acetone/ether and then resolubilized and used in an assay system containing [3H]triolein as substrate; [3H]fatty acids released after a 60 min incubation period were extracted and determined by the method of Nilsson-Ehle and Schotz [20]. LPL activity is expressed as nmol of fatty acid released/min per mg of acetone-dried powder. 2.4. Blood metabolites and plasma insulin Whole blood glucose was determined by the method of Slein [21] and lactate was determined by the method of Hohorst et al. [22]. Blood acetoacetate and b-hydroxybutyrate were determined fluorimetrically [23]. Plasma insulin was determined by radioimmunoassay, with a rat insulin standard [24]. Plasma triacylglycerols [25], glycerol [26] and non-esterified fatty acids [27] were also measured. 2.5. Statistics The results are expressed as mean ± SEM for the number of individual observations indicated in each experiment. Statistical significance was determined by Student’s t-test.
3. Results and discussion The Yoshida AH-130 is a highly cachectic rapidly growing rat tumour containing poorly differentiated cells, with a relatively short doubling time of 1 day [28]. After tumour inoculation, there is an exponentially growing phase which lasts until day 8, followed by a stationary growth phase which precedes the animals’ deaths [29]. Growth of the ascites hepatoma AH-130 in rats elicits an early and conspicuous loss
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of body weight and skeletal muscle mass, associated with a protein hypercatabolic state in host tissues [30], as well as a profound perturbation in hormonal homeostasis, an elevation of plasma prostaglandin E2 and the presence of circulating TNF [5]. Lipid metabolism is also disturbed, with increased levels of total cholesterol and triacylglycerols and reduced levels of esterified cholesterol in the blood plasma [5,31]. This tumour also induces hepatomegaly and a dramatic loss of adipose tissue [15]. These features have been observed in other rapidly growing tumour models in the rat such as the Walker 256 carcinosarcoma [4].
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concentrations of free glycerol (2.2 fold at day 4) as a result of tumour growth (Fig. 2). Glycerol is another indicator of an intense lipolytic activity, being preferentially used by the liver for glucose production during tumour burden [3]. 3.3. Lipoprotein lipase activity The implantation of the tumour resulted in alterations in tissue LPL activity (Fig. 3). Thus, LPL activ-
3.1. Body weight and tumour burden Previous results have shown that the presence of the Yoshida AH-130 ascites hepatoma induces a marked weight reduction in the tumour-bearing host, which is associated with decreases in both food intake and energy efficiency in the last period of tumour growth [29]. As shown in Fig. 1, the tumour induced a significant decrease in body weight (subtracting the tumour mass) in comparison with the control (day 0) group, already detectable at day 4 after implantation. At day 10 following tumour inoculation the decrease in body weight reached 30%. The decrease in body weight was accompanied by a very characteristic increase in tumour cell content (Fig. 1), as previously reported [29]. 3.2. Hypertriglyceridaemia As shown in Fig. 2, the presence of the tumour promoted important changes in the circulating concentration of triacylglycerols. Thus, at day 4 following inoculation the concentration was already elevated (2-fold increase), reaching a peak at day 6 (2.3-fold increase). Conversely, there was a tendency towards normalization of the triacylglycerol concentrations towards control levels at the end of the inoculation period, when the tumour reaches a stationary phase of growth [29]. The changes in triglyceridaemia were accompanied by increases in the plasma concentration of non-esterified fatty acids at the end of the period studied (Fig. 2). These results clearly indicate that the tumour-bearing host is under a strong lipolytic activity, as previously described [10,11]. Interestingly, an increase was also detected in the circulating
Fig. 1. Body weight and tumour cell number in rats bearing the Yoshida AH-130 hepatoma. For further details see Section 2. The results are mean values ± SEM for five different animals. Body weights exclude the weight of the tumour plus ascitic fluid. Values that are statistically significant differences (Student’s t-test, P , 0.05) from those of the control (day 0) group were already present at day 4 after tumour inoculation.
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LPL activity) are well-known uncouplers of oxidative phosphorylation in BAT [33]. Cardiac LPL was increased during tumour growth (Fig. 3); it may be suggested that an enhanced LPL activity in this organ may serve as an alternative strategy for substrate uptake (fatty acids) in a metabolic environment where there is a high glucose extraction by the tumour [3]. Hyperlipidaemia, particularly an elevation of circulant triacylglycerol concentration, is a general feature both in cancer patients [34] and animals bearing a number of different tumours [4,35]. The mechanism responsible for the development of the hypertriglyceridaemia is not fully understood, although a role of LPL deficiency in peripheral tissues has been suggested by several authors [11,12,36]. Other studies, however, did not find a correlation between the lipid levels in plasma and the activity of LPL in cancer patients [37]. In our study, changes in WAT LPL activity correlate well with the increase in circulating triacylglycerols (Fig. 2), thus suggesting that the hyperlipidaemia present in the Yoshida AH-130 bearing rats is partly due to a decreased LPL activity in WAT which seems to be only slightly influenced by changes in insulin circulating concentrations. Indeed, insulin activates LPL activity in WAT [38]. However,
Fig. 2. Plasma triacylglycerols, non-esterified fatty acids and glycerol concentrations in rats bearing the Yoshida AH-130 hepatoma. For further details see Section 2. The results are mean values ± SEM for five different animals. Plasma concentrations are expressed as mg/100 ml for triacylglycerols, mmol/l for glycerol and mmol/100 ml for non-esterified fatty acids. Values that are significantly different by Student’s t-test from those of the control (day 0) group are indicated by: *P , 0.05, **P , 0.01, ***P , 0.00l.
ity is considerably decreased in WAT, reaching the lowest value at day 6 (75% decrease). Interestingly, BAT LPL activity is subject to little changes in the tumour-bearing animals except for a punctual increase at day 6. This increase may possibly have a relation with the increased thermogenic activity found in this tissue during tumour growth [32], since fatty acids (their uptake being enhanced through the increased
Fig. 3. Tissue lipoprotein lipase (LPL) activity in rats bearing the Yoshida AH-130 hepatoma. For further details see Section 2. LPL activity is expressed as pmol of fatty acid released/min per mg of acetone-dried tissue. The results are mean values ± SEM for five different animals. Values that are significantly different by Student’s t-test from those of the control (day 0) group are indicated by: *P , 0.05, **P , 0.01, ***P , 0.001. WAT, white adipose tissue; BAT, brown adipose tissue.
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the decrease in WAT LPL activity was also observed when the insulin concentrations were close to control values (days 6–10) (Fig. 4), this probably being an indication of the insulin resistant status of the tumourbearing host [3]. 3.4. Blood metabolites Glycaemia was only maintained for the first 2 days
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after tumour inoculation, the circulating glucose concentrations being significantly reduced with respect to those from day 0 to day 4 onwards (Fig. 4). No changes were detected in blood lactate as a consequence of tumour burden. However, ketone bodies (acetoacetate and b-hydroxybutyrate) blood levels were significantly increased from day 4 onwards (Fig. 4). The increase was observed earlier for bhydroxybutyrate than for acetoacetate; the highest increases were detected at day 10, i.e. 6.8- and 17.3fold for b-hydroxybutyrate and acetoacetate, respectively. Plasma insulin levels were significantly reduced only on day 4 after tumour implantation.
4. Conclusion
Fig. 4. Blood metabolites and plasma insulin concentrations in rats bearing the Yoshida AH-130 hepatoma. For further details see Section 2. The results are mean values ± SEM for five different animals. Blood glucose and lactate concentrations are mM, plasma insulin is expressed as mU/100 ml, and plasma ketone bodies concentrations are mM. Values that are significantly different by Student’s t-test from those of the control (day 0) group are indicated by: *P , 0.05, **P , 0.01, ***P , 0.00l.
Some of the metabolic changes associated with tumour growth may be due to cytokines such as tumour necrosis factor-a (TNF). This peptide, mainly produced by activated macrophages, has a wide range of metabolic effects (see Ref [39] for review). Yoshida AH-130 bearing rats show important perturbations in hormonal homeostasis, which include the presence of circulating TNF [5]. Recently, we have demonstrated that the administration of antibodies against murine TNF can partially reverse the changes observed in LPL activity in this experimental model [40] as well as the accelerated protein turnover [41]. The alterations in lipid metabolism observed in the cancer patient, which contribute to the metabolic abnormalities observed in situations of cancer cachexia, cannot be solely attributed to the reduction in food intake, since they often precede the onset of anorexia [8]. On these lines, cancer cachexia, characterized by an accelerated depletion of peripheral protein and lipid stores, cannot be reversed by forced hyperalimentation. In future, therefore, the design of therapeutic strategies to counteract the cachexia found in the cancer patient will have to focus on an effective neutralization of the changes in lipid metabolism induced by the tumour in the host.
Acknowledgements This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social
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(F.I.S.) (94/1048) of the Spanish Health Ministry, from the DGICYT (PB 94-0938) of the Spanish Ministry of Education and Science and from the Fundacio´ Pi i Sunyer (E00667). The authors are very grateful to Dr Luciana Tessitore (Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Torino, Italy) for the generous sample of Yoshida cells given. In addition, we express our gratitude to Dr Roser Casamitjana (Servei d’Hormones, Hospital Clı´nic i Provincial, Barcelona, Spain) for her kind help in the determination of insulin concentrations.
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Sequential changes in lipoprotein lipase activity and lipaemia induced by the Yoshida AH-130 ascites hepatoma in rats Joaquı´n Lo´pez-Soriano, Josep M. Argile´s, Francisco J. Lo´pez-Soriano* Departament de Bioquı´mica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08071-Barcelona, Spain Received 14 January 1997; accepted 4 March 1997
Abstract The implantation of the Yoshida AH-130 ascites hepatoma to rats resulted in an exponential growth of the tumour cells followed by a late stationary phase. The tumour burden was accompanied by a dramatic decrease in body weight. Tumour growth was associated with a marked hypertriglyceridaemia during the period of exponential growth, while in the stationary phase the plasma triacylglycerol concentration was similar to that observed in the non-tumour-bearing animals. Similar increases were observed, following tumour inoculation, in the plasma concentrations of non-esterified fatty acids and glycerol, suggesting an intense lipolytic activity. These changes in lipaemia were associated with a marked decrease in LPL activity in white adipose tissue; in contrast, LPL activity was increased in the tumour-bearing animals in brown adipose tissue at day 6 following inoculation and in the heart during most of the period studied. Although the presence of the tumour did not induce any changes in blood lactate concentrations, it caused a decrease in circulating glucose; conversely, the tumour induced an important increase in the concentration of circulating ketone bodies, suggesting a metabolic adaptation of the tumour-bearing rats to glucose sparing and alternative fuel utilization. It may be suggested that the hyperlipidaemia present in the Yoshida AH-130 bearing rats is partly due to a decreased LPL activity in white adipose tissue which does not seem to be influenced by changes in insulin circulating concentrations. 1997 Elsevier Science Ireland Ltd. Keywords: Tumour growth; Circulating triglycerides; Lipoprotein lipase; Cancer cachexia; Rat
1. Introduction Hypertriglyceridaemia and depletion of fat stores have been observed both in patients [1,2] and experimental animals [3–5] during the growth of a wide variety of neoplasms. Understanding the mechanisms involved in the depletion of body fat may have clinical relevance because weight loss itself has a bad prognostic value [6] and the amount of remaining fat in * Corresponding author. Tel.: +34 3 4021525; fax: +34 3 4021559; e-mail: [email protected]
starved individuals closely correlates with the duration of survival [7]. Animal studies reveal that loss of fat is not caused by decreased food intake alone, since it precedes the onset of anorexia in mice [8] and it is more severe in tumour-bearing animals than in pair-fed controls [9]. Thus, increased catabolic events such as enhanced lipolytic rate [10,11] and a reduction in adipose tissue lipoprotein lipase (LPL) activity promote fat mobilization during tumour growth [4,11–13]. A decrease in LPL content or activity in a tissue usually correlates with a decreased uptake of triacylglycerol fatty acids
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by that tissue [14], which is also the case in tumourbearing animals [4]. In the latter, another contributing mechanism is an important decrease in the rate of lipogenesis in adipose tissue [11,15], which is consistent with a reduction in the activities of enzymes involved in this metabolic pathway [12]. Although a great deal of work concerning the metabolic effects caused by the presence of a malignant tumour has been carried out, the results concerning the effects of tumour burden on host lipid metabolism are not so clear, with contradictory results in both experimental animals and man. Lipid oxidation has been reported as either increased [8,16], unchanged [17] or decreased [18]. In addition, LPL activity decreases in adipose tissue [11,12], with a concomitant increase in plasma triacylglycerol levels [12]. Bearing all this in mind, the aim of the present investigation was to characterize the changes in LPL activity and lipaemia associated with tumour growth. In order to accomplish this objective, we have used the rat Yoshida AH-130 ascites hepatoma, a rapidly growing tumour.
2. Materials and methods 2.1. Animals and tumour implantation Female Wistar rats (12 weeks old) from our own colony were fed ad libitum on a chow diet (Panlab, Barcelona, Spain) consisting (by weight) of 54% carbohydrate, 17% protein and 5% fat (the residue was non-digestible material), with free access to drinking water, and were kept in individual polypropylene cages fitted with wire-mesh bottoms at an ambient temperature of 22 ± 2°C with a 12 h-light/12 h-dark cycle (lights on from 0800 h). Food intake and body weight were measured daily after tumour inoculation. A Yoshida AH-130 ascites hepatoma cell suspension (approx. 120 × 106 cells in 2 ml) was injected intraperitoneally, the control rats being injected with 2 ml of sterile 0.9% (w/v) NaCl solution. Total cell number was estimated using Trypan blue staining. 2.2. Biochemicals and radioactive compounds The biochemical and radioactive compounds were all reagent grade and obtained either from Boehringer
Mannheim S.A. (Barcelona, Spain) or from Sigma Chemical Co. (St. Louis, MO, USA). Radiochemicals were purchased from Amersham Int. (Amersham, Bucks., UK). 2.3. Lipoprotein lipase activity Perirenal white adipose tissue (WAT), heart and interscapular brown adipose tissue (BAT) LPL activities were measured by a modification of the technique of Nilsson-Ehle and Ekman [19]. Tissue samples were dried to a powder with acetone/ether and then resolubilized and used in an assay system containing [3H]triolein as substrate; [3H]fatty acids released after a 60 min incubation period were extracted and determined by the method of Nilsson-Ehle and Schotz [20]. LPL activity is expressed as nmol of fatty acid released/min per mg of acetone-dried powder. 2.4. Blood metabolites and plasma insulin Whole blood glucose was determined by the method of Slein [21] and lactate was determined by the method of Hohorst et al. [22]. Blood acetoacetate and b-hydroxybutyrate were determined fluorimetrically [23]. Plasma insulin was determined by radioimmunoassay, with a rat insulin standard [24]. Plasma triacylglycerols [25], glycerol [26] and non-esterified fatty acids [27] were also measured. 2.5. Statistics The results are expressed as mean ± SEM for the number of individual observations indicated in each experiment. Statistical significance was determined by Student’s t-test.
3. Results and discussion The Yoshida AH-130 is a highly cachectic rapidly growing rat tumour containing poorly differentiated cells, with a relatively short doubling time of 1 day [28]. After tumour inoculation, there is an exponentially growing phase which lasts until day 8, followed by a stationary growth phase which precedes the animals’ deaths [29]. Growth of the ascites hepatoma AH-130 in rats elicits an early and conspicuous loss
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of body weight and skeletal muscle mass, associated with a protein hypercatabolic state in host tissues [30], as well as a profound perturbation in hormonal homeostasis, an elevation of plasma prostaglandin E2 and the presence of circulating TNF [5]. Lipid metabolism is also disturbed, with increased levels of total cholesterol and triacylglycerols and reduced levels of esterified cholesterol in the blood plasma [5,31]. This tumour also induces hepatomegaly and a dramatic loss of adipose tissue [15]. These features have been observed in other rapidly growing tumour models in the rat such as the Walker 256 carcinosarcoma [4].
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concentrations of free glycerol (2.2 fold at day 4) as a result of tumour growth (Fig. 2). Glycerol is another indicator of an intense lipolytic activity, being preferentially used by the liver for glucose production during tumour burden [3]. 3.3. Lipoprotein lipase activity The implantation of the tumour resulted in alterations in tissue LPL activity (Fig. 3). Thus, LPL activ-
3.1. Body weight and tumour burden Previous results have shown that the presence of the Yoshida AH-130 ascites hepatoma induces a marked weight reduction in the tumour-bearing host, which is associated with decreases in both food intake and energy efficiency in the last period of tumour growth [29]. As shown in Fig. 1, the tumour induced a significant decrease in body weight (subtracting the tumour mass) in comparison with the control (day 0) group, already detectable at day 4 after implantation. At day 10 following tumour inoculation the decrease in body weight reached 30%. The decrease in body weight was accompanied by a very characteristic increase in tumour cell content (Fig. 1), as previously reported [29]. 3.2. Hypertriglyceridaemia As shown in Fig. 2, the presence of the tumour promoted important changes in the circulating concentration of triacylglycerols. Thus, at day 4 following inoculation the concentration was already elevated (2-fold increase), reaching a peak at day 6 (2.3-fold increase). Conversely, there was a tendency towards normalization of the triacylglycerol concentrations towards control levels at the end of the inoculation period, when the tumour reaches a stationary phase of growth [29]. The changes in triglyceridaemia were accompanied by increases in the plasma concentration of non-esterified fatty acids at the end of the period studied (Fig. 2). These results clearly indicate that the tumour-bearing host is under a strong lipolytic activity, as previously described [10,11]. Interestingly, an increase was also detected in the circulating
Fig. 1. Body weight and tumour cell number in rats bearing the Yoshida AH-130 hepatoma. For further details see Section 2. The results are mean values ± SEM for five different animals. Body weights exclude the weight of the tumour plus ascitic fluid. Values that are statistically significant differences (Student’s t-test, P , 0.05) from those of the control (day 0) group were already present at day 4 after tumour inoculation.
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LPL activity) are well-known uncouplers of oxidative phosphorylation in BAT [33]. Cardiac LPL was increased during tumour growth (Fig. 3); it may be suggested that an enhanced LPL activity in this organ may serve as an alternative strategy for substrate uptake (fatty acids) in a metabolic environment where there is a high glucose extraction by the tumour [3]. Hyperlipidaemia, particularly an elevation of circulant triacylglycerol concentration, is a general feature both in cancer patients [34] and animals bearing a number of different tumours [4,35]. The mechanism responsible for the development of the hypertriglyceridaemia is not fully understood, although a role of LPL deficiency in peripheral tissues has been suggested by several authors [11,12,36]. Other studies, however, did not find a correlation between the lipid levels in plasma and the activity of LPL in cancer patients [37]. In our study, changes in WAT LPL activity correlate well with the increase in circulating triacylglycerols (Fig. 2), thus suggesting that the hyperlipidaemia present in the Yoshida AH-130 bearing rats is partly due to a decreased LPL activity in WAT which seems to be only slightly influenced by changes in insulin circulating concentrations. Indeed, insulin activates LPL activity in WAT [38]. However,
Fig. 2. Plasma triacylglycerols, non-esterified fatty acids and glycerol concentrations in rats bearing the Yoshida AH-130 hepatoma. For further details see Section 2. The results are mean values ± SEM for five different animals. Plasma concentrations are expressed as mg/100 ml for triacylglycerols, mmol/l for glycerol and mmol/100 ml for non-esterified fatty acids. Values that are significantly different by Student’s t-test from those of the control (day 0) group are indicated by: *P , 0.05, **P , 0.01, ***P , 0.00l.
ity is considerably decreased in WAT, reaching the lowest value at day 6 (75% decrease). Interestingly, BAT LPL activity is subject to little changes in the tumour-bearing animals except for a punctual increase at day 6. This increase may possibly have a relation with the increased thermogenic activity found in this tissue during tumour growth [32], since fatty acids (their uptake being enhanced through the increased
Fig. 3. Tissue lipoprotein lipase (LPL) activity in rats bearing the Yoshida AH-130 hepatoma. For further details see Section 2. LPL activity is expressed as pmol of fatty acid released/min per mg of acetone-dried tissue. The results are mean values ± SEM for five different animals. Values that are significantly different by Student’s t-test from those of the control (day 0) group are indicated by: *P , 0.05, **P , 0.01, ***P , 0.001. WAT, white adipose tissue; BAT, brown adipose tissue.
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the decrease in WAT LPL activity was also observed when the insulin concentrations were close to control values (days 6–10) (Fig. 4), this probably being an indication of the insulin resistant status of the tumourbearing host [3]. 3.4. Blood metabolites Glycaemia was only maintained for the first 2 days
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after tumour inoculation, the circulating glucose concentrations being significantly reduced with respect to those from day 0 to day 4 onwards (Fig. 4). No changes were detected in blood lactate as a consequence of tumour burden. However, ketone bodies (acetoacetate and b-hydroxybutyrate) blood levels were significantly increased from day 4 onwards (Fig. 4). The increase was observed earlier for bhydroxybutyrate than for acetoacetate; the highest increases were detected at day 10, i.e. 6.8- and 17.3fold for b-hydroxybutyrate and acetoacetate, respectively. Plasma insulin levels were significantly reduced only on day 4 after tumour implantation.
4. Conclusion
Fig. 4. Blood metabolites and plasma insulin concentrations in rats bearing the Yoshida AH-130 hepatoma. For further details see Section 2. The results are mean values ± SEM for five different animals. Blood glucose and lactate concentrations are mM, plasma insulin is expressed as mU/100 ml, and plasma ketone bodies concentrations are mM. Values that are significantly different by Student’s t-test from those of the control (day 0) group are indicated by: *P , 0.05, **P , 0.01, ***P , 0.00l.
Some of the metabolic changes associated with tumour growth may be due to cytokines such as tumour necrosis factor-a (TNF). This peptide, mainly produced by activated macrophages, has a wide range of metabolic effects (see Ref [39] for review). Yoshida AH-130 bearing rats show important perturbations in hormonal homeostasis, which include the presence of circulating TNF [5]. Recently, we have demonstrated that the administration of antibodies against murine TNF can partially reverse the changes observed in LPL activity in this experimental model [40] as well as the accelerated protein turnover [41]. The alterations in lipid metabolism observed in the cancer patient, which contribute to the metabolic abnormalities observed in situations of cancer cachexia, cannot be solely attributed to the reduction in food intake, since they often precede the onset of anorexia [8]. On these lines, cancer cachexia, characterized by an accelerated depletion of peripheral protein and lipid stores, cannot be reversed by forced hyperalimentation. In future, therefore, the design of therapeutic strategies to counteract the cachexia found in the cancer patient will have to focus on an effective neutralization of the changes in lipid metabolism induced by the tumour in the host.
Acknowledgements This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social
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(F.I.S.) (94/1048) of the Spanish Health Ministry, from the DGICYT (PB 94-0938) of the Spanish Ministry of Education and Science and from the Fundacio´ Pi i Sunyer (E00667). The authors are very grateful to Dr Luciana Tessitore (Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale, Torino, Italy) for the generous sample of Yoshida cells given. In addition, we express our gratitude to Dr Roser Casamitjana (Servei d’Hormones, Hospital Clı´nic i Provincial, Barcelona, Spain) for her kind help in the determination of insulin concentrations.
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