Clinical Science and Molecular Medicine (1976) 50, 315-31 8.
SHORT COMMUNICATION
Lipoprotein lipase and hormone-sensitive lipase activities in human subcutaneous lipomas: comparison with normal subcutaneous adipose tissue Y. GIUDICELLI,* R. PECQUERY,* B. AGLI,* C. J A M I N ? A N D J. QUEVAUVILLIERSt *Service de Biochimie and ?Service de Mtdecine Interne, Centre Hospiralier Intercommunal, Poissy, France
(Received 7 January 1976)
summary 1. Lipoprotein lipase activity and hormonesensitive lipase activity were investigated in subcutaneous lipomas removed from two patients and compared with the enzyme activities in subcutaneous adipose tissue from two normal subjects. 2. Confirmation was obtained of the presence of lipoprotein lipase activity in lipomas with an activity fifteen to forty-five times that in the two control samples. 3. Hormonssensitive lipase activity was demonstrated in lipomas under basal conditions of assay as well as in the presence of adrenaline plus theophylline. However, compared with the non-lipomatous fat samples, these activities were lower, as was the magnitude of the lipolytic response to adrenaline plus theophylline. 4. The significance of these measurements of enzyme activity and their role in the pathogenesis of lipomas are briefly discussed.
Key words : adipose tissue, hormone-sensitive lipase, lipomas, lipoprotein lipase.
Introduction
Despite the numerous studies devoted to the metabolism of normal adipose tissue, the biochemicaldisturbancesinvolved in the pathogenesis of lipoma have not been extensivelystudied and remain therefore still obscure. The occurrence of lipomas may, in theory, result from localized increased lipogenesis, from decreased lipolysis or from both. Increased lipogenesis was formerly suggested by Gellhorn & Marks (1961), who first reported that lipomas incorporate acetate into mixed lipids at a greater rate than occurs in n o d adipose tissue. Lipoprotein lipase activity, an important factor regulating the tissue uptake and storage of blood triglyceride fatty acids, has been reported to be lacking (Marshall, 1965) or to be markedly higher in lipomas than in normal adipose tissue (Etienne, Van den Akker, Mafart, Pieron, Debray & Polonovski, 1974). Since fat mobilization and consequently fat storage are controlled by the so-called hormonesensitive lipase activity, modification of this activity could contribute to the pathogenesis of lipomas. In order to test this hypothesis and because of the contradictory data concerning lipoprotein lipase, we have compared the two enzymes activities in lipomas and in normal subcutaneous adipose tissue.
Lipoma is a benign, slow-growing fat tumour occurring at any site where adipose tissue exists, especially in the subcutaneoustissue, and which is distinguished from normal adipose tissue by the presence of a fibrous capsule.
Materialandmethods Four subjectswere studied. Their agesranged from 30 to 40 years. None was known to be diabetic, thin or obese. Fasting blood sugar, triglycerides and cholesterol values were within n o d ranges in all patients.
Correspondence: Dr Y. Giudicelli, Service de Biochimie, Centre HospitaIier Intercommunal, 78303 Poissy, France.
315
316
Y.Giudicelli et al.
Lipomas were removed from the anterior abdominal wall of two patients. Since the metabolic activity of human subcutaneous adipose tissue was reported to vary from one anatomical site to another (Hirsch & Goldrick, 1965), normal subcutaneous fat was also obtained from the anterior abdominal wall of the two other subjects. Since seasonal variations of lipoprotein lipase activity have also been reported in human subcutaneous adipose tissue (Persson, 1974), care was taken to obtain the different samples during the same period (January-February) of the year. Operations were performed after an overnight fast ; samples, excised under local anaesthesia induced with xylocaine (10 g/l), were immediately transferred in ice-cold NaCl (150 mmol/l), rinsed, blotted on filter paper and weighed. In each case informed consent of the patient was obtaihed. Lipoma samples were also examined by lightmicroscopy, with conventional inclusion and staining methods (haematoxylin, eosin, Safflor Yellow, Malachite Green, silver carbonate). For the determination of lipoprotein lipase activity, lipomas and normal subcutaneous adipose tissue were rapidly homogenized in sucrose (250 mmol/l), pH 8.4, containing heparin (2 i.u./ml). The lipoprotein lipase activity in the homogenates was determined with Intralipid (Vitrum) as substrate and by following the conditions described previously for rat adipose tissue (Giudicelli, Nordmann & Nordmann, 1975), except that the incubation
medium contained normal fasting human serum in lieu of rat serum and was adjusted to pH 8.4, which was found optimal in these experimental conditions. Five paired assays per tissue sample were incubated in a metabolic shaker at 37°C with air as gas phase. The two paired samples were removed after 5 min and 65 min respectively and the amount of nonesterified fatty acids in each assay was determined by the method of Dole & Meinertz (1960). The difference corrected for blank values was taken as a measure of lipoprotein lipase activity, which was expressed as pmol of non-esterified fatty acids released h-' g-' fresh weight at 37°C. Hormone-sensitive lipase activity was studied in the basal state as well as in the presence of both adrenaline and theophylline. Samples were divided into pieces weighing approximately 50-60 mg and preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate buffer, pH 7.4, containing glucose (5 mmol/l). After preincubation, 150-200 mg of tissue was transferred into stoppered flasks containing 5 ml of Krebs-Ringer bicarbonate buffer, pH 7.4, with bovine albumin (40 g/l) and glucose (5 mmol/l). Incubations were performed for 3 h at 37°C with gentle shaking and under air as gas phase. Addition of adrenaline (50 pmol/l) and theophylline (5 mmol/l) to the medium was done at zero time. At the end of the incubation, the glycerol and non-esterified fatty acids released into the medium were determined as previously described (Giudicelli, Provin & Nordmann, 1975) and taken as
TABLE1. Hormone-sensitive lipase and lipoprotein lipase activities in human subcutaneous lipomas Activities are compared with those of normal human subcutaneous adipose tissue. Mean values? SEM represent the assay of ten adipose tissue samples obtained from two normal subjects or of five lipoma samples removed from either one patient (case no. 1) or another patient (case no. 2). The statistical significance (P values) refers to the data for normal adipose tissue. NEFA= non-esterified fatty acids. Hormone-sensitive lipase activity (pmol 3 h-' g-' wet wt.) Lipoprotein lipase activity With adrenaline (50 pmol/l) and theophylline (5 mmol/l) @mol of NEFA h-' g-' wet wt.)
Basal Glycerol
NEFA
Glycerol
NEFA
Normal adipose tissue 2.35+ 0.43
6.07k 0.53
11.95+ 0.92
26.31 2.32
0.20k 0.07
Lipomatosis (case no. 1)
0.82k0.16 (P
SHORT COMMUNICATION
Lipoprotein lipase and hormone-sensitive lipase activities in human subcutaneous lipomas: comparison with normal subcutaneous adipose tissue Y. GIUDICELLI,* R. PECQUERY,* B. AGLI,* C. J A M I N ? A N D J. QUEVAUVILLIERSt *Service de Biochimie and ?Service de Mtdecine Interne, Centre Hospiralier Intercommunal, Poissy, France
(Received 7 January 1976)
summary 1. Lipoprotein lipase activity and hormonesensitive lipase activity were investigated in subcutaneous lipomas removed from two patients and compared with the enzyme activities in subcutaneous adipose tissue from two normal subjects. 2. Confirmation was obtained of the presence of lipoprotein lipase activity in lipomas with an activity fifteen to forty-five times that in the two control samples. 3. Hormonssensitive lipase activity was demonstrated in lipomas under basal conditions of assay as well as in the presence of adrenaline plus theophylline. However, compared with the non-lipomatous fat samples, these activities were lower, as was the magnitude of the lipolytic response to adrenaline plus theophylline. 4. The significance of these measurements of enzyme activity and their role in the pathogenesis of lipomas are briefly discussed.
Key words : adipose tissue, hormone-sensitive lipase, lipomas, lipoprotein lipase.
Introduction
Despite the numerous studies devoted to the metabolism of normal adipose tissue, the biochemicaldisturbancesinvolved in the pathogenesis of lipoma have not been extensivelystudied and remain therefore still obscure. The occurrence of lipomas may, in theory, result from localized increased lipogenesis, from decreased lipolysis or from both. Increased lipogenesis was formerly suggested by Gellhorn & Marks (1961), who first reported that lipomas incorporate acetate into mixed lipids at a greater rate than occurs in n o d adipose tissue. Lipoprotein lipase activity, an important factor regulating the tissue uptake and storage of blood triglyceride fatty acids, has been reported to be lacking (Marshall, 1965) or to be markedly higher in lipomas than in normal adipose tissue (Etienne, Van den Akker, Mafart, Pieron, Debray & Polonovski, 1974). Since fat mobilization and consequently fat storage are controlled by the so-called hormonesensitive lipase activity, modification of this activity could contribute to the pathogenesis of lipomas. In order to test this hypothesis and because of the contradictory data concerning lipoprotein lipase, we have compared the two enzymes activities in lipomas and in normal subcutaneous adipose tissue.
Lipoma is a benign, slow-growing fat tumour occurring at any site where adipose tissue exists, especially in the subcutaneoustissue, and which is distinguished from normal adipose tissue by the presence of a fibrous capsule.
Materialandmethods Four subjectswere studied. Their agesranged from 30 to 40 years. None was known to be diabetic, thin or obese. Fasting blood sugar, triglycerides and cholesterol values were within n o d ranges in all patients.
Correspondence: Dr Y. Giudicelli, Service de Biochimie, Centre HospitaIier Intercommunal, 78303 Poissy, France.
315
316
Y.Giudicelli et al.
Lipomas were removed from the anterior abdominal wall of two patients. Since the metabolic activity of human subcutaneous adipose tissue was reported to vary from one anatomical site to another (Hirsch & Goldrick, 1965), normal subcutaneous fat was also obtained from the anterior abdominal wall of the two other subjects. Since seasonal variations of lipoprotein lipase activity have also been reported in human subcutaneous adipose tissue (Persson, 1974), care was taken to obtain the different samples during the same period (January-February) of the year. Operations were performed after an overnight fast ; samples, excised under local anaesthesia induced with xylocaine (10 g/l), were immediately transferred in ice-cold NaCl (150 mmol/l), rinsed, blotted on filter paper and weighed. In each case informed consent of the patient was obtaihed. Lipoma samples were also examined by lightmicroscopy, with conventional inclusion and staining methods (haematoxylin, eosin, Safflor Yellow, Malachite Green, silver carbonate). For the determination of lipoprotein lipase activity, lipomas and normal subcutaneous adipose tissue were rapidly homogenized in sucrose (250 mmol/l), pH 8.4, containing heparin (2 i.u./ml). The lipoprotein lipase activity in the homogenates was determined with Intralipid (Vitrum) as substrate and by following the conditions described previously for rat adipose tissue (Giudicelli, Nordmann & Nordmann, 1975), except that the incubation
medium contained normal fasting human serum in lieu of rat serum and was adjusted to pH 8.4, which was found optimal in these experimental conditions. Five paired assays per tissue sample were incubated in a metabolic shaker at 37°C with air as gas phase. The two paired samples were removed after 5 min and 65 min respectively and the amount of nonesterified fatty acids in each assay was determined by the method of Dole & Meinertz (1960). The difference corrected for blank values was taken as a measure of lipoprotein lipase activity, which was expressed as pmol of non-esterified fatty acids released h-' g-' fresh weight at 37°C. Hormone-sensitive lipase activity was studied in the basal state as well as in the presence of both adrenaline and theophylline. Samples were divided into pieces weighing approximately 50-60 mg and preincubated for 30 min at 37°C in Krebs-Ringer bicarbonate buffer, pH 7.4, containing glucose (5 mmol/l). After preincubation, 150-200 mg of tissue was transferred into stoppered flasks containing 5 ml of Krebs-Ringer bicarbonate buffer, pH 7.4, with bovine albumin (40 g/l) and glucose (5 mmol/l). Incubations were performed for 3 h at 37°C with gentle shaking and under air as gas phase. Addition of adrenaline (50 pmol/l) and theophylline (5 mmol/l) to the medium was done at zero time. At the end of the incubation, the glycerol and non-esterified fatty acids released into the medium were determined as previously described (Giudicelli, Provin & Nordmann, 1975) and taken as
TABLE1. Hormone-sensitive lipase and lipoprotein lipase activities in human subcutaneous lipomas Activities are compared with those of normal human subcutaneous adipose tissue. Mean values? SEM represent the assay of ten adipose tissue samples obtained from two normal subjects or of five lipoma samples removed from either one patient (case no. 1) or another patient (case no. 2). The statistical significance (P values) refers to the data for normal adipose tissue. NEFA= non-esterified fatty acids. Hormone-sensitive lipase activity (pmol 3 h-' g-' wet wt.) Lipoprotein lipase activity With adrenaline (50 pmol/l) and theophylline (5 mmol/l) @mol of NEFA h-' g-' wet wt.)
Basal Glycerol
NEFA
Glycerol
NEFA
Normal adipose tissue 2.35+ 0.43
6.07k 0.53
11.95+ 0.92
26.31 2.32
0.20k 0.07
Lipomatosis (case no. 1)
0.82k0.16 (P
