253
Atherosclerosis, 33 (1979) 253-258 0 Elsevier/North-Holland Scientific Publishers, Ltd.
LIPOPROTEIN LIPASE ACTIVITY IN HUMAN ADIPOSE TISSUE DURING INDUCED HYPERTRIGLYCERIDAEMIA
G. HOLDSWORTH,
K.G. TAYLOR
* and D.J. GALTON
Diabetes and Lipid Research Laboratory, (Great Britain)
St Bartholomew’s
Hospital, London
ECIA
7BE
(Received 23 November, 1978) (Revised, received 27 December, 1978) (Accepted 5 January, 1979)
Summary Needle biopsies of adipose tissue and blood samples were obtained before and at short intervals after a “bolus” injection of 10% Intralipid. Lipoprotein lipase activities were measured in acetone-ether extracts of the tissue samples. Levels of serum triglyceride began to fall less than 5 min after the injection of the Intralipid with a half-life of 20 min. During this time interval, no significant changes were observed in the activities of lipoprotein lipase in adipose tissue. A patient with a severe hypertriglyceridaemia (Type V) underwent plasma exchange with a reduction in serum triglyceride levels from 11 to 4 mm/l. There was a parallel fall in adipose tissue lipoprotein lipase activity. We conclude that lipoprotein lipase activity in adipose tissue is unaltered during experimental hypertriglyceridaemia and that the activity of the enzyme in adipose tissue is probably not reduced as a secondary feature of an elevated plasma triglyceride level. Key words:
Hypertriglyceridaemia
-Lipoprotein
lipase -Plasma
exchange
Introduction Primary hypertriglyceridaemia probably comprises a heterogeneous group of metabolic disorders. Both an overproduction of VLDL by the liver and a failure of clearance mechanisms in the periphery have been proposed as important * Present address: Department of Diabetes, Birmingham General Hospital, Steelhouse Lane, Birmingham B4 6NH. Great Britain.
254
determinants of hypertriglyceridaemia in some patients [ 11. Lipoprotein lipase is a rate-determining enzyme in the hydrolysis and clearance of VLDL and chylomicron triglyceride, and measurements of the activity of this enzyme in adipose tissue or muscle may indicate whether a clearance defect is present in patients with hypertriglyceridaemia. However, studies involving the intravenous infusion of Intralipid, a synthetic analogue of triglyceride-rich lipoproteins, have suggested that experimental hypertriglyceridaemia can decrease the activity of the enzyme in both rat adipose tissue [ 21, and the post-heparin plasma lipolytic activity in man [ 31. If high levels of serum triglyceride can lead to a secondary depletion of tissue lipoprotein lipase activities, then the importance of measuring the enzyme to clarify the aetiology of the hypertriglyceridaemias would be reduced. To examine this point, we have treated 6 patients with a “bolus” injection of 10% Intralipid and then followed the subsequent changes in serum triglyceride and adipose tissue lipoprotein lipase by means of serial blood samples and tissue biopsies. A patient with a severe Type V hyperlipidaemia underwent plasma exchange to remove the large quantities of circulating VLDL and chylomicra. The changes in the tissue lipase activity of this patient were also measured before and after reduction of plasma triglyceride. Material and Methods Patients Six female patients were investigated (mean age 38 + 7 yr, mean weight 98.2 + 4.9 kg, mean fasting glucose 4.2 * 0.6 mm/l, mean fasting plasma triglyceride 2.02 + 0.32 mm/l). All had been referred to the Lipid and Diabetic Clinics of St Bartholomew’s Hospital and had undergone an overnight fast immediately prior to the study. None of the patients were receiving any drug therapy prior to the study. A “bolus” injection .of Intralipid (0.1 g/kg) was administered intravenously over about 2 min. Adipose tissue Adipose tissue samples were obtained by subcutaneous needle biopsy from the anterior abdominal wall after infiltration of the skin with 2% lignocaine 141. Plasma exchange (a) Patient: A male patient, age 66 yr, weight 94.7 kg, fasting glucose 7.6 mm/l, had been admitted to hospital to stabilize his diabetes mellitus and severe Type V hyperlipidaemia. Fasting plasma triglyceride on admission was 60 mm/l. (b) Procedure: Plasma exchange was carried out by an intermittent flow technique using a cell separator (Hemonetics model 30). Plasma was exchanged for a plasma protein fraction. 3.5 1 of plasma was exchanged in 2.5 h. Acidcitrate saline was used as the anticoagulant. The plasma triglyceride at the end of exchange was 4.2 mm/l.
255
Measurement of lipoprotein lipase activity of the (a) Acetone--ether extraction: This was carried out by a modification procedure of Salaman and Robinson [ 51. Weighed amounts of tissue were thoroughly homogenized in a 2% w/v aqueous solution of fatty acid free bovine serum albumin and then treated with an excess of cold acetone (4°C) to precipitate the proteins. After a further homogenization, the resulting suspensions were centrifuged in a bench centrifuge, and the pellets washed with several volumes of cold acetone, and dðyl ether. After the final ether wash, the pellets were allowed to dry. Immediately before the enzyme assay, the dried tissue powders were resuspended in 0.05 M NH4Cl buffer, pH 8.6. (b) Assay: The assay for lipoprotein lipase activity was based on the radiochemical method described by Krauss et al. [6]. 12.5 mg of glyceryl trioleate, together with 6 E.tCi of glyceryl tri-[ 1-14C]oleate and 1.25 mg of lysolecithin were ultrasonicated for 4 min at maximum power using an MSE 1OOW ultrasonic disintegrator with titanium probe, in a solution of 6 ml of 0.16 M TrisHCl buffer, pH 8.1, containing 20 mg of crystalline bovine serum albumin. A volume, 250 ~1, of this substrate emulsion was used in each assay, together with 100 ~1 of normal human serum taken when fasting, to activate the enzyme. Incubations were for 1 h at 37°C and then 200 ~1 was extracted in a BelfrageVaughan partition system. A volume, 1 ml, of the upper phase was counted in a Packard liquid scintillation counter, model 2420. Presentation of results Adipose cell size and triglyceride content were determined [7], enabling results of enzyme activity to be expressed as nmol free fatty acid/lo6 cells/h. Results are expressed as means f SEM, and the significance of differences calculated by the Student paired t-test.
TABLE 1 LIPOPROTEIN LIPASE ACTIVITIES IN ADIPOSE TISSUE AND SERUM TRIGLYCERIDE BEFORE AND AFTER A BOLUS INJECTION OF INTRALIPID
LEVELS
Results are means f SEM. Minutes 0 Adipose tissue
110.8
t 45.4
10
1
5
N.D.
159.5
6.71 r 1.95
(6) N.S. 5.12 +
(2) -
(6)
Atherosclerosis, 33 (1979) 253-258 0 Elsevier/North-Holland Scientific Publishers, Ltd.
LIPOPROTEIN LIPASE ACTIVITY IN HUMAN ADIPOSE TISSUE DURING INDUCED HYPERTRIGLYCERIDAEMIA
G. HOLDSWORTH,
K.G. TAYLOR
* and D.J. GALTON
Diabetes and Lipid Research Laboratory, (Great Britain)
St Bartholomew’s
Hospital, London
ECIA
7BE
(Received 23 November, 1978) (Revised, received 27 December, 1978) (Accepted 5 January, 1979)
Summary Needle biopsies of adipose tissue and blood samples were obtained before and at short intervals after a “bolus” injection of 10% Intralipid. Lipoprotein lipase activities were measured in acetone-ether extracts of the tissue samples. Levels of serum triglyceride began to fall less than 5 min after the injection of the Intralipid with a half-life of 20 min. During this time interval, no significant changes were observed in the activities of lipoprotein lipase in adipose tissue. A patient with a severe hypertriglyceridaemia (Type V) underwent plasma exchange with a reduction in serum triglyceride levels from 11 to 4 mm/l. There was a parallel fall in adipose tissue lipoprotein lipase activity. We conclude that lipoprotein lipase activity in adipose tissue is unaltered during experimental hypertriglyceridaemia and that the activity of the enzyme in adipose tissue is probably not reduced as a secondary feature of an elevated plasma triglyceride level. Key words:
Hypertriglyceridaemia
-Lipoprotein
lipase -Plasma
exchange
Introduction Primary hypertriglyceridaemia probably comprises a heterogeneous group of metabolic disorders. Both an overproduction of VLDL by the liver and a failure of clearance mechanisms in the periphery have been proposed as important * Present address: Department of Diabetes, Birmingham General Hospital, Steelhouse Lane, Birmingham B4 6NH. Great Britain.
254
determinants of hypertriglyceridaemia in some patients [ 11. Lipoprotein lipase is a rate-determining enzyme in the hydrolysis and clearance of VLDL and chylomicron triglyceride, and measurements of the activity of this enzyme in adipose tissue or muscle may indicate whether a clearance defect is present in patients with hypertriglyceridaemia. However, studies involving the intravenous infusion of Intralipid, a synthetic analogue of triglyceride-rich lipoproteins, have suggested that experimental hypertriglyceridaemia can decrease the activity of the enzyme in both rat adipose tissue [ 21, and the post-heparin plasma lipolytic activity in man [ 31. If high levels of serum triglyceride can lead to a secondary depletion of tissue lipoprotein lipase activities, then the importance of measuring the enzyme to clarify the aetiology of the hypertriglyceridaemias would be reduced. To examine this point, we have treated 6 patients with a “bolus” injection of 10% Intralipid and then followed the subsequent changes in serum triglyceride and adipose tissue lipoprotein lipase by means of serial blood samples and tissue biopsies. A patient with a severe Type V hyperlipidaemia underwent plasma exchange to remove the large quantities of circulating VLDL and chylomicra. The changes in the tissue lipase activity of this patient were also measured before and after reduction of plasma triglyceride. Material and Methods Patients Six female patients were investigated (mean age 38 + 7 yr, mean weight 98.2 + 4.9 kg, mean fasting glucose 4.2 * 0.6 mm/l, mean fasting plasma triglyceride 2.02 + 0.32 mm/l). All had been referred to the Lipid and Diabetic Clinics of St Bartholomew’s Hospital and had undergone an overnight fast immediately prior to the study. None of the patients were receiving any drug therapy prior to the study. A “bolus” injection .of Intralipid (0.1 g/kg) was administered intravenously over about 2 min. Adipose tissue Adipose tissue samples were obtained by subcutaneous needle biopsy from the anterior abdominal wall after infiltration of the skin with 2% lignocaine 141. Plasma exchange (a) Patient: A male patient, age 66 yr, weight 94.7 kg, fasting glucose 7.6 mm/l, had been admitted to hospital to stabilize his diabetes mellitus and severe Type V hyperlipidaemia. Fasting plasma triglyceride on admission was 60 mm/l. (b) Procedure: Plasma exchange was carried out by an intermittent flow technique using a cell separator (Hemonetics model 30). Plasma was exchanged for a plasma protein fraction. 3.5 1 of plasma was exchanged in 2.5 h. Acidcitrate saline was used as the anticoagulant. The plasma triglyceride at the end of exchange was 4.2 mm/l.
255
Measurement of lipoprotein lipase activity of the (a) Acetone--ether extraction: This was carried out by a modification procedure of Salaman and Robinson [ 51. Weighed amounts of tissue were thoroughly homogenized in a 2% w/v aqueous solution of fatty acid free bovine serum albumin and then treated with an excess of cold acetone (4°C) to precipitate the proteins. After a further homogenization, the resulting suspensions were centrifuged in a bench centrifuge, and the pellets washed with several volumes of cold acetone, and dðyl ether. After the final ether wash, the pellets were allowed to dry. Immediately before the enzyme assay, the dried tissue powders were resuspended in 0.05 M NH4Cl buffer, pH 8.6. (b) Assay: The assay for lipoprotein lipase activity was based on the radiochemical method described by Krauss et al. [6]. 12.5 mg of glyceryl trioleate, together with 6 E.tCi of glyceryl tri-[ 1-14C]oleate and 1.25 mg of lysolecithin were ultrasonicated for 4 min at maximum power using an MSE 1OOW ultrasonic disintegrator with titanium probe, in a solution of 6 ml of 0.16 M TrisHCl buffer, pH 8.1, containing 20 mg of crystalline bovine serum albumin. A volume, 250 ~1, of this substrate emulsion was used in each assay, together with 100 ~1 of normal human serum taken when fasting, to activate the enzyme. Incubations were for 1 h at 37°C and then 200 ~1 was extracted in a BelfrageVaughan partition system. A volume, 1 ml, of the upper phase was counted in a Packard liquid scintillation counter, model 2420. Presentation of results Adipose cell size and triglyceride content were determined [7], enabling results of enzyme activity to be expressed as nmol free fatty acid/lo6 cells/h. Results are expressed as means f SEM, and the significance of differences calculated by the Student paired t-test.
TABLE 1 LIPOPROTEIN LIPASE ACTIVITIES IN ADIPOSE TISSUE AND SERUM TRIGLYCERIDE BEFORE AND AFTER A BOLUS INJECTION OF INTRALIPID
LEVELS
Results are means f SEM. Minutes 0 Adipose tissue
110.8
t 45.4
10
1
5
N.D.
159.5
6.71 r 1.95
(6) N.S. 5.12 +
(2) -
(6)
