Acta anaesth. scand. 1976, 20, 327-333
Effects of Halothane on the Metabolism of Human Adipose Tissue JARL
BENNIS, JIM OLSONAND ULF SMITH
Department of Anaesthesiology, Eastern Hospital, Department of Physical Chemistry and Department of Medicine 11, Sahlgrens Hospital, University of Gothenburg, Gothenburg, Sweden
The metabolism of specimens of human adipose tissue exposed to different concentrations of halothane was studied. Halothane was added to the incubation medium directly or via the gas phase above the medium. The basal lipolysis was significantly increased by low concentrations of halothane. Higher concentrations clearly diminished the lipolysis, but here, in spite of the inhibitory effect on the basal lipolysis, the lipolytic effect of noradrenaline expressed as percent increment was increased. The rate of lipid synthesis from glucose was reduced when halothane was present in the gas phase. The effect of insulin on glucose metabolism was not affected by the presence of halothane, while the antilipolytic action was abolished by high concentrations of halothane. The results show that halothane may exert dual effects on the mobilization of lipids from human adipose tissue; at low concentrations halothane enhances the basal lipolysis, while at higher concentrations it exerts inhibitory effects. Received 9 January, accepted for publication I 5 February 1976
Halothane anaesthesia is often accompanied techniques used and to elucidate this, we by effects similar to those produced by carried out the present study on the effect of stimulation of the P-adrenergic receptors, different concentrations of halothane on the e.g. vasodilation, bronchial relaxation and lipolytic process. uterine hypotonia. An abstract of this work has appeared I t is now generally recognized that the previously (BENNIS & SMITH1975). lipolytic process in human adipose tissue is mediated by the level of cyclic adenosine 3',5'-monophosphate (cyclic AMP) (CARLSON MATERIAL et al. 1970) induced by the P-receptorThirteen patients undergoing laparotomy were adenyl cyclase system (BURNS& LANGLEY investigated. The patients suffered from isolated 1970, EFENDIC& OSTMAN 1970). abdominal disorders, but those with known jaundice, In a previous work (BENNIS & SMITH1973) malignant or endocrinological diseases were excluded. we studied the metabolism of specimens of Their age range was 29-69 years and their body human adipose tissue obtained before and weights varied from 100-225°k of ideal weight (METROPOLITAN LIFE INSURANCE Co. 1959). All after halothane anaesthesia. The basal lipo- patients fasted overnight before the operation. lytic rate was found to be reduced, while the effect of noradrenaline tended to be increased. Recently, MAKELAINEN et al. M E T HODS (1973b) reported that halothane stimulates the basal lipolysis in rat adipose tissue. The Anaesthetic management discrepancy in results between these in- The patients were anaesthetized according to the vestigations may be due to the different normal procedure of the anaesthetic department; i.e.
328
J. BENNIS, J. OLSSON AND
u.
SMITH
they were premedicated with diazepam and given atropinejust before anaesthesia was started. Anaesthesia was induced with a short-acting barbiturate (thiopental or hexobarbital) ; succinylcholine was given; the patients were intubated; and the anaesthesia was continued with nitrous-oxide, oxygen and halothane plus a muscle-relaxing agent. Immediately after skin incision a specimen of the subcutaneous tissue was taken.
phase according to the method of CARLSON(1959). The incorporation of labelled glucose into the lipids was determined on the chloroform phase in a Packard Tri Carb liquid scintillation spectrometer (Packard, La Grange, Ill., U.S.A.). I n accordance with the suggestion of several investigators (SALANSet al. 1968, BJORNTORP& KARLSSON1970), the metabolic parameters are expressed in terms of the cellularity of the tissue specimens. Mean cell diameter was determined as previously described (SMITHet al. 1972). The mean cellular volume was calculated according to GOLDRICK (1967) and the mean cellular weight as suggested by HIRSCH & GALLIAN(1968). The number of fat cells in the specimens was then determined by dividing the triglyceride content by the mean cellular weight. With the techniques used, the p H of the medium was maintained within 7.4k 0.2 throughout the experiments. Significance levels were calculated according to Student's t-test using an Olivetti table computer (Programma 101).
Incubation procedure in uitro The incubation procedure used is a further development of that described by SMITH (1970). Small fragments of the tissue specimens, weighing about 25-50 mg each, were incubated (in duplicate) in 2.0 ml Krebs-Ringer bicarbonate buffer, with albumin added a t a concentration of 4% (Bovine albumin, Fraction V, Armour Pharmaceutical Co., Eastbourne, England) and in the presence of 0.15 pC D-[1-'4C]glucose (New England Nuclear Corp., Frankfurt am Main, W. Germany) per 2 ml medium, with or without the addition of recrystallized pork insulin (Vitrum AB, Stockholm, Sweden) or noradrenaline (Astra AB, Sodertalje, Sweden) at concentrations of lo5 ,uU/ml and 5 x ~O-'M, respectively. RESULTS I n one set of experiments, halothane (Fluothane") was added to the medium a t an initial concentration The present results are expressed in terms of ~ I I - ~ M , ~O-'M, i O - 4 ~ , 1 0 - ~or~ 1 0 - 2 ~ ,re, of the cellularity of the specimens. It should spectively. All incubations were performed in tightly stoppered glass vials for 2 h at 37°C and p H 7.4. The be clear, however, that the mean fat cell gas phase was 5% v/v COZ and 95% v/v 0 2 . sizes differed between the individuals, thus I n another set of experiments, halothane was affecting the rates of metabolism (SALANS administered a t 23°C in the following concentrations 1970, et al. 1968, BJORNTORP & KARLSSON in a gas mixture: 0.5, 1.0, 1.5, 2.0, and 3.0% v/v. SMITHI970), as shown in the tables. T o obtain these gaseous concentrations, a gas mixture containing 95% v/v O2 and 5% v/v C 0 2 passed a Fluotec" vaporizer (Cyprane, Keighley, England) Halothane added to the incubation medium a t a flow rate of 10 l/min. After the addition of 1 . Lipid mobilization. When halothane was halothane, the gases went through a gas mixer and present in the medium it was observed to then on to an incubation chamber. From this chamber the gas mixture was evacuated to outdoor air. The have a stimulating effect on the lipolysis at concentration of halothane was repeatedly measured low concentrations (Table 1). This effect was at the inlet and outlet of the chamber with a Narcotest significant at concentrations of 1 0 - 6 ~and M@equipment (Dragerwerk, Liibeck, W. Germany). 10-5M (P~ 0 . 0 5 )However, . at concentrations Before halothane was administered all glass vials of 1 0 4 ~ or greater this effect was not were placed without stoppers in the chamber. After exposure for 10 min equilibrium was reached in the consistently found. I n fact, in 50% of the chamber. The biopsies were then placed in the vials incubations the lipolytic process was reduced and gassed via a tube with the gas mixture for another below the initial levels. Neither the lipolytic 30 s. The vials were tightly stoppered and incubated as effect of noradrenaline nor the antilipolytic mentioned above. I n order to avoid leakage of air into effect of insulin were consistently changed by the chamber when working within it, the operator the presence of halothane in the incubation used CUES around the forearms. Aliquots of the incubation media were taken at 0 medium. min and after incubation for 120 min for determination I t should be emphasized that the fat of glycerol, as described by LAURELL& TIBBLINC specimens were taken immediately after (1966). Tissue lipids were extracted with chloroformintubation; i.e. at a time when the patients et al. (1957). methanol (2: I), as described by FOLCH had only had halothane for a very short Glyceride-glycerol was determined on the chloroform
HALOTHANE AND LIPID METABOLISM
329
Table 1 Basal lipolysis in the presence of different concentrations of halothane in the medium. Halothane concentration
Glycerol release
~
nmol/105 cells 190.0+47.1 235.0 f64.4* 228.5 k 58.0* 226.3k72.0 190.5k54.8 209.7 k 57.6
0 10-6M 10-’M 10-4M 10-3M 10-’M
Results+_s.e. mean of seven incubations performed in duplicate. * Significant increase over basal values (P
Effects of Halothane on the Metabolism of Human Adipose Tissue JARL
BENNIS, JIM OLSONAND ULF SMITH
Department of Anaesthesiology, Eastern Hospital, Department of Physical Chemistry and Department of Medicine 11, Sahlgrens Hospital, University of Gothenburg, Gothenburg, Sweden
The metabolism of specimens of human adipose tissue exposed to different concentrations of halothane was studied. Halothane was added to the incubation medium directly or via the gas phase above the medium. The basal lipolysis was significantly increased by low concentrations of halothane. Higher concentrations clearly diminished the lipolysis, but here, in spite of the inhibitory effect on the basal lipolysis, the lipolytic effect of noradrenaline expressed as percent increment was increased. The rate of lipid synthesis from glucose was reduced when halothane was present in the gas phase. The effect of insulin on glucose metabolism was not affected by the presence of halothane, while the antilipolytic action was abolished by high concentrations of halothane. The results show that halothane may exert dual effects on the mobilization of lipids from human adipose tissue; at low concentrations halothane enhances the basal lipolysis, while at higher concentrations it exerts inhibitory effects. Received 9 January, accepted for publication I 5 February 1976
Halothane anaesthesia is often accompanied techniques used and to elucidate this, we by effects similar to those produced by carried out the present study on the effect of stimulation of the P-adrenergic receptors, different concentrations of halothane on the e.g. vasodilation, bronchial relaxation and lipolytic process. uterine hypotonia. An abstract of this work has appeared I t is now generally recognized that the previously (BENNIS & SMITH1975). lipolytic process in human adipose tissue is mediated by the level of cyclic adenosine 3',5'-monophosphate (cyclic AMP) (CARLSON MATERIAL et al. 1970) induced by the P-receptorThirteen patients undergoing laparotomy were adenyl cyclase system (BURNS& LANGLEY investigated. The patients suffered from isolated 1970, EFENDIC& OSTMAN 1970). abdominal disorders, but those with known jaundice, In a previous work (BENNIS & SMITH1973) malignant or endocrinological diseases were excluded. we studied the metabolism of specimens of Their age range was 29-69 years and their body human adipose tissue obtained before and weights varied from 100-225°k of ideal weight (METROPOLITAN LIFE INSURANCE Co. 1959). All after halothane anaesthesia. The basal lipo- patients fasted overnight before the operation. lytic rate was found to be reduced, while the effect of noradrenaline tended to be increased. Recently, MAKELAINEN et al. M E T HODS (1973b) reported that halothane stimulates the basal lipolysis in rat adipose tissue. The Anaesthetic management discrepancy in results between these in- The patients were anaesthetized according to the vestigations may be due to the different normal procedure of the anaesthetic department; i.e.
328
J. BENNIS, J. OLSSON AND
u.
SMITH
they were premedicated with diazepam and given atropinejust before anaesthesia was started. Anaesthesia was induced with a short-acting barbiturate (thiopental or hexobarbital) ; succinylcholine was given; the patients were intubated; and the anaesthesia was continued with nitrous-oxide, oxygen and halothane plus a muscle-relaxing agent. Immediately after skin incision a specimen of the subcutaneous tissue was taken.
phase according to the method of CARLSON(1959). The incorporation of labelled glucose into the lipids was determined on the chloroform phase in a Packard Tri Carb liquid scintillation spectrometer (Packard, La Grange, Ill., U.S.A.). I n accordance with the suggestion of several investigators (SALANSet al. 1968, BJORNTORP& KARLSSON1970), the metabolic parameters are expressed in terms of the cellularity of the tissue specimens. Mean cell diameter was determined as previously described (SMITHet al. 1972). The mean cellular volume was calculated according to GOLDRICK (1967) and the mean cellular weight as suggested by HIRSCH & GALLIAN(1968). The number of fat cells in the specimens was then determined by dividing the triglyceride content by the mean cellular weight. With the techniques used, the p H of the medium was maintained within 7.4k 0.2 throughout the experiments. Significance levels were calculated according to Student's t-test using an Olivetti table computer (Programma 101).
Incubation procedure in uitro The incubation procedure used is a further development of that described by SMITH (1970). Small fragments of the tissue specimens, weighing about 25-50 mg each, were incubated (in duplicate) in 2.0 ml Krebs-Ringer bicarbonate buffer, with albumin added a t a concentration of 4% (Bovine albumin, Fraction V, Armour Pharmaceutical Co., Eastbourne, England) and in the presence of 0.15 pC D-[1-'4C]glucose (New England Nuclear Corp., Frankfurt am Main, W. Germany) per 2 ml medium, with or without the addition of recrystallized pork insulin (Vitrum AB, Stockholm, Sweden) or noradrenaline (Astra AB, Sodertalje, Sweden) at concentrations of lo5 ,uU/ml and 5 x ~O-'M, respectively. RESULTS I n one set of experiments, halothane (Fluothane") was added to the medium a t an initial concentration The present results are expressed in terms of ~ I I - ~ M , ~O-'M, i O - 4 ~ , 1 0 - ~or~ 1 0 - 2 ~ ,re, of the cellularity of the specimens. It should spectively. All incubations were performed in tightly stoppered glass vials for 2 h at 37°C and p H 7.4. The be clear, however, that the mean fat cell gas phase was 5% v/v COZ and 95% v/v 0 2 . sizes differed between the individuals, thus I n another set of experiments, halothane was affecting the rates of metabolism (SALANS administered a t 23°C in the following concentrations 1970, et al. 1968, BJORNTORP & KARLSSON in a gas mixture: 0.5, 1.0, 1.5, 2.0, and 3.0% v/v. SMITHI970), as shown in the tables. T o obtain these gaseous concentrations, a gas mixture containing 95% v/v O2 and 5% v/v C 0 2 passed a Fluotec" vaporizer (Cyprane, Keighley, England) Halothane added to the incubation medium a t a flow rate of 10 l/min. After the addition of 1 . Lipid mobilization. When halothane was halothane, the gases went through a gas mixer and present in the medium it was observed to then on to an incubation chamber. From this chamber the gas mixture was evacuated to outdoor air. The have a stimulating effect on the lipolysis at concentration of halothane was repeatedly measured low concentrations (Table 1). This effect was at the inlet and outlet of the chamber with a Narcotest significant at concentrations of 1 0 - 6 ~and M@equipment (Dragerwerk, Liibeck, W. Germany). 10-5M (P~ 0 . 0 5 )However, . at concentrations Before halothane was administered all glass vials of 1 0 4 ~ or greater this effect was not were placed without stoppers in the chamber. After exposure for 10 min equilibrium was reached in the consistently found. I n fact, in 50% of the chamber. The biopsies were then placed in the vials incubations the lipolytic process was reduced and gassed via a tube with the gas mixture for another below the initial levels. Neither the lipolytic 30 s. The vials were tightly stoppered and incubated as effect of noradrenaline nor the antilipolytic mentioned above. I n order to avoid leakage of air into effect of insulin were consistently changed by the chamber when working within it, the operator the presence of halothane in the incubation used CUES around the forearms. Aliquots of the incubation media were taken at 0 medium. min and after incubation for 120 min for determination I t should be emphasized that the fat of glycerol, as described by LAURELL& TIBBLINC specimens were taken immediately after (1966). Tissue lipids were extracted with chloroformintubation; i.e. at a time when the patients et al. (1957). methanol (2: I), as described by FOLCH had only had halothane for a very short Glyceride-glycerol was determined on the chloroform
HALOTHANE AND LIPID METABOLISM
329
Table 1 Basal lipolysis in the presence of different concentrations of halothane in the medium. Halothane concentration
Glycerol release
~
nmol/105 cells 190.0+47.1 235.0 f64.4* 228.5 k 58.0* 226.3k72.0 190.5k54.8 209.7 k 57.6
0 10-6M 10-’M 10-4M 10-3M 10-’M
Results+_s.e. mean of seven incubations performed in duplicate. * Significant increase over basal values (P
