Acta physiol. scand. 1978. 103. 113-119 From Institute of Medical Biology, Section of Physiology, University of Tromsq Norway
Effects of sodium salicylate on plasma insulin concentration and fatty acid turnover in dogs BY HARALD VIK-Mo, KNUTHOVEand OLED. M ~ 0 s Received 13 June 1977
Abstract VIK-Mo, H., K. Hove and 0. D. MJ0s. Effects of sodium salicylate on plasma insulin corrcentration and fatty acid turnover in dogs. Acta physiol. scand. 1978. 103. 113-119. The effects of intravenous sodium salicylate administration on plasma concentrations of insulin, free fatty acids (FFA) and glucose were studied in intact, anaesthetized dogs both during basal and isoprenaline stimulated lipolysis. In both situations sodium salicylate reduced the plasma concentrations of insulin. The reduction was associated with decreased plasma FFA concentrations and FFA turnover rate, while plasma glucose concentrations remained unaltered. The reduced plasma insulin concentrations effected by sodiuni salicylate is most likely secondary to the concomitant fall in plasma FFA concentrations due to inhibition of FFA mobilization from adipose tissue.
Key words; Fatty acids, fatty acid turnover, insulin, isoprenaline, sodium salicylate.
New interest in the mode of action of salicylates has been created by recent findings that acetylsalicylic acid (ASA) might reduce the incidence of acute myocardial infarction in man (Boston Collaborative Drug Surveillance Group 1974, Elwood et al. 1974), and that ASA (Moschos et af. 1975, Vik-Mo and M j ~ 1977) s and sodium salicylate (SS) (Vik-Mo and M j ~ s1977) reduced the size of an acute myocardial ischemic injury in dogs. These beneficial effects have been related to the well known reduction of plasma free fatty acid (FFA) concentrations induced by salicylates (Carlson and 0stman 1961, Bizzi, Garattini and Veneroni 1965). The mechanism for the plasma FFA lowering effect of salicylates is not clear, however, and whether insulin is involved is not known. Although an antilipolytic effect of salicylates on adipose tissue has been demonstrated in vitro (Schonhofer et al. 1973, Stone, Brown and Steele 1969), the plasma FFA lowering effect of salicylates in viuo can also be due to other mechanisms. Since insulin is an important regulator of lipolysis, an elevation of plasma insulin concentration by salicylates, as reported by Field, Boyle and Remer (1967) in man, would reduce FFA mobilization from adipose tissue. On the other hand, changes in plasma concentrations of insulin might be a consequence of alterations in plasma concen8 - 785876
113
114
HARALD VIK-MO. KNUT HOVE AND OLE D. MJ0S
trations of some of the substrates known to affect insulin regulation, i.e. glucose, amino acids, ketone bodies and FFA (Goodman 1974). In the present study the effects of sodium salicylate infusion on plasma concentration of insulin, glucose and FFA were investigated in order to establish possible relationships between the plasma FFA lowering effect of salicylates and the plasma insulin concentration. To obtain a wide range of FFA levels anesthetized dogs were given sodium salicylate before and during isoprenaline-stimulated lipolysis. Tn addition to the possible effects of salicylates on lipolysis in adipose tissue the reduction of plasma FFA concentration induced by salicylates could be effected by an increase in tissue FFA extraction. Since salicylates irt rifro have been reported to reduce fatty acid binding to albumin (Dawkins, McArthur and Smith 1970), a mechanism for the reduced plasma FFA concentrations might be a higher uptake of FFA in peripheral tissue at a given FFA concentration, as a consequence of lower albumin binding. A second aim of this study was therefore to examine if the plasma FFA lowering effect of sodium salicylate could alternatively be explained through a change in FFA turnover rate.
Methods Aifiuiul prc,pplrrirtiori
Experiments were performed in 7 mongrel dogs of either sex, mean b.wt. 20 kg. After fasting overnight the dog5 were anesthetized by a n i.v. injection of sodium pentobarbital, 25 mg/kg b.wt. Supplementary doses were given at interbals when necessary. A femoral artery was cannulated for blood sampling and a femoral vein for infusion. Heparin, I00 i.u.’kg b.wt., was given i.v. to avoid clotting of catheters.
MPfubolic I I I C ~ I S N ~ C I ~ I C I ~ ~ S Arterial Mood \\as collected into precooled heparinized plasma tubes. T h e blood was centrifuged immediately at 4 C. and the plasma was frozen a n d stored at - 20-C until analyscd. All analyses were done in duplicate. f’lasma glucose concentration was estimated by the glucose oxidase method (Bochringer, Mannheim, Germany). Plasma insulin \*as measured by a radioimmunoassay (Poznanski a n d Pornanski 1969). HoLine insulin was used both as a standard and for the production of anti-insulin antibodies. An antibody concentration gi\ing a maximal sensitivity in the range of insulin concentrations was chosen. Assays were performed to determine whether the presence of salicylate in the assay mixture alter the binding of labelled inbulin to the antisera. Within the range of salicylate concentrations found in plasma n o effect o n binding was observed. The concentrations of FFA in plasma were determined by the titrimetric method of Dole (1956) a s modified by Trout, Estes and Friedberg (1960). T h e presence of salicylate in plasma slightly influenced the FFA titration, thus 67 {tmolil per nimo1;l of salicylate in plasma had to be subtracted from actually measured plasma FFA concentration. Plasma salicylate concentration was assayed by a spectrophotometric method based on a colour reaction with ferric nitrate (Trinder 1954). Isolation of 3H-palmitic acid from the plasma was performed in a two phahe extraction system as described by Kaplan (19701. The radioactivity was determined i n a Packard TriCarti Iiqiiid-scintillation spectrometer (,Model 3320 Packard Instruments Co., Inc., Dowers Grove, USA). €.vpcritirenral procedure T h e effects of sodium salicylate infusion o n arterial plasma concentrations of insulin, FFA and glucose during basal and isoprenaline stimulated lipolysis were studied in 7 dogs. T h e following experimental situations were studied in each dog: I ) Control situation I. Immediately after the blood sampling a continuous infusion of isoprenaline a t a rate of 0.10-0.20 !tg/kg min. 2 ) Isoprenaline situation: Blood sampling was performed 10 minutes after the start of isoprenaline infusion. 3 ) Control situation 11 30 min after cessation of the isoprenaline infusion.
115
SALICYLATE ON PLASMA lNSULIN AND FFA
4) Salicylate situation: Blood sampling was performed 30 min after a slow i.v. injection of sodium salicylate, 60 mg/kg b.w., freshly dissolved in saline and adjusted to p H 7.40, and followed by the continuous infusion of 0.15 mg/kg min during the rest of the experiment. 5) Isoprenaline-salicylate: Blood sampling was performed 10 min after start of isoprenaline infusion in the same dose as in 2).
In 4 of the 7 expts. the effects of sodium salicylate o n FFA turnover during basal and isoprenaline stimulated lipolysis were studied. 3H-plamitic acid with specific activity 500 pCi/,Lmol (The Radiochemical Centre, Amersham, England) was bound to 5 % bovine albumin (No. A-6003, Sigma Chemical Comp., St. Louis, USA) in saline as described by Lassers, Kaijser and Carlson (1972). A continuous i.v. infusion of 0.1 ml/min (0.5 pCi/min) was given. The albumin-fatty acid infusion was started 90 min before the first control registration to give a constant 3H-palmitic acid concentration. The radioactivity in the SH-palmitic acid infusate was determined in duplicate samples of the infusate.
Calculations Specific activity of plasma FFA was calculated by dividing the radioactivity of each sample by the chemically measured F F A concentr5tii;n. FFA turnover was d e k 5 n e d from the equation: radioactivity infusion rate (cpm/min) -
mean F F A specific activity (cpm/pmol)
in pmol/min. Fractional turnover of FFA (per cent/min) was calculated as FFA turnover x 100 FFA pool where FFA pool is F F A concentration X plasma volume. Plasma volume was assumed to be 5 per cent of body weight.
Statistics Wilcoxon non-parametric test for paired data (two-tailed) and Spearman rank correlation test were used to calculate probabilities (Snedecor and Cochran 1967) P j 0.05 was regarded as not statistically significant.
Results The effects of sodium salicylate on arterial concentrations of insulin, FFA and glucose before and during isoprenaline-stimulated lipolysis are presented in Table I. Plasma insulin concentration increased from 0.82 k0.13 to 2.87 k0.49 ng/ml following isoprenaline infusion (mean k S.E.M., P
Effects of sodium salicylate on plasma insulin concentration and fatty acid turnover in dogs BY HARALD VIK-Mo, KNUTHOVEand OLED. M ~ 0 s Received 13 June 1977
Abstract VIK-Mo, H., K. Hove and 0. D. MJ0s. Effects of sodium salicylate on plasma insulin corrcentration and fatty acid turnover in dogs. Acta physiol. scand. 1978. 103. 113-119. The effects of intravenous sodium salicylate administration on plasma concentrations of insulin, free fatty acids (FFA) and glucose were studied in intact, anaesthetized dogs both during basal and isoprenaline stimulated lipolysis. In both situations sodium salicylate reduced the plasma concentrations of insulin. The reduction was associated with decreased plasma FFA concentrations and FFA turnover rate, while plasma glucose concentrations remained unaltered. The reduced plasma insulin concentrations effected by sodiuni salicylate is most likely secondary to the concomitant fall in plasma FFA concentrations due to inhibition of FFA mobilization from adipose tissue.
Key words; Fatty acids, fatty acid turnover, insulin, isoprenaline, sodium salicylate.
New interest in the mode of action of salicylates has been created by recent findings that acetylsalicylic acid (ASA) might reduce the incidence of acute myocardial infarction in man (Boston Collaborative Drug Surveillance Group 1974, Elwood et al. 1974), and that ASA (Moschos et af. 1975, Vik-Mo and M j ~ 1977) s and sodium salicylate (SS) (Vik-Mo and M j ~ s1977) reduced the size of an acute myocardial ischemic injury in dogs. These beneficial effects have been related to the well known reduction of plasma free fatty acid (FFA) concentrations induced by salicylates (Carlson and 0stman 1961, Bizzi, Garattini and Veneroni 1965). The mechanism for the plasma FFA lowering effect of salicylates is not clear, however, and whether insulin is involved is not known. Although an antilipolytic effect of salicylates on adipose tissue has been demonstrated in vitro (Schonhofer et al. 1973, Stone, Brown and Steele 1969), the plasma FFA lowering effect of salicylates in viuo can also be due to other mechanisms. Since insulin is an important regulator of lipolysis, an elevation of plasma insulin concentration by salicylates, as reported by Field, Boyle and Remer (1967) in man, would reduce FFA mobilization from adipose tissue. On the other hand, changes in plasma concentrations of insulin might be a consequence of alterations in plasma concen8 - 785876
113
114
HARALD VIK-MO. KNUT HOVE AND OLE D. MJ0S
trations of some of the substrates known to affect insulin regulation, i.e. glucose, amino acids, ketone bodies and FFA (Goodman 1974). In the present study the effects of sodium salicylate infusion on plasma concentration of insulin, glucose and FFA were investigated in order to establish possible relationships between the plasma FFA lowering effect of salicylates and the plasma insulin concentration. To obtain a wide range of FFA levels anesthetized dogs were given sodium salicylate before and during isoprenaline-stimulated lipolysis. Tn addition to the possible effects of salicylates on lipolysis in adipose tissue the reduction of plasma FFA concentration induced by salicylates could be effected by an increase in tissue FFA extraction. Since salicylates irt rifro have been reported to reduce fatty acid binding to albumin (Dawkins, McArthur and Smith 1970), a mechanism for the reduced plasma FFA concentrations might be a higher uptake of FFA in peripheral tissue at a given FFA concentration, as a consequence of lower albumin binding. A second aim of this study was therefore to examine if the plasma FFA lowering effect of sodium salicylate could alternatively be explained through a change in FFA turnover rate.
Methods Aifiuiul prc,pplrrirtiori
Experiments were performed in 7 mongrel dogs of either sex, mean b.wt. 20 kg. After fasting overnight the dog5 were anesthetized by a n i.v. injection of sodium pentobarbital, 25 mg/kg b.wt. Supplementary doses were given at interbals when necessary. A femoral artery was cannulated for blood sampling and a femoral vein for infusion. Heparin, I00 i.u.’kg b.wt., was given i.v. to avoid clotting of catheters.
MPfubolic I I I C ~ I S N ~ C I ~ I C I ~ ~ S Arterial Mood \\as collected into precooled heparinized plasma tubes. T h e blood was centrifuged immediately at 4 C. and the plasma was frozen a n d stored at - 20-C until analyscd. All analyses were done in duplicate. f’lasma glucose concentration was estimated by the glucose oxidase method (Bochringer, Mannheim, Germany). Plasma insulin \*as measured by a radioimmunoassay (Poznanski a n d Pornanski 1969). HoLine insulin was used both as a standard and for the production of anti-insulin antibodies. An antibody concentration gi\ing a maximal sensitivity in the range of insulin concentrations was chosen. Assays were performed to determine whether the presence of salicylate in the assay mixture alter the binding of labelled inbulin to the antisera. Within the range of salicylate concentrations found in plasma n o effect o n binding was observed. The concentrations of FFA in plasma were determined by the titrimetric method of Dole (1956) a s modified by Trout, Estes and Friedberg (1960). T h e presence of salicylate in plasma slightly influenced the FFA titration, thus 67 {tmolil per nimo1;l of salicylate in plasma had to be subtracted from actually measured plasma FFA concentration. Plasma salicylate concentration was assayed by a spectrophotometric method based on a colour reaction with ferric nitrate (Trinder 1954). Isolation of 3H-palmitic acid from the plasma was performed in a two phahe extraction system as described by Kaplan (19701. The radioactivity was determined i n a Packard TriCarti Iiqiiid-scintillation spectrometer (,Model 3320 Packard Instruments Co., Inc., Dowers Grove, USA). €.vpcritirenral procedure T h e effects of sodium salicylate infusion o n arterial plasma concentrations of insulin, FFA and glucose during basal and isoprenaline stimulated lipolysis were studied in 7 dogs. T h e following experimental situations were studied in each dog: I ) Control situation I. Immediately after the blood sampling a continuous infusion of isoprenaline a t a rate of 0.10-0.20 !tg/kg min. 2 ) Isoprenaline situation: Blood sampling was performed 10 minutes after the start of isoprenaline infusion. 3 ) Control situation 11 30 min after cessation of the isoprenaline infusion.
115
SALICYLATE ON PLASMA lNSULIN AND FFA
4) Salicylate situation: Blood sampling was performed 30 min after a slow i.v. injection of sodium salicylate, 60 mg/kg b.w., freshly dissolved in saline and adjusted to p H 7.40, and followed by the continuous infusion of 0.15 mg/kg min during the rest of the experiment. 5) Isoprenaline-salicylate: Blood sampling was performed 10 min after start of isoprenaline infusion in the same dose as in 2).
In 4 of the 7 expts. the effects of sodium salicylate o n FFA turnover during basal and isoprenaline stimulated lipolysis were studied. 3H-plamitic acid with specific activity 500 pCi/,Lmol (The Radiochemical Centre, Amersham, England) was bound to 5 % bovine albumin (No. A-6003, Sigma Chemical Comp., St. Louis, USA) in saline as described by Lassers, Kaijser and Carlson (1972). A continuous i.v. infusion of 0.1 ml/min (0.5 pCi/min) was given. The albumin-fatty acid infusion was started 90 min before the first control registration to give a constant 3H-palmitic acid concentration. The radioactivity in the SH-palmitic acid infusate was determined in duplicate samples of the infusate.
Calculations Specific activity of plasma FFA was calculated by dividing the radioactivity of each sample by the chemically measured F F A concentr5tii;n. FFA turnover was d e k 5 n e d from the equation: radioactivity infusion rate (cpm/min) -
mean F F A specific activity (cpm/pmol)
in pmol/min. Fractional turnover of FFA (per cent/min) was calculated as FFA turnover x 100 FFA pool where FFA pool is F F A concentration X plasma volume. Plasma volume was assumed to be 5 per cent of body weight.
Statistics Wilcoxon non-parametric test for paired data (two-tailed) and Spearman rank correlation test were used to calculate probabilities (Snedecor and Cochran 1967) P j 0.05 was regarded as not statistically significant.
Results The effects of sodium salicylate on arterial concentrations of insulin, FFA and glucose before and during isoprenaline-stimulated lipolysis are presented in Table I. Plasma insulin concentration increased from 0.82 k0.13 to 2.87 k0.49 ng/ml following isoprenaline infusion (mean k S.E.M., P
