Bk~chimica et Biophysica Attn. 1086 ( 1991 ) 191 - 196
I9 l
~,', 1991 Elsevier Science Publishers B.V. All rights reserved (NIO5-2760/ql/$ID.50
BBALIP 53762
Comparison of effects of platelet-activating factor and tumour necrosis factor-a on lipid metabolism in adrenalectomized rats in vivo Rhys D. Evans
I
and Dermot H. Wiiliamson -'
t Nuffield l~,partment of Anaesthesia. Radcliffi, InfirmaD'. Oxh~rd (U.K) and : Metabolk" Re~,'arch I.ahoratory, Nuffield Department of Clinical Medicine. Raddiffi, Infirnlut3". ()xfi~rd (U.K.) (Received 3 April Ib'91)
Key words: Platelet activating factor; Tumor necrosis factor a: Liver lipogenesis: Lipid metal'adism; (Adrenalcclomized rat)
The acute metabolic effects of platelet-activating factor (PAF) and tumour necrosis factor-e (TNF.o) were compared in sham-operated and adrenalectomized rats. PAF caused hyperglycaemia in sham-operated rats, whereas with TNF-a there was a slight decrease in blood glucose. Both PAF and TNF-a resulted in marked hypoglycaemia in the adrenalectomized rats. Plasma insulin was depressed (about 50%) by PAF and TNF-a in sham-operated rats, whereas in the adrenalectomized rats the already low plasma insulin concentration was not significantly altered. Liver glycogen content was the same in control and treated sham-operated rats, but was considerably decreased (about 50%) in the adrenalectomized rats. In sham-operated rats, PAF and TNF.a increased plasma non-esterified fatty acids and triacylglycerols, suggesting increased lipolysis, whereas in udrenalectomized rats there was no significant increase in non-esterifled fatty acids with PAF, although it still occurred with TNF-a. This suggests that the lipolytic effect of TNF-a may be direct, whereas that of PAF is indirect, possibly via increased catecbolamines in the sham-operated rats. The stimulation (about 3-foid) of hepatic fatty acid synthesis in vivo by PAl: and TNF-a in sham-operated rats was still evident in the adrenalectomized rats, although the absolute increase was smaller. PAF, hut not TNF-a increased (100%1 sterol synthesis in adrenalectomized rats. it is concluded that PAl: and TNF-a can increase hepatic lipagenesis in t,it'o in the absence of adrenal hormones and in the presence of a low plasma insulin.
Introduction
Sepsis and other 'stress' states arc accompanied by characteristic and well-documented changes in carbohydrate, protein and lipid metabolism (for review, see Ref. 1). These changes are mostly directed at substrate mobilization, and include increased lipolysis and decreased lipoprotein lipase (EC 3.1.1.341 activity in adipose tissue with hypertriglyceridaemia [2,3], and increased hepatic glycogenolysis with hyperglycaemia [4].
Abbreviatkms: ACTtt. ,drcnocorticotropic horrmme: IF'N-y. interferon-y; IL-I/L interleukin-I~: LPL. lipopn)tein lipase: mRNA, messenger ribonucleic acid; NEFA. non-esterified fany itcids: PAF. plalelet-activating factor: TAG. triacylglycerol: TNF,tx. tumour necrosis factor-a. Correspondence: D.H. Williamw,m, Metabolic Research Laboratory. Nuffield Department of Clinical Medicine. Radcliffe Infirmary. Wtmdstock Road. Oxford OX2 6HE. U.K.
However, liver lipid synthesis (lipogenesis) is paradoxically increased in otherwise catabolic septic animal models [5], and this effect can be mimicked by administration of putative mediators of inflammation and septic shock, including the cytokines, tumour necrosis factor-a (TNF-a), intcrlcukin-I/3 (IL-I/3) and interferon-,/ (IFN-y) [6] and the lipid autacoid, platelet-activating factor ( I-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) [7]. Elevated bhx~d levels of TNF-a and PAF have been detected during sepsis [8,9]. To date. the mechanism for this stimulation of hepatic lipogenesis is unknown; neither TNF-a nor PAF has been shown to increase lipid synthesis directly in t'itro. PAF does increase hepatic glycogenolysis in the perfu~d rat liver [ 10] and decrease hepatic glycogen in t'it'o in the rat [7], leading to the possibility that increased hepatic lipogenesis is secondary to increased intrahepatic glucose availability However, this seems unlikely because in fed rats in t'il'o PAF stimulates hepatic lipogenesis at doses lower than those required
192 to decrease hcpatic glycogen [7]. Administration of either PAF or TNF-a also results in hypcrtriglyccridacmia [7,11], although whether this is mainly due to increased secretion of VLDL and/or decreased peripheral utilization of triacylglycerol is unknown in the case of PAF and still open for TNF-a. Characteristic hormonal changes accompany the metabolic derangement of sepsis; thus the 'stress' hormones such as catecholamines, adrenocorticotropic hormone (ACTH) and adrenal steroids are markedly increased, whils~ insulin is modestly elevated, with accompanying insulin resistance. The effects of endocrine and paracrine systems may be interrelated in that TNF-a and PAF stimulate catecholamine [12,13] and corticostcroid release [14,15], whilst dexamethasone potently decreases TNF-a mRNA in cultured macrophages [ 16]. Increased concentrations of catecholamines would be expected to decrease hepatic lipogenesis and stimulate glycogenolysis and adipose ti'~,sue lipolysis. Consequently, we have compared the effects of TNF-a and PAF in adrenalectomized rats, to examine whether adrenal hormones modify the action of these inflammatory mediators on glycogen and lipid metabolism in viro. Methods
Rats. Female Wistar rats (1611-200 g) were fed ad libitum on a commercial chow diet (by wt.) consisting of approx. 52% carbohydrate, 2iv& protein and 4% fat plus added salts and vitamins (the residue was non-digestible material. Special Diet Services, Witham, Es~x, U.K.). They had free access to drinking water and were maintained at an ambient temperature of 22 + 2 °C with a 12-h light/12-h dark cycle (light from 07:00 hi. Where indicated, adrenalectomy was performed by a dorsal approach by the method of Zarrow et al. [ 17] 6 days before experimentation; drinking water was replaced by (I.9% (w/v) NaCI solution in the adrenalectomized groups. A group of rats was sham-operated (intact adrenals) to act as controls to the adrenalectomized animals. All surgical wounds were inspected after killing for macroscopic evidence of infection. Food intake was not significantly different between the sham-operated and adrenalectomized group. Experiments were commenced between 09:(10 h and 10:00 h. Biochemicals. PAF prepared from bovine heart lecithin was obtained from Sigma Chemical Co. Ltd., Poole, Dorset, U.K. and recombinant human TNF-a was generously given by BASF/Knoil AG, Ludwigshafen, Germany. All enzymes and coenzymes were from Boehringer Corp. (London) Ltd., Lewes, Sussex, U.K. 3H,O was obtained from Amersham International, Amersham, Bucks, U.K.
Measurement of lipogenesis. Lipogenic rate in vivo was determined with 3H20 as previously described [18]. Animals were injected subcutaneously ((I.3 ml) with PAF (25 ~ g / k g body wt. in 0.25% bovine serum albumin), TNF-a (150 ~ g / k g body wt. in 0.5 ml phosphate-buffered saline (50 mM potassium phosphate/ 0.9% NaCI pH 8.5) (contamination with endotoxin
I9 l
~,', 1991 Elsevier Science Publishers B.V. All rights reserved (NIO5-2760/ql/$ID.50
BBALIP 53762
Comparison of effects of platelet-activating factor and tumour necrosis factor-a on lipid metabolism in adrenalectomized rats in vivo Rhys D. Evans
I
and Dermot H. Wiiliamson -'
t Nuffield l~,partment of Anaesthesia. Radcliffi, InfirmaD'. Oxh~rd (U.K) and : Metabolk" Re~,'arch I.ahoratory, Nuffield Department of Clinical Medicine. Raddiffi, Infirnlut3". ()xfi~rd (U.K.) (Received 3 April Ib'91)
Key words: Platelet activating factor; Tumor necrosis factor a: Liver lipogenesis: Lipid metal'adism; (Adrenalcclomized rat)
The acute metabolic effects of platelet-activating factor (PAF) and tumour necrosis factor-e (TNF.o) were compared in sham-operated and adrenalectomized rats. PAF caused hyperglycaemia in sham-operated rats, whereas with TNF-a there was a slight decrease in blood glucose. Both PAF and TNF-a resulted in marked hypoglycaemia in the adrenalectomized rats. Plasma insulin was depressed (about 50%) by PAF and TNF-a in sham-operated rats, whereas in the adrenalectomized rats the already low plasma insulin concentration was not significantly altered. Liver glycogen content was the same in control and treated sham-operated rats, but was considerably decreased (about 50%) in the adrenalectomized rats. In sham-operated rats, PAF and TNF.a increased plasma non-esterified fatty acids and triacylglycerols, suggesting increased lipolysis, whereas in udrenalectomized rats there was no significant increase in non-esterifled fatty acids with PAF, although it still occurred with TNF-a. This suggests that the lipolytic effect of TNF-a may be direct, whereas that of PAF is indirect, possibly via increased catecbolamines in the sham-operated rats. The stimulation (about 3-foid) of hepatic fatty acid synthesis in vivo by PAl: and TNF-a in sham-operated rats was still evident in the adrenalectomized rats, although the absolute increase was smaller. PAF, hut not TNF-a increased (100%1 sterol synthesis in adrenalectomized rats. it is concluded that PAl: and TNF-a can increase hepatic lipagenesis in t,it'o in the absence of adrenal hormones and in the presence of a low plasma insulin.
Introduction
Sepsis and other 'stress' states arc accompanied by characteristic and well-documented changes in carbohydrate, protein and lipid metabolism (for review, see Ref. 1). These changes are mostly directed at substrate mobilization, and include increased lipolysis and decreased lipoprotein lipase (EC 3.1.1.341 activity in adipose tissue with hypertriglyceridaemia [2,3], and increased hepatic glycogenolysis with hyperglycaemia [4].
Abbreviatkms: ACTtt. ,drcnocorticotropic horrmme: IF'N-y. interferon-y; IL-I/L interleukin-I~: LPL. lipopn)tein lipase: mRNA, messenger ribonucleic acid; NEFA. non-esterified fany itcids: PAF. plalelet-activating factor: TAG. triacylglycerol: TNF,tx. tumour necrosis factor-a. Correspondence: D.H. Williamw,m, Metabolic Research Laboratory. Nuffield Department of Clinical Medicine. Radcliffe Infirmary. Wtmdstock Road. Oxford OX2 6HE. U.K.
However, liver lipid synthesis (lipogenesis) is paradoxically increased in otherwise catabolic septic animal models [5], and this effect can be mimicked by administration of putative mediators of inflammation and septic shock, including the cytokines, tumour necrosis factor-a (TNF-a), intcrlcukin-I/3 (IL-I/3) and interferon-,/ (IFN-y) [6] and the lipid autacoid, platelet-activating factor ( I-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) [7]. Elevated bhx~d levels of TNF-a and PAF have been detected during sepsis [8,9]. To date. the mechanism for this stimulation of hepatic lipogenesis is unknown; neither TNF-a nor PAF has been shown to increase lipid synthesis directly in t'itro. PAF does increase hepatic glycogenolysis in the perfu~d rat liver [ 10] and decrease hepatic glycogen in t'it'o in the rat [7], leading to the possibility that increased hepatic lipogenesis is secondary to increased intrahepatic glucose availability However, this seems unlikely because in fed rats in t'il'o PAF stimulates hepatic lipogenesis at doses lower than those required
192 to decrease hcpatic glycogen [7]. Administration of either PAF or TNF-a also results in hypcrtriglyccridacmia [7,11], although whether this is mainly due to increased secretion of VLDL and/or decreased peripheral utilization of triacylglycerol is unknown in the case of PAF and still open for TNF-a. Characteristic hormonal changes accompany the metabolic derangement of sepsis; thus the 'stress' hormones such as catecholamines, adrenocorticotropic hormone (ACTH) and adrenal steroids are markedly increased, whils~ insulin is modestly elevated, with accompanying insulin resistance. The effects of endocrine and paracrine systems may be interrelated in that TNF-a and PAF stimulate catecholamine [12,13] and corticostcroid release [14,15], whilst dexamethasone potently decreases TNF-a mRNA in cultured macrophages [ 16]. Increased concentrations of catecholamines would be expected to decrease hepatic lipogenesis and stimulate glycogenolysis and adipose ti'~,sue lipolysis. Consequently, we have compared the effects of TNF-a and PAF in adrenalectomized rats, to examine whether adrenal hormones modify the action of these inflammatory mediators on glycogen and lipid metabolism in viro. Methods
Rats. Female Wistar rats (1611-200 g) were fed ad libitum on a commercial chow diet (by wt.) consisting of approx. 52% carbohydrate, 2iv& protein and 4% fat plus added salts and vitamins (the residue was non-digestible material. Special Diet Services, Witham, Es~x, U.K.). They had free access to drinking water and were maintained at an ambient temperature of 22 + 2 °C with a 12-h light/12-h dark cycle (light from 07:00 hi. Where indicated, adrenalectomy was performed by a dorsal approach by the method of Zarrow et al. [ 17] 6 days before experimentation; drinking water was replaced by (I.9% (w/v) NaCI solution in the adrenalectomized groups. A group of rats was sham-operated (intact adrenals) to act as controls to the adrenalectomized animals. All surgical wounds were inspected after killing for macroscopic evidence of infection. Food intake was not significantly different between the sham-operated and adrenalectomized group. Experiments were commenced between 09:(10 h and 10:00 h. Biochemicals. PAF prepared from bovine heart lecithin was obtained from Sigma Chemical Co. Ltd., Poole, Dorset, U.K. and recombinant human TNF-a was generously given by BASF/Knoil AG, Ludwigshafen, Germany. All enzymes and coenzymes were from Boehringer Corp. (London) Ltd., Lewes, Sussex, U.K. 3H,O was obtained from Amersham International, Amersham, Bucks, U.K.
Measurement of lipogenesis. Lipogenic rate in vivo was determined with 3H20 as previously described [18]. Animals were injected subcutaneously ((I.3 ml) with PAF (25 ~ g / k g body wt. in 0.25% bovine serum albumin), TNF-a (150 ~ g / k g body wt. in 0.5 ml phosphate-buffered saline (50 mM potassium phosphate/ 0.9% NaCI pH 8.5) (contamination with endotoxin
