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Phosphoinositide metabolism in adipocytes from hypothyroid rats
European Journal of Pharmacology - Molec:dar Pharmacology Sect:on. 206 (1991)8!-85
81
~: 1991 ElsevierScience Publisher~ B.V.0922~41C6/91/$03.50 A D O N I S 0922410691000614
EJPMOL 90133
Phosphoinositide metabolism in adipocytes from hypothyroid r a t s Per .Heden Andersen, H e n n i n g JuhI, Steen Bon!okke Pedersen, Bj~rn Richelsen Umt,ers~ty Chnic of End~crmoloKv m~d InternM Medicine, Aarhus a m ~ s ~ e h ~ , DK-80OO Aarhus C, Denmark
Received 6 September1990.accepted25 September1990
The effect of hypothyroidism on insulin- and epinephrine-stimulated phosphoinositide metabolism was investigated in rat adipocytes. Insulin-mediated phosphoinositide synthesis was enahaneed by h~q3othyroidism(t4.5 =' 1.5% above basal level, control vs. 22.5 *_.2.0% above basal level, h3pothyroid, P < 0.05). However, insulin did not stimulate hydrolysis of phosphoinositid~ ~o inositot phosphates, neither in control nor m hypothyroid rats. The ai-adrenoceptor agonist (e~& epinephrine) siN-Jficamly stimulated the incorporation of myo-[3H]inositolinto phosphoinositides (P < 0.01) and hydrolysis of phosphoinosi*ides {P < 0.0l L but this stimulatory action was unaffected by the hypothyroid state. The iesuhs suggest that hypoth5Tnidism has differentiated effects on the hormone-regnlaled phosphoinositide metabolism and that the presumptive G-protein coupled to the a~-adrenoceptor seems to be unaffected by hypothyroidism. Adipose tissue; Hypothyroidism: Insulin: Epinephrine; Phosphoinosifides
L Introduction
Phosphoinositide metaboiism has attracted much attention during recent years, as it has become evident that phosphoinositide turnover is involved in lransmembrahe signalling. Phosphoinositide turnover is stimulated by aa-adrenoceptor agonists in various cells such as myecytes (Farese et at., 1986a), hepatocytes (Uhing etal., 1986) and adipocytes (Pennington and Martin, 1985). Previously, it has been shown that insulin is able to stimulate phosphoinositide synthesis in rat adipocytes (Farese et al., 1985; Pennington and Martin, 1985; Augert and Extort, 1988). According to the current hypothesis, hydrolysis of phosphatidylinositol-4,5-biphosphate to inositol triphosphate and diacylglycerol, is stimulated by activation of phospholipase C, which seems to be coupled to a wide variety of surface receptors via Goproteins (Litogch and Fain, 1986). Thyroid status markedly affects the fl-adrenoceptor response of catecholamines in rat adipocytes. Hypothyroidism impairs and hyperthyroidism enhances the lipolytic response of catecholamines (fl-adreno~ptor effect) (Armstrong et al., 1974; Goswami and Rosenberg, 1978; Malbon et at., 1978; Fain, 1981). The ira-
Correspondence to: Per Heden Andersen, UniversityClinic of Endocringtogyand Imemal Medicine,Aaxhusamtss~ehus,Tage tfansens Gade, DK-8000 Aarhus C, Denmark.
pact of hypothyroidism on the catecholamine-induced phosphoinositide turnover (a~-adrenoceptor effect) has been investigated in only a few studies (Garcia-Samz and Fain, 1980; Fain, i981), and in these studies only phosphoinositide synthesis was examined. In both studies phosphoinositide synthesis was found to be unaffected by thyroid status. Insulin action on ~ucose metabolism and lipolysis in adipocytes also has been s h o w to be affected by thyroid status (Seihet et at.. I978: Rifici and Kip!an, 1984; McKenzie et at.. t987). However, the influence of thyroid ho~,xnones on the insulin-stimulated phosphoinositide metabolism has not been examined. In the present study, the effect of hypothyroidism on insulin- and a~-adrenoceptor-stimulated phosphoinositide metabolism was investigated. Both synthesis and hydrolysis of pho~vphoinositides were assessed. In addition, some aspects of the lipolytic system were investigated in order to confirm the hypothyroid state.
2. Materials and methods 2. L M a t e r i a l s
D-myo-[3H]inositol (specific activity 10-20 Ci/mmol) was purchased from A-mersham (U.K.). Dowex-l-X8 resin (formate form, 100-200 mesh) v-as obtained from Bio-Rad (U.S.A.). Epinephrine and propranotol were obtained from Sigma Chemicals (U.S.A.). Crystalfine porcine insulin was a gift from Novo (DK). 6-N-propyl-
82 2-thionracil was a gift from DAK-laboratofies (DK}. Prazosin was obtained from Ercopharm (DK). All other reagents were obtained as described in references (Pedersen and Hjollund, I982; R!chelsen and BeckNielsen, 198,1).
2.2. Ammals Male Wistar rals weiglling 125-150 g were divided into two groups (littermates): control and hypothyroid rats. Hypothyroidism was induced by keeping the rats for 1,1-18 days on an iodine-deficient diet and supplying the drinking water with 0.00625% 6-N-propyl-2thiouracil. Control rats were maintained on a similar diet containing iodine, and on tap water. Thyroid status was assessed.by measuring S-triiodthyronine (T3) and S-thyroxine (T4) by the ELISA technique (Amerlite, Amersbam).
Z3. Adipocyte preparation
packed with Dowex-l-X8. lnosilo! 1-phosphate (IPl), inositol 1,4-biphospbate ( I ~ ) and inositol 1.4,5-triphosphate (IP3) were sequentially eluted from the column w-ith buffers containing increasing formate concentrations (Be,ridge et al., 1983). Incorporation of myo-[3H]inositol into total phosphomos~tides was measured as described (Pennington and Martin, 1985).
2.5. Lipotysis studies Glycerol release was taken as an index of lipolysis. The glycerol release ,,,,'as determined enzymaticaliy (Peridochrom, Boehringer Mannheim) by a fluorometric method (Chernick, 1969). In experiments where the anti!ipolytic effect of insulin was examined, lipolysis was stimulated v¢ith adenosine deaminase ( A D A ) (0.5 u/~).
2.6. Statistics
Adipocytes were isolated from epididymal fat pads by collagenase digestion (Pedersen and Hjollund. 1982). The volume of the adipocytes was calculated (Pedersen and Hjollund, 1982). Adipocytes were finally washed four times and resuspended in a buffer containing (mM): 10 Hepes, 135 NaCI, 4.8 KC1, 1.7 MgSO4. 2.5 CaCI,, 0.2 N a H 2 P O 4, 1 N a 2 H P O 4. 5 glucose, 2.5% BSA, p H 7.4.
2.4. Pkosphoinositide metabolism Isolated adipocytes were incubated in HEPES buffer supplemented with myo-[3H]inositol (20 ,uCi/rnl) and LiCI (10 raM) for 90 min at 37°C. The cells were then washed three times in an isotope-flee buffer containing 10 m M LiC1 and diluted to 1-2 × 10 s cells/nil. Aliquots of 1 ral were preincubated for 15 rain and finally incubated in the absence or in the presence of hormone in a shaking waterbath for 60 rain at 37°C. Incubations •,,,'ere stopped by adding 1 ml ice-cold 10% trichloracetic acid and vigorous mixing. The samples ",,,'erecentrifuged 4000 x g for 5 rain and the supernatant was removed and neutralized by 10 m M sodiumborate. Radioactive inositol phosphates were chromatographed on a column
Data are given as means _+ S.E.M. P values were determined using Student's paired t-test. P < 0.05 was considered significant. N o statistical significance = N.S.
3. Results The hypothyroid state was confirmed by a significant decrease in thyroid hormones. In addition, the eninephrine-stimulated lipolysis was greatly reduced as expected (table 1). Further characteristics of the rats are shown in table t. Insulln-stimulated incorporation of myo-[3H]inositol into phosphoinositides was significantly increased in the hypothyroid state (14.5 +_ 1.5% above basal level, control vs. 22.5 + 2.0% above basal level, hypothyroid, P < 0.05) (fig. 1). In contrast to Farese et al. (1986b), we were not able to show an effect of insulin on the hydrolysis of phosphoinositides to inositol phosphates neither in the control nor in the hypothyroid animals (fig. 2). The increased effect of insulin on phosphoinositide synthesis could have been caused by enhanced insulin binding; however, we found insulin bi-
TABLE l Characteristics of euthyroid and hypothyroid rats. Data are given as rneans±S.E.M, for (n) paired experiments. * P<0.05; ** P<0.001; • ** P < 0.0001. n Basic characteristics Weight (g) 22 Fatcdlvolume(pl) 10 Trfiodth3,xonine(nM) 5 Th~lroxine(nlVO 6 Lipolysis~glycerolrelease(nmol/1Os cells) Basal 5 Epinephrine(12.5 t~M) 5
PTUdreated 199.77+ 127.00+0.10+2.60+116 540
4_43 15.00 0.04 2.10
± 30 ± 111
Control
P value
256.14+- 8.05 150.70+- 24.00 0.64-- 0.22 64.50 +- 5.90 254 1550
+- 52 +-326
N.S.
i
40 l
Fig. 1. Eff~'t of hypothyroidism on myo-[3H]inositol incorporation in phosphoino~tides. Adipocytes were incubated with: I, in~uhn (10 ruM); II, epinephrine (5 ,~M)+pmpranolot (5 taM); lit, epinephrine {5 ttM)+propranolol (5 ,uM)+prazosin (5 taM). Experiments were performed as described in Materials and methods. Black and white bars represent adipoeytes from euthyroid and hypothyroid rats, respectively. Data are expressed as percent above basal value (11150 ± 1959 epm/10 + cells, control vs. 125805:2124 epm/10 ~ cells, hypothyroid, N.S.) and are given as means±S.E.M, for five paired experiments. * P < 0.05.
nding to be unaffected b y the hypothyroid state (dala not shown). Furthermore, we investigated the antilipolytic effect of insulin. The sensitbAty of insulin was enhanced fivefold in adipocytes from the hypothyroid rats (IC+,0, 0.51 _+ 0.14 n M , control vs. 0.11 ± 0.05 n M ,
¢' 1000 o O,a
m~°°°t '~ -~ 5000t IPl 4oot?
+S t I li l[I Fig. 2. FAfnir of hypothyroidism on inositol phosphate produ~+ion. Adipocytes were incubated with: L no addition; 1t, insulin (10 nM); III, epinephrine (5 FM)+propranoiol (5 ,~M). Experiments were performed as described in Materials and methods. Black and white bars represent adilxx--ytes from euthyroid mad hypothyroid rats, respectively. Data are expressed as epm/106 cells and are given as means+ S.E.M. for four paired experiments_
Fig 3. ,~ti]Jpot~dc effect of insulin. Adipocytes from euth>Toid {6) and h>~othyToid rats {G) were incubzted for 90 ~rl with insuhn. Lipolysis w~ s~3a~aulated-*ith ADA f~).5 U/wA). Data a++eexpressed as percentage of the ADA-s+.imulaledva~ue and are given as means_+ S.F-M. for five paired experiments.
hypothyroid, P < 0.05), whereas the maximal inhibition was unchanged (fig. 3). Activation o f the al-adrenoceptors (epinephrine (5 # M ) in the presence of propranolol (5 ,uM)) significantly enhanced the incorporation of myc-[3H]inositol into phosphoinositides, both ~n adipocytes from contro_+ and hypothyroid rats. However, no difference was observed between the two groups (3t.0 +_+_4.0% above basal value, control vs. 27_5 _+ 2.0% above basal value, hypothyroid, N.S.) (fig. 1). Additionally, a marked hydrolysis of phosphoinositides to inositol phosphates was observed during %-adrcmc~x'ptor stimulation, but just like phosphoinositide synthesis this pathway was unchanged by hypothyroidism (fig. 2). T h e a~-adrenoceptor specificity was demonstrated b y addition of the specific al-adrenoceptor antagonist prazosin (5 # M ) , which both inbdbited the a F a d r e n o ceptor-induced phosphoLnusitide hydrolysis (data not shown) and synthesis (fig. I). ,'I2ae residual phosphoinositide synthesis observed in connection with the cq-adrenoceptor blockade with prazosin, was due to a nonspecific effect of propranolot (Juhl etal., 1990). Furthermore, c A M P concentrazinn was reduced in the hypothyroid rats (431.0 ~ 35.5 p m o l / 1 0 ~ cells, control vs. 286.5 ± 55.0 p m o t / 1 0 + cells, hypothyroid, n = 3) when stimulated w'2th epinephrine (10.0 #M).
4. Discussion In the present study, it was demonstrated that insulin-stimulated phosphoinositide synthesis waS enhanced by hypothyroidism. In accordance with Czech et al. (1980), we found that insulin binding was unchanged b? hypothyroidism. Thus, the enhanced action of insulin was nut du= to an alteration in receptor binding. A s the ant[lipolytic effect of insulin was increased also in the hypothyroid animals, there seems to be a general postreceptor effect in the hypothyroid state which on-
hances insulin action. Since cAMP is aMe to inhibit insulin action in ~dipocytes (Taylor e t a l . . 1976; Lonnroth etaL. 19875, the decreased cA~ 2P levels found in adipocytes from hypothyroid ~ais could be involved in the increased effect of insulin, The insulin-stimulated pliosphoinositidc synthesis in adipocytes is consistent with previous studies (Pennington and Martin. 1985: Farese el al., 1986b; Augert and Exton, 19885. The precise site of action of insulin on phosphoinositide metabolism however, is unknown. Phosphoinositides are derived from CDP-diacylglycerol formed from phosphatidic acid. Glycerol-3-phosphate acyltransferase is involved in the synthesis of phosphatidic acid and, in adipose tissue, insulin has been reported to reverse the intfbitory effect of epinephrine on glycern~-3-phosphate acyltransferase a c t i v i t y (Sooranna and Saggerson 1976; Rider and Saggerson, 19835. Thus. the increased insulin-stimulated phosphoinositide synthesis in the hypothyroid state might be due to the lower content of cAMP, resulting in increased synthesis of phosphatidic acid. During ~ e last couple of years, contradictory data concerning insulin-induced hydrolysis of phosphoinositides have appeared in the literature. In contrast to Farese et al. (1986b), but in line with other groups (Pennington and Martin, 1985; Augert and Exton, 1988). we were not able to detect an), effect of insulin on phosphoinositide hydrolysis. Even in the hypothyroid rats where insulin action was augmented, we were still not able to find an effect_ of insulin on this pathway. The at-adrenoceptor effects on phosphoinositide syntt, esis and hydrolysis were found to be unaffected by thyroid status. Gare~a-Sainz_ and Fain (19805 and Fain ( I 9 8 D found simila: results in rat and hamster adipocytes concerning phosphoinositide synthesis; however, the at-adrenoceptor effect on phosphoinositide hydrolysis in hypothyroid animals has not been investigated before. Phosphoinositide hydrolysis involves activation of phospholipase C, which in turn is supposed to be coupled to the cq-adrenoceptor ~ a a G-protein (Williamson, 1986). G~-proteins associated with the adenylate cyclase complex have been found to be increased in adipocytes from hypothyroid rats (Ros et al., 19885, a finding which might explain some of the decreased /3-adrenoceptor effects seen in hypothyroid rats. Since the ~h-adrenoceptor effect on phosphoinositide turnover was unaffected by hypothyroidism, we suggest that the presumptive G-protein coupled to the at-adrenoceptor is not affected by hypothyroidism. In conclusion, the insulin-stimulated phosphoinositide synthesis in rat adipocytes was enhanced by hypothyroidism. Neither in adipocytes from normal nor hypothyroid rats did insulin activate phosphoinositide breakdown. Epinephrine ( a i - a d r e n o c e p t o r effect) stimulated both phosphoinositide synthesis and hydrol-
ysis. bu t. hypothyroidism did not affect these processes. Thus. hypothyroidism ha~ differentiated effects on the hormone-regulated phosphoinositide metabolism.
Acknowledgements We are Indebted to J. Sohoh. T. Skrumsager and P. Sonne for skillful technical ~ork The study was supported by grants from the Danish Medical Research Council. the Danish Diabetic Association. Jeppe og Ovita Juhls Mindelegat. P. Carl Petersens Fond. Novo" Fond. Nordisk lnsulinfond and Fonden tii L~egevidenskabens Fremme.
References Armstrong. K.3.. J.E. Stouffer. R.G. Van lnwegen, WJ Thompson and G.A Robinson, 1974. Effects of thyroid hormoh~ defi,:/ency on cyclic adenosine 3':5"-monopbosphate and control of !igolysis in fat cells, J. Biol.Chem. 249. 4226. Auger/. G. and J.H. Exton. 1988. Insulin and oxytocin effects on phosphoinositide metabolism in adipc,-'-ytes, J. Biol. Chem. 263, 3600. Berridge. M.J., RM.C Dawson, C.P. Downes, J.P. Heslop and R.F. Irvnie. 1983. Changes in the levels of mositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides, Biochem. f. 212, 473. Cherniek. S.S.. 1969, Determination of glycerol in aeyl glycerols, Methods Enzymol. 14. 627. Czech. M.P.. C.C. Malbon, K. Kerman, W. Gitomer and P.F. Pileh, 1980. Effect of thyroid status on insulin action in rat adipocytes and skeletal music, J. Clin. Invest. 66. 574. Fain, J., 1981. Catecholamine-thyroid hormone interaction in liver and adipo~ tissue, Life Sci. 28, 1745. Farese. R.V.. J.S. Davis. D.E. Barnes, M.L. Standaert. 3.S. Babinschkin, R. Hock, M.K. Rosic and R.3. Poltet, 1985, The de novo phosphohpid effect of insulin is associated ~ith increases in diacylglycerol, but not inositol phosphates or cytosolic Ca:÷. BiDchem. J. 231. 269. Farese. R.V., N. Rosic, M. Staendaert, J. Babischldn, D.R. Cooper, J.S Da~4s and RJ. Pollet. 1986a. Further evidence implicating diacytglycerol generation and protein kinase C activation in agonist-induced increases in glucose uptake, insulin-likeeffects of phenylephrine in BC3H-I myocytes, Diabetes 35, 951. Farese, R.V., J.Y. Kuo, J.S. Babischkinand J.S. Davis, 1986b. Insulin pro,,-okes a transient activation of phospholipase C in the rat epididymal fat pad. J; Biol. Chem. 261, 8589. Garcia-Sainz, J.A. and J.N. Fain. 1980, Effect of adrenergic amines on phosphatidylinositollabeling and glycogensynthase activity in fat cells from euthyroid and hypothyroid rats, Mol. Pharmacol. 18, 72. Goswami. A. and I.N. Rosenberg, 1978. Thyroid hormone modulation of ephiephrine-induced lipolysis in rat adipocytes: a possible role of calcium, Endoctinol. 103, 2223. Juhl. H., P.H. Andersen and B. RJchelsen. 1990, Phosphoinosilide metabolism in adipocb'tes from fed and fasted rats, Biochem. Int. 20~ 1189. Litosch, L and J.N. Fain. 1986, Regulation of phosphoinositide breakdown by guanine nucleotides. Life Sci. 39, 187. Lermroth. p.. J.1. Davies, I. L~nnroth and U. Smith. 1987, The interaction between the adenylate c~'clase system and insulh~stimulated glucose transport, Biochem. J. 243, 789. Matbon, C.C.. F.J. Moreno, J. Cabelli and J. Fain, I978. Fat cell adenylate cyclase and fl-adrenergic recep*ors in altered thyroid states. J. Biol. Chem. 253, 671.
McKer,zie, M.L.. K-G. Tnomakutty, M1 S,.6rt ._~,~ R H. Pointer, 1987. Interactinn be!wean Lns,,fin ~qd ~hyr~id }~3~cn~; ~n ~.hz control of carbohydrate and lipid metabdlsm in rat adipose t;asu~, Biecliem. Med. Metab Biol. 38, 81. Pedersen, O. and E. Hjdiund, 1982, Insulin receptor binding to fat and blood cells and insulin action in fat ceils from insulin-dependent diabetics, Diabetes 31.70@ Penningmn, S.R. al~d B.R. Marlin, !985. Insulin-stimulated phosphoinositide metabolism m isolated fat cells, J. Biul. Chem. 209, 11039. Richdsen, B. and H- Beck-Nielsen. 198~, Decrease of prostaglandin E~ receptor binding is accompanied by reduced antilipol~dc effects of prostaglandin E 2 in i~olated rat adipocytm, J. Lipid. Res. 26, 127. Rider, M.H. and E.D. Saggerson, 1983, Regulation by noradrenalLne of tube mJlochondtial and ll0Jcrosoma] for/D£ of glycerol phosphate acyltransferase in rat adipocytes, Biochem. J. 214, 235. Rifici, V.A. and M.L. Kaplan, 1984, Darect effect of tffiodthyronine on glucose utilisation in adipocytes from obese and nonobcse Zucker rats, Int. L Binchem. 16, 353.
Ros, M., J.K Nertup and C.C. Malbon, 1988. Siead)-stat~ !e~e]~ of Chem. 263, 4362 SeibeL M.J., M. L~ob~a and E. Herrera, ]978. Effect>. of g]ucose, • -. ,Jlin and ad-enatin en glycCrot melah~o~sm L, ~dipo~e ~*s~,ue from i,ST-,~yr~id rats, Me*l. Cell Endocv~no]. 10. 307. Sooranna. S.R and E . D Sagger~on. 1976, Studies ol the effect of adrenaline on glycerol phosphate acyltransferase act ~at) in rat adipocytes, FEBS Leu. 90, 141. Tayinr, W.M., M . L Mak and M.L Halperm. 1976. E~'Iect of Y:5"cyclic AMP on glucose transporl m. adipe~}~es. Proc Nail. Acad SoL U.S.A. 73, 4359. Uhing. RA.. V. P~pi¢, H. Jiang and J.H. ]:-xton. ]986. Horm~nestimuIated pol3.-phosphoinosidde breakdo~,~na in rat lker plasma membranes. J. BinL Chem. 261.2i40. Williamson. J.R.. ,gg6, Role of mositol br -eekdo~ in the generation of intraceltular s~gmals. Hypertension (S~ppl iii 8. 1"-4~.
81
~: 1991 ElsevierScience Publisher~ B.V.0922~41C6/91/$03.50 A D O N I S 0922410691000614
EJPMOL 90133
Phosphoinositide metabolism in adipocytes from hypothyroid r a t s Per .Heden Andersen, H e n n i n g JuhI, Steen Bon!okke Pedersen, Bj~rn Richelsen Umt,ers~ty Chnic of End~crmoloKv m~d InternM Medicine, Aarhus a m ~ s ~ e h ~ , DK-80OO Aarhus C, Denmark
Received 6 September1990.accepted25 September1990
The effect of hypothyroidism on insulin- and epinephrine-stimulated phosphoinositide metabolism was investigated in rat adipocytes. Insulin-mediated phosphoinositide synthesis was enahaneed by h~q3othyroidism(t4.5 =' 1.5% above basal level, control vs. 22.5 *_.2.0% above basal level, h3pothyroid, P < 0.05). However, insulin did not stimulate hydrolysis of phosphoinositid~ ~o inositot phosphates, neither in control nor m hypothyroid rats. The ai-adrenoceptor agonist (e~& epinephrine) siN-Jficamly stimulated the incorporation of myo-[3H]inositolinto phosphoinositides (P < 0.01) and hydrolysis of phosphoinosi*ides {P < 0.0l L but this stimulatory action was unaffected by the hypothyroid state. The iesuhs suggest that hypoth5Tnidism has differentiated effects on the hormone-regnlaled phosphoinositide metabolism and that the presumptive G-protein coupled to the a~-adrenoceptor seems to be unaffected by hypothyroidism. Adipose tissue; Hypothyroidism: Insulin: Epinephrine; Phosphoinosifides
L Introduction
Phosphoinositide metaboiism has attracted much attention during recent years, as it has become evident that phosphoinositide turnover is involved in lransmembrahe signalling. Phosphoinositide turnover is stimulated by aa-adrenoceptor agonists in various cells such as myecytes (Farese et at., 1986a), hepatocytes (Uhing etal., 1986) and adipocytes (Pennington and Martin, 1985). Previously, it has been shown that insulin is able to stimulate phosphoinositide synthesis in rat adipocytes (Farese et al., 1985; Pennington and Martin, 1985; Augert and Extort, 1988). According to the current hypothesis, hydrolysis of phosphatidylinositol-4,5-biphosphate to inositol triphosphate and diacylglycerol, is stimulated by activation of phospholipase C, which seems to be coupled to a wide variety of surface receptors via Goproteins (Litogch and Fain, 1986). Thyroid status markedly affects the fl-adrenoceptor response of catecholamines in rat adipocytes. Hypothyroidism impairs and hyperthyroidism enhances the lipolytic response of catecholamines (fl-adreno~ptor effect) (Armstrong et al., 1974; Goswami and Rosenberg, 1978; Malbon et at., 1978; Fain, 1981). The ira-
Correspondence to: Per Heden Andersen, UniversityClinic of Endocringtogyand Imemal Medicine,Aaxhusamtss~ehus,Tage tfansens Gade, DK-8000 Aarhus C, Denmark.
pact of hypothyroidism on the catecholamine-induced phosphoinositide turnover (a~-adrenoceptor effect) has been investigated in only a few studies (Garcia-Samz and Fain, 1980; Fain, i981), and in these studies only phosphoinositide synthesis was examined. In both studies phosphoinositide synthesis was found to be unaffected by thyroid status. Insulin action on ~ucose metabolism and lipolysis in adipocytes also has been s h o w to be affected by thyroid status (Seihet et at.. I978: Rifici and Kip!an, 1984; McKenzie et at.. t987). However, the influence of thyroid ho~,xnones on the insulin-stimulated phosphoinositide metabolism has not been examined. In the present study, the effect of hypothyroidism on insulin- and a~-adrenoceptor-stimulated phosphoinositide metabolism was investigated. Both synthesis and hydrolysis of pho~vphoinositides were assessed. In addition, some aspects of the lipolytic system were investigated in order to confirm the hypothyroid state.
2. Materials and methods 2. L M a t e r i a l s
D-myo-[3H]inositol (specific activity 10-20 Ci/mmol) was purchased from A-mersham (U.K.). Dowex-l-X8 resin (formate form, 100-200 mesh) v-as obtained from Bio-Rad (U.S.A.). Epinephrine and propranotol were obtained from Sigma Chemicals (U.S.A.). Crystalfine porcine insulin was a gift from Novo (DK). 6-N-propyl-
82 2-thionracil was a gift from DAK-laboratofies (DK}. Prazosin was obtained from Ercopharm (DK). All other reagents were obtained as described in references (Pedersen and Hjollund, I982; R!chelsen and BeckNielsen, 198,1).
2.2. Ammals Male Wistar rals weiglling 125-150 g were divided into two groups (littermates): control and hypothyroid rats. Hypothyroidism was induced by keeping the rats for 1,1-18 days on an iodine-deficient diet and supplying the drinking water with 0.00625% 6-N-propyl-2thiouracil. Control rats were maintained on a similar diet containing iodine, and on tap water. Thyroid status was assessed.by measuring S-triiodthyronine (T3) and S-thyroxine (T4) by the ELISA technique (Amerlite, Amersbam).
Z3. Adipocyte preparation
packed with Dowex-l-X8. lnosilo! 1-phosphate (IPl), inositol 1,4-biphospbate ( I ~ ) and inositol 1.4,5-triphosphate (IP3) were sequentially eluted from the column w-ith buffers containing increasing formate concentrations (Be,ridge et al., 1983). Incorporation of myo-[3H]inositol into total phosphomos~tides was measured as described (Pennington and Martin, 1985).
2.5. Lipotysis studies Glycerol release was taken as an index of lipolysis. The glycerol release ,,,,'as determined enzymaticaliy (Peridochrom, Boehringer Mannheim) by a fluorometric method (Chernick, 1969). In experiments where the anti!ipolytic effect of insulin was examined, lipolysis was stimulated v¢ith adenosine deaminase ( A D A ) (0.5 u/~).
2.6. Statistics
Adipocytes were isolated from epididymal fat pads by collagenase digestion (Pedersen and Hjollund. 1982). The volume of the adipocytes was calculated (Pedersen and Hjollund, 1982). Adipocytes were finally washed four times and resuspended in a buffer containing (mM): 10 Hepes, 135 NaCI, 4.8 KC1, 1.7 MgSO4. 2.5 CaCI,, 0.2 N a H 2 P O 4, 1 N a 2 H P O 4. 5 glucose, 2.5% BSA, p H 7.4.
2.4. Pkosphoinositide metabolism Isolated adipocytes were incubated in HEPES buffer supplemented with myo-[3H]inositol (20 ,uCi/rnl) and LiCI (10 raM) for 90 min at 37°C. The cells were then washed three times in an isotope-flee buffer containing 10 m M LiC1 and diluted to 1-2 × 10 s cells/nil. Aliquots of 1 ral were preincubated for 15 rain and finally incubated in the absence or in the presence of hormone in a shaking waterbath for 60 rain at 37°C. Incubations •,,,'ere stopped by adding 1 ml ice-cold 10% trichloracetic acid and vigorous mixing. The samples ",,,'erecentrifuged 4000 x g for 5 rain and the supernatant was removed and neutralized by 10 m M sodiumborate. Radioactive inositol phosphates were chromatographed on a column
Data are given as means _+ S.E.M. P values were determined using Student's paired t-test. P < 0.05 was considered significant. N o statistical significance = N.S.
3. Results The hypothyroid state was confirmed by a significant decrease in thyroid hormones. In addition, the eninephrine-stimulated lipolysis was greatly reduced as expected (table 1). Further characteristics of the rats are shown in table t. Insulln-stimulated incorporation of myo-[3H]inositol into phosphoinositides was significantly increased in the hypothyroid state (14.5 +_ 1.5% above basal level, control vs. 22.5 + 2.0% above basal level, hypothyroid, P < 0.05) (fig. 1). In contrast to Farese et al. (1986b), we were not able to show an effect of insulin on the hydrolysis of phosphoinositides to inositol phosphates neither in the control nor in the hypothyroid animals (fig. 2). The increased effect of insulin on phosphoinositide synthesis could have been caused by enhanced insulin binding; however, we found insulin bi-
TABLE l Characteristics of euthyroid and hypothyroid rats. Data are given as rneans±S.E.M, for (n) paired experiments. * P<0.05; ** P<0.001; • ** P < 0.0001. n Basic characteristics Weight (g) 22 Fatcdlvolume(pl) 10 Trfiodth3,xonine(nM) 5 Th~lroxine(nlVO 6 Lipolysis~glycerolrelease(nmol/1Os cells) Basal 5 Epinephrine(12.5 t~M) 5
PTUdreated 199.77+ 127.00+0.10+2.60+116 540
4_43 15.00 0.04 2.10
± 30 ± 111
Control
P value
256.14+- 8.05 150.70+- 24.00 0.64-- 0.22 64.50 +- 5.90 254 1550
+- 52 +-326
N.S.
i
40 l
Fig. 1. Eff~'t of hypothyroidism on myo-[3H]inositol incorporation in phosphoino~tides. Adipocytes were incubated with: I, in~uhn (10 ruM); II, epinephrine (5 ,~M)+pmpranolot (5 taM); lit, epinephrine {5 ttM)+propranolol (5 ,uM)+prazosin (5 taM). Experiments were performed as described in Materials and methods. Black and white bars represent adipoeytes from euthyroid and hypothyroid rats, respectively. Data are expressed as percent above basal value (11150 ± 1959 epm/10 + cells, control vs. 125805:2124 epm/10 ~ cells, hypothyroid, N.S.) and are given as means±S.E.M, for five paired experiments. * P < 0.05.
nding to be unaffected b y the hypothyroid state (dala not shown). Furthermore, we investigated the antilipolytic effect of insulin. The sensitbAty of insulin was enhanced fivefold in adipocytes from the hypothyroid rats (IC+,0, 0.51 _+ 0.14 n M , control vs. 0.11 ± 0.05 n M ,
¢' 1000 o O,a
m~°°°t '~ -~ 5000t IPl 4oot?
+S t I li l[I Fig. 2. FAfnir of hypothyroidism on inositol phosphate produ~+ion. Adipocytes were incubated with: L no addition; 1t, insulin (10 nM); III, epinephrine (5 FM)+propranoiol (5 ,~M). Experiments were performed as described in Materials and methods. Black and white bars represent adilxx--ytes from euthyroid mad hypothyroid rats, respectively. Data are expressed as epm/106 cells and are given as means+ S.E.M. for four paired experiments_
Fig 3. ,~ti]Jpot~dc effect of insulin. Adipocytes from euth>Toid {6) and h>~othyToid rats {G) were incubzted for 90 ~rl with insuhn. Lipolysis w~ s~3a~aulated-*ith ADA f~).5 U/wA). Data a++eexpressed as percentage of the ADA-s+.imulaledva~ue and are given as means_+ S.F-M. for five paired experiments.
hypothyroid, P < 0.05), whereas the maximal inhibition was unchanged (fig. 3). Activation o f the al-adrenoceptors (epinephrine (5 # M ) in the presence of propranolol (5 ,uM)) significantly enhanced the incorporation of myc-[3H]inositol into phosphoinositides, both ~n adipocytes from contro_+ and hypothyroid rats. However, no difference was observed between the two groups (3t.0 +_+_4.0% above basal value, control vs. 27_5 _+ 2.0% above basal value, hypothyroid, N.S.) (fig. 1). Additionally, a marked hydrolysis of phosphoinositides to inositol phosphates was observed during %-adrcmc~x'ptor stimulation, but just like phosphoinositide synthesis this pathway was unchanged by hypothyroidism (fig. 2). T h e a~-adrenoceptor specificity was demonstrated b y addition of the specific al-adrenoceptor antagonist prazosin (5 # M ) , which both inbdbited the a F a d r e n o ceptor-induced phosphoLnusitide hydrolysis (data not shown) and synthesis (fig. I). ,'I2ae residual phosphoinositide synthesis observed in connection with the cq-adrenoceptor blockade with prazosin, was due to a nonspecific effect of propranolot (Juhl etal., 1990). Furthermore, c A M P concentrazinn was reduced in the hypothyroid rats (431.0 ~ 35.5 p m o l / 1 0 ~ cells, control vs. 286.5 ± 55.0 p m o t / 1 0 + cells, hypothyroid, n = 3) when stimulated w'2th epinephrine (10.0 #M).
4. Discussion In the present study, it was demonstrated that insulin-stimulated phosphoinositide synthesis waS enhanced by hypothyroidism. In accordance with Czech et al. (1980), we found that insulin binding was unchanged b? hypothyroidism. Thus, the enhanced action of insulin was nut du= to an alteration in receptor binding. A s the ant[lipolytic effect of insulin was increased also in the hypothyroid animals, there seems to be a general postreceptor effect in the hypothyroid state which on-
hances insulin action. Since cAMP is aMe to inhibit insulin action in ~dipocytes (Taylor e t a l . . 1976; Lonnroth etaL. 19875, the decreased cA~ 2P levels found in adipocytes from hypothyroid ~ais could be involved in the increased effect of insulin, The insulin-stimulated pliosphoinositidc synthesis in adipocytes is consistent with previous studies (Pennington and Martin. 1985: Farese el al., 1986b; Augert and Exton, 19885. The precise site of action of insulin on phosphoinositide metabolism however, is unknown. Phosphoinositides are derived from CDP-diacylglycerol formed from phosphatidic acid. Glycerol-3-phosphate acyltransferase is involved in the synthesis of phosphatidic acid and, in adipose tissue, insulin has been reported to reverse the intfbitory effect of epinephrine on glycern~-3-phosphate acyltransferase a c t i v i t y (Sooranna and Saggerson 1976; Rider and Saggerson, 19835. Thus. the increased insulin-stimulated phosphoinositide synthesis in the hypothyroid state might be due to the lower content of cAMP, resulting in increased synthesis of phosphatidic acid. During ~ e last couple of years, contradictory data concerning insulin-induced hydrolysis of phosphoinositides have appeared in the literature. In contrast to Farese et al. (1986b), but in line with other groups (Pennington and Martin, 1985; Augert and Exton, 1988). we were not able to detect an), effect of insulin on phosphoinositide hydrolysis. Even in the hypothyroid rats where insulin action was augmented, we were still not able to find an effect_ of insulin on this pathway. The at-adrenoceptor effects on phosphoinositide syntt, esis and hydrolysis were found to be unaffected by thyroid status. Gare~a-Sainz_ and Fain (19805 and Fain ( I 9 8 D found simila: results in rat and hamster adipocytes concerning phosphoinositide synthesis; however, the at-adrenoceptor effect on phosphoinositide hydrolysis in hypothyroid animals has not been investigated before. Phosphoinositide hydrolysis involves activation of phospholipase C, which in turn is supposed to be coupled to the cq-adrenoceptor ~ a a G-protein (Williamson, 1986). G~-proteins associated with the adenylate cyclase complex have been found to be increased in adipocytes from hypothyroid rats (Ros et al., 19885, a finding which might explain some of the decreased /3-adrenoceptor effects seen in hypothyroid rats. Since the ~h-adrenoceptor effect on phosphoinositide turnover was unaffected by hypothyroidism, we suggest that the presumptive G-protein coupled to the at-adrenoceptor is not affected by hypothyroidism. In conclusion, the insulin-stimulated phosphoinositide synthesis in rat adipocytes was enhanced by hypothyroidism. Neither in adipocytes from normal nor hypothyroid rats did insulin activate phosphoinositide breakdown. Epinephrine ( a i - a d r e n o c e p t o r effect) stimulated both phosphoinositide synthesis and hydrol-
ysis. bu t. hypothyroidism did not affect these processes. Thus. hypothyroidism ha~ differentiated effects on the hormone-regulated phosphoinositide metabolism.
Acknowledgements We are Indebted to J. Sohoh. T. Skrumsager and P. Sonne for skillful technical ~ork The study was supported by grants from the Danish Medical Research Council. the Danish Diabetic Association. Jeppe og Ovita Juhls Mindelegat. P. Carl Petersens Fond. Novo" Fond. Nordisk lnsulinfond and Fonden tii L~egevidenskabens Fremme.
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