Biochimica et Biaphysica Acta, 1137(1992) 208-214 © 1992ElsevierSciencePublishersB.V. All rightsre.fred 0167-4889/92/${15.0{)

Insulin effect on isolated rat hepatocytes: diacylglycerol - - phosphatidic acid interrelationship Patrizia M. Baldini, Antonella Zannetti, Victoria Donehenko, Luciana Dini and Paolo Luly Department of ~iology, Uni~'ersiO"of Rotor; "Tor Vergata; Rome (Italy)

(Received24 February 1992) (Revisedmanuscriptreceived23 June 1992) Keywords: Insulin;Phospholipase;Phosphatidylcholine;Diacylglycerul:Phosphatidicacid; (Hepatocyte) it is widely accepted that insulin action does not involve inositol phospholipid hydrolysis through the stimulation of a phosphatidylinositol-spc(ific phospholipase C (PI-PLC). This consideration prompted us tc, investigate the insulin effect on the mechanism leading to the accumulation of diacylglyccrol (DAG) and phosphatidic ~cid (PAl in ~'at hcpatocytcs. Basically, insulin induces: (i) a significant increase of both [SH]glyccrol and fatty acid labelling of DAG; (ii) a significant increase of PA labelling prccceding DAG labelling and paralleled by a decrease of phosphatidylcholinc (PC) labellin~ These observations, which suggest an insulin-dependent involvemcn' of a phospholipas¢ D. arc strengthened by the increase of PC-derived phosph,:didylethanol in presence of ethanol. Finally, th~"t~bservatinn that the PA levcls do not return to basal suggests that other mechanisms diffcr~mt from PC hydrolysis, such as the stimulation of direct synthesis of PA, may be activated. Introduction Some hormones, neurotransmitters and growth factors increase diacy~glycerol (DAG) levels by phospholipase C-mediated hydrolysis of phosphatldylinositol bisphosphate (PIP2), thus inducing protein kinase C (PKC) translocation from cytosoi to the plasma membrane: this pher.omenon is interpreted as evidence of PKC activation [1], but seems to be unnecessary for insulin-dependent stimulation of PKC isoforms in cultured neurons [2]. Insulin has been reported to induce the increase of DAG levels in BC3H-I myocytes [3], in rat diaphragm [4], in rat adipose tissue [5], in skeletal muscle [6] and in rat hepatocytes [7]. Insulin does not stimulate el P2 hydrolysis significantly [8,9], but it seems that DAG may arise from sources other than inositol phospholipid hydrolysis: (i) de novo synthesis of phosphatidic acid (PAl [10]; (ii) hydrolysis of phosphatidylinositol glycan [11,12]; (iii) hydrolysis of non-inositol

Correspondenceto: P.M. Baldini,Departmentof Biolosy,University of Rome. "Tor Vergata',00173 Rome, Italy. Abbreviations: DAG. 1,2-sn-diacylglycerol;PC, phosphatidylcholine; PE. phosphatidylethanolamine;Pl, phosphatidylinositol;PA, phosphatidic acid; TAG, triacYlslycerul;MAG, monoacylglycerul;PIP2, 4.5-phosphatidylinositolbisphosphate;PKC, protein kinase C; IP.~. 1,~,.5.inosi!'sl trisphosphate; BSA. bovine serum albumin: Hepes, 4-(2-hydroxyethyl)-I-I:iperazincethanesulphonieacid" PetOH. phosphatidylethanol;G3PAT,glycerul-3-phosphateacyltlansferase.

iipids, such as phosphatidylcholine (PC) [13,14]. These different mechanisms may lead to the production of structurally distinct species of DAG without inositol tris!~hosphate (IP3)-induced calcium mobilization [15] and in this connection it was suggested that insulin might cause a selective activation of PKC isoforms by structurally distinct molecules. 8aito and Kanfer [16] reported the presence of a phospholipase D in mammalian tissues; the enzsme has been purified from rat brain [17] and human eosinophils [18] being characterized in many tissues [19]. The hydrolysis of PC by phospholipase D leads to the formation of phosphatidic acid and choline; the phosphatidic acid, in turn, may be hydrolyzed by a phosphatase to yield diacylglycerol [20]. In the presence of primary alcohols, phospholipase D can also catalyze a transphnsphatidylation reaction, i.e., the exchange of the polar head group of the phospholipid substrate with the given ~l,:ohol to form the corresponding t~hogph~tidyta;f,.,hol ['2.1,22]. Reports have appe.~red on vasopressin-induced diacyiglycerol and phosphatidie acid production in rat hepatocytes with special reference to qualitative fatty acid content [23]; a more or less generalized increase of diacylglycerols upon vasopressin treatment was reported [24,25] together with an increase of arachidonic and stearic acid as well as a decrease of oleic acid presence in diacylglyderols [26]. Finally, Augert et al, [27] reported the presence of twelve molecular species of" dia~lglycerol

209 but only three species out of twelve (containing 16:0, 18:0, 18:2, 20:4) were altered regarding their fractional percentage following vasopressin treatment. The present study was aimed to investigate, in rat hepatocytes, th~ insulin effect on the mechanism leading to the aecu~nulation of diacylglyeerol and phosphatidic ac!d, i,ainly derived from plasma membrane phosphatidylcholine hydrolysis; however, in addition, the possibility of a direct insulin-dependent stimulation of PA synthesis from sources other than membrane phospholipids has [een taken into account. Materials and Mett~ods

Isolation and treatment of hepatocytes Hepatocytes wetc isolated as described by Moldeus et al. [28] with minor modification from male rats (Wistar, average boc~. wt. 120-150 g) anesthetized with sodium pentoba~bital (1 mg/100 g body weigh0. Briefly, liver was peffused first through the portal vein with Ca2+-tree Hanks' balanced salt solution for 5 min; men the tissue was perfused with collagenase (type I from BoehringeroMannheim, Germany) 90 U / m l in Hanks' buffer containing 5 mM Ca 2+ and 0.1 M Hepes at 390C until the liver was soft, (usually 4 to 7 rain). The liver capsule was removed and the isolated hepatocytes were shaken into fresh Krebs-Henseleit medium. Parenchymal cells were washed by two centrifugations at 600 x g for 5 rain. Cell viability, throughout the experimental procedure, was always higher than 95% as assessed by Trypan blue exclusion. 2.106 cells were pre-labelled with 5 ,¢Ci of [3H]glycerol by incubation 37°C for 60 min in 100 mM Hepes .4-0.2% bovine serum albumin (BSA) before addition of porcine insulin; after 60 min, labelled hepatocytes were washed twice, as indicated above, to remoee free radioactivity and then resuspended in the same buffer before insulin treatment. At various times, after addition of the hormone, 0.8 ml samples were removed and extracted with 3 ml CHCI3/MetOH (1 : 2); 1 ml of CHC! 3 and 1 ml of 0.1 M KCI were then added with vigorous mixing. After centrifugafion (15 rain at 2000×g), the aqueous layer was discarded and the CHCI 3 phase was filtered, transferred to new tubes and dried under N 2. For fatty acid labelling experiments, 2- I0 ~' hepatocytes were prelabelled for 60 rain at 37°C before insulin addition as lollows: with 5 p.Ci of [3H]myristic acid; with 0.5/zCi [14C]palmitic acid, [~4C]stearic acid, [14C]oleic acid; with 0.25 p.Ci [t4C]araehidonic acid. Thia-layer chromatography of lipids The dried lipids ',,ere redissotved in 20 p.l of CHCi 3. Giycerolipids were separated from other iipids by chromatography on 20 x 10 cm silica gel 60 plates, activated at I10~C for 1 h immediately before use. The

solvent system was: light petroleum (b.p. 40/60°C)/ diethyl ether/acetic acid (80: 20:1, by eel.). Phospholipids were identified by two-dimensional chromatography on 20 × 20 cm silica gel 60 coated glass plates activated as above. The solvent system was: CHCl3/ MetOH/25% NH 4OH/H 20 (58: 34.7: 3.5 : 3.5, by eel.) in the first dimen~km, and C H C l 3 / M e t O H / a c e t o n e / acetic acid/H20 (49:16.4:19.7:9.8:5, by eel.) in the second. At the end of chromatography, the plates were dried under N 2 and the spots revealed under iodine vapours, scraped off and cluted with 0.2 ml EtOH and counted for radioactivity after addition of 5 ml Optiflour (Pt, ckard Instruments, Downers Grove, IL). Lipids were identified by comparing their R r values with those of authentic standards obtained from Supeico (Bellefonte, PA).

Fatty acids analysis Fatty acids were methylated according to Morrison and Smith [29] and analyzed in a Perkin Elmer 8310 gas chromatograph equipped with a flame ionization detector and interfaced with a Perkin-EImer LCI-100 integrator. Analysis of fatty acid methyl esters was carried out using a GP 3% SP-2310/2% SP 2300 on 100-120 Chromosorb W AV column (6' × 1/8", from Supelco). The column temperature was programmed as follows: 5 rain at 170°C, then a stepwise (3C°/min) increase to 250°C and finally 10 rain at 2500C; the nitrogen flow ra~e was 25 ml/min. Fatty acids were identified by comparing their peak retention times with those of authentic standards obtained from Sigma (St. Louis, Me). DAG mass estimation Diacylglycerol content was measured by incubating aliquots of lil~id extract with DAG kinase as described by Preiss et al. [30]. After incubation [3~'P]phosphatidie acid was purified by thin-layer chromatography [31] and DAG content .vas calculated by comparing samples to DAG stan~,~rds. Transphosphatidylation reaction In the experiments for determination of phospholipase D activity, 2.104 cells were prclabelled with 1 /tCi of [3H]myristic acid for 60 rain in the presence or absence of 2% ethanol before illsulin addition. Phosphatidylethanol and phosphatidie acid were separated oy thin-layer chromatography on 20 x 20 silica gel 60. The plates were developed with the solvent system ethyl acetate/iso-octane/acetic acid/H20 (130: 20: 30:100, by eel.). At the end of chromatography the plates were dried under N, and the spots revealed under iodine vapours, ~:eraped off and eluted with 0.2 ml EtOH and counted fol radio.~ctivity after addition of 5 ml Optifluor. PA was i¢ie~ltified by eomp'-~,'ing its R v value with that of an authentic standard. PetOH

210 was identified by comparing its R r value with that of a standard prepared according to Chattopadhyay et al.


~o _ ~


Determitlation o f G 3 P A T activity

G3PAT activity was measured according to Lawson e t a l . [33]. The incubation mixture contained 250 mM KCI, 50 mM Tris-HCl (pH 7.4), 0.7 mM dithiothreitol, 0.2 mM sn-[1,3-3H]glycerol 3-phosphate (1 p.Ci), 136 p.M palmitoyl-CoA, 6 m g / m l of fatty acid-free bovine serum albumin and 500/~g of liver homogenate protein in a final volume of 0.8 ml. The reaction was stopped after 3 miu at 37~C by adding 1 ml of water-saturated butanol followed by 0.75 ml of butanol-saturated water. Radioactivity in the bottom phase was determined by liquid-scintillation counting.

o^o ~'V

,i.i______i_ r 0~

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~ 0

tlme (min)

Fig. 1. Time-courseof inqulineffect on [3H]glycerolincorporation into DA,'3, PA and PC. Cells were prelabelled for I h with [3H]glycerolbefore adding10-9 M insulin.Each point is the average +S.D. of duplicate measurement,carried out on four independeut preparations. * P < 0.05, at least, as from both paired data analys~s with respect to controlgand the Wilcoxontest. The absenceof bars indicatesa S.D. below2% of reported values.

StatL~tical analysis

Data were analyzed for statistical significance using the SPSS/PC + statistical package (SPSS Inc., 19841985) on an IBM-PS/2 computer. Materials

Radioactive ma,'erials were from Amersham International (Amersham, UK): l(3)-[3H]-glycerol (3 C i / retool); 9,10(n)-[3H]myfistic acid (53 Ci/mmoD; 1[t~C]ulcic acid (52 mCi/mmol); l-[t4C]stearic acid (55.3 mCi/mmoi); !-[t4C]palmitic acid (56.7 mCi/mmol); 1[t4C]arachidonic acid (55 mCi/mmol); [',/- 32P]A'TP (3 Ci/mmol); L-[U-t4C]glycerol 3-phosphate (155 m C i / m m o ! ) . Plates for thin-layer chromatography were obtained from Merck, Darmstadt (Germany). Diacyiglycerol kinase was from l,ipidex (Westficld, N J). All other chemicals were of the purest reagent grade. Insulin and vasopressin were front Sigma," St. Louis, MO. Results Preliminary experiments on- [3H]glycerol incorporation into hepatocytes indi~.ated that, in a time range up to 90 min, optimal incorporation was obtained after 6~D rain (not shown); the ["H]glycerol labelling of hepatc,cytes was routinely carried out for 60 rain before testing horwoue effect. After 60 min of [3H]glycerol prelabelling, insulin ( 1 . 1 0 -9 M) treatment induced a significant increase of D A G and PA label!~ng as well as a significant decrease of PC labelling (Fig. 1); in particular, the insulin effect on PC and PA was significant at 30 s, whereas DAG was positively affected aftel' 1 rain. At the same time we observed, after insulin t~'eatment, a slight 5at .-lot significant increase of T A G and PE labelling after 2 and 5 rain, respectively, and no appreciable alteration of PI and MAG (not shown~. We then carried out experiments in order to assess the insulin effect on D A G mass in isolated hepal:ocytes

(Table I). Insulin, at any concentration tested, significantly enhanced D A G levels already at 30 s ( P < 0.001, with respect to basal level) with a maximal effect at 2 rain (significant with respect to 1 and 5 rain, P < 0.002 at least, at 10 -9 M and 10 -1° M insulin): this peculiar behaviour is stressed by the different effect of vasopressin which induced a steady increase of DAG mass. The dose-responsiveness of insulin effect appears to be biphasic as already reported in different systems [3:,,35]. In Table II we report the major fatty acid pattern of hepatocyte diacyiglycerol before and after a brief insulin treatment selected on the basis of previously reported data (see Fig. 1 and Table I). Then we labelled hepatocytes with different fatty acids for 60 rain to follow their turnover in diacylglycerol, and the same approach was also applied to monitor PA and PC labelling after insulin stimulation: myr~stic (14:0), palm±tic (16:0), stearic (18:0), oh:ic (18:1) and arachidonic acid (20:4)were employed. We observed (Fig. 2) a significant and time-dependent labelling in diacylglycerol, due to insulin stimulation, only for 16:0, 18:0 and 20:4 whereas a very transient increase was observed for 14:0; for all labelled fatty TABLE I Time-course of insulin effect on DAG mass of isolated rat hepatocytes

Resultsare reported as % increaseover basal level(7.20+ 1.09amol DAG/106 cells) and are averages+S.D, of 4 independent experiments carried out in dupli~-ate. Time(min)



10 - s

10 -y


0.5 I

80:1:12 1614-16

92:1:6 94:1:10 2{)7-I-17 158:1:12

2 5 15

175±!2 270±24 242±21 164±115 182±21 163±i8 158+16 176+20 133+t5

(10 - s M)


82:1: 6 159±14 165:t:18 170+20

211 acids employed we observed an increased turnover in PA starting at 1 min and a decreased labelling of PC at 30 s followed by a late normalization after 2 ~ 5 min. Finally, as far as Pl is concerned, we did not observe any significant alteration of labelling for all fatty acids tested (not shown). Fig. 3 shows ,*he time-course of [3H]phosphatidyb ethanol and of [:~H]phnsphatidic acid formation in the presence of 2% ethanol in isolated hepatocytes after insulin addition. [3H]Phosphatidylethanol increased

around 2 min up to 30 rain, whereas [3H]phosphatidic acid increased at early time points and then decreased at around 2 rain. The D A G production after insulin stimulation in the presence of ethanol was basically unaffected (not shown). The effect of insulin on another possible pathway of P A formation, the glycerol-3-phosphate acyitransferase (G3PAT) activity was also tested. In Fig. 4 we report the time-course of insulin (10 -9 M) on G 3 P A T activity of homogenates obtained from isolated hepatocytes

,ot i 5oi. /

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. . . . . 4







6 8 10 lima (mln)



ofi~ ~ .,o[

° 14


time ( , , i . )







m 4

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FiB. 2. Tim~-course of insulin effect on PC (E]), EA (o) and DAG (11) after hepatocyte labelling with different fatty acids. 2.10° cells were prelabelled for l h with [3H]myrlst_ieacid (t4:0), l:4C]palmitic acid (16:0), [aaC}stearleacid (18:0), [laC]oleit' a~.id(18:1) and [14C]arachidonic acid (20:4). RcsulLsate reported as % of variation with respect to basal levels which did not change significantlythroughout the experimental period. Basal I~'els were (dpm/2-10e cells, averages±S.D, of dupiic~te measurements carried out on 4 independent preparations): PC 29017:1:2375, PA 5490±450, DAG 8390±677 for 14:0; PC 22775± 1270, PA 490±41, DAG 327+52 for 16:0; PC 13420+ 1252, PA 189:J:26, DAG 362+16 for 18:0: PC 11153.-1:1908, PA 900+52, DAG 857+143 for 18:1; PC 55704±7089, PA 2125:1:284, DAG 932±83 for 20:4. * P < 0.05, at least, as from both paired data analysis with respect to contro|s and the Wilcoxontest. The absence of bars indicates a S.D. below 2% of the reported values.

212 TABLE II Effect of insulin (10- 9 M) on DAG fatty acid composition

Kesuhs. represent the % compositkm (avcrage±S.D.) obtained in 4 indepe~zdent experiments. Fatty acid relative percentages below I% were not considered. Insulin treatment was carried out for 2 rain before starting the procedure for gas chromatographic analysis. Fatty acid 14:0 16:0 16:1 18:0 18:! 18:2 20:4 22:6

Control 4.0+0.5 29.0± 1.2 10.8±0.9 13.6±0.4 27.8+_2.4 2.0±0.2 4.6±0.3 3.8:/:0.6

Insulin 5.4+0.6 * 31.6"i:1.4 * 10.0± 1.0 15.4~0.7 * 25.6+2.2 2.6±0.3 * 5.3±0.4 * 4.9+-0.2 *

P < 0.05 at least, from paired data analysis.

15 20 25. aO time (mic,) Fig. 3. Time-course of [:tH]phosphatidylethanolformation in isolated h,:patocytes after insulin addition. Cells were prelabclled with [3H]myristicacid for 1 h before adding 10-'~ M insulin for different times in the presence ( • ) and absence {,a ) of 2% ethanol. Results are reported as % of increase over basal values which were (dpm/2i0 s cells, averages± S.D. of duplicate measurements carried oat on 4 independent preparations): 5100=1:402 ~or [3H]PA; 1785+2it) for [3H]PetOH. * P < G.05. at Icas¢. as frGm both paired data analysis with respect to controls and WUcoxon test. The absence of bars indicates a S.D. below 2% of reported values. 5

[ 0.9~/ o


. !


. 2


. 3

. 4


t,me (s*nl

Fig. 4. Time-course of insulin effect on G3PAT activity. Cells were treated with insulin (10-9 M) for the times indicated; the activitywas then assessed on cell homagcnates. Each Imint is the average ± S.D. of duplicate n:easuremen~scarried out o, 4 independent preparations. * P < tit)5, at least, as from both paired data analys;s with respect to controls and Wil,:oxontest

previot~sly treated with the hormone. G 3 P A T reached a maximura after 1 rain of stimulation and then declined s;doothly. Discussion We report, in isolated rat hepatocytes, a significant increase of D A G and P A as well as a significant decrease of PC labelling at early times after insulin treatment; in addition, the early D A G increase has al~o been studied regarding the assessment of the hormonal effect on D A G mass: i.e., the overall cellular D A G content under steady-state conditions. Our results suggest that the insulin-induced increase of D A G levels, since the hormone does not increase inositol phospholipids hydrolysis in hepatocytes [27], can be derived directly from PC hydrolysis and, indirectly, from the increased P A which, in turn, could undergo a phosphatase-mediated hydrolysis. A more dynamic study after cellular labelling with [3H]glycerol (Fig. l ) shows that the increase of P A (0.5 m i n i precedes D A G increase (1 mini, being paralleled by PC decrease. In hepatocytes t r e a t e d with Ca2+-mobilizing agents (vasopressin, angiotensin II, epinephrine, epidermal growth factor) the P A accumulation occurs through tbe activity of a phospbolipase D [23]: in particular, following vasopressin stimulation, P A is formed much more rapidly than DAG, mainly within the plasma membrane [36]. in fact, it is known that plasma membranes contain a phospholipase D activity stimulated through a G protein system [37] as well as by I)2 purinergic agonists [38] and by the receptors for Ca2+-mobilizing agents [39]; moreover, phorbol diesters, with insolin-like effects, reportedly induce, in different cellular systems, PC degradation through the activation of both phospholipase C [40,41] and phospholipase D [42]. On the basis of our results, the activation of the phospholipase D pathway seems to be likely: only the phospholipase 13. ~ known to catalyze the phosphatidylethanol formation from PC in the presence of ethanol, and insulin increases the phosphatidylethanol formation in i ~ i a t e d hepatocytes. P A decrease is paralleled by the ~ormation of phosphatidylethanol, even if basal P A levels are not completely restored following insulin stimulation (Fig. 3). This observation is in accordance with the increase of P A levels, after insulin stimulation of G3PAT, involving de novo synthesis of P A [43,44]. The activation of the phospholipase D pathway a n d / o r of the phospbolipase C pathway in hepatocytes after insulin stimularion appears to be a likely possibility: the relative contribution of the two pathways to D A G increase is still unknown and probably the relevance of a particular phospholipase depends on both the e~ll ~.,pe and agonist utilized, as suggested by Cabot et aL [45].

213 The changes in the D A G species generated in response to CaZ+-mobilizing agents and other agonists [46] are in accordance with previous observations [474o] which suggest that inositol phospholipids are not the only source of DAG. In hepatocytes, inositol phospholipid composition differs from that of D A G and P A after vasopressin stimulation [23,24]; Augert et al. [27] have shown that P I P 2 is a limited source of D A G and PA, thus demonstrating the presence of unique pool of hormone-sensitive PI/'2 and indicating that D A G accumulation can be mainly due to PC breakdown, possibly in relation with the regulation of protein kinase C. In the present work, we tried to gain information on the fatty acid profile of bepatocyte diacylglycerols, te~ting the insulin effect on the basal composition as well as on fatty acid turnover after specific fatty acids labelling: our gas-chromatographic analysis indicates an increase of some fatty acids in diacylglycerols after 2 rain of insulin stimulation and this observation is strengthened by the increased turnover of the same fatty acids in diacylglycerols in prelabelled hepatocytes (Table 1I and Fig. 2). Moreover, after phospholipid fatty acid labelling, we have observed, for all fatty acids tested, a n increased turnover of P A at early ~imes of insulin stimulation, together with a decrease labelling of PC, but we did not observe any significant variation of PI labelling (not shown). These findings are in agreement with increasing evidence [50] that some agonisls stimulate both PC breakdown, involving an early activation of phospholipase D to give P A and, at a later time, an activation of phospholipase C to give DAG. W e suppose that insulin increases early P A labelling througil the stimulation of phospholipase D without 'ruling out a later contribution of PC-dependent phospholipase C to D A G increase; in addition, the observation that the P A levels did not return to basal suggests that [3H]PA can derive also from sources other than PC breakdown. In this respect, we demonstrated that P A can be formed by a direct stimulation of de n e r o synthesis, through the activation of the G 3 P A T pathway, as has also been reported in myocytes and ad[pocytes [43,44]. In conclusion, we have presented evidence that, on the one hand, points to a relevant role of PC hydrolysis through the activation of a phospholipase D for the generatio.l of putative mediators of insulin action, and on the other does not rule out significant contributions by mechanisms not strictly dependent on membrane phospholipid breakdown.

Aelmowledgements This study was supported by grants from the Italian Ministry of University and Scientific-Technological Research ( 4 0 - 6 0 % funds).

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Insulin effect on isolated rat hepatocytes: diacylglycerol-phosphatidic acid interrelationship.

It is widely accepted that insulin action does not involve inositol phospholipid hydrolysis through the stimulation of a phosphatidylinositol-specific...
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