Insulin effect on the phospholipid organization and some enzyme activities of rat liver membrane fractions

Comp. Biochem. PhysioL Vol.95B, No. 4, pp. 685-689, 1990 Printed in Great Britain

0305-0491/90$3.00+ 0.00 © 1990PergamonPress plc

INSULIN EFFECT ON THE PHOSPHOLIPID ORGANIZATION A N D SOME ENZYME ACTIVITIES OF RAT LIVER M E M B R A N E FRACTIONS DIANA H. PETKOVA,MARIANAN. NIKOLOVA,ALBENAB. MOMCHILOVA-PANKOVAand KAMEN S. KOUMANOV Central Laboratory of Biophysics, Bulgarian Academy of Sciences, I 113 Sofia, Bulgaria (Received 2 August 1989)

Abstract--1. The influence of insulin on rat liver membrane lipid composition, fluidity, some enzyme activities and asymmetry of microsomal phospholipids were investigated. 2. The total phospholipids and cholesterol were increased in microsomes and reduced in plasma membranes from insulin-treated rats. 3. Of all the investigated enzymesparticipating in the lipid metabolism, only the neutral sphingomyelinase activity was observed to be enhanced, whereas the ceramide--phosphatidylethanolamine (PE) synthetase and phospholipase A2 activities remained unchanged. 4. Insulin administration caused translocation of phosphatidylserine (PS) and PE to the outer leaflet and of phosphatidylinositol (PI) to the inner leaflet of microsomal membranes.

INTRODUCTION It is largely accepted that the mechanism of insulin effect is mediated by interaction with highly specific receptors, situated in liver plasma membranes (Cuatrecasas, 1974; Olefsky, 1976). This hormone is an important regulator of the synthesis of proteins, carbohydrates and lipids. There is evidence indicating that insulin stimulates lipogenesis in the liver (Topping and Mayes, 1972, 1982, 1987 et al.) and adipose tissue (Kiechle et al., 1986; Sandra and Marshal, 1986), as well as the turnover of phospholipids and particularly of phosphoinositides (Farese et al., 1982; Farese et al., 1983). Recently, insulin was reported to reduce methylation of phosphatidylethanolamine (PE) to produce phosphatidylcholine (PC) by inhibition of the glucagen-dependent methyltransferase (Merida and Mato, 1987). Moreover, insulin is known to inhibit t-oxidation of fatty acids (Topping and Mayes, 1982; Topping et al., 1987), thus providing possibilities for elevation of lipogenesis and lipoprotein secretion. The insulin-induced alterations in the lipid composition of different membrane fractions might influence their physico-chemical functions. For example, Lully and Shinitzky (1979) reported that the addition of 10 -9 M insulin to a suspension of rat liver plasma membranes increased the overall lipid microviscosity by approx. 10-20%. In contrast, Dutta-Ray et al. (1985) reported a significant reduction of membrane microviscosity using erythrocytes, treated with physiological doses of insulin. It is well known that many membrane-bound enzyme activities depend on the composition and physico-chemical properties of the surrounding lipids (Coleman, 1973). In previous papers we reported that the activities of liver plasma membrane-bound phospholipase A 2 (PLase A2) (Momchilova et al., 1986) and neutral sphingomyelinase (SMase) (Petkova

et al., 1986) depend on the structural order and composition of the membrane lipid bilayer. In this regard, we investigated the influence of in vivo treatment with insulin on the phospholipid composition and organization of rat liver plasma and microsomal membranes as well as on the activities of some enzymes, related to phospholipid metabolism. MATERIALS AND METHODS

Animals Male Wistar rats (Ratus norvegicus) weighingabout 200 g and fed a standard laboratory diet were separated into two groups. The animals in the first group were injected with 6 IU insulin per kg body weight and were decapitated 6 hr after insulin administration. The animals in the control group were injected with 0.9% NaC1. Reagents The reagents used were phospholipase C (PLase C) from Clostridium welchii (Sigma), L-~-phosphatidylethanolamine, sphingomyelin(SM) (Sigma), 2,4,6-trinitrobensenesulfonate (TNBS) (Fluka), 1-acyl-[2-t4C]linoleoyl-sn-glycerophosphoethanolamine and [methylJ4C]choline sphingomyelin (Amersham). Isolation of rat liver membrane fractions Liver plasma membranes (PM) were isolated according to the procedure described by Wisher and Evans (1975). This method includes membrane flotation through a discontinuous sucrose gradient in a Sorvall OTD-50 ultracentrifuge, rotor AH-627 at 96,000g for 3 hr. The purity of the membrane fractions was controlled by the specific activities of marker enzymes as well as by electron microscopy. 5'-Nucleotidase (Michell and Hawthorne, 1965) was used as the marker enzyme for PM, glucose-6-phosphatase (Duttera et al., 1968) for microsomes and succinate cytochrome c reductase (Tisdal, 1967) for mitochondria. The microsomal fraction was isolated by differential centrifugation at 105,000g for 1 hr in 100mM Tris-HCl, pH 7.4.

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Table 1. Influence of in vivo insulin treatment on liver plasma membrane and microsomal phospholipids and cholesterol (/~g/mg protein), steady-state fluorescence anisotropy (rs) and structural order parameter of DPH (SopH) Plasma membranes Microsomes Lipids Control Insulin Control Insulin TPL 254.31 ± 2.47 211.04 ± 3.14+ 257.04 ± 4.53 460.13 + 3.68I CH 87.15 ± 1.23 24.54 ± 0.34"t 35.83±1.65 41.74_+2.11" CH/PL 0.342 0.116 0.139 0.090 SM/PC 0,449 0,378 0.084 0.080 PE/PC 0.418 0,562 0.538 0.531 rs 0.189±0.011 0.165±0.016 0.123±0.016 0.120±0.025 SDpH 0.620 ± 0.036 0.551± 0.053 0.403± 0.032 0.390 ± 0.041 * p < 0.01, tP < 0.001; Mean + SD (n = 10). TPL, total phospholipids; CH, cholesterol; CH/PL, cholesterol/phospholipid molar ratio; SM/PC, sphingomyelin/phosphatidylcholine molar ratio; PE/PC, phosphatidylethanolamine/phosphatidylcholine molar ratio.

Incorporation o f [1D4C]oleic acid into microsomal phospholipids Rats were injected i.v. with 10 #Ci/rat [1-14C]oleic acid (Amersham; specific activity of 50 mCi/mmol). One hour after injection the livers were removed and the membrane fractions were isolated as described above. Phospholipase A 2 assay The activity of membrane-bound phospholipase A 2 was determined by the method of Colard-Torquebiau et al. (1975). As substrate we used 1-acyl-[2-14C]linoleoyl-sn glycero-phosphoethanolamine (Amersham). The incubation medium contained 100#g plasma membrane protein, 170 nmol 14C-PE in 100 m M Tris-HCl, 5 m M CaCI2, pH 8.6 in a total vol. of 0.2 ml. The released radioactivity labeled fatty acids were extracted twice by hexan and counted on a Rackbeta II liquid scintillation counter. The specific activity was calculated as nmol released fatty acids per min/mg protein.

Determination o f enzyme activities participating in sphingomyelin metabolism The incubation medium for determination of the endogenous neutral sphingomyelinase activity contained 1 mg membrane prelabeled with [methyl-~4C]choline sphingomyelin by the aid of SM-transfer protein ( K o u m a n o v et al., 1982), 5 0 # g Triton X-100, 4 0 m M MgCI2, 1 0 m M Tris-HC1, p H 7.4 at a total vol. of 1 ml (Petkova et al., 1989). The incubation was performed for 2 h r at 37°C under continuous shaking. The reaction was terminated by addition of 100 m M EDTA. The liberated phosphorylcholine was extracted with ether and counted on an LKB-1215 Rackbeta II scintillation counter. The specific activity was expressed as nmol hydrolyzed SM/mg protein per hr. SM synthetase activity was estimated by the method of Malgat et al. (1986) using an incubation medium containing as substrate, L-~-phosphatidyl [2J4C]ethanolamine, dioleoyl (Amersham) with a specific activity of 1260 dpm/nmol in a final vol. of 350 #1.

Assay of microsomal membrane phospholipid asymmetry (Bollen and Higgins, 1980) The distribution of the membrane phospholipids was investigated using phospholipase C from Clostridium welchii (Sigma). The incubations were performed for 15 min at 37'C at a ratio of 1:0.1 (membrane protein:enzyme protein) in 100 m M Tris buffer (pH 7.4). The reaction was terminated by chilling and centrifugation of microsomes at 105,000 g. The pellet was washed twice with the same buffer and the phospholipids were extracted and fractionated by TLC. We measured the latency of glucose 6-phosphate (Arion et al., 1972) for testing the microsomal membrane integrity. TNBS was also used for assessing the aminophospholipid distribution in the microsomal membranes (Duijn et al., 1986). The individual phospholipid fractions were separated by two-dimensional chromatography (Nortlund et al., 1981).

Analytical methods. Lipids were extracted from PM and microsomes by the method of Folch et al. (1957). The phospholipid (Kahovkova and Odavic, 1969) and cholesterol content (Sperry and Webb, 1950) was determined in the total lipid extract. The individual phospholipids were chromatographed on silica gel 60 thin-layer plates (Merck). Chloroform/methanol/isopropanol/0.25 % KC1/triethylamine (30:9:25:6:18 by vol) were used as developing solvents (Touchstone et aL, 1980) in the case of uni-dimensional chromatography. Chloroform/methanol/NH4OH/ water (70: 30: 4:1, by vol) and chloroform/methanol/acetic acid/water (90:40:12:2, by vol) were used for the first and second dimensions consecutively for the two-dimensional chromatography (Vale, 1977). The protein content of PM and microsomes was also determined (Lowry et al., 1951).

Fluorescence assays 1,6-Diphenyl-1,3,5-hexatriene (DPH) (Fluka) was used as a fluorescent probe for estimation of plasma and microsomal membrane fluidity. Fluorescent measurements were performed at 37°C on a Perkin Elmer 3000 fluorescence

Table 2. Phospholipid composition of liver plasma membranes and microsomes (ltg/mg protein) Plasma membranes Microsomes Phospholipids Control Insulin Control Insulin SM 37.88 + 1.80 25.90 + 0.86¢ 10.86 ± 0.86 18.80 + 0.24:~ PC 84.26 - 0.39 68.58 ± 0.35:~ 129.70_+7.82 235.01+ 4.66"t PS 47.73 + 1.67 29.51 ± 0.43~ 9.11 ± 1.77 13.83 ± 2.41 PA 19.44+0.05 14.91 + 0.84~ 7.01 ± 1 . 3 5 15.05+ 2.71:~ PI 17.25 ± 1.68 19.69 ± 1.82 25.90 ± 3.50 44.18 + 5.69:~ PE 35.20 ± 0.73 38.54 ± 1.45" 69.79± 5.21 124.82± 5.43:~ PG + DPG 12.57±0.04 13.91 ±0.07 4.67±0.49 8.44 ± 0.56~ *P <0.05, +P < 0.01; ~P < 0.001; Mean + SD (n = 10).

Insulin effect on rat liver membrane Table 3. Incorporationof 14C-oleicacid in microsomalphospholipids(dpm/mg) Phospholipids Control Insulin PC 3600 4600 PE 1800 1900

PC

RESULTS Insulin was observed to increase the total phospholipids (78%) and cholesterol (17%) in microsomes, whereas in plasma membranes both the total phospholipids and cholesterol were reduced by 16 and 72%, respectively (Table 1). The elevation of the microsomal phospholipids was due to the increase of all phospholipid fractions, whereas the diminution of the plasma membrane total phospholipids was due only to the decrease of SM, PC, phosphatidylserine (PS) and phosphatidic acid (PA) (Table 2). However, in both microsomal and plasma membranes the CH/PL ratio was diminished (Table 1). Moreover, insulin administration also provoked a decrease of the structural order parameter of plasma membranes. As shown in Table 3, the incorporation of radioactive precursor (14C-oleic acid) into the molecules of microsomal PC and PE was increased in insulintreated rats by about 25 and 15%, respectively. The eventual influence of the insulin-induced alterations in phospholipid synthesis is discussed later. Our data did not indicate any significant changes in plasma membrane and microsomal PLase A2 activity (Table 4). Statistically significant alterations of the enzymes participating in SM metabolism in plasma membranes from insulin-treated rats was observed only for the endogenous SMase activity, whereas SM synthetase remained almost unchanged. In contrast, SM synthesis in microsomal membranes was elevated (Table 4). As already mentioned, we investigated the asymmetric phospholipid distribution between the two monolayers of microsomal membranes using phospholipase C and TNBS (Fig. 1). The hydrolysis of microsomal phospholipids using phospholipase C was approx. 48%. The results showed that the phospholipids were asymmetrically distributed between the outer and inner leaflets of the microsomal membranes as reported by some other authors (Roelofsen,

PI

PE

PG

80

spectrophotometer at 355 nm (excitation beam) and 425 nm (emission beam). The steady-state fluorescence anisotropy (rs) was estimated according to the following equation (Shinitzky and Barenholz, 1974): The lipid structural order parameter (SopH) was calculated using the empirical method described by van Blitterswijk e t al., (1981).

PS

,°°f 60

r, = I . -- I l ~Ill + 21 z

687

40 20

%

1

i

OUT

IN

100 L

Fig. 1. Asymmetrical distribution of the microsomal phospholipids. Control rats (K]). Insulin-treated rats (@). 1982). Phosphatidylinositol (PI) and PC were mainly located in the outer leaflet (72 and 68%, respectively) and PS, phosphatidylglycerol (PG) and PE in the inner leaflet (91, 80 and 63%, respectively). However, insulin induced alterations in the asymmetric distribution of the microsomal phospholipids. The PC and PI content were decreased in the outer leaflet, whereas PE and PS were increased to 60 and 25%, respectively. DISCUSSION In general, the increase of the microsomal phospholipids could be due to their intensified synthesis, reduced catabolism or slowed down lipid transfer towards the membrane fractions. Since the incorporation of ~4C-oleic acid into the molecules of the two major microsomal phospholipids, PC and PE was elevated, it seems quite probable that the synthesis in the endoplasmic reticulum is enhanced (Table 3). Moreover, insulin is known to stimulate lipogenesis in liver (Topping and Mayer, 1972, 1982; Topping e t a l . , 1987). The fact that PLase A 2 activity remained almost unchanged indicated that the phospholipid hydrolysis in microsomal membranes, obtained from insulin-treated animals was not reduced compared to controls (Table 3). In contrast, most of the phospholipid fractions as well as the total phospholipid content were reduced in liver plasma

Table 4. Specificactivityof phospholipaseA2 (nmolmin t mg ~) neutralsphingomyelinaseand sphingomyelinsynthetase(nmolhr-I mg t) Plasma membranes Microsomes Enzyme Control Insulin Control Insulin PLase A2 3.34_+0.26 3.72_+0.37 2.96+ 0.31 3.42_+0.20t SMase 6.63 + 0.19 8.46___0.36~ --SM synthetase* 3.55 4-0.60 4.30 ___0.61 2.17_+0.17 3.01 + 0.40t tP < 0.01, ~P < 0.001; Mean+ SD (n = 10, *n = 5).

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DIANAH. PETKOVAet al.

membranes of insulin-treated rats. This could be due either to the elevated activity of some enzymes responsible for their catabolism and/or to the diminution of the phospholipid transfer from their biosynthesis site, the endoplasmic reticulum, up to the plasma membranes. The latter seems more probable, since the results shown in Table 4 did not reveal any enhancement of PLase A 2 activity in liver plasma membranes. Phospholipid synthesis is known to be carried out mainly in the endoplasmic reticulum, and very few phospholipid classes are synthesized in other subcellular membranes (van den Bosh, 1974). Plasma membranes are provided with phospholipids mainly by the aid of lipid transfer proteins (Wirts and Zilversmit, 1968). Thus, if plasma membranes become unable to complete their phospholipid composition as a result of insulin treatment, the level of some phospholipid fractions could be altered, which might induce changes in the membranes' physical state. The structural order parameter of the membrane phospholipids in insulin-treated animals was in fact significantly decreased, as shown in Table 1. This was caused by the decrease of SM/PC (due to some extent to the increase of the endogenous activity of SMase) and CH/PL, and the elevation of the PE/PC ratio. These ratios are known to significantly influence membrane fluidity (Barenholz and Thompson, 1980; Shinitzky and Lubar, 1974; Cullis and De Kruijff, 1979). It should be pointed out that the SM level was elevated in microsomes and reduced in plasma membranes. Therefore, the lowered SM content in plasma membranes from insulin-treated rats could be a result of both the unchanged SM-synthetase activity and/or the augmented SM hydrolysis (Table 4). Similarly, the enhanced SM synthesis and the lack of SM hydrolytic activity could explain the augmented SM level in microsomal membranes'. Furthermore, treatment of rats with insulin caused translocation of some phospholipids in the membrane bilayer. As shown in Fig. 1, the outer leaflet was enriched with PE and PS, and the inner leaflet with PC and PI. Nevertheless, these alterations influenced neither the microsomal fluidity nor the activity of the investigated membrane-bound enzymes. Since the lipid composition of the donor membranes significantly affects the transfer of lipids towards the acceptor membranes (Helmkap, 1983), it is not unlikely that changes in the outer monolayer of the microsomal membranes could influence lipid transfer towards the plasma membranes. Apparently, insulin administration induced different alterations in the composition and organization of liver membranes. However, the intimate mechanisms of this effect, as well as the eventual participation of other factors, such as the altered phospholipid synthesis and/or the lipid transfer activity need further clarification. CONCLUSION The influence of insulin on the phospholipid composition, organization and some membrane-bound enzymes in liver plasma and microsomal membranes has been investigated.

The total phospholipids were increased in microsomes and decreased in plasma membranes of insulintreated rats. The specific activity of phospholipase A 2 in plasma membranes and microsomes was not changed significantly, whereas the endogenous plasma membrane neutral sphingomyelinase activity was enhanced due to insulin treatment. Alterations in the asymmetric distribution of microsomal phospholipids were also observed. Phosphoatidylethanolamine and phosphatidylserine underwent translocation towards the outer monolayer of the membrane, whereas the inner monolayer was enriched with phosphatidylinositol.

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