Glycosaminoglycans are important macromolecular components. surface of cell membranes and also membranes of subcellular

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1 Chapter VII EFFECT OF GLYCOSAMINOGLYCANS ON THE SYNTHESIS AND SECRETION OF APOLIPOPROTEIN B BY RAT HEPATOCYTES IN CULTURE Glycosaminoglycans are important macromolecular components of intercellular matrix where they are present as proteoglycans ' These substances are also present on the surface of cell membranes and also membranes of subcellular particles 258,259. Since glycosaminoglycans are present both in the intercellular matrix and on the cell membrane surface, it is interesting to note whether they have any effect on the synthesis and secretion of apob by rat hepatocytes in culture. In the previous chapters, the effect of exogenous fatty acids and.other lipids like cholesterol, VLDL, LDL, phospholipid and bile acids on the synthesis and secretion of apob by rat hepatocytes in cuiture was discussed. It was found that oleic acid stimulated the synthesis and secretion of apob, cholesterol and lipoproteins had a similar effect while the phospholipids stimulated the release of apob.into the medium, without affecting the synthesis. The bile acids showed an increased production of apob.

2 113 It is known that both VLDL and LDL form complexes with 26,262 sulphated glycosaminoglycans which according to many reports, may play a role in the pathogenesis of atherosclerosis. The apoproteins of lipoproteins, particularly apob and E have also been shown to bind glycosaminoglycans such as heparin. The fact that some of the glycosaminoglycans such as heparin or heparan sulphate and chondroitin sulphate are also present in the cell surface may suggest that they may possibly have a role in the release of these. lipoproteins by the hepatocytes. However no report seems to be available in this respect. In view of these, the effect of addition of some of the glycosaminoglycans (heparin, chondroitin-4- sulphate, and hyaluronic acid) in vitro on the synthesis and secretion of apob by rat hepatocytes in culture, was studied and the results are discussed in this chapter. Materials and methods Heparin, chondroitin-4-sulphate and hyaluronic acid were purchased from Sigma Chemicals, (St. Louis, USA). The procedures used for the preparation of hepatocytes from normal rats and their culture in vitro, metabolic labelling with 3 ( H)-leucine, immunoprecipitation of apob and all other procedures used are the same as described in chapter II.

3 114 Results 1. Effect of heparin on the synthesis and secretion of apob: Results (25a). are given in table (25) and 't' values are given in table 3 The average values of ( H)-apoB secreted into the medium are also plotted against concentration of heparin added and presented in figure (Fig. 2). Heparin stimulated the secretion of (3H)-apOB into the medium. The effect was maximum at a concentration of 1pg/ml, at 2pg, the stimulatory effect was the same but further increase in the concentration of heparin to 5fg/ml decreased the effect. Cell associated apob was more at 1fg/ml but comparable at 2 and 5pg/ml. Total apob (media + cell associated) was significantly more. indicating increased synthesis of apob. 2. Pulse chase experiment: 3 Cells were pulsed with 1pCi of ( H)- leucine for:3 hours. The medium was removed, cells were washed with nonradioactive MEM and then reincubated in a medium containing nonradioactive leucine in presence of heparin (WOfg/ml). Heparin caused significant increase in the release of newly synthesised apob into the medium (Fig. 21) 3. Effect of chondroitln-4-sulphate: Results are given in table (26) and I t I values are given in table (26a).

4 Table.2 Effect of heparin on the synthesis,and secretion of apob by cultured rat hepatocytes: rt'1 I X s::.~ 4-.S U iu '... p.. ~ M M iuu S.' 8- S." ::E Pol U... S.2 >- +' 3.~ >.~ 2.8 +' u I'd.5 'rl 'tl I'd P::: e.~ I.c<) ::r: 1 too BlPUD <1&1> bo lu Cells were!ncubated with different concentrations of heparin in ( H}-leucine (2pei) containing media for 12 hours. The apob secreted into the media was immudoprecipitated and quantitated as described in the text. The values given are the mean of four experiments.

5 Fig. 21 Pulse chase of a pcb in presence of heparin:... ~ >c: s:: 1"'1 Q) +' M P. 4 t-- r-i r-i Q) CJ bo 8-::E III 2 u- >- - +' "1"'1 > "1"'1 +' CJ III 1"'1 't1 III t:t:: I ~ (Y") C T Cells were pulsed with 1pCi of (3H)-leucine for 3 hours. The medium was removed, cell monolayer was washed with nonradioactive medium and reincubated with medium containing nonradioactive leucine for 6 hours in presence of heparin (IOOpg). The apob secreted into the medium was immunoprecipitated and quantitated as described in the text. The values given are the average of triplicate analysis. C-control, T-test.

6 115 Table. 25 Incorporation of (3H)-leucine into apob - Effect of heparin Heparin Media Cell layer [CPM I mg cell protein] Control 1pg 2<f1g 5pg 2487± ±253 a 4457±237 a 351±195 a 24± ±115 a 2612± ±148 Hepatocytes were incubated with different concentrations of haparin in medium containing (3H)-leucine (2pCi) for 12 hours. The radioactivity associated with secreted and cell associated apob was measured. The results giyen are the mean of four different experiments ± SD. On comparison with control t a=p<o.ol No symbol - not significant. Table. 25a It I values I t I between control 8«heparin Media Cell layer

7 116 Table. 26 Incorporation of (3H)-leucine into apob - chondraitin-4-sulphate Effect of Chondroitin sulphate Media [CPM/mg cell Cell layer protein] Control 1fg 5pg 2487± ±162 a 2857±14 b 24± ±63 a 2158±96 b Hepatocytes were incubated with different concentrations of chondroitin-4-sulphate in medium containing (3H)-leucine (2fCi) for 12 hours. The radioactivity associated with secreted and cell associated apob was measured. The results given are the mean of four different experiments ± SD. On comparison with control, a = p<..1 b = p between.1 and.5 Table. 26a ItI values I t I between control & chondroitin sulphate Media Cell layer

8 117 Table. 27 Incorporation of (3H)-leucine into apob - Effect of hyaluronic acid Hyaluronic acid Media Cell layer [ CPM I mg cell protein] control 2487± ± ±143 24± ±84 148±93 Hepatocytes were incubated with different concentrations of hyaluronic acid in medium containing (3H)-leucine (2fCi) for 12 hours. The radioactivity associated with secreted and cell associated apob was measured. The results given are the mean of four different experiments ± SO. On comparison with control, p.c..1 in all cases. Table. 27a ItI values I t I between control & hyaluronic acid Media Cell layer

9 118 Addition of chondroitin-4-sulphate to the medium caused an increase in the release of apob into the medium. The stimulatory effect was maximum at a concentration of 1rg/ml but was lower at 5o,g/mi. Cell associated apob was lower but total apob (media+cell associated) apob. was more at 1pg/ml indicating increased synthesis of 4. Effect of hyaluronic acid: Results are given in table (27) and It I values are also given in table (27a). Hyaluronic acid also caused significant increase in the secretion of apob into the medium. The effect was more at a concentration of 1opg/ml than at 5arg/ml. Cell associated apob showed a decrease but the total apob (media + cell associated) was more. Discussion The results now obtained indicate that glycosaminoglycans stimulate the release of apob into the medium and also the synthesis of apob. The latter is evident from the fact that the total apob (media + cell associated) was increased in the presence of the glycosaminoglycans. The stimulatory effect of apob synthesis and secretion was found to be more with heparin than with the other glycosaminoglycans.

10 119 Condroitin-4-sulphatEl had. lower effect than hyaluronic acid. The decrease in the cell associated apob in the case of hyaluronic acid, chondroitin sulphate and also in the case of heparin may indicate that the rate of release of apob is more than the rate of synthesis. As mentioned earlier many glycosaminoglycans are known to be present on the surface of cell mem branes and mem branes of subcellular particles. For example, heparan sulphate is known to be present on the surface of the membranes of several cells particularly hepatocytes It is possible that the newly synthesised apob may remain attached to the heparan SUlphate before being released into the medium. The attachment may involve the binding domains of apob with the glyco&aminoglycans. No information seems to be available in the literature on this aspect. But an analogy can be the binding of lipoprotein lipase on the cell surface by heparan sulphate. Externally added heparin releases the enzyme from the bound heparan 27 sulphate. A similar situation is possible in the case of hepatocytes also, the externally added glycosaminoglycans serving to release the bound lipoproteins from the cell surface. Addition of heparin has been shown to release part of the heparan su1ph a te proteog1ycans from hepatocyte cell surface 267, 271. Pulse chase experiments showed that there is an increase in secretion 3 of ( H)-apoB into the medium in presence of heparin.

11 12 In this connection Williams et a1 272 put forward an interesting hypothesis that local uptake of apob from the unstressed water layer sorrounding the cells may be a general mechanism in secretory control. If this hypothesis is correct, it is possible that the added glycosaminoglycans may bind apob in the medium thus preventing its uptake by the cells. This can probably result in the accumulation of apob containing lipoproteins in the medium and may also lead to disruption of secretory control causing more release. The stimulation of apob synthesis by the cells in the presence of added glycosaminoglycans cannot be explained at present. It is known that externally added glycosaminoglycans are taken up by the hepatocytes and inside the cell, they are cleaved by the lysosomal enzymes Whether the degradation products of glycosaminoglycans or the unchanged glycosaminoglycans can exert an effect on apob synthesis at a co-translation and/or post-translational level is to be investigated.

Plasma lipoproteins & atherosclerosis by. Prof.Dr. Maha M. Sallam

Biochemistry Department Plasma lipoproteins & atherosclerosis by Prof.Dr. Maha M. Sallam 1 1. Recognize structures,types and role of lipoproteins in blood (Chylomicrons, VLDL, LDL and HDL). 2. Explain