CHAPTER EFFECT OF CHRONIC ADMINISTRATION OF GLUCAGON ON THE METABOLISM OF GLYCOSAMINOGLYCANS

Transcription

1 "f CHAPTER IV EFFECT OF CHRONIC ADMINISTRATION OF GLUCAGON ON THE METABOLISM OF GLYCOSAMINOGLYCANS

2 SECTION A INTRODUCTION Glycosaminoglycans are complex carbohydrate molecules which are important components of the intercellular matrix. It is known that the metabolism of these macromolecules is influenced by hormones. This chapter discusses the results of studies on the effect of administration of glucagon on the metabolism of these substances. As will be discussed later, no information is available on the effect of glucagon on the metabolism of glycosaminoglycans. Glycosaminoglycans are present in the tissues covalently linked to proteins to form proteoglycans. 97 However not 'all connective tissue glycosaminoglycans are bound to proteins. Whether hyaluronic acid (HA) is covalently linked to protein is still an open question. In the case of some glycosaminoglycans, although they may occur as protein free chains, they have in all likelihood passed through a Sta08 of attachment to protein. This is true particularly in the case of heparin which is probably synthesised as a proteoglycan

3 130 and subsequently degraded to a mixture of fragments, some of which are attached to small peptides while others hav~ residues. free reducing terminal occupied by glucuronosyl The proteoglycans are most abundant in the extracellular matrix. But they are also found intracellularly and in close association with the cell surface. sulfate (HS) Heparan is known to be a constituent of cell surface of most cells and heparin is typically stored in intracellular granules of mast cells, from where in response to specific stimuli. it is released The proteoglycans are polyanionic substances of high molecular weight and are distinguished from other carbohydrate protein compounds (glycoproteins) by the presence of relatively large polysaccharide chains containing repeating disaccharide units as their most characteristic feature. These disaccharide units except in keratan sulfate, are composed of a hexosamine and a uronic acid. D-glucuronic acid and L-iduronic acid are the uronic acids while D-glucosamine and D-galactosamine are the hexosamine. The amino group of the hexosamine is always acetylated and in many case sulphated. Sulphate is also present in several glycosaminoglycans as O-sulphate. The structures of the ca~bohydrate In repeating units of glycosaminoglycans are givenlfigures II, III and IV.

4 n CHONDROITIN-4-SULFATE. (Ch- 4-S) n CHONDROITIN- 6- SULFATE. (Ch-6-S) H

5 eooh eooh HEPARIN (H) AND HEPARAN SULFATE DERMATAN SULFATE (OS) Fig_III

6 COOH COOH CH OH HYALURONIC ACID (HA) KE RATO SULFATE (K S)

7 134 Different glycosaminoglycans vary considerahly in size with molecular weights ranging from about 10 4 for h~parin upto 10 7 for hyaluronic acid. Individual glycosaminoglycans, as isolated, may contain a mixture of chains of varying lengths. Small amounts of sugars other thon those found in the disacchar1~e repeating units are also found in some of the proteoglycans and are involved in structures linking the polysaccharide chains to the protein moiety. The proteoglycans show ~arying degrees of heterogeneity. Different polysaccharide chains of varying length may be attached to the same protein core. For example chondrcitin-4-sulphate, chondroitin-6-sulphate and keratan sulphate may be present attached to the same protein in varying proportion. In the same polysaccharide chain, sulphate may be present in carbon-4- pr carbon-6-of the hexosamine resulting in the occurance of 4-sulphate and 6-sulphate as hybrids. The degree of sulphation may also vary considerably.98 Sulphate may be present in both C 4 or C 6 of the hexosamine in the same disaccharide repeating unit, or an additional sulphate may be present in position C 3 or C 2 of the uronic acid moiety. SUlphate may also be absent in certain disaccharide units.

8 135 Although L-iduronic acid has been idertified as the main uronic acid component of dermatan sulphate, small amounts of D-glucuronic acid are also present in this glycosaminoglycan. Dermatan sulphate is believed to be a hybrid molecule with both L-iduronic acid and D-glucuronic acid residues. Keratan Sulphate is unusual in that it does not contain uronic acid instead galactose. Keratan Sulphate 1 and II are found in different tissues, the former in the cornea and the latter in the skeletal tissue. Heparin is found on the surface of many cells but it is an intracellular constituent of mast cells. Heparan sulfate resemble heparin in its chemical composition. The carbohydrate backbone of heparin and heparan sulphate is found to be similar. The two glycosaminoglycans differ with respect to sulphate and acetate content. Heparan sulphate has more N-acetyl group, fewer N-sulphate groups and a lower degree of 4-sulphation. Moreover, like dermatan sulphate, these glycosaminoglycans also contain some L-iduronic acid in place of D-glucuronic acid. As mentioned before, the glycosaminoglycans are present in the tissues covalently linked to proteins to form the macromolecular proteoglycans. Three types of carbohydrate protein linkage regions have been recognised in the connective tissue proteoglycan. These are:

9 a-glycosidic linkage between xylose and serine hydr~xyl group of the polypeptide as found in the proteoglycans of chondroitin-4-sulphate, dermatan sulphate, heparin and heparan sulphate. 2. a-glycosidic linkage between N-acetyl-gaLactosamine and hydroxyl group of serine or threonine as found in skeletal keratan sulphate (Keratan sulphate II). 3. N-glycosylamino linkage between N-acetyl glucosamine and the amide group of asparagine as found in corneal keratan sulphate (Keratan sulphate 1). In the case of chondroitin sulphate proteoglycan, the repeating disaccharide chain is covalently linked to serine in the polypeptide backbone by glycosidic attachment to a trisaccharide Ga!-Gal-Xyl-Ser as shown below. [Glc UA ~ I ~ 3 Ga I NAC or 65] n - ~ I -> 4 Glc UA ~l ~ 3 Gal ~l --> 3 Gal ~l ~4 Xyl ~-a-ser The mechanisms that have been established for the biosynthesis of glycoproteins also apply by and large to the synthesis of connective tissue proteoglycans. The formation of protein core precedes the addition of the

10 D7 carbohydrate groups. In the case of glycoproteins, the addition of the carbohydrate groups may be initiated in two different ways, depending on the nature of the carbohydrate - protein linkage. 1. Direct transfer of the first monosaccharide from the corresponding nucleotide sugar to an amino acid residue in the protein core. This occurs in the glycoproteins which are linked by glycosidic bonds bet~een N-acetyl galactosamine and serine or threonine residues and also in collagen and related glycoproteins which have a galactosehydroxylysine linkage. 2. N-glycosylamine linkage between N-acetyl glucosamine and asparagine residues formed via polyprenol lipid intermediates. N-acetyl glucosamine-l-phosphate is first transferred from UDP-N-acetyl glucosamine to dolichol phosphate. Following the addition of a second N-acetyl glucosamine residue and several mannose units, the entire oligosaccharide is transferred enbloc to the core protein of the glycoprotein. In proteoglycan synthesis, the evidence is that the first mode of conjugation, tak~place, ie., by direct transfer from the corresponding nucleotide sugar. This is exemplified by the synthesis of chondroitin sulphate -

11 138 proteoglycan which is initiated by the transfer of xylose, from,udp protein (Figure V) at a xylose to serine hydroxyl group of the core The oligosaccharide chains are elongated one residue time, each additional sugar being added to the nonreducing ~nd of another. Six different glycosyl transferases and one sulfotransferase are involved. The transferases have strict specificity. For ego two independent UDP-galactosyl transferases are required to I incorporate the two galactose residues into the growing chain. It is believed that glycosylation occurs in the golgi apparatus of secretory cells such as the chondroyctes of cartilage. After the polypeptide chain of a proteoglycan is released from the ribosomes, it traverses the channels of the endoplasmic reticulum to the golgi apparatus, where the membrane-bound transferases begin the sequential synthesis of the oligosaccharide groups. Sulfation apparently occurs during chain elongation, utilising 3'-phosphoadenosine-5'-phosphosulphate as substrate, and the extent of sulfation may help signal termination of synthesis. Molecules with completed chains pass from the g01gi apparatus to the cell plasma membrane and are then secreted. At present, there appear to be no unique regulatory mechanisms at the enzyme level that

12 (j)---.. UCF ~ CD Xyl-transferase Q) Gal-transferase I Q) Gal-transferase II G) GlcUA-transferase N Ac-transferase (ID GIcUA transferase II (i) Sulfotransferase Protein core Xylose Galactose Glucuronic acid N-Acetylgal actosam ine Sulfate ō i I Fig - V

13 140 control proteoglycan synthesis, and different proteoglycans are synthesised because of the strict substrate specificity of the enzyme present in a cell. other xylose linked proteoglycan like DS-pg, HS-pg etc. are presumably formed by the same route. Similarly the linkage between N-acetyl galactosamine and serine or threonine residues in skeletal keratan sulphate (Keratan ~lphate II) is in all likelihood formed by direct transfer in an analogous manner. The N-acetyl glucosamine-asparagine linkage is present only in one connective tissue polysacchariae, ie., corneal keratan sulphate.-pg (Keratan sulphate I). It appears that initiation of the proteoglycan synthesis in this case may follow the lipid intermediate pathway as indicated by inhibition of keratan sulphate synthesis by tunicamyc in. 99, Initially glucuronic acid from UDP glucuronic acid is incorporated into the disaccharide repeating units of glycosaminoglycans such as dermatan sulphate and after the polymer of an ap~ropriate size is synthesised, the D-glucuronic acid is epimerized to,l-iduronic acid by epimerase action.

14 141 Proteoglycan Aggregates, The proteoglycans exist in the cartilage of several tissues as proteoglycan aggregates. These aggregates can be dissociated and fractionated by caesium chloride densitygradient ultra centrifugation into hyaluronic acid proteoglycan subunits and low-molecular weight proteins. These aggregates; have molecular weights ranging from about 30 to 210 x 10 6 The low-molecular weight fraction, however, contains two hydrophobic proteins, designated as the link proteins. Most of the mass of the aggregate is accounted for by the proteoglycan subunits, with hyaluronic acid and the link proteins representing only about one percent of the total. It has been suggested that the proteoglycan aggregates have a 'bottle-brush' structure (Figure VI). The proteoglycan subunits are non-covalently bound to a \ long filamentous hyaluronic acid molecule with the aid of link proteins. Functions of Proteoglycans: The most striking property of these diverse structures is that all are large polyvalent anions that attract and + + tightly bind cations. Even Na and K may be bound so effectively as to appear nonionic. All have a tendency to aggregate and this is enhanced by polyvalent cations like ca 2 ;

15 --=-----.~y(lhjiitqli1liit uii~ liilllik pltolttbla,~~ lktlt~u.fil $luuate Core pmteill1l Subunits

16 142 The physiological functions of proteoglycans in the connective tissue are not clearly understood. It has been suggested by ~orfmanloo that the glycosaminoglycans of the connective tissue have a role in a number of physiological and! lpat.ihho1.ogii.cal process es including calcific ation, cont.rol of electrolytes and water in the intracellular fluids, ~):liouoo lheallifjl9" lubrication and maintenance of the stable tr,ai)!1lspltql:rt~ledhjl!!i!"jlof the eye. The func tion 0 f glycosaljl1un09jlycans in all these processes is associated with their polyanionic nature resulting from the presence of a large lnlljlmitner of carboxyl and sulphate residues. The connective t.issue provides a continuous local matrix through wjlhich water, electrolytes and various metabolites diffuse from the blood into the cells and through which exchange of these substances occurs. The l:ilijrge chains of glycosaminoglycans, particularly that of hyaluronic acid, coil in a relatively random way and hence may occupy a volume largely filled with solvent water, to which small molecules or ions have access but from which larger molecules, e.g., serum albumin, may be excluded. In less concentrated solutions, this volume, the domain of the polysaccharide, would exist as separate zones In more concentrated solutions, the domains would collapse

17 143 and interpenetrate, thus accounting for the very high viscosity of such solutions. The high viscosity of hya1uronic acid suggests that it serves as a lubricant in the joints, and the changes in viscous behaviour of joint fluids in rheumatic diseases may reflect alterations in proteoglycan structure. The proteoglycans also impede the flow of water to an external pressure and give the tissue elasticity and resistance to compression. Moreover, they serve as molecular sieves in much the same manner as agarose and dextrans, restrict the movement of large cations, and deny access to molecules the size of albumin and immunoglobulin, which are virtually unable to penetrate the proteoglycan domains. Although these properties of proteoglycans are thought to reflect their physiological functions, there is no explanation for the differences in glycosaminoglycan content in different tissues. Hyaluronic acid also functions in morphogenesis and appears to control mesenchymal cell aggregation in embryogenesis. Its removal by the action of endogenous hyaluronidase is coincident with aggregation and subsequent differentiation of mesenchymal cells in the regenerating newt limb and in the developing chick limb and skeleton. During development of the chick cornea, the hyaluronic acid content diminishes as the chondroitin sulfate content,

18 144 increases during the stage that transparency to light increases. The sulphated glycosaminoglycans interact with various macromolecules like lipoproteins, glycoproteins, collagen and even elastin. The interaction with lipoproteins is considered to be particularly important in the accumulation of lipids in the arterial wall in atherosclerosis. Some of the glycosaminoglycans present on the cell surface particularly heparan sulphate have special role. The enzyme lipoprotein lipase is believed to exist in the surface bound to heparan sulphate. Some of the glycosaminoglycans partitularly heparin and heparan sulphate may playa role in blood coagulation acting as anticoagulant. The glycosaminoglycans interact with other cell surface and intercellular matrix' glycoproteins like fibronectin and these interactions are believed to play a very important role in cell to cell interactions. As stated above, physiological functions of these important rna cromolecules are not yet fully understood at this time.

19 145 Turnover of Proteoglycans studies with labelled sugars, sulfate and amino acids indicate that the major proteoglycans are subject to constant degradation and resynthesis at varying rates characteristic of individual proteoglycans in specific tissues. The halflife of cartilage chondroitin sulfates in 9 week old rats is 7 to 9 days. Hyaluronic acid is known to have a halflife of 2.4 to 4 days. Degradation is initially the result of proteolysis, probably by the protease cathepsin-o, which is found in cartilage from many tissues. Once degraded extensively by proteolysis, the proteoglycans may be further degraded by lysosomal proteases and glycosidases in chondrocytes or other nearby cells, or they may diffuse out of the tissue into the circulation. Liver lysosomes degrade chondroitin sulfate completely, presumably by the sequential action of a sulfatase and several glycosidases. Removal of sulfate is a key step since lysosomal glycosidases do not hydrolyse sulfated oligosaccharides. Degraded chondroitin sulfate and other glycosaminoglycans are excreted in the urine; their size suggests that they have been partially degraded by glycosidases. It has been estimated that human adults catabolize about 250 mg. of proteoglycans per day, but since normal urine contains only a few milligrams of the partially degraded glycosaminoglycans, considerable degradation must occur in tissues. Moreover, from the known half-life

20 146 of chondroitin sulfate and from the results of liver perfusion experiments, it is estimated that the liver ca~ completely degrade all chondroitin sulfate that is turned over daily. Mammalian hyaluronidase hydrolyses the r1l~4 glycosidic bond between the disaccharide repeating units in hyaluronic acid and chondroitin sulfate to give the tetrasaccharide, which is degraded to monosaccharides by lysosomal glycosidases. Hyaluronidases of bacteria and invertebrates have different substrate specificities. The proteoglycan composition of animal tissues varies in a rather consistent manner with aging. ThUS, the keratosulfate concentration of tissues that contain this glycosaminoglycan increases throughout life, whereas the chondroitin sulfate content of cartilage and intervertebral d~sks and the hyaluronic acid of skin decrease with age. Effect of hormones on proteoglycan metabolism Hormones have an influence on the metabolism of these molecules but little definite information is available in this respect. Administration of growth hormone at any age has been reported to result in a pattern of proteoglycan synthesis and composition resembling that of the extremely

21 147 young animal. At least part of the effects of growth hormone are the result of somatomedins, one of which, formerly termed sulfation factor and now called somatomedin A, promotes proliferation of cartilage cells and stimulates incorporation of sulfate into proteoglycans. Administration of testosterone appears, specifically, to increase markedly the rate of hyaluronic acid synthesis in such loci as heart valves, skin, the comb of the rooster, and the sex skin of the monkey. On the other hand, administration of certain adrenal cortical steroids has been reported to result in rapid repolymerization of existing hyaluronic acid while abruptly inhibiting further de novo synthesis. Glycosaminoglycans of the skin of the alloxan-diabetic rat, has been reported to exhibit a turnover rate approximately one third of that found in normal animals. This diminished rates can be restored to normal by administration of insulin. Since in diabetes mellitus there is a significantly greater than normal susceptibility to infection, with retarded wound healing and accelerated vascular regeneration, these characteristics may reflect, in part, the decreased ability to synthesize glycosaminoglycans when the insulin supply is inadequate. Recent work in this laboratory has shown that administration of insulin to rabbits resulted in an increase in the concentration of various glycosaminoglycans

22 14B in the aorta. IOI Chondroitin-4-sulphate and heparin have been found to increase in the aortic arch and thoracic and abdominal aorta, while dermatan sulphate and chondroitin-6 sulphate increased only in the aortic arch and abdominal region b\ut was not affected in the thoracic aorta. Heparan Sulphate and hyaluronic acid increased only in the abdominal aorta. In the liver significant increase occured in all the glycosaminoglycan fractions. Some of the enzymes involved in the biosynthesis of precursors of glycosaminoglycans i.e. glue osamine..:..6-phos phate isomerase (glutamine forming) and UDP glucose dehydrogenase increased in the animals given insulin while the activity of enzymes involved in the degradation viz. hyaluronoglucosan!inidase, p-glucuronidase, aryl sulphatase, p-n-acetyl-glucosaminidase and cathepsin D decreased. Concentration of hepatic 3'-:phosphoadenos1nes 5'- phosphoy sulphate, activity of sulphate activating system and sulphotransferase increased on administration of ~nsulin. It J.5 evident from this review that much remains to be kno\'in concpining th,=' h01'ii:onnj control of proteoglycan metabolism. Almost nothing r~ known about the effect of glucagon on the metabolism of glycosaminoglycans. In view of these, the effect of chronic administration of the hormone on some

23 149 aspects of the metabolism of these macromolecules has been studied in rats. Investigations carried out in this respect include effect of the hormone administration on the concentration of different g1ycosaminoglycan fractions in the liver, activity of some enzymes involved in the biosynthesis of precursors of glycosaminoglyc~ns and degradation of glycosaminoglycans and biological sulphation. The results of these studies are discussed in this chaptnr. SECTION B EFFECT OF CHRONIC ADMINISTRATION OF GLUCAGON ON THE CONCENTRATION OF DIFFERENT GLYCOSAMINOGLYCAN FRACTIONS IN THE LIVER The effect of administration of glucagon for seven days on the concentration of different g1ycosaminoglycan fractions - Hyaluronic Acid (HA). Heparan SUlphate (HS). Chondroitin-4-Sulphate + Chondroitin-6-sulphate (Ch-4S+6S), Dermatan Sulphate (OS) and Heparin ('N) in the liver has been studied. Materials and Methods Male albino rats (Sprague-Dawley strain, weighing g) were divided into two groups of twelve rats each.

24 150 Group 1. Control rats 2. Experimental rats given glucagon All the experimental details are the same as described in Chapter III - Section A-II. Rats of the experimental group were injected with glucagon in physiological saline (0.1 mg/loo 9 body weight, SUbcutaneously) twice daily. Control rats received the same volume of physiological saline. The duration of the experiment was seven days. After the experimental period, the rats were killed as described before. Serum and tissues were removed to ice-cold containers for various estimations. Details of the procedure used for the estimation of total and different glycosaminoglycan fractions are given in chapter II. Results 1. Blood glucose and plasma free fatty acids Results are given in table 30. t-values are given in table 30a. Rats administered glucagon showed elevated blood glucose and plasma free fatty acid when compared to control rats, as in previous exper~mgnts.

25 151 TABLE 30 CONCENTRATION OF BLOOD GLUCOSE AND PLASMA FREE FATTY ACID Groups 1. Control 2. Experimental ----_._-- ~ Values are the mean ± SEM Blood glucose Free fatty acid (mg/l00ml blood) (mg/l00ml serum) ± ± ± 3.60 b ± 4.51 a for 6 rats Group 1 has been compared with group 2 b - P between 0.01 and 0.05 t a - p < 0.01 TABLE 30a 't' VALUES TO TABLE 30 It' between groups Blood glucose Free fatty acid and

26 Concentration of total glycosaminoglycans(gag) in the liver and aorta. Results are given in table 31. t values are given in table 3la. Concentration of total glycosaminoglycans in the liver showed significant decrease in the rats administered glucagon when compared to control rats. A similar decrease in the concentration of total glycosaminoglycans in the aorta was also observed in the rats administered glucagon. 3. Changes in the different glycosaminoglycan fractions in the liver. Results are given in table 32. t values are given in table 32a. The ~oncentration of HA, HS, Ch-4S + 6S and OS showed significant decrease in the liver in the rats administered glucagon when compared to control rats. There was no significant alteration in the concentration of heparin. Thus administration of glucagon for seven days resulted in significant decrease in the concentration of total glycosaminoglycans and all the glycosarninoglycan' fractims except heparin in the liver.

27 153 TABLE 31 CONCENTRATION OF TOTAL GAG IN THE LIVER AND AORTA Groups Liver Aorta,.L~...J:Ironic ac2:s!.l.9 dr.y...-.gefatted tissue ) 1. Control 2. Experimental 976 ± ± a 5612 ± ± 1.29 a Values are the mean ± SEM for 6 rats Group 1 has been compared with group 2 a - p < 0.01 TABLE 31a 't t ~ALUES TO TABLE 31 't' betweon groups Liver Aorta 1 and 2 --,

28 154 TABLE 32 CONCENTRATION OF DIFFEhENT GLYCOSAMINCGLYCANS IN THE LIVER Groups To: t', Dr.. HS Ch-4S.otCh-6-S DS H ( ~g uronic acid /9 dry defatted tissue....) 1 Control 134 ± ± ± ± ± Experimental 104 ± 3.14 a 128 ± 4.9 a 257 ± 7.53 a 98 ± 3.08 a 138 ± 3.86 Values are the mean ± SEM for 6 rats Group 1 has been compared with group 2 a - p < No symbol - no significant difference TABLE 32a 't' VALUES TO TABIE 32 It' between groups HA HS Ch-4S+Ch-6-S DS H 1 and :

29 155 SECTION C EFFECT OF CHRONIC ADMINISTRATION OF GLUCAGON ON THE ACTIVITY OF SONE ENZYMES INVOLVED IN THE SYNTHESIS OF PRECURSORS OF GLYCOSAMINOGLYCANS The effect of administration of the hormone for seven days on the activity of two important biosynthetic enzymes ie. glucosamine-6-phosphate isomerase and UDP dehydrogenase in the liver was studied in rats. Materials and Methods The liver tissue from the rats of the experiment was used for the study. glucose previous Details of the procedure for the determination of the activity of glucosamine-6- phosphate isomerase and UDP glucose dehydrogenase are described in chapter 11. Protein was estimated after TCA precipitation by the method of Lowry ~!!.69 Results 1. Activity of glucosamine-6-phosphate isomerase in the liver. Results are given in table 33. t-values are given in table 33a. There was no significant alteration in the activity of the enzyme in the liver in the rats administered glucagon when compared to control rats. 2. Activity of UDP glucose dehydrogenase in the liver. Results are given in table 34 t-values are given in table 34a.

30 156 TABLE 33 ACTIVITY OF D-GLUCOSAMINE-6-PHOSPHATE ISOMERASE IN THE LIVER -====---_._---, Groups D-glucosamine-6-phosphate isomerase (micromoles of hexosamine liberated/ hr/g protein) 1. Control 2. Experimental ± 0.99 Values a:ce--the mean :l: SEM for 6 rats Group 1 has been c~pared with group 2 No symbol - no signif~cant difference It' between groups TABLE 33a I t I VALUES TO TABLE 33 D-glucosamine-6-phosphate isomerase(glutamine forming) land ,..._',. '.'~'~._

31 157 TABLE 34 ACTIVITY OF UDPG DEHYDROGENASE ln THE LIVER ,---- Groups UDPG dehydrogenase (Units*/g protein) 1. Control 2. Experimental 2016 ± ± Values are the mean ± SEM for 6 rats Group 1 has been compared with group 2 No symbol - no significant difference *The amount of enzyme required to give an increase in optional density of O.Ol/min TABLE 34a, t' VALUES TO TABLE 34 It' between groups 1 and 2 - UBPG dehydrogenase.\ \ _._-_...--,----

32 158 Rats administered glucagon showed no significant alteration in the activity of UDP glucose dehydrogenase in the liver when compared to control rats. Thus administration of the hormone for seven days to rats resulted in no significant alteration in the activity of the biosynthetic enzymes. SECTION D EFFECT OF CHRONIC ADMINISTRATION OF GLUCAGON ON THE ACTIVITY OF SOME ENZYMES INVOLVED ll~ THE DEGRADATION OF GLYCOSAMINO GLYCANS ON THE LIVER The effect of administration of glucagon for seven days on the activity of p-glucuronidase, hyaluronoglucosaminidase, aryl sulphatase, p-n-acetyl-glucosaminidase and cathepsin-d in the liver was studied. Materials and Methods Male albino ~ts (Sprague-Dawley strain, weighing 80-l00g) were divided into two groups of twelve rats ~each Group 1 Control rats 2 Experimental rats given glucagon All the experimental details are the same as described in Chapter III Section A-II.

33 159 Rats of the experimental group were injected with glucagon in physiological saline (0.1 mg/loo g body weight, subcutaneously) twice daily. Control rats received the same volume of physiological saline. The duration of the experiment was seven days. After the experimental period, the rats were killed as described before. Serum and tissues were removed to 1ce-cold containers for various estimations. Details of the procedure used for the determination of the activity of hyaluronoglucosaminidase ~-N~acetyl glucosam1nidase, aryl sulphatase and cathepsin-d are described in chapter II. Prote1n was estimated after TCA precipitation by the method of Lowry ~1!1. 69 Res~lts 1. Rats administered glucagon showed elevated blood glucose and plasma free fatty acid when compared to fontrol rats (table 30)~ as in previous experiments. 2. Activity of degrading enzymes. Results are given in table 35.. \ t-values are g1ven in table 35a. \ The activity of ~-glucuronidase, hyaluronoglucosaminidase,~-n-acetylglucosaminidase, aryl sulfatase and cathepsin-d showed significant increase in the liver in

34 TABLE 35 ACTIVITY OF ENZYME INVOLVED IN THE DEGRADATION OF GAG IN THE LIVER Groups ~-glucuronidase ~-hexosaminidase (~g p-nitrophe-' (~g p-nitrophenol/ nol!hj/mg pro- hr/mg protein) tein) Hyaluronoglucosaminisase r~g glucosamine hr/mg 'protein) Aryl Sulfatase p-nitrocatechol/hi/ mg protein (~g Cathepsin D tyros ine/ hr/mg protein) (~g 1. Control 82.4 ± ± ± ± ±5.l9 2. Experimental a a 92.4 ± 3.75 a 85.7 ± 3.26 a a 260.0± ±7.33 Values are the mean ± SEM for 6 rats Group 1 has been compared with group 2 a - p < 0.01 TABLE 35a! t I VALUES TO TABLE 35 I t I behveen groups p-glucuronidase ~-hexosamj..- nidase Hyaluronoglucosaminidase,t.TVJ. sulfat ase Cathepsin D 1 and

35 161 the rats administered glucagon when compared to control rats. Thus administration of glucagon resulted in significant increase in the activity of all degrading enzymes. SECTION E EFFECT OF CHRONIC ADMINISTRATION OF GLUCAGON ON THE CONCENTRATION OF PAPS, ACTIVITY OF SULPHATE ACTIVATING SYSTEM AND ARYL SULFOTRANSFERASE IN THE LIVER The effect of administration of glucagon for seven days on the concentration of PAPS, activity of SUlphate activating system and aryl sulfotransferase was studied in rats. Materials and Methods The liver tissue from the rats of the previous experiment (SECTION D) was used for this study. Procedures for the determination of the conce~tration of PAPS, activity of sulphate activating system and aryl sulfotransferase are described in chapter II. Protein was estimated after TCA precipitation by the method of Lowry ~!l.69

36 162 Results 1. Concentration of PAPS in the liver Results are given in table 36. t-values are given in table 36a. Administration of glucagon for seven days resulted in significant decrease in the concentration of PAPS in the liver when compared to control r~ts. 2. Activity of sulphate activating system. Results are given in table 37. t-values are given in table 37a. A significant decrease in the activity of the sulphate activating system was observed in the liver when compared to control rats. 3. Activity of aryl sulfotransferase Results are given in table 38 t-values are given in table 38a. The activity of sulfotransferase showed significant decrease in the liver in the rats administered glucagon when compared to control rats. Thus rats administered glucagon showed significant decrease in the sulphate metabolism in the liver. The

37 163 TABLE 36 CONCENTRATION OF PAPS Groups Concentration of PAPS ( micromoles ~f methyl umbelliferone sulphate formed/hr/g protein. J 1. Control 2. Experimental ± :t 2.62 a Values are the mean ± SEM for 6 rats Group 1 has been compared with group 2 a - p < 0.01 TABLE 36a It' VALUES TO TABLE 36 ItI between groups Concentration of PAPS 1 and

38 164 TABLE 37 ACTIVITY OF SULPHATE ACTIVATING SYSTEM Groups Sulphate activating system (.. micromoles of methyl umbelliferone sulphate formed/dr/g protein.. ) 1. Control ~. Experimental 26.5 ± ± O.4S a Values are the mean ± SEM for 6 rats Group 1 has been compared with group 2 a - p < 0.01 TABLE 37a 't' VALUES TO TABLE 37 't' between groups land 2 Sulphate activating system -_._-,

39 165 TABLE 38 ACTIVITY OF ARYL SULPHOTRANSFERASE Groups Aryl Sulphotransferase ( micromoles of methyl umbe11iferone sulphate formed/hr/g protein ) 1. Control 2. Exper imenta ± ± 0.46 a Values are the mean ± SEM for 6 rats Group 1 has been compared with group 2 a - p < 0.01 TABLE 38a Itt VALUES TO TABLE 38 ttl ~etween groups Aryl, SulphGtransferase --'-- 1 and

40 166 concentration of PAPS, activity of sulphate activating\ system and sulfotransferase all decreased in the livex on adm'inistration of the hormone. DISCUSSION The results obtained indicate that administration of glucagon for seven days produced significant alteration in the metabolism of glycosaminoglycans in the liver. The concentration of total glycosaminoglycans showed 5ign1f1- fant decrease in the liver in the rats administered glucagon, All the individual glycosaminoglycan fractions except heparin (H) also showed significant decrease while the concentration of heparin did not show any significant change. In this connection the results obtained on administratiod of glucagon, are opposite to those obtained on administration of insulin. It has been previously reported from this laboratory that administration of ' insulin to rabbits for a period of 45 days resulted in significant increase in the concentration of all the glyco-. ]01 saminoglycans fractions of the liver: Decreased ability to synthesise glycosaminoglycans has also been reported in the diabetic state.

41 167 The activity of biosynthetic enzymes now studied, namely glucosamine-6-phosphate isomerase and UDP glucose dehydrogenase, is not affected by the administration of glucagon. Both these enzymes catalyse r~te~limitin~ steps in the synthesis of precursors of glycosaminoglycans. Glucosamine-6-phosphate isomerase is an important enzyme in the biosynthetic pathway of hexosamine precursors of glycosaminoglycans. It supplies glucosamine-6-phosphate which is the precursor of UDP - glucosam~ne,udp N-acetyl glucosamine and UDP-N-acetyl galactosamine, the forms in which the amino sugar is incorporated into glycosaminoglycans. This enzyme is a site of metabolic regulation, since its activity is subject to feedback inhibition by UDP-N-acety1 glucosamine. UDP glucose dehydrogenase is another important enzyme in the giosynthetic pathway of uronic acid precursors. It forms UDP-glucuronic acid from UDP-glucose. UDP-glucuronic acid and UDP-iduronic acid (formed from UDP-glucuronic acid by 5 t epimerase reaction) are the forms in which the uronic acid moiety is incorporated into glycosaminoglycans. This enzyme is also a site of metabolic regulation since its activity is inhibited by feedback inhibition by UDP-xylose. The activity of these two enzymes generally reflect the rate of biosynthesis of glycosaminoglycans. The fact that the activity of these two enzymes is not affected on glucagon

42 168 administration, may indicate that the hormone may hot affect the rate of biosynthesis of glycosaminog1ycans. In this connection, administration of insulin to rabbits has been previously reported to result in increased activity of these enzymes in the 1iver. 101 The activity of all the enzymes degradation of glycosaminoglycans J rats administered glucagon. cathepsin-d cleaves proteoglycans liberating glycosaminoglycans. studied) which.catalyse the showed an increase in the Aryl sulphatase are non-specific enzymes which bring about desulphation of sulphated molecules including glycosaminoglycans. p-glucuronidase and p-n-acetyl glucosaminidase are both exoenzymes which cleaves uronic acid and hexosamines which form the constituents of glycosaminoglycans. The increased activity of these enzymes may mean increased degradation of glycosaminoglycans in the liver. The decrease in the concentration of glycosaminoglycans observed in the rats administered glucagon may be due to their increased degradation. Administration of insulin to rabbits has been reported previously from this laboratory to result in decreased activity of all these enzymes in the liver:0 1 The effect of glucagon on th(-~ deq.l'adinq enzymes is opposite to that of insulin.

43 169 Administration of glucagon also significsntly alters the sulphate metabolism in the liver. The concentration of PAPS decreased in the liver in the rats administered the hormone. PAPS is the biological sulfating agent and its decreased concentration may be the result of the decreased sulphate activating system (which includes sulphate adenylyl transferase and adenylyl sulphate kinase) which generate it. The activity of sulphate activating system decreased on administration of glucagon. Sulfotransferase activity is also decreased on glucb90n administration. Eventhough the sulfotransferase now studied is aryl sulfotransferase, PAPS is also the sulphate donor for the specific transferases involved in the sulphation of glycosaminoglycans. Decreased concentration of PAPS may therefore result in decreased sulphation of glycosaminoglycans. In this connection, insulin administration has been found from this laboratory to increase sulphate 1m metabolism in rabbits. The activity of PAPS, ~ulphate activating system and aryl sulfotransferase all increased in the liver on administration of insulin. Thus the effect of glucagon on the metabolism of glycosaminoglycans in the liver appears to be opposite to that of insulin.

44 170 Glucagon has also an effect on the concent~ation of glycosaminoglycans in the aorta. There is significant decrease in the concentration of total glycosaminoglycan in the aorta in rats administered the hormone. In this connection there are a number of reports which indicate that the concentration of glycosaminoglycans is significantly altered in a theros clerosis. However the results reported have been conflictory.102_184 Majority of reports indicate an increase in the glycosaminoglycans in the early stages of atherosclerosis. In this connection the results obtained on the effect of glucagon on the concentration of total glycosaminoglycans in the aorta are significant. The decrease in the glycosaminoglycans caused by the hormone indicates that the hormone may probably exert an antiatherogenic effect, as far as the metabolism of glycosaminoglycans is concerned.' However, as indicated in a previous chapter (Chapter 111), it has no effect on aortic cholesterol on chronic administration, eventhough serum LDL+VLDL cholesterol is decreased.