4:187−196, 2006

The effect of succinic acid monoethyl ester on plasma and tissue
glycoproteins in streptozotocin-nicotinamide induced diabetic
Leelavinothan Pari, Ramalingam Saravanan

Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar,
Received 28th July 2006. Revised 18th August 2006. Published online 22nd October 2006. Summary
Succinic acid monoethyl ester (EMS) was recently proposed as an insulinotropic agent for the treatment
of non-insulin dependent diabetes mellitus. The present study investigated the effect of EMS and
Metformin on dearrangement in glycoprotein levels in the streptozotocin-nicotinamide induced type 2
diabeteic model. Succinic acid monoethyl ester was administered intraperitoneally for 30 days to normal
and diabetic rats. The effect of EMS on glucose, insulin, and plasma and tissue glycoproteins were
studied. The effect of EMS was compared with Metformin, a reference drug. The levels of glucose,
glycosylated haemoglobin and plasma glycoproteins containing hexose, hexosamine and fucose were
increased significantly whereas the level of plasma insulin and haemoglobin were decreased significantly
in diabetic rats. There was a significant decrease in the level of sialic acid and elevated levels of hexose,
hexosamine and fucose in the liver and kidney of streptozotocin-nicotinamide diabetic rats.
Administration of EMS to diabetic rats was followed by a decreased level of glucose, glycosylated
haemoglobin and plasma glycoproteins. The levels of plasma insulin, haemoglobin and tissue sialic acid
were increased whereas the levels of tissue hexose, hexosamine and fucose were near normal. The present
study indicates that the EMS possesses a significantly beneficial effect on the glycoprotein moiety in
addition to its antidiabetic effect.
Keywords: succinic acid monoethyl ester − nicotinamide − streptozotocin − glycoprotein components
insulin deficiency; insulin resistance accompanied by decreased glucose transport into muscle and fat Type 2 diabetes mellitus typically involves an cells, and increased hepatic glucose output. All of abnormal beta-cell function that results in relative these contribute to hyperglycaemia, resulting in the impairment of the metabolism of glucose, lipids, proteins and glycoproteins (Hawan et al. 1996). * L. Pari, Department of Biochemistry and The level of different types of serum glycoproteins Biotechnology, Faculty of Science, Annamalai are maintained within a narrow range in health University, Annamalai Nagar-608 002, Tamil (Sharma and Sur 1967), but is elevated in many pathological conditions, cardiovascular disease (Tandon et al. 1983), diabetes mellitus (Anand et al. 1985). Defects in insulin secretion and insulin action are universally present in type 1 diabetes, and also type 2 diabetes, in both human patients Pari and Saravanan : The effect of succinic acid monoethyl ester on plasma and animal models. Glycoproteins are carbohydrate of a number of new oral agents for the treatment of linked protein macromolecules found in the cell type 2 diabetes with the hope of achieving better surface, which is the principal component of animal glycemic control. Metformin is a biguanide, which cells. Abnormal levels of glycoproteins are has been in clinical use for the treatment of type 2 important in the pathogenesis of liver and kidney diabetes for over 40 years. Metformin enhances the sensitivity of both hepatic and peripheral tissues to Glycoproteins are rich in extra cellular matrix insulin. The drug also inhibits gluconeogenesis in and they contribute a major source to the structure of the matrix (Begum et al. 1978). It is well documented that the oligosaccharide moieties of investigations had been carried out on the effect of glycoproteins: hexose, hexosamine, fucose and succinic acid monoethyl ester in streptozotocin- sialic acid have an important role in protein nicotinamide diabetic rats on glycoproteins status, stability, function, and turnover (Wiese et al. 1997). so the present investigation was carried out to study In the diabetic state, glucose is utilized by the the effect of a nonglucidic nutrient such as EMS on insulin independent pathways leading to the plasma and tissue streptozotocin- synthesis of glycoproteins, and even a mild deficiency of insulin influences the thickening of the basement membrane (Konukoglu et al. 1999). The raised levels of glycoproteins in diabetics may also be a predictor of angiopathic complications MATERIALS AND METHODS
(Konukoglu et al. 1999). The therapy of non-insulin dependent diabetes mellitus presently relies upon compounds from a number of chemical classes: Succinic acid monoethyl ester (Fig. 1), and all other sulfonylureas, non-sulfonylureas and biguanides biochemicals and chemicals used in this experiment etc. A wide variety of structurally distinct were purchased from Sigma, USA. The chemicals molecules stimulate insulin secretion from pancreatic â cells by different mechanisms of Esters of succinic acid are new potent insulin secretagogues (Fahien et al.1988, MacDonald and Fahien 1988, Picton et al. 2001) and have been proposed as a novel antidiabetic agent for type 2 diabetes. It has been previously shown that succinic acid ester can be taken up and metabolized by pancreatic â cells, leading to increased pro-insulin Fig.1. Succinic acid monoethyl ester
biosynthesis (Alarcon and Wicksteed 2002), insulin secretion and lower blood glucose (Zawalich and Zawalich 1992, GarciaMartinez et al. 1998). The possible mechanism of action of EMS could be Male albino Wistar strain rats (200 − 220g b.w. correlated with the reminiscent effect of the obtained from Central Animal House, Rajah hypoglycaemic sulphonylureas that promote insulin Muthiah Medical College, Annamalai University secretion by closure of K+-ATP channels, were used in the present study. The rats were fed on membrane depolarization and stimulation of Ca2+ pellet diet (Hindustan Lever Limited, Mumbai, influx − an initial key step in insulin secretion India) and water ad libitum. The rats used in the (Fahien et al.1988, MacDonald and Fahien 1988). present study were maintained in accordance with These esters also protect pancreatic islets in vivo the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, streptozotocin (Akkan and Malaisse 1993), India and the study approved by the ethical interleukin 1â ( (Eizirik et al. 1994), and nitric committee (Vide. No: 285, 2005), Annamalai oxide donors (Eizirik et al. 1996). Previously we had reported that EMS has a plasma glucose lowering effect in experimental type 2 diabetes Experimental Induction of Type 2 Diabetes in Rats Non-Insulin dependent diabetes mellitus (NIDDM) In non-insulin-dependent or type-2 diabetes was induced in rats fasted overnight by a single mellitus, oral hypoglycaemic agents are used to intraperitonial injection of 45 mg/kg streptozotocin, stimulate the pancreatic beta cells to secrete insulin 15 min after the i.p. administration of 110 mg/kg and/or increase the sensitivity of peripheral insulin b.w. of nicotinamide. Streptozotocin (STZ) was receptors to the action of endogenous insulin (Elson dissolved in citrate buffer (pH 4.5) and and Meredith 1998, Reusch 1998, Lebovitz 2001). nicotinamide was dissolved in normal saline. The The last few years have witnessed the introduction elevated glucose levels in plasma determined at Pari and Saravanan : The effect of succinic acid monoethyl ester on plasma 72 h, and then on day 7 after injection, confirmed contained 0.5 ml of aliquot/ plasma, 0.5 ml of 5% hyperglycaemia. The rats found with permanent phenol and 2.5 ml of conc. H2SO4 and boiled for 20 NIDDM were used for the study (Masiello et al. Hexosamine was estimated by the method of Elson and Morgan (1933) with slight modifications by Niebes (1972). Briefly, the reaction mixture In the experiment, a total of 42 rats (36 surviving contained 0.5 ml plasma/ 1.0 ml aliquot, and 2.5 ml diabetic rats, and 6 control rats) were used. The rats of 3N HCl. It was boiled over 6 h and neutralized were divided into seven groups of six rats each. with 6N NaOH. To 0.8 ml of the neutralized Group I were control rats (vehicle treated). sample was added 0.6 ml of acetyl acetone reagent and it was boiled for 30 min. The mixture was intraperitoneally with EMS 8 µmol/g bw for 30 treated with 2.0 ml of Ehrlich’s reagent. The colour days. Group III were diabetic control rats, and developed was read at 540 nm colorimetrically. Sialic acid and fucose were determined by the intraperitoneally with EMS 2 µmol/g bw for 30 method of Warren (1959), Dische and Shettles days. Group V were diabetic rats administered (1948). In brief, 0.5 ml of aliquot/ plasma, was intraperitoneally with EMS 4 µmol/g bw for 30 treated with 0.5 ml of water and 0.25 ml of periodic days, and GroupVI were diabetic rats administered acid, and incubated at 37 °C for 30 min. 0.2 ml of intraperitoneally with EMS 8 µ mol/g bw for 30 sodium meta arsenate and 2.0 ml of thiobarbituric days (Pari and Saravanan 2005). Group VII were acid were added to the reaction mixture which was diabetic rats given Metformin 25 mg/kg bw/day in heated for 6 min. 5.0 ml of acidified butanol was 1 ml of saline for 30 days (Yanardag et al. 2005). then added. The absorbance was read at 540 nm. At the end of the experimental period, the rats For fucose estimation, 0.5 ml of aliquot/ were deprived of food overnight and blood was plasma were treated with 4.5 ml of H2SO4 and collected in a tube containing potassium oxalate boiled for 3 min. 0.1 ml of cysteine hydrochloride and sodium fluoride for the estimation of plasma reagent was then added. After 75 min in the dark, the absorbance was read at 393 and 430 nm. haemoglobin. Plasma was separated for the assay of insulin. Liver and kidney were dissected out, washed in ice-cold saline, patted dry and weighed. The data for various biochemical parameters were analyzed using analysis of variance (ANOVA) and the group means were compared by Duncan’s Determination of plasma glucose and insulin Plasma glucose was estimated colorimetrically considered statistically significant when P< 0.05. Diagnostics (I) Pvt Ltd., Baroda, India) (Trinder 1969). Plasma insulin was assayed using an enzyme linked immunosorbent assay (ELISA) kit (Roche Determination of haemoglobin and glycosylated Plasma glucose and insulin levels Fig. 2 demonstrates the levels of plasma glucose The level of haemoglobin was estimated by using and insulin in control and experimental animals. In the cyanmethaemoglobin method described by diabetic rats the level of plasma glucose was Drabkin and Austin (1932). The glycosylated significantly increased whereas the plasma insulin haemoglobin level was estimated according to the was significantly decreased. The administration of method of Sudhakar Nayak and Pattabiraman EMS significantly reversed the changes in a dose (1981) with modifications according to Bannon dependent manner. EMS at a dose of 8 µmol/g bw showed a highly significant effect compared to 2 and 4 µmol/g bw. Administration of EMS was Determination of glycoproteins levels compared with metformin, a reference drug. The For the estimation of glycoproteins, the tissues effect of EMS at a dose 8 µmol/g bw was used for were defatted by the method of Folch et al. (1957) and the defatted tissues were treated with 0.1N Haemoglobin and glycosylated haemoglobin levels 2SO4, hydrolysed at 80 °C and aliquot was used for sialic acid estimation. To the remaining Fig. 3 shows the levels of haemoglobin and solution, 0.1 N NaOH was added. The aliquots glycosylated haemoglobin in the blood of control were used for fucose, hexose, and hexosamine and experimental rats. The diabetic rats showed a estimation. Hexose was estimated by the method significant decrease in the level of total described by Niebes (1972). The reaction mixture haemoglobin and a significant increase in the level Pari and Saravanan : The effect of succinic acid monoethyl ester on plasma of glycosylated haemoglobin. The administration of changes in total haemoglobin and glycosylated EMS and metformin to diabetic rats reversed the C N+EMS DC D+EMS D+EMS D+EMS D+ Met (8 µmol/g) (2 µmol/g) (4 µmol/g) ( 8 µmol/g) (25 mg/kg)
Fig. 2. Changes in the levels of plasma glucose and insulin in control and experimental animals. C, control; N, normal;
DC, diabetic control; EMS, succinic acid monoethyl ester; MET, Metformin.Values are given as mean ± S.D for 6 rats in
each group. Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT).
Effect of EMS on plasma and tissue glycoproteins
this disorder increases, more appropriate therapies Fig. 4 shows the changes in the level of plasma are required (Bailey and Flalt 1986). The esters of glycoproteins of control and experimental rats. selected carboxylic metabolites, which are There was a significant increase of plasma mediating the Krebs cycle, or their precursors such glycoproteins in diabetic rats. Administration of as pyruvic acid, succinic acid, and glutamic acid, EMS and Metformin significantly decreased the are currently under investigation as potent insulinotropic tools in the treatment of non insulin The levels of liver and kidney glycoprotein of control and experimental rats are shown in Figs. insulinotropic capacity of succinic acid monoethyl 5−8. The levels of glycoprotens containing hexose, ester was first disclosed in pancreatic islets hexosamine and fucose were significantly increased (Malaisse 1995). The nutritional value of this ester whereas the level of sialic acid was significantly when infused into starved rats has been recently decreased in diabetic rats. Administration of EMS documented (Ladriere and Malaisse 1997). In the and metformin significantly reversed these changes present investigation, treatment with EMS showed in the glycoproteins levels in the liver and kidney significant antihyperglycaemic activity. The of diabetic rats. The effect of EMS was compared administration of EMS and metformin to decrease the increased blood glucose concentration to normal glycaemic concentration is an essential trigger for the liver to revert to its normal homeostasis during experimental diabetes. DISCUSSION
It is well documented that EMS triggers a proinsulin synthesis and insulin release similar to Diabetes mellitus is a heterogeneous endocrine glucose induced insulin synthesis and release disorder in which hyperglycaemia is the unifying (Maechler and Wollheim 2000). MacDonald and feature and, as knowledge of the heterogeneity of Fahien (1988) found that initiation of insulin Pari and Saravanan : The effect of succinic acid monoethyl ester on plasma release by esters of succinate by mitochondrial 1979). Glycosylated haemoglobin is found to be metabolisms is sufficient to initiate and support significantly increased in diabetic animals and the insulin release from β cells (Ainscow et al. 2000). amount of this increase is directly proportional to Hyperglycaemia is the clinical hallmark of poorly the fasting blood glucose level (Peters et al. 1996, controlled diabetes, which is known to cause Koening et al. 1978). The level of total protein glycation, also known as non - enzymatic haemoglobin is found to be decreased in the glycosylation (Zhang and Swaan 1999). It has been diabetic group and this may be due to the increased reported that various proteins, including formation of glycosylated haemoglobin. This was haemoglobin, albumin, collagen, low-density well correlated with earlier studies, which reported lipoprotein, a crystalline of lens and fibronectin, that there was a decrease in the level of undergo non enzymatic glycation in diabetes haemoglobin in experimental diabetic rats (Koyama et al. 1998, Kumar et al. 2005). In long- (Shirwaukar et al. 2006). The increase in the level term diabetes, the glycosylated form of Hb has an of haemoglobin in animals given EMS may be due altered affinity for oxygen and this may be a factor to the decreased level of blood glucose. in tissue anoxia (Yiping et al. 2004, Bunn et al. C N+EMS DC D+EMS D+ Met (8 µmol/g) (8 µmol/g) (25mg/kg) Fig. 3. Changes in the levels of plasma hemoglobin and glycosylated hemoglobin in control and experimental animals.
Symbols as in Fig. 2.
protein degradation and renewal. Glycation of the glucose and other saccharide derivatives with extracellular matrix produces changes in proteins, nucleotides and lipids (Brownlee 2001). macromolecular structure affecting matrix-matrix Non-enzymatic glycation (Maillard reaction) is a and matrix cell interactions associated with complex series of reactions between reducing decreased elasticity and increased fluid filtration sugars and amino groups of proteins, which leads to across the arterial wall, and endothelial cell browning, fluorescence and cross-linking of the proteins. The reaction is initiated by the reversible concentration of AGEs increased above a critical formation of a Schiff base, which undergoes a level, cell surface AGE receptors become activated. rearrangement to form a relatively stable Amadori This is associated with increased expression of product. The Amadori product further undergoes a extracellular matrix proteins, vascular adhesion series of reactions through dicarbonyl intermediates molecules, cytokines and growth factor. Depending to form AGE (advanced glycation end-products). on the cell type and concurrent signalling, this can Formation of some AGEs combines both the be associated with chemotaxis, angiogenesis, glycation and oxidative steps in a process termed oxidative stress, and cell proliferation or apoptosis glycoxidation (Thornalley 2002). Glycation occurs (Thornalley 2004). These processes are thought to inside and outside cells. Glycation of cellular contribute to disease mechanisms associated with proteins produces changes in structure and loss of the development of diabetic complications (Vlassar enzymatic activity. These effects are countered by Pari and Saravanan : The effect of succinic acid monoethyl ester on plasma The biochemical markers hexose, hexosamine, secreted into the blood. The present experiment sialic acid, and fucose have been measured in the attempts to extract the protective role of EMS on liver and serum because liver is responsible for the glycoprotein levels in control and experimental synthesis of all major proteins, which are then
Fig. 4. Changes in the levels of plasma glycoproteins in control and experimental animals. Symbols as in Fig. 2.

Fig. 5. Changes in the levels of tissues hexose in control and experimental animals. Symbols as in Fig. 2.
Generalized abnormalities in the metabolism of significant modification in the connective tissue glycoproteins are observed in both naturally macromolecule. The requirement of insulin for the occurring and experimental diabetes (Mc Millan biosynthesis of the carbohydrate moiety of 1970, Latha and Pari 2005). Berenson et al (1972) mucoproteins from glucose is thus evident. reported that streptozotocin diabetic rats exhibited a Decreased incorporation of the carbohydrate moieties in diabetic rats may be due to insulin structure and composition to those in circulation deficiency. The increases in plasma glycoprotein (Latha and Pari 2003). Therefore, vascular components have been reported to be associated complications that involve complex protein- with the severity and duration of diabetes. carbohydrate molecules could contribute to an Glycoproteins found in a variety of tissues including the arterial wall are very similar in Pari and Saravanan : The effect of succinic acid monoethyl ester on plasma
Fig. 6. Changes in the levels of tissues hexosamine in control and experimental animals. Symbols as in Fig. 2.

Fig. 7. Changes in the levels of tissues sialic acid in control and experimental animals. Symbols as in Fig. 2.
The biosynthesis of the carbohydrate moieties in the hyperglycaemic state accelerates the of glycoprotein forms the insulin independent synthesis of basement membrane components, i.e., pathways for the utilization of glucose-6-phosphate. glycoproteins (Radhakrishnamoorthy et al. 1973). But the deficiency of insulin during diabetes This is due to the depressed utilization of glucose produces a dearrangement of glycoprotein by insulin dependent pathways, thereby enhancing metabolism, resulting in the thickening of basal the formation of hexose, hexosamine and fucose for membranes. The increased availability of glucose the accumulation of glycoproteins (Spiro et al. fucose content. Yorek (1993) found that fucose was An earlier study has shown that in normal significantly increased in serum from a more circulation, fucose concentration increase as much severely diabetic set of rats compared with the as 8 fold in diabetes (Radhakrishnamurthy et al. more moderately diabetic rats. Recent reports have 1976). In diabetes, three serum proteins also indicated that hepatic and serum fucosidase (haptoglobin, á-1 acid glycoprotein and activities are increased in streptozotocin induced á1-antitrypsin) synthesized in the liver are mainly diabetic rats. Our results suggest that the increased responsible for the increase in bound fucose levels fucosylated proteins in diabetic rats could be due to an increase in the synthesis and/or decrease in metabolism and synthesis of these proteins may be altered in diabetes leading to changes in serum Pari and Saravanan : The effect of succinic acid monoethyl ester on plasma
Fig. 8. Changes in the levels of tissues fucose in control and experimental animals. Symbols as in Fig. 2.
Sialic acid is an acylated derivative of levels of the carbohydrate moieties of glycoproteins neuraminic acid and exists as a terminal component with inhibition of the excessive glycosylation, of the non-reducing end of carbohydrate chains of which indicates the therapeutic value of glycoprotein in mammals. Their implications in a nonglucidic nutrient EMS in type 2 diabetes. variety of surface-related vital cell function in numerous tissues are well documented (Patti et al. 1999). The sialic acid moiety of carbohydrate epitope is important for biological interactions REFERENCES
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