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BioscienceHorizons Volume 6 2013 10.1093/biohorizons/hzt002 Research article The potential effect of vanadium compounds on glucose-6-phosphatase Saima Shehzad* *Corresponding author: Email: email@example.com Supervisor: Dr Sue Bird, S516, Mellor Building, Stoke Campus Stafforshire University. Type 2 diabetes is a major chronic health condition in which hyperglycaemia has significant impact on morbidity and mortality, and its ever-increasing incidence has made the production of therapeutic agents for type 2 diabetes more necessary. Vanadium compounds are known to control hyperglycaemia but the exact focus of where they work is a matter of debate. A proposed mechanism of action is that it inhibits glucose-6-phosphatase, a key enzyme in the development of insulin resistance and thus type 2 diabetes. This paper looks at the inhibitory effects of vanadium salts on glucose-6-phosphatase and also studies the mechanism of inhibition, the hypothesis being that the two vanadium compounds, vanadyl sulphate (VOSO ) and vanadyl acetylacetonate (Vace) will inhibit glucose-6-phosphatase. This was achieved by using a proof of principle study by extracting glucose-6-phosphatase from bovine liver microsomes using differential centrifugation, and then the enzyme was assayed in the presence and absence of vanadium compounds. The study found that vanadyl compounds inhibit glucose-6-phosphatase, as mean specific enzyme activity was calculated which showed that VOSO at a concentration of 48 µ M inhibited glucose- 6-phosphatase activity by 36.9% (P < 0.0003) and Vace at a concentration of 200 µ M inhibited glucosse-6-phosphatase activity by 50% (P < 0.0001) similar to findings in the previous research. However, this study also found using Lineweaver–Burk plot analysis that VOSO is a competitive inhibitor of glucose-6-phophatase and Vace is a mixed: non-competitive and uncompeti- tive inhibitors of glucose-6-phosphatase. The mechanism of inhibitory action of these vanadyl compounds had not been reported previously. The difference in the concentration of inhibitor required may be due to the type of inhibition. This supports the hypothesis to some extent as the results were found to be statistically significant; however, further data will be required to clarify these findings. Keywords: glucose-6-phosphatase, vanadyl sulphate, vanadyl acetylacetonate, type 2 diabetes, inhibition, therapeutics Received 27 August 2012; revised 17 February 2013; accepted 28 February 2013 Introduction required for blood glucose regulation. Type 2 diabetes is the more common type and will be the focus of this paper. Diabetes mellitus, a disorder of blood glucose regulation, is known to occur in over 2.6 million people in the UK, and According to the National Institute for Health and Clinical over half a million of people are unaware that they have this Excellence (NICE, 2008) direct mortality attributable to type 2 condition (NICE, 2008). This generic term encompasses two diabetes is 4.2% in men and 7.7% in women within the UK. It pathogenically different conditions, Type 1 and Type 2 diabe- suggests that a 60-year old man without any other arterial dis- tes. Type 1 diabetes is known to be auto-immune mediated, ease is expected to lose 8–10 years of his life without proper where the body attacks its own β-cells within the pancreas; management. Its burden on the economy comprises its direct these cells are responsible for the production of insulin cost to the National Health Service due to managing it, indirect Institution where research was carried out: Mellor Building, Stoke Campus Stafforshire University. © The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 6 2013 Table 1. Drugs for the treatment of type 2 diabetes and their mechanisms of action Effect on Drug name Mechanism of action insulin It decreases the amount of glucose released by the liver into the Biguanide drug (Metformin) Resistance bloodstream and also increases the sensitivity of body’s cells to insulin Sulphonylurea drugs (gliclazide, glimepiride They function by increasing the insulin amount which pancreas makes Secretion and glipizide) Nateglinide and repaglinide Drugs action is similar to sulphonylurea and increases insulin level Secretion Dipeptidyl peptidase 4 (DDP4) inhibitors or These drugs improve the effects of incretins as they stop DDP4 Secretion incretin enhancers (sitagliptin and vildagliptin) functioning Thiazolidinediones (commonly called They function by increasing the body’s cells sensitivity to insulin Resistance glitazones, e.g. pioglitazone) Acarbose Acarbose works by delaying carbohydrate absorption Resistance Secretion and Insulin Injections for lowering blood glucose level for people with type 2 diabetes resistance It works similarly to the naturally occurring hormone GLP-1. The actions Exenatide—a glucagon-like peptide-1 (GLP-1) Secretion and involved are stimulating insulin secretion in response to glucose and mimetic resistance prevent glucagons Source: Patient UK (2009). effect on the economy through early mortality and lost produc - tivity and impact of this debilitating condition on patients and families, which places it on high priority to manage this condi- tion effectively. NICE (2008) states that Type 2 diabetes occurs Figure 1. Glucose-6-phosphatase reaction. due to impaired sensitivity of insulin within the body plus inabil- ity by the pancreas to secrete insulin to compensate for this. Another characteristic found in type 2 diabetic patients is obe- increase the production of insulin to compensate for this per- sity, which has led to the understanding of a syndrome, known ceived lack of insulin. Table 1 shows the current agents used as metabolic syndrome. The cluster of abnormalities found to control type 2 diabetes and how resistance and secretion within this population includes hyperglycaemia, hyperinsulinae- are the recognized defects. mia, dyslipidaemia and hypertension. These are thought to be due to a genetic defect resulting in insulin resistance, a phenom- The mechanism of insulin resistance has been widely enon which is exacerbated by obesity. If the pancreas cannot debated. A genetic component has been accepted as shown in compensate for the apparent reduction in insulin then type 2 Pima Indians where it is estimated that 30% of the variance diabetes results. The mechanism by which these result remains in insulin insensitivity can be accounted by familial clustering in question (German and Masharani, 2007). (German and Masharani, 2007). Many genes have been implicated including the impaired activities of key enzymes Insulin resistance was first studied by Himsworth in the involved in tissue glucose metabolism in insulin-responding 1930s who introduced the first standardized approach to tissues. One of these key enzymes is glucose-6-phosphatase, quantifying insulin sensitivity in vivo (Kim, 2011). He carried found within the liver and kidney cortex and involved in the out two oral glucose tolerance tests, one with and one with- final steps of gluconeogenesis, the production of glucose from out exogenous insulin in diabetics, and showed the insensitiv- non-carbohydrate carbon substances and glycogenolysis, ity of this population to insulin. Plasma measurement of where it completes the transformation to glucose from glu- insulin was later made possible by radioimmunoassay for cose-6-phosphate, so that it can be utilized within the body. insulin in 1960s. Yalow and Berson (1960) demonstrated a As shown in Fig. 1, it hydrolyses the intermediate, glucose- delayed response to an oral glucose challenge in diabetic 6-phosphate, resulting in glucose and a free phosphate. patients. The hyperglycaemia present following the glucose challenge proved the insensitivity towards insulin. This enzyme is a target of insulin action, where it is inhib- ited in states of hyperglycaemia to prevent the production of With this knowledge, the treatment of type 2 diabetes has glucose. In type 2 diabetes the resistance of liver to insulin been to regulate the body’s response to insulin and also to 2 Bioscience Horizons • Volume 6 2013 Research article leads to uncontrolled gluconeogenesis. Clore, Stillman and this it was also found to inhibit various essential enzyme sys- Sugerman (2000) have shown in type 2 diabetes that there is tems such as Na and K ATPases (Cantley et al., 1978), an overexpression of glucose-6-phosphatase, leading to Ca-ATPase (O’Neal, Rhoads and Racker 1979), dynein increased endogenous glucose production. Additionally, Cori ATPase (Kobayashi et al., 1978) and alkaline phosphatase and Cori (1952) demonstrated that deficiency of this enzyme (Lopez, Stevens and Lindquist, 1976). is responsible for glycogen storage disease type 1, which leads Vanadium is a member of group VB of the periodic table. to profound hypoglycaemia indicating its importance in Two common salts of vanadium, vanadyl sulphate (VOSO ) plasma glucose homeostasis. Arion et al. (1972) showed this and vanadyl acetylacetonate (Vace), are shown in Figs 3 and enzyme is found within the endoplasmic reticulum and its 4, respectively. Although most food contains low amounts of functionality was lost when extraction attempts were made, vanadium (<1 ng/g), food is the major source of exposure to it requires an intact microsome for the enzyme to function vanadium for the general population (Barceloux, 1999). which initially provided difficulties in studying this enzyme system (van Schaftingen and Gerin, 2002). It was found to Cam, Brownsey, and McNeill (2000) demonstrated blood catalyse specifically the hydrolysis of glucose-6-phosphate. glucose concentration in insulin-dependent diabetic rats and The enzyme is 10 times more efficient in hydrolysing glucose- humans was almost normalized after the administration of 6-phosphate than mannose-6-phosphate and Nordlie and vanadium compounds. These effects on glucose metabolism Arion (1964) showed it is capable of hydrolysing other sub- are consistent with the observed lowering of blood glucose strates other than sugar phosphates such as pyrophosphate level of diabetic rats into which vanadate and vanadyl deriva- and carbamoyl phosphate. Due to its reaction mechanism tives have been orally administered (Bevan et al., 1995). shown in Fig. 2, Luecke and Nordlie (1970) demonstrated Tsiani et al. (1998) showed the mechanism by which vana- that glucose-6-phosphatase also has phosphotransferase dium salts reduce hyperglycaemia and improved insulin activity, being able to synthesize glucose-6-phosphate from action is by increasing the glucose transporters activity via donors which includes glucose. insulin receptor substrates 1 and 2 (IRS1/2), phophatidylino- sitol 3-kinase (PI 3-kinase). Vanadium has also been found to Due to the role of glucose-6-phosphatase in plasma glu- activate serine/threonine kinases involved in intracellular cose homeostasis, it gives inhibitors of phospho-transferases insulin signalling at sites distal to the insulin receptor, thereby the ability to control hyperglycaemia, and though there are preventing protein dephosphorylation through inhibition of many treatments for type 2 diabetes, as listed in Table 1, tyrosine phosphatases (Krentz, 2008). there is potential for improvement in glycaemic control, thus phospho-transferase inhibitors are potential therapeutic agents. Lindquist, Lynn and Lienhard (1973) found vanadate to be a potent inhibitor of phospho-transferases and following Figure 3. Molecular structure of VOSO . Vanadyl(IV ) sulphate (VOSO ) 4 4 is a well-known inorganic compound of vanadium. This blue solid is one of the most common sources of vanadium in the laboratory, reflecting its high stability. It features the vanadyl ion, VO +. Figure 2. Reaction mechanism of glucose 6 phosphatase. In the presence of an adequate phosphate donor, the enzyme (E) forms a Figure 4. Molecular structure of Vace (Source: Google Images). Vace phosphoenzyme, which can be hydrolysed or can serve to (Vace) is the chemical compound with the formula VO(C H O ) . This 5 7 2 2 phosphorylate another substrate. Under physiological conditions, only blue–green coordination complex consists of the vanadyl group, glucose-6-phosphatase serves as a substrate and the phosphoenzyme VO + , bound to two acetylacetonate anions, acac−. Like other charge- is hydrolysed. Source: Schaftingen and Gerin (2002). neutral acetylacetonates, this complex is soluble in organic solvents. 3 Research article Bioscience Horizons • Volume 6 2013 As well as controlling hyperglycaemia, vanadium has also ice. Tissue samples were minced using a scalpel after rinsing been shown to decrease triglyceride levels in the plasma, with ice-cold 0.25 M sucrose in 25 mM potassium phosphate which is an important risk factor for the development of buffer (pH 7.4). Then a 20–21 g portion of minced liver was major chronic health conditions such as hypertension and transferred to a homogenizing tube and 10 ml ice-cold coronary heart disease. Manninen et al. (1992) showed that 0.25 M sucrose in 25 mM potassium phosphate buffer (pH the initial plasma triglyceride values of diabetic rats were 7.4) was added and homogenized for ~3 min per portion. markedly higher (P < 0.05) than those of non-diabetic rats. In Using a Sigma 3K30 centrifuge with 12156-H rotor the diabetic rat groups receiving physiological saline (diabetic homogenate was centrifuged at 4°C for 10 min at 1000g and control), the plasma triglyceride levels remained at the initial then for 15 min at 12 000g and 4°C. After each centrifuga- higher triglyceride levels. After treatment using Vace, the tion the thin floating lipid layer was removed by filtering plasma triglyceride levels in diabetic rats gradually decreased through muslin into another centrifuge tube and the pellet to the initial triglyceride values of non-diabetic rats. Similar was discarded. The post-mitochondrial fraction (PMF) was to the triglyceride, the initial values of plasma cholesterol lev- diluted with 7.5 times its volume of ice-cold 8 mM calcium els in diabetic rats were evidently higher (P < 0.05) than those chloride, added dropwise and constantly stirred. PMF was of non-diabetic rats, and after the treatment with Vace, the left to stir at 4°C for 15 min and centrifuged at 8000g for high plasma cholesterol levels in diabetic rats gradually 10 min at 4°C. For each gram of original tissue, 0.15 ml of reversed to a normal range. 0.25 M sucrose was added to re-suspend the pellet after removing the supernatant. The suspension called the micro- Fewer papers, including Kiersztan et al. (2004), have studied some isolation was stored at −80°C until required. the effect of vanadium salts on glucose-6-phosphatase and have found it has an inhibitory effect; however, they have not com- Glucose-6-phosphatase assay mented on the mechanism of inhibition. There are three main Microsomes were diluted using sodium cholate (0.5% w/v mechanisms of inhibition, which are competitive, non-compet- final concentration). The assay was performed in duplicate itive and uncompetitive, illustrated in Fig. 5a–c: (a) shows the by adding 100 µ l of 160 mM sodium cacodylate as a buffer competitive inhibition model, (b) the non-competitive inhibi- solution at pH 6.5, 15 µ l of water and 25 µ l of glucose- tion model and (c) the uncompetitive inhibition model. 6-phosphate (final concentrations of 0, 1.56, 3.13, 4.69 and The aim of the current study is to verify the inhibition of 6.25 mM) and finally 20 µ l of diluted microsomes were glucose-6-phophatase by vanadium compounds. It has been added to initiate the assay. shown that vanadium sulphate exerts its effect at a lower The inhibitor 15 µ l of VOSO was added to give a final concentration than Vace. This may be explained in different 4 concentration of 6, 12, 24 and 48 µ M, whereas 15 µ l of Vace types of inhibition. To investigate this, a proof of principle was added to give a final concentration of 50, 100, 150 and study will be made using bovine liver microsome, which 200 µ M. both added instead of water. To some tubes no would be isolated using the modification of the method used microsomes were added and these were the control tubes. by Woodward (2008), based on that of Ernster, Siekevitz and Mixtures were vortexed and incubated at 30°C for 20 min. Palade (1962) and Hamilton et al. (1999). Glucose-6- The reaction was stopped with the addition of 1.6 ml of phosphatase can be assayed using a modification to the Burchell’s reagent (made at a 6:2:1 ratio by volume, by add- method stated by Woodward (2008), Taussky and Shorr ing 0.42% w/v acid molybdate, 5% w/v sodium dodecyl sul- (1953) and Nordlie and Arion (1966) and the tissue protein phate and 10% w/v ascorbic acid, respectively). Microsomes will be determined by a Lowry assay (Lowry et al., 1951). in the control tubes (blanks) were added after adding the Singh, Nordlie and Jorgenson (1981) found sodium vanadate Burchell’s reagent. to be a competitive inhibitor and seemed to generalize it for all vanadium compounds. No data could be found on the A standard curve was constructed using 0.5 mM monopo- mechanism of action of VOSO and Vace on glucose-6-phos- tassium dihydrogen phosphate at volumes of 0–150 µ l (final phatase. Therefore, the proposed hypothesis is that VOSO phosphate concentrations 0, 20, 25, 40, 50 and 75 nM) and and Vace will competitively inhibit glucose-6-phosphatase. up to a final volume of 160 µ l with 160 mM sodium cacodyl- ate at pH 6.5. Finally, 1.6 ml of Burchell reagent was added and all solutions were incubated (enzyme assay and standard Materials and Methods curve tubes) in water bath at 47°C for 2 h. Then absorbance All materials and reagents were provided by Staffordshire was measured using a Cecil CE1021 spectrophotometer at University and chemicals were purchased from Sigma-Aldrich 820 nm. Using direct interpolation from the standard curve Co. (UK). Bovine liver samples were supplied from a local the inorganic phosphate concentration was estimated. abattoir. Tissue protein determination Liver microsome isolation The microsome isolation was diluted with 0.5 M sodium Liver tissue samples of 80–90 g were cut from whole bovine hydroxide at a ratio of 1:100, and 1 ml of this solution was liver, delivered within ~2–4 h of animal death and kept on used. A blank contained 1 ml of 0.5 M NaOH. Then 5 ml of 4 Bioscience Horizons • Volume 6 2013 Research article Figure 5. (a) Competitive inhibition model. Source: http://www.chm.davidson.edu/erstevens/Lineweaver/Lineweaver.html. In this mechanism, the inhibitor and substrates compete for the same site on the enzyme. The presence of the inhibitor will decrease the affinity between the enzyme and the substrate, reflected by an increase in the K value for the reaction. However as there is competition, if enough substrate is added it can out-compete the inhibitor which means the maximum rate of reaction can be reached and so V value is reached despite the addition of max inhibitor. (b) Non-competitive inhibition model. Source: http://www.chm.davidson.edu/erstevens/Lineweaver/Lineweaver.html. In non- competitive the inhibitor binds allosterically at a different site. With a pure non-competitive inhibitor, the K value is unchanged as the substrate has equal affinity for the enzyme as well as the enzyme–inhibitor complex; however, as there is no competition between the inhibitor and the substrate V value will be lowered as there will be always an enzyme–inhibitor complex present which will not be able to catalyse any reaction. max (c) Uncompetitive inhibition model. Source: http://www.chm.davidson.edu/erstevens/Lineweaver/Lineweaver.html. In uncompetitive inhibition the inhibitor will only bind to an enzyme–substrate complex. The enzyme–inhibitor–substrate complex cannot form any product, as in non- competitive inhibition, but here the K value would actually increase, converse to what we would see in non-competitive inhibition. This could be understood using Le Chatlier’s principle, because due to the inhibitor effect, the equilibrium will shift towards forming more enzyme– substrate complex, therefore the enzyme shows a higher affinity for the substrate even though this increased affinity does not lead to a higher V value as there is no competition between substrate and inhibitor. max copper reagent at 100:1:1 ratio by volume (containing 2% absorbance was read using a Cecil CE1021 spectrophotom- w/v sodium carbonate in 0.1 M NaOH, 1% w/v copper sul- eter at 750 nm. By using direct interpolation from the stan- phate in water and 2% w/v sodium potassium tartrate in dard curve, protein determination was estimated. water) was added to 1 ml diluted microsomal solution and to A bovine serum albumin stock solution was prepared at a blanks also. The mixture was thoroughly mixed by vortexing −1 concentration of 1 mg ml of 0.5 M NaOH and a standard and left to stand at room temperature for 10 min. Then −1 curve of 0–1 mg ml protein was prepared in 0.5 M NaOH. 0.5 ml of 1 M Folin reagent was added to all samples includ- The standard curve solution was then processed as detailed ing blanks, mixed immediately by vortexing and left to stand for the tissue protein assay. at room temperature for 30 min. After zeroing on the blank, 5 Research article Bioscience Horizons • Volume 6 2013 Effect of various inhibitor concentrations on enzyme The standard deviations are represented in the graph, but as activity was analysed by analysis of variance test (ANOVA). the differences were so small (±0.03), they are not very clear in the graph. This shows that in the presence of VOSO there is The mean specific enzyme activity was calculated for glu - inhibition and as the concentration of VOSO increases there is cose-6-phosphatase in the absence or presence of VOSO and greater inhibition as shown by the increasing slope of the graph. Vace, which is shown in Table 2. Analysis of variance showed that the inhibition by VOSO was highly significant ( P < 0.0001). Results −1 −1 This graph shows the V value to be 1.22 µ mol min mg max The results indicate that both vanadyl compounds inhibit of protein. glucose-6-phosphatase. For VOSO and no inhibitor, the two-tailed P value is <0.0003 and for Vace and no inhibitor From this graph K and K values were calculated, which m i it is <0.0001. are shown in Table 3. Following the assay of microsomal fraction in a range of In this assay V value was achieved and K was increas- max m glucose-6-phosphate with differing VOSO concentrations, ing as the concentration of VOSO was increased; this shows 4 4 the following results were obtained. Figure 6 shows the that VOSO conforms to competitive inhibition Consequently, results in the form of a Lineweaver–Burk plot and so it could K would be expected to decrease as increasing the concentra- be ascertained whether there was any inhibition and what tion of VOSO would mean increased affinity between the mechanism of inhibition there was. VOSO and glucose-6-phosphatase. Table 2. Mean specific enzyme activity for glucose-6-phosphatase in Table 3. K and K values for VOSO m i 4 the absence or presence of inhibitor indicating extent of inhibition at the maximum concentration of the inhibitor VOSO K (mM) K (mM) m i concentration (mM) Specific enzyme activity Inhibition −1 −1 (pmol mgprotein min ) (%) 0 6.46 — Control 134.4 (±19.2, n = 5) — 0.006 6.75 0.13 84.8 (±12.8, n = 5), 0.012 8.67 0.04 VOSO (48 µ M) 36.9 P < 0.0003 0.024 8.85 0.06 67.2 (±14.4, n = 5), Vace (200 µ M) 50 P < 0.0001 0.048 13.36 0.04 Figure 6. Lineweaver–Burk plot of the mean (±SD, n = 5) inverse rate of reaction of glucose-6-phosphatase against inverse concentrations of VOSO . 6 Bioscience Horizons • Volume 6 2013 Research article Figure 7. Lineweaver–Burk plot of the mean (±SD, n = 5) inverse rate of reaction of glucose-6-phosphatase with inverse different concentrations of Vace. Similarly, a Lineweaver–Burk plot was constructed when Table 4. K values for Vace Vace was added in increasing concentrations to the micro- some preparations containing glucose-6-phosphate, as shown Vace (mM) K (mM) in Fig. 7. 0 6.46 Again the standard deviations are shown in the graph, but as the differences were small (±0.03), these are not shown 0.05 15.35 clearly. This plot shows that Vace inhibits glucose-6-phos- 0.1 15.58 phatase. Analysis of variance showed that the inhibition by Vace was highly significant ( P < 0.0014). 0.15 8.38 From this graph K and K values were calculated, which m i 0.2 6.54 are tabulated in Table 4. As K varies, and V is reduced, the results suggest that m max Table 5. K values for vanadyl acetylacteonate Vace has mixed competitive/uncompetitive inhibitory effect i on glucose-6-phosphatase. As it is mixed, the inhibitor will have the affinity to glucose-6-phosphatase on its own and also Vace (mM) K (mM) (mM) the glucose-6-phosphatase and glucose-6-phosphate complex, which means two K values can be deduced (shown in Table 5) 0 — — as K and . 0.05 0.07 0.00 The first set of K results indicate the affinity between Vace and the enzyme–substrate and this value increases as the 0.1 0.08 0.00 concentration of inhibitor increases, the second set of K 0.15 0.36 1.58 results show varying values as the concentration of Vace is increased. The first column suggests uncompetitive inhibition 0.2 0.72 0.28 as K values are increasing as the K values increase. i m (2008), but this was sufficient to proceed with the study in con - Discussion fidence that microsomes were present. Microsome isolations were deemed to be successful as the Initial studies focused on determination of the kinetic marker for microsome glucose-6-phosphatase was present. parameters for bovine glucose-6-phosphatase. The results The isolations showed glucose-6-phosphatase activities of indicated a K value of 6.46 mM and a V value of m max −1 −1 −1 0.42 pmol mg min of protein (SD ± 0.03, n = 5), slightly 1.22 µ mol min of protein. The K value stated by Nordlie −1 −1 <0.44 pmol mg min of protein recorded in Woodward et al. (1992) is 3.1 ± 0.4 mM; however, the K value reported 7 Research article Bioscience Horizons • Volume 6 2013 by Waddell and Burchell (1988) was much lower, but their plot is both to move the x-intercept and increase the slope, tissue samples were obtained from rats, 0.5 ± 0.08 mM. and from Fig. 6 it can be seen that the slope is greater when Waddell and Burchell (1988) reported a V value of there is VOSO added to microsome compared with the con- max 4 −1 0.52 ± 0.03 µ mol min , whereas Nordlie et al. (1992) report trol microsome preparation without any inhibitor and also −1 −1 a V value of 0.24 ± 0.3 µ mol min mg of protein. The that the different concentrations intercept the x-axis at differ- max discrepancy between the results could be due to the differ- ent points. The decrease in the K value can be explained by ences in the source tissue. The samples reported in this study the fact that as the concentration of inhibitor increases, the were bovine in origin and the results from the above studies affinity between VOSO and glucose-6-phosphatase would were from rat liver samples. increase as it is a competitive inhibitor and due to its increased availability it is more likely to out-compete the glucose- On the contrary, Arion et al. (1972) found the V value of max 6-phosphate for the active site on glucose-6-phosphatase. −1 −1 glucose-6-phosphatase to be 1.2 µ mol min mg of protein, −1 −1 Similarly, a Lineweaver–Burk plot was constructed when which was similar to the V value of 1.22 µ mol min mg max varying concentrations of Vace were added to separate micro- of protein reported here; however, Gonzalez-Mujica et al. −1 −1 some preparations containing glucose-6-phosphate, which is (2005) reported a V value of 7.59 ± 0.91 µ mol h mg of max −1 shown in Fig. 7. This plot shows that Vace inhibits glucose- protein (equivalent to 0.127 µ mol min mg of protein) and a 6-phosphatase and increasing the concentration of Vace K value of 4.78 ± 1.10 mM from glucose-6-phosphatase shows greater inhibition as shown by the increasing slopes. from intact rat hepatic microsomes. The discrepancy in these Subsequently, K and K values were deduced which are tabu- findings can be explained as their samples were intact and m i lated in Tables 4 and 5. These show that the K values vary from rat microsomes, whereas here they were disrupted m as the concentration of the inhibitor increases, and as the and bovine. However, the values of K of 0.79 ± 0.27 mM and −1 −1 graph shows that V value is also decreasing as the inhibi- V of 11.57 ± 1.55 µ mol h mg of protein are equivalent max max −1 −1 tor increases in concentration, it suggests that Vace is a to 0.193 µ mol h mg of protein in disrupted microsomes. In mixed: non-competitive and uncompetitive inhibitor of glu- this case the differences from the results found in this study cose-6-phosphatase. This can be understood by looking at were seemingly from the different source of microsome (they the model of uncompetitive inhibition (shown in Fig. 5c). The used rat and this paper looked at bovine samples). K values tend to decrease, converse to what we see in non- The preparations were incubated with 48 µ M of VOSO 4 competitive inhibition (Fig. 5b). This can be understood and 200 µ M of Vace separately; these concentrations were using Le Chatlier’s principle, because due to the inhibitor used as Kiersztan et al. (2004) had reported significant inhibi - effect, the equilibrium will shift towards forming more tion of glucose-6-phosphatase with these concentrations. In enzyme– substrate complex; therefore, the enzyme shows a the control microsome preparation, the glucose 6 phosphatase higher affinity for the substrate even though this increased −1 −1 activity was 134.4 pmol mg protein min (SD ± 19.2, n = 5), affinity does not lead to a higher V value as there is no max whereas in the preparation containing 48 µ M of VOSO and 4 competition between substrate and inhibitor. As expected in −1 −1 200 µ M of Vace, 84.8 pmol mg protein min (SD ± 12.8, the Lineweaver–Burk plot, uncompetitive inhibitors shift the −1 −1 n = 5) and 67.2 pmol mg protein min (SD ± 14.4, n = 5) of line higher with a raised y-intercept, which is seen here as the phosphate, respectively, was found, respectively. This shows microsome preparation without any inhibitor intercept the −1 that VOSO at a concentration of 48 µ M causes an inhibition 4 y-axis at 0.82 µ M min , whereas the preparation with −1 of 36.9% on glucose-6-phosphatase and Vace at a concentra- 200 µ M Vace intercepts the y-axis at 2.16 µ M min . tion of 200 µ M causes an inhibition of 50% on glucose- However, the decrease in K values is not uniform and as the 6-phosphatase, showing both vanadium salts act as inhibitors. V value is lowered it suggest that Vace also has non-com- max petitive inhibitor properties. The K values in the first column The results in Fig. 6 indicate that VOSO is a competitive of Table 5 increase, which again can be understood by Le inhibitor of glucose-6-phosphatase; this can be explained by Chatlier’s principle as an increase in uncompetitive inhibitor looking at what happens during competitive inhibition, the concentration, would shift the equilibrium to more forma- model is shown in Fig. 5a. From the Lineweaver–Burk plot, tion of glucose-6-phosphate and glucose-6-phosphatase com- the proposed theory is that VOSO competes against glucose- plex, which means reduced affinity between Vace and 6-phosphate for glucose-6-phosphatase. The inhibitor and glucose-6-phosphatase which here is represented by increas- the substrate both compete for the same site; therefore, when ing K values in the first column of Table 5. The second col- the inhibitor is present the affinity of the enzyme towards the umn in Table 5 shows the affinity between Vace and substrate is decreased, this would be seen as an increase in the glucose-6-phosphatase varies, and as the V value decreases max K value as the inhibitor is added and its concentration is it suggest there is also non-competitive inhibition which gives increased, as shown in Fig. 6. Also if the inhibition was com- the inhibitor mixed properties. petitive, increasing the concentration of the substrate would mean it can out-compete the inhibitor and so V value If it were proposed the inhibition mechanism for Vace is max could be achieved, which is why the Lineweaver–Burk plot uncompetitive you would expect the K values to decrease, the shows that V value is achieved despite adding inhibitor. V value would not be achieved and the K values would be max max i The effect of a competitive inhibitor on the Lineweaver–Burk expected to increase. If data from 0.1 mM were not included it 8 Bioscience Horizons • Volume 6 2013 Research article may fit this pattern, therefore it may be concluded that errors dose of 100 mg (from 141.37 to 114.92 unit/dl). This sug- were made during the preparation of this concentration which gests that 100 mg dose of VOSO causes liver damage and has led to inaccurate results at that concentration, this is also due to the longevity of treatment required to manage diabe- shown by the zero K values found which would suggest that tes, it could result in complications such as liver failure and Vace is not an inhibitor which we know from the other results so understandably therapeutic agents which do not carry this is incorrect. Five liver samples were used in an attempt to get risk, e.g. long-term insulin, would be more preferable. accurate readings, and the mean of these was used to assay the However, Boden et al. (1996) showed that the administration enzyme; however, more can be used to gain more accuracy. of VOSO at a dose of 50 mg twice daily for 4 weeks in eight patients (four men and four women) with non-insulin-depen- There is no previously reported data on the mechanism of dent diabetes mellitus was well tolerated without any toxic action of VOSO or Vace on glucose-6-phosphatase; how- manifestations. They also showed that VOSO was associ- ever, Li et al. (2008), looked at another vanadium compound, ated with a 20% decrease in fasting glucose concentration bis(maltolato)oxovanadium (BMOV) (IV) and its action on and a decrease in the hepatic glucose output during hyperin- different phosphatase systems, protein tyrosine phosphatase sulinaemia. Dai et al. (1994) provided evidence for the long- 1B (PTP1B) and alkaline phosphatase. BMOV showed mixed term safety of VOSO as supplementation of it at competitive and non-competitive inhibition on PTP1B and it −1 concentrations of 0.5–1.5 mg ml in water for a year did not competitively inhibited alkaline phosphatase. It also noted show any significant toxic manifestations in streptozotocin- that BMOV was more potent towards PTP1B than alkaline induced diabetic rats and their normal counterparts. VOSO phosphatase. These data along with those found in this study did not produce persistent changes in plasma aspartate ami- suggest vanadium is a reversible inhibitor of phosphatases; notransferase, alanine aminotransferase and urea level, however, the mechanism can vary depending on the vana- equating to no long-term liver damage, in those animals and dium compound and also the phosphatase system it targets. non-specific morphological abnormalities were detected in any organs in this study. Fawcett et al. (1997) studied the There has been much reported data that vanadium exhibits effects of oral VOSO (0.5 mg/kg per day) for a period of 12 insulin-like properties. Several clinical studies, such as weeks in 31 weight training athletes on their haematological Mukherjee et al. (2004), have shown that vanadium salts parameters, including red and white cells, platelet counts, improve insulin action and reduce hyperglycaemia by potent haemoglobin level, haematocrit, plasma viscosity, blood vis- inhibition of phosphatases that dephosphorylate and deacti- cosity, lipids and indices of liver and kidney function. They vate insulin receptor tyrosine kinase activity and provide a reported that there was no effect of VOSO treatment on hae- mechanism for enhanced insulin action. These compounds matological indices and biochemistry of the organs studied. increase glucose transport in the skeletal muscle when the rec- ognized pathway of insulin stimulated glucose transport via This paper confirms the results of Clore, Stillman and IRS1/2, PI 3-kinase and protein kinase B has been blocked. Sugerman (2000) that vanadium is an inhibitor of glucose- Accordingly, vanadium compounds have been reported to act 6-phosphatase, as well as confirming VOSO is a competitive via an alternative signalling pathway to increase the transloca- inhibitor of glucose-6-phosphatase, reported by Singh, tion or activity of glucose transporters. However, this study Nordlie and Jorgenson (1981). Vace shows mixed uncom- also suggests that this is probably not the only mechanism by petitive/non-competitive inhibition, the mechanism of action which vanadium compounds enhance insulin action. A few on this has not been mentioned in previous research and studies, such as Kiersztan et al. (2004), support the results future studies to clarify this would be beneficial. Once the found in this paper, that vanadium inhibits glucose-6-phospha- mechanism of action is confirmed further work to look at the tase, but this paper has also described the mechanism of this in vivo effect of Vace would ensure the in vitro results are action. For future studies, if these compounds can be given in consistent. Overall, the results reported here uphold the quantifiable amounts to see how much would be required to hypothesis and also support what has been discovered previ- control hyperglycaemia, and along with the knowledge of its ously. From the results obtained in this study together with mechanism of action, potential therapeutic agents can be cre- the evidence published regarding the benefit of vanadium ated which attempt to regulate hyperglycemia and treat type 2 compounds in lowering plasma triglycerides, vanadium com- diabetes differently than the treatments currently available pounds are potentially important therapeutic agents. Further which are shown in Table 1 in the Introduction section. research into the mode of action of these compounds and evidence of toxicity would be an important step to make in One of the obstacles in using vanadium for glucose man- developing treatments for type 2 diabetes. agement is that it is known to be harmful to humans. Glutamate pyruvate transaminase is an enzyme used to mon- itor liver function and if the levels of this enzyme in the Acknowledgements plasma are raised, it indicates liver cell damage. Refat and El-Shazly (2010) have shown that the activity of this enzyme The author acknowledges the Faculty of Science’s supervisor is slightly increased in a treated group with VOSO at a dose and staff’s contribution, support, guidance, expertise and of 100 mg (from 141.37 to 153.25 unit/dl) and slightly facilities of Staffordshire University. Special thanks to Dr Sue decreased in the group treated with vanadyl(II) sulphate at a Bird and Dr Rob Manning for their constant support, 9 Research article Bioscience Horizons • Volume 6 2013 German, M. S. and Masharani, U. (2007) Pancreatic hormones & diabetes supervision and advice throughout the completion of project. mellitus, in D. Gardner and D. Shoback, eds, Greenspan’s Basic & Acknowledgements to Dr Stephen Merry for his assistance Clinical Endocrinology, 8th edn, McGraw-Hill Medical Publishing and Staffordshire University’s technical staff for providing Division, USA, pp. 661–743. the liver samples and equipments. 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Bioscience Horizons – Oxford University Press
Published: May 2, 2013
Keywords: glucose-6-phosphatase vanadyl sulphate vanadyl acetylacetonate type 2 diabetes inhibition therapeutics
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