316 Short Communication
Authors
Z. Kender1*, T. Fleming2*, S. Kopf2, P. Torzsa3, V. Grolmusz1, S. Herzig4, E. Schleicher5, K. Rácz1, P. Reismann1, P. P. Nawroth2
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ type 2 diabetes ● ▶ metformin ● ▶ methylglyoxal ● ▶ glyoxalase I ●
Abstract
▼
The effect of metformin on methylglyoxal (MG) metabolism was studied in a prospective nonrandomized 24 weeks trial in patients with type 2 diabetes. Metformin treatment, in addition to life style intervention, significantly reduced morning glucose and HbA1c whilst body weight and BMI were only marginally reduced during the 24 week trial. Treatment significantly reduced both plasma MG and carboxymethyl-lysine (CML), a marker of oxidative stress. The reduction in MG
Introduction
▼ received 24.11.2013 first decision 24.11.2013 accepted 28.02.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1371818 Published online: April 7, 2014 Exp Clin Endocrinol Diabetes 2014; 122: 316–319 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence Dr. T. Fleming Medizinische Klinik I und klinische Chemie Universität Heidelberg Im Neuenheimer Feld 410 F02 Room 02.414-02.434 69120 Heidelberg Tel.: + 49/6221/5638 490 Fax: + 49/6221/566 934 Thomas.Fleming@med. uni-heidelberg.de
Metformin, due to its cardio-vascular protective effects, has become one of the most widely prescribed oral glucose-lowering agents for the treatment of type 2 diabetes. It is a disubstituted biguanide and although the exact mechanism of action is not completely understood, its main blood glucose-lowering activity is believed to be primarily through suppression of hepatic glucose output [1]. The results of large-scale clinical investigations have also provided evidence that metformin has preventive effects on diabetic complications [2]. One such reported effect is as a scavenger of reactive carbonyl species, such as the dicarbonyl, methylglyoxal (MG). MG is formed exclusively from the spontaneous degradation of trioses phosphates (glyceraldehyde-3-phosphate and dihydroxyacetonephosphate) [3]. It has been shown for both type 1 and type 2 diabetic patients, that they are more susceptible to the accumulation of trioses phosphates and MG [4]. Once formed, MG can react with proteins, specifically lysine, arginine and cysteine residues to form advanced glycation end products (AGEs) [3].
was paralleled by a significant increase in the activity of Glyoxalase 1 (Glo1), the major route of MG detoxification, in peripheral blood mononuclear cells and red blood cells. Multivariate analysis showed that the changes in MG were dependent upon the metformin treatment. This study supports previous findings that metformin can reduce plasma MG in type 2 diabetic patients. However, given the observed increase in Glo1 activity, this reduction is due not only to the scavenging properties of metformin, but the restoration of Glo1 activity.
It has been shown that metformin treatment ( > 1 g per day) was able to reduce systemic plasma MG levels in patients with type 2 diabetes by 16 % [5]. It was suggested that the mechanism for the lowering of plasma MG was related to the scavenging properties of metformin. It has been previously demonstrated that metformin, through the guanidine group, can bind to MG to form hydroimidazolone, triazepinone and other adducts [6, 7]. Compared to aminoguanidine, a structurally similar scavenger of MG, it has a significantly lower rate constant; the rate constant for metformin and MG is 0.034 M − 1min − 1 compared to 155 M − 1min − 1 for aminoguanidine [7]. This would suggest that whilst metformin can bind MG, it is not the primary means by which it reduces systemic MG. It was also shown that in addition to the lowering of plasma MG, the levels of D-lactate the stable endproduct of MG detoxification by the Glyoxalase system [3], increased. This suggests that an alternative mechanism for the reduction of MG by metformin is through enhancement of detoxification. In this study, we investigate the effect of metformin treatment on MG metabolism in a prospective non-randomized 24 week trial in patients with type 2 diabetes, as to establish
* Both authors contributed equally to the work.
Kender Z et al. Effect of Metformin on Methylglyoxal Metabolism … Exp Clin Endocrinol Diabetes 2014; 122: 316–319
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Effect of Metformin on Methylglyoxal Metabolism in Patients with Type 2 Diabetes
Short Communication
317
Table 1 Patient Characteristics at baseline (0 week) and at end of trail (24 weeks). Data represents mean ± SD; Students t-test was used for comparison. Analysis were done unadjusted and adjusted (*) for age and gender. 51.3 ± 10.3 7/5 Baseline (n = 12) Body-Mass-Index Fasting glucose (mmol/L) HbA1c ( %) MG (nM) CML (ng/ml) Glo1 pBMC (mU/mg) Glo1 RBC (mU/mg) Glo2 pBMC (mU/mg) Glo2 RBC (mU/mg) Total TPI RBC
35.4 ± 6.2 7.3 ± 2.0 7.1 ± 0.92 653.3 ± 155.3 283.4 ± 63.5 0.51 ± 0.25 0.26 ± 0.18 8.01 ± 3.6 0.17 ± 0.13 102.3 ± 60.9
Unadjusted 24 weeks (n = 11) 32.9 ± 6.3 6.3 ± 2.0 6.3 ± 0.6 444.5 ± 172.3 206.3 ± 70.5 1.02 ± 0.44 0.36 ± 0.17 6.70 ± 3.3 0.19 ± 0.11 106.7 ± 70.3
whether changes in the glyoxalase system underlie the MG reducing capacity of metformin.
Adjusted
T
p
3.4 2.2 3.6 3.8 2.4 − 4.1 − 1.4 1.4 − 0.3 0.3
< 0.01 < 0.05 < 0.01 < 0.01 < 0.05 < 0.01 n.s. n.s. n.s. n.s.
T* 1.7 2.0 3.6 4.2 2.9 − 6.4 − 1.4 – – –
p* 0.11 0.08 < 0.01 < 0.01 < 0.05 < 0.001 0.19 – – –
Determination of MG The concentration of MG in deproteinized plasma samples was measured by HPLC after a derivatisation with 1,2-diamino-4,5dimethoxybenzene, as previously described [4].
Material & Methods
▼
Determination trioses phosphates intermediates
Participants 12 patients with type 2 diabetes mellitus were recruited from the 2nd Department of Medicine, Semmelweis University, Buda▶ Table 1. Inclusion cripest. Patient characteristics are given in ● teria were newly diagnosed type 2 diabetes, no previous anti-diabetic treatment, age between 20–80 years, no pregnancy, no malignant disease and no alcohol or drug abuse. Subjects were treated with a high dose metformin (1 000–2 000 mg/ day) for 24 weeks. The starting dose was 500 mg of metformin twice a day and then increased to 1 000 mg 2 times a day. Lifestyle intervention was performed monthly by a dietetic counselor in a combination of group and individual sessions. The patients were given specific caloric consumption and exercise goals. The study protocol was approved by the Hungarian Ethic Committee and all patients gave written informed consent.
The concentration of trioses phosphate intermediates (Glyceraldehyde-3-phosphate, dihydroxyacetonephosphate and fructose1,6-bisphosphate), in deproteinized haemolysate samples was determined, as previously described [4].
Determination of plasma Nε-(Carboxymethyl)-Lysine (CML)-modified protein concentration The measurement of CML-modified protein concentration in plasma samples was performed by direct ELISA as previously described [10] using an antibody against CML (Biologo, Kronshagen, Germany).
Data analysis
Sample processing
Comparison of baseline variables with 24-weeks follow-up variables were performed unadjusted and adjusted (age and gender) with paired t-test. Changes of variables were calculated by 24-weeks values minus baseline values (∆-variable). For prediction of ∆MG linear regression analysis were performed stepwise forward and backward with following independent variables: age, gender, ∆BMI, ∆fasting glucose, ∆Glo-1 activity in RBC and pBMCs. The differences were considered to be significant when p < 0.05. Statistical analyses were performed using IBM SPSS Statistics 19 (Armonk, USA).
For the determination of metabolites in RBCs, samples were processed as previously described [4]. Cytoplasmic extracts from the pBMCs were prepared as previously described [8].
Results
Isolation of human red blood cells (RBC) & peripheral blood mononuclear cells (pBMCs) Blood was sampled in the fasting state. RBCs and pBMC were isolated from venipuncture EDTA anticoagulant, processed within and 60 min after sampling, as previously described [4, 8].
Assay of Glyoxalase 1 (Glo1) and Glyoxalase 2 (Glo2) activity The activity of Glo1 determined using the spectrometric method, which monitors the initial rate of change in absorbance at 240 nm caused by the formation of S-D-lactoylglutathione [4]. The activity of Glo2 was also determined spectrophotometrically, by measuring the initial rate of decrease in absorbance at 235 nm over 10 min, as S-D-lactoylglutathione is hydrolyzed to D-lactate and water [9].
▼
Effect of metformin on metabolic parameters In the unadjusted analyses, metformin treatment significantly reduced plasma levels of glucose (7.3 ± 2.0 vs. 6.3 ± 2.0 mmol/l; p = 0.04), HbA1c (7.1 ± 0.92 vs. 6.3 ± 0.6 %; p < 0.01) and BMI (35.4 ± 6.2 vs. 32.9 ± 6.3 kg/m²; p < 0.01). In the adjusted analyses for age and gender, HbA1c reduction was significant (p < 0.01), while reduction of fasting glucose (p = 0.08) and BMI (p = 0.11) ▶ Table 1). were not significant (●
Effect of metformin on trioses phosphate intermediates Metformin treatment had no effect on the levels of trioses phos▶ Table 1). No correlation was observed phate intermediates (● Kender Z et al. Effect of Metformin on Methylglyoxal Metabolism … Exp Clin Endocrinol Diabetes 2014; 122: 316–319
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Age (years) Female/male
318 Short Communication
n = 11 − 195.3 ± 172.1
Independent variables Age Gender Δ BMI Δ fasting glucose Δ Glo1 pBMC Δ Glo1 RBC
51.3 ± 10.3 7/5 − 2.6 ± 2.8 − 1.0 ± 1.6 0.55 ± 0.44 0.10 ± 0.23
Univariable r
p
0.06 0.30 − 0.01 0.34 − 0.70 0.11
0.44 0.19 0.49 0.15 0.08 0.38
between trioses phosphates intermediates, plasma glucose, HbA1c or MG levels.
Effect of metformin on MG, glyoxalase activity and CML Metformin treatment significantly reduced plasma MG levels (653.3 ± 155.3 vs. 444.5 ± 172.3 nM; p < 0.01) as well as plasma levels of CML (283.4 ± 63.5 vs. 206.3 ± 70.5 ng/ml; p = 0.016, ▶ Table 1). The reduction in plasma MG and CML was paralleled ● by an increase in the activity of Glo1 in both the pBMCs (0.51 ± 0.25 vs. 1.02 ± 0.44) and RBCs (0.26 ± 0.18 vs. 0.36 ± 0.17). No significant differences were found in Glo2 activity in either ▶ Table 1). At baseline, it was found that the pBMCs or RBCs (● Glo1 activity significantly correlated with plasma MG in a negative relationship (r = − 0.431; P = 0.0003). Patients at the start of the trial could therefore be characterized as having low pBMC Glo1 activity and high plasma MG whereas at the end of the trial they had high activity and low plasma MG levels. Multivariable regression analysis demonstrated that the observed reduction in plasma MG was dependent only on changes in Glo-1 activity in ▶ Table 2). pBMC and RBC only (●
Discussion
▼
In this study, it has been shown that high dose metformin treatment, in addition to life style intervention, is an effective treatment for the reduction of blood glucose and HbA1c in type 2 diabetic patients. Furthermore, it has been shown that metformin treatment is associated with a reduction of plasma MG. It has previously been suggested that this reduction is due to the scavenging properties of metformin [5], however, given its poor scavenging properties it is likely that the observed increase in Glo1 activity, both in the RBCs and pBMCs, is responsible for the observed reduction in plasma MG. However, the mechanism by which metformin increases the activity of Glo1 and whether this affect is systemic, particularly given that glyoxalase system is ubiquitous in tissues, remains unclear. Increased oxidative stress is considered to be a hallmark of the diabetic state. This can lead to the reduction of glutathione (GSH). Glo1 is a glutathione-dependent enzyme and previous studies have shown that increased production of reactive oxygen species (ROS) can result in the depletion of GSH and NADPH, which can in turn decrease in the in situ activity of Glo1 [11], and thereby increase the concentration of MG. It has previously been shown that metformin can improve the GSSH/GSH balance in diabetes [12] and prevent the accumulation of ROS and ROS induced compounds such as malondialdehyde [13, 14]. In this study, significant reduction in the level of CML-modified proteins, a marker for oxidative stress was observed. This would suggest that the anti-oxidant capacity of metformin may enhance the increases in Glo1 activity by ensuring that sufficient
Multivariable beta − 0.15 0.17 − 0.08 0.29 − 425.3 − 451.7
T
p‡
− 0.6 0.8 − 0.4 1.7 − 4.3 − 2.4
n.s. n.s. n.s. 0.14 < 0.01 < 0.05
Table 2 Multivariable linear Regression analysis for prediction of the changes of methylglyoxal. Data represents mean ± SD; Stepwise forward and backward regression model was used (R2 = 0.70).
GSH is available for detoxification of MG. However, as the activity measurements in this study were performed under saturated substrate conditions, the relative differences observed in activity would be due to an increase in the level of the protein or specific activity. It is interesting to note that they was no change in plasma D-lactate levels (Data not shown), the stable endproduct of glyoxalase metabolism. It would have been expected that an increase in Glo1 activity would lead to increased D-lactate, however, the production of D-lactate is dependent upon Glo2 activity and no affect on activity was observed following treatment with metformin. It has been shown that at the transcriptional level, metformin was able to restore diabetes associated decreases in key antioxidant defense enzymes, such as Glutathione-S-Transferase, NAD(P)H quinone oxidoreductase and catalase, whereas proinflammatory genes such as TNFα and IL-6 were inhibited [15]. It is currently unknown whether this restoration arises indirectly from the anti-oxidant capacities of metformin reducing the levels of oxidative stress sufficiently to allow for the recovery of the cell’s defences systems, or more directly, through interaction and activation of signalling pathways and transcription factors. Further in vitro studies are required to establish the underlying molecular mechanisms by which metformin has a positive effect in diabetes, particularly with respect to the activation of redox-sensitive transcription factors such AP1, NFκB and Nrf2; that latter of which has been shown to positively regulate Glo1 transcription [16].
Author Contributions
▼
Z.K & T.F: performed the experiments, analyzed the data and were involved in data interpretation and writing the manuscript. K.S: was involved in statistical analysis P.T., V.G., K.R., P.R: were involved in planning of experiments & data interpretation S.H. & E.S: was involved in planning of experiments, data interpretation and writing the manuscript. P.P.N: planned and supervised all experiments, was responsible for data interpretation and wrote the manuscript together with T.F.
Acknowledgements
▼
The study was supported by a grant from the Deutsche Forschungsgemeinschaft (BI-1281/3-1 & NA 138 /7-1), the Deutsches Zentrum für Diabetesforschung (DZD; funded by the Federal Ministry of Education and Research (BMBF).) in cooperation with the Institute for Diabetes Research and Metabolic Disease
Kender Z et al. Effect of Metformin on Methylglyoxal Metabolism … Exp Clin Endocrinol Diabetes 2014; 122: 316–319
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Δ methylglyoaxal
(Prof. H-U. Häring, University Hospital Tübingen, Germany), the Dietmar-Hopp-Stiftung (to A. Bierhaus and P.P. Nawroth) and the EFSD Albert Renold Fellowship Programme, supported by an unrestricted educational grant from Merck Sharp & Dohme (to Z. Kender).
Disclosure statement: Part of this work was presented at the 49th Annual meeting of EASD in Barcelona, Spain. Affiliations 2 Department of Medicine, Semmelweis University, Budapest, Hungary 2 Department of Internal Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany 3 Department of Family Medicine, Semmelweis University, Budapest, Hungary 4 Joint Research Division, Molecular Metabolic Control, German Cancer Research Center DKFZ, Network Aging Research, ZMBH, Heidelberg, Germany 5 Division of Clinical Chemistry/Central Laboratory, Department of Internal Medicine, University of Tubingen, Tubingen, Germany 1 nd
References
4 Fleming T, Cuny J, Djuric Z et al. Diabetologia 2012; 55: 1151–1155 5 Beisswenger PJ, Howell SK, Touchette AD et al. Diabetes 1999; 48: 198–202 6 Ruggiero-Lopez D, Lecomte M, Moinet G et al. Biochem Pharmacol 1999; 58: 1765–1773 7 Battah S, Ahmed N, Thornalley PJ. International Congress Series 2002; 1245: 355–356 8 Hofmann MA, Schiekofer S, Isermann B et al. Diabetologia 1999; 42: 222–232 9 Allen RE, Theodore WCL, Thornalley PJ. Eur J Biochem 1993; 213: 1261–1267 10 Horiuchi S, Araki N, Morino Y. J BioChem 1991; 266: 7329–7332 11 Abordo EA, Minhas HS, Thornalley PJ. Biochem Pharmacol 1999; 58: 641–648 12 Correia S, Carvalho C, Santos MS et al. Medicinal chemistry 2008; 4: 358–364 13 Algire C, Moiseeva O, Deschenes-Simard X et al. Cancer prevention research 2012; 5: 536–543 14 Batandier C, Guigas B, Detaille D et al. Journal of bioenergetics and biomembranes 2006; 38: 33–42 15 Alhaider AA, Korashy HM, Sayed-Ahmed MM et al. Chem Biol Interact 2011; 192: 233–42 16 Xue M, Rabbani N, Momiji H et al. Biochem J 2012; 443: 213–222
1 Viollet B, Guigas B, Sanz Garcia N et al. Clinical science 2012; 122: 253–270 2 UK Prospective Diabetes Study (UKPDS) Group. Lancet 1998; 352: 854–865 3 Thornalley PJ. Drug Metabol Drug Interact 2008; 23: 125–150
Kender Z et al. Effect of Metformin on Methylglyoxal Metabolism … Exp Clin Endocrinol Diabetes 2014; 122: 316–319
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Short Communication
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Authors
Z. Kender1*, T. Fleming2*, S. Kopf2, P. Torzsa3, V. Grolmusz1, S. Herzig4, E. Schleicher5, K. Rácz1, P. Reismann1, P. P. Nawroth2
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ type 2 diabetes ● ▶ metformin ● ▶ methylglyoxal ● ▶ glyoxalase I ●
Abstract
▼
The effect of metformin on methylglyoxal (MG) metabolism was studied in a prospective nonrandomized 24 weeks trial in patients with type 2 diabetes. Metformin treatment, in addition to life style intervention, significantly reduced morning glucose and HbA1c whilst body weight and BMI were only marginally reduced during the 24 week trial. Treatment significantly reduced both plasma MG and carboxymethyl-lysine (CML), a marker of oxidative stress. The reduction in MG
Introduction
▼ received 24.11.2013 first decision 24.11.2013 accepted 28.02.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1371818 Published online: April 7, 2014 Exp Clin Endocrinol Diabetes 2014; 122: 316–319 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence Dr. T. Fleming Medizinische Klinik I und klinische Chemie Universität Heidelberg Im Neuenheimer Feld 410 F02 Room 02.414-02.434 69120 Heidelberg Tel.: + 49/6221/5638 490 Fax: + 49/6221/566 934 Thomas.Fleming@med. uni-heidelberg.de
Metformin, due to its cardio-vascular protective effects, has become one of the most widely prescribed oral glucose-lowering agents for the treatment of type 2 diabetes. It is a disubstituted biguanide and although the exact mechanism of action is not completely understood, its main blood glucose-lowering activity is believed to be primarily through suppression of hepatic glucose output [1]. The results of large-scale clinical investigations have also provided evidence that metformin has preventive effects on diabetic complications [2]. One such reported effect is as a scavenger of reactive carbonyl species, such as the dicarbonyl, methylglyoxal (MG). MG is formed exclusively from the spontaneous degradation of trioses phosphates (glyceraldehyde-3-phosphate and dihydroxyacetonephosphate) [3]. It has been shown for both type 1 and type 2 diabetic patients, that they are more susceptible to the accumulation of trioses phosphates and MG [4]. Once formed, MG can react with proteins, specifically lysine, arginine and cysteine residues to form advanced glycation end products (AGEs) [3].
was paralleled by a significant increase in the activity of Glyoxalase 1 (Glo1), the major route of MG detoxification, in peripheral blood mononuclear cells and red blood cells. Multivariate analysis showed that the changes in MG were dependent upon the metformin treatment. This study supports previous findings that metformin can reduce plasma MG in type 2 diabetic patients. However, given the observed increase in Glo1 activity, this reduction is due not only to the scavenging properties of metformin, but the restoration of Glo1 activity.
It has been shown that metformin treatment ( > 1 g per day) was able to reduce systemic plasma MG levels in patients with type 2 diabetes by 16 % [5]. It was suggested that the mechanism for the lowering of plasma MG was related to the scavenging properties of metformin. It has been previously demonstrated that metformin, through the guanidine group, can bind to MG to form hydroimidazolone, triazepinone and other adducts [6, 7]. Compared to aminoguanidine, a structurally similar scavenger of MG, it has a significantly lower rate constant; the rate constant for metformin and MG is 0.034 M − 1min − 1 compared to 155 M − 1min − 1 for aminoguanidine [7]. This would suggest that whilst metformin can bind MG, it is not the primary means by which it reduces systemic MG. It was also shown that in addition to the lowering of plasma MG, the levels of D-lactate the stable endproduct of MG detoxification by the Glyoxalase system [3], increased. This suggests that an alternative mechanism for the reduction of MG by metformin is through enhancement of detoxification. In this study, we investigate the effect of metformin treatment on MG metabolism in a prospective non-randomized 24 week trial in patients with type 2 diabetes, as to establish
* Both authors contributed equally to the work.
Kender Z et al. Effect of Metformin on Methylglyoxal Metabolism … Exp Clin Endocrinol Diabetes 2014; 122: 316–319
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Effect of Metformin on Methylglyoxal Metabolism in Patients with Type 2 Diabetes
Short Communication
317
Table 1 Patient Characteristics at baseline (0 week) and at end of trail (24 weeks). Data represents mean ± SD; Students t-test was used for comparison. Analysis were done unadjusted and adjusted (*) for age and gender. 51.3 ± 10.3 7/5 Baseline (n = 12) Body-Mass-Index Fasting glucose (mmol/L) HbA1c ( %) MG (nM) CML (ng/ml) Glo1 pBMC (mU/mg) Glo1 RBC (mU/mg) Glo2 pBMC (mU/mg) Glo2 RBC (mU/mg) Total TPI RBC
35.4 ± 6.2 7.3 ± 2.0 7.1 ± 0.92 653.3 ± 155.3 283.4 ± 63.5 0.51 ± 0.25 0.26 ± 0.18 8.01 ± 3.6 0.17 ± 0.13 102.3 ± 60.9
Unadjusted 24 weeks (n = 11) 32.9 ± 6.3 6.3 ± 2.0 6.3 ± 0.6 444.5 ± 172.3 206.3 ± 70.5 1.02 ± 0.44 0.36 ± 0.17 6.70 ± 3.3 0.19 ± 0.11 106.7 ± 70.3
whether changes in the glyoxalase system underlie the MG reducing capacity of metformin.
Adjusted
T
p
3.4 2.2 3.6 3.8 2.4 − 4.1 − 1.4 1.4 − 0.3 0.3
< 0.01 < 0.05 < 0.01 < 0.01 < 0.05 < 0.01 n.s. n.s. n.s. n.s.
T* 1.7 2.0 3.6 4.2 2.9 − 6.4 − 1.4 – – –
p* 0.11 0.08 < 0.01 < 0.01 < 0.05 < 0.001 0.19 – – –
Determination of MG The concentration of MG in deproteinized plasma samples was measured by HPLC after a derivatisation with 1,2-diamino-4,5dimethoxybenzene, as previously described [4].
Material & Methods
▼
Determination trioses phosphates intermediates
Participants 12 patients with type 2 diabetes mellitus were recruited from the 2nd Department of Medicine, Semmelweis University, Buda▶ Table 1. Inclusion cripest. Patient characteristics are given in ● teria were newly diagnosed type 2 diabetes, no previous anti-diabetic treatment, age between 20–80 years, no pregnancy, no malignant disease and no alcohol or drug abuse. Subjects were treated with a high dose metformin (1 000–2 000 mg/ day) for 24 weeks. The starting dose was 500 mg of metformin twice a day and then increased to 1 000 mg 2 times a day. Lifestyle intervention was performed monthly by a dietetic counselor in a combination of group and individual sessions. The patients were given specific caloric consumption and exercise goals. The study protocol was approved by the Hungarian Ethic Committee and all patients gave written informed consent.
The concentration of trioses phosphate intermediates (Glyceraldehyde-3-phosphate, dihydroxyacetonephosphate and fructose1,6-bisphosphate), in deproteinized haemolysate samples was determined, as previously described [4].
Determination of plasma Nε-(Carboxymethyl)-Lysine (CML)-modified protein concentration The measurement of CML-modified protein concentration in plasma samples was performed by direct ELISA as previously described [10] using an antibody against CML (Biologo, Kronshagen, Germany).
Data analysis
Sample processing
Comparison of baseline variables with 24-weeks follow-up variables were performed unadjusted and adjusted (age and gender) with paired t-test. Changes of variables were calculated by 24-weeks values minus baseline values (∆-variable). For prediction of ∆MG linear regression analysis were performed stepwise forward and backward with following independent variables: age, gender, ∆BMI, ∆fasting glucose, ∆Glo-1 activity in RBC and pBMCs. The differences were considered to be significant when p < 0.05. Statistical analyses were performed using IBM SPSS Statistics 19 (Armonk, USA).
For the determination of metabolites in RBCs, samples were processed as previously described [4]. Cytoplasmic extracts from the pBMCs were prepared as previously described [8].
Results
Isolation of human red blood cells (RBC) & peripheral blood mononuclear cells (pBMCs) Blood was sampled in the fasting state. RBCs and pBMC were isolated from venipuncture EDTA anticoagulant, processed within and 60 min after sampling, as previously described [4, 8].
Assay of Glyoxalase 1 (Glo1) and Glyoxalase 2 (Glo2) activity The activity of Glo1 determined using the spectrometric method, which monitors the initial rate of change in absorbance at 240 nm caused by the formation of S-D-lactoylglutathione [4]. The activity of Glo2 was also determined spectrophotometrically, by measuring the initial rate of decrease in absorbance at 235 nm over 10 min, as S-D-lactoylglutathione is hydrolyzed to D-lactate and water [9].
▼
Effect of metformin on metabolic parameters In the unadjusted analyses, metformin treatment significantly reduced plasma levels of glucose (7.3 ± 2.0 vs. 6.3 ± 2.0 mmol/l; p = 0.04), HbA1c (7.1 ± 0.92 vs. 6.3 ± 0.6 %; p < 0.01) and BMI (35.4 ± 6.2 vs. 32.9 ± 6.3 kg/m²; p < 0.01). In the adjusted analyses for age and gender, HbA1c reduction was significant (p < 0.01), while reduction of fasting glucose (p = 0.08) and BMI (p = 0.11) ▶ Table 1). were not significant (●
Effect of metformin on trioses phosphate intermediates Metformin treatment had no effect on the levels of trioses phos▶ Table 1). No correlation was observed phate intermediates (● Kender Z et al. Effect of Metformin on Methylglyoxal Metabolism … Exp Clin Endocrinol Diabetes 2014; 122: 316–319
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Age (years) Female/male
318 Short Communication
n = 11 − 195.3 ± 172.1
Independent variables Age Gender Δ BMI Δ fasting glucose Δ Glo1 pBMC Δ Glo1 RBC
51.3 ± 10.3 7/5 − 2.6 ± 2.8 − 1.0 ± 1.6 0.55 ± 0.44 0.10 ± 0.23
Univariable r
p
0.06 0.30 − 0.01 0.34 − 0.70 0.11
0.44 0.19 0.49 0.15 0.08 0.38
between trioses phosphates intermediates, plasma glucose, HbA1c or MG levels.
Effect of metformin on MG, glyoxalase activity and CML Metformin treatment significantly reduced plasma MG levels (653.3 ± 155.3 vs. 444.5 ± 172.3 nM; p < 0.01) as well as plasma levels of CML (283.4 ± 63.5 vs. 206.3 ± 70.5 ng/ml; p = 0.016, ▶ Table 1). The reduction in plasma MG and CML was paralleled ● by an increase in the activity of Glo1 in both the pBMCs (0.51 ± 0.25 vs. 1.02 ± 0.44) and RBCs (0.26 ± 0.18 vs. 0.36 ± 0.17). No significant differences were found in Glo2 activity in either ▶ Table 1). At baseline, it was found that the pBMCs or RBCs (● Glo1 activity significantly correlated with plasma MG in a negative relationship (r = − 0.431; P = 0.0003). Patients at the start of the trial could therefore be characterized as having low pBMC Glo1 activity and high plasma MG whereas at the end of the trial they had high activity and low plasma MG levels. Multivariable regression analysis demonstrated that the observed reduction in plasma MG was dependent only on changes in Glo-1 activity in ▶ Table 2). pBMC and RBC only (●
Discussion
▼
In this study, it has been shown that high dose metformin treatment, in addition to life style intervention, is an effective treatment for the reduction of blood glucose and HbA1c in type 2 diabetic patients. Furthermore, it has been shown that metformin treatment is associated with a reduction of plasma MG. It has previously been suggested that this reduction is due to the scavenging properties of metformin [5], however, given its poor scavenging properties it is likely that the observed increase in Glo1 activity, both in the RBCs and pBMCs, is responsible for the observed reduction in plasma MG. However, the mechanism by which metformin increases the activity of Glo1 and whether this affect is systemic, particularly given that glyoxalase system is ubiquitous in tissues, remains unclear. Increased oxidative stress is considered to be a hallmark of the diabetic state. This can lead to the reduction of glutathione (GSH). Glo1 is a glutathione-dependent enzyme and previous studies have shown that increased production of reactive oxygen species (ROS) can result in the depletion of GSH and NADPH, which can in turn decrease in the in situ activity of Glo1 [11], and thereby increase the concentration of MG. It has previously been shown that metformin can improve the GSSH/GSH balance in diabetes [12] and prevent the accumulation of ROS and ROS induced compounds such as malondialdehyde [13, 14]. In this study, significant reduction in the level of CML-modified proteins, a marker for oxidative stress was observed. This would suggest that the anti-oxidant capacity of metformin may enhance the increases in Glo1 activity by ensuring that sufficient
Multivariable beta − 0.15 0.17 − 0.08 0.29 − 425.3 − 451.7
T
p‡
− 0.6 0.8 − 0.4 1.7 − 4.3 − 2.4
n.s. n.s. n.s. 0.14 < 0.01 < 0.05
Table 2 Multivariable linear Regression analysis for prediction of the changes of methylglyoxal. Data represents mean ± SD; Stepwise forward and backward regression model was used (R2 = 0.70).
GSH is available for detoxification of MG. However, as the activity measurements in this study were performed under saturated substrate conditions, the relative differences observed in activity would be due to an increase in the level of the protein or specific activity. It is interesting to note that they was no change in plasma D-lactate levels (Data not shown), the stable endproduct of glyoxalase metabolism. It would have been expected that an increase in Glo1 activity would lead to increased D-lactate, however, the production of D-lactate is dependent upon Glo2 activity and no affect on activity was observed following treatment with metformin. It has been shown that at the transcriptional level, metformin was able to restore diabetes associated decreases in key antioxidant defense enzymes, such as Glutathione-S-Transferase, NAD(P)H quinone oxidoreductase and catalase, whereas proinflammatory genes such as TNFα and IL-6 were inhibited [15]. It is currently unknown whether this restoration arises indirectly from the anti-oxidant capacities of metformin reducing the levels of oxidative stress sufficiently to allow for the recovery of the cell’s defences systems, or more directly, through interaction and activation of signalling pathways and transcription factors. Further in vitro studies are required to establish the underlying molecular mechanisms by which metformin has a positive effect in diabetes, particularly with respect to the activation of redox-sensitive transcription factors such AP1, NFκB and Nrf2; that latter of which has been shown to positively regulate Glo1 transcription [16].
Author Contributions
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Z.K & T.F: performed the experiments, analyzed the data and were involved in data interpretation and writing the manuscript. K.S: was involved in statistical analysis P.T., V.G., K.R., P.R: were involved in planning of experiments & data interpretation S.H. & E.S: was involved in planning of experiments, data interpretation and writing the manuscript. P.P.N: planned and supervised all experiments, was responsible for data interpretation and wrote the manuscript together with T.F.
Acknowledgements
▼
The study was supported by a grant from the Deutsche Forschungsgemeinschaft (BI-1281/3-1 & NA 138 /7-1), the Deutsches Zentrum für Diabetesforschung (DZD; funded by the Federal Ministry of Education and Research (BMBF).) in cooperation with the Institute for Diabetes Research and Metabolic Disease
Kender Z et al. Effect of Metformin on Methylglyoxal Metabolism … Exp Clin Endocrinol Diabetes 2014; 122: 316–319
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Δ methylglyoaxal
(Prof. H-U. Häring, University Hospital Tübingen, Germany), the Dietmar-Hopp-Stiftung (to A. Bierhaus and P.P. Nawroth) and the EFSD Albert Renold Fellowship Programme, supported by an unrestricted educational grant from Merck Sharp & Dohme (to Z. Kender).
Disclosure statement: Part of this work was presented at the 49th Annual meeting of EASD in Barcelona, Spain. Affiliations 2 Department of Medicine, Semmelweis University, Budapest, Hungary 2 Department of Internal Medicine I and Clinical Chemistry, University of Heidelberg, Heidelberg, Germany 3 Department of Family Medicine, Semmelweis University, Budapest, Hungary 4 Joint Research Division, Molecular Metabolic Control, German Cancer Research Center DKFZ, Network Aging Research, ZMBH, Heidelberg, Germany 5 Division of Clinical Chemistry/Central Laboratory, Department of Internal Medicine, University of Tubingen, Tubingen, Germany 1 nd
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