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* Department of Pharmacology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; and
National Research Council of Canada, Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada
1Correspondence: Department of Pharmacology, College of Medicine, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, SK S7N-5E5, Canada. E-mail: liw070{at}duke.usask.ca
SPECIFIC AIMS
Although previous studies indicated increased methylglyoxal (MG) levels in different insulin resistance states, no direct evidence for a link between MG and dysfunction of insulin has been found. This study aimed to investigate whether MG causes structural modification on insulin molecule and whether the modification changes the biological function of insulin.
PRINCIPAL FINDINGS
1. MG induced mass changes of insulin
To determine whether MG can induce a mass change on insulin molecules, human insulin was analyzed by MALDI-TOF MS after incubating with MG. Incubation of 1 µg/µl insulin with 10 µM MG for 3 d resulted in additional peaks in the MS spectrum that provided clear evidence for the formation of MG-insulin adducts.
2. Amino acid target(s) for MG modification of insulin
To determine the position(s) of MG attachment, native insulin and MG-insulin were separated permanently into A- and B-chains and then analyzed by MALDI-T of MS. The mass spectrum of reduced insulin alone contained major peaks at m/z 2611 and 3543 (Fig. 1
A), corresponding to the reduced and alkylated A- and B-chains, respectively. Incubation with MG produced additional peaks that showed progressive enlargement of the B-chain with increasing MG concentrations (Fig. 1B-D
).
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The most prominent B-chain adducts that appeared at MG concentrations above 15 µM corresponded to a mass increase of 54 dalton (Da) (m/z 3597). MS/MS analysis of the corresponding 4+ ion (m/z 900.2) generated by nanoES produced a spectrum in which all the observed b-ions were shifted by +54 Da, relative to the corresponding spectrum for the unmodified insulin B-chain (m/z 886.7). In contrast, all the observed y-ions remained unchanged, a result consistent with attachment of MG (as R–N=CH–C(CH3)O) to the N terminus of the insulin B-chain. MS/MS analysis of the +108 Da adduct (m/z 913.7) also showed a b-ion shift of +54 Da. However, a shift of +54 Da was also observed in the larger observable y-ions, whereas the smaller observable y-ions remained unchanged. The intervening region contained arginine as the only basic residue, the side chain of which is known to increase by 54 Da on reaction with MG. These observations indicated that MG adduction occurred both at the N terminus of the insulin B-chain and at an internal, arginine residue.
3. MG-insulin impaired normal glucose uptake by different insulin sensitive cells
[3H]-2-DOG uptake by 3T3-L1 adipocytes was determined after the cells were treated with insulin (1, 10, and 100 nM) or MG-insulin, which was generated by incubating insulin at the same concentrations as for native insulin with 1, 10, or 100 µM MG, respectively. As shown in Fig. 2
A, 3T3-L1 cells showed a significantly increase of glucose uptake induced by insulin in a concentration-dependent manner. However, a significant lower uptake of glucose was observed after the cells were treated with MG-insulin, compared with the cells treated with concentration-matched native insulin. Similarly, lower glucose uptake induced by MG-insulin was also observed in L8 skeletal muscle cells (Fig. 2B
).
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To determine whether MG directly interfered with glucose insulin-induced glucose uptake rather than via formation of MG-insulin, we cotreated 3T3-L1 cells with MG (3–300 µM) and native insulin (100 nM) for 30 min before measuring glucose uptake. No difference was observed between the co-treated groups and the insulin treated group. In one group of experiments, 3T3-L1 cells were pretreated with MG (1 or 10 µM) for 48 h and then exposed to insulin (100 nM). Insulin-induced glucose uptake was not affected by MG. There was also a similar increase in insulin-stimulated glucose uptake by 3T3-L1 cells with or without 24 h pretreatment of MG (3–30 µM).
The effect of MG on the expression of insulin receptor at mRNA concentration was also explored in 3T3-L1 cells. After 3T3-L1 cells were treated with MG (3 or 30 µM) for 24 h, mRNA expression concentration (
Ct) of insulin receptor was 12.3 ± 0.2 or 12.1 ± 0.15, which was not significantly different from that of MG-untreated group.
4. Effects of MG-insulin on C-peptide secretion from INS-1E cells
Whether MG-insulin impairs the insulin-induced feedback inhibition on insulin release from pancreatic ßbeta;-cells was investigated. When INS-1E cells were coexposed to native insulin (100 nM) and glucose (16.7 mM) for 2 h, the C-peptide release dropped to 76.5% of the C-peptide released from cells treated only with glucose at the same concentration. When INS-1E cells were cotreated with MG-insulin and glucose, the insulin-induced inhibition of C-peptide secretion disappeared, in comparison with that from cells treated with native insulin and glucose. Without the formation of MG-insulin adducts, a mere coapplication of MG and insulin did not alter the insulin-induced inhibition of C-peptide release.
5. Decreased degradation of MG-insulin through hepatocytes
As the main site for insulin clearance, liver removes 
50% of the circulating insulin through a receptor-mediated endocytosis process. To investigate whether MG-insulin was still removed by hepatocytes properly, we treated H4-II-E cells with insulin (200 nM) or the same concentration of MG-insulin for 15 min and then detected the insulin clearance in culture medium. We found a significantly higher insulin clearance rate (51.5%±5.6) in the cells treated with native insulin, compared with that from cells treated with MG-insulin (32.0%±3.6).
CONCLUSION AND SIGNIFICANCE
An increase in plasma MG concentration and MG-induced irreversible advanced glycated endproducts (AGEs) have been observed in different insulin resistant status, including diabetes and hypertension. In our study, mass spectrometry provided strong evidence for the formation of MG-insulin adducts. The peak at m/z 5880 corresponded to the addition of one MG molecule (72 Da) to insulin, while the peak at m/z 5934 corresponded to the addition of two MG molecules with concomitant loss of a single water molecule (72+54 Da). Previous studies reported that MG derivatized lysine or arginine residues of human serum albumin in vitro. These findings are consistent with our observations. For example, addition of MG to lysine forms the monolysyl adduct CEL, resulting in a mass increase of 72 Da. In contrast, MG undergoes a condensation reaction with arginine to form one of three hydroimidazolone isomers (MG-H1, H2, and H3), resulting in a net mass increase of 54 Da. However, other peaks observed during MS analysis suggested that multiple additions of MG occur both with and without concomitant loss of water, despite the fact that insulin contains a single lysine and arginine residue. MG adductions at both the N terminus of the insulin B-chain and at internal arginine residue were subsequently confirmed by tandem MS analysis of insulin B-chain adducts.
Our study showed that MG-insulin adducts induced a significant and concentration-dependent decrease in glucose uptake in insulin-sensitive adipocytes and skeletal muscle cells compared with intact insulin. Neither the basal nor the insulin-stimulated glucose uptake was changed significantly by this MG pretreatment. An unchanged transcriptional expression of insulin receptor was observed after the cells were treated with or without MG. It is, therefore, unlikely that the lower glucose uptake observed was resulted by the unattached MG in the solution. It was the formation of MG-insulin adducts that reduced the capability of native insulin to stimulate glucose uptake.
The autocrine control of insulin release by the extracellular insulin concentration has attracted great attention since the discovery of insulin receptors and insulin receptor substrates in pancreatic ßbeta;-cells. Our results suggest that the formation of MG-insulin could no longer inhibit insulin (C-peptide) secretion. In addition, a significant lower MG-insulin clearance rate was also observed through hepatocytes, which indicated that MG-insulin adducts could not go through endocytosis properly, or there was an aggregation of MG-insulin.
In summary, our results showed that MG modifies the B-chain of human insulin in vitro and that modification occurred predominantly at the N terminus and arginine residue via Schiff base formation. The extent of modification increases with the relative concentration of MG. The formation of MG-insulin adducts led to the reduction of insulin-mediated glucose uptake by its target cells or tissues, impaired autocrine control of insulin release from pancreatic ßbeta;-cells, and decreased hepatic clearance of insulin from liver cells. Therefore, a chronic increase in the circulating MG concentration, with enhanced formation of MG-insulin adducts, might play an important role in the development of insulin resistance. Hence, clarification of the role of MG in the development of insulin resistance may lead to a discovery of new mechanisms and methods for the management and prevention of insulin-resistance syndrome, including diabetes and hypertension.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5478fje
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  A.-V. Cantero, M. Portero-Otin, V. Ayala, N. Auge, M. Sanson, M. Elbaz, J.-C. Thiers, R. Pamplona, R. Salvayre, and A. Negre-Salvayre Methylglyoxal induces advanced glycation end product (AGEs) formation and dysfunction of PDGF receptor-{beta}: implications for diabetic atherosclerosis FASEB J, October 1, 2007; 21(12): 3096 - 3106. [Abstract] [Full Text] [PDF]
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