Glycation and the Maillard Reaction In Vitro: Implications for Diabetes Mellitus

A colorimetric microassay for protein glycation based on the periodate method has been developed. This microassay has improved sensitivity, speed and yield of chromophore enabling it to be used on 0.1 mg aliquots of intact protein. However this assay is unsuitable for collagen and so a second microassay based on the TBA method was developed. This TBA microassay has an improved yield of HMF, is less cumbersome, requires less protein than earlier versions, and is suitable as a rapid assay for glycated collagen. Fructation induces AGE formation considerably faster than glucation in vitro probably because of the highly reactive fructose-derived aldehydic AP. However glycation assays designed for the glucose-AP (a ketose) were found to underestimate the true extent of fructation. For this reason a microassay based on the DNPH method was developed to quantify fructation. Comparative rates of protein fluorescence generated by different sugars were identical to their comparative rates of SB formation but not crosslinking. Using model proteins and different sugars, this study has revealed that fluorescent-AGE and crosslinked-AGE may be formed by independent pathways ie some proteins are more susceptible to crosslinked-AGE and vice versa. Phosphate, pH and calcium have been found to increase glucose and fructose-derived fluorescent-AGE in BSA. Contrary to current dogma, no evidence for an accelerated rise in AGE levels in reincubated proteins after removal of free sugars was found. Only a small increase in fluorescence of reincubated glucated-BSA but not fructated-BSA was detectable. These studies suggest that free sugars have a significant role in fluorescence generation and indeed using chemically modified proteins, evidence has been presented for a reaction between the AP and free sugars to form fluorescent products. This secondary glycation is more pronounced for fructose and explains in part the higher fluorescence generated by fructated proteins. Comparative studies between inhibitors with different modes of action have revealed that aminoguanidine is the most effective at reducing fluorescence and crosslinking of proteins in vitro on a molar basis. This is probably because the compound has multiple sites of action and more than one amino group. Phenylenediamine reacts with 3-DG preventing formation of AGE and was found to have a greater inhibitory effect on fructose compared to glucose-derived AGE. In this study, no evidence was found for sugar-induced protein fragmentation in vitro unless transition metals were included. Furthermore binding of the transition metal to protein appears to be important for significant fragmentation to occur and in vivo this autoxidative glycation may be restricted to the limited number of copper binding proteins only

Cheiradone: a vascular endothelial cell growth factor receptor antagonist

<p>Abstract</p> <p>Background</p> <p>Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing) and pathological conditions (tumour development). Vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) are the major angiogenic regulators. We have identified a natural product (cheiradone) isolated from a <it>Euphorbia </it>species which inhibited <it>in vivo </it>and <it>in vitro </it>VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in <it>in vitro </it>(proliferation, wound healing, invasion in Matrigel and tube formation) and <it>in vivo </it>(the chick chorioallantoic membrane) models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC₅₀) was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated.</p> <p>Results</p> <p>Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC₅₀values in the range 5.20–7.50 μM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC₅₀values of 2.9 and 0.61 μM respectively.</p> <p>Conclusion</p> <p>Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.</p

Effect of puerarin on glutamine synthetase activity in rat retina following acute intraocular hypertension

This study was conducted to demonstrate whether puerarin regulates glutamine synthetase (GS) activity following intraocular hypertension and has therapeutic potential in ophthalmology for the protection of optic nerves in patients with glaucoma. This study used a Wistar rat model of acute closed-angle glaucoma to investigate the effect of puerarin on GS activity in rat retina following intraocular hypertension. Acute intraocular hypertension was induced by increasing anterior chamber pressure to 110 mmHg for 30 min in the left eyes of 50 Wistar rats, while 5 additional Wistar rats lacking intraocular hypertension were used as a control group. Retinal GS activity was measured at 4, 12, 24, 36 and 72 h after induction of acute intraocular hypertension with/without puerarin treatment. Compared to the control group that lacked intraocular hypertension, GS activity in the intraocular hypertension group significantly decreased at 4 and 12 h (P<0.01), before increasing at 24 to 36 h and restoring to a level similar to the control group at 72 h. However, puerarin significantly (P<0.05) prevented the loss of GS activity seen in the intraocular hypertension group at 4 and 12 h, with no significant (P<0.05) difference in GS activity noted between the control group and rats treated with puerarin at these early time points. GS activity significantly (P<0.05) increased above control values at 24 and 36 h in the puerarin-treated group before eventually restoring to control levels at 72 h. These findings suggest puerarin protects GS activity in the early stages of retinal acute intraocular hypertension and may be of potential therapeutic benefit in acute closed-angle glaucoma

Momordica charantia extracts protect against inhibition of endothelial angiogenesis by advanced glycation endproducts in vitro.

Diabetes mellitus characterized by hyperglycemia favors formation of advanced glycation endproducts (AGEs) capable of triggering vascular complications by interfering with imbalanced inflammation and angiogenesis to eventually impede wound-healing. Momordica charantia (MC, bitter melon) has been shown to prevent AGE formation and to promote angiogenesis in diabetic wounds in animal models. However, the mechanism underlying its effects on angiogenesis is unclear. We investigated the effects of methanolic extracts of MC pulp (MCP), flesh (MCF) and charantin (active component of MC) using an in vitro model of angiogenesis. MC extracts or low concentrations of bovine serum albumin-derived AGEs (BSA-AGEs) stimulated proliferation, migration (using wound-healing assay) and tube formation (using Matrigel™-embedded 3D culture) of bovine aortic endothelial cells (BAEC) together with increases in the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, the key angiogenic signaling cytoplasmic protein. Blocking the receptor for AGEs (RAGE) inhibited low BSA-AGE- and MC extract-induced ERK1/2 phosphorylation and tube formation, indicating the crucial role of RAGE in the pro-angiogenic effects of MC extracts. Moreover, inhibitory effects of high BSA-AGE concentration on cell proliferation and migration were reduced by the addition of MC extracts, which reversed the BSA-AGE anti-angiogenic effect on tube formation. Thus, MC extracts exert direct pro-angiogenic signaling mediated via RAGE to overcome the anti-angiogenic effects of high BSA-AGEs, highlighting the biphasic RAGE-dependent mechanisms involved. This study enhances our understanding of the mechanisms underlying the pro-angiogenic effects of MC extracts in improvement of diabetes-impaired wound-healing

Biophysical, Biochemical, and Molecular Docking Investigations of Anti-Glycating, Antioxidant, and Protein Structural Stability Potential of Garlic.

Garlic has been reported to inhibit protein glycation, a process that underlies several disease processes, including chronic complications of diabetes mellitus. Biophysical, biochemical, and molecular docking investigations were conducted to assess anti-glycating, antioxidant, and protein structural protection activities of garlic. Results from spectral (UV and fluorescence) and circular dichroism (CD) analysis helped ascertain protein conformation and secondary structure protection against glycation to a significant extent. Further, garlic showed heat-induced protein denaturation inhibition activity (52.17%). It also inhibited glycation, advanced glycation end products (AGEs) formation as well as lent human serum albumin (HSA) protein structural stability, as revealed by reduction in browning intensity (65.23%), decrease in protein aggregation index (67.77%), and overall reduction in cross amyloid structure formation (33.26%) compared with positive controls (100%). The significant antioxidant nature of garlic was revealed by FRAP assay (58.23%) and DPPH assay (66.18%). Using molecular docking analysis, some of the important garlic metabolites were investigated for their interactions with the HSA molecule. Molecular docking analysis showed quercetin, a phenolic compound present in garlic, appears to be the most promising inhibitor of glucose interaction with the HSA molecule. Our findings show that garlic can prevent oxidative stress and glycation-induced biomolecular damage and that it can potentially be used in the treatment of several health conditions, including diabetes and other inflammatory diseases

Antiglycation and antioxidant properties of Momordica charantia

The accumulation of advanced glycation endproducts (AGEs) and oxidative stress underlie the pathogenesis of diabetic complications. In many developing countries, diabetes treatment is unaffordable, and plants such as bitter gourd (or bitter melon; Momordica charantia) are used as traditional remedies because they exhibit hypoglycaemic properties. This study compared the antiglycation and antioxidant properties of aqueous extracts of M. charantia pulp (MCP), flesh (MCF) and charantin in vitro. Lysozyme was mixed with methylglyoxal and 0–15 mg/ml of M. charantia extracts in a pH 7.4 buffer and incubated at 37°C for 3 days. Crosslinked AGEs were assessed using gel electrophoresis, and the carboxymethyllysine (CML) content was analyzed by enzyme-linked immunosorbent assays. The antioxidant activities of the extracts were evaluated using assays to assess DPPH (1,1-diphenyl-2-picryl-hydrazyl) and hydroxyl radical scavenging activities, metal-chelating activity and reducing power of the extracts. The phenolic, flavonol and flavonoid content of the extracts were also determined. All extracts inhibited the formation of crosslinked AGEs and CML in a dose-dependent manner, with MCF being the most potent. The antioxidant activity of MCF was higher than that of MCP, but MCP showed the highest metal-chelating activity. MCF had the highest phenolic and flavonoid contents, whereas MCP had the highest flavonol content. M. charantia has hypoglycaemic effects, but this study shows that M. charantia extracts are also capable of preventing AGE formation in vitro. This activity may be due to the antioxidant properties, particularly the total phenolic content of the extracts. Thus, the use of M. charantia deserves more attention, as it may not only reduce hyperglycaemia but also protect against the build-up of tissue AGEs and reduce oxidative stress in patients with diabetes

Aged garlic extract and S-allyl cysteine prevent formation of advanced glycation endproducts

Hyperglycaemia causes increased protein glycation and the formation of advanced glycation endproducts which underlie the complications of diabetes and ageing. Glycation is accompanied by metal-catalysed oxidation of glucose and Amadori products to form free radicals capable of protein fragmentation. Aged garlic extract is a potent antioxidant with established lipid-lowering effects attributed largely to a key ingredient called S-allyl cysteine. This study investigated the ability of aged garlic extract and S-allyl cysteine to inhibit advanced glycation in vitro. Bovine serum albumin (BSA) was glycated in the presence of Cu2+ ions and different concentrations of aged garlic extract and protein fragmentation was examined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Lysozyme was glycated by glucose or methylglyoxal in the presence of different concentrations of aged garlic extract or S-allyl cysteine with subsequent analysis of glycation-derived crosslinking using SDS-PAGE. Amadori-rich protein was prepared by dialysing lysozyme that had been glycated by ribose for 24 h. This ribated lysozyme was reincubated and the effects of aged garlic extract, S-allyl cysteine and pyridoxamine on glycation-induced crosslinking was monitored. Aged garlic extract inhibited metal-catalysed protein fragmentation. Both aged garlic extract and S-allyl cysteine inhibited formation of glucose and methylglyoxal derived advanced glycation endproducts and showed potent Amadorin activity when compared to pyridoxamine. S-allyl cysteine inhibited formation of carboxymethyllysine (CML), a non-crosslinked advanced glycation endproduct derived from oxidative processes. Further studies are required to assess whether aged garlic extract and S-allyl cysteine can protect against the harmful effects of glycation and free radicals in diabetes and ageing

Effects of different concentrations of MCF (A), MCP (B) and charantin (C) on the formation of crosslinked AGEs.

<p>A representative SDS-PAGE gel showing lysozyme (10 mg/ml) incubated alone (lane 1) or in the presence of 0.1 M methylglyoxal (lane 2) in 0.1 M sodium phosphate buffer of pH 7.4 for 3 days at 37°C. The dimer formation resulting from protein crosslinking was determined using different concentrations of the MCF (A₁) and MCP (B₁) extracts or charantin (C₁): 5 mg/ml (lane 3), 10 mg/ml (lane 4) and 15 mg/ml (lane 5). Lane 0 contains the marker proteins. The bar charts show the effects of different concentrations of MCF (A₂), MCP (B₂) and charantin (C₂) on the formation of crosslinked AGEs relative to the control. The results are presented as means ± SDs (n = 3). *: <i>p</i> < 0.05, **: <i>p</i> < 0.01, ***: <i>p</i> < 0.001 vs control.</p