Ferulic acid prevents methylglyoxal-induced protein glycation,DNA damage,and apoptosis in pancreatic β-cells

Methylglyoxal (MG) can react with amino acids of proteins to induce protein glycation and consequently the formation of advanced glycation end-products (AGEs). Previous studies reported that ferulic acid (FA) prevented glucose-, fructose-, and ribose-induced protein glycation. In this study, FA (0.1–1 mM) inhibited MG-induced protein glycation and oxidative protein damage in bovine serum albumin (BSA). Furthermore, FA (0.0125–0.2 mM) protected against lysine/MG-mediated oxidative DNA damage, thereby inhibiting superoxide anion and hydroxyl radical generation during lysine and MG reaction. In addition, FA did not have the ability to trap MG. Finally, FA (0.1 mM) pretreatment attenuated MG-induced decrease in cell viability and prevented MG-induced cell apoptosis in pancreatic β-cells. The results suggest that FA is capable of protecting β-cells from MG-induced cell damage during diabetes.     

Role of the glycolytic protein,glyceraldehyde-3-phosphate dehydrogenase,in normal cell function and in cell pathology

The glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) appeared to be an archtypical protein of limited excitement. However, independent studies from a number of different laboratories reported a variety of diverse biological properties of the GAPDH protein. As a membrane protein, GAPDH functions in endocytosis; in the cytoplasm, it is involved in the translational control of gene expression; in the nucleus, it functions in nuclear tRNA export, in DNA replication, and in DNA repair. The intracellular localization of GAPDH may be dependent on the proliferative state of the cell. Recent studies identified a role for GAPDH in neuronal apoptosis. GAPDH gene expression was specifically increased during programmed neuronal cell death. Transfection of neuronal cells with antisense GAPDH sequences inhibited apoptosis. Lastly, GAPDH may be directly involved in the cellular phenotype of human neurodegenerative disorders, especially those characterized at the molecular level by the expansion of CAG repeats. In this review, the current status of ongoing GAPDH studies are described (with the exception of its unique oxidative modification by nitric oxide). Consideration of future directions are suggested. J. Cell. Biochem. 66:133-140, 1997. © 1997 Wiley-Liss, Inc.     

Effects of thermal denaturation on protein glycation

Protein denaturation occurs at sites of inflammation. We hypothesized that denatured protein may provide a more susceptible target for glycation, which is a known mediator of inflammation. We examined the effects of thermal denaturation on the susceptibility of protein glycation using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and aspartate aminotransferase (AAT) as our target proteins. GAPDH and AAT are ubiquitous proteins that exhibited very different thermal stabilities. Glycating agents, methylglyoxal (MG) and glyceraldehyde (Glyc), caused an increase in the formation of advanced glycation endproducts (AGEs) in native and denatured GAPDH and AAT. The effects of the glycating agents were more pronounced with the denatured proteins. In addition to nitroblue tetrazolium (NBT)- reactivity, our measured endpoints were absorbance (lambda = 365 nm) and fluorescence (lambda(ex) = 370 nm; lambda(em) = 470 nm) properties that are typically associated with protein glycation. We also looked at carnosine's ability to prevent glycation of native and denatured protein. Carnosine, an endogenous histidine dipeptide, exhibits anti-inflammatory activity presumably due to its anti-oxidant and anti-glycation properties. Carnosine prevented Glyc-induced AGE formation in both native and denatured AAT suggesting that carnosine's anti-inflammatory activity may be due in part to carnosine's ability to prevent glycation of denatured protein.     

糖基化3－磷酸甘油醛脱氢酶的分离纯化及糖基代位点的探讨

应用糖基化蛋白亲和层析技术对兔肌及人红细胞的３－磷酸甘油醛脱氢酶的分离分析表明,兔肌非糖基化ＧＡＰＤＨ的比活为１８０—２００单位,而糖基化ｇＧＡＰＤＨ的为４０—５０单位,并占该酶蛋白总量的４０％。人类红细胞糖基化ｇＧＡＰＤＨ的活力占其总活力的５５％左右。以上结果表明：哺乳动物体内存在糖基化３－磷酸甘油醛脱氢酶。由于（１）糖基化明显影响ＧＡＰＤＨ的活力;（２）糖基化酶活性部位的巯基（Ｃｙｓ－１４９）空间位置发生了改变;（３）糖基化影响活性部位的空间构象及（４）ＯＰＴ对糖基化及非糖基化酶的修饰无论在动力学上还是在ＫＩ淬灭时都有明显差异,因此,糖基化的位点可能与赖氨酸残基有关,并且接近或位于酶的活性部位。     

糖基化3－磷酸甘油醛脱氢酶的分离纯化及糖基代位点的探讨

应用糖基化蛋白亲和层析技术对兔肌及人红细胞的３－磷酸甘油醛脱氢酶的分离分析表明,兔肌非糖基化ＧＡＰＤＨ的比活为１８０—２００单位,而糖基化ｇＧＡＰＤＨ的为４０—５０单位,并占该酶蛋白总量的４０％。人类红细胞糖基化ｇＧＡＰＤＨ的活力占其总活力的５５％左右。以上结果表明：哺乳动物体内存在糖基化３－磷酸甘油醛脱氢酶。由于（１）糖基化明显影响ＧＡＰＤＨ的活力;（２）糖基化酶活性部位的巯基（Ｃｙｓ－１４９）空间位置发生了改变;（３）糖基化影响活性部位的空间构象及（４）ＯＰＴ对糖基化及非糖基化酶的修饰无论在动力学上还是在ＫＩ淬灭时都有明显差异,因此,糖基化的位点可能与赖氨酸残基有关,并且接近或位于酶的活性部位。     

Nitric oxide and NAD-dependent protein modification

Nitric oxide (NO) has been suggested to act as a regulator of endogenous intracellular ADP-ribosylation, based on radiolabelling of proteins in tissue homogenates incubated with [³²P]NAD and No. After the NO-stimulated modification was replicated in a defined system containing only the purified acceptor protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the hypothesis of NO-stimulation of an endogenous ADP-ribosyltransferase became moot. The NO-stimulated, NAD-dependent modification of GAPDH was recently characterized as covalent binding of the whole NAD molecule to the enzyme, not ADP-ribosylation. With this result, along with the knowledge that GAPDH is stoichiometrically S-nitrosylated, the role of NO in protein modification with NAD may be viewed as the conferring of an unexpected chemical reactivity upon GAPDH, possibly due to nitrosylation of a cysteine in the enzyme active site.     

糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小,但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明,ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变,糖基化位点可能发生在酶活性部位附近。     

糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小,但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明,ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变,糖基化位点可能发生在酶活性部位附近。     

Glycation of collagen: the basis of its central role in the late complications of ageing and diabetes

The most serious late complications of ageing and diabetes mellitus follow similar patterns in the dysfunction of retinal capillaries, renal tissue, and the cardiovascular system. The changes are accelerated in diabetic patients owing to hyperglycaemia and are the major cause of premature morbidity and mortality. These tissues and their optimal functioning are dependent on the integrity of their supporting framework of collagen. It is the modification of these properties by glycation that results in many of the damaging late complications. Initially glycation affects the interactions of collagen with cells and other matrix components, but the most damaging effects are caused by the formation of glucose-mediated intermolecular cross-links. These cross-links decrease the critical flexibility and permeability of the tissues and reduce turnover. In contrast to the renal and retinal tissue, the cardiovascular system also contains a significant proportion of the other fibrous connective tissue protein elastin, and its properties are similarly modified by glycation. The nature of these glycation cross-links is now being unravelled and this knowledge is crucial in any attempt to inhibit these deleterious glycation reactions.     

Kinetic analysis of glycation as a tool for assessing the half-life of proteins

Glycation of proteins, a common postribosomal modification, proceeds via Amadori rearrangement to yield a stable ketoamine linkage of glucose with the protein. Kinetic analysis of the reaction shows that the amount of glycation at steady state is proportional to the glucose concentration, to protein half-life and to the rate of glycation. Thus, when the rate of glycation is determined in vitro and the extent of glycation of a given protein isolated from euglycemic subjects is measured, the half-life may be calculated. As the in vivo situation may not be simulated accurately in vitro, the calculated values may be considered as approximation. When the calculated values were compared with values reported in the literature fairly good agreement was found except for hemoglobin. Studies on stability of glycated albumin show that ketoamine decreases by about 20% when incubated under physiological conditions for 20 days. The method described by us is especially valuable when turnover of proteins in normal and pathophysiological states are compared. The half-life of plasma low-density lipoprotein is longer in patients with hypothyroidism or a high plasma low-density lipoprotein level than in normal subjects. Extending our studies to tissue proteins we did not find a significant increase in half-life of tendon collagen with age. Basement membrane collagen turnover is faster in diabetic patients in bad metabolic control. Thus, the procedure using fructosylamine as endogenous label of protein offers a method of great potential to study the turnover of human body proteins.     

Dicarbonyl stress in clinical obesity

The glyoxalase system in the cytoplasm of cells provides the primary defence against glycation by methylglyoxal catalysing its metabolism to D-lactate. Methylglyoxal is the precursor of the major quantitative advanced glycation endproducts in physiological systems - arginine-derived hydroimidazolones and deoxyguanosine-derived imidazopurinones. Glyoxalase 1 of the glyoxalase system was linked to anthropometric measurements of obesity in human subjects and to body weight in strains of mice. Recent conference reports described increased weight gain on high fat diet-fed mouse with lifelong deficiency of glyoxalase 1 deficiency, compared to wild-type controls, and decreased weight gain in glyoxalase 1-overexpressing transgenic mice, suggesting a functional role of glyoxalase 1 and dicarbonyl stress in obesity. Increased methylglyoxal, dicarbonyl stress, in white adipose tissue and liver may be a mediator of obesity and insulin resistance and thereby a risk factor for development of type 2 diabetes and non-alcoholic fatty liver disease. Increased methylglyoxal formation from glyceroneogenesis on adipose tissue and liver and decreased glyoxalase 1 activity in obesity likely drives dicarbonyl stress in white adipose tissue increasing the dicarbonyl proteome and related dysfunction. The clinical significance will likely emerge from on-going clinical evaluation of inducers of glyoxalase 1 expression in overweight and obese subjects. Increased transcapillary escape rate of albumin and increased total body interstitial fluid volume in obesity likely makes levels of glycation of plasma protein unreliable indicators of glycation status in obesity as there is a shift of albumin dwell time from plasma to interstitial fluid, which decreases overall glycation for a given glycemic exposure.     

Inhibitory effect of paeoniflorin on methylglyoxal-mediated oxidative stress in osteoblastic MC3T3-E1 cells

PurposeMethylglyoxal (MG) has been suggested to be one major source of intracellular reactive carbonyl compounds. In the present study, the effect of paeoniflorin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells.MethodsOsteoblastic MC3T3-E1 cells were pre-incubated with paeoniflorin before treatment with MG, and markers of oxidative damage and mitochondrial function were examined.ResultsPretreatment of MC3T3-E1 cells with paeoniflorin prevented the MG-induced cell death and formation of intracellular reactive oxygen species, cardiolipin peroxidation, and protein adduct in osteoblastic MC3T3-E1 cells. In addition, paeoniflorin increased glutathione level and restored the activity of glyoxalase I to almost the control level. These findings suggest that paeoniflorin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing MG detoxification system. Pretreatment with paeoniflorin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss. Additionally, the nitric oxide and nuclear respiratory factor 1 levels were significantly increased by paeoniflorin, suggesting that paeoniflorin may induce mitochondrial biogenesis. Paeoniflorin treatment decreased the levels of proinflammatory cytokines such as TNF-α and IL-6.ConclusionsThese findings indicate that paeoniflorin might exert its therapeutic effects via upregulation of glyoxalase system and mitochondrial function. Taken together, paeoniflorin may prove to be an effective treatment for diabeteic osteopathy.     

The assessment of protein glycation in human atherosclerotic plaques by affinity chromatography

SUMMARY

Human atherosclerotic plaques are characterized by a massive deposition of lipid within arterial walls. The lipids accumulated are partly oxidized, as assessed by gas chromatography of lipids and their oxidation products. Both advancing age and diabetes mellitus are associated with an increased prevalence and severity of atherosclerosis.

In diabetes mellitus the development of secondary complications appear to be increased by poor glucose control. Indeed, the post-translational modification of protein by non-enzymatic glycation may provide the link between abnormal glucose control and diabetic complications. For atherosclerosis however, the relationship between glucose control and disease is unclear, with evidence available to support and discount such a link. To study protein glycation in a condition associated with a significant level of lipid oxidation products poses several methodological problems, most of which are associated with interference by lipid-derived aldehydes. Many chemical assays of protein glycation monitor aldehydic products common to the chemistry of both protein glycation and lipid oxidation. Studies of protein glycation in human atheroma, obtained at necropsy, are presented which make use of a commercially available boronic acid affinity-based chromatographic assay of glycated protein. The commercially available affinity-based chromatographic assay of glycated protein appears to be free from such interference and may well prove useful in the study of other conditions in which the non-enzymatic glycation of protein is suspected.     

Aging of human glyceraldehyde-3-phosphate dehydrogenase is dependent on its subcellular localization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), long considered a traditional glycolytic protein, displays multiple activities independent of its role in energy generation. This functional diversity is dependent on its membrane, cytoplasmic or nuclear localization. GAPDH is encoded by one active gene and is synthesized as a single 37 kDa protein without alternate splicing. Accordingly, the identical protein would be present in each subcellular fraction. The accumulation of post-translational errors in protein structure as a function of oxidative stress is thought to provide a basic molecular mechanism for the aging process. Thus, during aging, the GAPDH protein should contain the identical degree of oxidative sequence alteration irrespective of its distribution. This would result in equivalent effects on GAPDH activity. However, conformational differences in GAPDH structure due to its subcellular protein, nucleic acid or membrane interactions could affect its degree of modification thereby selectively affecting its function. For that reason, we examined the subcellular expression and intracellular activity of GAPDH as a function of human aging. Subcellular GAPDH expression was quantitated by immunoblot analysis in fetal and senior human cells (postnuclear, nuclear, perinuclear). GAPDH activity was determined by in vitro assay. We now report that the aging of human GAPDH was subcellular dependent. Reductions of nuclear and postnuclear GAPDH activity in senior cells were twofold lower than that observed for the perinuclear protein. In contrast, the subcellular expression of the GAPDH protein was age-independent. These results suggest the possibility that subcellular interactions may mitigate oxidative stress-induced GAPDH modification in human aging. Such selective effects on GAPDH could affect its functional diversity.     

Effect of (-)-epigallocatechin-3-gallate on glucose-induced human serum albumin glycation

Abstract

(-)-Epigallocatechin-3-gallate (EGCg) is a naturally occurring polyphenol found in plant-based foods and beverages such as green tea. Although EGCg can eliminate carbonyl species produced by glucose autoxidation and thus can inhibit protein glycation, it is also reported to be a pro-oxidant that stimulates protein glycation in vitro. To better understand the balance between antioxidant and pro-oxidant features of EGCg, we evaluated EGCg-mediated bioactivities in a human serum albumin (HSA)/glucose model by varying three different parameters (glucose level, EGCg concentration, and time of exposure to EGCg). Measurements of glycation-induced fluorescence, protein carbonyls, and electrophoretic mobility showed that the level of HSA glycation was positively related to the glucose level over the range 10–100 mM during a 21-day incubation at 37°C and pH: 7.4. Under mild glycemic pressure (10 mM), long exposure to EGCg enhanced HSA glycation, while brief exposure to low concentrations of EGCg did not. Under high glycemic pressure (100 mM glucose), long exposure to EGCg inhibited glycation. For the first time we showed that brief exposure to EGCg reversed glycation-induced fluorescence, indicating a restorative effect. In conclusion, our research identified glucose level, EGCg concentration, and time of exposure as critical factors dictating EGCg bioactivities in HSA glycation. EGCg did not affect HSA glycation under normal physiological conditions but had a potential therapeutic effect on HSA severely damaged by glycation.     

Vasculopathy in type 2 diabetes mellitus: role of specific angiogenic modulators

Type 2 diabetes mellitus (T2DM) is largely defined by hyperglycemia that promotes vascular complications. Abnormal angiogenesis has been claimed to have a role in this disease. This study aimed to investigate serum levels of both conventional and other markers of angiogenesis not well studied before in diabetes, and to correlate findings with age of the patients, glycemic control, presence of microvascular complications, and oxidative stress. Thirty-eight patients with T2DM and 13 age- and sex-matched healthy persons representing controls were recruited. Serum levels of basic fibroblast growth factor (b-FGF) was measured by immunosorbent assay kit; advanced glycosylation end products, platelet-derived endothelial cell growth factor (PD-ECGF), cathepsin-D (CD), gangliosides, hyaluronic acid (HA), nitric oxide (NO), lipid peroxides (LPER), superoxide dismutase, and total thiols by chemical methods; and copper (Cu) by atomic absorption flame photometry. Advanced glycosylation end products and angiogenic factors (b-FGF, PD-ECGF, CD, gangliosides, HA, and Cu) were significantly higher in patients than controls. Oxidative stress markers, NO, and LPER were significantly higher while total thiols were significantly lower in patients than controls. These changes were more pronounced with age, poor glycemic control, and presence of microvascular complications. Angiogenesis dysfunction coinciding with elevated levels of many angiogenic growth factors may point to their malfunctioning due to oxidative stress and/or protein glycation at the factor and the receptor levels. This necessitates further investigations.     

Enhanced iron availability by protein glycation may explain higher infection rates in diabetics

Serum proteins exist in a state of higher glycation among individuals with poor glycemic control, notably diabetics. These non-enzymatic modifications via the Maillard reaction have far reaching effects on metabolism and regulation, and may be responsible for increased infection rates within this population. Here we explore the effects of glycation on iron metabolism and innate immunity by investigating the interaction between siderophores and bovine serum albumin (BSA). Using a quartz crystal microbalance with dissipation monitoring to quantify association rates, glycated BSA exhibited a significantly reduced affinity for apo and holo enterobactin compared to a non-glycated BSA standard. Bacterial growth assays in the presence of BSA and under iron-limited conditions indicated the growth rate of enterobactin-producing E. coli increased significantly when the BSA was in a glycated form. The results, in addition to data in the literature, support the hypothesis that glycation of serum proteins may effectively increase the available free iron pool for bacteria in blood serum and weaken our innate immunity. This phenomenon may be partially responsible for higher infection rates in some diabetics, especially those with poor glycemic control.     

The role of posttranslational modification in moonlighting glyceraldehyde-3-phosphate dehydrogenase structure and function

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting protein exhibiting distinct activities apart from its classical role in glycolysis. Regulation of its moonlighting functions and its subcellular localization may be dependent on its posttranslational modification (PTM). The latter include its phosphorylation, which is required for its role in intermembrane trafficking, synaptic transmission and cancer survival; nitrosylation, which is required for its function in apoptosis, heme metabolism and the immune response; acetylation which is necessary for its modulation of apoptotic gene regulation; and N-acetylglucosamine modification which may induce changes in GAPDH oligomeric structure. These findings suggest a structure function relationship between GAPDH posttranslational modification and its diverse moonlighting activities.