Effect of exogenous free N^(ε)-(carboxymethyl)lysine on diabetes-associated cognitive dysfunction:neuroinflammation,and metabolic disorders

Diabetes-associated cognitive dysfunction has already been attracted considerable attention.Advanced glycation end products(AGEs)from daily diets are thought to be a vital contributor to the development of this diseases.However,the effect of one of the best-characterized exogenous AGEs N^(ε)-(carboxymethyl)lysine(CML)on cognitive function is not fully reported.In the present study,diabetical Goto-Kakizaki(GK)rats were treated with free CML for 8-weeks.It was found that oral consumption of exogenous CML significantly aggravated diabetes-associated cognitive dysfunction in behavioral test.In details,exogenous CML increased levels of oxidative stress,promoted the activation of glial cells in the brain,up-regulated the release of inflammatory cytokines interleukin-6,inhibited the protein expression of the brain-derived neurotrophic factor and thus led to neuroinflammation.Furthermore,exogenous CML promoted the amyloidogenesis in the brain of GK rats,and inhibited the expression of GLUT4.Additionally,several tricarboxylic acid cycle and glutamate-glutamine/γ-aminobutyric acid cycle intermediates including pyruvate,succinic acid,glutamine,glutamate were significantly changed in brain of GK rats treated with exogenous free CML.In conclusion,exogenous free CML is a potentially noxious compounds led to aggravated diabetes-associated cognitive dysfunction which could be possibly explained by its effects on neuroinflammation,energy and neurotransmitter amino acid homeostasis.

Dietary Nε-(carboxymethyl)lysine affects cardiac glucose metabolism and myocardial remodeling in mice

BACKGROUND Myocardial remodeling is a key factor in the progression of cardiovascular disease to the end stage.In addition to myocardial infarction or stress overload,dietary factors have recently been considered associated with myocardial remodeling.Nε-(carboxymethyl)lysine(CML)is a representative foodborne toxic product,which can be ingested via daily diet.Therefore,there is a marked need to explore the effects of dietary CML on the myocardium.AIM To explore the effects of dietary CML(dCML)on the heart.METHODS C57 BL/6 mice were divided into a control group and a dCML group.The control group and the dCML group were respectively fed a normal diet or diet supplemented with CML for 20 wk.Body weight and blood glucose were recorded every 4 wk.^(18)F-fluorodeoxyglucose(FDG)was used to trace the glucose uptake in mouse myocardium,followed by visualizing with micro-positron emission tomography(PET).Myocardial remodeling and glucose metabolism were also detected.In vitro,H9C2 cardiomyocytes were added to exogenous CML and cultured for 24 h.The effects of exogenous CML on glucose metabolism,collagen I expression,hypertrophy,and apoptosis of cardiomyocytes were analyzed.RESULTS Our results suggest that the levels of fasting blood glucose,fasting insulin,and serum CML were significantly increased after 20 wk of dCML.Micro-PET showed that ^(18)F-FDG accumulated more in the myocardium of the dCML group than in the control group.Histological staining revealed that dCML could lead to myocardial fibrosis and hypertrophy.The indexes of myocardial fibrosis,apoptosis,and hypertrophy were also increased in the dCML group,whereas the activities of glucose metabolism-related pathways and citrate synthase(CS)were significantly inhibited.In cardiomyocytes,collagen I expression and cellular size were significantly increased after the addition of exogenous CML.CML significantly promoted cellular hypertrophy and apoptosis,while pathways involved in glucose metabolism and level of Cs mRNA were significantly inhibited.CONCLUSION This study reveals that dCML alters myocardial glucose metabolism and promotes myocardial remodeling.

Nε-(carboxymethyl)lysine promotes lipid uptake of macrophage via cluster of differentiation 36 and receptor for advanced glycation end products

BACKGROUND Advanced glycation end products(AGEs)are diabetic metabolic toxic products that cannot be ignored.Nε-(carboxymethyl)lysine(CML),a component of AGEs,could increase macrophage lipid uptake,promote foam cell formation,and thereby accelerate atherosclerosis.The receptor for AGEs(RAGE)and cluster of differentiation 36(CD36)were the receptors of CML.However,it is still unknown whether RAGE and CD36 play key roles in CML-promoted lipid uptake.AIM Our study aimed to explore the role of RAGE and CD36 in CML-induced macrophage lipid uptake.METHODS In this study,we examined the effect of CML on lipid uptake by Raw264.7 macrophages.After adding 10 mmol/L CML,the lipid accumulation in macrophages was confirmed by oil red O staining.Expression changes of CD36 and RAGE were detected with immunoblotting and quantitative real-time polymerase chain reaction.The interaction between CML with CD36 and RAGE was verified by immunoprecipitation.We synthesized a novel N-succinimidyl-4-18Ffluorobenzoate-CML radioactive probe.Radioactive receptor-ligand binding assays were performed to test the binding affinity between CML with CD36 and RAGE.The effects of blocking CD36 or RAGE on CML-promoting lipid uptake were also detected.RESULTS The study revealed that CML significantly promoted lipid uptake by macrophages.Immunoprecipitation and radioactive receptor-ligand binding assays indicated that CML could specifically bind to both CD36 and RAGE.CML had a higher affinity for CD36 than RAGE.ARG82,ASN71,and THR70 were the potential interacting amino acids that CD36 binds to CML Anti-CD36 and anti-RAGE could block the uptake of CML by macrophages.The lipid uptake promotion effect of CML was significantly attenuated after blocking CD36 or RAGE.CONCLUSION Our results suggest that the binding of CML with CD36 and RAGE promotes macrophage lipid uptake.

The initial phase of oxidative stress in a steroid-induced osteonecrosis rabbit model

Using a rabbit model, we examined N epsilon-(hexanoyl) lysine (HEL) levels in bone and urine to detect when peroxidative reaction first occurs after steroid administration. Japanese white rabbits weighing about 3.5 kg each were injected with a single intramuscular dose of methylprednisolone 40 mg/kg and divided into groups consisting of 10 rabbits each, which were killed after 1, 3, 5 and 14 days (groups A, B, C and D respectively). As a control, 10 untreated rabbits (group N) were also studied. The proximal femurs were examined histopathologically and immunohistochemically using monoclonal antibody HEL, which is a highly specific antibody against N epsilon-(hexanoyl) lysine, an early peroxidation marker. In addition, urinary levels of HEL were measured by enzyme-linked immunosorbent assay in group N, A, B and C. Osteonecrosis was detected only in group D (90%). Increase of positive reaction of HEL in the bone was observed in group A and D. HEL expression in group D was judged to be a secondary reaction resulting from the development of osteonecrosis. Urinary level of HEL showed a significant increase in only group A (P < 0.001). The present findings suggest that peroxidation in bone occurred within 24 hours after steroid administration in a rabbit model and that it is possible to noninvasively grasp the timing of this peroxidative reaction by measuring the urinary level of HEL.

Acetylproteomics analyses reveal critical features of lysine-ε-acetylation in Arabidopsis and a role of 14-3-3 protein acetylation in alkaline response

Lysine-ε-acetylation(Kac)is a post-translational modification(PTM)that is critical for metabolic regulation and cell signaling in mammals.However,its prevalence and importance in plants remain to be determined.Employing high-resolution tandem mass spectrometry,we analyzed protein lysine acetylation in five representative Arabidopsis organs with 2~3 biological replicates per organ.A total of 2887 Kac proteins and 5929 Kac sites were identified.This comprehensive catalog allows us to analyze proteome-wide features of lysine acetylation.We found that Kac proteins tend to be more uniformly expressed in different organs,and the acetylation status exhibits little correlation with the gene expression level,indicating that acetylation is unlikely caused by stochastic processes.Kac preferentially targets evolutionarily conserved proteins and lysine residues,but only a small percentage of Kac proteins are orthologous between rat and Arabidopsis.A large portion of Kac proteins overlap with proteins modified by other PTMs including ubiquitination,SUMOylation and phosphorylation.Although acetylation,ubiquitination and SUMOylation all modify lysine residues,our analyses show that they rarely target the same sites.In addition,we found that“reader”proteins for acetylation and phosphorylation,i.e.,bromodomain-containing proteins and GRF(General Regulatory Factor)/14-3-3 proteins,are intensively modified by the two PTMs,suggesting that they are main crosstalk nodes between acetylation and phosphorylation signaling.Analyses of GRF6/14-3-3λreveal that the Kac level of GRF6 is decreased under alkaline stress,suggesting that acetylation represses plant alkaline response.Indeed,K56ac of GRF6 inhibits its binding to and subsequent activation of the plasma membrane H+-ATPase AHA2,leading to hypersensitivity to alkaline stress.These results provide valuable resources for protein acetylation studies in plants and reveal that protein acetylation suppresses phosphorylation output by acetylating GRF/14-3-3 proteins.

噬菌体裂解酶Lysin1902原核表达及其与ε-聚赖氨酸联用效果评价

【目的】研究裂解酶Lysin1902与ε-聚赖氨酸(ε-PL)对大肠杆菌O157:H7的协同抗菌作用。【方法】通过Mega构建进化树、使用在线工具等分析大肠杆菌噬菌体裂解酶Lysin1902氨基酸序列组成和结构等;原核表达并纯化Lysin1902;通过平板裂解实验检测Lysin1902对大肠杆菌O157:H7灭活菌株的裂解活性;用96孔板法检测Lysin1902或ε-PL的活菌裂解能力;棋盘法验证Lysin1902和ε-PL联用效果。【结果】体外成功表达并纯化了Lysin1902。Lysin1902对大肠杆菌O157:H7灭活菌株具有裂解活性,但不能有效裂解活的大肠杆菌。噬菌体裂解酶与ε-PL联用结果表明,加入Lysin1902后,ε-PL能够完全控制大肠杆菌O157:H7增殖的使用浓度由0.7mg/mL降低到0.1 mg/mL。【结论】体外原核表达并纯化Lysin1902,其单独使用对活的大肠杆菌O157:H7无裂解活性,但与ε-PL联用可显著提高ε-PL对大肠杆菌O157:H7的裂解能力。

增生性瘢痕中羰基应激水平的研究

目的:观察增生性瘢痕中丙二醛(MDA)、4-羟基壬烯醛(4-HNE)及己酰赖氨酸加合物(HEL)含量,探讨羰基应激在瘢痕过度增生中的作用。方法:收集手术切除标本31例,其中增生性瘢痕12例,成熟瘢痕标本10例以及正常皮肤标本9例,取组织匀浆上清液应用分光光度法检测MDA,ELISA法检测4-HNE及HEL含量。结果:与正常皮肤比较,增生性瘢痕中MDA、4-HNE及HEL含量显著变化(P<0.01或P<0.05),呈上升趋势,成熟瘢痕组与正常皮肤组比,则无明显变化(P>0.05)。结论:在增生性瘢痕组织中脂质过氧化来源的醛类物质升高,由这些醛类引起的羰基应激可能在胶原沉积、瘢痕过度增生中发挥作用。

Advanced glycation end products induce neural tube defects through elevating oxidative stress in mice

Our previous study showed an association between advanced glycation end products （AGEs） and neural tube defects （NTDs）. To understand the molecular mechanisms underlying the effect of AGEs on neural tube development, C57BL/6 female mice were fed for 4 weeks with com- mercial food containing 3% advanced glycation end product bovine serum albumin （AGE-BSA） or 3% bovine serum albumin （BSA） as a control. After mating mice, oxidative stress markers including malondialdehyde and H202 were measured at embryonic day 7.5 （E7.5） of ges- tation, and the level of intracellular reactive oxygen species （ROS） in embryonic cells was determined at E8.5. In addition to evaluating NTDs, an enzyme-linked immunosorbent assay was used to determine the effect of embryonic protein administration on the N-（carboxymethyl） lysine reactivity of acid and carboxyethyl lysine antibodies at E10.5. The results showed a remarkable increase in the incidence of NTDs at El0.5 in embryos of mice fed with AGE-BSA （no hyperglycemia） compared with control mice. Moreover, embryonic protein administration resulted in a noticeable increase in the reactivity of N-（carboxymethyl） lysine and N（ε）-（carboxyethyl） lysine antibodies. Malondialdehyde and H2O2 levels in embryonic cells were increased at E7.5, followed by increased intracellular ROS levels at E8.5. Vitamin E supplementation could partially recover these phenomena. Collectively, these results suggest that AGE-BSA could induce NTDs in the absence of hyperglycemia by an underlying mechanism that is at least partially associated with its capacity to increase embryonic oxidative stress levels.

Synthesis and Gelation Capability of Fmoc and Boc Mono-substituted Cyclo（L-Lys-L-Lys）s

Fmoc or Boc mono-substituted cyclo（L-Lys-L-Lys）s were synthesized via the reaction of lysine cyclic dipeptide with Fmoc N-hydroxysuccinimide este（Fmoc-OSu） and di-tert-butyl dicarbonate[（Boc）20], respectively. The resulted mono-substituted cyclo（L-Lys-L-Lys）s（2-4） by means of test tube inversion method served as organogelators enabled to form stable thermo-reversible organogels in alcoholic, substituted benzene and chlorinated solvents, with the minimum gelation concentration（MGC） in a range of 1%-4%（mass fraction）. The transmission electron microscopy（TEM） and scanning electron microscopy（SEM） observations reveal that these gelators self-assembled into 3D nanofiber, nanoribbon or nanotube network structures. The rheological measurement exhibited that the sto- rage modulus of gels is higher than the loss one, and the complex viscosity is reduced linearly with the increasing of scanning frequency. The fluorescence spectrum of compound 2 in 1,2-dichloroethane and benzene demonstrates that the emission peak of Fmoc at 320 nm has red-shifted and the intensity decreases gradually, while the intensity of the emission peak at 460 nm substantially enhances as a function of concentration, indicating the existence of π-π stacking interactions and the formation of J-type aggregates. Meanwhile, compound 4 self-assembled into nanotubes via the stacking of multiple bilayer membranes. Fmoc and Boc disubstituted cyclo（L-Lys-L-Lys）（3） holds the relatively lower MGC values, showing the stronger gelation ability in most selected organic solvents due to the presence of both Fmoc and Boc groups.