Glycated Insulin Exacerbates the Cytotoxicity of Human Islet Amyloid Polypeptides: a Vicious Cycle in Type 2 Diabetes.

The aggregation of human islet amyloid polypeptide (hIAPP) is one of the triggering factors of type 2 diabetes mellitus (T2DM). hIAPP is cosynthesized, costored, and cosecreted with insulin in pancreatic ß-cells, and insulin inhibits hIAPP aggregation. In T2DM patients, long-term hyperglycemia causes glycation of near 10% of total insulin. The glycation not only modifies insulin but also cross-links insulin into oligomers. However, the effect of glycated human insulin on hIAPP aggregation is unknown. In this study, four physiologically relevant monosaccharides, methylglyoxal, glucose, fructose, and ribose were used to glycate human insulin and two C-terminus truncated insulin analogues. Glycated insulin monomers or low molecular weight oligomers such as dimers significantly exacerbated the cytotoxicity of hIAPP. Notably, glycation-induced cross-linking of insulin inhibited the aggregation, membrane disruption, and cytotoxicity of hIAPP, which was corroborated by a control study using EGS-induced cross-linking of insulin or lysozyme. Removal of B29Lys on the C terminus of the insulin B chain not only abolished glycation-induced cross-linking but also attenuated the aggravation effect of glycated insulin on hIAPP cytotoxicity. Taken together, this study reveals a vicious cycle in T2DM, that hyperglycemia-driven insulin glycation exacerbates the cytotoxicity of hIAPP, which accelerates ß-cells death and further deteriorates T2DM.

ANGPTL8 negatively regulates NF-κB activation by facilitating selective autophagic degradation of IKKγ.

Excessive nuclear factor-κB (NF-κB) activation mediated by tumor necrosis factor α (TNFα) plays a critical role in inflammation. Here we demonstrate that angiopoietin-like 8 (ANGPTL8) functions as a negative feedback regulator in TNFα-triggered NF-κB activation intracellularly. Inflammatory stimuli induce ANGPTL8 expression, and knockdown or knockout of ANGPTL8 potentiates TNFα-induced NF-κB activation in vitro. Mechanistically, upon TNFα stimulation, ANGPTL8 facilitates the interaction of IKKγ with p62 via forming a complex, thus promoting the selective autophagic degradation of IKKγ. Furthermore, the N-terminal domain mediated self-oligomerization of ANGPTL8 is essential for IKKγ degradation and NF-κB activation. In vivo, circulating ANGPTL8 level is high in patients diagnosed with infectious diseases, and the ANGPTL8/p62-IKKγ axis is responsive to inflammatory stimuli in the liver of LPS-injected mice. Altogether, our study suggests the ANGPTL8/p62-IKKγ axis as a negative feedback loop that regulates NF-κB activation, and extends the role of selective autophagy in fine-tuned inflammatory responses.

How the imidazole ring modulates amyloid formation of islet amyloid polypeptide: A chemical modification study.

BACKGROUND: The misfolding of human islet amyloid polypeptide (hIAPP) is an important pathological factor on the onset of type 2 diabetes. A number of studies have been focused on His(18), the only histidine of hIAPP, whose imidazole ring and the protonation state might impact hIAPP amyloid formation, but the exact mechanism remains unclear. METHODS: We used diethylpyrocarbonate (DEPC) to specifically modify His(18) and obtained mono-ethyloxyformylated hIAPP (DMI). Thioflavin T based fluorescence, transmission electronic microscopy, circular dichroism spectroscopy, fluorescence dye leakage, Fourier transform infrared spectroscopy and replica-exchange molecular dynamics (REMD) simulation were applied to study the impact of DEPC-modification on hIAPP amyloid formation. RESULTS: After an ethyl-acetate group was introduced to the His(18) of hIAPP by diethylpyrocarbonate (DEPC) modification, the pH dependent hIAPP fibrillation went to the opposite order and the number of intra-molecular hydrogen bonds decreased, while the possibility of His(18) participating in the formation of α-helical structures increased. Furthermore, the membrane-peptide interaction and ion-peptide interaction were both impaired. CONCLUSIONS: The intramolecular hydrogen bond formation by His(18) and the possibility of His(18) participating in the formation of α-helical structures greatly modulated the manner of hIAPP amyloid formation. The imidazole ring directly participates in the hIAPP-membrane/ion interaction. GENERAL SIGNIFICANCE: DEPC modification is an alternative approach to investigate the role of the imidazole ring during amyloid formation.

How our bodies fight amyloidosis: effects of physiological factors on pathogenic aggregation of amyloidogenic proteins.

The process of protein aggregation from soluble amyloidogenic proteins to insoluble amyloid fibrils plays significant roles in the onset of over 30 human amyloidogenic diseases, such as Prion disease, Alzheimer's disease and type 2 diabetes mellitus. Amyloid deposits are commonly found in patients suffered from amyloidosis; however, such deposits are rarely seen in healthy individuals, which may be largely attributed to the self-regulation in vivo. A vast number of physiological factors have been demonstrated to directly affect the process of amyloid formation in vivo. In this review, physiological factors that influence amyloidosis, including biological factors (chaperones, natural antibodies, enzymes, lipids and saccharides) and physicochemical factors (metal ions, pH environment, crowding and pressure, etc.), together with the mechanisms underlying these proteostasis effects, are summarized.

Effect of single nucleotide polymorphism in thrombin-activatable fibrinolysis inhibitor on the risk of diabetic macrovascular disease.

Hypofibrinolysis is commonly found in patients with diabetes mellitus and is associated with the increased risk for many diabetic complications. An important inhibitor of fibrinolysis, thrombin-activatable fibrinolysis inhibitor (TAFI), participates in hypofibrinolysis in diabetes mellitus and may be involved in diabetic macrovascular disease. The present study was designed to determine whether TAFI polymorphisms (505G/A and 1040C/T) and TAFI levels are correlated with the development of type 2 diabetes mellitus (T2DM) and macrovascular diseases (MVDs). A total of 249 clinical samples were collected, including 102 healthy individuals (H group), 44 T2DM patients without MVD (T group) and 103 T2DM patients with MVD (M group). The 505G/A polymorphism was equally represented in the three groups. In contrast, analysis of the 1040C/T polymorphism revealed a statistically lower percentage of the T allele in the M group than in the H group (P = 0.014). This difference was due to decreased T/T homozygotes in the M groups compared with the H group (P = 0.029). The antigen TAFI level was 31.72 ±â€Š13.64% in the H group, 62.56 ±â€Š18.77% in the T group (P < 0.05, compared with the H group) and 63.70 ±â€Š15.76% in the M group (P < 0.05, compared with the H group). As high plasma TAFI level is associated with the increasing risk of T2DM, it may thus serve as a potential marker for the diagnosis of T2DM.

Diethylpyrocarbonate modification reveals HisB5 as an important modulator of insulin amyloid formation.

More than 30 amyloid proteins are reported to be associated with amyloidosis diseases. Studies have implicated histidine may be critically involved in amyloid formation. Here, we used diethylpyrocarbonate (DEPC) modification to obtain a His(B5) mono-ethyloxyformylated insulin (DMI-B(5)). The secondary structure, amyloidogenicity, metal ion interaction, and cytotoxicity of DMI-B(5) and insulin were compared. DMI-B(5) was less prone to aggregation in acidic condition but easier to aggregate at neutral pH. DEPC modification resulted in attenuated inhibitory effect of Zn(2+) on aggregation, whereas DMI-B(5) fibrils induced more severe erythrocytes haemolysis compared to insulin fibrils. This study not only provides a fast new approach for studying the impact of imidazole ring in amyloid formation, but also reveals the critical modulating role of histidine imidazole ring on the amyloidogenicity of insulin.

Inhibitory effect of leonurine on the formation of advanced glycation end products.

Long-term hyperglycemia is a typical symptom of diabetes mellitus (DM) which can cause a high level of protein glycation and lead to the formation of advanced glycation end products (AGEs). The accumulation of AGEs in turn deteriorates DM and its complications. Insulin, the only hormone that directly decreases blood sugar in vivo, is vulnerable to glycation which causes the loss of its biological activity. In this study, we used a porcine insulin (PI)-methylglyoxal (MGO) model to investigate the inhibitory effect of leonurine (LN), a natural alkaloid extracted from Herba leonuri, on AGE formation. Assays including AGE-specific fluorescence, and fructosamine level and carbonyl group content determination showed that LN can dose-dependently suppress PI glycation. A significantly decreased cross-linking level on the glycated PI was also proven by SDS-PAGE electrophoresis. A further liquid chromatography-mass spectrometry study suggested that LN may inhibit PI glycation through trapping MGO and keeping it from reacting with PI. Our results thus indicate that LN is a promising anti-glycation agent for the prevention of diabetes and its complications via inhibiting AGE formation.

The effect of exposing a critical hydrophobic patch on amyloidogenicity and fibril structure of insulin.

It is widely accepted that the formation of amyloid fibrils is one of the natural properties of proteins. The amyloid formation process is associated with a variety of factors, among which the hydrophobic residues play a critical role. In this study, insulin was used as a model to investigate the effect of exposing a critical hydrophobic patch on amyloidogenicity and fibril structure of insulin. Porcine insulin was digested with trypsin to obtain desoctapeptide-(B23-B30) insulin (DOI), whose hydrophilic C-terminal of B-chain was removed and hydrophobic core was exposed. The results showed that DOI, of which the ordered structure (predominantly α-helix) was markedly decreased, was more prone to aggregate than intact insulin. As to the secondary structure of amyloid fibrils, DOI fibrils were similar to insulin fibrils formed under acidic condition, whereas under neutral condition, insulin formed less polymerized aggregates by showing decreased ß-sheet contents in fibrils. Further investigation on membrane damage and hemolysis showed that DOI fibrils induced significantly less membrane damage and less hemolysis of erythrocytes compared with those of insulin fibrils. In conclusion, exposing the hydrophobic core of insulin can induce the increase of amyloidogenicity and formation of higher-order polymerized fibrils, which is less toxic to membranes.

Proanthocyanidins are the major anti-diabetic components of cinnamon water extract.

Cinnamon consumption has been found to associate with the attenuation of diabetes mellitus. The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as a causative factor of type 2 diabetes mellitus (T2DM). Here, we investigated whether cinnamon has any beneficial effect on the toxic aggregation of hIAPP. We found that cinnamon water extract (CWE) inhibited the amyloid formation of hIAPP in a dose-dependent manner, and identified proanthocyanidins as the major anti-amyloidogenic compounds of CWE. Proanthocyanidins affected the secondary structures of hIAPP and delayed the structural transition from unstructured coils to ß-sheet-rich structures. Further studies showed that proanthocyanidins not only inhibited the formation of hIAPP oligomers, but also significantly attenuated the membrane damaging and cytotoxic effects caused by the hIAPP aggregation. Together, these results suggest a possible way by which cinnamon shows beneficial effects on T2DM, and indicate a potential pharmacological usage of proanthocyanidins as an anti-diabetic drug candidate.

Coffee components inhibit amyloid formation of human islet amyloid polypeptide in vitro: possible link between coffee consumption and diabetes mellitus.

Global epidemic studies have suggested that coffee consumption is reversely correlated with the incidence of type 2 diabetes mellitus (T2DM), a metabolic disease. The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of T2DM. Coffee extracts have three major active components: caffeine, caffeic acid (CA), and chlorogenic acid (CGA). In this study, the effects of these major coffee components, as well as dihydrocaffeic acid (DHCA) (a major metabolite of CGA and CA), on the amyloidogenicity of hIAPP were investigated by thioflavin-T based fluorescence emission, transmission electronic microscopy, circular dichroism, light-induced cross-linking, dynamic light scattering, and MTT-based cell viability assays. The results suggest that all components show varied inhibitory effects on the formation of toxic hIAPP amyloids, in which CA shows the highest potency in delaying the conformational transition of the hIAPP molecule with the most prolonged lag time, whereas caffeine shows the lowest potency. At a 5-fold excess molar ratio of compound to hIAPP, all coffee-derived compounds affect the secondary structures of incubated hIAPP as suggested by the circular dichroism spectra and CDPro deconvolution analysis. Further photoinduced cross-linking based oligomerization and dynamic light scattering studies suggested CA and CGA significantly suppressed the formation of hIAPP oligomers, whereas caffeine showed no significant effect on oligomerization. Cell protection effects were also observed for all three compounds, with the protection efficiency being greatest for CA and least for CGA. These findings suggest that the beneficial effects of coffee consumption on T2DM may be partly due to the ability of the major coffee components and metabolites to inhibit the toxic aggregation of hIAPP.