Glycated insulin exacerbates the cytotoxicity of human islet amyloid

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Glycated insulin exacerbates the cytotoxicity of human islet amyloid polypeptides: a vicious cycle in type 2 diabetes Liang Ma, Chen Yang, Lianqi Huang, Yuchen Chen, Yang Li, Cheng Cheng, Biao Cheng, Ling Zheng, and Kun Huang ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b01128 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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ACS Chemical Biology

Diabetic hyperglycemia causes a vicious cycle between insulin glycation and pancreatic β-cell death

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ACS Chem. Biol.

Glycated Insulin exacerbates the cytotoxicity of human islet amyloid polypeptides: a vicious cycle in type 2 diabetes Liang Maa#, Chen Yanga#, Lianqi Huanga, Yuchen Chena, Yang Lia, Cheng Chenga, Biao Chengb, Ling Zhengc and Kun Huanga*

a

Tongji School of Pharmacy, Tongji Medical College, Huazhong University of Science

and Technology, Wuhan, China, 430030; b

Department of Pharmacy, The Central Hospital of Wuhan, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan, China, 430014; c

Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan

University, Wuhan, China, 430072

# These

authors contribute equally

*Correspondence Author Kun Huang, PhD Tongji School of Pharmacy Tongji Medical College Huazhong University of Science and Technology Wuhan, China, 430030 [email protected]

1


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Abstract The aggregation of human islet amyloid polypeptide (hIAPP) is one of the triggering factors of type 2 diabetes mellitus (T2DM). hIAPP is co-synthesized, co-stored and co-secreted with insulin in pancreatic -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 crosslinks 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 weights 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 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 cells death and further deteriorates T2DM.

Keywords Advanced glycation end products; human islet polypeptides; human insulin; amyloidogenicity; cytotoxicity; crosslinking

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Abbreviations AGEs, advanced glycation end products; CD, circular dichroism; DLS, dynamic light scattering; DKI, desoctapeptide-(B23–B30) insulin; DKRI, desnonapeptide-(B22-B30) insulin; EGS, ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester); HMW, high molecular weight; hIns, human insulin; hIAPP, human islet amyloid polypeptide; LMW, low molecular weight; MGO, methylglyoxal; NBT, nitroblue-tetrazolium; POPG, 2-Oleoyl-1-palmitoyl-sn-glycerol-3-phospho-rac (1-glycerol) sodium salt; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RP-HPLC, reversed phase high performance liquid chromatography; T2DM, type 2 diabetes mellitus; TCA, trichloroacetic acid; ThT, thioflavin-T; TEM, transmission electron microscopy

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Introduction Diabetes mellitus, characterized by hyperglycemia, is a global metabolic disease with 451 million diagnosed patients in 2017, and type 2 diabetes mellitus (T2DM) comprises 90% of diabetic patients (1). The presence of amyloid fibrils in pancreatic -cells, arising from the aggregation of human islet amyloid polypeptide (hIAPP), is a hallmark of T2DM (2-5). hIAPP is a 37-residue natively unstructured polypeptide that is prone to aggregate into amyloid fibrils (5-8). Increasing evidence shows that hIAPP aggregates induce pancreatic β-cell dysfunction, cell death and loss of islet β-cell mass, therefore the aggregation of hIAPP is considered as one of the major causes of T2DM (9-14). Insulin is a major hormonal peptide synthesized by the -cells (Figure 1A & B). Physiologically, hIAPP is co-expressed, co-packaged and co-secreted with human insulin by -cells (15-17). Many factors have been suggested that may inhibit hIAPP from aggregation in vivo, such as low intra granular pH, C-peptide, metal ions, and insulin (8, 18, 19). Studies have shown that insulin inhibits the aggregation of hIAPP at equal molar ratio in vitro (20, 21), which may partly explain why hIAPP stored at ultra-high concentration (0.8-4 mM) in -cells without showing abnormal aggregates (22).

Under long-term hyperglycemia induced by T2DM, a large number of proteins, including insulin, are subjected to glycation (23). Glycation occurs between the carbonyl of reducing sugar and amino group of the protein. Initially, Schiff bases form reversibly, which further undergo reversible rearrangement to form the Amadori products (e.g. fructosamine) that can be irreversibly oxidized to yield the advanced glycation end products (AGEs; Figure 1C) (24-26). In T2DM patients, approximately 9-10% of total 4



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insulin is glycated (27, 28). A1Gly, B1Phe, B22Arg and B29Lys are major glycation sites of insulin (Figure 1A) (29, 30). The biological activity of glycated insulin is drastically compromised by nearly 90% (31). However, the effect of glycated insulin on the aggregation and cytotoxicity of co-localized hIAPP remains unknown.

Here, we investigated the effects of insulin glycation on the kinetic and cytotoxicity of hIAPP aggregation. Four physiological relevant monosaccharides were used to induce insulin glycation (Figure 1D). Glucose (Glu) is the most abundant reducing sugar in vivo with its plasma concentrations range from 3.9 to 7.8 mM in healthy individuals, while 4-times higher in T2DM patients (32). Methylglyoxal (MGO), the most reactive AGE precursor (33), is formed by oxidative decomposition of polyunsaturated fatty acids and fructose, the concentration of MGO in healthy human plasma may increase by 4 fold in T2DM patients (34, 35). Fructose (Frc) is 10 times more reactive than glucose, despite of its relatively low plasma concentration of 10-35 M (36, 37). Ribose (Rib) is a highly active monosaccharide involved in the formation of AGEs presented in all cells (38, 39). We prepared glycated human insulin or C-terminus truncated insulin analogues with these monosaccharides, and assessed the impacts of glycated insulin or analogues on hIAPP aggregation and cytotoxicity.

Results Four monosaccharides glycate human insulin Long-term hyperglycemia in diabetic patients promotes insulin glycation and reduces its biological activity (24, 31). Previous studies have shown that glucose- or 5


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methylglyoxal-induced insulin glycation led to impaired insulin signaling and reduced insulin sensitivity (40, 41). However, the relationship between glycated insulin and hIAPP has not been reported. Here, we first investigated the effect of glycated insulin on hIAPP aggregation and cytotoxicity. Four physiological relevant monosaccharides (MGO, Glu, Frc, Rib) that have been reported to glycate insulin were applied (32, 37, 42, 43). AGEs-specific fluorescence and fructosamine level were used to evaluate glycation status before and after dialysis. Compared to untreated insulin, the fluorescence intensity and fructosamine concentration of glycated insulin samples were at least 2-fold higher (Figure 2A & B), indicating glycation of insulin. After dialysis, the fluorescence intensity of glycated samples were still higher than untreated insulin, while the concentration of fructosamine showed no difference between glycated samples and the untreated insulin (Figure 2B), suggesting that intermediate glycation products turned back to non-glycated state after dialysis, and only advanced glycation end products remained (Figure 1C). Far-UV CD spectroscopy was applied to assess the secondary structure of glycated samples. Insulin and its glycated products shared similar CD spectra results with two negative bands at 208 nm and 222 nm, indicating typical -helical structures (Figure 2C). SDS-PAGE analysis suggested that glucose glycated insulin (Glu-hIns) existed mainly as monomers and dimers; whereas cross-linked oligomers of higher molecular weights were observed in ribose-glycated insulin (Rib-hIns), fructose-glycated insulin (Frc-hIns) and MGO-glycated insulin (MGO-hIns) (Figure 2D).

Glycated human insulin attenuates the aggregation of hIAPP 6



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Amyloid deposition of hIAPP in the islet is closely associated with T2DM (2). It has been proved that hIAPP rapidly aggregates into toxic prefibrillar intermediates, and eventually turned to mature ribbon-like fibrils (44). Human insulin, a major hormonal peptide synthesized by the -cells (8), have been shown to inhibit the aggregation of hIAPP at equal molar ratio in vitro. In our experiments, the amyloid formation of hIAPP in the presence of glycated insulin was monitored by thioflavin-T (ThT) fluorescence, which is a fluorescent dye binds specifically to -sheet structures (45, 46). hIAPP alone reached plaque stage after 4 h incubation, with T50 and lag time of 2.43 ± 0.26 h and 1.43 ± 0.12 h, respectively (Figure 3A and Table 1). Compared to the hIAPP, the presence of insulin prolonged the T50 and lag time to 5.17 ± 0.70 h (p < 0.01) and 3.34 ± 0.95 h (p < 0.05) (Figure 3A and Table 1), which agreed with previous reports that insulin inhibits the aggregation of hIAPP (21). Meanwhile, in the presence of Glu-hIns, Mgo-hIns, Frc-hIns or Rib-hIns, the T50 was further increased to 5.87 ± 0.79 h (p < 0.01), 6.53 ± 0.29 h (p < 0.001), 6.37 ± 0.42 h (p < 0.001) and 6.77 ± 0.21 h (p < 0.001), respectively (Table 1); and the lag time was prolonged to 3.88 ± 1.20 h (p < 0.05), 4.34 ± 0.32 h (p < 0.001), 4.14 ± 0.87 h (p < 0.05), and 5.07 ± 0.58 h (p < 0.001), respectively (Table 1). These results suggest that glycated insulin significantly inhibited the aggregation of hIAPP. Notably, compared to the inhibitory effect of insulin on hIAPP aggregation, glycated insulin (MGO-hIns, Glu-hIns, Frc-hIns and Rib-hIns) showed even stronger inhibitory effects (Figure 3A and Table 1). The morphology of hIAPP aggregates in the presence or absence of insulin or glycated insulin were examined by TEM. hIAPP showed typical linear fibrils, while in the presence of equimolar insulin, only amorphous deposits were observed (Figure 3B). 7


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Punctiform aggregates were found in hIAPP samples co-incubated with MGO-hIns, Glu-hIns, Frc-hIns or Rib-hIns (Figure 3B). As the controls, only protein particles were observed for native or glycated insulin samples (Figure S1).

Glycated insulin prevents particle size growth of hIAPP Dynamic light scattering (DLS) was applied to monitor the size and distribution of particles formed during aggregation (47). The particle size was undetectable in all groups at 0 h (Figure 4A); after 2 h incubation, the mean diameter of hIAPP increased to over 500 nm; for samples incubated for 4 h and 8 h, particles with diameters over 10000 nm were detected, which exceeded the detection limit, suggesting the formation of large fibrils. For hIAPP co-incubated with insulin or glycated insulin samples, the particle sizes increased at much slower rates than that of hIAPP group (Figure 4A), and the mean diameters were less than 5000 nm even after 8 h incubation (Figure 4B). These results indicated that insulin or glycated insulin inhibited the formation of large hIAPP aggregates.

Low- and high-molecular weight glycated insulin differently affect the cytotoxicity of hIAPP Prefibrillar intermediates of hIAPP during aggregation are reported to be the most toxic components which may induce membrane disruption and cytotoxicity (48-50). Dye leakage and trypan blue assays were applied to examine how glycated insulin influence the membrane disruption effect and cytotoxicity of hIAPP. Glycated insulin showed no membrane disruption capacity and cytotoxicity (Figure S2). hIAPP severely damaged the 8



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membrane (82.5 ± 3.4%) (Figure 5A). Compared with hIAPP alone, the presence of different insulin samples decreased the membrane damage by 32.2 ± 5.2% (hIns), 35.3 ± 4.1% (Glu-hIns), 50.4 ± 4.3% (MGO-hIns), 42.5 ± 5.8% (Frc-hIns), and 53.5 ± 3.1% (Rib-hIns), respectively (p < 0.01) (Figure 5A). Trypan blue assays showed that hIAPP decreased the cell viability by 37.0 ± 4.6%, and the presence of equimolar of hIns, Mgo-hIns, Frc-hIns or Rib-hIns reduced the cytotoxicity of hIAPP by 22.7 ± 7.0%, 20.4 ± 11.2%, 24.1 ± 8.9%, and 28.1 ± 6.0%, respectively (p < 0.01) (Figure 5B). However, the presence of Glu-hIns exacerbated the cytotoxicity of hIAPP by 20.6 ± 4.4% (p < 0.001) (Figure 5B). SDS electrophoresis analysis suggested that Glu-hIns mainly existed as glycated insulin monomers or dimers while other forms of glycated insulin also showed high molecular weight cross-linked insulin (Figure 2D). Samples with high molecular weight (cross-linked

insulin

molecules)

or

low

molecular

weight

(glycated

insulin

monomers/dimers) were separated with ultracentrifugal filters (10 kD cutoff) and characterized by SDS-PAGE (Figure 5C). AGEs-specific fluorescence and fructosamine assays demonstrated that the low- or high-molecular weight samples both contain advanced glycation end products (Figure S3). Moreover, ThT fluorescence assays indicated that low molecular weight glycated samples showed comparable inhibitory effect with nonglycated insulin on hIAPP aggregation, while high molecular weight glycated samples exhibited stronger inhibitory capacity compared to nonglycated insulin (Figure S3). Meanwhile, no membrane disruption and cytotoxicity were found for these low- or high-molecular weight glycated samples (Figure S3). However, high molecular weight glycated samples reduced the membrane disruption and cytotoxicity of hIAPP 9


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more effectively than insulin, while low molecular weight glycated samples enhanced the cytotoxicity of hIAPP (Figure 5D & E). These results indicated that cross-linked insulin molecules protect cells from toxic hIAPP aggregation, whereas the glycated insulin monomers or dimers exacerbate the cytotoxicity of hIAPP.

EGS-induced cross-linking inhibits the aggregation and cytotoxicity of hIAPP To evaluate the influence of cross-linked insulin on the aggregation and cytotoxicity of hIAPP, EGS was used to induce chemical cross-linking of insulin, and lysozyme was used as a control. Successful cross-linking triggered by EGS was seen for insulin and lysozyme (Figure 6A). ThT fluorescence was performed to evaluate the effect of EGS-hIns/EGS-Lyz on hIAPP aggregation. The presence of human insulin prolonged the T50 and lag time to 5.53 ± 0.12 h (p < 0.001) and 3.39 ± 0.59 h (p < 0.001), respectively (Table S1). But lysozyme only decreased the maximum fluorescence intensity of hIAPP while showed no significant effect on T50 and lag time (Table S1). Notably, compared with non-crosslinking insulin and lysozyme, EGS-hIns and EGS-Lyz both exhibited stronger inhibitory effects on the aggregation of hIAPP. EGS-hIns prolonged the T50 and lag time to 5.77 ± 0.12 h and 3.41 ± 0.57 h, respectively; whereas EGS-Lyz almost abolished the aggregation of hIAPP (Figure 6B & Table S1). Under TEM, hIAPP formed typical ribbon like fibril structure, and the presence of EGS-hIns or EGS-Lyz inhibited the formation of fibrils (Figure 6C). Dye leakage results suggested the presence of human insulin, EGS-hIns or EGS-Lyz all decreased the membrane disruption ability of hIAPP (Figure 6D). Trypan blue results suggested the presence of human insulin, EGS-hIns or EGS-Lyz all decreased the cytotoxicity of hIAPP, while the control lysozyme showed no 10



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effect (Figure 6E). These data implicated that it was the glycation-induced oligomerization in MGO-, Frc-, Rib-hIns that inhibits the aggregation and cytotoxicity of hIAPP.

The C-terminus of insulin B chain is crucial for the glycation-exacerbated cytotoxicity of hIAPP A1Gly, B1Phe, B22Arg and B29Lys have been reported as crucial glycation sites of the insulin (29, 30). DKI (removal of B29Lys) and DKRI (removal of both B22Arg and B29Lys) were generated to study how they affect hIAPP aggregation and cytotoxicity. AGEs-specific fluorescence and fructosamine assays showed that DKI and DKRI could still be glycated by MGO, Glu, Frc and Rib, indicating A1Gly and B1Phe as the glycation site (Figure 7A & B). The SDS-PAGE results suggested that glycated DKI samples existed mainly as monomers and dimers, while glycated DKRI mainly existed as monomers (Figure 7C). This result indicated that glycation on LysB29 and ArgB22 are of vital importance to induce cross-linking during glycation. ThT fluorescence results showed that the inhibitory effects of glycated DKI and DKRI were similar to that of untreated insulin (Figure 7D & E, Figure S4). We investigated the cytotoxicity and membrane disruption capacity of hIAPP in the presence of glycated DKI or DKRI. Glycated DKI and DKRI displayed no obvious effect on -TC6 cells and membrane disruption (Figure S5). hIAPP alone induced 82.5 ± 3.4% dye leakage and decreased the cell viability by 34.9 ± 3.7% (Figure 7F). Compared with that of hIAPP, the presence of DKI and DKRI decreased the membrane disruption by 28.9 ± 11.4% and 29.6 ± 6.0% (p < 0.05) (Figure 7F) and decreased the cytotoxicity of 11


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hIAPP by 19.7 ± 12.5% and 18.7 ± 9.1% (p < 0.01) (Figure 7G). Notably, among nonglycated and Glu-, MGO-, Frc-, Rib-glycated DKI and DKRI, no significant difference were observed on their effects on hIAPP-induced dye leakage and decreased cell viability (Figure 7F & G). Removal of LysB29 abolished the cytotoxicity-promoting effect of glycated human insulin, which implicated that the modification of B29Lys may be important in the pathogenesis of T2DM.

Discussion Insulin, which could reduce the blood glucose level, has been reported to stabilize hIAPP in monomers, thus inhibiting the kinetics and cytotoxicity of hIAPP aggregation (21). In T2DM patients, long-term hyperglycemia results in glycation of ~10% total insulin (27, 28). However, the effects of glycated insulin on hIAPP aggregation and cytotoxicity remain unclear. In this work, we found four monosaccharides (glucose, methylglyoxal, fructose and ribose) glycated insulin and the resulting products attenuated the aggregation of hIAPP (Figures 3-5). Notably, high molecular weight glycated insulin (cross-linked insulin) and low molecular weight glycated insulin (insulin monomers/dimers) seem to play different roles. Cross-linked insulin molecules alleviated the aggregation, membrane disruption and cytotoxicity of hIAPP, while glycated insulin monomers/dimers exacerbated the cytotoxicity of hIAPP (Figure 5). It has been reported that the most abundant form of glycated insulin in the plasma of T2DM patients is monoglycated monomers (27), which is consistent with the much lower physiological concentration of glucose compared with that used for in vitro experiments, it is reasonable to speculate that the glycated insulin in 12



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vivo may exist mainly as low molecular weight forms, which may thus contributing to the development of T2DM through affecting the cytotoxicity of hIAPP as our results suggested. B22Arg and B29Lys have been reported as crucial glycation sites of insulin, the removal of which attenuate insulin glycation (29, 30). Our observations that glycated DKI (removal of B29Lys) and DKRI (removal B22Arg and B29Lys) existed mostly as monomers/dimers, and did not exacerbate the cytotoxicity of hIAPP (Figure 7), suggest that B29Lys plays center roles in glycation and the resulted crosslinking of insulin, as well as affects the cytotoxicity of hIAPP. In summary, our results suggest a potential vicious cycle in T2DM. Long-term hyperglycemia in T2DM results in insulin glycation, and glycated insulin monomers or dimers exacerbate the cytotoxicity of hIAPP, thus damaging β-cells and deteriorating T2DM. However, the present study is limited by lacking in vivo studies that directly demonstrate glycation of insulin in -cells in hyperglycemic condition and its association with hIAPP aggregation. It will be highly interesting in future studies using hIAPP transgenic mouse or human pancreas samples of diabetic patients to confirm the physiological interaction of glycated insulin and hIAPP. Moreover, it will be interesting to find out whether hyperglycemia-induced glycation participates in the pathogenesis of other amyloidosis diseases.

Materials and Methods Materials Human insulin (hIns, catalog number 91077C) was obtained from Sigma-Aldrich (St. 13


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Louis, MO, USA). hIAPP was purchased from Shanghai Science Peptide Biological Technology (Shanghai, China). ThT, NBT, 1-DMF, cholesterol and ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) (EGS) were obtained from Sigma-Aldrich (St. Louis, USA). POPG (2-Oleoyl-1-palmitoyl-sn-glycerol-3-phosphorac

(1-glycerol)

sodium

salt)

and

POPC

(1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphocholine) were obtained from Avanti Polar Lipids (Alabaster, Alabama, USA). Amicon Ultra-15 centrifugal filter was purchased from Millipore (Massachusetts, USA). Pancreatic  cells derived from insulinoma mouse (-TC6) was obtained from the China Center for Type Culture Collection (CCTCC). All other chemicals were of the highest grade available.

Protein preparation Protein preparation is conducted as previously reported (51). Zinc-free hIns was prepared on a Hitachi L-2000 HPLC system (Hitachi, Tokyo, Japan) with an Apollo C18 column (Welch Materials, Maryland, USA). Desoctapeptide-(B23–B30) insulin (DKI) was prepared as we previously described (17). Briefly, trypsin (5 mg/mL) was mixed with hIns solution to reach hIns to trypsin molar ratio of 25:1, then the mixture was incubated at 37 °C for 12 h followed by HPLC purification. Carboxypeptidase B (CPB) which catalyzes the hydrolysis of Lys/Arg from the C-terminus was used to prepare desnonapeptide-(B22-B30) insulin (DKRI) from DKI. The stock solution of CPB (0.5 mg/mL) was prepared with 50 mM NaAc with 1 M NaCl (pH 5.0) containing 0.01% NaN3. DKI (1 mg/mL) was dissolved in 10 mM HEPES (pH 7.6) for digestion, CPB was added to a final concentration of 2 g/mL (DKI to CPB 500:1, w/w). After incubation at 14



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37 °C for 6 h, the solution was filtered through a 0.22 m Millipore filter, and then purified by HPLC with a Kromasil C18 semi-preparative reverse-phase column (NY, USA). Mobile phases were water (A) and acetonitrile (B), a linear gradient 30–34% B for 34 min was used. The products were confirmed by matrix-assisted laser desorption ionization mass (MALDI-TOF).

Glycation 130 M hIns/DKI/DKRI was incubated with 5 mM MGO for 2 days as we previously described (26), or with 1 M glucose/fructose/ribose for 7 days as reported (52). After sample harvesting, dialysis was immediately performed to remove unconjugated free sugars and stored in -80 °C. All samples were filtered with a 0.22 μm Millipore filter before use.

EGS induced chemical cross-linking EGS was dissolved in DMSO (1 mg/mL) as stock solution. 80 M insulin or lysozyme was mixed with EGS (molar ratio of EGS to protein, 50:1) for 12 h and gently rocked with a rotary shaker at room temperature. Harvested samples were dialyzed against 10 mM PBS to remove extra EGS, and then stocked in -20 °C before use.

AGEs-specific fluorescence Glycated samples were diluted 30-times with phosphate buffer saline (pH 7.4) to measure AGEs-specific fluorescence on a Hitachi FL-2700 fluorometer (Hitachi, Tokyo, Japan). The excitation and emission wavelengths were set at 370 nm and 440 nm, respectively. 15


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All experiments were performed at least three times.

Fructosamine measurement The concentration of fructosamine was quantified using the nitroblue-tetrazolium (NBT) assays as we previously described (26). Briefly, 0.1 mg/mL glycated samples were incubated with 0.25 mM NBT in carbonate buffer (100 mM, pH 10.4) at 37 °C for 1 h. Absorbance at 530 nm was measured, and 1-deoxy-1-morpholino-fructose (1-DMF) was used as standard sample.

SDS-PAGE electrophoresis The cross-linking property of glycated samples was studied by gel electrophoresis. Briefly, 10 μL glycated samples were mixed with 5 μL loading buffer, followed by denaturation at 95 °C for 10 min. The samples were separated on a 20% tricine-urea gel and visualized by a fast silver staining kit (Beyotime, Jiangsu, China).

Circular dichroism (CD) CD spectra assays were conducted as previously reported (53). CD spectra were corded at 25 °C under a constant flow of N2 by using a JASCO-810 spectropolarimeter (JASCO, Japan). Sample solutions containing 20 M hIAPP (dissolved in 25 mM PBS and 50 mM NaCl) in the presence or absence of glycated hIns and/or insulin were recorded for CD spectra. Data were recorded from 260 to 200 nm with a 1 mm path length, 2 nm bandwidth, 200 nm/min scanning speed and 1 s response. The reported spectrum for each sample was the average of at least 3 measurements and the background was subtracted 16



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using appropriate controls as previously described (54).

Thioflavin-T (ThT) fluorescence assays Freshly prepared hIAPP solution (20 M) was incubated at 25 °C for amyloid formation in the absence or presence of glycated insulin at equal molar ratio. ThT fluorescence assays were performed on a Hitachi FL-2700 fluorometer at designated time points. The final assays solution contains 25 mM PBS (pH 7.4), 50 mM NaCl and 20 M ThT. ThT fluorescence was recorded at 482 nm with an excitation wavelength of 450 nm. Glycated human insulin was measured as the controls. All experiments were performed in triplicates, and the lag times were calculated as we previously described (55).

Transmission electron microscopy (TEM) TEM was performed as described (56). Briefly, 5 L incubated sample (20 M) was applied onto a 300-mesh formvar-carbon coated copper grid, followed by staining with 1% fresh prepared uranylformate. Samples were air-dried and observed under a transmission microscope (Hitachi, Tokyo, Japan) operating at an accelerating voltage of 100 kV.

Dye leakage assays POPC/POPG/ cholesterol (w/w 5:1:4) was dissolved in chloroform at a concentration of 5 mg/mL. Chloroform was removed under a stream of N2, and freeze-dried overnight. Multilamellar vesicles were made by mixing dry lipid films with 25 mM PBS (pH 7.4) containing 40 mM carboxyfluorescein. PD-10 columns (Sangon, Shanghai, China) were 17


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used to remove nonencapsulated carboxyfluorescein (56). Lipid vesicles containing carboxyfluorescein were diluted in 25 mM PBS (pH 7.4) for fluorescence measurements. Samples were added to lipid vesicles at a final concentration of 0.33 M, after 30 s incubation, the fluorescence was measured with excitation and emission wavelength set at 493 nm and 518 nm, respectively. Lipid vesicles alone were used as the baseline and the signals of lipid vesicles treated with 0.2% (v/v) TritonX-100 (for complete membrane leakage) were set as a positive control of 100%. All measurements were repeated at least three times.

Glycated insulin separation Glycated samples were loaded onto a 10 kD centrifugal filter (Massachusetts, USA) and centrifuged at 4000 g for 1 h to obtain low molecular weight fractions (< 10 kD; L) and high molecular weight fractions (> 10 kD; H). The concentration of different fractions was measured by BCA assay and diluted to suitable concentrations before use.

Dynamic light scattering (DLS) analysis DLS was performed by using a zeta pals potential analyzer (Brookhaven Instruments, New York, USA) as previously reported (57). Samples containing 20 M hIAPP prepared in 25 mM PBS and 50 mM NaCl, in the presence or absence of insulin, glycated insulin or EGS cross-linked samples, were incubated at 25 °C. Insulin, glycated insulin or EGS cross-linked samples alone in 25 mM PBS buffer (containing 50 mM NaCl) were used as the controls. All samples were scanned for 3 times (30 s per scan), the mean particle size and multimodal size distribution were recorded every 15 min. 18



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Trypan blue assays -TC6 cells (mouse insulinoma  cell) were cultured in DMEM high glucose medium (containing 15% FBS, 1% penicillin-streptomycin solution and 1% sodium pyruvate). Before experiment, cells were seeded in a 12 well plate at a density of 1*105 cells per well and incubated overnight. Cell-culture medium was then replaced with fresh medium containing hIAPP (0.5 M) with equal molar of glycated hIns or hIns/DKI/DKRI for further 36 h incubation. Cells treated with glycated hIns or hIns/DKI/DKRI were used as controls. After incubation, cells were harvested and resuspended with PBS, stained with trypan blue for 3 min, then applied to a cytometer (Nexcelom, Massachusetts, USA) to count live cells. All experiments were repeated at least three times.

Data analysis All data were expressed as mean ± SEM. Each treatment was repeated at least three times. Data were analyzed by the nonparametric Kruskal-Wallis test followed by the Mann-Whitney test. p < 0.05 was considered significant.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the internet at http://pubs.acs.org at DOI:

Amyloidogenic properties of hIAPP in the presence or absence of non-crosslinked and 19


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EGS-crosslinked insulin and lyszome (Table S1). TEM images of human insulin, glycated insulin samples and EGS-crosslinking samples (Figure S1). The membrane disruption capacity and cytotoxicity of glycated insulin samples (Figure S2). AGEs-specific fluorescence intensities and fructosamine levels of low- or high-molecular weight fractions of glycated insulin samples, the effects of low- or high-molecular weight fractions of glycated insulin samples on the aggregation, membrane disruption capacity and cytotoxicity of hIAPP (Figure S3). The effects of hIns, DKI and DKRI on the aggregation of hIAPP (Figure S4). Membrane disruption capacity and cytotoxicity of glycated DKI and DKRI (Figure S5).

Author Information Corresponding Author *E-mail: [email protected] Author Contributions K.H. and L.Z. designed the research; L.M., C.Y, L.Q.H. and Y.L. performed experiments;; L.M., C.Y, L.Q.H., C.C., Y.C.C. and B.C. analyzed data; L.M., C.Y, L.Q.H., Y.C.C. and K.H. wrote the manuscript. Notes The authors declare no competing financial interest.

Acknowledgements 20



Page 22 of 38

This work was supported by the Natural Science Foundation of China (NSFC No. 31471208, 31671195 and 31871381), the Natural Science Foundation of Hubei Province (2014CFA021), the Front Youth Academic Team Program of HUST, and Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College, HUST. The work is technically supported by the Analytical and Testing Core of College of Life Sciences, Wuhan University, and by the Analytical and Testing Center of Huazhong University of Science and Technology.

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Table 1. Amyloidogenic properties of hIAPP in the presence or absence of native and glycated human insulin. T50 (h)

Lag Time (h)

hIAPP 2.43 ± 0.26 1.43 ± 0.12 hIAPP + hIns 5.17 ± 0.70** 3.34 ± 0.95* hIAPP + MGO-hIns 5.87 ± 0.79** 3.88 ± 1.20* hIAPP + Glu-hIns 6.53 ± 0.29*** 4.34 ± 0.32*** hIAPP + Frc-hIns 6.37 ± 0.42*** 4.14 ± 0.87* hIAPP + Rib-hIns 6.77 ± 0.21*** 5.07 ± 0.58*** *p < 0.05, **p < 0.01, ***p < 0.001, compared to hIAPP group.

26



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Figure Legends Figure 1. (A) Primary structure of human insulin (hIns), known possible glycated sites are colored in red and marked with asterisk. (B) 3D structure of hIns/DKI/DKRI, possible glycated sites are marked with stick. (C) Main steps in AGE formation. (D) Chemical structure of MGO, Glu, Frc and Rib. Figure 2. Characteristics of hIns glycated by Glu, MGO, Frc and Rib, respectively. (A) AGEs-specific fluorescence intensities of four glycated insulin samples before/after dialysis. (B) Fructosamine levels of four glycated insulin samples before/after dialysis. (C) CD spectra of hIns and four glycated insulin samples. (D) SDS electrophoresis analysis of glycated samples before or after dialysis. **p < 0.01, ***p < 0.001. Figure 3. Inhibitory effects of native and glycated insulin on the amyloid formation of hIAPP. (A) ThT fluorescence results of hIAPP incubated with or without different native and glycated insulin. (B) TEM images of fibrils formation of hIAPP treated with or without native and glycated insulin. Figure 4. Particle distributions of hIAPP co-incubated with native and glycated insulin. (A) Dynamic light scattering detected hIAPP particle distributions in the presence or absence of native and glycated insulin. (B) Mean diameter distributions of hIAPP particles in the presence or absence of native and glycated insulin. 10,000 represents the detection limit of the instrument. ***p < 0.001. Figure 5. Membrane disruption and cytotoxicity of hIAPP co-incubated with different fractions of glycated insulin. (A) Dye leakage assays of hIAPP in the presence of 27


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glycated insulin. (B) Trypan blue assays of hIAPP in the presence of glycated insulin. (C) SDS electrophoresis analysis of high molecular weight (H) and low molecular weight (L) fractions of glycated insulin samples. (D) Dye leakage assays of hIAPP in the presence of high molecular weight (H) and low molecular weight (L) fractions of glycated insulin. (E) Trypan blue assays of hIAPP in the presence of high molecular weight (H) and low molecular weight (L) fractions of glycated insulin. *p < 0.05, **p < 0.01, ***p < 0.001, compared to hIAPP group. Figure 6. Characteristics of EGS triggered cross-linking of insulin and lysozyme (Lyz) on hIAPP aggregation and cytotoxicity. (A) SDS electrophoresis analysis of EGS triggered cross-linking. (B) ThT fluorescence results of hIns, Lyz, EGS-hIns and EGS-Lyz incubated with hIAPP. (C) TEM images of fibrils formation of hIAPP incubated with hIns, Lyz, EGS-hIns, EGS-Lyz. (D) Dye leakage assays of hIAPP incubated with hIns, Lyz, EGS-hIns and EGS-Lyz. (E) Trypan blue results of hIAPP treated with hIns, lyz, EGS-hIns, and EGS-Lyz. *p < 0.05, **p < 0.01, ***p < 0.001, compared to hIAPP group. Figure 7. Effects of glycated DKI/DKRI on the aggregation, membrane disruption and cytotoxicity of hIAPP. (A) AGEs-specific fluorescence intensities of glycated DKI/DKRI. (B) Fructosamine levels of four glycated DKI/DKRI. (C) SDS electrophoresis analysis of glycated DKI/DKRI. (D) ThT fluorescence assays of hIAPP incubated with glycated DKI. (E) ThT fluorescence assays of hIAPP incubated with glycated DKRI. (F) Trypan blue assays of -TC6 cells treated with hIAPP in the presence of glycated DKI/DKRI. (G) 28



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Dye leakage assays of hIAPP in the presence of glycated DKI/DKRI. *p < 0.05, **p < 0.01, ***p < 0.001, compared to hIAPP group. Figure 8. Working model for a potential vicious cycle of T2DM

29


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Figure 1. (A) Primary structure of human insulin (hIns), known possible glycated sites are colored in red and marked with asterisk. (B) 3D structure of hIns/DKI/DKRI, possible glycated sites are marked with stick. (C) Main steps in AGE formation. (D) Chemical structure of MGO, Glu, Frc and Rib.



Figure 2. Characteristics of hIns glycated by Glu, MGO, Frc and Rib, respectively. (A) AGEs-specific fluorescence intensities of four glycated insulin samples before/after dialysis. (B) Fructosamine levels of four glycated insulin samples before/after dialysis. (C) CD spectra of hIns and four glycated insulin samples. (D) SDS electrophoresis analysis of glycated samples before or after dialysis. **p < 0.01, ***p < 0.001.


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Figure 3. Inhibitory effects of native and glycated insulin on the amyloid formation of hIAPP. (A) ThT fluorescence results of hIAPP incubated with or without different native and glycated insulin. (B) TEM images of fibrils formation of hIAPP treated with or without native and glycated insulin.



Figure 4. Particle distributions of hIAPP co-incubated with native and glycated insulin. (A) Dynamic light scattering detected hIAPP particle distributions in the presence or absence of native and glycated insulin. (B) Mean diameter distributions of hIAPP particles in the presence or absence of native and glycated insulin. 10,000 represents the detection limit of the instrument. ***p < 0.001.


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Figure 5. Membrane disruption and cytotoxicity of hIAPP co-incubated with different fractions of glycated insulin. (A) Dye leakage assays of hIAPP in the presence of glycated insulin. (B) Trypan blue assays of hIAPP in the presence of glycated insulin. (C) SDS electrophoresis analysis of high molecular weight (H) and low molecular weight (L) fractions of glycated insulin samples. (D) Dye leakage assays of hIAPP in the presence of high molecular weight (H) and low molecular weight (L) fractions of glycated insulin. (E) Trypan blue assays of hIAPP in the presence of high molecular weight (H) and low molecular weight (L) fractions of glycated insulin. *p < 0.05, **p < 0.01, ***p < 0.001, compared to hIAPP group.



Figure 6. Characteristics of EGS triggered cross-linking of insulin and lysozyme (Lyz) on hIAPP aggregation and cytotoxicity. (A) SDS electrophoresis analysis of EGS triggered cross-linking. (B) ThT fluorescence results of hIns, Lyz, EGS-hIns and EGS-Lyz incubated with hIAPP. (C) TEM images of fibrils formation of hIAPP incubated with hIns, Lyz, EGS-hIns, EGS-Lyz. (D) Dye leakage assays of hIAPP incubated with hIns, Lyz, EGS-hIns and EGS-Lyz. (E) Trypan blue results of hIAPP treated with hIns, lyz, EGS-hIns, and EGS-Lyz. *p < 0.05, **p < 0.01, ***p < 0.001, compared to hIAPP group.


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Figure 7. Effects of glycated DKI/DKRI on the aggregation, membrane disruption and cytotoxicity of hIAPP. (A) AGEs-specific fluorescence intensities of glycated DKI/DKRI. (B) Fructosamine levels of four glycated DKI/DKRI. (C) SDS electrophoresis analysis of glycated DKI/DKRI. (D) ThT fluorescence assays of hIAPP incubated with glycated DKI. (E) ThT fluorescence assays of hIAPP incubated with glycated DKRI. (F) Trypan blue assays of β-TC6 cells treated with hIAPP in the presence of glycated DKI/DKRI. (G) Dye leakage assays of hIAPP in the presence of glycated DKI/DKRI. *p < 0.05, **p < 0.01, ***p < 0.001, compared to hIAPP group.



Figure 8. Working model for a potential vicious cycle of T2DM


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Glycated insulin exacerbates the cytotoxicity of human islet amyloid

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