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Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic because they activate apoptotic signaling pathways. First, fibril formation by glucagon in acidic solution is discussed in light of morphological and structural changes during elapsed time. Second, we provide kinetic analyses using a two-step autocatalytic reaction mechanism; the first step is a homogeneous nuclear formation process, and the second step is an autocatalytic heterogeneous fibril elongation process. Third, the processes of fibril formation by glucagon in a membrane environment are discussed based on the structural changes in the fibrils. In the presence of bicelles in acidic solution, glucagon interacts with the bicelles and forms fibril intermediates on the bicelle surface and grows into elongated fibrils. Glucagon-dimyristoylphosphatidylcholine (DMPC) bilayers in neutral solution mimic the environment for fibril formation by glucagon under near-physiological condition. Under these conditions, glucagon forms fibril intermediates that grow into elongated fibrils inside the lipid bilayer. Many days after preparing the glucagon-DMPC bilayer sample, the fibrils form networks inside and outside the bilayer. Furthermore, fibril intermediates strongly interact with lipid bilayers to form small particles.
Graduate School of Engineering, Yokohama National University, Yokohama, Japan
*Address all correspondence to: naito@ynu.ac.jp
1. Introduction
Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs, where it activates the glycogenolysis and gluconeogenic pathways, resulting in raised blood glucose levels [1, 2, 3]. Glucagon tends to form gel-like fibrillar aggregates in acidic condition [4]. These aggregates are cytotoxic due to the activation of apoptotic signaling pathways [5]. These fibrils are similar to those of other therapeutic peptides and proteins such as human calcitonin (hCT) [6] and insulin [7] and pathologically related fibrils such as prion [8], amylin (type 2 diabetes) [9], β-amyloid (Alzheimer’s disease) [10], and polyglutamine [11].
Some non-fibrillar proteins and peptides have been observed by electron microscopy to form amyloid fibrils with similar morphologies [12]. Several characteristics of these fibrils are related to the misfolding of proteins, leading to severe conditions such as fibril deposits in the brains of Alzheimer’s disease patients [13] and in the pancreas of patients with type 2 diabetes [9].
Kinetic analyses of fibril formation by the therapeutic peptide human calcitonin indicate that hCT molecules associate to form fibril intermediates via a two-step autocatalytic reaction mechanism. The first step of kinetic reaction (rate constant, k1) is a homogeneous reaction from micelle-like oligomers to fibril intermediates. These intermediates react with monomeric molecules to elongate into longer fibrils via a heterogeneous fibril elongation process (rate constant, k2) [14, 15, 16, 17]. Elucidating the molecular structure of amyloid fibrils is important for understanding the mechanism of self-aggregation, but it is difficult to determine high-resolution molecular structures using typical spectroscopic methods because fibrils are heterogeneous solids. Solid-state NMR spectroscopy has demonstrated advantages for the conformational determination of Alzheimer’s amyloid β-peptides (Aβ), which mainly comprise 40 or 42 amino acid residues and are the main component of the amyloid plaques found in Alzheimer’s disease patients [12, 18]. Both the intra-chain conformation of the Aβ molecule in fibrils and their intermolecular alignment have been analyzed to explore the mechanism of molecular association underlying the formation of Aβ (1–40) [19, 20, 21] and the more toxic Aβ (1–42) [22, 23, 24] fibrils.
The primary structure of the glucagon peptide is His1-Ser-Gln-Gly-Thr5-Phe-Thr-Ser-Asp-Tyr10-Ser-Lys-Tyr-Leu-Asp15-Ser-Arg-Arg-Ala-Gln20-Asp-Phe-Val-Gln-Trp25-Leu-Met-Asn-Thr-OH.
An X-ray crystallographic study showed that glucagon adopts a trimeric α-helix structure stabilized by hydrophobic interactions between molecules related by threefold symmetry [25], whereas a solution NMR study showed that glucagon in dilute aqueous solution may not form a specific structure, with the exception of the 22–25 region [26]. The secondary structure of glucagon in the presence of dodecylphosphocholine micelles comprises three turns of an irregular α-helix formed by residues 17 to 29 near the C-terminus, a stretch of extended polypeptide chain from residues 14 to 17, an α-helix-like turn formed by residue 10 to 14, and another extended region from residue 5 to 10 [27].
Fibril formation by glucagon molecules was observed by Beaven et al. in undisturbed aqueous solution at pH 2 [4]. The viscosity initially increased and a birefringent gel is formed. With time, a precipitate appeared comprising long fibrils, as determined using electron microscopy. Infrared spectra of the gel, solid film, and precipitate showed that in all these states, glucagon is in the form of antiparallel β-sheet chains [5, 28]. Kinetic analysis of fibril formation by glucagon under acidic conditions demonstrated a complex fibrillation mechanism in which suitable changes in the fibrillation condition can alter the type of fibril formed or result in the formation of a mixture of several types of fibrils [29, 30]. Furthermore, the fibrils come in two forms: one composed entirely of glucagon monomers and the other entirely of glucagon trimers [31]. Studies of fibril formation typically use acidic pH solutions because of the low solubility of glucagon in neutral solution.
Understanding the cytotoxicity of amyloid-forming peptides requires investigating the interaction of these peptides with membranes because lipid bilayer components dramatically alter most aspects of amyloid aggregation [32, 33, 34, 35]. We previously reported glucagon fibrillation in the presence of dimyristoylphosphatidylcholine/1,2-dihexanoyl-sn-glycero-3-phophocholine (DMPC-DHPC) bicelles in acidic solution. The glucagon structure in the fibril in the presence of these bicelles is different from that in their absence [36]: the N- and C-termini both change from α-helix to β-sheet in acidic solution, while the N-terminus remains in an α-helical conformation, whereas the C-terminus changes from α-helix to β-sheet in the presence of bicelles. The nucleation rate is slower, and the fibril elongation rate is faster in acidic solution than in the presence of bicelles.
In neutral conditions, glucagon molecules are incorporated into lipid bilayers above the phase transition temperature, and the properties of the lipids appear to remain unperturbed. Below the phase transition temperature, glucagon forms discoidal particle with DMPC [37, 38] and induces closer packing of the phospholipid bilayers [39]. Similar peptide-lipid interaction to form discoidal particles below the phase transition temperature is seen in the melittin-DMPC bilayer system [40, 41].
The time course behavior of glucagon fibril formation inside a DMPC bilayer under neutral conditions, which approximates the physiological condition, and the kinetic behavior of glucagon under these conditions have been investigated to understand the fibrillation process under near-physiological conditions [42].
2. Fibril formation by glucagon in acidic solution
Gel formation by glucagon in the β-sheet conformation in acid solutions is a relatively slow process at room temperature [4] and can be followed by observing the change in viscosity as shown in Figure 1. The most marked feature of the change in viscosity in these time profiles is the presence of a substantial lag phase during which oligomeric nuclei are likely formed and function as initiation sites. These phenomena were confirmed by adding a small seed of preformed glucagon gel on the end of a wire into a viscometer containing a fresh acidic glucagon solution. The viscosity increased immediately with essentially no lag (Figure 1a). After a prolonged reaction time, the viscosity began to decrease and fibrils sometimes precipitated. These fibrils represent a variant of the β-structure of glucagon.
Figure 1.
Time course behavior of glucagon aggregation in 0.01 M hydrochloric acid at 26°C and a glucagon concentration of 2.5 mg/ml. (a) Time course of reduced viscosity (msp/c); glucagon with no addition (○); glucagon seeded with preformed gel (□); glucagon solution containing 5% by volume dioxin (●). (b) Effect of glucagon concentration at 4 mg/ml (□); 2.5 mg/ml (○); and 1 mg/ml (●). (c) Effect of temperature on the aggregation rate of glucagon at a glucagon concentration of 2.5 mg/ml at 26°C (○); 30°C (□); and 35°C (∇) (ref. [4]).
Fibril formation strongly depends on the peptide concentration, proceeding very slowly at less than about 1.5 mg/ml and occurring more readily at higher concentration (Figure 1b). Increasing the ionic strength of the solution results in both an increasing aggregation rate and more rapid production of fibrils. The change in viscosity with time in even 0.01 M sodium chloride occurs much more rapidly than in the absence of salt, and viscosity decreases quickly.
The effect of temperature on the polymerization rate is also very marked. Both the aggregation rate and the size of the aggregates as reflected in the maximal values of the reduced viscosity show a strong temperature maximum around 30°C (Figure 1c). An addition of 5% (v/v) of the nonaqueous solvent dioxin completely inhibits the aggregation (Figure 1a).
Transmission electron microscopy (TEM) time-elapsed pictures were obtained during fibril formation by glucagon dissolved in 0.015 M acetic acid solution (18 mg/ml) at pH 3.3 [36]. TEM pictures were measured approximately 2 hrs, then 1 week, and 6 months after the dissolution of glucagon (Figure 2). A small number of spherical-shaped fibril intermediates appeared after approximately 2 hrs, as shown in Figure 2a. After 1 week, the number of spherical fibril intermediates had increased, and elongated fibrils had appeared (Figure 2b). After 6 months, long mature fibrils about 10 nm in diameter were observed, and the spherical fibril intermediates had completely disappeared (Figure 2c).
Figure 2.
Transmission electron micrographs of glucagon (18 mg/ml) in 0.015 M acetic acid solution at pH 3.3. (a) Taken approximately 2 hrs after the dissolution of glucagon. The bar indicates 20 nm. (b) Taken 1 week after dissolution of glucagon. The bar indicates 50 nm. (c) Taken 6 months after dissolution of glucagon. The bar indicates 50 nm (ref. [36]).
The α-helical content of aged glucagon at 5.0 mg/ml decreased significantly to approximately 1%. The CD spectrum of a β-sheet structure typically shows an intense positive band at 198 nm and a negative band at 218 nm [5]. The CD spectral pattern of aged glucagon was that of a β-sheet structure, indicating a conformational transition from α-helical to β-sheet structure under these conditions.
The FTIR spectrum of glucagon immediately after dissolution showed a low-intensity β-sheet band at 1620–1630 cm−1, and aging resulted in progressively greater amounts of β-sheet [28]. Deconvolution and curve fitting of the amide I band showed that unaged glucagon contained 54% α-helix/random coil structure and 2% β-sheet structure, whereas aged glucagon comprised 22% α-helix/random coil structure and 49% β-sheet structure.
Detailed kinetic, spectroscopic, and morphological studies have revealed that glucagon can form several types of fibrils that differ at the level of molecular packing of the peptide [29, 30, 31, 43, 44, 45, 46]. Each type forms through distinct nucleation-dependent aggregation pathways influenced by the solution conditions and can be self-propagated by seeding. Type A fibrils that form at high glucagon concentration (>5 mg/ml, pH 2.5) represent the least stable fibril type with a low melting midpoint (Tmapp < 32°C) and single protofilament fibrils observable by TEM (Figure 3a). Type B (Bunagitated and Bagitated) fibrils form under low glucagon concentration (<0.5 mg/ml, pH 2.5). Type Bunagitated fibrils grow by branching in the absence of agitation and appear as branched twisted fibrils by TEM (Figure 3b). Type Bagitated glucagon fibrils form when the solution is agitated, suggesting that agitation breaks the fibril creating more free fibril ends that align as parallel pairs (Figure 3c). Type D fibrils grow under low glucagon concentration (<0.5 mg/ml, pH 2.5) in the presence of 150–250 mM Cl− and are twisted and tightly packed (Figure 3d). Type S fibrils grow under low glucagon concentration (<0.5 mg/ml, pH 2.5) in the presence of 1 mM Na2SO4 (7:1 ratio with glucagon) and appear as twisted mature fibrils by TEM (Figure 3e).
Figure 3.
Electron microscope image of morphology of different types of glucagon fibrils. The scale bar is 50 nm. (a) Type A: The glucagon concentration, >5 mg/ml, 50 mM glycine, pH 2.5, low agitation. (b) Type Bunagitated: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, pH 2.5, low agitation. (c) Type Bagitated: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, vigorous agitation. (d) Type D: Glucagon concentration, <0.5 mg/ml, 50 mM glycine, pH 2.5+, 150–250 mM Cl−. (e) Type S: Glucagon concentration, <0.5 mg/ml, 0.01 N HCl, 1 mM SO42− (ref. [30]).
Solid-state 13C NMR spectra were observed for 18 mg/ml [1-13C]Gly4 and [3-13C]Ala19-glucagon in 0.015 M acetic acid solution, pH 3.3 [36]. The 13C direct excitation with dipolar decoupling and magic angle spinning (DD-MAS) signal indicates monomeric glucagon, and the 13C cross polarization with magic angle spinning (CP-MAS) signal indicates fibril glucagon. The DD-MAS spectra (Figure 4A and C) of [1-13C]Gly4 and [3-13C]Ala19 exhibit signals at 171.7 and 16.4 ppm, respectively, consistent with the monomeric state and indicate that the region near the Gly4 and Ala19 residues forms α-helix structures, as shown by the conformationally dependent chemical shift values [47, 48, 49]. The experimentally determined chemical shift values and secondary structures are summarized in Table 1. The 13C CP-MAS spectra of [1-13C]Gly4 and [3-13C]Ala19 (Figure 4B and D) of glucagon in the fibril state exhibit signals at 167.2 and 21.0 ppm, respectively, and indicate that the vicinities of Gly4 and Ala19 form β-sheet structures.
Figure 4.
13C DD-MAS and CP-MAS NMR spectra of [1-13C]Gly4 (A and B) and [3-13C]Ala19 (C and D) of [1-13C]Gly4 and [3-13C]Ala19-glucagon in acetic acid solution at pH 3.3. Schematic structures of glucagon in the monomeric and fibril states (right panels) (ref. [36]).
Fibril formation condition
Fibril type
[1-13C]Gly4 (structure)
[3-13C]Ala19 (structure)
Ref.
Acidic solution (0.015 M acetic acid solution pH 3.3)
Structural transitions during glucagon fibrillation in various conditions as determined by conformation-dependent 13C chemical shifts (ppm)*.
The structure around each amino acid residues was determined by comparing the experimentally obtained 13C chemical shift values (δiso) with typical 13C chemical shift values (δiso) of α-helix and β-sheet, reported as 171.6 and 168.5 for [1-13C]Gly and 14.9 and 19.9 for [3-13C]Ala, respectively [47, 48, 49].
Conformationally dependent chemical shift values [47, 48, 49] clearly indicate that the N-terminus of monomeric glucagon forms an α-helix structure, the center portion forms a random coil, and the C-terminus forms an α-helix structure, as shown in Table 1 and Figure 4 (right panels) in acetic acid solution. When the glucagon monomer aggregates to form fibrils, the N-terminal and C-terminal regions change from an α-helix to a β-sheet as seen with other amyloid-forming peptides such as human calcitonin [14] in acetic acid solution.
The cytotoxicity of the glucagon fibril was assessed by exposing PC12 and NIH-3 T3 cells to 0.1–100 μM peptide aggregate for 72 hrs followed by cell viability determination under the WST-8 assay and released lactate dehydrogenase (LDH) [5]. A significant decrease in cell viability was observed in cultures exposed to 10–100 μM aged glucagon (P < 0.01) but not in cultures treated with 100 μM nonaged glucagon. It was determined whether the loss of cell viability was due to cell death by measuring the release of LDH. Treatment with 10 μM aged glucagon induced a significant increase in LDH release compared to control, whereas no significant increase in LDH release was observed in cultures treated with 100 μM nonaged glucagon or 1 μM or lower aged glucagon. Thus, glucagon fibrils were found to be highly toxic to PC12 cells, similar to the case of aged prion protein fragment (PrP)106–126 [50] and β-amyloid (Aβ)1–42 [51] (>10 mM). Aged salmon calcitonin also displayed significant cytotoxicity in PC12 cells, whereas nonaged salmon calcitonin did not induce significant cell death [5].
Next, signaling pathways for the cytotoxicity of peptide fibrils were investigated [5]. Caspase-3 activation is required for the early stages of apoptosis that include DNA fragmentation and morphological changes. To determine whether aged glucagon induces caspase-3 activation in PC12 cells, cells were exposed to 50 μM aged glucagon, and the caspase-3-like activities of the cell lysates were measured by cleavage of the fluorometric caspase-3 substitute Z-DEVD-rhodamine 110. The activity increased prior to the loss of membrane integrity, and 24 hrs after incubation, maximum caspase-3 activity was detected (160% of the control level). In contrast, no significant elevation of casepase-3 activity was observed in cells treated with 50 μM nonaged glucagon. These results indicate that the exposure of PC12 cells to peptide fibrils induces a rapid (within 24 hrs) and significant elevation in casepase-3 activity prior to the loss of cell viability 72 hrs after exposure.
In summary, the misfolding of the therapeutic peptide glucagon generates amyloidogenic fibrils, leading to cytotoxicity mediated by the activation of the apoptotic enzyme caspase-3 in vitro.
4. Kinetic analysis of the glucagon fibrillation process
As shown in Figure 5, glucagon monomers (A) first aggregate to form weakly coupled oligomers (An) akin to the micelle state. Next, glucagon oligomers (An) form fibril intermediates (nuclei) (Bn) through a homogeneous nucleation process with a rate constant k1. Fibril intermediates (Bn) then react with monomer (A) to form elongated fibrils with a rate constant k2. This is called the inhomogeneous fibril elongation process. The B form plays a role in the catalysis of A to B, and therefore this is an autocatalytic reaction. Since the nucleation and elongation processes are rate-determining steps, fibril formation is a two-step autocatalytic reaction.
Figure 5.
Schematic of the glucagon fibril formation process in acidic solution. Several monomers (A) aggregate to form weakly coupled micelles (An). Micelles (An) form a fibril nucleus (Bn) through a homogeneous nucleation process with a rate constant k1. Fibril nuclei react with monomers (A) to form elongated fibrils (Bn + 1) with a rate constant k2. In this reaction, B acts as a catalyst to change the A form to the B form. Overall, this fibril formation reaction is a two-step autocatalytic reaction mechanism.
The rate constants of glucagon fibril formation were determined by observing the signal intensities of [1-13C]Gly4 in [1-13C]Gly4 and [3-13C]Ala19-glucagons by 13C CP-MAS NMR spectroscopy with time (Figure 6A and B). The signal intensities increased after a delay time. The increase in 13C CP-MAS signal intensities corresponds to the increase in fibril components, and thus we obtained the rate constants, k1 and k2, for the two-step autocatalytic reaction mechanism, in which k1 is the rate constant for the fibril nucleation process and k2 is the rate constant for the fibril elongation process [14].
Figure 6.
(A) Time course of changes in the 13C CP-MAS and DD-MAS signals of [1-13C]Gly-glucagon during the fibril formation processes [36]. (B) Plot of normalized CP-MAS signal intensity against elapsed time for glucagon fibril formation in acidic solution at pH 3.3 [36]. (C) Plot of normalized turbidity intensities against elapsed time for glucagon fibril formation in neutral solution (20% acetonitrile solution, pH 7.5) [42].
The first reaction step is homogeneous nuclear formation given by
Ano→k1Bno,E1
where Ano is the micelles formed by no number of A-form glucagon monomers and Bno is the fibrils formed by no number of B-form glucagon fibrils. The kinetic equation for Reaction (1) can be given by
df/dt1=k11−f,E2
where f is the fraction of B-glucagon fibrils in the system.
The second heterogeneous fibril elongation reaction can be given by k2
A+Bn→k2Bn+1,E3
where Bn and Bn + 1 are elongated fibrils with n and n + 1 number of B-form glucagons. The relevant kinetic equation is given by
df/dt2=k2af1−f,E4
where a is the initial concentration of glucagon. The overall kinetic equation can be given by
where f is the fraction of glucagon molecules in the fibril form at time t and ρ represents a dimensionless value describing the ratio of k1 (the rate constant for the first nucleation process) to k, namely, ρ = k1/k and k = ak2 (k2 is the rate constant for the second elongation process of the fibrils, and a is the initial peptide concentration) [14]. The best fits of Eq. (6) are shown in Figure 6B (solid lines), and the analyzed rate constants are summarized in Table 2. The k1 and k2 values were obtained experimentally from the intensity variation of the 13C CP-MAS NMR signals of [1-13C]Gly4 as shown in Figure 6B and Table 2 for glucagon in acidic solution. In comparison, rate constants of fibril formation were obtained for glucagon in neutral solution by plotting the turbidity of the solution against the elapsed time by using an established protocol [52] (Figure 6C).
Rate constants for glucagon fibril nucleation (k1) and fibril elongation (k2) for a two-step autocatalytic reaction mechanism under a variety of conditions.
The intensity of the 13C CP-MAS NMR signal of [1-13C]Gly4 was plotted against the elapsed time for glucagon in the presence of bicelles in acidic solution and for glucagon embedded inside lipid bilayers in neutral solution. The obtained rate constants are given in Table 2.
5. Fibril formation by glucagon in the presence of bicelles in acidic solution
In the presence of bicelles (DMPC-DHPC; 3:1), the N-terminus of glucagon forms an α-helix, the center portion forms a random coil, and the C-terminus forms an α-helix in the monomeric state (Table 1). In contrast to glucagon in acetic acid solution, the aggregation of monomers into the fibrils in the presence of bicelles results in the N-terminus maintaining an α-helix structure and the center portion remaining in a random coil structure, whereas the C-terminus changes from an α-helix to a β-sheet structure (Figure 7 and Table 1). There is therefore significant difference in the structural transition between monomer and fibril in the presence and absence of bicelles, since the N-terminus maintains an α-helix structure in the process of fibril formation in the presence of bicelles. This result suggests that the N-terminal portion of a glucagon fibril significantly interacts with the lipid bilayer surface.
Figure 7.
Schematic diagram of the fibrillation processes of glucagon in acetic acid solution (left) and acidic solution in the presence of bicelles (center). (A) Monomeric form. (B) Weakly coupled oligomer. (C) Fibril intermediate. (D) Elongated fibril. TEM pictures of glucagon fibril intermediates with ellipsoidal shapes and of disk-type bicelles are seen in the top right photo. An elongated fibril is seen attached by its end to a bicelle in the bottom right photo (ref. [36]).
The above findings provide insights into the mechanism of fibril formation in the presence and absence of lipid bilayers, as shown in Figure 7. In the absence of lipid bilayers, monomers may aggregate with each other to form oligomeric intermediates (similar to micelles) through a homogeneous reaction (Figure 7B; left), likely driven by the amphipathic natures of the N-terminal and C-terminal α-helices. These oligomeric intermediates then change into spherical fibril intermediates (Figure 7C; left) as observed by TEM (Figure 2a and b). Subsequently, these spherical fibril intermediates may form fibril nuclei and interact with monomeric glucagon to allow elongation of the fibril by changing from an α-helix to a β-sheet through a heterogeneous elongation process (Figure 7D; left).
In the presence of lipid bilayers, monomers form a structure similar to that in the absence of lipid bilayers. The monomers likely associate quickly with the lipid bilayer and subsequently associate with other monomers to form weakly coupled oligomeric intermediates (Figure 7B; right). These oligomeric intermediates may change their structure to form fibril intermediates on the surface of the lipid bilayer and are observed as ellipsoid-shaped fibril intermediates (Figure 7C; right and TEM picture) on the surface of the lipid bilayer. The elliptical shape is due to the N-terminal region retaining an α-helix structure even in the fibril intermediates. The fibril intermediates grow into longer fibrils on the surface of the lipid bilayer and protrude outside the lipid bilayers, as shown schematically in Figure 7D (right) and in the TEM picture (Figure 7D; right bottom).
The k1 rate constant for nuclear formation in the presence of lipid bilayers is faster than in the absence of lipid bilayers (Table 2) because glucagon monomers associate with the surface of the lipid bilayer, migrate laterally on the surface of the lipid bilayer to form oligomeric intermediates, and then subsequently change to fibril intermediate through a homogeneous nucleation reaction. This two-dimensional process may be faster than nuclear formation in the three-dimensional solution state.
The k2 rate constant for fibril elongation in the presence of lipid bilayers is slower than in the absence of lipid bilayers. As discussed previously, the N-terminal part of the glucagon molecule in a fibril in the presence of a lipid bilayer remains in an α-helix which may be stabilized when the helix interacts with the lipid bilayer. However, after the fibril grows and is released from the lipid bilayer, the N-terminal α-helix becomes more unstable than the N-terminal β-sheet formed in a fibril that protrudes from the lipid bilayer. This unstable fibril can grow to the outside of the lipid bilayer because it potentially acts as a template to form structures identical to the fibril nuclei formed on the surface of the lipid bilayer. The instability of the fibril state outside the lipid bilayer results in a decrease in the k2 value for fibril elongation as compared to the absence of a lipid bilayer.
These results show that glucagon molecules significantly interact with lipid bilayers during the fibril formation processes. To understand the fibril formation process under physiological condition, a variety of lipid bilayer systems including a lipid bilayer in neutral solution were investigated.
6. Fibrillation mechanism of glucagon inside the lipid bilayer in the neutral condition
Fibril formation processes were investigated for glucagon embedded inside a lipid bilayer in neutral solutions (i.e., essentially physiological conditions) [42]. Glucagon-induced morphological changes of lipid bilayers and fibril formation process in a glucagon containing lipid bilayer are shown in Figure 8. The monomers may aggregate with each other to form oligomers (Figure 8B) likely driven by the amphipathic nature of the N-terminal and C-terminal α-helices. These oligomers then change into ellipsoidal fibril intermediates (nuclei) (Figure 8C) through a homogeneous reaction, as observed by TEM (Figure 8C, bottom). At this stage, the glucagon intermediates strongly interact with the lipid bilayer to form discoidal lipid bilayer particles. Subsequently, this ellipsoidal fibril intermediate (nucleus) interacts with monomeric glucagon inside the lipid bilayer to allow elongation of the fibril by changing from an α-helix to a β-sheet through a heterogeneous elongation process (Figure 8D). After a long time standing, fibril networks are formed, and lipid molecules are compartmentalized (Figure 8E), resulting in increased lipid molecule mobility and the induction of a gel-like state throughout the sample, now in an amyloidogenic gel state [53, 54].
Figure 8.
Schematic diagrams of the morphological states of glucagon-DMPC bilayer systems (DMPC/glucagon; 50/1) at temperatures above and below the phase transition temperature (Tc = 23°C for DMPC). (A) Glucagon-DMPC bilayer state 1 day after sample preparation. (B) Glucagon-DMPC bilayer state 2 days after sample preparation. (C) Glucagon-DMPC bilayer state 4 days after sample preparation with the corresponding TEM picture shown below. (D) Glucagon-DMPC bilayer state 7 days after sample preparation with the corresponding TEM picture shown below. (E) Glucagon-DMPC bilayer system 10 days after sample preparation. The corresponding TEM picture is shown below (ref. [42]).
The kinetic of fibrillation was thus analyzed using a two-step autocatalytic mechanism as summarized in Table 2. The k1 rate constant for nuclear formation in neutral solution is much slower than that in a DMPC bilayer in neutral conditions (Table 2). In a DMPC/glucagon bilayer, glucagon molecules are condensed inside the DMPC bilayer, and thus the nucleation rate of glucagon in a DMPC/glucagon bilayer is much faster than that in neutral solution. The k1 rate constant for nuclear formation in a lipid bilayer in a neutral solution is slower than in a lipid bilayer in an acidic solution. In an acidic solution, glucagon locates on the surface of lipid bilayer and then migrates laterally on the surface of the lipid bilayer to form oligomers and subsequently changes to fibril nuclei through homogeneous nucleation reaction. In contrast, glucagon is deeply embedded inside a lipid bilayer in neutral solution and thus migrates more slowly inside the lipid bilayer, as reflected in the lower k1 value.
The k2 rate constant for the elongation of glucagon fibrils in a DMPC/glucagon bilayer is much slower than that in neutral solution. Glucagon molecules in a DMPC/glucagon bilayer interact strongly with the DMPC bilayer. It therefore takes a long time to disrupt the interaction with the lipid bilayer and form interaction with glucagon fibrils. The k2 rate constant for fibril elongation in the presence of lipid bilayers under neutral condition is significantly slower than that in lipid bilayers under acidic conditions. Under neutral conditions, glucagon is embedded deep inside the lipid bilayer, and hence it takes longer to release monomeric glucagon from the lipid bilayer. Therefore, the k2 values for fibril elongation decrease as compared to the case in a lipid bilayer in acidic conditions.
It is demonstrated that glucagon forms fibrils in acidic solution and in the presence of lipid bilayer (bicelle) in acidic solution. Glucagon aggregates to form fibril intermediates that glow into elongated fibrils. Glucagon intermediates are formed on the surface of the lipid bilayer in the presence of bicelle. These fibrils are cytotoxic through their activation of apoptotic processes, similar to β-amyloid and salmon calcitonin. Kinetic analyses of glucagon fibril formation are performed using a two-step autocatalytic reaction mechanism comprising fibril nucleation and elongation processes. It is revealed that glucagon forms fibril intermediates and grows into elongated fibrils inside the lipid bilayer under neutral conditions. These properties of glucagon fibril formation indicate that the interaction of the glucagon fibril with lipid bilayers is strongly dependent on the process of fibril formation. A neutral system is thus considered to reflect the fibril formation process in biological cells and provides insight into the mechanism underlying cytotoxicity of glucagon fibrils.
This work was supported by Grants-in-Aid for Scientific Research in an Innovative Area (JP16H00756 to AN) and by Grants-in-Aid for Scientific Research (C) (JP15K06963 to AN) from the Ministry of Culture, Sports, Science and Technology of Japan. The author wishes to thank Izuru Kawamura and Yoshiteru Makino for the discussion on this study and Izumi Yamane, Ayano Momose, Hideki Fujita, Eri Yoshimoto, Akie Kikuchi-Kinoshita, and Kazumi Haya for their experimental assistances.
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Written By
Akira Naito
Submitted: 17 November 2019Reviewed: 10 February 2020Published: 02 April 2020
Open access peer-reviewed chapter
