Amylin (rat)

The localization and expression of amylin protein in the rodent brain and mouse neuroblastoma Neuro-2a (N2a) are less widely known. Thus, this study investigated the expression distribution of amylin in the rat brain and N2a treated with steroid hormones. Amylin protein was identified in the olfactory bulb, cerebral cortex, dentate gyrus, thalamus, hypothalamus, ventral tegmental area (VTA), cerebellum, and brain stem in the rat brain. Additionally, the amylin protein was localized with the mature neurons of the cerebral cortex and dopaminergic neurons of the VTA. Progesterone (P4) and dexamethasone (Dex) significantly decreased, and 17β-estradiol (E2) increased the amylin protein level in the cerebral cortex. The P4 receptor antagonist RU486 significantly influenced the effects of P4 and Dex, and the E2 receptor antagonist ICI 182,780 slightly changed E2's effect. Amylin protein expression was significantly reduced in the VTA by P4 and Dex, and its expression was changed only following P4 plus RU486 treatment. It was confirmed for the first time that amylin protein is strongly expressed in the cytoplasm in N2a cells using immunofluorescent staining. P4 increased the levels of amylin, and RU486 treatment decreased them. Dex significantly increased the levels of amylin protein. RU486 treatment reversed the effects of Dex. Therefore, amylin protein is expressed in the cerebral cortex neurons and dopaminergic neurons of the VTA of the immature rat brain. P4 and Dex influence the expression of amylin protein in the rat brain and N2a cells.

The aggregation of human amylin has been strongly implicated in the progression of Type II diabetes. This 37-residue peptide forms a variety of secondary structures, including random coils, α-helices, and β-hairpins. The balance between these structures depends on the chemical environment, making amylin an ideal candidate to examine inherent biases in force fields. Rat amylin differs from human amylin by only 6 residues; however, it does not form fibrils. Therefore it provides a useful complement to human amylin in studies of the key events along the aggregation pathway. In this work, the free energy of rat and human amylin was determined as a function of α-helix and β-hairpin content for the Gromos96 53a6, OPLS-AA/L, CHARMM22/CMAP, CHARMM22*, Amberff99sb*-ILDN, and Amberff03w force fields using advanced sampling techniques, specifically bias exchange metadynamics. This work represents a first systematic attempt to evaluate the conformations and the corresponding free energy of a large, clinically relevant disordered peptide in solution across force fields. The NMR chemical shifts of rIAPP were calculated for each of the force fields using their respective free energy maps, allowing us to quantitatively assess their predictions. We show that the predicted distribution of secondary structures is sensitive to the choice of force-field: Gromos53a6 is biased towards β-hairpins, while CHARMM22/CMAP predicts structures that are overly α-helical. OPLS-AA/L favors disordered structures. Amberff99sb*-ILDN, AmberFF03w and CHARMM22* provide the balance between secondary structures that is most consistent with available experimental data. In contrast to previous reports, our findings suggest that the equilibrium conformations of human and rat amylin are remarkably similar, but that subtle differences arise in transient alpha-helical and beta-strand containing structures that the human peptide can more readily adopt. We hypothesize that these transient states enable dynamic pathways that facilitate the formation of aggregates and, eventually, amyloid fibrils.

Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have been associated with calcium dysregulation. In the heart, accumulating evidence suggests that human amylin oligomers form moderately cation-selective [4,5] channels that embed in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentration-dependent manner [5], which is correlated with elevated cytosolic Ca2+. These findings motivate our core hypothesis that non-selective inward Ca2+ conduction afforded by human amylin oligomers increase cytosolic and sarcoplasmic reticulum (SR) Ca2+ load, which thereby magnifies intracellular Ca2+ transients. Questions remain however regarding the mechanism of amylin-induced Ca2+ dysregulation, including whether enhanced SL Ca2+ influx is sufficient to elevate cytosolic Ca2+ load [6], and if so, how might amplified Ca2+ transients perturb Ca2+-dependent cardiac pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca2+ signaling to reflect experimentally-measured changes in SL membrane permeation and decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from acute and transgenic human amylin peptide exposure. With this model, we confirmed the hypothesis that increasing SL permeation alone was sufficient to enhance Ca2+ transient amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca2+ loading of the SR and that greater fractional release may contribute to the Ca2+-dependent activation of calmodulin, which could prime the activation of myocyte remodeling pathways. Importantly, elevated Ca2+ in the SR and dyadic space collectively drive greater fractional SR Ca2+ release for human amylin expressing rats (HIP) and acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca2+ transients. These findings suggest that increased membrane permeation induced by oligomeratization of amylin peptide in cell sarcolemma contributes to Ca2+ dysregulation in pre-diabetes.