Structural Insights into Seeding Mechanisms of hIAPP Fibril Formation.

The deposition of islet amyloid polypeptide (hIAPP) fibrils is a hallmark of ß-cell death in type II diabetes. In this study, we employ state-of-the-art MAS solid-state spectroscopy to investigate the previously elusive N-terminal region of hIAPP fibrils, uncovering both rigidity and heterogeneity. Comparative analysis between wild-type hIAPP and a disulfide-deficient variant (hIAPPC2S,C7S) unveils shared fibril core structures yet strikingly distinct dynamics in the N-terminus. Specifically, the variant fibrils exhibit extended ß-strand conformations, facilitating surface nucleation. Moreover, our findings illuminate the pivotal roles of specific residues in modulating secondary nucleation rates. These results deepen our understanding of hIAPP fibril assembly and provide critical insights into the molecular mechanisms underpinning type II diabetes, holding promise for future therapeutic strategies.

Exploring the Role of Metabolic Hormones in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive loss of motor neurons. Emerging evidence suggests a potential link between metabolic dysregulation and ALS pathogenesis. This study aimed to investigate the relationship between metabolic hormones and disease progression in ALS patients. A cross-sectional study was conducted involving 44 ALS patients recruited from a tertiary care center. Serum levels of insulin, total amylin, C-peptide, active ghrelin, GIP (gastric inhibitory peptide), GLP-1 active (glucagon-like peptide-1), glucagon, PYY (peptide YY), PP (pancreatic polypeptide), leptin, interleukin-6, MCP-1 (monocyte chemoattractant protein-1), and TNFα (tumor necrosis factor alpha) were measured, and correlations with ALSFRS-R, evolution scores, and biomarkers were analyzed using Spearman correlation coefficients. Subgroup analyses based on ALS subtypes, progression pattern of disease, and disease progression rate patterns were performed. Significant correlations were observed between metabolic hormones and ALS evolution scores. Insulin and amylin exhibited strong correlations with disease progression and clinical functional outcomes, with insulin showing particularly robust associations. Other hormones such as C-peptide, leptin, and GLP-1 also showed correlations with ALS progression and functional status. Subgroup analyses revealed differences in hormone levels based on sex and disease evolution patterns, with male patients showing higher amylin and glucagon levels. ALS patients with slower disease progression exhibited elevated levels of amylin and insulin. Our findings suggest a potential role for metabolic hormones in modulating ALS progression and functional outcomes. Further research is needed to elucidate the underlying mechanisms and explore the therapeutic implications of targeting metabolic pathways in ALS management.

Catalysis driven by an amyloid-substrate complex.

Amine modification through nucleophilic attack of the amine functionality is a very common chemical transformation. Under biorelevant conditions using acidic-to-neutral pH buffer, however, the nucleophilic reaction of alkyl amines (pKa ≈ 10) is not facile due to the generation of ammonium ions lacking nucleophilicity. Here, we disclose a unique molecular transformation system, catalysis driven by amyloid-substrate complex (CASL), that promotes amine modifications in acidic buffer. Ammonium ions attached to molecules with amyloid-binding capability were activated through deprotonation due to the close proximity to the amyloid catalyst formed by Ac-Asn-Phe-Gly-Ala-Ile-Leu-NH2 (NL6), derived from islet amyloid polypeptide (IAPP). Under the CASL conditions, alkyl amines underwent various modifications, i.e., acylation, arylation, cyclization, and alkylation, in acidic buffer. Crystallographic analysis and chemical modification studies of the amyloid catalysts suggested that the carbonyl oxygen of the Phe-Gly amide bond of NL6 plays a key role in activating the substrate amine by forming a hydrogen bond. Using CASL, selective conversion of substrates possessing equivalently reactive amine functionalities was achieved in catalytic reactions using amyloids. CASL provides a unique method for applying nucleophilic conversion reactions of amines in diverse fields of chemistry and biology.

Neuroinflammation induced by amyloid-forming pancreatic amylin: Rationale for a mechanistic hypothesis.

Amylin is a systemic neuroendocrine hormone co-expressed and co-secreted with insulin by pancreatic ß-cells. In persons with thype-2 diabetes, amylin forms pancreatic amyloid triggering inflammasome and interleukin-1ß signaling and inducing ß-cell apoptosis. Here, we summarize recent progress in understanding the potential link between amyloid-forming pancreatic amylin and Alzheimer's disease (AD). Clinical data describing amylin pathology in AD alongside mechanistic studies in animals are reviewed. Data from multiple research teams indicate higher amylin concentrations are associated with increased frequency of cognitive impairment and amylin co-aggregates with ß-amyloid in AD-type dementia. Evidence from rodent models further suggests cerebrovascular amylin accumulation as a causative factor underlying neurological deficits. Analysis of relevant literature suggests that modulating the amylin-interleukin-1ß pathway may provide an approach for counteracting neuroinflammation in AD.

Experimental and computational investigation of the effect of Hsc70 structural variants on inhibiting amylin aggregation.

The misfolding and aggregation of human islet amyloid polypeptide (hIAPP), also known as amylin, have been implicated in the pathogenesis of type 2 diabetes (T2D). Heat shock proteins, specifically, heat shock cognate 70 (Hsc70), are molecular chaperones that protect against hIAPP misfolding and inhibits its aggregation. Nevertheless, there is an incomplete understanding of the mechanistic interactions between Hsc70 domains and hIAPP, thus limiting their potential therapeutic role in diabetes. This study investigates the inhibitory capacities of different Hsc70 variants, aiming to identify the structural determinants that strike a balance between efficacy and cytotoxicity. Our experimental findings demonstrate that the ATPase activity of Hsc70 is not a pivotal factor for inhibiting hIAPP misfolding. We underscore the significance of the C-terminal substrate-binding domain of Hsc70 in inhibiting hIAPP aggregation, emphasizing that the removal of the lid subdomain diminishes the inhibitory effect of Hsc70. Additionally, we employed atomistic discrete molecular dynamics simulations to gain deeper insights into the interaction between Hsc70 variants and hIAPP. Integrating both experimental and computational findings, we propose a mechanism by which Hsc70's interaction with hIAPP monomers disrupts protein-protein connections, primarily by shielding the ß-sheet edges of the Hsc70-ß-sandwich. The distinctive conformational dynamics of the alpha helices of Hsc70 potentially enhance hIAPP binding by obstructing the exposed edges of the ß-sandwich, particularly at the ß5-ß8 region along the alpha helix interface. This, in turn, inhibits fibril growth, and similar results were observed following hIAPP dimerization. Overall, this study elucidates the structural intricacies of Hsc70 crucial for impeding hIAPP aggregation, improving our understanding of the potential anti-aggregative properties of molecular chaperones in diabetes treatment.

The Co-oligomers of Aß42 and Human Islet Amyloid Polypeptide Exacerbate Neurotoxicity and Alzheimer-like Pathology at Cellular Level.

The Aß hypothesis has long been central to Alzheimer's disease (AD) theory, with a recent surge in attention following drug approvals targeting Aß plaque clearance. Aß42 oligomers (AßO) are key neurotoxins. While ß-amyloid (Aß) buildup is a hallmark of AD, postmortem brain analyses have unveiled human islet amyloid polypeptide (hIAPP) deposition in AD patients, suggesting a potential role in Alzheimer's pathology. This study investigates the neurotoxic effects of co-aggregates of Aß42 and hIAPP, specifically focusing on their impact on cell survival, apoptosis, and AD-like pathology. We analyzed and compared the impact of AßO and Aß42-hIAPP on cell survival in SH-SY5Y cells, apoptosis and inducing AD-like pathology in glutamatergic neurons. Aß42-hIAPP co-oligomers exhibited significantly greater toxicity, causing 2.3-3.5 times higher cell death compared to AßO alone. Furthermore, apoptosis rates were significantly exacerbated in glutamatergic neurons when exposed to Aß42-hIAPP co-oligomers. The study also revealed that Aß42-hIAPP co-oligomers induced typical AD-like pathology in glutamatergic neurons, including the presence of Aß deposits (detected by 6E10 and 4G8 immunofluorescence) and alterations in tau protein (changes in total tau HT7, phosphorylated tau AT8, AT180). Notably, Aß42-hIAPP co-oligomers induced a more severe AD pathology compared to AßO alone. These findings provide compelling evidence for the heightened toxicity of Aß42-hIAPP co-oligomers on neurons and their role in exacerbating AD pathology. The study contributes novel insights into the pathogenesis of Alzheimer's disease, highlighting the potential involvement of hIAPP in AD pathology. Together, these findings offer novel insights into AD pathogenesis and routes for constructing animal models.

Identification and utility exploration of a highly potent and long-acting bullfrog GLP-1 analogue in GLP-1 and amylin combination therapy.

This study assesses the efficacy of an innovative therapeutic approach that combines GLP-1 and amylin analogues for weight reduction. Focusing on GLP-1 analogues from bullfrog (Rana catesbeiana), we designed ten bGLP-1 analogues with various modifications. Among them, bGLP-10 showed high potency in binding and activating GLP-1 receptors, with superior albumin affinity. In diet-induced obesity (DIO) mice fed a high-fat diet, bGLP-10 demonstrated significant superiority over semaglutide in reducing blood sugar and food intake at a dose of 10 nmol/kg (P < 0.001). Notably, in a chronic study involving DIO mice, the combination of bGLP-10 with the amylin analogue cagrilintide led to a more substantial weight loss (-38.4%, P < 0.001) compared to either the semaglutide-cagrilintide combination (-23.0%) or cagrilintide (-5.7%), bGLP-10 (-16.1%), and semaglutide (-10.9%) alone. Furthermore, the bGLP-10 and cagrilintide combination exhibited superior glucose control and liver lipid management compared to the semaglutide-cagrilintide combination (P < 0.001). These results highlight bGLP-10's potential in GLP-1 and amylin-based therapies and suggest exploring more GLP-1 analogues from natural sources for anti-obesity and anti-diabetic treatments.

Calcitonin receptor, calcitonin gene-related peptide and amylin distribution in C1/2 dorsal root ganglia.

BACKGROUND: The upper cervical dorsal root ganglia (DRG) are important for the transmission of sensory information associated with the back of the head and neck, contributing to head pain. Calcitonin receptor (CTR)-based receptors, such as the amylin 1 (AMY1) receptor, and ligands, calcitonin gene-related peptide (CGRP) and amylin, have been linked to migraine and pain. However, the contribution of this system to nociception involving the cervical DRG is unclear. Therefore, this study aimed to determine the relative distribution of the CTR, CGRP, and amylin in upper cervical DRG. METHODS: CTR, CGRP, and amylin immunofluorescence was examined relative to neural markers in C1/2 DRG from male and female mice, rats, and human cases. Immunofluorescence was supported by RNA-fluorescence in situ hybridization examining amylin mRNA distribution in rat DRG. RESULTS: Amylin immunofluorescence was observed in neuronal soma and fibres. Amylin mRNA (Iapp) was also detected. Amylin and CGRP co-expression was observed in 19% (mouse), 17% (rat), and 36% (human) of DRG neurons in distinct vesicle-like neuronal puncta from one another. CTR immunoreactivity was present in DRG neurons, and both peptides produced receptor signalling in primary DRG cell cultures. CTR-positive neurons frequently co-expressed amylin and/or CGRP (66% rat; 84% human), with some sex differences. CONCLUSIONS: Amylin and CGRP could both be local peptide agonists for CTR-based receptors in upper cervical DRG, potentially acting through autocrine and/or paracrine signalling mechanisms to modulate neuron function. Amylin and its receptors could represent novel pain targets.

Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

The processing intermediate of human amylin, pro-amylin(1-48), has in vivo and in vitro bioactivity.

Amylin is released by pancreatic beta-cells in response to a meal and its major soluble mature form (37 amino acid-peptide) produces its biological effects by activating amylin receptors. Amylin is derived from larger propeptides that are processed within the synthesizing beta-cell. There are suggestions that a partially processed form, pro-amylin(1-48) is also secreted. We tested the hypothesis that pro-amylin(1-48) has biological activity and that human pro-amylin(1-48) may also form toxic pre-amyloid species. Amyloid formation, the ability to cross-seed and in vitro toxicity were similar between human pro-amylin(1-48) and amylin. Human pro-amylin(1-48) was active at amylin-responsive receptors, though its potency was reduced at rat, but not human amylin receptors. Pro-amylin(1-48) was able to promote anorexia by activating neurons of the area postrema, amylin's primary site of action, indicating that amylin can tolerate significant additions at the N-terminus without losing bioactivity. Our studies help to shed light on the possible roles of pro-amylin(1-48) which may be relevant for the development of future amylin-based drugs.

Composition and Conformation of Hetero- versus Homo-Fluorinated Triazolamers Influence their Activity on Islet Amyloid Polypeptide Aggregation.

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units mixed with natural amino acids from the hIAPP sequence reduces it, and more efficiently than the parent natural peptide. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded ß-sheets have no effect. This work demonstrates that a good balance between the structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

Liraglutide versus pramlintide in protecting against cognitive function impairment through affecting PI3K/AKT/GSK-3ß/TTBK1 pathway and decreasing Tau hyperphosphorylation in high-fat diet- streptozocin rat model.

The American Diabetes Association guidelines (2021) confirmed the importance of raising public awareness of diabetes-induced cognitive impairment, highlighting the links between poor glycemic control and cognitive impairment. The characteristic brain lesions of cognitive dysfunction are neurofibrillary tangles (NFT) and senile plaques formed of amyloid-ß deposition, glycogen synthase kinase 3 beta (GSK3ß), and highly homologous kinase tau tubulin kinase 1 (TTBK1) can phosphorylate Tau proteins at different sites, overexpression of these enzymes produces extensive phosphorylation of Tau proteins making them insoluble and enhance NFT formation, which impairs cognitive functions. The current study aimed to investigate the potential contribution of liraglutide and pramlintide in the prevention of diabetes-induced cognitive dysfunction and their effect on the PI3K/AKT/GSK-3ß/TTBK1 pathway in type 2 diabetic (T2D) rat model. T2D was induced by administration of a high-fat diet for 10 weeks, then injection of a single dose of streptozotocin (STZ); treatment was started with either pramlintide (200 µg/kg/day sc) or liraglutide (0.6 mg/kg/day sc) for 6 weeks in addition to the HFD. At the end of the study, cognitive functions were assessed by novel object recognition and T-maze tests. Then, rats were sacrificed for biochemical and histological assessment of the hippocampal tissue. Both pramlintide and liraglutide treatment revealed equally adequate control of diabetes, prevented the decline in memory function, and increased PI3K/AKT expression while decreasing GSK-3ß/TTBK1 expression; however, liraglutide significantly decreased the number of Tau positive cells better than pramlintide did. This study confirmed that pramlintide and liraglutide are promising antidiabetic medications that could prevent associated cognitive disorders in different mechanisms.

Amylin in Alcohol Addiction: A Potential New Treatment Target or an Adjuvant to Other Treatments?

Amylin is a neuroendocrine hormone with a potential role in addictive disorders, including alcohol use disorder (AUD). In addition to reducing appetitive behavior, amylin has been shown to affect alcohol-related behaviors in rodents. Delineating the biobehavioral correlates of amylin in relation to alcohol seeking and consumption has the potential of identifying new treatment targets for AUD, yet additional translational and human research is needed.

Chronic pramlintide decreases feeding via a reduction in meal size in male rats.

Amylin, a pancreatic hormone, is well-established to suppress feeding by enhancing satiation. Pramlintide, an amylin analog that is FDA-approved for the treatment of diabetes, has also been shown to produce hypophagia. However, the behavioral mechanisms underlying the ability of pramlintide to suppress feeding are unresolved. We hypothesized that systemic pramlintide administration in rats would reduce energy intake, specifically by reducing meal size. Male rats were given b.i.d. administration of intraperitoneal pramlintide or vehicle for 1 week, and chow intake, meal patterns, and body weight were monitored throughout the test period. Consistent with our hypothesis, pramlintide decreased chow intake mainly via suppression of meal size, with corresponding reductions in meal duration on several days. Fewer effects on meal number or feeding rate were detected. Pramlintide also reduced weight gain over the 1-week study. These results highlight that the behavioral mechanisms by which pramlintide produces hypophagia are similar to those driven by amylin itself, and provide important insight into the ability of this pharmacotherapy to promote negative energy balance over a period of chronic administration.

Robust Chemical Synthesis of "Difficult Peptides" via 2-Hydroxyphenol-pseudoproline (ψ2-hydroxyphenolpro) Modifications.

The challenging preparation of "difficult peptides" has always hindered the development of peptide-active pharmaceutical ingredients. Pseudoproline (ψpro) building blocks have been proven effective and powerful tools for the synthesis of "difficult peptides". In this paper, we efficiently prepared a set of novel 2-(oxazolidin-2-yl)phenol compounds as proline surrogates (2-hydroxyphenol-pseudoprolines, ψ2-hydroxyphenolpro) and applied it in the synthesis of many well-known "difficult peptides", including human thymosin α1, amylin, and ß-amyloid (1-42) (Aß42).

Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins.

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.

Are insulin sensitizers the new strategy to treat Type 1 diabetes? A long-acting dual amylin and calcitonin receptor agonist improves insulin-mediated glycaemic control and controls body weight.

BACKGROUND AND PURPOSE: Insulin therapies for Type 1 diabetes (T1D) have limitations, such as glucose fluctuations, hypoglycaemia, and weight gain. Only pramlintide is approved with insulin. However, its short half-life limits efficacy, requiring multiple daily injections and increasing hypoglycaemia risk. New strategies are needed to improve glycaemic control. Dual amylin and calcitonin receptor agonists are potent insulin sensitizers developed for Type 2 diabetes (T2D) as they improve glucose control, reduce body weight, and attenuate hyperglucagonemia. However, it is uncertain if they could be used to treat T1D. EXPERIMENTAL APPROACH: Sprague Dawley rats received a single intravenous injection of streptozotocin (STZ) (50 mg·kg-1) to induce T1D. Humulin (1 U/200 g·day-1 or 2 U/200 g·day-1) was continuously infused, while half of the rats received additional KBP-336 (4.5 nmol·kg-1 Q3D) treatment. Bodyweight, food intake, and blood glucose were monitored throughout the study. An oral glucose tolerance test was performed during the study. KEY RESULTS: Treatment with Humulin or Humulin + KBP-336 improved the health of STZ rats. Humulin increased body weight in STZ rats, but KBP-336 attenuated these increases and maintained a significant weight loss. The combination exhibited greater blood glucose reductions than Humulin-treated rats alone, reflected by improved HbA1c levels and glucose control. The combination prevented hyperglucagonemia, reduced amylin levels, and increased pancreatic insulin content, indicating improved insulin sensitivity and beta-cell preservation. CONCLUSION AND IMPLICATIONS: The insulin sensitizer KBP-336 lowered glucagon secretion while attenuating insulin-induced weight gain. Additionally, KBP-336 may prevent hypoglycaemia and improve insulin resistance, which could be a significant advantage for individuals with T1D seeking therapeutic benefits.

Analyzing Gene Expression After Administration of Low-Intensity Therapeutic Ultrasound in Human Islet Cells.

OBJECTIVES: Diabetes mellitus is a complex heterogenous metabolic disease that significantly affects the world population. Although many treatments exist, including medications such as metformin, sulfonylureas, and glucagon-like peptide-1 (GLP) receptor agonist, there is growing interest in finding alternative methods to noninvasively treat this disease. It has been previously shown that low-intensity ultrasound stimulation of pancreatic ß-cells in mice can elicit insulin secretion as a potential treatment for this disease. This is desirable as therapeutic ultrasound has the ability to induce bioeffects while selectively focusing deep within tissues, allowing for modulation of hormone secretion in the pancreas to mitigate insufficient levels of insulin. METHODS: Exactly 800 kHz ultrasound with intensity 0.5 W/cm2 was administered 5 minutes continuously, that is, 100% duty cycle, to donor pancreatic human islets, followed by 1 hour incubation and RT-qPCR to assess the effect of ultrasound stimulation on gene expression. The genes were insulin (INS), glucagon (Glu), amylin (Amy), and binding immunoglobulin protein (BiP). Nine donor pancreatic human islets were used to assess insulin and glucagon secretion, while eight samples were used for amylin and BiP. Fold change (FC) was calculated to analyze the effect of ultrasound stimulation on the gene expression of the donor islet cells. High-glucose and thapsigargin-treated islets were utilized as positive controls. Cell viability testing was done using a Trypan Blue Exclusion Test. RESULTS: Ultrasound stimulation did not cause a statistically significant upregulation in any of the tested genes (INS FC = 1.15, P-value = .5692; Glu FC = 1.60, P-value = .2231; Amy FC, P-value = .2863; BiP FC = 2.68, P-value = .3907). CONCLUSIONS: The results of this study show that the proposed ultrasound treatment parameters do not appear to significantly affect gene expression of any gene tested.

On the importance of being amidated: Analysis of the role of the conserved C-terminal amide of amylin in amyloid formation and cytotoxicity.

The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to ß-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO-) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured ß-cells, although hAmylin-COO- is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132-49 (2018)) shows that Amylin- COO- exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO- exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.