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Frontiers in Immunology 2024There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of β-cell antigens, including hybrid insulin...
There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of β-cell antigens, including hybrid insulin peptides (HIPs). The ligands for several CD4 T-cell clones derived from the NOD mouse are HIPs composed of a fragment of proinsulin joined to peptides from endogenous β-cell granule proteins. The diabetogenic T-cell clone BDC-6.9 reacts to a fragment of C-peptide fused to a cleavage product of pro-islet amyloid polypeptide (6.9HIP). In this study, we used a monoclonal antibody (MAb) to the 6.9HIP to determine when and where HIP antigens are present in NOD islets during disease progression and with which immune cells they associate. Immunogold labeling of the 6.9HIP MAb and organelle-specific markers for electron microscopy were employed to map the subcellular compartment(s) in which the HIP is localized within β-cells. While the insulin B9-23 peptide was present in nearly all islets, the 6.9HIP MAb stained infiltrated islets only in NOD mice at advanced stages of T1D development. Islets co-stained with the 6.9HIP MAb and antibodies to mark insulin, macrophages, and dendritic cells indicate that 6.9HIP co-localizes within insulin-positive β-cells as well as intra-islet antigen-presenting cells (APCs). In electron micrographs, the 6.9HIP co-localized with granule structures containing insulin alone or both insulin and LAMP1 within β-cells. Exposing NOD islets to the endoplasmic reticulum (ER) stress inducer tunicamycin significantly increased levels of 6.9HIP in subcellular fractions containing crinosomes and dense-core granules (DCGs). This work demonstrates that the 6.9HIP can be visualized in the infiltrated islets and suggests that intra-islet APCs may acquire and present HIP antigens within islets.
Topics: Animals; Mice; Mice, Inbred NOD; Diabetes Mellitus, Type 1; Islets of Langerhans; Peptides; Insulin-Secreting Cells; Antigens
PubMed: 38455055
DOI: 10.3389/fimmu.2024.1348131 -
The processing intermediate of human amylin, pro-amylin(1-48), has in vivo and in vitro bioactivity.Biophysical Chemistry May 2024Amylin 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 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.
Topics: Humans; Rats; Animals; Islet Amyloid Polypeptide; Receptors, Islet Amyloid Polypeptide; Amyloid
PubMed: 38452520
DOI: 10.1016/j.bpc.2024.107201 -
Annals of the New York Academy of... Apr 2024This review consolidates current knowledge on mammalian parental care, focusing on its neural mechanisms, evolutionary origins, and derivatives. Neurobiological studies... (Review)
Review
This review consolidates current knowledge on mammalian parental care, focusing on its neural mechanisms, evolutionary origins, and derivatives. Neurobiological studies have identified specific neurons in the medial preoptic area as crucial for parental care. Unexpectedly, these neurons are characterized by the expression of molecules signaling satiety, such as calcitonin receptor and BRS3, and overlap with neurons involved in the reproductive behaviors of males but not females. A synthesis of comparative ecology and paleontology suggests an evolutionary scenario for mammalian parental care, possibly stemming from male-biased guarding of offspring in basal vertebrates. The terrestrial transition of tetrapods led to prolonged egg retention in females and the emergence of amniotes, skewing care toward females. The nocturnal adaptation of Mesozoic mammalian ancestors reinforced maternal care for lactation and thermal regulation via endothermy, potentially introducing metabolic gate control in parenting neurons. The established maternal care may have served as the precursor for paternal and cooperative care in mammals and also fostered the development of group living, which may have further contributed to the emergence of empathy and altruism. These evolution-informed working hypotheses require empirical validation, yet they offer promising avenues to investigate the neural underpinnings of mammalian social behaviors.
Topics: Humans; Animals; Female; Male; Parenting; Brain; Mammals; Social Behavior; Neurons; Maternal Behavior
PubMed: 38426943
DOI: 10.1111/nyas.15111 -
Journal of Ultrasound in Medicine :... Jun 2024Diabetes mellitus is a complex heterogenous metabolic disease that significantly affects the world population. Although many treatments exist, including medications such...
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 β-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/cm 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.
Topics: Humans; Islets of Langerhans; Ultrasonic Therapy; Insulin; Glucagon; Gene Expression; Islet Amyloid Polypeptide
PubMed: 38414281
DOI: 10.1002/jum.16441 -
Life (Basel, Switzerland) Jan 2024The mammary gland of mammals can generate numerous bioactive proteins. To express the human amylin protein in the mammary glands of domestic animals, we engineered a...
The mammary gland of mammals can generate numerous bioactive proteins. To express the human amylin protein in the mammary glands of domestic animals, we engineered a transgenic mammary gland bioreactor. For this study, we produced transgenic mice through prokaryotic microinjection. RT-PCR, qPCR, and Western blotting confirmed the presence of transgenes in the mice. The ELISA assay indicated an amylin yield of approximately 1.44 μg/mL in the mice milk. Further research revealed that consuming milk containing amylin resulted in a slight, but insignificant enhancement in food consumption, blood sugar equilibrium, and glucose tolerance. The influence of amylin-fortified milk on the abundance of fecal strains in mice was examined, and a significant difference in the quantity of strains needed for fatty acid synthesis and metabolism was discovered. The amylin protein gathered from humans is safe to consume, as no harmful effects were detected in the mice. Our study examined the production of human amylin using a new safety strategy that could potentially alleviate diabetic symptoms in the future through oral administration of milk containing amylin.
PubMed: 38398700
DOI: 10.3390/life14020191 -
International Journal of Molecular... Feb 2024The primary treatment for autoimmune Diabetes Mellitus (Type 1 Diabetes Mellitus-T1DM) is insulin therapy. Unfortunately, a multitude of clinical cases has demonstrated... (Review)
Review
The primary treatment for autoimmune Diabetes Mellitus (Type 1 Diabetes Mellitus-T1DM) is insulin therapy. Unfortunately, a multitude of clinical cases has demonstrated that the use of insulin as a sole therapeutic intervention fails to address all issues comprehensively. Therefore, non-insulin adjunct treatment has been investigated and shown successful results in clinical trials. Various hypoglycemia-inducing drugs such as Metformin, glucagon-like peptide 1 (GLP-1) receptor agonists, dipeptidyl peptidase-4 (DPP-4) inhibitors, amylin analogs, and Sodium-Glucose Cotransporters 2 (SGLT-2) inhibitors, developed good outcomes in patients with T1DM. Currently, SGLT-2 inhibitors have remarkably improved the treatment of patients with diabetes by preventing cardiovascular events, heart failure hospitalization, and progression of renal disease. However, their pharmacological potential has not been explored enough. Thus, the substantial interest in SGLT-2 inhibitors (SGLT-2is) underlines the present review. It begins with an overview of carrier-mediated cellular glucose uptake, evidencing the insulin-independent transport system contribution to glucose homeostasis and the essential roles of Sodium-Glucose Cotransporters 1 and 2. Then, the pharmacological properties of SGLT-2is are detailed, leading to potential applications in treating T1DM patients with automated insulin delivery (AID) systems. Results from several studies demonstrated improvements in glycemic control, an increase in Time in Range (TIR), a decrease in glycemic variability, reduced daily insulin requirements without increasing hyperglycemic events, and benefits in weight management. However, these advantages are counterbalanced by increased risks, particularly concerning Diabetic Ketoacidosis (DKA). Several clinical trials reported a higher incidence of DKA when patients with T1DM received SGLT-2 inhibitors such as Sotagliflozin and Empagliflozin. On the other hand, patients with T1DM and a body mass index (BMI) of ≥27 kg/m treated with Dapagliflozin showed similar reduction in hyperglycemia and body weight and insignificantly increased DKA incidence compared to the overall trial population. Additional multicenter and randomized studies are required to establish safer and more effective long-term strategies based on patient selection, education, and continuous ketone body monitoring for optimal integration of SGLT-2 inhibitors into T1DM therapeutic protocol.
Topics: Humans; Diabetes Mellitus, Type 1; Dipeptidyl-Peptidase IV Inhibitors; Glucose; Hypoglycemic Agents; Insulin; Multicenter Studies as Topic; Risk Assessment; Sodium; Sodium-Glucose Transporter 2 Inhibitors
PubMed: 38396657
DOI: 10.3390/ijms25041972 -
ChemPlusChem Jun 2024Protein misfolding and amyloid formation are implicated in the protein dysfunction, but the underlying mechanism remains to be clarified due to the lack of effective... (Review)
Review
Protein misfolding and amyloid formation are implicated in the protein dysfunction, but the underlying mechanism remains to be clarified due to the lack of effective tools for detecting the transient intermediates. Sum frequency generation vibrational spectroscopy (SFG-VS) has emerged as a powerful tool for identifying the structure and dynamics of proteins at the interfaces. In this review, we summarize recent SFG-VS studies on the structure and dynamics of membrane-bound proteins during misfolding processes. This paper first introduces the methods for determining the secondary structure of interfacial proteins: combining chiral and achiral spectra of amide A and amide I bands and combining amide I, amide II, and amide III spectral features. To demonstrate the ability of SFG-VS in investigating the interfacial protein misfolding and amyloid formation, studies on the interactions between different peptides/proteins (islet amyloid polypeptide, amyloid β, prion protein, fused in sarcoma protein, hen egg-white lysozyme, fusing fusion peptide, class I hydrophobin SC3 and class II hydrophobin HFBI) and surfaces such as lipid membranes are discussed. These molecular-level studies revealed that SFG-VS can provide a unique understanding of the mechanism of interfacial protein misfolding and amyloid formation in real time, in situ and without any exogenous labeling.
Topics: Protein Folding; Membrane Proteins; Spectrum Analysis; Amyloid; Humans; Vibration; Animals; Protein Structure, Secondary
PubMed: 38380553
DOI: 10.1002/cplu.202300684 -
British Journal of Pharmacology Jun 2024Insulin therapies for Type 1 diabetes (T1D) have limitations, such as glucose fluctuations, hypoglycaemia, and weight gain. Only pramlintide is approved with insulin....
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) to induce T1D. Humulin (1 U/200 g·day or 2 U/200 g·day) was continuously infused, while half of the rats received additional KBP-336 (4.5 nmol·kg 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.
Topics: Animals; Rats, Sprague-Dawley; Receptors, Calcitonin; Male; Diabetes Mellitus, Type 1; Rats; Insulin; Body Weight; Hypoglycemic Agents; Blood Glucose; Glycemic Control; Diabetes Mellitus, Experimental; Amylin Receptor Agonists; Islet Amyloid Polypeptide; Streptozocin
PubMed: 38378168
DOI: 10.1111/bph.16329 -
The Journal of Organic Chemistry Mar 2024The challenging preparation of "difficult peptides" has always hindered the development of peptide-active pharmaceutical ingredients. Pseudoproline (ψpro) building...
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, ψpro) and applied it in the synthesis of many well-known "difficult peptides", including human thymosin α1, amylin, and β-amyloid (1-42) (Aβ42).
Topics: Humans; Catechols; Proline; Islet Amyloid Polypeptide; Thiazoles
PubMed: 38373048
DOI: 10.1021/acs.joc.3c02576 -
Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins.Biophysical Journal Mar 2024Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a...
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.
Topics: Rats; Animals; Humans; Amyloid; Cryoelectron Microscopy; HEPES; Ice; Islet Amyloid Polypeptide
PubMed: 38368506
DOI: 10.1016/j.bpj.2024.02.009