Insulin-Binding Peptide Probes Provide a Novel Strategy for Pancreatic Beta Cell Imaging

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Pancreatic Beta-Cell Membrane Fluidity and Toxicity Induced by Human Islet Amyloid Polypeptide Species.

Aggregation of human islet amyloid polypeptide (hIAPP) into fibrils and plaques is associated with pancreatic β-cell loss in type 2 diabetes (T2D). However, due to the rapidness of hIAPP conversion in aqueous phase, exactly which hIAPP species is responsible for the observed toxicity and through what mechanisms remains ambiguous. In light of the importance of understanding hIAPP toxicity for T2D here we show a biophysical scheme based on the use of a lipophilic Laurdan dye for examining MIN6 cell membranes upon exposure to fresh and oligomeric hIAPP as well as mature amyloid. It has been found that all three hIAPP species, especially fresh hIAPP, enhanced membrane fluidity and caused losses in cell viability. The cell generation of reactive oxygen species (ROS), however, was the most pronounced with mature amyloid hIAPP. The correlation between changes in membrane fluidity and cell viability and their lack of correlation with ROS production suggest hIAPP toxicity is elicited through both physical and biochemical means. This study offers a new insight into β-cell toxicity induced by controlled hIAPP species, as well as new biophysical methodologies that may prove beneficial for the studies of T2D as well as neurological disorders.info:eu-repo/semantics/publishe

p53-upregulated-modulator-of-apoptosis (PUMA) deficiency affects food intake but does not impact on body weight or glucose homeostasis in diet-induced obesity.

BCL-2 proteins have been implicated in the control of glucose homeostasis and metabolism in different cell types. Thus, the aim of this study was to determine the role of the pro-apoptotic BH3-only protein, p53-upregulated-modulator-of-apoptosis (PUMA), in metabolic changes mediated by diet-induced obesity, using PUMA deficient mice. At 10 weeks of age, knockout and wild type mice either continued consuming a low fat chow diet (6% fat), or were fed with a high fat diet (23% fat) for 14-17 weeks. We measured body composition, glucose and insulin tolerance, insulin response in peripheral tissues, energy expenditure, oxygen consumption, and respiratory exchange ratio in vivo. All these parameters were indistinguishable between wild type and knockout mice on chow diet and were modified equally by diet-induced obesity. Interestingly, we observed decreased food intake and ambulatory capacity of PUMA knockout mice on high fat diet. This was associated with increased adipocyte size and fasted leptin concentration in the blood. Our findings suggest that although PUMA is dispensable for glucose homeostasis in lean and obese mice, it can affect leptin levels and food intake during obesity.info:eu-repo/semantics/publishe

Lipotoxic Stress Induces Pancreatic β-Cell Apoptosis through Modulation of Bcl-2 Proteins by the Ubiquitin-Proteasome System

Pancreatic β-cell loss induced by saturated free fatty acids (FFAs) is believed to contribute to type 2 diabetes. Previous studies have shown induction of endoplasmic reticulum (ER) stress, increased ubiquitinated proteins, and deregulation of the Bcl-2 family in the pancreas of type 2 diabetic patients. However, the precise mechanism of β-cell death remains unknown. In the present study we demonstrate that the FFA palmitate blocks the ubiquitin-proteasome system (UPS) and causes apoptosis through induction of ER stress and deregulation of Bcl-2 proteins. We found that palmitate and the proteasome inhibitor MG132 induced ER stress in β-cells, resulting in decreased expression of the prosurvival proteins Bcl-2, Mcl-1, and Bcl-XL, and upregulation of the prodeath BH3-only protein PUMA. On the other hand, pharmacological activation of the UPS by sulforaphane ameliorated ER stress, upregulated prosurvival Bcl-2 proteins, and protected β-cells from FFA-induced cell death. Furthermore, transgenic overexpression of Bcl-2 protected islets from FFA-induced cell death in vitro and improved glucose-induced insulin secretion in vivo. Together our results suggest that targeting the UPS and Bcl-2 protein expression may be a valuable strategy to prevent β-cell demise in type 2 diabetes

Graphene oxide inhibits hIAPP amyloid fibrillation and toxicity in insulin-producing NIT-1 cells.

Human islet amyloid polypeptide (hIAPP or amylin) aggregation is directly associated with pancreatic β-cell death and subsequent insulin deficiency in type 2 diabetes (T2D). Since no cure is currently available for T2D, it is of great benefit to devise new anti-aggregation molecules, which protect β-cells against hIAPP aggregation-induced toxicity. Engineered nanoparticles have been recently exploited as anti-aggregation nanomedicines. In this work, we studied graphene oxide (GO) nanosheets for their potential for hIAPP aggregation inhibition by combining computational modeling, biophysical characterization and cell toxicity measurements. Using discrete molecular dynamics (DMD) simulations and in vitro studies, we showed that GO exhibited an inhibitory effect on hIAPP aggregation. DMD simulations indicated that the strong binding of hIAPP to GO nanosheets was driven by hydrogen bonding and aromatic stacking and that the strong peptide-GO binding efficiently inhibited hIAPP self-association and aggregation on the nanosheet surface. Secondary structural changes of hIAPP upon GO binding derived from DMD simulations were consistent with circular dichroism (CD) spectroscopy measurements. Transmission electron microscopy (TEM) images confirmed the reduction of hIAPP aggregation in the presence of GO. Furthermore, we carried out a cell toxicity assay and found that these nanosheets protected insulin-secreting NIT-1 pancreatic β-cells against hIAPP-induced toxicity. Our multidisciplinary study suggests that GO nanosheets have the potential to be utilized as an anti-aggregation nanomedicine itself in addition to a biosensor or delivery vehicle for the mitigation of T2D progression.info:eu-repo/semantics/publishe

α-MSH stimulates glucose uptake in mouse muscle and phosphorylates Rab-GTPase-activating protein TBC1D1 independently of AMPK

The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure regulation, which are mainly mediated by MC4R. The pituitary gland is the source of various POMC-derived hormones released to the circulation, which raises the possibility that there may be actions of the melanocortins on peripheral energy homeostasis. In this study, we examined the molecular signaling pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate -MSH stimulation in both wild type and AMPK deficient mice. We found that -MSH significantly induces phosphorylation of TBC1 domain (TBC1D) family member 1 (S237 and T596), which is independent of upstream PKA and AMPK. We find no evidence to support that -MSH-stimulated glucose uptake involves TBC1D4 phosphorylation (T642 and S704) or GLUT4 translocation

Inhibition of hIAPP Amyloid Aggregation and Pancreatic β‐Cell Toxicity by OH‐Terminated PAMAM Dendrimer

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137295/1/smll201502317.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137295/2/smll201502317-sup-0001-S1.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137295/3/smll201502317_am.pd

Immune-phenotype and bone marrow (BM) progenitor status of naïve <i>ngr1-/-</i> and WT mice.

<p>(<b>A</b>) Comparative flow cytometric analysis of single cell suspension from spleen, BM, thymus, blood, lymph nodes and central nervous system (CNS) associated mononuclear cells. Proportion and total number of CD3⁺CD8⁺ and CD3⁺CD4⁺ T cells, B220⁺ B cells, Gr-1⁺ granulocytes and F4/80 (Gr-1^loF4/80⁺) monocyte/macrophage are shown. Data represent mean ± SEM (n = 8-11). *p&lt;0.05 Mann-Whitney test. (<b>B</b>) Quantification of BM-derived colony number in naïve <i>ngr1-/-</i> and WT mice<b>.</b> After 8 days on methylcellulose culture, BM-derived progenitors from <i>ngr1-/-</i> animals were capable of producing granulocytes (G); macrophages (M) and mixed (GM) colonies at comparable numbers to WT mice. Bars represent mean colony number/plate ± SEM (n = 3).</p

Immune-phenotype of <i>ngr1-/-</i> mice during EAE induced with rMOG.

<p><i>ngr1-/-</i> and WT animals were immunized with rMOG and the mononuclear cells isolated at 18 (peak) and 45 (chronic) days post-immunization (dpi) were analyzed by flow cytometry. Proportion and total number of lymphocytes, granulocytes and monocyte/macrophages of bone marrow (BM; <b>A</b>); spleen, (<b>B</b>); lymph nodes, (<b>C</b>); thymus (<b>D</b>) and central nervous system (CNS; <b>E</b>) are shown. No significant differences were found between <i>ngr1-/-</i> and WT for all organs and time points examined. Data represent mean ± SEM (n = 6-11). (<b>F</b>) Further analysis of microglia (CD45^loCD11b⁺) and macrophages (CD45^hiCD11b⁺) in the CNS of <i>ngr1-/-</i> and WT mice was performed. <i>ngr1-/-</i> animals presented an increased proportion of microglial cells at 18 dpi and a decreased number of macrophages at 45 dpi (n = 3-4; *p&lt; 0.05 Mann-Whitney test).</p