isolated-pancreatic-islet-treatment-and-apoptosis-measurement

Isolated Pancreatic Islet Treatment and Apoptosis Measurement

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing &#946;-cells, using a combination of ...

Colorado School of Mines

University of Colorado Anschutz Medical Campus

In type 1 diabetes (T1D), islet dysfunction occurs prior to diabetes onset. Pro-inflammatory cytokines can disrupt insulin secretion and Ca homeostasis. Connexin36 (Cx36) gap junctions electrically couple β-cells to coordinate glucose-stimulated Ca and insulin secretion. Cx36 gap junction coupling can also protect against cytokine-induced apoptosis. Our goal was to determine how islet gap junction coupling and Ca dynamics are altered in mouse models of T1D prior to diabetes. Glucose tolerance was assessed in NOD and immunodeficient NOD-RAG1KO mice at 6-12 weeks age. Glucose-stimulated insulin secretion, Ca dynamics, and gap junction coupling were measured in islets isolated at each age. Gap junction coupling was also measured in islets from mice that underwent transfer of diabetogenic splenocytes and from chromograninA knockout NOD mice. Cell death was measured in islets isolated from wild-type, Cx36 knockout or Cx36 over-expression mice, each treated with a cocktail of pro-inflammatory cytokines and K or SERCA activators/inhibitors. NOD mice over-expressing Cx36 were also monitored for diabetes development, and islets assessed for insulitis and apoptosis. NOD and NOD-RAG1KO controls showed similar glucose tolerance at all ages. Ca dynamics and gap junction coupling were disrupted in islets of NOD mice at 9 weeks, compared to controls. Transfer of diabetogenic splenocytes also decreased gap junction coupling. Islets from chromograninA knockout mice displayed normal coupling. Overexpression of Cx36 protected islets from cytokine-induced apoptosis. A knockout of Cx36 amplified cytokine-induced apoptosis, which was reversed by K activation or SERCA activation. Cx36 overexpression in NOD mice delayed diabetes development compared to NOD controls. However, apoptosis and insulitis were not improved. Decreases in islet gap junction coupling occur prior to T1D onset. Such decreases alter islet susceptibility to apoptosis due to altered Ca. Future studies will determine if increasing Cx36 gap junction coupling in combination with restoring Ca homeostasis protects against islet decline in T1D.

Modulation of Gap Junction Coupling Within the Islet of Langerhans During the Development of Type 1 Diabetes.

The pancreatic islet is surrounded by ECM that provides both biochemical and mechanical cues to the islet β-cell to regulate cell survival and insulin secretion. Changes in ECM composition and mechanical properties drive β-cell dysfunction in many pancreatic diseases. While several studies have characterized changes in islet insulin secretion with changes in substrate stiffness, little is known about the mechanotransduction signaling driving altered islet function in response to mechanical cues. We hypothesized that increasing matrix stiffness will lead to insulin secretion dysfunction by opening the mechanosensitive ion channel Piezo1 and disrupting intracellular Ca dynamics in mouse and human islets. To test our hypothesis, mouse and human cadaveric islets were encapsulated in a biomimetic reverse thermal gel (RTG) scaffold with tailorable stiffness that allows formation of islet focal adhesions with the scaffold and activation of Piezo1 in 3D. Our results indicate that increased scaffold stiffness causes insulin secretion dysfunction mediated by increases in Ca influx and altered Ca dynamics via opening of the mechanosensitive Piezo1 channel. Additionally, inhibition of Piezo1 rescued glucose-stimulated insulin secretion (GSIS) in islets in stiff scaffolds. Overall, our results emphasize the role mechanical properties of the islet microenvironment plays in regulating function. It also supports further investigation into the modulation of Piezo1 channel activity to restore islet function in diseases like type 2 diabetes (T2D) and pancreatic cancer where fibrosis of the -islet ECM leads to increased tissue stiffness and islet dysfunction.

Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca dynamics.

In type 1 diabetes (T1D), autoreactive immune cells infiltrate the pancreas and secrete proinflammatory cytokines that initiate cell death in insulin producing islet β-cells. Protein kinase C δ (PKCδ) plays a role in mediating cytokine-induced β-cell death; however, the exact mechanisms are not well understood. To address this, we used an inducible β-cell specific PKCδ KO mouse as well as a small peptide inhibitor of PKCδ. We identified a role for PKCδ in mediating cytokine-induced β-cell death and have shown that inhibiting PKCδ protects pancreatic β-cells from cytokine-induced apoptosis in both mouse and human islets. We determined that cytokines induced nuclear translocation and activity of PKCδ and that caspase-3 cleavage of PKCδ may be required for cytokine-mediated islet apoptosis. Further, cytokine activated PKCδ increases activity both of proapoptotic Bax with acute treatment and C-Jun N-terminal kinase with prolonged treatment. Overall, our results suggest that PKCδ mediates cytokine-induced apoptosis via nuclear translocation, cleavage by caspase-3, and upregulation of proapoptotic signaling in pancreatic β-cells. Combined with the protective effects of PKCδ inhibition with δV1-1, the results of this study will aid in the development of novel therapies to prevent or delay β-cell death and preserve β-cell function in T1D.

Cleavage of protein kinase c δ by caspase-3 mediates proinflammatory cytokine-induced apoptosis in pancreatic islets.

Targeting current therapies to treat or prevent the loss of pancreatic islet β-cells in Type 1 Diabetes (T1D) may provide improved efficacy and reduce off-target effects. Current efforts to target the β-cell are limited by a lack of β-cell-specific targets and the inability to test multiple targeting moieties with the same delivery vehicle. Here, we fabricate a tailorable polycaprolactone nanocapsule (NC) in which multiple different targeting peptides can be interchangeably attached for β-cell-specific delivery. Incorporation of a cationic surfactant in the NC shell allows for the attachment of Exendin-4 and an antibody for ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3) for β-cell-specific targeting. The average NC size ranges from 250 to 300 nm with a polydispersity index under 0.2. The NCs are nontoxic, stable in media culture, and can be lyophilized and reconstituted. NCs coated with a targeting peptide were taken up by human cadaveric islet β-cells and human stem cell-derived β-like cells (sBC) in vitro with a high level of specificity. Furthermore, NCs successfully delivered both hydrophobic and hydrophilic cargo to human β-cells. Additionally, Exendin-4-coated NCs were stable and targeted the mouse pancreatic islet β-cell in vivo. Overall, our tailorable NCs have the potential to improve cell-targeted drug delivery and can be utilized as a screening platform to test the efficacy of cell-targeting peptides.

Peptide-Coated Polycaprolactone-Benzalkonium Chloride Nanocapsules for Targeted Drug Delivery to the Pancreatic β-Cell.

Here, we demonstrate that the spatial distribution of lipophilic cations governs the complexation pathways, serum stability, and biological performance of polymer-pDNA complexes (polyplexes). Previous research focused on block/statistical copolymers, whereas gradient copolymers, where the density of lipophilic cations diminishes (gradually or steeply) along polymer backbones, remain underexplored. We engineered gradient copolymers that combine the polyplex colloidal stability of block copolymers with the transfection efficiency of statistical copolymers. We synthesized length- and compositionally equivalent gradient copolymers (-) along with statistical () and block () copolymers of 2-(diisopropylamino)ethyl methacrylate and 2-hydroxyethyl methacrylate. We mapped how polymer microstructure governs pDNA loading per polyplex, pDNA conformational changes, and polymer-pDNA binding thermodynamics via static light scattering, circular dichroism spectroscopy, and isothermal titration calorimetry, respectively. While gradient steepness is a powerful design handle to improve polyplex physical properties, augment pDNA delivery capacity, and attenuate polycation-triggered hemolysis, microstructural contrasts did not elicit differences in complement activation.

The Spatial Distribution of Lipophilic Cations in Gradient Copolymers Regulates Polymer-pDNA Complexation, Polyplex Aggregation, and Intracellular pDNA Delivery.

Type 1 diabetes is an autoimmune disease characterised by the destruction of pancreatic beta cells, resulting in lifelong insulin dependence. Although exogenous insulin can maintain glycaemic control, this approach does not protect residual or replacement pancreatic beta cells from immune-mediated death. Current therapeutics designed to protect functional beta cell mass or promote beta cell proliferation and regeneration can have off-target effects, resulting in higher dose requirements and adverse side effects. Targeted drug delivery using nanocarriers has demonstrated potential for overcoming these limitations. The critical bottleneck limiting the development of beta cell-targeted therapies is a lack of highly specific beta cell markers. This review provides an overview of the use of nanocarriers for cell-targeted delivery and the current state of the field of beta cell targeting. Technologies such as systematic evolution of ligands by exponential enrichment (SELEX) aptamer selection, phage display screening, and omics datasets from human samples are highlighted as tools to identify novel beta cell-specific targets that can be combined with nanocarriers for targeted delivery of therapeutics. Ultimately, beta cell-targeted therapies using nanocarriers present a unique opportunity to develop tailored treatments for each stage of type 1 diabetes with the goal of providing individuals with treatment options that prevent further progression or reverse the course of the disease.

Active targeting of type 1 diabetes therapies to pancreatic beta cells using nanocarriers.

We present the synthesis, properties, and imaging applications of a new class of diazaborine-based probes (Peroxynitrite Probe-1, PNP-1) for selective peroxynitrite (ONOO) imaging in live cells. PNP-1 features a diazaborine-based reaction motif that provides excellent discrimination between HO and ONOO, solving a persistent challenge of organoboron-based fluorescent probes for oxidative metabolite imaging. We demonstrate the utility of PNP-1 to detect endogenously produced ONOO in live RAW 264.7 macrophages by fluorescence microscopy, with probe selectivity confirmed with inhibition of NADPH oxidases and nitric oxide synthase, the requisite enzymatic machinery for ONOO production. Co-localization studies unexpectedly reveal preferential mitochondrial localization, which we show is dependent on the naphthalimide scaffold. Taken together, our results show that diazaborines are a novel motif for selective ONOO reactivity, positioning them for incorporation into other ONOO-specific chemical biology tools.

Article	Year
Modulation of Gap Junction Coupling Within the Islet of Langerhans During the Development of Type 1 Diabetes. Frontiers in physiology\| PubMed ID: 35837011	2022
Extracellular matrix stiffness mediates insulin secretion in pancreatic islets via mechanosensitive Piezo1 channel regulated Ca dynamics. Matrix biology plus\| PubMed ID: 38803329	2024
Cleavage of protein kinase c δ by caspase-3 mediates proinflammatory cytokine-induced apoptosis in pancreatic islets. The Journal of biological chemistry\| PubMed ID: 39074637	2024
Peptide-Coated Polycaprolactone-Benzalkonium Chloride Nanocapsules for Targeted Drug Delivery to the Pancreatic β-Cell. ACS applied bio materials\| PubMed ID: 39315885	2024
The Spatial Distribution of Lipophilic Cations in Gradient Copolymers Regulates Polymer-pDNA Complexation, Polyplex Aggregation, and Intracellular pDNA Delivery. Biomacromolecules\| PubMed ID: 39318335	2024
Active targeting of type 1 diabetes therapies to pancreatic beta cells using nanocarriers. Diabetologia\| PubMed ID: 39847085	2025
A Diazaborine Activity-Based Sensing Fluorescent Probe Reports Endogenously Produced Mitochondrial Peroxynitrite in Live Macrophages. Chembiochem : a European journal of chemical biology\| PubMed ID: 40064607	2025

Nikki L. Farnsworth

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