Biology subjects

Delgadillo, L.

Publications and source records attributed to Delgadillo, L..

4 recordsLinked to original sources

bioRxiv2025

Exploration of individual beta cell function over time in vivo: effects of hyperglycemia and glucagon-like peptide-1 receptor (GLP1R) agonism

The coordinated function of beta cells within the pancreatic islet is required for the normal regulation of insulin secretion and is partly controlled by specialized "leader" and highly connected "hub" beta-cell subpopulations. Whether cells within these subpopulations are functionally stable in vivo remains unclear. Here, we establish an approach to monitor Ca2+ dynamics within individual beta cells over time, after engraftment into the anterior eye chamber, where continuous blood perfusion and near normal innervation pertain. Under normoglycemic conditions, islet network dynamics, and the behavior of individual leaders and hubs, remain stable for at least seven days. Hyperglycemia, resulting from high-fat diet feeding or the loss of a host Gck allele, caused engrafted islets to display incomplete and abortive Ca2+ waves and overall connectivity was diminished. Whereas hub cell numbers were lowered profoundly in both disease models, leaders largely persisted. Treatment with the GLP1R agonist Exendin-4 led to a recovery of islet-wide Ca2+ dynamics and the re-emergence of hub cells within minutes, with the effects of the incretin mimetic being more marked than those observed after analogous treatments in vitro. Similar observations were made using 3-dimensional imaging across the whole islet. Our findings thus suggest that incretins may act both directly and indirectly on beta cells in vivo. The approach described may provide broad applicability to the exploration of individual cell function over time in the living animal.

physiology↗

bioRxiv2025

ER calcium stores contribute to glucose-induced Ca2+ waves and intercellular connectivity in mouse pancreatic islets

Defective insulin secretion is a hallmark of diabetes mellitus. Glucose-induced Ca2+ oscillations are critical for the stimulation of insulin secretion, though the mechanisms through which these propagate across the islet are poorly understood. Here, we use beta cell-targeted GCaMP6f to explore the role of endoplasmic reticulum (ER) Ca2+ mobilization in response to submaximal (11mM) and hyperglycemic (25mM) glucose concentrations. Inhibition of inositol 1,4,5 trisphosphate (IP3) receptors, and other ion channels, with 2-aminoethoxydiphenyl borate (2-APB) had minimal effects on the initial peak or intercellular connectivity provoked by 11mM glucose. However, 2-APB lowered subsequent glucose-induced cytosolic Ca2+ increases and connectivity at both 11 and 25mM glucose. Unexpectedly, the activation of IP3 receptors with the muscarinic acetylcholine receptor agonist carbachol had minimal impact on the initial peak elicited by 11 mM glucose, but Ca2+ waves at 11 and 25 mM glucose were more poorly coordinated. To determine whether ER calcium mobilization was sufficient to initiate Ca2+ waves we next blocked sarco(endo)plasmic Ca2+ ATPase (SERCA) pumps with thapsigargin, whilst preventing plasma membrane depolarization with the KATP-channel opener, diazoxide. Under these conditions, an initial cytosolic Ca2+ increase was followed by secondary Ca2+ waves that slowly subsided. The application of carbachol alongside diazoxide still enhanced Ca2+ dynamics, though this activity was uncoordinated and beta cells were poorly connected. Our results show that ER Ca2+ mobilization plays a relatively minor role in the initiation and propagation of Ca2+ waves in response to glucose. On the other hand, ER stores are required to transition to sustained Ca2+ waves. HighlightsO_LIIP3R inhibition or activation perturbs glucose-induced Ca2+ waves in islets C_LIO_LIER store mobilization is insufficient to generate Ca2+ waves C_LIO_LIER Ca2+ stores are required for sustained Ca2+ waves and beta cell connectivity C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/643121v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e7ed00org.highwire.dtl.DTLVardef@78db10org.highwire.dtl.DTLVardef@7627e4org.highwire.dtl.DTLVardef@1351300_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Robust and Gradient Thickness Porous Membranes for In Vitro Modeling of Physiological Barriers

Porous membranes are fundamental elements for tissue-chip barrier and co-culture models. However, the exaggerated thickness of commonly available membranes impedes an accurate in vitro reproduction of the biological multi-cellular continuum as it occurs in vivo. Existing techniques to fabricate membranes such as solvent cast, spin-coating, sputtering and PE-CVD result in uniform thickness films. To understand critical separation distances for various barrier and co-culture models, a gradient thickness membrane is needed. Here, we developed a robust method to generate ultrathin porous parylene C (UPP) membranes not just with precise thicknesses down to 300 nm, but with variable gradients in thicknesses, while at the same time having porosities up to 25%. We also show surface etching and increased roughness lead to improved cell attachment. Next, we examined the mechanical properties of UPP membranes with varying porosity and thickness and fit our data to previously published models, which can help determine practical upper limits of porosity and lower limits of thickness. Lastly, we validate a straightforward approach allowing the successful integration of the UPP membranes into a prototyped 3D-printed scaffold enabling in vitro barrier modeling and investigation of cell-cell interplay over variable distances using thickness gradients.

bioengineering↗

bioRxiv2019

Altered Cell-Substrate Behavior on Microporous Membranes is a Result of Disruption and Grip

Porous membranes are ubiquitous in cell co-culture and tissue-on-a-chip studies. These materials are predominantly chosen for their semi-permeable and size exclusion properties to restrict or permit transmigration and cell-cell communication. However, previous studies have shown pore size, spacing and orientation affect cell behavior including extracellular matrix production and migration. The mechanism behind this behavior is not fully understood. In this study, we fabricated micropatterned non-fouling polyethylene glycol (PEG) islands to mimic pores in order to decouple the effect of surface discontinuity from grip provided by pore wall edges. Similar to porous membranes, we found that the PEG islands hindered fibronectin fibrillogenesis with cells on patterned substrates producing shorter fibrils. Additionally, cell migration speed over micropatterned PEG islands was greater than unpatterned controls, suggesting that disruption of cell-substrate interactions by PEG islands promoted a more dynamic and migratory behavior, similarly to cells migrating on microporous membranes. Preferred cellular directionality during migration was nearly identical between substrates with identically patterned PEG islands and micropores, further confirming disruption of cell-substrate interactions as a common mechanism behind the cellular responses on these substrates. Interestingly, cell spreading and the magnitude of migration speed was significantly greater on porous membranes compared to PEG islands with identical feature size and spacing, suggesting pore edges enhanced cellular grip. These results provide a more complete picture on how porous membranes affect cells which are grown on them in an increasing number of cellular barrier and co-culture studies.

bioengineering↗