Measuring the rate of NADPH consumption by glutathione reductase in the cytosol and mitochondria.

Background: NADPH is an essential co-factor supporting the function of enzymes that participate in both inflammatory and anti-inflammatory pathways in myeloid cells, particularly macrophages. Although individual NADPH-dependent pathways are well characterized, how these opposing pathways are co-regulated to orchestrate an optimized inflammatory response is not well understood. To investigate this, techniques to track the consumption of NADPH need to be applied.

Beta cell specific ZnT8 gene deficiency and resulting loss in zinc content significantly improve insulin secretion.

Zinc transporter 8 (ZnT8) is highly expressed in pancreatic beta cells, localizes to insulin secretory granules (ISG), and regulates zinc content. ZnT8 gene polymorphisms have revealed a relationship between ZnT8 activity and type 2 diabetes (T2D) risk, however, the role of beta-cell ZnT8 is not well understood. A beta cell specific ZnT8 knockout (ZnT8 BKO) mouse model was investigated.

Twenty years of islet-on-a-chip: microfluidic tools for dissecting islet metabolism and function.

Pancreatic islets are metabolically active micron-sized tissues responsible for controlling blood glucose through the secretion of insulin and glucagon. A loss of functional islet mass results in type 1 and 2 diabetes. Islet-on-a-chip devices are powerful microfluidic tools used to trap and study living human and murine pancreatic islets and potentially stem cell-derived islet organoids.

Insulin C-peptide secretion on-a-chip to measure the dynamics of secretion and metabolism from individual islets.

First-phase glucose-stimulated insulin secretion is mechanistically linked to type 2 diabetes, yet the underlying metabolism is difficult to discern due to significant islet-to-islet variability. Here, we miniaturize a fluorescence anisotropy immunoassay onto a microfluidic device to measure C-peptide secretion from individual islets as a surrogate for insulin (InsC-chip). This method measures secretion from up to four islets at a time with ∼7 s resolution while providing an optical window for real-time live-cell imaging.

Apollo-NADP reveals in vivo adaptation of NADPH/NADP metabolism in electrically activated pancreatic β cells.

Several genetically encoded sensors have been developed to study live cell NADPH/NADP dynamics, but their use has been predominantly in vitro. Here, we developed an in vivo assay using the Apollo-NADP sensor and microfluidic devices to measure endogenous NADPH/NADP dynamics in the pancreatic β cells of live zebrafish embryos. Flux through the pentose phosphate pathway, the main source of NADPH in many cell types, has been reported to be low in β cells.

Targeting Apollo-NADP to Image NADPH Generation in Pancreatic Beta-Cell Organelles.

NADPH/NADP redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome.

Design of a versatile microfluidic device for imaging precision-cut-tissue slices.

Precision-cut-tissues (PCTs), which preserve many aspects of a tissue's microenvironment, are typically imaged using conventional sample dishes and chambers. These can require large amounts of reagent and, when used for flow-through experiments, the shear forces applied on the tissues are often ill-defined. Their physical design also makes it difficult to image large volumes and repetitively image smaller regions of interest in the living slice.

Laminin matrix regulates beta-cell FGFR5 expression to enhance glucose-stimulated metabolism.

We previously showed that pancreatic beta-cells plated on laminin matrix express reduced levels of FGFR1, a receptor linked to beta-cell metabolism and differentiation. Due to recent evidence that adult beta-cells also express FGFR5, a co-receptor for FGFR1, we now aim to determine the effect of laminin on FGFR5 expression and consequent effects on beta-cell metabolism. Using a genetically encoded sensor for NADPH/NADP redox state (Apollo-NADP), we show overexpression of FGFR5 enhances glucose-stimulated NADPH metabolism in beta-cell lines as well as mouse and human beta-cells.

Mitochondrial Efflux of Citrate and Isocitrate Is Fully Dispensable for Glucose-Stimulated Insulin Secretion and Pancreatic Islet β-Cell Function.

The defining feature of pancreatic islet β-cell function is the precise coordination of changes in blood glucose levels with insulin secretion to regulate systemic glucose homeostasis. While ATP has long been heralded as a critical metabolic coupling factor to trigger insulin release, glucose-derived metabolites have been suggested to further amplify fuel-stimulated insulin secretion. The mitochondrial export of citrate and isocitrate through the citrate-isocitrate carrier (CIC) has been suggested to initiate a key pathway that amplifies glucose-stimulated insulin secretion, though the physiological significance of β-cell CIC-to-glucose homeostasis has not been established.

Pancreatic β cell-selective zinc transporter 8 insufficiency accelerates diabetes associated with islet amyloidosis.

GWAS have shown that the common R325W variant of SLC30A8 (ZnT8) increases the risk of type 2 diabetes (T2D). However, ZnT8 haploinsufficiency is protective against T2D in humans, counterintuitive to earlier work in humans and mouse models. Therefore, whether decreasing ZnT8 activity is beneficial or detrimental to β cell function, especially under conditions of metabolic stress, remains unknown.

Distinct roles of UVRAG and EGFR signaling in skeletal muscle homeostasis.

Objective: Autophagy is a physiological self-eating process that can promote cell survival or activate cell death in eukaryotic cells. In skeletal muscle, it is important for maintaining muscle mass and function that is critical to sustain mobility and regulate metabolism. The UV radiation resistance-associated gene (UVRAG) regulates the early stages of autophagy and autophagosome maturation and plays a key role in endosomal trafficking.

Leveraging multimodal microscopy to optimize deep learning models for cell segmentation.

Deep learning provides an opportunity to automatically segment and extract cellular features from high-throughput microscopy images. Many labeling strategies have been developed for this purpose, ranging from the use of fluorescent markers to label-free approaches. However, differences in the channels available to each respective training dataset make it difficult to directly compare the effectiveness of these strategies across studies.

Flow Rate Affects Nanoparticle Uptake into Endothelial Cells.

Nanoparticles are commonly administered through systemic injection, which exposes them to the dynamic environment of the bloodstream. Injected nanoparticles travel within the blood and experience a wide range of flow velocities that induce varying shear rates to the blood vessels. Endothelial cells line these vessels, and have been shown to uptake nanoparticles during circulation, but it is difficult to characterize the flow-dependence of this interaction in vivo.

Hypoxia induction in cultured pancreatic islets enhances endothelial cell morphology and survival while maintaining beta-cell function.

Background: Pancreatic islets are heavily vascularized in vivo yet lose this vasculature after only a few days in culture. Determining how to maintain islet vascularity in culture could lead to better outcomes in transplanting this tissue for the treatment of type 1 diabetes as well as provide insight into the complex communication between beta-cells and endothelial cells (ECs). We previously showed that islet ECs die in part due to limited diffusion of serum albumin into the tissue.

Fibroblast growth factor receptor 5 (FGFR5) is a co-receptor for FGFR1 that is up-regulated in beta-cells by cytokine-induced inflammation.

Fibroblast growth factor receptor-1 (FGFR1) activity at the plasma membrane is tightly controlled by the availability of co-receptors and competing receptor isoforms. We have previously shown that FGFR1 activity in pancreatic beta-cells modulates a wide range of processes, including lipid metabolism, insulin processing, and cell survival. More recently, we have revealed that co-expression of FGFR5, a receptor isoform that lacks a tyrosine-kinase domain, influences FGFR1 responses.

Isolation of Phenotypically Distinct Cancer Cells Using Nanoparticle-Mediated Sorting.

Isolating subpopulations of heterogeneous cancer cells is an important capability for the meaningful characterization of circulating tumor cells at different stages of tumor progression and during the epithelial-to-mesenchymal transition. Here, we present a microfluidic device that can separate phenotypically distinct subpopulations of cancer cells. Magnetic nanoparticles coated with antibodies against the epithelial cell adhesion molecule (EpCAM) are used to separate breast cancer cells in the microfluidic platform.

Dynamin-Related Protein 1-Dependent Mitochondrial Fission Changes in the Dorsal Vagal Complex Regulate Insulin Action.

Mitochondria undergo dynamic changes to maintain function in eukaryotic cells. Insulin action in parallel regulates glucose homeostasis, but whether specific changes in mitochondrial dynamics alter insulin action and glucose homeostasis remains elusive. Here, we report that high-fat feeding in rodents incurred adaptive dynamic changes in mitochondria through an increase in mitochondrial fission in parallel to an activation of dynamin-related protein 1 (Drp1) in the dorsal vagal complex (DVC) of the brain.

Clarifying intact 3D tissues on a microfluidic chip for high-throughput structural analysis.

On-chip imaging of intact three-dimensional tissues within microfluidic devices is fundamentally hindered by intratissue optical scattering, which impedes their use as tissue models for high-throughput screening assays. Here, we engineered a microfluidic system that preserves and converts tissues into optically transparent structures in less than 1 d, which is 20× faster than current passive clearing approaches. Accelerated clearing was achieved because the microfluidic system enhanced the exchange of interstitial fluids by 567-fold, which increased the rate of removal of optically scattering lipid molecules from the cross-linked tissue.

Single-Molecule Analysis of the Supramolecular Organization of the M2 Muscarinic Receptor and the Gαi1 Protein.

G protein-coupled receptors constitute the largest family of transmembrane signaling proteins and the largest pool of drug targets, yet their mechanism of action remains obscure. That uncertainty relates to unresolved questions regarding the supramolecular nature of the signaling complex formed by receptor and G protein. We therefore have characterized the oligomeric status of eGFP-tagged M2 muscarinic receptor (M2R) and Gi1 by single-particle photobleaching of immobilized complexes.

Highly efficient adenoviral transduction of pancreatic islets using a microfluidic device.

Tissues are challenging to genetically manipulate due to limited penetration of viral particles resulting in low transduction efficiency. We are particularly interested in expressing genetically-encoded sensors in ex vivo pancreatic islets to measure glucose-stimulated metabolism, however poor viral penetration biases these measurements to only a subset of cells at the periphery. To increase mass transfer of viral particles, we designed a microfluidic device that holds islets in parallel hydrodynamic traps connected by an expanding by-pass channel.

Allosteric modulation in monomers and oligomers of a G protein-coupled receptor.

The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects.

Tailoring nanoparticle designs to target cancer based on tumor pathophysiology.

Nanoparticles can provide significant improvements in the diagnosis and treatment of cancer. How nanoparticle size, shape, and surface chemistry can affect their accumulation, retention, and penetration in tumors remains heavily investigated, because such findings provide guiding principles for engineering optimal nanosystems for tumor targeting. Currently, the experimental focus has been on particle design and not the biological system.

Jagn1 Is Induced in Response to ER Stress and Regulates Proinsulin Biosynthesis.

The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches.

Apollo-NADP(+): a spectrally tunable family of genetically encoded sensors for NADP(+).

NADPH-dependent antioxidant pathways have a critical role in scavenging hydrogen peroxide (H2O2) produced by oxidative phosphorylation. Inadequate scavenging results in H2O2 accumulation and can cause disease. To measure NADPH/NADP(+) redox states, we explored genetically encoded sensors based on steady-state fluorescence anisotropy due to FRET (fluorescence resonance energy transfer) between homologous fluorescent proteins (homoFRET); we refer to these sensors as Apollo sensors.