Wen Hong Li

Lipotoxicity disrupts incretin-regulated human β cell connectivity

Pancreatic β cell dysfunction is pathognomonic of type 2 diabetes mellitus (T2DM) and is driven by environmental and genetic factors. β cell responses to glucose and to incretins such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are altered in the disease state. While rodent β cells act as a coordinated syncytium to drive insulin release, this property is unexplored in human islets. In situ imaging approaches were therefore used to monitor in real time the islet dynamics underlying hormone release. We found that GLP-1 and GIP recruit a highly coordinated subnetwork of β cells that are targeted by lipotoxicity to suppress insulin secretion. Donor BMI was negatively correlated with subpopulation responses to GLP-1, suggesting that this action of incretin contributes to functional β cell mass in vivo. Conversely, exposure of mice to a high-fat diet unveiled a role for incretin in maintaining coordinated islet activity, supporting the existence of species-specific strategies to maintain normoglycemia. These findings demonstrate that β cell connectedness is an inherent property of human islets that is likely to influence incretin-potentiated insulin secretion and may be perturbed by diabetogenic insults to disrupt glucose homeostasis in humans.

Critical role of PIP5KIγ87 in InsP3-mediated Ca2+ signaling

Phosphatidylinositol 4,5-bisphosphate (PIP2) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP3 or IP3) and is therefore critical to intracellular Ca2+ signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase γ (PIP5KIγ87) as the major contributor of the PIP2 pool that supports G protein–coupled receptor (GPCR)-mediated IP3 generation. PIP5KIγ87 RNAi decreases the histamine-induced IP3 response and Ca2+ flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP2 mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIγ87s PIP2 pool that supports GPCR-mediated Ca2+ signaling is functionally compartmentalized from those generated by the other PIP5KIs.

Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn2+, and that Zn2+ is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn2+ chelation and bound Zn2+ with high selectivity against Ca2+ and Mg2+. When added to cultured β cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn2+ concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled β cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn2+/insulin release occurred largely in small groups of adjacent β cells, with each forming a “secretory unit.” Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca2+ elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca2+ signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn2+-containing granules, ZIMIR may find applications in β-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.

LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling.

Using a new class of photo-activatible fluorophores, we have developed a new imaging technique for measuring molecular transfer rates across gap junction connexin channels in intact living cells. This technique, named LAMP, involves local activation of a molecular fluorescent probe, NPE-HCCC2/AM, to optically label a cell. Subsequent dye transfer through gap junctions from labeled to unlabeled cells was quantified by fluorescence microscopy. Additional uncagings after prior dye transfers reached equilibrium enabled multiple measurements of dye transfer rates in the same coupled cell pair. Measurements in the same cell pair minimized variation due to differences in cell volume and number of gap junctions, allowing us to track acute changes in gap junction permeability. We applied the technique to study the regulation of gap junction coupling by intracellular Ca2+ ([Ca2+]i). Although agonist or ionomycin exposure can raise bulk [Ca2+]i to levels higher than those caused by capacitative Ca2+ influx, the LAMP assay revealed that only Ca2+ influx through the plasma membrane store-operated Ca2+ channels strongly reduced gap junction coupling. The noninvasive and quantitative nature of this imaging technique should facilitate future investigations of the dynamic regulation of gap junction communication.

Versatile Synthesis and Rational Design of Caged Morpholinos

Embryogenesis is regulated by genetic programs that are dynamically executed in a stereotypic manner, and deciphering these molecular mechanisms requires the ability to control embryonic gene function with similar spatial and temporal precision. Chemical technologies can enable such genetic manipulations, as exemplified by the use of caged morpholino (cMO) oligonucleotides to inactivate genes in zebrafish and other optically transparent organisms with spatiotemporal control. Here we report optimized methods for the design and synthesis of hairpin cMOs incorporating a dimethoxynitrobenzyl (DMNB)-based bifunctional linker that permits cMO assembly in only three steps from commercially available reagents. Using this simplified procedure, we have systematically prepared cMOs with differing structural configurations and investigated how the in vitro thermodynamic properties of these reagents correlate with their in vivo activities. Through these studies, we have established general principles for cMO design and successfully applied them to several developmental genes. Our optimized synthetic and design methodologies have also enabled us to prepare a next-generation cMO that contains a bromohydroxyquinoline (BHQ)-based linker for two-photon uncaging. Collectively, these advances establish the generality of cMO technologies and will facilitate the application of these chemical probes in vivo for functional genomic studies.

Photoactivatable fluorophores and techniques for biological imaging applications

Photoactivatable fluorophores (PAFs) are powerful imaging probes for tracking molecular and cellular dynamics with high spatiotemporal resolution in biological systems. Recent developments in biological microscopy have raised new demands for engineering new PAFs with improved properties, such as high two photon excitation efficiency, reversibility, cellular delivery and targeting. Here we review the history and some of the recent developments in this area, emphasizing our efforts in developing a new class of caged coumarins and related imaging methods for studying dynamic cell-cell communication through gap junction channels, and in extending the application of these caged coumarins to new areas including spatiotemporal control of microRNA activity in vivo.

ADCY5 Couples Glucose to Insulin Secretion in Human Islets

Single nucleotide polymorphisms (SNPs) within the ADCY5 gene, encoding adenylate cyclase 5, are associated with elevated fasting glucose and increased type 2 diabetes (T2D) risk. Despite this, the mechanisms underlying the effects of these polymorphic variants at the level of pancreatic β-cells remain unclear. Here, we show firstly that ADCY5 mRNA expression in islets is lowered by the possession of risk alleles at rs11708067. Next, we demonstrate that ADCY5 is indispensable for coupling glucose, but not GLP-1, to insulin secretion in human islets. Assessed by in situ imaging of recombinant probes, ADCY5 silencing impaired glucose-induced cAMP increases and blocked glucose metabolism toward ATP at concentrations of the sugar >8 mmol/L. However, calcium transient generation and functional connectivity between individual human β-cells were sharply inhibited at all glucose concentrations tested, implying additional, metabolism-independent roles for ADCY5. In contrast, calcium rises were unaffected in ADCY5-depleted islets exposed to GLP-1. Alterations in β-cell ADCY5 expression and impaired glucose signaling thus provide a likely route through which ADCY5 gene polymorphisms influence fasting glucose levels and T2D risk, while exerting more minor effects on incretin action.

Temporal and spatial regulation of microRNA activity with photoactivatable cantimirs.

MicroRNAs (miRNAs) are small non-coding RNAs that play numerous important roles in physiology and human diseases. During animal development, many miRNAs are expressed continuously from early embryos throughout adults, yet it is unclear whether these miRNAs are actually required at all the stages of development. Current techniques of manipulating microRNA function lack the required spatial and temporal resolution to adequately address the functionality of a given microRNA at a specific time or at single-cell resolution. To examine stage- or cell-specific function of miRNA during development and to achieve precise control of miRNA activity, we have developed photoactivatable antisense oligonucleotides against miRNAs. These caged oligonucleotides can be activated with 365 nm light with extraordinarily high efficiency to release potent antisense reagents to inhibit miRNAs. Initial application of these caged antimirs in a model organism (C. elegans) revealed that the activity of a miRNA (lsy-6) is required specifically around the comma stage during embryonic development to control a left/right asymmetric differentiation program in the C. elegans nervous system. This suggests that a transient input of lsy-6 during development is sufficient to specify the neuronal cell fate.

Imaging dynamic cell-cell junctional coupling in vivo using Trojan-LAMP

To study the physiological regulation and function of cell-cell gap junction communication in vivo, we developed a bioconjugate of caged dye, named dextran-CANPE-HCC, for imaging cell coupling in small model organisms. In vitro, the compound was photolyzed efficiently with robust fluorescence enhancement. Dextran-CANPE-HCC delivered into Caenorhabditis elegans oocytes was retained in cells throughout development. Using local uncaging, we photolyzed dextran-CANPE-HCC to release the small HCC dye and imaged the dynamics of intercellular dye transfer through gap junction channels, a technique we named Trojan–local activation of molecular fluorescent probes (LAMP). Early during embryonic development, the pattern of cell coupling undergoes dramatic remodeling and imaging revealed that the germ cell precursors, P2, P3 and P4, were isolated from the somatic cell communication compartment. As dextran-CANPE-HCC is chemically and metabolically stable, labeled worms showed very bright signal upon photoactivation after hatching, which allowed us to examine cell coupling in living worms noninvasively.

Intercellular calcium signaling in a gap junction-coupled cell network establishes asymmetric neuronal fates in C. elegans

The C. elegans left and right AWC olfactory neurons specify asymmetric subtypes, one default AWCOFF and one induced AWCON, through a stochastic, coordinated cell signaling event. Intercellular communication between AWCs and non-AWC neurons via a NSY-5 gap junction network coordinates AWC asymmetry. However, the nature of intercellular signaling across the network and how individual non-AWC cells in the network influence AWC asymmetry is not known. Here, we demonstrate that intercellular calcium signaling through the NSY-5 gap junction neural network coordinates a precise 1AWCON/1AWCOFF decision. We show that NSY-5 gap junctions in C. elegans cells mediate small molecule passage. We expressed vertebrate calcium-buffer proteins in groups of cells in the network to reduce intracellular calcium levels, thereby disrupting intercellular communication. We find that calcium in non-AWC cells of the network promotes the AWCON fate, in contrast to the autonomous role of calcium in AWCs to promote the AWCOFF fate. In addition, calcium in specific non-AWCs promotes AWCON side biases through NSY-5 gap junctions. Our results suggest a novel model in which calcium has dual roles within the NSY-5 network: autonomously promoting AWCOFF and non-autonomously promoting AWCON.