Alejandro Caicedo

A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations

Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA‐binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA‐binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining β‐cell viability.

Noninvasive in vivo imaging of pancreatic islet cell biology

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.

Innervation Patterns of Autonomic Axons in the Human Endocrine Pancreas

The autonomic nervous system regulates hormone secretion from the endocrine pancreas, the islets of Langerhans, thus impacting glucose metabolism. The parasympathetic and sympathetic nerves innervate the pancreatic islet, but the precise innervation patterns are unknown, particularly in human. Here we demonstrate that the innervation of human islets is different from that of mouse islets and does not conform to existing models of autonomic control of islet function. By visualizing axons in three dimensions and quantifying axonal densities and contacts within pancreatic islets, we found that, unlike mouse endocrine cells, human endocrine cells are sparsely contacted by autonomic axons. Few parasympathetic cholinergic axons penetrate the human islet, and the invading sympathetic fibers preferentially innervate smooth muscle cells of blood vessels located within the islet. Thus, rather than modulating endocrine cell function directly, sympathetic nerves may regulate hormone secretion in human islets by controlling local blood flow or by acting on islet regions located downstream.

Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming beta cell function in humans

Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-secreting pancreatic beta cell. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to the case in mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of human islets provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. Cholinergic signaling within islets represents a potential therapeutic target in diabetes, highlighting the relevance of this advance to future drug development.

Individual mouse taste cells respond to multiple chemical stimuli

Sensory organs are specialized to detect and decode stimuli in terms of intensity and quality. In the gustatory system, the process of identifying and distinguishing taste qualities (e.g. bitter versus sweet) begins in taste buds. A central question in gustatory research is how information about taste quality is extracted by taste receptor cells. For instance, whether and how individual taste cells respond to multiple chemical stimuli is still a matter for debate. A recent study showed that taste cells expressing bitter‐responsive taste receptors do not also express sweet‐responsive taste receptors and vice versa. These results suggest that the gustatory system may use separate cellular pathways to process bitter and sweet signals independently. Results from electrophysiological studies, however, reveal that individual taste receptor cells respond to stimuli representing multiple taste qualities. Here we used non‐invasive Ca2+ imaging in slices of lingual tissue containing taste buds to address the issue of quality detection in murine taste receptor cells. We recorded calcium transients elicited by chemical stimuli representing different taste qualities (sweet, salty, sour and bitter). Many receptor cells (38 %) responded to multiple taste qualities, with some taste cells responding to both appetitive (‘sweet’) and aversive (‘bitter’) stimuli. Thus, there appears to be no strict and separate detection of taste qualities by distinct subpopulations of taste cells in peripheral gustatory sensory organs in mice.

Glutamate is a positive autocrine signal for glucagon release.

An important feature of glucose homeostasis is the effective release of glucagon from the pancreatic alpha cell. The molecular mechanisms regulating glucagon secretion are still poorly understood. We now demonstrate that human alpha cells express ionotropic glutamate receptors (iGluRs) that are essential for glucagon release. A lowering in glucose concentration results in the release of glutamate from the alpha cell. Glutamate then acts on iGluRs of the AMPA/kainate type, resulting in membrane depolarization, opening of voltage-gated Ca(2+) channels, increase in cytoplasmic free Ca(2+) concentration, and enhanced glucagon release. In vivo blockade of iGluRs reduces glucagon secretion and exacerbates insulin-induced hypoglycemia in mice. Hence, the glutamate autocrine feedback loop endows the alpha cell with the ability to effectively potentiate its own secretory activity. This is a prerequisite to guarantee adequate glucagon release despite relatively modest changes in blood glucose concentration under physiological conditions.

Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye

There is clearly a demand for an experimental platform that enables cell biology to be studied in intact vascularized and innervated tissue in vivo. This platform should allow observations of cells noninvasively and longitudinally at single-cell resolution. For this purpose, we use the anterior chamber of the mouse eye in combination with laser scanning microscopy (LSM). Tissue transplanted to the anterior chamber of the eye is rapidly vascularized, innervated and regains function. After transplantation, LSM through the cornea allows repetitive and noninvasive in vivo imaging at cellular resolution. Morphology, vascularization, cell function and cell survival are monitored longitudinally using fluorescent proteins and dyes. We have used this system to study pancreatic islets, but the platform can easily be adapted for studying a variety of tissues and additional biological parameters. Transplantation to the anterior chamber of the eye takes 25 min, and in vivo imaging 1–5 h, depending on the features monitored.

Sour Taste Stimuli Evoke Ca2+ and pH Responses in Mouse Taste Cells

Sour taste is elicited by acids. How taste cells transduce sour taste is controversial because acids (specifically protons) have diverse effects on cell membranes. Consequently, it is difficult to differentiate between events related to sour taste transduction per se and unrelated effects of protons. We have studied acid taste transduction in mouse taste buds using a lingual slice preparation where it is possible to measure changes in pH and [Ca2+]i simultaneously in taste cells. Focal application of citric acid or HCl to the apical tips of taste buds produced widespread acidification of the entire taste bud. Citric acid was effective at a pH of ∼4, but HCl only at a pH of ∼1.5. Despite acidification of the whole taste bud, only a select few taste cells exhibited Ca2+ responses. Acid‐evoked Ca2+ responses were dose dependent in a range consistent with them being sour‐taste responses. Cells exhibiting acid‐evoked Ca2+ responses also responded to KCl depolarization. Acid‐evoked Ca2+ responses were blocked by Ba2+ (2 mm) and Cd2+ (500 μm), suggesting that acid responses are generated by Ca2+ influx through depolarization‐gated Ca2+ channels. Removing extracellular Ca2+ reduced acid‐evoked Ca2+ responses, but depleting intracellular Ca2+ stores with thapsigargin had no effect, suggesting that acid taste responses are generated by an influx of extracellular Ca2+. Neither Cs+ (500 μm) nor amiloride (100 μm) affected acid‐evoked Ca2+ responses, suggesting that neither hyperpolarization‐activated cyclic nucleotide‐gated cation (pacemaker) channels nor epithelial Na+ channels, respectively, transduce sour taste. Collectively, the results indicate that acids, especially weak acids, acidify the taste bud and evoke depolarization‐induced Ca2+ entry into a select subset of taste cells. The primary transducer protein(s) for sour taste remain undiscovered.

ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic β cell

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human β cells sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca2+ concentration, [Ca2+]i, and hormone release in vitro, we show that human β cells express ionotropic ATP receptors of the P2X3 type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X3 receptors in the β-cell plasma membrane, resulting in increased [Ca2+]i and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the β cells secretory machinery. This may explain how the human pancreatic β cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.

Role of the G-Protein Subunit α-Gustducin in Taste Cell Responses to Bitter Stimuli

Many bitter stimuli are believed to bind to specific G-protein-coupled membrane receptors on taste cells. Despite the compelling evidence for its pivotal role in bitter taste sensation, a direct involvement of the G-protein subunit α-gustducin in bitter taste transduction in taste cells has not been demonstrated in situ at the cellular level. We recorded activation of taste cells by bitter stimuli using Ca2+ imaging in lingual slices and examinedα-gustducin immunoreactivity in the same cells. In mice vallate papillae, many, but not all, bitter-responsive cells expressed α-gustducin. In agreement with this correlation, the incidence of cells responding to bitter stimuli was reduced by 70% in mutant mice lacking α-gustducin. Nevertheless, some taste cells lacking α-gustducin responded to bitter stimuli, suggesting that other G-protein α subunits are involved. We found that the G-protein α subunit Gαi2 is present in most bitter-responsive cells and thus may also play a role in bitter taste transduction. The reduced behavioral sensitivity to bitter stimuli in α-gustducin knock-out mice thus appears to be the consequence of a reduced number of bitter-activated taste cells, as well as reduced sensitivity.