Amicia D. Elliott

NFI transcription factors interact with FOXA1 to regulate prostate-specific gene expression

Androgen receptor (AR) action throughout prostate development and in maintenance of the prostatic epithelium is partly controlled by interactions between AR and forkhead box (FOX) transcription factors, particularly FOXA1. We sought to identity additional FOXA1 binding partners that may mediate prostate-specific gene expression. Here we identify the nuclear factor I (NFI) family of transcription factors as novel FOXA1 binding proteins. All four family members (NFIA, NFIB, NFIC, and NFIX) can interact with FOXA1, and knockdown studies in androgen-dependent LNCaP cells determined that modulating expression of NFI family members results in changes in AR target gene expression. This effect is probably mediated by binding of NFI family members to AR target gene promoters, because chromatin immunoprecipitation (ChIP) studies found that NFIB bound to the prostate-specific antigen enhancer. Förster resonance energy transfer studies revealed that FOXA1 is capable of bringing AR and NFIX into proximity, indicating that FOXA1 facilitates the AR and NFI interaction by bridging the complex. To determine the extent to which NFI family members regulate AR/FOXA1 target genes, motif analysis of publicly available data for ChIP followed by sequencing was undertaken. This analysis revealed that 34.4% of peaks bound by AR and FOXA1 contain NFI binding sites. Validation of 8 of these peaks by ChIP revealed that NFI family members can bind 6 of these predicted genomic elements, and 4 of the 8 associated genes undergo gene expression changes as a result of individual NFI knockdown. These observations suggest that NFI regulation of FOXA1/AR action is a frequent event, with individual family members playing distinct roles in AR target gene expression.

Somatostatin and insulin mediate glucose-inhibited glucagon secretion in the pancreatic α-cell by lowering cAMP

The dysregulation of glucose-inhibited glucagon secretion from the pancreatic islet α-cell is a critical component of diabetes pathology and metabolic disease. We show a previously uncharacterized [Ca(2+)]i-independent mechanism of glucagon suppression in human and murine pancreatic islets whereby cAMP and PKA signaling are decreased. This decrease is driven by the combination of somatostatin, which inhibits adenylyl cyclase production of cAMP via the Gαi subunit of the SSTR2, and insulin, which acts via its receptor to activate phosphodiesterase 3B and degrade cytosolic cAMP. Our data indicate that both somatostatin and insulin signaling are required to suppress cAMP/PKA and glucagon secretion from both human and murine α-cells, and the combination of these two signaling mechanisms is sufficient to reduce glucagon secretion from isolated α-cells as well as islets. Thus, we conclude that somatostatin and insulin together are critical paracrine mediators of glucose-inhibited glucagon secretion and function by lowering cAMP/PKA signaling with increasing glucose.

Real-time hyperspectral fluorescence imaging of pancreatic β-cell dynamics with the image mapping spectrometer.

Summary The development of multi-colored fluorescent proteins, nanocrystals and organic fluorophores, along with the resulting engineered biosensors, has revolutionized the study of protein localization and dynamics in living cells. Hyperspectral imaging has proven to be a useful approach for such studies, but this technique is often limited by low signal and insufficient temporal resolution. Here, we present an implementation of a snapshot hyperspectral imaging device, the image mapping spectrometer (IMS), which acquires full spectral information simultaneously from each pixel in the field without scanning. The IMS is capable of real-time signal capture from multiple fluorophores with high collection efficiency (∼65%) and image acquisition rate (up to 7.2 fps). To demonstrate the capabilities of the IMS in cellular applications, we have combined fluorescent protein (FP)-FRET and [Ca2+]i biosensors to measure simultaneously intracellular cAMP and [Ca2+]i signaling in pancreatic &bgr;-cells. Additionally, we have compared quantitatively the IMS detection efficiency with a laser-scanning confocal microscope.

Interleukin-6 amplifies glucagon secretion: coordinated control via the brain and pancreas

Inappropriate glucagon secretion contributes to hyperglycemia in inflammatory disease. Previous work implicates the proinflammatory cytokine interleukin-6 (IL-6) in glucagon secretion. IL-6-KO mice have a blunted glucagon response to lipopolysaccharide (LPS) that is restored by intravenous replacement of IL-6. Given that IL-6 has previously been demonstrated to have a transcriptional (i.e., slow) effect on glucagon secretion from islets, we hypothesized that the rapid increase in glucagon following LPS occurred by a faster mechanism, such as by action within the brain. Using chronically catheterized conscious mice, we have demonstrated that central IL-6 stimulates glucagon secretion uniquely in the presence of an accompanying stressor (hypoglycemia or LPS). Contrary to our hypothesis, however, we found that IL-6 amplifies glucagon secretion in two ways; IL-6 not only stimulates glucagon secretion via the brain but also by direct action on islets. Interestingly, IL-6 augments glucagon secretion from both sites only in the presence of an accompanying stressor (such as epinephrine). Given that both adrenergic tone and plasma IL-6 are elevated in multiple inflammatory diseases, the interactions of the IL-6 and catecholaminergic signaling pathways in regulating GCG secretion may contribute to our present understanding of these diseases.

Hyperspectral imaging for simultaneous measurements of two FRET biosensors in pancreatic β-cells

Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS’s capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic β-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic β-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.

Real-time hyperspectral imaging of pancreatic β-cell dynamics with Image Mapping Spectrometer (IMS)

Real-time hyperspectral imaging of pancreatic β-cell dynamics is achieved by utilizing an Image Mapping Spectrometer (IMS). The calcium signal was successfully monitored during caspase-3 mediated FRET in cellular apoptosis.

Image Mapping Spectrometer (IMS) for Real Time Hyperspectral Fluorescence Microscopy

Image Mapping Spectrometer is a non-scanning hyperspectral imaging technique providing a complete spectral-spatial information simultaneously. IMS acquires, analyzes and displays data at 5-10 frame/sec rates. Imaging results for cells expressing GFP/YFP/CFP are presented.

Monitoring the [ATP]/[ADP] Ratio in Beta-Cells During Glucose Stimulated Insulin Secretion Using the Genetically Encoded Fluorescent Reporter Perceval

Pancreatic beta-cells secrete insulin in response to elevated blood glucose levels. Glucose stimulated insulin secretion depends on glucose metabolism that produces ATP. The resulting increase in [ATP]/[ADP] ratio closes ATP-sensitive potassium (KATP) channels, which leads to membrane depolarization and opening of voltage-dependent Ca2+ channels. This causes an elevation of intracellular free Ca2+ and insulin exocytosis. Insulin is secreted in a pulsatile manner, which is thought to be regulated in part by oscillations in glucose metabolism. Such metabolic oscillations would also lead to oscillations in the [ATP]/[ADP] ratio and hence regulate KATP channel activity.Oscillations in [ATP]/[ADP] ratio have been demonstrated using biochemical and luciferase assays, but neither approach allows measurements of such oscillations in single cells. Perceval is a recently developed fluorescent protein biosensor for [ATP]/[ADP] ratio, and it permits direct measurement of [ATP]/[ADP] ratios inside living cells. We use Perceval in combination with quantitative confocal and two-photon excitation microscopy for direct measurement of the [ATP]/[ADP] ratio in beta-cells during glucose stimulated insulin secretion. For this purpose we have developed an adenoviral vector to express Perceval specifically in the beta-cells of intact mouse islets. Dynamic changes in [ATP]/[ADP] ratio can be correlated with glucose metabolism (by simultaneous imaging of Perceval fluorescence and NAD(P)H autofluorescence) and with intracellular free Ca2+ levels (by simultaneous imaging of Perceval fluorescence and the calcium sensor, FuraRed). This data allows us to test hypotheses regarding the role of localized subcellular signaling complexes and putative microdomains of glucose metabolism, [ATP]/[ADP] ratio, and Ca2+ dynamics in the regulation of glucose stimulated insulin secretion.