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Beta cell replacement strategies hold promise for permanently treating type 1 diabetes. In Cell Stem Cell, Xiao et al. (2018) restore pancreatic beta cell mass and normalize blood glucose in diabetic mice by reprogramming pancreatic alpha to beta cells using Pdx1- and Mafa-expressing adeno-associated virus infused into the pancreatic duct.
Copyright © 2017 Elsevier Inc. All rights reserved.
Excessive glucocorticoid exposure has been shown to be deleterious for pancreatic β-cell function and insulin release. However, glucocorticoids at physiological levels are essential for many homeostatic processes, including glycemic control. We show that corticosterone and cortisol and their less active precursors 11-dehydrocorticosterone (11-DHC) and cortisone suppress voltage-dependent Ca channel function and Ca fluxes in rodent as well as in human β-cells. However, insulin secretion, maximal ATP/ADP responses to glucose, and β-cell identity were all unaffected. Further examination revealed the upregulation of parallel amplifying cAMP signals and an increase in the number of membrane-docked insulin secretory granules. Effects of 11-DHC could be prevented by lipotoxicity and were associated with paracrine regulation of glucocorticoid activity because global deletion of 11β-hydroxysteroid dehydrogenase type 1 normalized Ca and cAMP responses. Thus, we have identified an enzymatically amplified feedback loop whereby glucocorticoids boost cAMP to maintain insulin secretion in the face of perturbed ionic signals. Failure of this protective mechanism may contribute to diabetes in states of glucocorticoid excess, such as Cushing syndrome, which are associated with frank dyslipidemia.
© 2017 by the American Diabetes Association.
Apicobasal polarity is known to affect epithelial morphogenesis and cell differentiation, but it remains unknown how these processes are mechanistically orchestrated. We find that ligand-specific EGFR signalling via PI(3)K and Rac1 autonomously modulates apicobasal polarity to enforce the sequential control of morphogenesis and cell differentiation. Initially, EGF controls pancreatic tubulogenesis by negatively regulating apical polarity induction. Subsequently, betacellulin, working via inhibition of atypical protein kinase C (aPKC), causes apical domain constriction within neurogenin3 endocrine progenitors, which results in reduced Notch signalling, increased neurogenin3 expression, and β-cell differentiation. Notably, the ligand-specific EGFR output is not driven at the ligand level, but seems to have evolved in response to stage-specific epithelial influences. The EGFR-mediated control of β-cell differentiation via apical polarity is also conserved in human neurogenin3 cells. We provide insight into how ligand-specific EGFR signalling coordinates epithelial morphogenesis and cell differentiation via apical polarity dynamics.
Stimulus-secretion coupling in pancreatic β-cells requires Ca influx through voltage-dependent Ca channels, whose activity is controlled by the plasma membrane potential (V ). Here, we present a method of measuring fluctuations in the β-cell V and Ca influx simultaneously, which provides valuable information about the ionic signaling mechanisms that underlie insulin secretion. This chapter describes the use of perforated patch clamp electrophysiology on cells loaded with a fluorescent intracellular Ca indicator, which permits the stable recording conditions needed to monitor the V and Ca influx in β-cells. Moreover, this chapter describes the protocols necessary for the preparation of mouse and human islet cells for the simultaneous recording of V and Ca as well as determining the specific islet cell type assessed in each experiment.
Ca handling by the endoplasmic reticulum (ER) serves critical roles in controlling pancreatic β cell function and becomes perturbed during the pathogenesis of diabetes. ER Ca homeostasis is determined by ion movements across the ER membrane, including K flux through K channels. We demonstrated that K flux through ER-localized TALK-1 channels facilitated Ca release from the ER in mouse and human β cells. We found that β cells from mice lacking TALK-1 exhibited reduced basal cytosolic Ca and increased ER Ca concentrations, suggesting reduced ER Ca leak. These changes in Ca homeostasis were presumably due to TALK-1-mediated ER K flux, because we recorded K currents mediated by functional TALK-1 channels on the nuclear membrane, which is continuous with the ER. Moreover, overexpression of K-impermeable TALK-1 channels in HEK293 cells did not reduce ER Ca stores. Reduced ER Ca content in β cells is associated with ER stress and islet dysfunction in diabetes, and islets from TALK-1-deficient mice fed a high-fat diet showed reduced signs of ER stress, suggesting that TALK-1 activity exacerbated ER stress. Our data establish TALK-1 channels as key regulators of β cell ER Ca and suggest that TALK-1 may be a therapeutic target to reduce ER Ca handling defects in β cells during the pathogenesis of diabetes.
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
AIMS/HYPOTHESIS - Tissue-resident macrophages sense the microenvironment and respond by producing signals that act locally to maintain a stable tissue state. It is now known that pancreatic islets contain their own unique resident macrophages, which have been shown to promote proliferation of the insulin-secreting beta cell. However, it is unclear how beta cells communicate with islet-resident macrophages. Here we hypothesised that islet macrophages sense changes in islet activity by detecting signals derived from beta cells.
METHODS - To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells.
RESULTS - Islet-resident macrophages expressed functional purinergic receptors, making them exquisite sensors of interstitial ATP levels. Indeed, islet-resident macrophages responded selectively to ATP released locally from beta cells that were physiologically activated with high levels of glucose. Because ATP is co-released with insulin and is exclusively secreted by beta cells, the activation of purinergic receptors on resident macrophages facilitates their awareness of beta cell secretory activity.
CONCLUSIONS/INTERPRETATION - Our results indicate that islet macrophages detect ATP as a proxy signal for the activation state of beta cells. Sensing beta cell activity may allow macrophages to adjust the secretion of factors to promote a stable islet composition and size.
PDX1/NKX6-1 pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of β cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2 cells generate β-like cells (C-PEPTIDE/NKX6-1) more efficiently compared to GP2 and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.
The transcription factor encoded by the gene is a critical transcriptional regulator, as it has fundamental actions in the formation of all pancreatic cell types, islet β-cell development, and adult islet β-cell function. Transgenic- and cell line-based experiments have identified 5'-flanking conserved sequences that control pancreatic and β-cell type-specific transcription, which are found within areas I (bp -2694 to -2561), II (bp -2139 to -1958), III (bp -1879 to -1799), and IV (bp -6200 to -5670). Because of the presence in area IV of binding sites for transcription factors associated with pancreas development and islet cell function, we analyzed how an endogenous deletion mutant affected expression embryonically and postnatally. The most striking result was observed in male mutant mice after 3 weeks of birth (i.e., the onset of weaning), with only a small effect on pancreas organogenesis and no deficiencies in their female counterparts. Compromised Pdx1 mRNA and protein levels in weaned male mutant β-cells were tightly linked with hyperglycemia, decreased β-cell proliferation, reduced β-cell area, and altered expression of Pdx1-bound genes that are important in β-cell replication, endoplasmic reticulum function, and mitochondrial activity. We discuss the impact of these novel findings to gene regulation and islet β-cell maturation postnatally.
© 2017 by the American Diabetes Association.
We used mice lacking , a key component of the β-cell K-channel, to analyze the effects of a sustained elevation in the intracellular Ca concentration ([Ca]) on β-cell identity and gene expression. Lineage tracing analysis revealed the conversion of β-cells lacking into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified β-cells confirmed an increase in gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca signaling, the maintenance of β-cell identity, and cell adhesion. The expression of and , two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca] Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, , was confirmed to be directly controlled by Ca influx in β-cells. Interestingly, among the upregulated genes is , a putative marker of β-cell dedifferentiation, and other genes associated with β-cell failure. Taken together, our results suggest that chronically elevated β-cell [Ca] in islets contributes to the alteration of β-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca-regulated genes.
© 2017 by the American Diabetes Association.
Glucose-stimulated insulin secretion (GSIS) relies on β-cell Ca2+ influx, which is modulated by the two-pore-domain K+ (K2P) channel, TALK-1. A gain-of-function polymorphism in KCNK16, the gene encoding TALK-1, increases risk for developing type-2 diabetes. While TALK-1 serves an important role in modulating GSIS, the regulatory mechanism(s) that control β-cell TALK-1 channels are unknown. Therefore, we employed a membrane-specific yeast two-hybrid (MYTH) assay to identify TALK-1-interacting proteins in human islets, which will assist in determining signaling modalities that modulate TALK-1 function. Twenty-one proteins from a human islet cDNA library interacted with TALK-1. Some of these interactions increased TALK-1 activity, including intracellular osteopontin (iOPN). Intracellular OPN is highly expressed in β-cells and is upregulated under pre-diabetic conditions to help maintain normal β-cell function; however, the functional role of iOPN in β-cells is poorly understood. We found that iOPN colocalized with TALK-1 in pancreatic sections and coimmunoprecipitated with human islet TALK-1 channels. As human β-cells express two K+ channel-forming variants of TALK-1, regulation of these TALK-1 variants by iOPN was assessed. At physiological voltages iOPN activated TALK-1 transcript variant 3 channels but not TALK-1 transcript variant 2 channels. Activation of TALK-1 channels by iOPN also hyperpolarized resting membrane potential (Vm) in HEK293 cells and in primary mouse β-cells. Intracellular OPN was also knocked down in β-cells to test its effect on β-cell TALK-1 channel activity. Reducing β-cell iOPN significantly decreased TALK-1 K+ currents and increased glucose-stimulated Ca2+ influx. Importantly, iOPN did not affect the function of other K2P channels or alter Ca2+ influx into TALK-1 deficient β-cells. These results reveal the first protein interactions with the TALK-1 channel and found that an interaction with iOPN increased β-cell TALK-1 K+ currents. The TALK-1/iOPN complex caused Vm hyperpolarization and reduced β-cell glucose-stimulated Ca2+ influx, which is predicted to inhibit GSIS.