A Versatile, Portable Intravital Microscopy Platform for Studying Beta-cell Biology In Vivo

The pancreatic islet is a complex micro-organ containing numerous cell types, including endocrine, immune, and endothelial cells. The communication of these systems is lost upon isolation of the islets, and therefore the pathogenesis of diabetes can only be fully understood by studying this organized, multicellular environment in vivo. We have developed several adaptable tools to create a versatile platform to interrogate β-cell function in vivo. Specifically, we developed β-cell-selective virally-encoded fluorescent protein biosensors that can be rapidly and easily introduced into any mouse. We then coupled the use of these biosensors with intravital microscopy, a powerful tool that can be used to collect cellular and subcellular data from living tissues. Together, these approaches allowed the observation of in vivo β-cell-specific ROS dynamics using the Grx1-roGFP2 biosensor and calcium signaling using the GcAMP6s biosensor. Next, we utilized abdominal imaging windows (AIW) to extend our in vivo observations beyond single-point terminal measurements to collect longitudinal physiological and biosensor data through repeated imaging of the same mice over time. This platform represents a significant advancement in our ability to study β-cell structure and signaling in vivo, and its portability for use in virtually any mouse model will enable meaningful studies of β-cell physiology in the endogenous islet niche

Platelet-type 12-lipoxygenase deletion provokes a compensatory 12/15-lipoxygenase increase that exacerbates oxidative stress in mouse islet β cells

In type 1 diabetes, an autoimmune event increases oxidative stress in islet β cells, giving rise to cellular dysfunction and apoptosis. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids that can form lipid metabolites involved in several biological functions, including oxidative stress. 12-Lipoxygenase and 12/15-lipoxygenase are related but distinct enzymes that are expressed in pancreatic islets, but their relative contributions to oxidative stress in these regions are still being elucidated. In this study, we used mice with global genetic deletion of the genes encoding 12-lipoxygenase (arachidonate 12-lipoxygenase, 12S type [Alox12]) or 12/15-lipoxygenase (Alox15) to compare the influence of each gene deletion on β cell function and survival in response to the β cell toxin streptozotocin. Alox12−/− mice exhibited greater impairment in glucose tolerance following streptozotocin exposure than WT mice, whereas Alox15−/− mice were protected against dysglycemia. These changes were accompanied by evidence of islet oxidative stress in Alox12−/− mice and reduced oxidative stress in Alox15−/− mice, consistent with alterations in the expression of the antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12−/− mice displayed a compensatory increase in Alox15 gene expression, and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. Collectively, these results indicate that Alox12 loss activates a compensatory increase in Alox15 that sensitizes mouse β cells to oxidative stress

Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Differential stimulation of insulin secretion by GLP-1 and Kisspeptin-10.

β-cells in the pancreatic islet respond to elevated plasma glucose by secreting insulin to maintain glucose homeostasis. In addition to glucose stimulation, insulin secretion is modulated by numerous G-protein coupled receptors (GPCRs). The GPCR ligands Kisspeptin-10 (KP) and glucagon-like peptide-1 (GLP-1) potentiate insulin secretion through Gq and Gs-coupled receptors, respectively. Despite many studies, the signaling mechanisms by which KP and GLP-1 potentiate insulin release are not thoroughly understood. We investigated the downstream signaling pathways of these ligands and their affects on cellular redox potential, intracellular calcium activity ([Ca(2+)]i), and insulin secretion from β-cells within intact murine islets. In contrast to previous studies performed on single β-cells, neither KP nor GLP-1 affect [Ca(2+)]i upon stimulation with glucose. KP significantly increases the cellular redox potential, while no effect is observed with GLP-1, suggesting that KP and GLP-1 potentiate insulin secretion through different mechanisms. Co-treatment with KP and the Gβγ-subunit inhibitor gallein inhibits insulin secretion similar to that observed with gallein alone, while co-treatment with gallein and GLP-1 does not differ from GLP-1 alone. In contrast, co-treatment with the Gβγ activator mSIRK and either KP or GLP-1 stimulates insulin release similar to mSIRK alone. Neither gallein nor mSIRK alter [Ca(2+)]i activity in the presence of KP or GLP-1. These data suggest that KP likely alters insulin secretion through a Gβγ-dependent process that stimulates glucose metabolism without altering Ca(2+) activity, while GLP-1 does so, at least partly, through a Gα-dependent pathway that is independent of both metabolism and Ca(2+)

Differential Stimulation of Insulin Secretion by GLP-1 and Kisspeptin-10 - Figure 1

<p><b>A</b>. Percent of insulin content secreted from intact islets after static incubation at 2.8, 10, or 16.7 mM glucose with and without KP (1 µM, dark gray) or GLP-1 (20 nM, light gray). Secretion from untreated control islets is shown in white. Data are the mean ± S.E. <i>n</i> = 4–19. *(<i>p</i><0.05) and **(<i>p</i><0.001) indicate significance compared to untreated control. <b>B</b>, Glucose-dependent percent change in NAD(P)H from untreated intact islets (circles) and islets treated with KP (1 µM, squares) or GLP-1 (20 nM, triangles) compared to values at 2 mM glucose. Data are the mean ± S.E. <i>n</i> = 9–11. *<i>p</i><0.01.</p

Percent of insulin content secreted from intact islets after static incubation at 2.8, 10, and 16.7 mM glucose with and without treatment.

<p>Untreated control samples are shown in white. <b>A</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations in the presence and absence of GLP-1 (20 nM, light gray), the G_βγ inhibitor, gallein (10 µM, checked), or combination treatment with gallein and GLP-1 (striped). <b>B,</b> Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations with GLP-1 (20 nM, light gray), the G_βγ-activating peptide mSIRK (30 µM, checked), or combination treatment with mSIRK and GLP-1 (striped). Data are the mean ± S.E. <i>n</i> = 4–19. *(<i>p</i><0.05) and **(<i>p</i><0.001) indicate significance compared to untreated control, GLP-1 only, or gallein alone.</p

Percent of insulin content secreted from intact islets after incubation at 2.8, 10, or 16.7 mM glucose with and without treatment.

<p>Untreated control samples are shown in white. <b>A</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations in the presence and absence of KP (10 µM, dark gray), gallein (10 µM, checked), or gallein+KP (striped). <b>B</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations with and without KP (10 µM, dark gray), mSIRK (30 µM, checked), or mSIRK+KP (striped). Data are the mean ± S.E. <i>n</i> = 4–19. *<i>p</i><0.05 and **<i>p</i><0.001 compared to untreated control, KP alone, or mSIRK only.</p

Changes in Fluo4 signal in dispersed β-cells recorded at 10 mM glucose in the presence and absence of KP (1 µM) or with GLP-1 (20 nM) to measure the frequency and amplitude of [Ca²⁺]_i oscillations.

<p><b>A & C</b>, Representative oscillations in [Ca²⁺]_i recorded from dispersed β-cells before (dotted line) and after (solid line) treatment with KP (<b>A</b>) or GLP-1 (<b>C</b>). <b>B & D</b>, The normalized [Ca²⁺]_i oscillation frequency measured pre- and post-treatment with KP (<b>B</b>, dark gray) or GLP-1 (<b>D</b>, light gray). Data are normalized to the data collected from the dispersed β-cells prior to ligand treatment (white). Data are the mean ± S.E. <i>n</i> = 4. <i>p</i><0.05.</p

Fluo4 signal recorded at 10 mM glucose in the presence and absence of GLP-1 (20 nM) alone or combined with gallein (10 µM) or mSIRK (30 µM) to detect changes in the frequency and amplitude of [Ca²⁺]_i oscillations.

<p><b>A, C &, E</b>, Representative [Ca²⁺]_i oscillations from intact islets recorded pre- (dotted line) and post-treatment (solid line) with GLP-1 (<b>A</b>), gallein and GLP-1 (<b>C</b>) or mSIRK and GLP-1 (<b>E</b>). <b>B, D, & F</b>, The normalized [Ca²⁺]_i oscillation frequency measured pre- and post-treatment with GLP-1 (<b>B</b>, light gray), gallein and GLP-1 (<b>D</b>, light gray stripes) or mSIRK and GLP-1 (<b>F</b>, light gray checks). Data are normalized to the data collected from the islet prior to ligand/G_βγ modulator treatment (white). Data are the mean ± S.E. <i>n</i> = 4–5. <i>p</i>>0.1.</p