Publications by authors named "Yu Hsuan Carol Yang"

12 Publications

  • Page 1 of 1

Bax and Bak jointly control survival and dampen the early unfolded protein response in pancreatic β-cells under glucolipotoxic stress.

Sci Rep 2020 07 3;10(1):10986. Epub 2020 Jul 3.

Diabetes Research Group, BC Children's Hospital Research Institute, Vancouver, BC, Canada.

ER stress and apoptosis contribute to the loss of pancreatic β-cells under pro-diabetic conditions of glucolipotoxicity. Although activation of canonical intrinsic apoptosis is known to require pro-apoptotic Bcl-2 family proteins Bax and Bak, their individual and combined involvement in glucolipotoxic β-cell death are not known. It has also remained an open question if Bax and Bak in β-cells have non-apoptotic roles in mitochondrial function and ER stress signaling, as suggested in other cell types. Using mice with individual or combined β-cell deletion of Bax and Bak, we demonstrated that glucolipotoxic β-cell death in vitro occurs by both non-apoptotic and apoptotic mechanisms, and the apoptosis could be triggered by either Bax or Bak alone. In contrast, they had non-redundant roles in mediating staurosporine-induced apoptosis. We further established that Bax and Bak do not affect normal glucose-stimulated β-cell Ca responses, insulin secretion, or in vivo glucose tolerance. Finally, our experiments revealed that combined deletion of Bax and Bak amplified the unfolded protein response in islets during the early stages of chemical- or glucolipotoxicity-induced ER stress. These findings shed new light on roles of the core apoptosis machinery in β-cell survival and stress signals of importance for the pathobiology of diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-67755-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335194PMC
July 2020

Disruption of the pancreatic vasculature in zebrafish affects islet architecture and function.

Development 2019 11 4;146(21). Epub 2019 Nov 4.

Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, 61231 Bad Nauheim, Germany

A dense local vascular network is crucial for pancreatic endocrine cells to sense metabolites and secrete hormones, and understanding the interactions between the vasculature and the islets may allow for therapeutic modulation in disease conditions. Using live imaging in two models of vascular disruption in zebrafish, we identified two distinct roles for the pancreatic vasculature. At larval stages, expression of a dominant negative version of Vegfaa (dnVegfaa) in β-cells led to vascular and endocrine cell disruption with a minor impairment in β-cell function. In contrast, expression of a soluble isoform of Vegf receptor 1 (sFlt1) in β-cells blocked the formation of the pancreatic vasculature and drastically stunted glucose response, although islet architecture was not affected. Notably, these effects of dnVegfaa or sFlt1 were not observed in animals lacking , , , or function, indicating that they interfere with multiple ligands and/or receptors. In adults, disrupted islet architecture persisted in dnVegfaa-expressing animals, whereas sFlt1-expressing animals displayed large sheets of β-cells along their pancreatic ducts, accompanied by impaired glucose tolerance in both models. Thus, our study reveals novel roles for the vasculature in patterning and function of the islet.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.173674DOI Listing
November 2019

Screening for insulin-independent pathways that modulate glucose homeostasis identifies androgen receptor antagonists.

Elife 2018 12 6;7. Epub 2018 Dec 6.

Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

Pathways modulating glucose homeostasis independently of insulin would open new avenues to combat insulin resistance and diabetes. Here, we report the establishment, characterization, and use of a vertebrate 'insulin-free' model to identify insulin-independent modulators of glucose metabolism. knockout zebrafish recapitulate core characteristics of diabetes and survive only up to larval stages. Utilizing a highly efficient endoderm transplant technique, we generated viable chimeric adults that provide the large numbers of mutant larvae required for our screening platform. Using glucose as a disease-relevant readout, we screened 2233 molecules and identified three that consistently reduced glucose levels in mutants. Most significantly, we uncovered an insulin-independent beneficial role for androgen receptor antagonism in hyperglycemia, mostly by reducing fasting glucose levels. Our study proposes therapeutic roles for androgen signaling in diabetes and, more broadly, offers a novel in vivo model for rapid screening and decoupling of insulin-dependent and -independent mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.42209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300353PMC
December 2018

Whole-Organism Chemical Screening Identifies Modulators of Pancreatic β-Cell Function.

Diabetes 2018 11 16;67(11):2268-2279. Epub 2018 Aug 16.

Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

β-Cell loss and dysfunction play a critical role in the progression of type 1 and type 2 diabetes. Identifying new molecules and/or molecular pathways that improve β-cell function and/or increase β-cell mass should significantly contribute to the development of new therapies for diabetes. Using the zebrafish model, we screened 4,640 small molecules to identify modulators of β-cell function. This in vivo strategy identified 84 stimulators of expression, which simultaneously reduced glucose levels. The promoter activation kinetics for 32 of these stimulators were consistent with a direct mode of action. A subset of stimulators, including the antidiabetic drug pioglitazone, induced the coordinated upregulation of gluconeogenic expression, suggesting functional response to increased insulin action in peripheral tissues. Notably, Kv1.3 inhibitors increased β-cell mass in larval zebrafish and stimulated β-cell function in adult zebrafish and in the streptozotocin-induced hyperglycemic mouse model. In addition, our data indicate that cytoplasmic Kv1.3 regulates β-cell function. Thus, using whole-organism screening, we have identified new small-molecule modulators of β-cell function and glucose metabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db17-1223DOI Listing
November 2018

A new mode of pancreatic islet innervation revealed by live imaging in zebrafish.

Elife 2018 06 19;7. Epub 2018 Jun 19.

Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.

Pancreatic islets are innervated by autonomic and sensory nerves that influence their function. Analyzing the innervation process should provide insight into the nerve-endocrine interactions and their roles in development and disease. Here, using in vivo time-lapse imaging and genetic analyses in zebrafish, we determined the events leading to islet innervation. Comparable neural density in the absence of vasculature indicates that it is dispensable for early pancreatic innervation. Neural crest cells are in close contact with endocrine cells early in development. We find these cells give rise to neurons that extend axons toward the islet as they surprisingly migrate away. Specific ablation of these neurons partly prevents other neurons from migrating away from the islet resulting in diminished innervation. Thus, our studies establish the zebrafish as a model to interrogate mechanisms of organ innervation, and reveal a novel mode of innervation whereby neurons establish connections with their targets before migrating away.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.34519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039180PMC
June 2018

High-content screening identifies a role for Na(+) channels in insulin production.

R Soc Open Sci 2015 Dec 2;2(12):150306. Epub 2015 Dec 2.

Department of Cellular and Physiological Sciences University of British Columbia Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.

Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 β-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of β-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect β-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and β-cell differentiation status. In particular, our unbiased screen identified a novel role for a β-cell sodium channel gene in insulin production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1098/rsos.150306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807443PMC
December 2015

A live-cell, high-content imaging survey of 206 endogenous factors across five stress conditions reveals context-dependent survival effects in mouse primary beta cells.

Diabetologia 2015 Jun 14;58(6):1239-49. Epub 2015 Mar 14.

Department of Cellular and Physiological Sciences, Faculty of Medicine, Diabetes Research Group, Life Sciences Institute, University of British Columbia, 5358-2350 Health Sciences Mall, Vancouver, BC, Canada, V6T 1Z3.

Aims/hypothesis: Beta cell death is a hallmark of diabetes. It is not known whether specific cellular stresses associated with type 1 or type 2 diabetes require specific factors to protect pancreatic beta cells. No systematic comparison of endogenous soluble factors in the context of multiple pro-apoptotic conditions has been published.

Methods: Primary mouse islet cells were cultured in conditions mimicking five type 1 or type 2 diabetes-related stresses: basal 5 mmol/l glucose, cytokine cocktail (25 ng/ml TNF-α, 10 ng/ml IL-1β, 10 ng/ml IFN-γ), 1 μmol/l thapsigargin, 1.5 mmol/l palmitate and 20 mmol/l glucose (all in the absence of serum). We surveyed the effects of a library of 206 endogenous factors (selected based on islet expression of their receptors) on islet cell survival through multi-parameter, live-cell imaging.

Results: Our survey pointed to survival factors exhibiting generalised protective effects across conditions meant to model different types of diabetes and stages of the diseases. For example, our survey and follow-up experiments suggested that OLFM1 is a novel protective factor for mouse and human beta cells across multiple conditions. Most strikingly, we also found specific protective survival factors for each model stress condition. For example, semaphorin4A (SEMA4A) was toxic to islet cells in the serum-free baseline and serum-free 20 mmol/l glucose conditions, but protective in the context of lipotoxicity. Rank product testing supported the consistency of our observations.

Conclusions/interpretation: Collectively, our survey reveals previously unidentified islet cell survival factors and suggest their potential utility in individualised medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00125-015-3552-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415993PMC
June 2015

Effects of insulin on human pancreatic cancer progression modeled in vitro.

BMC Cancer 2014 Nov 6;14:814. Epub 2014 Nov 6.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada.

Background: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined.

Methods: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model.

Results: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling.

Conclusions: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2407-14-814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4233074PMC
November 2014

Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

Nat Biotechnol 2014 Nov 11;32(11):1121-33. Epub 2014 Sep 11.

1] Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada. [2] Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada.

Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nbt.3033DOI Listing
November 2014

Multiparameter screening reveals a role for Na+ channels in cytokine-induced β-cell death.

Mol Endocrinol 2014 Mar 17;28(3):406-17. Epub 2014 Jan 17.

Department of Cellular and Physiological Sciences (Y.H.C.Y., J.D.J.), Department of Anesthesiology, Pharmacology, and Therapeutics (Y.Y.V., H.T.K.), and Department of Biochemistry and Molecular Biology (M.R.), University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

Pancreatic β-cell death plays a role in both type 1 and type 2 diabetes, but clinical treatments that specifically target β-cell survival have not yet been developed. We have recently developed live-cell imaging-based, high-throughput screening methods capable of identifying factors that modulate pancreatic β-cell death, with the hope of finding drugs that can intervene in this process. In the present study, we used a high-content screen and the Prestwick Chemical Library of small molecules to identify drugs that block cell death resulting from exposure to a cocktail of cytotoxic cytokines (25 ng/mL TNF-α, 10 ng/mL IL-1β, and 10 ng/mL IFN-γ). Data analysis with self-organizing maps revealed that 19 drugs had profiles similar to that of the no cytokine condition, indicating protection. Carbamazepine, an antiepileptic Na(+) channel inhibitor, was particularly interesting because Na(+) channels are not generally considered targets for antiapoptotic therapy in diabetes and because the function of these channels in β-cells has not been well studied. We analyzed the expression and characteristics of Na(+) currents in mature β-cells from MIP-GFP mice. We confirmed the dose-dependent protective effects of carbamazepine and another use-dependent Na(+) channel blocker in cytokine-treated mouse islet cells. Carbamazepine down-regulated the proapoptotic and endoplasmic reticulum stress signaling induced by cytokines. Together, these studies point to Na(+) channels as a novel therapeutic target in diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2013-1257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414926PMC
March 2014

Intraislet SLIT-ROBO signaling is required for beta-cell survival and potentiates insulin secretion.

Proc Natl Acad Sci U S A 2013 Oct 24;110(41):16480-5. Epub 2013 Sep 24.

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.

We previously cataloged putative autocrine/paracrine signaling loops in pancreatic islets, including factors best known for their roles in axon guidance. Emerging evidence points to nonneuronal roles for these factors, including the Slit-Roundabout receptor (Robo) family, in cell growth, migration, and survival. We found SLIT1 and SLIT3 in both beta cells and alpha cells, whereas SLIT2 was predominantly expressed in beta cells. ROBO1 and ROBO2 receptors were detected in beta and alpha cells. Remarkably, even modest knockdown of Slit production resulted in significant beta-cell death, demonstrating a critical autocrine/paracrine survival role for this pathway. Indeed, recombinant SLIT1, SLIT2, and SLIT3 decreased serum deprivation, cytokine, and thapsigargin-induced cell death under hyperglycemic conditions. SLIT treatment also induced a gradual release of endoplasmic reticulum luminal Ca(2+), suggesting a unique molecular mechanism capable of protecting beta cells from endoplasmic reticulum stress-induced apoptosis. SLIT treatment was also associated with rapid actin remodeling. SLITs potentiated glucose-stimulated insulin secretion and increased the frequency of glucose-induced Ca(2+) oscillations. These observations point to unexpected roles for local Slit secretion in the survival and function of pancreatic beta cells. Because diabetes results from a deficiency in functional beta-cell mass, these studies may contribute to therapeutic approaches for improving beta-cell survival and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1214312110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799350PMC
October 2013

Multi-parameter single-cell kinetic analysis reveals multiple modes of cell death in primary pancreatic β-cells.

J Cell Sci 2013 Sep 10;126(Pt 18):4286-95. Epub 2013 Jul 10.

Department of Cellular and Physiological Sciences, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver BC V6T 1Z3, Canada.

Programmed β-cell death plays an important role in both type 1 and type 2 diabetes. Most of what is known about the mechanisms of β-cell death comes from single time-point, single parameter measurements of bulk populations of mixed cells. Such approaches are inadequate for determining the true extent of the heterogeneity in death mechanisms. Here, we characterized the timing and order of molecular events associated with cell death in single β-cells under multiple diabetic stress conditions, including hyperglycemia, cytokine exposure, nutrient deprivation and endoplasmic reticulum (ER) stress. We simultaneously measured the kinetics of six distinct cell death mechanisms by using a caspase-3 sensor and three vital dyes, together with brightfield imaging. We identified several cell death modes where the order of events that usually define apoptosis were not observed. This we termed 'partial apoptosis'. Remarkably, complete classical apoptosis, defined as cells with plasma membrane blebbing, caspase-3 activity, nuclear condensation and membrane annexin V labeling prior to loss of plasma membrane integrity, was found in only half of the cytokine-treated primary β-cells and never in cells stressed by serum removal. By contrast, in the MIN6 cell line, death occurred almost exclusively through complete classical apoptosis. Ambient glucose modulated the cell death mode and kinetics in primary β-cells. Taken together, our data define the kinetic progression of β-cell death mechanisms under different conditions and illustrate the heterogeneity and plasticity of cell death modes in β-cells. We conclude that apoptosis is not the primary mode of adult primary β-cell death.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.133017DOI Listing
September 2013