β-Cell Differentiation from a Human Pancreatic Cell Line in Vitro and in Vivo

Abstract Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human β-cells, inducibly transformed pancreatic β-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of β-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional β-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human β-cells is a major step toward developing a cell transplantation therapy for diabetes.

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Perilipin2 down-regulation in β cells impairs insulin secretion under nutritional stress and damages mitochondria

10.1101/2020.10.01.322974 ◽

2020 ◽

Author(s):

Akansha Mishra ◽

Siming Liu ◽

Joseph Promes ◽

Mikako Harata ◽

William Sivitz ◽

...

Keyword(s):

Insulin Secretion ◽

Cell Function ◽

Islet Cells ◽

Cell Metabolism ◽

Nutritional Stress ◽

Β Cells ◽

Β Cell ◽

Down Regulation

Perilipin 2 (PLIN2) is the lipid droplet (LD) protein in β cells that increases under nutritional stress. Down-regulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was down-regulated in mouse β cells, INS1 cells, and human islet cells. β cell specific deletion of PLIN2 in mice on a high fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Down-regulation of PLIN2 in INS1 cells blunted GSIS after 24 h incubation with 0.2 mM palmitic acids. Down-regulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Down-regulation of PLIN2 decreased specific OXPHOS proteins in all three models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2 deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells have an important role in preserving insulin secretion, β cell metabolism and mitochondrial function under nutritional stress.

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Downregulation of lncRNA TUG1 Affects Apoptosis and Insulin Secretion in Mouse Pancreatic β Cells

Cellular Physiology and Biochemistry ◽

10.1159/000373999 ◽

2015 ◽

Vol 35 (5) ◽

pp. 1892-1904 ◽

Cited By ~ 68

Author(s):

Dan-dan Yin ◽

Er-bao Zhang ◽

Liang-hui You ◽

Ning Wang ◽

Lin-tao Wang ◽

...

Keyword(s):

Insulin Secretion ◽

Cell Function ◽

Annexin V ◽

Pancreatic Tissue ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Lncrna Tug1

Background: Increasing evidence indicates that long noncoding RNAs (IncRNAs) perform specific biological functions in diverse processes. Recent studies have reported that IncRNAs may be involved in β cell function. The aim of this study was to characterize the role of IncRNA TUG1 in mouse pancreatic β cell functioning both in vitro and in vivo. Methods: qRT-PCR analyses were performed to detect the expression of lncRNA TUG1 in different tissues. RNAi, MTT, TUNEL and Annexin V-FITC assays and western blot, GSIS, ELISA and immunochemistry analyses were performed to detect the effect of lncRNA TUG1 on cell apoptosis and insulin secretion in vitro and in vivo. Results: lncRNA TUG1 was highly expressed in pancreatic tissue compared with other organ tissues, and expression was dynamically regulated by glucose in Nit-1 cells. Knockdown of lncRNA TUG1 expression resulted in an increased apoptosis ratio and decreased insulin secretion in β cells both in vitro and in vivo . Immunochemistry analyses suggested decreased relative islet area after treatment with lncRNA TUG1 siRNA. Conclusion: Downregulation of lncRNA TUG1 expression affected apoptosis and insulin secretion in pancreatic β cells in vitro and in vivo. lncRNA TUG1 may represent a factor that regulates the function of pancreatic β cells.

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Cellular plasticity of the pancreas

Biological Chemistry ◽

10.1515/bc.2009.117 ◽

2009 ◽

Vol 390 (10) ◽

Cited By ~ 7

Author(s):

Luc Baeyens ◽

Luc Bouwens

Keyword(s):

Diabetes Mellitus ◽

Cell Differentiation ◽

Cell Types ◽

Β Cells ◽

Cell Replacement Therapy ◽

Β Cell ◽

Pancreatic Cell ◽

Cell Replacement

Abstract Cell replacement therapy holds promises for treatment of patients suffering from diabetes mellitus. When determining the appropriate strategies to amplify the amount of transplantable β-cells, sufficient knowledge of the developmental programs regulating β-cell differentiation is crucial. Here, we describe the plasticity of the different pancreatic cell types in vivo and in vitro and their potential to serve as β-cell progenitor.

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The HDAC Inhibitor Butyrate Impairs β Cell Function and Activates the Disallowed Gene Hexokinase I

International Journal of Molecular Sciences ◽

10.3390/ijms222413330 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13330

Author(s):

Stephanie Bridgeman ◽

Gaewyn Ellison ◽

Philip Newsholme ◽

Cyril Mamotte

Keyword(s):

Insulin Secretion ◽

Low Dose ◽

Cell Function ◽

High Dose ◽

Hdac Inhibition ◽

Β Cells ◽

Β Cell ◽

Β Cell Function

Histone deacetylase (HDAC) inhibitors such as butyrate have been reported to reduce diabetes risk and protect insulin-secreting pancreatic β cells in animal models. However, studies on insulin-secreting cells in vitro have found that butyrate treatment resulted in impaired or inappropriate insulin secretion. Our study explores the effects of butyrate on insulin secretion by BRIN BD-11 rat pancreatic β cells and examined effects on the expression of genes implicated in β cell function. Robust HDAC inhibition with 5 mM butyrate or trichostatin A for 24 h in β cells decreased basal insulin secretion and content, as well as insulin secretion in response to acute stimulation. Treatment with butyrate also increased expression of the disallowed gene hexokinase I, possibly explaining the impairment to insulin secretion, and of TXNIP, which may increase oxidative stress and β cell apoptosis. In contrast to robust HDAC inhibition (>70% after 24 h), low-dose and acute high-dose treatment with butyrate enhanced nutrient-stimulated insulin secretion. In conclusion, although protective effects of HDAC inhibition have been observed in vivo, potent HDAC inhibition impairs β cell function in vitro. The chronic low dose and acute high dose butyrate treatments may be more reflective of in vivo effects.

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Inhibition of Small Maf Function in Pancreatic β-Cells Improves Glucose Tolerance Through the Enhancement of Insulin Gene Transcription and Insulin Secretion

Endocrinology ◽

10.1210/en.2014-1906 ◽

2015 ◽

Vol 156 (10) ◽

pp. 3570-3580 ◽

Cited By ~ 9

Author(s):

Hiroshi Nomoto ◽

Takuma Kondo ◽

Hideaki Miyoshi ◽

Akinobu Nakamura ◽

Yoko Hida ◽

...

Keyword(s):

Insulin Secretion ◽

Glucose Tolerance ◽

High Fat Diet ◽

Cell Function ◽

High Fat ◽

Β Cells ◽

Β Cell ◽

Β Cell Function

The large-Maf transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) has been found to be crucial for insulin transcription and synthesis and for pancreatic β-cell function and maturation. However, insights about the effects of small Maf factors on β-cells are limited. Our goal was to elucidate the function of small-Maf factors on β-cells using an animal model of endogenous small-Maf dysfunction. Transgenic (Tg) mice with β-cell-specific expression of dominant-negative MafK (DN-MafK) experiments, which can suppress the function of all endogenous small-Mafs, were fed a high-fat diet, and their in vivo phenotypes were evaluated. Phenotypic analysis, glucose tolerance tests, morphologic examination of β-cells, and islet experiments were performed. DN-MafK-expressed MIN6 cells were also used for in vitro analysis. The results showed that DN-MafK expression inhibited endogenous small-Maf binding to insulin promoter while increasing MafA binding. DN-MafK Tg mice under high-fat diet conditions showed improved glucose metabolism compared with control mice via incremental insulin secretion, without causing changes in insulin sensitivity or MafA expression. Moreover, up-regulation of insulin and glucokinase gene expression was observed both in vivo and in vitro under DN-MafK expression. We concluded that endogenous small-Maf factors negatively regulates β-cell function by competing for MafA binding, and thus, the inhibition of small-Maf activity can improve β-cell function.

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Reduced synchroneity of intra-islet Ca2+ oscillations in vivo in Robo-deficient β cells

eLife ◽

10.7554/elife.61308 ◽

2021 ◽

Vol 10 ◽

Author(s):

Melissa T Adams ◽

JaeAnn M Dwulet ◽

Jennifer K Briggs ◽

Christopher A Reissaus ◽

Erli Jin ◽

...

Keyword(s):

Stem Cells ◽

Insulin Secretion ◽

Cell Differentiation ◽

Intravital Microscopy ◽

Β Cells ◽

Β Cell ◽

Unique Model ◽

Spatial Architecture ◽

Intrinsic Function

The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

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Regulation of islet function and gene expression by prolactin during pregnancy

10.1101/830836 ◽

2019 ◽

Author(s):

Vipul Shrivastava ◽

Megan Lee ◽

Marle Pretorius ◽

Guneet Makkar ◽

Carol Huang

Keyword(s):

Gene Expression ◽

Insulin Secretion ◽

Serum Insulin ◽

Prolactin Receptor ◽

Cell Mass ◽

Insulin Level ◽

Β Cells ◽

Β Cell

AbstractPancreatic islets adapt to insulin resistance of pregnancy by up regulating β-cell proliferation and increase insulin secretion. Previously, we found that prolactin receptor (Prlr) signaling is important for this process, as heterozygous prolactin receptor-null (Prlr+/−) mice are glucose intolerant, had a lower number of β cells and lower serum insulin levels than wild type mice during pregnancy. However, since Prlr expression is ubiquitous, to determine its β-cell specific effects, we generated a transgenic mouse with a floxed Prlr allele under the control of an inducible promoter, allowing conditional deletion of Prlr from β cells in adult mice. In this study, we found that β-cell-specific Prlr reduction resulted in elevated blood glucose during pregnancy. Similar to our previous finding in mouse with global Prlr reduction, β-cell-specific Prlr loss led to a lower β-cell mass and a lower in vivo insulin level during pregnancy. However, these islets do not have an intrinsic insulin secretion defect when tested in vitro. Interestingly, when we compared the islet gene expression profile, using islets isolated from mice with global versus β-cell-specific Prlr reduction, we found some important differences in genes that regulate apoptosis and insulin secretion. This suggests that Prlr has both cell-autonomous and non-cell-autonomous effect on β cells, beyond its regulation of pro-proliferative genes.

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β-Cell-specific ablation of sirtuin 4 does not affect nutrient-stimulated insulin secretion in mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00170.2020 ◽

2020 ◽

Vol 319 (4) ◽

pp. E805-E813

Author(s):

Frank K. Huynh ◽

Brett S. Peterson ◽

Kristin A. Anderson ◽

Zhihong Lin ◽

Aeowynn J. Coakley ◽

...

Keyword(s):

Insulin Secretion ◽

Glucose Tolerance ◽

Cell Function ◽

Whole Body ◽

Β Cells ◽

Β Cell ◽

Direct Role ◽

Normal Glucose

Sirtuins are a family of proteins that regulate biological processes such as cellular stress and aging by removing posttranslational modifications (PTMs). We recently identified several novel PTMs that can be removed by sirtuin 4 (SIRT4), which is found in mitochondria. We showed that mice with a global loss of SIRT4 [SIRT4-knockout (KO) mice] developed an increase in glucose- and leucine-stimulated insulin secretion, and this was followed by accelerated age-induced glucose intolerance and insulin resistance. Because whole body SIRT4-KO mice had alterations to nutrient-stimulated insulin secretion, we hypothesized that SIRT4 plays a direct role in regulating pancreatic β-cell function. Thus, we tested whether β-cell-specific ablation of SIRT4 would recapitulate the elevated insulin secretion seen in mice with a global loss of SIRT4. Tamoxifen-inducible β-cell-specific SIRT4-KO mice were generated, and their glucose tolerance and glucose- and leucine-stimulated insulin secretion were measured over time. These mice exhibited normal glucose- and leucine-stimulated insulin secretion and maintained normal glucose tolerance even as they aged. Furthermore, 832/13 β-cells with a CRISPR/Cas9n-mediated loss of SIRT4 did not show any alterations in nutrient-stimulated insulin secretion. Despite the fact that whole body SIRT4-KO mice demonstrated an age-induced increase in glucose- and leucine-stimulated insulin secretion, our current data indicate that the loss of SIRT4 specifically in pancreatic β-cells, both in vivo and in vitro, does not have a significant impact on nutrient-stimulated insulin secretion. These data suggest that SIRT4 controls nutrient-stimulated insulin secretion during aging by acting on tissues external to the β-cell, which warrants further study.

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Glucose-dependent expansion of pancreatic β-cells by the protein p8 in vitro and in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00436.2005 ◽

2006 ◽

Vol 291 (6) ◽

pp. E1168-E1176 ◽

Cited By ~ 13

Author(s):

Günter Päth ◽

Anne Opel ◽

Martin Gehlen ◽

Veit Rothhammer ◽

Xinjie Niu ◽

...

Keyword(s):

Insulin Secretion ◽

Blood Glucose ◽

Protein Expression ◽

Cell Expansion ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

C Peptide

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic β-cells. Here we demonstrate that glucose-induced INS-1 β-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 β-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although β-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses β-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 β-cells in response to 0–25 mM glucose is not altered by preceding p8-induced β-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human β-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic β-cell expansion in vitro and in vivo and support the notion of existing β-cell replication in the adult organism.

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Synchronized β cell response to glucose is lost concomitant with loss of islet architecture in Robo deficient islets of Langerhans in vivo

10.1101/2019.12.11.873471 ◽

2019 ◽

Cited By ~ 1

Author(s):

Melissa T. Adams ◽

Christopher A. Reissaus ◽

JaeAnn M. Dwulet ◽

Erli Jin ◽

Joseph M. Szulczewski ◽

...

Keyword(s):

Stem Cells ◽

Insulin Secretion ◽

Cell Differentiation ◽

Islets Of Langerhans ◽

Β Cells ◽

Β Cell ◽

Unique Model ◽

Spatial Architecture ◽

Intrinsic Function

AbstractThe spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion between β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets lose synchronized intra-islet Ca2+ oscillations between β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, loss of Connexin36 gap junctions, or changes in islet vascularization, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

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