Expression of a tetracycline-controlled transactivator (Tet-On/Off system) in beta cells reduces insulin expression and secretion in mice

ABSTRACTControllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However, in beta cells, transgene silencing, low inducibility, ectopic expression and off-targets effects on cell function and glucose homeostasis are a persistent challenge. In this study, we investigated whether an inducible, Tet-Off system with beta-cell specific MIP-itTA driven expression of TetO-CreJaw/J could circumvent previous issues of specificity, efficacy and toxicity. Following assessment of tissue-specific gene recombination; beta cell architecture; in vitro and in vivo glucose-stimulated insulin secretion (GSIS); and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g. itTA, rtTA or tTA) in beta cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying a MIP-itTA transgene, independent of Cre-recombinase expression or doxycycline treatment. These results raise significant concern regarding the use of Tet-On or Tet-Off systems for genome editing in beta cells and emphasize the need to control for effects of transactivator expression. Our study echoes ongoing challenges faced by fundamental researchers focused on beta cells and highlights the need for consistent and careful control of experiments using these research tools.

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The Tetracycline-Controlled Transactivator (Tet-On/Off) System in Beta Cells Reduces Insulin Expression and Secretion in Mice

10.2337/figshare.16713151 ◽

2021 ◽

Author(s):

Nathalie Jouvet ◽

Khalil Bouyakdan ◽

Scott A. Campbell ◽

Cindy Baldwin ◽

Shannon E. Townsend ◽

...

Keyword(s):

Insulin Secretion ◽

Beta Cell ◽

Beta Cells ◽

Cell Biology ◽

Genetic Manipulation ◽

Ectopic Expression ◽

Whole Body ◽

Specific Gene

Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in beta cells, transgene silencing, low inducibility, ectopic expression and off-targets effects are persistent challenges. In this study, we investigated whether an inducible, Tet-Off system with beta-cell specific MIP-itTA driven expression of TetO-CreJaw/J could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination; beta cell architecture; in vitro and in vivo glucose-stimulated insulin secretion; and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g. itTA, rtTA or tTA) in beta cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with beta cells and highlights the need for consistent and comprehensive controls when using the Tet-On or Tet-Off systems for genome editing.

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The Tetracycline-Controlled Transactivator (Tet-On/Off) System in Beta Cells Reduces Insulin Expression and Secretion in Mice

10.2337/figshare.16713151.v1 ◽

2021 ◽

Author(s):

Nathalie Jouvet ◽

Khalil Bouyakdan ◽

Scott A. Campbell ◽

Cindy Baldwin ◽

Shannon E. Townsend ◽

...

Keyword(s):

Insulin Secretion ◽

Beta Cell ◽

Beta Cells ◽

Cell Biology ◽

Genetic Manipulation ◽

Ectopic Expression ◽

Whole Body ◽

Specific Gene

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Effects of Extra Virgin Olive Oil Polyphenols on Beta-Cell Function and Survival

Plants ◽

10.3390/plants10020286 ◽

2021 ◽

Vol 10 (2) ◽

pp. 286

Author(s):

Nicola Marrano ◽

Rosaria Spagnuolo ◽

Giuseppina Biondi ◽

Angelo Cignarelli ◽

Sebastio Perrini ◽

...

Keyword(s):

Insulin Secretion ◽

Beta Cell ◽

Olive Oil ◽

Beta Cells ◽

Intracellular Signaling ◽

Beta Cell Function ◽

Cell Function ◽

Virgin Olive Oil ◽

Extra Virgin Olive Oil ◽

Insulin Biosynthesis

Extra virgin olive oil (EVOO) is a major component of the Mediterranean diet and is appreciated worldwide because of its nutritional benefits in metabolic diseases, including type 2 diabetes (T2D). EVOO contains significant amounts of secondary metabolites, such as phenolic compounds (PCs), that may positively influence the metabolic status. In this study, we investigated for the first time the effects of several PCs on beta-cell function and survival. To this aim, INS-1E cells were exposed to 10 μM of the main EVOO PCs for up to 24 h. Under these conditions, survival, insulin biosynthesis, glucose-stimulated insulin secretion (GSIS), and intracellular signaling activation (protein kinase B (AKT) and cAMP response element-binding protein (CREB)) were evaluated. Hydroxytyrosol, tyrosol, and apigenin augmented beta-cell proliferation and insulin biosynthesis, and apigenin and luteolin enhanced the GSIS. Conversely, vanillic acid and vanillin were pro-apoptotic for beta-cells, even if they increased the GSIS. In addition, oleuropein, p-coumaric, ferulic and sinapic acids significantly worsened the GSIS. Finally, a mixture of hydroxytyrosol, tyrosol, and apigenin promoted the GSIS in human pancreatic islets. Apigenin was the most effective compound and was also able to activate beneficial intracellular signaling. In conclusion, this study shows that hydroxytyrosol, tyrosol, and apigenin foster beta-cells’ health, suggesting that EVOO or supplements enriched with these compounds may improve insulin secretion and promote glycemic control in T2D patients.

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Multi-Organ Crosstalk with Endocrine Pancreas: A Focus on How Gut Microbiota Shapes Pancreatic Beta-Cells

Biomolecules ◽

10.3390/biom12010104 ◽

2022 ◽

Vol 12 (1) ◽

pp. 104

Author(s):

Elisa Fernández-Millán ◽

Carlos Guillén

Keyword(s):

Beta Cell ◽

Beta Cells ◽

Cell Biology ◽

Pancreatic Beta Cells ◽

Metabolic Diseases ◽

Cell Function ◽

Cell Mass ◽

Beta Cell Mass ◽

Short Chain Fatty Acids

Type 2 diabetes (T2D) results from impaired beta-cell function and insufficient beta-cell mass compensation in the setting of insulin resistance. Current therapeutic strategies focus their efforts on promoting the maintenance of functional beta-cell mass to ensure appropriate glycemic control. Thus, understanding how beta-cells communicate with metabolic and non-metabolic tissues provides a novel area for investigation and implicates the importance of inter-organ communication in the pathology of metabolic diseases such as T2D. In this review, we provide an overview of secreted factors from diverse organs and tissues that have been shown to impact beta-cell biology. Specifically, we discuss experimental and clinical evidence in support for a role of gut to beta-cell crosstalk, paying particular attention to bacteria-derived factors including short-chain fatty acids, lipopolysaccharide, and factors contained within extracellular vesicles that influence the function and/or the survival of beta cells under normal or diabetogenic conditions.

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Bergenin protects pancreatic beta cells against cytokine-induced apoptosis in INS-1E cells

PLoS ONE ◽

10.1371/journal.pone.0241349 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0241349

Author(s):

Sajid Ali Rajput ◽

Munazza Raza Mirza ◽

M. Iqbal Choudhary

Keyword(s):

Insulin Secretion ◽

Beta Cell ◽

Beta Cells ◽

Proinflammatory Cytokines ◽

Cell Apoptosis ◽

Pancreatic Beta Cells ◽

Cell Function ◽

Beta Cell Apoptosis ◽

Atp Levels ◽

Induced Apoptosis

Beta cell apoptosis induced by proinflammatory cytokines is one of the hallmarks of diabetes. Small molecules which can inhibit the cytokine-induced apoptosis could lead to new drug candidates that can be used in combination with existing therapeutic interventions against diabetes. The current study evaluated several effects of bergenin, an isocoumarin derivative, in beta cells in the presence of cytokines. These included (i) increase in beta cell viability (by measuring cellular ATP levels) (ii) suppression of beta cell apoptosis (by measuring caspase activity), (iii) improvement in beta cell function (by measuring glucose-stimulated insulin secretion), and (iv) improvement of beta cells mitochondrial physiological functions. The experiments were carried out using rat beta INS-1E cell line in the presence or absence of bergenin and a cocktail of proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon- gamma) for 48 hr. Bergenin significantly inhibited beta cell apoptosis, as inferred from the reduction in the caspase-3 activity (IC50 = 7.29 ± 2.45 μM), and concurrently increased cellular ATP Levels (EC50 = 1.97 ± 0.47 μM). Bergenin also significantly enhanced insulin secretion (EC50 = 6.73 ± 2.15 μM) in INS-1E cells, presumably because of the decreased nitric oxide production (IC50 = 6.82 ± 2.83 μM). Bergenin restored mitochondrial membrane potential (EC50 = 2.27 ± 0.83 μM), decreased ROS production (IC50 = 14.63 ± 3.18 μM), and improved mitochondrial dehydrogenase activity (EC50 = 1.39 ± 0.62 μM). This study shows for the first time that bergenin protected beta cells from cytokine-induced apoptosis and restored insulin secretory function by virtue of its anti-inflammatory, antioxidant and anti-apoptotic properties. To sum up, the above mentioned data highlight bergenin as a promising anti-apoptotic agent in the context of diabetes.

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Endothelial and beta cell composite aggregates for improved function of a bioartificial pancreas encapsulation device

The International Journal of Artificial Organs ◽

10.1177/0391398817752295 ◽

2018 ◽

Vol 41 (3) ◽

pp. 152-159 ◽

Cited By ~ 8

Author(s):

Katarzyna Skrzypek ◽

Yazmin Brito Barrera ◽

Thomas Groth ◽

Dimitrios Stamatialis

Keyword(s):

Endothelial Cells ◽

Insulin Secretion ◽

Beta Cell ◽

Pancreatic Islets ◽

Beta Cells ◽

Cell Function ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

Min6 Cells ◽

Umbilical Vein Endothelial Cells

Introduction: Encapsulation of pancreatic islets or beta cells is a promising strategy for treatment of type 1 diabetes by providing an immune isolated environment and allowing for transplantation in a different location than the liver. However, islets used for encapsulation often show lower functionality due to the damaging of islet endothelial cells during the isolation procedure. Factors produced by endothelial cells have great impact on beta cell insulin secretion. Therefore, mutual signaling between endothelial cells and beta cells should be considered for the development of encapsulation systems to achieve high insulin secretion and maintain beta cell viability. Here, we investigate whether co-culture of beta cells with endothelial cells could improve beta cell function within encapsulation devices. Materials and methods: Mouse insulinoma MIN6 cells and human umbilical vein endothelial cells were used for creating composite aggregates on agarose microwell platform. The composite aggregates were encapsulated within flat poly(ether sulfone)/polyvinylpyrrolidone device. Their functionality was assessed by glucose-induced insulin secretion test and compared to non-encapsulated free-floating aggregates. Results: We created composite aggregates of 80–100 µm in diameter, closely mimicking pancreatic islets. Upon glucose stimulation, their insulin secretion is improved in comparison to aggregates consisting of only MIN6 cells. Moreover, the composite aggregates encapsulated within a device secrete more insulin than aggregates consisting of only MIN6 cells. Conclusion: Composite aggregates of MIN6 cells with human umbilical vein endothelial cells have improved insulin secretion in comparison to MIN6 aggregates showing that the interaction of beta cell and endothelial cell is crucial for a functional encapsulation system.

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AAV8-mediated gene transfer of microRNA-132 improves beta cell function in mice fed a high-fat diet

Journal of Endocrinology ◽

10.1530/joe-18-0287 ◽

2019 ◽

Vol 240 (2) ◽

pp. 123-132 ◽

Cited By ~ 4

Author(s):

Niels L Mulder ◽

Rick Havinga ◽

Joost Kluiver ◽

Albert K Groen ◽

Janine K Kruit

Keyword(s):

Cell Proliferation ◽

Insulin Secretion ◽

Gene Transfer ◽

Beta Cell ◽

Beta Cells ◽

High Fat Diet ◽

Beta Cell Function ◽

Cell Function ◽

Beta Cell Proliferation ◽

High Fat

MicroRNAs have emerged as essential regulators of beta cell function and beta cell proliferation. One of these microRNAs, miR-132, is highly induced in several obesity models and increased expression of miR-132 in vitro modulates glucose-stimulated insulin secretion. The aim of this study was to investigate the therapeutic benefits of miR-132 overexpression on beta cell function in vivo. To overexpress miR-132 specifically in beta cells, we employed adeno-associated virus (AAV8)-mediated gene transfer using the rat insulin promoter in a double-stranded, self-complementary AAV vector to overexpress miR-132. Treatment of mice with dsAAV8-RIP-mir132 increased miR-132 expression in beta cells without impacting expression of miR-212 or miR-375. Surprisingly, overexpression of miR-132 did not impact glucose homeostasis in chow-fed animals. Overexpression of miR-132 did improve insulin secretion and hence glucose homeostasis in high-fat diet-fed mice. Furthermore, miR-132 overexpression increased beta cell proliferation in mice fed a high-fat diet. In conclusion, our data show that AAV8-mediated gene transfer of miR-132 to beta cells improves beta cell function in mice in response to a high-fat diet. This suggests that increased miR-132 expression is beneficial for beta cell function during hyperglycemia and obesity.

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ER stress increases store-operated Ca2+ entry (SOCE) and augments basal insulin secretion in pancreatic beta cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012721 ◽

2020 ◽

Vol 295 (17) ◽

pp. 5685-5700

Author(s):

Irina X. Zhang ◽

Jianhua Ren ◽

Suryakiran Vadrevu ◽

Malini Raghavan ◽

Leslie S. Satin

Keyword(s):

Type 2 Diabetes ◽

Insulin Secretion ◽

Beta Cell ◽

Er Stress ◽

Beta Cells ◽

Beta Cell Function ◽

Cell Function ◽

Critical Role ◽

Beta Cell Apoptosis

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress–inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion–activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

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Activation of Aldehyde Dehydrogenase 2 Ameliorates Glucolipotoxicity of Pancreatic Beta Cells

Biomolecules ◽

10.3390/biom11101474 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1474

Author(s):

Shiau-Mei Chen ◽

Siow-Wey Hee ◽

Shih-Yun Chou ◽

Meng-Wei Liu ◽

Che-Hong Chen ◽

...

Keyword(s):

Insulin Secretion ◽

Beta Cell ◽

Beta Cells ◽

Aldehyde Dehydrogenase ◽

Mitochondrial Function ◽

Beta Cell Function ◽

Pancreatic Beta Cells ◽

Cell Function ◽

Aldehyde Dehydrogenase 2 ◽

Min6 Cells

Chronic hyperglycemia and hyperlipidemia hamper beta cell function, leading to glucolipotoxicity. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) detoxifies reactive aldehydes, such as methylglyoxal (MG) and 4-hydroxynonenal (4-HNE), derived from glucose and lipids, respectively. We aimed to investigate whether ALDH2 activators ameliorated beta cell dysfunction and apoptosis induced by glucolipotoxicity, and its potential mechanisms of action. Glucose-stimulated insulin secretion (GSIS) in MIN6 cells and insulin secretion from isolated islets in perifusion experiments were measured. The intracellular ATP concentrations and oxygen consumption rates of MIN6 cells were assessed. Furthermore, the cell viability, apoptosis, and mitochondrial and intracellular reactive oxygen species (ROS) levels were determined. Additionally, the pro-apoptotic, apoptotic, and anti-apoptotic signaling pathways were investigated. We found that Alda-1 enhanced GSIS by improving the mitochondrial function of pancreatic beta cells. Alda-1 rescued MIN6 cells from MG- and 4-HNE-induced beta cell death, apoptosis, mitochondrial dysfunction, and ROS production. However, the above effects of Alda-1 were abolished in Aldh2 knockdown MIN6 cells. In conclusion, we reported that the activator of ALDH2 not only enhanced GSIS, but also ameliorated the glucolipotoxicity of beta cells by reducing both the mitochondrial and intracellular ROS levels, thereby improving mitochondrial function, restoring beta cell function, and protecting beta cells from apoptosis and death.

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