Browsing by Author "Robertson, Morgan A."

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    A Translational Regulatory Mechanism Mediated by Hypusinated Eukaryotic Initiation Factor 5A Facilitates β-Cell Identity and Function
    (American Diabetes Association, 2024) Connors, Craig T.; Villaca, Catharina B. P.; Anderson-Baucum, Emily K.; Rosario, Spencer R.; Rutan, Caleb D.; Childress, Paul J.; Padgett, Leah R.; Robertson, Morgan A.; Mastracci, Teresa L.; Biology, School of Science
    As professional secretory cells, β-cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic β-cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional β-cells is not well defined. In this study, we have identified a translational regulatory mechanism mediated by the specialized mRNA translation factor eukaryotic initiation factor 5A (eIF5A), which facilitates the maintenance of β-cell identity and function. The mRNA translation function of eIF5A is only active when it is posttranslationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of β-cell DHPS in mice reduces the synthesis of proteins critical to β-cell identity and function at the stage of β-cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the β-cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand.
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    Cigarette smoke exposure impairs β-cell function through activation of oxidative stress and ceramide accumulation
    (Elsevier, 2020-07) Tong, Xin; Chaudhry, Zunaira; Lee, Chih-Chun; Bone, Robert N.; Kanojia, Sukrati; Maddatu, Judith; Sohn, Paul; Weaver, Staci A.; Robertson, Morgan A.; Petrache, Irina; Evans-Molina, Carmella; Kono, Tatsuyoshi; Medicine, School of Medicine
    Objectives Epidemiological studies indicate that first- and second-hand cigarette smoke (CS) exposure are important risk factors for the development of type 2 diabetes (T2D). Additionally, elevated diabetes risk has been reported to occur within a short period of time after smoking cessation, and health risks associated with smoking are increased when combined with obesity. At present, the mechanisms underlying these associations remain incompletely understood. The objective of this study was to test the impact of CS exposure on pancreatic β-cell function using rodent and in vitro models. Methods Beginning at 8 weeks of age, C57BL/6 J mice were concurrently fed a high-fat diet (HFD) and exposed to CS for 11 weeks, followed by an additional 11 weeks of smoking cessation with continued HFD. Glucose tolerance testing was performed during CS exposure and during the cessation period. Cultured INS-1 β-cells and primary islets were exposed ex vivo to CS extract (CSE), and β-cell function and viability were tested. Since CS increases ceramide accumulation in the lung and these bioactive sphingolipids have been implicated in pancreatic β-cell dysfunction in diabetes, islet and β-cell sphingolipid levels were measured in islets from CS-exposed mice and in CSE-treated islets and INS-1 cells using liquid chromatography-tandem mass spectrometry. Results Compared to HFD-fed, ambient air-exposed mice, HFD-fed and CS-exposed mice had reduced weight gain and better glucose tolerance during the active smoking period. Following smoking cessation, CS-mice exhibited rapid weight gain and had accelerated worsening of their glucose tolerance. CS-exposed mice had higher serum proinsulin/insulin ratios, indicative of β-cell dysfunction, significantly lower β-cell mass (p = 0.017), reduced β-cell proliferation (p = 0.006), and increased islet ceramide content compared to non-smoking control mice. Ex vivo exposure of isolated islets to CSE was sufficient to increase islet ceramide levels, which was correlated with reduced insulin gene expression and glucose-stimulated insulin secretion, and increased β-cell oxidative and endoplasmic reticulum (ER) stress. Treatment with the antioxidant N-acetylcysteine markedly attenuated the effects of CSE on ceramide levels, restored β-cell function and survival, and increased cyclin D2 expression, while also reducing activation of β-cell ER and oxidative stress. Conclusions Our results indicate that CS exposure leads to impaired insulin production, processing, secretion and reduced β-cell viability and proliferation. These effects were linked to increased β-cell oxidative and ER stress and ceramide accumulation. Mice fed HFD continued to experience detrimental effects of CS exposure even during smoking cessation. Elucidation of the mechanisms by which CS exposure impairs β-cell function in synergy with obesity will help design therapeutic and preventive interventions for both active and former smokers.
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    Deoxyhypusine synthase, an essential enzyme for hypusine biosynthesis, is required for proper exocrine pancreas development
    (Wiley, 2021-05) Padgett, Leah R.; Robertson, Morgan A.; Anderson-Baucum, Emily K.; Connors, Craig T.; Wu, Wenting; Mirmira, Raghavendra G.; Mastracci, Teresa L.; Biology, School of Science
    Pancreatic diseases including diabetes and exocrine insufficiency would benefit from therapies that reverse cellular loss and/or restore cellular mass. The identification of molecular pathways that influence cellular growth is therefore critical for future therapeutic generation. Deoxyhypusine synthase (DHPS) is an enzyme that post-translationally modifies and activates the mRNA translation factor eukaryotic initiation factor 5A (eIF5A). Previous work demonstrated that the inhibition of DHPS impairs zebrafish exocrine pancreas development; however, the link between DHPS, eIF5A, and regulation of pancreatic organogenesis remains unknown. Herein we identified that the conditional deletion of either Dhps or Eif5a in the murine pancreas results in the absence of acinar cells. Because DHPS catalyzes the activation of eIF5A, we evaluated and uncovered a defect in mRNA translation concomitant with defective production of proteins that influence cellular development. Our studies reveal a heretofore unappreciated role for DHPS and eIF5A in the synthesis of proteins required for cellular development and function.
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    Glucagon is essential for alpha cell transdifferentiation and beta cell neogenesis
    (The Company of Biologists, 2015-04-15) Ye, Lihua; Robertson, Morgan A.; Hesselson, Daniel; Stainier, Didier Y. R.; Anderson, Ryan M.; Department of Pediatrics, IU School of Medicine
    The interconversion of cell lineages via transdifferentiation is an adaptive mode of tissue regeneration and an appealing therapeutic target. However, its clinical exploitation is contingent upon the discovery of contextual regulators of cell fate acquisition and maintenance. In murine models of diabetes, glucagon-secreting alpha cells transdifferentiate into insulin-secreting beta cells following targeted beta cell depletion, regenerating the form and function of the pancreatic islet. However, the molecular triggers of this mode of regeneration are unknown. Here, using lineage-tracing assays in a transgenic zebrafish model of beta cell ablation, we demonstrate conserved plasticity of alpha cells during islet regeneration. In addition, we show that glucagon expression is upregulated after injury. Through gene knockdown and rescue approaches, we also find that peptides derived from the glucagon gene are necessary for alpha-to-beta cell fate switching. Importantly, whereas beta cell neogenesis was stimulated by glucose, alpha-to-beta cell conversion was not, suggesting that transdifferentiation is not mediated by glucagon/GLP-1 control of hepatic glucose production. Overall, this study supports the hypothesis that alpha cells are an endogenous reservoir of potential new beta cells. It further reveals that glucagon plays an important role in maintaining endocrine cell homeostasis through feedback mechanisms that govern cell fate stability.
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    An insulin signaling feedback loop regulates pancreas progenitor cell differentiation during islet development and regeneration
    (Elsevier, 2016-01-15) Ye, Lihua; Robertson, Morgan A.; Mastracci, Teresa L.; Anderson, Ryan M.; Department of Pediatrics, IU School of Medicine
    As one of the key nutrient sensors, insulin signaling plays an important role in integrating environmental energy cues with organism growth. In adult organisms, relative insufficiency of insulin signaling induces compensatory expansion of insulin-secreting pancreatic beta (β) cells. However, little is known about how insulin signaling feedback might influence neogenesis of β cells during embryonic development. Using genetic approaches and a unique cell transplantation system in developing zebrafish, we have uncovered a novel role for insulin signaling in the negative regulation of pancreatic progenitor cell differentiation. Blocking insulin signaling in the pancreatic progenitors hastened the expression of the essential β cell genes insulin and pdx1, and promoted β cell fate at the expense of alpha cell fate. In addition, loss of insulin signaling promoted β cell regeneration and destabilization of alpha cell character. These data indicate that insulin signaling constitutes a tunable mechanism for β cell compensatory plasticity during early development. Moreover, using a novel blastomere-to-larva transplantation strategy, we found that loss of insulin signaling in endoderm-committed blastomeres drove their differentiation into β cells. Furthermore, the extent of this differentiation was dependent on the function of the β cell mass in the host. Altogether, our results indicate that modulation of insulin signaling will be crucial for the development of β cell restoration therapies for diabetics; further clarification of the mechanisms of insulin signaling in β cell progenitors will reveal therapeutic targets for both in vivo and in vitro β cell generation.
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    Polyamine biosynthesis is critical for growth and differentiation of the pancreas
    (Nature Publishing Group, 2015-08-24) Mastracci, Teresa L.; Robertson, Morgan A.; Mirmira, Raghavendra G.; Anderson, Ryan M.; Department of Pediatrics, IU School of Medicine
    The pancreas, in most studied vertebrates, is a compound organ with both exocrine and endocrine functions. The exocrine compartment makes and secretes digestive enzymes, while the endocrine compartment, organized into islets of Langerhans, produces hormones that regulate blood glucose. High concentrations of polyamines, which are aliphatic amines, are reported in exocrine and endocrine cells, with insulin-producing β cells showing the highest concentrations. We utilized zebrafish as a model organism, together with pharmacological inhibition or genetic manipulation, to determine how polyamine biosynthesis functions in pancreatic organogenesis. We identified that inhibition of polyamine biosynthesis reduces exocrine pancreas and β cell mass, and that these reductions are at the level of differentiation. Moreover, we demonstrate that inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, phenocopies inhibition or knockdown of the enzyme deoxyhypusine synthase (DHS). These data identify that the pancreatic requirement for polyamine biosynthesis is largely mediated through a requirement for spermidine for the downstream posttranslational modification of eIF5A by its enzymatic activator DHS, which in turn impacts mRNA translation. Altogether, we have uncovered a role for polyamine biosynthesis in pancreatic organogenesis and identified that it may be possible to exploit polyamine biosynthesis to manipulate pancreatic cell differentiation.
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    Targeting polyamine biosynthesis to stimulate beta cell regeneration in zebrafish
    (Taylor & Francis, 2020-07-25) Robertson, Morgan A.; Padgett, Leah R.; Fine, Jonathan A.; Chopra, Gaurav; Mastracci, Teresa L.; Biology, School of Science
    Type 1 diabetes (T1D) is a disease characterized by destruction of the insulin-producing beta cells. Currently, there remains a critical gap in our understanding of how to reverse or prevent beta cell loss in individuals with T1D. Previous studies in mice discovered that pharmacologically inhibiting polyamine biosynthesis using difluoromethylornithine (DFMO) resulted in preserved beta cell function and mass. Similarly, treatment of non-obese diabetic mice with the tyrosine kinase inhibitor Imatinib mesylate reversed diabetes. The promising findings from these animal studies resulted in the initiation of two separate clinical trials that would repurpose either DFMO (NCT02384889) or Imatinib (NCT01781975) and determine effects on diabetes outcomes; however, whether these drugs directly stimulated beta cell growth remained unknown. To address this, we used the zebrafish model system to determine pharmacological impact on beta cell regeneration. After induction of beta cell death, zebrafish embryos were treated with either DFMO or Imatinib. Neither drug altered whole-body growth or exocrine pancreas length. Embryos treated with Imatinib showed no effect on beta cell regeneration; however, excitingly, DFMO enhanced beta cell regeneration. These data suggest that pharmacological inhibition of polyamine biosynthesis may be a promising therapeutic option to stimulate beta cell regeneration in the setting of diabetes.