IU Indianapolis ScholarWorks :: Browsing by Author "Mastracci, Teresa L."

Browsing by Author "Mastracci, Teresa L."

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Abnormalities in proinsulin processing in islets from individuals with longstanding T1D
(Elsevier, 2019-11) Sims, Emily K.; Syed, Farooq; Nyalwidhe, Julius; Bahnson, Henry T.; Haataja, Leena; Speake, Cate; Morris, Margaret A.; Balamurugan, Appakalai N.; Mirmira, Raghavendra G.; Nadler, Jerry; Mastracci, Teresa L.; Arvan, Peter; Greenbaum, Carla J.; Evans-Molina, Carmella; Pediatrics, School of Medicine
We recently described the persistence of detectable serum proinsulin in a large majority of individuals with longstanding type 1 diabetes (T1D), including individuals with undetectable serum C-peptide. Here, we sought to further explore the mechanistic etiologies of persistent proinsulin secretion in T1D at the level of the islet, using tissues obtained from human donors. Immunostaining for proinsulin and insulin was performed on human pancreatic sections from the Network for Pancreatic Organ Donors with Diabetes (nPOD) collection (n = 24). Differential proinsulin processing enzyme expression was analyzed using mass spectrometry analysis of human islets isolated from pancreatic sections with laser capture microdissection (n = 6). Proinsulin processing enzyme mRNA levels were assessed using quantitative real-time PCR in isolated human islets (n = 10) treated with or without inflammatory cytokines. Compared to nondiabetic controls, immunostaining among a subset (4/9) of insulin positive T1D donor islets revealed increased numbers of cells with proinsulin-enriched, insulin-poor staining. T1D donor islets also exhibited increased proinsulin fluorescence intensity relative to insulin fluorescence intensity. Laser capture microdissection followed by mass spectrometry revealed reductions in the proinsulin processing enzymes prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE) in T1D donors. Twenty-four hour treatment of human islets with inflammatory cytokines reduced mRNA expression of the processing enzymes PC1/3, PC2, and CPE. Taken together, these data provide new mechanistic insight into altered proinsulin processing in long-duration T1D and suggest that reduced β cell prohormone processing is associated with proinflammatory cytokine-induced reductions in proinsulin processing enzyme expression.
Cilia Associated Signaling in Adult Energy Homeostasis
(2022-05) Bansal, Ruchi; Berbari, Nicolas F.; Perrin, Benjamin J.; Mastracci, Teresa L.; Baucum, Anthony J.; Dunn, Kenneth W.
Primary cilia are solitary cellular appendages that function as signaling centers for cells in adult energy homeostasis. Here in chapter 1, I introduce cilia and how dysfunction of these conserved organelles results in ciliopathies, such as Bardet-Biedl Syndrome (BBS), which present with childhood obesity. Furthermore, conditional loss of primary cilia from neurons in the hypothalamus leads to hyperphagia and obesity in mouse models of ciliopathies. Classically, cilia coordinate signaling often through specific G-protein coupled receptors (GPCRs) as is the case in both vision and olfaction. In addition, neurons throughout the brain including hypothalamic neurons possess primary cilia whose dysfunction contributes to ciliopathy-associated obesity. How neuronal cilia regulate the signaling of GPCRs remains unclear and many fundamental cell biology questions remain about cilia mediated signaling. For example, how cilia coordinate signaling to influence neuronal activity is unknown. To begin to address some of these cell biology questions around neuronal cilia, chapter 2, describes the development and use of a system for primary neuronal cultures from the hypothalamus. Using this system, we found that activation of the cilia regulated hedgehog pathway, which is critical in development, influenced the ability of neurons to respond to GPCR ligands. This result highlights the role of the developmentally critical hedgehog pathway on terminally differentiated hypothalamic neurons. One challenge facing the cilia field is our ability to assess cilia in large numbers without potential bias. This is especially true in tissues like the brain, where cilia appear to have region-specific characteristics. Work included in Chapter 3 describes the use of a computer-assisted artificial intelligence (Ai) approach to analyze cilia composition and morphology in a less biased and high throughput manner. Cilia length and intensities are important parameters for evaluation of cilia signaling. Evidence suggests that activation of some ciliary GPCRs results in shortening of cilia whereas deviations from normal cilia length in mutant phenotypes affects normal physiological processes such as decreased mucociliary clearance. Therefore, to analyze a large number of cilia, we describe the use of the Ai module from in vitro and in vivo samples in a reproducible manner that minimizes user bias. Using this approach, we identified that Mchr1 expression is significantly stronger in the cilia of paraventricular nucleus than that in the arcuate nucleus of adult mice. Work in Chapter 4 continues to explore the integration between hedgehog pathway and ciliary GPCR signaling in the central nervous system, and its relevance with energy homeostasis. We evaluated the hedgehog ligand in the plasma of mice in acute and long-term metabolic changes and identified that the activity of the ligand changed under altered metabolic conditions. We also developed a genetic mouse model where hedgehog signaling was constitutively active in neuronal cilia. These mice become hyperphagic and obese. These results further emphasize the potential role of the hedgehog signaling pathway in regulation of feeding behavior in adult vertebrates. Overall, results from this work will provide a better understanding of the defects not only underlying ciliopathy-associated obesity but may also reveal more common mechanisms of centrally mediated obesity. In addition, the tools I have developed will help in understanding how neuronal cilia are used for intercellular communications and ultimately how they regulate behaviors like feeding.
Distinct gene expression pathways in islets from individuals with short‐ and long‐duration type 1 diabetes
(Wiley, 2018) Mastracci, Teresa L.; Turatsinze, Jean-Valery; Book, Benita K.; Restrepo, Ivan A.; Pugia, Michael J.; Weibke, Eric A.; Pescovitz, Mark D.; Eizirik, Decio L.; Mirmira, Raghavendra G.; Biochemistry and Molecular Biology, School of Medicine
Aims Our current understanding of the pathogenesis of type 1 diabetes (T1D) arose, in large part, from studies using the non‐obese diabetic (NOD) mouse model. In the present study, we chose a human‐focused method to investigate T1D disease mechanisms and potential targets for therapeutic intervention by directly analysing human donor pancreatic islets from individuals with T1D. Materials and Methods We obtained islets from a young individual with T1D for 3 years and from an older individual with T1D for 27 years and performed unbiased functional genomic analysis by high‐depth RNA sequencing; the T1D islets were compared with islets isolated from 3 non‐diabetic donors. Results The islets procured from these T1D donors represent a unique opportunity to identify gene expression changes in islets after significantly different disease duration. Data analysis identified several inflammatory pathways up‐regulated in short‐duration disease, which notably included many components of innate immunity. As proof of concept for translation, one of the pathways, governed by IL‐23(p19), was selected for further study in NOD mice because of ongoing human trials of biologics against this target for different indications. A mouse monoclonal antibody directed against IL‐23(p19) when administered to NOD mice resulted in a significant reduction in incidence of diabetes. Conclusion While the sample size for this study is small, our data demonstrate that the direct analysis of human islets provides a greater understanding of human disease. These data, together with the analysis of an expanded cohort to be obtained by future collaborative efforts, might result in the identification of promising novel targets for translation into effective therapeutic interventions for human T1D, with the added benefit of repurposing known biologicals for use in different indications.
Hypusinated eIF5A is expressed in the pancreas and spleen of individuals with type 1 and type 2 diabetes
(Public Library of Science, 2020) Mastracci, Teresa L.; Colvin, Stephanie C.; Padgett, Leah R.; Mirmira, Raghavendra G.; Biochemistry and Molecular Biology, School of Medicine
The gene encoding eukaryotic initiation factor 5A (EIF5A) is found in diabetes-susceptibility loci in mouse and human. eIF5A is the only protein known to contain hypusine (hydroxyputrescine lysine), a polyamine-derived amino acid formed post-translationally in a reaction catalyzed by deoxyhypusine synthase (DHPS). Previous studies showed pharmacologic blockade of DHPS in type 1 diabetic NOD mice and type 2 diabetic db/db mice improved glucose tolerance and preserved beta cell mass, which suggests that hypusinated eIF5A (eIF5AHyp) may play a role in diabetes pathogenesis by direct action on the beta cells and/or altering the adaptive or innate immune responses. To translate these findings to human, we examined tissue from individuals with and without type 1 and type 2 diabetes to determine the expression of eIF5AHyp. We detected eIF5AHyp in beta cells, exocrine cells and immune cells; however, there was also unexpected enrichment of eIF5AHyp in pancreatic polypeptide-expressing PP cells. Interestingly, the presence of eIF5AHyp co-expressing PP cells was not enhanced with disease. These data identify new aspects of eIF5A biology and highlight the need to examine human tissue to understand disease.
Hypusine biosynthesis in β cells links polyamine metabolism to facultative cellular proliferation to maintain glucose homeostasis
(American Association for the Advancement of Science, 2019-12-03) Levasseur, Esther M.; Yamada, Kentaro; Piñeros, Annie R.; Wu, Wenting; Syed, Farooq; Orr, Kara S.; Anderson-Baucum, Emily; Mastracci, Teresa L.; Maier, Bernhard; Mosley, Amber L.; Liu, Yunlong; Bernal-Mizrachi, Ernesto; Alonso, Laura C.; Scott, Donald; Garcia-Ocaña, Adolfo; Tersey, Sarah A.; Mirmira, Raghavendra G.; Pediatrics, School of Medicine
Deoxyhypusine synthase (DHPS) utilizes the polyamine spermidine to catalyze the hypusine modification of the mRNA translation factor eIF5A and promotes oncogenesis through poorly-defined mechanisms. Because germline deletion of Dhps is embryonically lethal, its role in normal postnatal cellular function in vivo remains unknown. We generated a mouse model that enabled the inducible, postnatal deletion of Dhps specifically in postnatal islet β cells, which function to maintain glucose homeostasis. Removal of Dhps did not have an effect under normal physiologic conditions. However, upon development of insulin resistance, which induces β-cell proliferation, Dhps deletion caused alterations in proteins required for mRNA translation and protein secretion, reduced production of the cell cycle molecule cyclin D2, impaired β-cell proliferation, and induced overt diabetes. We found that hypusine biosynthesis was downstream of protein kinase C-ζ and was required for c-Myc-induced proliferation. Our studies reveal a requirement for DHPS in β cells to link polyamines to mRNA translation to effect facultative cellular proliferation and glucose homeostasis.
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.
Pancreatic and Islet Development and Function: The Role of Thyroid Hormone
(JSciMed Central, 2014) Mastracci, Teresa L.; Evans-Molina, Carmella; Department of Pediatrics, IU School of Medicine
A gradually expanding body of literature suggests that Thyroid Hormone (TH) and Thyroid Hormone Receptors (TRs) play a contributing role in pancreatic and islet cell development, maturation, and function. Studies using a variety of model systems capable of exploiting species-specific developmental paradigms have revealed the contribution of TH to cellular differentiation, lineage decisions, and endocrine cell specification. Moreover, in vitro and in vivo evidence suggests that TH is involved in islet β cell proliferation and maturation; however, the signaling pathway(s) connected with this function of TH/TR are not well understood. The purpose of this review is to discuss the current literature that has defined the effects of TH and TRs on pancreatic and islet cell development and function, describe the impact of hyper- and hypothyroidism on whole body metabolism, and highlight future and potential applications of TH in novel therapeutic strategies for diabetes.
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.
Proinsulin Secretion Is a Persistent Feature of Type 1 Diabetes
(American Diabetes Association, 2019-02) Sims, Emily K.; Bahnson, Henry T.; Nyalwidhe, Julius; Haataja, Leena; Davis, Asa K.; Speake, Cate; DiMeglio, Linda A.; Blum, Janice; Morris, Margaret A.; Mirmira, Raghavendra G.; Nadler, Jerry; Mastracci, Teresa L.; Marcovina, Santica; Qian, Wei-Jun; Yi, Lian; Swensen, Adam C.; Yip-Schneider, Michele; Schmidt, C. Max; Considine, Robert V.; Arvan, Peter; Greenbaum, Carla J.; Evans-Molina, Carmella; T1D Exchange Residual C-peptide Study Group; Pediatrics, School of Medicine
OBJECTIVE: Abnormally elevated proinsulin secretion has been reported in type 2 and early type 1 diabetes when significant C-peptide is present. We questioned whether individuals with long-standing type 1 diabetes and low or absent C-peptide secretory capacity retained the ability to make proinsulin. RESEARCH DESIGN AND METHODS: C-peptide and proinsulin were measured in fasting and stimulated sera from 319 subjects with long-standing type 1 diabetes (≥3 years) and 12 control subjects without diabetes. We considered three categories of stimulated C-peptide: 1) C-peptide positive, with high stimulated values ≥0.2 nmol/L; 2) C-peptide positive, with low stimulated values ≥0.017 but <0.2 nmol/L; and 3) C-peptide <0.017 nmol/L. Longitudinal samples were analyzed from C-peptide-positive subjects with diabetes after 1, 2, and 4 years. RESULTS: Of individuals with long-standing type 1 diabetes, 95.9% had detectable serum proinsulin (>3.1 pmol/L), while 89.9% of participants with stimulated C-peptide values below the limit of detection (<0.017 nmol/L; n = 99) had measurable proinsulin. Proinsulin levels remained stable over 4 years of follow-up, while C-peptide decreased slowly during longitudinal analysis. Correlations between proinsulin with C-peptide and mixed-meal stimulation of proinsulin were found only in subjects with high stimulated C-peptide values (≥0.2 nmol/L). Specifically, increases in proinsulin with mixed-meal stimulation were present only in the group with high stimulated C-peptide values, with no increases observed among subjects with low or undetectable (<0.017 nmol/L) residual C-peptide. CONCLUSIONS: In individuals with long-duration type 1 diabetes, the ability to secrete proinsulin persists, even in those with undetectable serum C-peptide.
Recessive Rare Variants in Deoxyhypusine Synthase, an Enzyme Involved in the Synthesis of Hypusine, Are Associated with a Neurodevelopmental Disorder
(Elsevier, 2019-02-07) Ganapathi, Mythily; Padgett, Leah R.; Yamada, Kentaro; Devinsky, Orrin; Willaert, Rebecca; Person, Richard; Au, Ping-Yee Billie; Tagoe, Julia; McDonald, Marie; Karlowicz, Danielle; Wolf, Barry; Lee, Joanna; Shen, Yufeng; Okur, Volkan; Deng, Liyong; LeDuc, Charles A.; Wang, Jiayao; Hanner, Ashleigh; Mirmira, Raghavendra G.; Park, Myung Hee; Mastracci, Teresa L.; Chung, Wendy K.; Pediatrics, School of Medicine
Hypusine is formed post-translationally from lysine and is found in a single cellular protein, eukaryotic translation initiation factor-5A (eIF5A), and its homolog eIF5A2. Biosynthesis of hypusine is a two-step reaction involving the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). eIF5A is highly conserved throughout eukaryotic evolution and plays a role in mRNA translation, cellular proliferation, cellular differentiation, and inflammation. DHPS is also highly conserved and is essential for life, as Dhps-null mice are embryonic lethal. Using exome sequencing, we identified rare biallelic, recurrent, predicted likely pathogenic variants in DHPS segregating with disease in five affected individuals from four unrelated families. These individuals have similar neurodevelopmental features that include global developmental delay and seizures. Two of four affected females have short stature. All five affected individuals share a recurrent missense variant (c.518A>G [p.Asn173Ser]) in trans with a likely gene disrupting variant (c.1014+1G>A, c.912_917delTTACAT [p.Tyr305_Ile306del], or c.1A>G [p.Met1?]). cDNA studies demonstrated that the c.1014+1G>A variant causes aberrant splicing. Recombinant DHPS enzyme harboring either the p.Asn173Ser or p.Tyr305_Ile306del variant showed reduced (20%) or absent in vitro activity, respectively. We co-transfected constructs overexpressing HA-tagged DHPS (wild-type or mutant) and GFP-tagged eIF5A into HEK293T cells to determine the effect of these variants on hypusine biosynthesis and observed that the p.Tyr305_Ile306del and p.Asn173Ser variants resulted in reduced hypusination of eIF5A compared to wild-type DHPS enzyme. Our data suggest that rare biallelic variants in DHPS result in reduced enzyme activity that limits the hypusination of eIF5A and are associated with a neurodevelopmental disorder.

Browsing by Author "Mastracci, Teresa L."

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