Browsing by Subject "Insulin"
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Item The activin ligand dawdle links diet and metabolism via receptor isoform-specific signaling in Drosophila.(2009-07) Jensen, Philip AnthonyAbstract not available.Item Diseases of New World Camelids(2014-05) O'Conor Dowd, Mary CatherineKnowledge of medical conditions that affect camelids has increased greatly in recent years. However, sometimes the first signs of illness in camelids can be subtle and nonspecific or animals may be found dead with no abnormalities observed. The purpose of this study was to catalog the fatal conditions seen in camelids in the Upper Midwest in order to recognize trends and ultimately to prevent disease. The reports of 234 alpacas and 125 llamas submitted to the University of Minnesota Veterinary Diagnostic Laboratory (UM VDL) for post‐mortem examination from 2001 to 2011 were reviewed. The cause of death was identified in 73% of the cases. The digestive system was the most common site of the major disease condition, accounting for 29% of alpacas and 31% of llamas. In adults, gastrointestinal parasitism associated with emaciation, neoplasia, and hepatic lipidosis were among the most common diagnoses identified. Meningeal worm (P. tenuis) was the most commonly identified cause of death associated with the nervous system in adults, since 13% of llamas and 2.4% of alpacas had central nervous system lesions consistent with verminous migration. The most common cause of death or euthanasia among alpaca crias was choanal atresia, which accounted for mortality in 19% of alpacas under 6 months of age. Other congenital abnormalities and bacterial infections were also common in neonates. In older crias (age 2 weeks to 6 months), enteritis and septicemia were most common. In conclusion, necropsy evaluation was successful in identifying the cause of death in the majority of camelids submitted to the UM VDL. The risk of morbidity or mortality related to many of the most common diseases identified in this study can be reduced by taking steps to ensure good husbandry practices, including close monitoring of body condition score, regular fecal testing to identify parasite burdens, basic biosecurity measures to limit spread of pathogens, and IgG measurement to ensure adequate passive transfer of immunity.Item IGF-1 stimulates glycolytic ATP production in MCF-7L cells(2023-07) Rajoria, BhumikaThe Insulin-like Growth Factor (IGF) system in breast cancer progression has been a matter of interest for decades, but targeting this system did not result in a successful clinical strategy. The system’s complexity, and homology of its two receptors - insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) are possible causes. The IGF system maintains cell proliferation and regulates metabolism, making it a pathway to explore. To understand the metabolic phenotype of breast cancer cells, we quantified their real-time ATP production rate upon acute stimulation with ligands – insulin-like growth factor 1 (1GF-1) and insulin. MCF-7L cells express both IGF-1R and IR, while tamoxifen-resistant MCF-7L (MCF-7L TamR) cells have downregulated IGF-1R with unchanged IR levels. Treating MCF-7L cells with 5nM IGF-1 increased the glycolytic ATP production rate while 10nM insulin did not affect metabolism when compared with control. Neither treatment altered ATP production in MCF-7L TamR cells. The study provides evidence of the relationship between metabolic dysfunction, cancer, and the IGF axis. In these cells, IGF-1R, and not IR, regulates ATP production.Item Insulin signals through IGF-IR in insulin receptor knockout breast cancer cell line(2020-07) Monteiro, MarvisBreast cancer is a common malignancy observed more in females than in males. In breast cancer there is an upregulation of the IGF system. Upregulation of insulin and InsR are associated with poor patient prognosis. In order to understand the role of InsR in breast cancer biology, an InsR knockout cell line was created from MCF-7L breast cancer cells. Clone 35 showed loss of InsR expression, despite this loss, clone 35 showed activation of p-Akt and p-MAPK on stimulation with insulin. The hypothesis was developed that in the InsR knockout cell line insulin bound to IGF-IR and activated signaling. This hypothesis was proven by developing a knockout model, then using InsR and IGF-IR specific inhibitors on clone 35 to suggest the involvement of IGF-IR in activation through insulin. The following research substantiate the claims and provide a new understanding in the role of InsR and IGF-IR in breast cancer biology.Item INSULIN TRAFFICKING PERTURBATIONS AT THE BLOOD-BRAIN BARRIER IN ALZHEIMER’S DISEASE MODELS(2017-11) Sarma, VidurAlzheimer’s disease (AD) is recognized as a multifactorial disease and a major cause of dementia in the elderly. Pathological hallmarks of AD include brain amyloid beta (Aβ) deposits and intraneuronal tangles of hyperphosphorylated tau protein. Reduction of brain Aβ burden is widely considered as a viable therapeutic strategy for AD, and developing methods to promote brain Aβ clearance has been at the forefront of AD research. Recent clinical trials conducted in a small group of AD patients demonstrated the efficacy of intranasally-administered insulin in improving memory and reducing brain amyloid burden (Craft et al., 2012). Although the mechanism of insulin action is not clear, epidemiological studies have linked type 2 diabetes (T2DM), characterized by hyperinsulinemia and peripheral insulin resistance, with cognitive decline and amyloid burden in AD (Matsuzaki et al., 2010). One possibility is that hyperinsulinemia damages the cerebrovascular endothelium, referred to as the blood brain barrier (BBB). The BBB is a major signaling and material trafficking pathway between plasma and brain that not only delivers glucose and insulin to brain but also serves as the primary clearance portal for brain Aβ (Storck et al., 2016; Yuede et al., 2016). Hence, T2DM could augment AD pathology by inhibiting insulin delivery and disrupting brain Aβ clearance. Although, intranasal insulin may remedy the situation, this approach may limited, due to variable absorption of insulin into CNS. Moreover, insulin is a growth factor with multiple physiological targets; hence, chronic insulin therapy will have off-target effects. As a fundamental approach, critical insulin-responsive cellular and molecular pathways that facilitate Aβ trafficking should be identified. Only through elucidating these dysfunctions in AD/T2DM can novel therapeutic targets be rationally sought. In this work, the key questions concern the dynamics of insulin transport from systemic circulation into brain via the BBB and the disruption in insulin trafficking caused by Aβ peptides in AD brain. Importantly, molecular components in the insulin trafficking/signaling pathway that are disturbed by Aβ exposure were identified as well as the perturbed insulin delivery and brain insulin resistance. This new knowledge will facilitate the search for novel therapeutic targets for AD. I have been investigating mechanisms triggering insulin scarcity in AD brains by conducting kinetic biodistribution and SPECT/CT imaging assays in Aß peptide over-producing transgenic mice (APP/PS1), insulin resistant db/db (leptin receptor deficient) and aged WT mice. I analyzed plasma pharmacokinetic and dynamic brain uptake data by 3-compartmental analysis on SAAM© and discovered differences in distribution rate constants between WT and the insulin-resistant mouse groups. Upon simulating tissue distribution of a given dose of insulin in a 3-compartmental Stella© model with the estimated rate constants, I discovered that APP/PS1, db/db aged, and Aβ40/Aβ42 pre-treated WT mouse display higher plasma AUC and lower brain AUC levels of insulin, as compared to healthy, young WT mice. This led us to a hypothesis that Aß40 and Aß42 interfere with the transport of insulin into the brain parenchyma. Now, the appearance of systemic insulin in the brain parenchyma is contingent upon insulin receptors (IR) expressed by BBB endothelial cells. Therefore, I employed human cerebrovascular microendothelial cells (hCMEC) and over-expressed hCMEC monolayers with IR to investigate mechanistic interactions between Aß peptides and insulin transcytosis across the BBB. The processes involved in insulin transcytosis across BBB endothelial cells are postulated to operate in tandem with downstream signaling cascades. I studied variables in IR-mediated transport processes and correlated the results with shifts in phosphorylation of proteins expressed in downstream insulin signaling pathways, as a consequence of Aß peptide exposure. Through flow cytometry and radioactive Transwell© transport assays, I discovered that the IR-mediated uptake, permeability and exocytosis of insulin across hCMEC/D3 cells were significantly impaired by pre-exposure to Aβ40 and Aβ42. With help of FRAP/FLIP imaging analyses, I found that the half-life of lateral diffusion and exocytic recycling of IR in response to insulin was increased in presence of Aβ40 and Aβ42. With respect to hCMEC/D3 signal transduction downstream of IR, Western Blot analyses confirmed that Aβ40 and Aβ42 excessively activated the phosphorylation of the IR-β subunit at Tyr1162/1163 and decreased the phosphorylation of Akt at Ser 473. In addition, reverse-phase protein array (RPPA) assays revealed that phosphorylation of MAPK3 was increased whereas that of caveolin-1 was decreased due to Aβ40 and Aβ42 exposure. My results lead to conclude that Aβ peptides interfere with insulin signaling/trafficking at the BBB and reduce insulin availability in the AD brain. It is therefore imperative to rectify motifs which result in impaired insulin signaling at the BBB to improve its transport into the brain parenchyma.Item ISLET O-GLCNACYLATION IN THE CONTEXT OF BETA CELL SECRETORY ADAPTATION TO OBESITY(2020-07) Lockridge, AmberObesity is the primary risk factor for the development of type 2 diabetes, primarily through the induction of insulin resistance which leads to a dysregulation of glucose homeostasis. Nevertheless, the majority of obese individuals avoid diabetic hyperglycemia by upregulating insulin output through adaptive mechanisms that tune pancreatic β-cell secretory function, insulin biosynthesis, cell growth, differentiation and proliferation in accordance with the duration of overnutrition. For example, glucose-stimulated insulin secretion of β-cell-containing islets is potentiated in mice within a few weeks of high fat diet (HFD) feeding but transitions to secretory impairment after several months, even as β-cell mass expansion continues to drive hyperinsulinemia. The cooperative dynamism of this adaptation strategy, which appears to balance insulin demand against β-cell overwork, suggests an (unknown) coordinated regulatory system that is capable of differentiating between acute, prolonged and chronic overnutrition and transducing multi-factorial cellular effects. The premise of this thesis is that post-translational protein O-GlcNAcylation, under the central control of the OGT attachment enzyme and nutrient-driven substrate supply, is well-positioned to provide this missing link. In the current studies, we found that islet O-GlcNAcylation is dynamically responsive to different phases of obesity compensation in both mice and humans, with elevations positively correlated to secretory hyperinsulinemia. β-cell specific OGT loss mice failed to develop in vivo hyperinsulinemia during early HFD and showed impairments in both HFD- and palmitate-induced in vitro potentiation of islet insulin secretion. Proinsulin processing and β-cell fate maintenance were also impaired. An unbiased RNAseq approach identified differentially expressed genes (DEGs) in βOGT KO islets, which were cross-referenced with a published transcriptome of HFD-adapted islets. Among the common DEGs, over 40% were upregulated by HFD but downregulated by OGT loss, including the ER Ca2+ ATPase SERCA2 protein. Allosteric activation of SERCA2, which was O-GlcNAcylated in both human islets and mouse β-cells, rescued palmitate-potentiation of insulin secretion in both constitutive and induced βOGT KO islets. These findings show that islet protein O-GlcNAcylation is uniquely and dynamically sensitive to obesity duration and selectively regulates hyperlipidemia-driven insulin secretory potentiation, consistent with a governing role in anti-diabetic β-cell adaptations during early obesity.Item Reprogramming of different cell types into pancreatic beta cells by using transcription factor genes Pdx1, Ngn3 and MafA(2012-08) Akinci, ErsinIn this dissertation we studied the effect of the pancreatic transcription factor genes Pdx1, Ngn3 and MafA, all of which were cloned into single adenoviral construct. First, we investigated the reprogramming competency to Pdx1, Ngn3 and MafA of different cell types at different developmental stages from mouse and rat. Second, we looked at the effect of these three genes on a pancreatic rat exocrine cell line AR42J-B13 to see if this gene combination provides a true beta cell reprogramming event. Next, we investigated the effect of some small molecules to see if they increased the reprogramming efficiency of this gene combination. Finally we tried to sort the small fraction of insulin-positive cells formed after Pdx1, Ngn3, MafA transduction to enrich the insulin-positive cell population hence getting more accurate data from them.Transduction of different cell types with Ad-PNM showed that the Pdx1, Ngn3 and MafA gene combination is enough to stimulate the expression of Insulin genes with varying intensities depending on cell type. Rat cell lines responded to Ad-PNM in a better way than mouse cell lines by activating more beta cell genes. The number of responding cells to Ad-PNM was also higher for the rat cell lines than the mouse cell lines. Moreover progenitor-like cells as well as the cells developmentally related to beta cells are prone to be reprogrammed into beta-like state. Supporting this final statement, embryonic mouse heptoblasts from Pdx1-GFP transgenic mice gave the most promising result by activating the endogenous Pdx1 as well as many other important beta cell genes upon transduction with Ad-PNM.Further characterization of the effect of Ad-PNM on the most resposive cell type AR42J-B13 cells showed that after Ad-PNM the cells became post-mitotic, began to express Insulin genes together with many other beta cell genes, produced mature insulin hormone, changed the epigenetic state of their chromatin, and rescued diabetic mice if transplanted into the kidney capture. However, some important beta cell genes were not activated thereby making these cells unresponsive to glucose which is an indispensable beta cell quality. It is obvious that even though the changes are dramatic, the combination of Pdx1, Ngn3 and MafA did not provide a true beta cell reprogramming in vitro at least for this cell system. Among some of the small molecules which had been reported to favor beta cell formation, regeneration and/or survival, three of them including DAPT, BIX-01294 and NECA increased the effect of Ad-PNM on mouse hepatocyte-derived small cells. Moreover when all three were used together they showed a better efficiency. Even though there was a significant increase in the number of insulin positive cells, the fraction of the Ad-PNM-responding cells was still low after the small molecules. For that reason the effect of small molecules on reprogramming efficiency of Ad-PNM was compared by counting the fraction of insulin-positive cells out of Ad-PNM-bearing cells between control and experimental cell groups instead of performing qRT-PCR.Of the attempts that we performed to increase the low fraction of insulin-positive cells via different sorting techniques to get more accurate and reliable results from Ad-PNM responding cells, only fluorescence-activated cell sorting of insulin-immunostained cells worked. Even though these cells were enriched through the FACS, because the cells were nonviable not alive we could not perform any downstream application that necessitate living cells. This system allows us to do qRT-PCR only.Item Timing, growth and homeostasis: an anthology of three novel players in Drosophila melanogaster(2013-08) Ghosh, ArpanDevelopmental timing, growth and homeostasis form the cornerstones that shape the genesis and subsequent maintenance of a healthy adult for all multicellular organisms. Understanding the mechanisms that regulate these processes has important implications both from the perspective of our understanding of basic biology and also for our understanding of complex biological disorders involving these processes. My work explores the mechanisms regulating developmental timing, homeostasis and growth using the model organism Drosophila melanogaster. In this thesis I report involvement of three novel players in the regulation of timing, homeostasis and growth in the Drosophila larvae.Firstly, my work unearths the mechanism by which the developmental gap gene, giant, regulates developmental timing in Drosophila larvae. While the effect of Giant (Gt) on larval developmental timing was long known, the mechanism by which Gt exerted this effect was not known. I find that Gt affects developmental timing by influencing the developmental fate of the prothoracicotropic hormone producing PG neurons that are essential for determining the timing of larval developmental transitions. Additionally, I show that Gt is required for PG neuron axon targeting. Secondly, I show that TGF-beta/Activin signaling mediated by the Activin-like ligand Dawdle (Daw) regulates sugar homeostasis, pH balance and mitochondrial metabolism in Drosophila larvae. Canonical signaling by Daw regulates sugar homeostasis primarily by affecting release of insulin in the larvae. The effect of Daw on pH is mediated independently by Daw's action on mitochondrial metabolism and production of metabolic acids. Interestingly, Daw affects both phenotypes in a dose-dependent manner, as demonstrated by both loss-of-function and over-expression/gain-of-function experiments, thereby providing evidence for a hormonal role of Daw in regulating systemic homeostasis. Lastly, I show that eukaryotic uracil salvaging enzyme uracil phospho-ribosyltransferase (UPRT), that was considered inactive in higher eukaryotes including Drosophila, is active in the Drosophila larvae. The Drosophila UPRT homologue, Krishah, can actively incorporate a uracil derivative (4TU) into RNA indicating that the enzyme is active in vivo. Interestingly, I find that Krishah is also essential for larval growth as knocking out the gene leads to impaired larval growth and increased larval and pupal lethality.Item Type II Diabetes: The Importance of Electronic Medical Records(2012-07-26) Heser, Justin