Novel Mechanisms In The Sorting Of Proglucagon To The Secretory Granules Of The Regulated Secretory Pathway

The prohormone proglucagon encodes for multiple peptide hormones, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, produced through tissue-specific processing by prohormone convertase (PC) 1/3 and PC2. In alpha cells, PC2 yields glucagon, the major counter-regulatory hormone to insulin, which together, control glucose homeostasis. In contrast, GLP-1 and GLP2 are mainly produced in intestinal L-cells by PC1/3. GLP-1 stimulates insulin secretion following a meal, and therefore has opposing function to glucagon regulating glucose homeostasis; in contrast, GLP-2 enhances gut nutrient absorption. Efficient sorting of proglucagon to secretory granules is required for nutrient-regulated secretion. The aim of this thesis is to discover the molecular mechanisms by which proglucagon is targeted to secretory granules, which ensures that proglucagon is correctly processed to mature hormones, and is necessary for prompt physiologic response to nutrient status. In this thesis, we identify several sorting signals within the hormone domains of proglucagon that encode targeting information. Using quantitative immunofluorescence microscopy and co-localization analyses, I was able to determine the molecular nature by which glucagon and GLP-1 enter granules. Despite these two hormones sharing a large degree of structural homology, it is their particular alpha-helix structures that enable the sorting of proglucagon. Further, I provide evidence that proglucagon is first sorted to granules prior to being processed to active hormones. Furthermore, I have identified carboxypeptidase E in the mechanism by which glucagon sorts within alpha cells. Together, each hormone carries with it a unique sorting “signature” to efficiently reach its destination, and allows alpha and L-cells to tightly regulate nutrient homeostasis

Cargo Transport By Myosin Va Molecular Motors Within Three-Dimensional In Vitro Models Of The Intracellular Actin Cytoskeletal Network

Intracellular cargo transport involves the movement of critical cellular components (e.g. vesicles, organelles, mRNA, chromosomes) along cytoskeletal tracks by tiny molecular motors. Myosin Va motors have been demonstrated to play a vital role in the transport of cargos destined for the cell membrane by navigating their cargos through the three-dimensional actin networks of the cell. Transport of cargo through these networks presents many challenges, including directional and physical obstacles which teams of myosin Va-bound to a single cargo must overcome. Specifically, myosin Va motors are presented with numerous actin-actin intersections and dense networks of filaments which can act as a physical barrier to transport. Due to the complexities of studying myosin Va cargo transport in cells, much effort has been focused on the in vitro observation and analysis of myosin Va transport along single actin filaments or simple actin cytoskeletal models. However, these model systems often rely on non-physiological cargos (e.g. beads, quantum dots) and two-dimensional arrangements of actin attached to glass surfaces. Interestingly, a disconnect exists between the transport of cargo on these simple model systems and studies of myosin Va transport on suspended 3D actin arrangements or cellular networks which show longer run lengths, increased velocities, and straighter, more directed trajectories. One solution to this discrepancy is that the cell may use the fluidity of the cargo surface, the recruitment of myosin Va motor teams, and the 3D geometry of the actin, to finely tune the transport of intracellular cargo depending on cellular need. To understand how myosin Va motors transport their cargo through 3D networks of actin, we investigated myosin Va motor ensembles transporting fluorescent 350 nm lipid-bilayer cargo through arrangements of suspended 3D actin filaments. This was accomplished using single molecule fluorescent imaging, three-dimensional super resolution Stochastic Optical Reconstruction Microscopy (STORM), optical tweezers, and in silico modeling. We found that when moving along 3D actin filaments, myosin motors could be recruited from across the fluid lipid cargo’s surface to the filaments which enabled dynamic teams to be formed and explore the full actin filaments binding landscape. When navigating 3D actin-actin intersections these teams capable of maneuvering their cargo through the intersection in a way that encouraged the vesicles to continue straight rather than switch filaments and turn at the intersection. We hypothesized that this finding may be the source of the relatively straight directed runs by myosin Va-bound cargo observed in living cells. To test this, we designed 3D actin networks where the vesicles interacted with 2-6 actin filaments simultaneously. Actin forms polarized filaments, which, in cells, generally have their plus-ends facing the exterior of the cell; the same direction in which myosin Va walks. We found that to maintain straight directed trajectories and not become stationary within the network, vesicles needed to move along filaments with a bias in their polarity. This allows for cargo-bound motors to align their motion along the polarized networks and produced productive motion despite physical and directional obstacles. Together this work demonstrates the physical properties of the cargo, the geometric arrangement of the actin, and the mechanical properties of the motor are all critical aspects of a robust myosin Va transport system

Pancreatic beta-cell insulin signaling in genetic and dietary models of obesity and insulin resistance

Type 2 Diabetes Mellitus (T2DM) is a heterogeneous metabolic disease characterized by elevated blood glucose levels that has reached pandemic proportions. Genome-wide association studies have linked T2DM to the function of the insulin-producing pancreatic βcell residing in the micro-organ islet of Langerhans. An individual´s risk to develop T2DM depends on genetic predisposition and environmental factors, e.g. life style. Central for disease development is the interplay between insulin resistance in insulin target tissues like muscle, liver and fat and deficient β-cell insulin secretion. Since the β-cell is an insulin target itself, βcell insulin resistance can contribute to β-cell dysfunction and the development of T2DM. This was shown in several genetic (knockout) mouse models, however the dynamics of β-cell insulin resistance and its relevance in a diet-induced context has so far not been explored. Furthermore the consequences of diet-induced β-cell insulin resistance for β-cell function remain to be understood. The difficulty to study β-cell insulin resistance in vivo has partly been due to the lack of a technique to monitor β-cell insulin resistance non-invasively and longitudinally in the living organism. In my thesis I employed the anterior chamber of the eye of mice as a transplantation site for biosensor-expressing reporter islets and the cornea as a natural body window to monitor β-cell insulin resistance non-invasively and longitudinally by microscopic imaging. The β-cell insulin resistance biosensor is based on GFP-labeled FoxO1, that changes its intracellular localization from cytoplasmic (insulin responsive) to nuclear (insulin resistant). With this technique we investigated β-cell insulin resistance dynamics in ob/ob and NZO mice and demonstrated that β-cell insulin resistance dynamics vary in animal models of insulin resistance and obesity. Furthermore, we showed that β-cell insulin resistance developed in the presence of whole-body insulin resistance, impaired glucose tolerance and increased body weight, but independently from liver insulin resistance. To study the relevance of β-cell insulin resistance in diet-induced T2DM development, we treated diabetes-prone male C57BL/6J mice with different combinations of solid high fat diet and drinking water containing either sucrose or fructose. Employing our new monitoring technique we showed that only mice that were fed a High-FatHigh-Sucrose-Diet developed β-cell insulin resistance. This demonstrated the importance of βcell insulin resistance in a model of diet-induced obesity and insulin resistance and highlighted the importance of diet composition for the development of T2DM. The β-cell insulin resistance was accompanied by a decreased functional β-cell mass and impaired insulin secretion downstream of glucose-stimulated Ca2+ influx, due to a reduction of syntaxin-1A. We were also able to show that β-cell insulin resistance in one insulin signaling cascade can re-route the insulin signal, thus allowing the co-existence of reduced and increased insulin response in the same cell. In conclusion, my in vivo studies of diet-induced β-cell insulin resistance and its consequences on β-cell function and survival contribute to better understanding of the development of T2DM

Studium zásobních forem insulinu v sekrečních granulích β buněk pankreatu

V této disertační práci jsme se zaměřili na komplexní výzkum zaměřený na produkci, skladování a sekreci inzulínu -buňkami pankreatu. Úspěšně jsme vyvinuli nový test pro rychlé a citlivé stanovení koncentrace inzulínu v biologických vzorcích. Tento test, založený na kompetici měřeného vzorku s radioligandem o inzulínový receptor, nám pomohl určit vliv různých nízkomolekulárních látek, ale i peptidů, na sekreci inzulinu. Zjistili jsme, že arginin a ornitin mají na dávce závislý stimulační účinek na glukózou stimulovanou sekreci inzulínu z −buněk, ale že dopamin sekreci inzulinu inhibuje. Účinek serotoninu na sekreci inzulinu byl nejednoznačný. Studovali jsme také účinky kostního proteinu osteokalcinu a jeho fragmentů na sekreci inzulínu. Zjistili jsme, že tyto peptidy nestimulují sekreci inzulinu z -buněk, ale že osteokalcin může mít proliferační vlastnosti. Testovali jsme také vliv tryptofanu a jeho metabolitů a zjistili jsme, že tyto sloučeniny nestimulují sekreci inzulinu, ale že některé z nich ji mohou ve vyšších koncentracích sekreci inhibovat. Významným výsledkem studie je experimentální potvrzení přítomnosti krystalického inzulinu v sekrečních granulích -buněk. Jedná se o první přímý důkaz, že za nativních podmínek může být inzulín v buňkách uložen ve formě mikrokrystalů. Celkově jsme...In this dissertation, we focused on a comprehensive investigation of insulin production, storage and secretion by pancreatic -cells. We successfully developed a new assay for the rapid and sensitive determination of insulin concentration in biological samples. This assay, based on the competition of the measured sample with a radioligand for the insulin receptor, helped us to determine the influence of several low molecular weight compounds, as well as peptides, on insulin secretion. We found that arginine and ornithine have a dose-dependent stimulatory effect on glucose- stimulated insulin secretion from -cells, but that dopamine inhibits insulin secretion. The effect of serotonin on insulin secretion was ambiguous. We also studied the effects of the bone protein osteocalcin and its fragments on insulin secretion. We found that these peptides do not stimulate insulin secretion from -cells, but that osteocalcin may have proliferative properties. We also tested the effect of tryptophan and its metabolites and found that these compounds do not stimulate insulin secretion but that some of them may inhibit secretion at higher concentrations. An important result of the study is the experimental confirmation of the presence of crystalline insulin in the secretory granules of -cells. This is the first...Katedra biochemieDepartment of BiochemistryPřírodovědecká fakultaFaculty of Scienc

Regulation of endoplasmic reticulum calcium homeostasis in pancreatic β cells

Indiana University-Purdue University Indianapolis (IUPUI)Diabetes mellitus is a group of metabolic diseases characterized by disordered insulin secretion from the pancreatic β cell and chronic hyperglycemia. In order to maintain adequate levels of insulin secretion, the β cell relies on a highly developed and active endoplasmic reticulum (ER). Calcium localized in this compartment serves as a cofactor for key proteins and enzymes involved in insulin production and maturation and is critical for ER health and function. The ER Ca2+ pool is maintained largely through activity of the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) pump, which pumps two Ca2+ ions into the ER during each catalytic cycle. The goal of our research is to understand the molecular mechanisms through which SERCA2 maintains β cell function and whole body glucose metabolism. Our previous work has revealed marked dysregulation of β cell SERCA2 expression and activity under diabetic conditions. Using a mixture of pro-inflammatory cytokines to model the diabetic milieu, we found that SERCA2 activity and protein stability were decreased through nitric oxide and AMP-activated protein kinase (AMPK)mediated signaling pathways. Moreover, SERCA2 expression, intracellular Ca2+ storage, and β cell death under diabetic conditions were rescued by pharmacologic or genetic inhibition of AMPK. These findings provided novel insight into pathways leading to altered β cell Ca2+ homeostasis and reduced β cell survival in diabetes. To next define the role of SERCA2 in the regulation of whole body glucose homeostasis, SERCA2 heterozygous mice (S2HET) were challenged with high fat diet (HFD). Compare to wild-type controls, S2HET mice had lower serum insulin and significantly reduced glucose tolerance with similar adiposity and systemic and tissue specific insulin sensitivity, suggesting an impairment in insulin secretion rather than insulin action. Consistent with this, S2HET mice exhibited reduced β cell mass, decreased β cell proliferation, increased ER stress, and impaired insulin production and processing. Furthermore, S2HET islets displayed impaired cytosolic Ca2+ oscillations and reduced glucose-stimulated insulin secretion, while a small molecule SERCA2 activator was able to rescue these defects. In aggregate, these data suggest a critical role for SERCA2 and the maintenance of ER Ca2+ stores in the β cell compensatory response to diet induced obesity

Insulin Secretory Granule biogenesis and VPS41

The pancreatic beta cells are not only the fundamental source of insulin production but also the predominant regulator of its storage and release upon appropriate stimuli to maintain glucose homeostasis. Insulin is stored in membrane-bounded structures called secretory granules (SGs), which are specialized secretory units of the regulated secretory pathway. Synthesized proinsulin as an inactive precursor form transports into the Golgi apparatus, where proinsulin is sorted and packaged into immature SGs. Soluble cargo proteins, membrane proteins, as well as ions are critical components for insulin production as well as insulin storage in pancreatic beta cells. Type 2 diabetes (T2D) is a prevalent and complex disease that is defined by chronic hyperglycemia and insulin resistance, which are strongly correlated with dysregulated insulin production machinery in beta cells. Recently, vacuolar protein sorting-associated protein 41 (VPS41) was identified as a regulator in the trafficking pathways of synthesized secretory proteins. In this thesis, I show that VPS41 plays a critical role in insulin storage capacity and insulin granule biogenesis in beta cells using both an in vitro rat insulinoma beta cell VPS41 knockout line and an in vivo mouse model with a conditional deletion of VPS41 in beta cells. I show for the first time that deletion of VPS41 in a mouse model leads to severe diabetes associated with extensive depletion of insulin in pancreatic beta cells. Together, my in vivo and in vitro data illustrate that VPS41 is a potent regulator of insulin secretory biology and is required for the maintenance of normal glucose homeostasis

Embryonic Stem Cells

Embryonic stem cells are one of the key building blocks of the emerging multidisciplinary field of regenerative medicine, and discoveries and new technology related to embryonic stem cells are being made at an ever increasing rate. This book provides a snapshot of some of the research occurring across a wide range of areas related to embryonic stem cells, including new methods, tools and technologies; new understandings about the molecular biology and pluripotency of these cells; as well as new uses for and sources of embryonic stem cells. The book will serve as a valuable resource for engineers, scientists, and clinicians as well as students in a wide range of disciplines

Degradomics : a study of the cellular proteolytic landscape in enterovirus infections

The common human pathogen, enteroviruses (EVs) are a genus of single stranded RNA viruses that include Coxsackie B virus (CVBs). The viral RNA encodes a polyprotein containing two viral proteases 2Apro and 3Cpro, that process the polyprotein into structural and non-structural proteins during virus replication. Previous research has shown that aside from their role in polyprotein processing, the proteases also cleave host proteins in order to modulate different cell functions such as translation, transcription and innate immunity. The innate immune response, and in particular the type 1 interferons (IFN), have an important role in controlling virus spread and protecting neighbouring cells from infection. Similarly, type III IFNs modulate the permissiveness of cells to CVBs. In paper I, we found that CVB3 has evolved a mechanism to evade the type III IFN response in a comparable manner to that previously shown for the type I IFNs. This perturbation is likely mediated via the proteolytic cleavage of the signal transduction proteins IPS-1 and TRIF by 2Apro. When investigating virus-associated diseases, high quality reagents are essential especially when the aim is to detect virus in serum or tissue samples. We recognised a need for reagents capable of detecting CVBs and in paper II we described the development of CVB specific antibodies against 2Apro/3Cpro and show their utility in western blotting, confocal microscopy, immunohistochemistry and flow cytometry. Several observations have suggested a role for CVB infection in the development of type 1 diabetes (T1D). Interestingly, β-cells infected with CVBs in vitro have defective insulin secretion. The aim of paper III was to investigate whether β-cell dysfunction observed during CVB infection could be attributed to the activity of the viral proteases. We found that the viral proteins 2Apro, 3Cpro and 3A individually exert negative effects on glucose stimulated insulin release (GSIS) and voltage stimulated exocytosis in β-cells. Based on the results in paper I and III, it is evident that 2Apro and 3Cpro have multifaceted roles in the viral replication cycle and in the modulation of the host cell. In paper IV we wanted to define the viral proteases specific targets in Coxsackeivirus B3 (CVB3) permissive cell lines of varied origin (HeLa, CaCo-2 and EndoC-βH1). By enriching for the N-terminal peptides using subtiligase labelling combined with LS-MS/MS, we identified and validated 81 host cell protease substrates. Among the substrates we identified, the protein TCF7L2 was a target of 2Apro mediated cleavage. TCF7L2 is an important transcription factor involved in maintaining β-cell functionality and glucose stimulated insulin secretion. This finding provides a potential mechanistic explanation for the observation that β-cells infected with CVBs in vitro are defective in insulin secretion. The studies presented in this thesis increase our understanding the molecular virology of enteroviruses. Moreover, they open a new chapter of research in examining how disease pathology might be caused by the activity of virally encoded proteases