Insulin and glucagon are released in pulses from pancreatic β- and α-cells, respectively. Both cell types are electrically excitable, and elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) due to depolarization with voltage-dependent entry of the cation is the main trigger of hormone secretion. Store-operated Ca2+ entry  (SOCE) also contributes to the [Ca2+]i elevation and this process has been suggested to be particularly important for glucagon secretion. cAMP is another important messenger that amplifies Ca2+-triggered secretion of both hormones, but little is known about cAMP dynamics in islet cells. In type-2 diabetes, there is deteriorated β-cell function associated with elevated concentrations of fatty acids, but the underlying mechanisms are largely unknown. To clarify the processes that regulate insulin and glucagon secretion, cAMP signalling and the store-operated pathway were investigated in β- and α-cells, primarily within their natural environment in intact mouse and human islets of Langerhans. Fluorescent biosensors and total internal reflection microscopy were used to investigate signalling specifically at the plasma membrane (PM). Adrenaline increased and decreased the sub-PM cAMP concentration ([cAMP]pm) in immuno-identified α-cells and β-cells, respectively, which facilitated cell identification. Glucagon elicited [cAMP]pm oscillations in α- and β-cells, demonstrating both auto- and paracrine effects of the hormone. Whereas glucagon-like peptide 1 (GLP-1) consistently elevated [cAMP]pm in β-cells, only few α-cells responded, indicating that GLP-1 regulates glucagon secretion without changes of α-cell [cAMP]pm. Both α- and β-cells responded to glucose with pronounced oscillations of [cAMP]pm that were partially Ca2+-dependent and synchronized among islet β-cells. The glucose-induced cAMP formation was mediated by plasma membrane-bound adenylyl cyclases. Several phosphodiesterases (PDEs), including the PDE1, -3, -4, and -8 families, were required for shaping the [cAMP]pm signals and pulsatile insulin secretion. Prolonged exposure of islets to the fatty acid palmitate deteriorated glucose-stimulated insulin secretion with loss of pulsatility. This defect was associated with impaired cAMP generation, while [Ca2+]i signalling was essentially unaffected. Stromal interacting molecule 1 (STIM1) is critical for activation of SOCE by sensing the Ca2+ concentration in the endoplasmic reticulum (ER). ER Ca2+ depletion caused STIM1 aggregation, co-clustering with the PM Ca2+ channel protein Orai1 and SOCE activation. Glucose, which inhibits SOCE by filling the ER with Ca2+, reversed the PM association of STIM1. Consistent with a role of the store-operated pathway in glucagon secretion, this effect was maximal at the low glucose concentrations that inhibit glucagon release, whereas considerably higher concentrations were required in β-cells. Adrenaline induced STIM1 translocation to the PM in α-cells and the reverse process in β-cells, partially reflecting the opposite effects of adrenaline on cAMP in the two cell types. However, cAMP-induced STIM1 aggregates did not co-cluster with Orai1 or activate SOCE, indicating that STIM1 translocation can occur independently of Orai1 clustering and SOCE.

Insulin-like growth factor-1 (IGF-1), a known secretory product of intestinal subepithelial myofibroblasts (ISEMF), is essential for the intestinotrophic effects of glucagon-like peptide-2(GLP-2). I hypothesized that GLP-2 increases the production of IGF-1 by primary murine ISEMF in culture. Immunocytochemistry showed that the ISEMF stained appropriately for α smooth muscle actin and vimentin but not for desmin. The ISEMF also expressed GLP-2 receptor and IGF-1 mRNA transcripts. ISEMF treated with GLP-2 revealed a maximal increase in IGF-1 mRNA transcript levels at 10-8 M GLP-2 and 2hr. Interestingly, immunoblotting revealed an increase in P-AKT/T-AKT with GLP-2, but no changes in cAMP, P-ERK/T-ERK or calcium were detected. PI3K inhibition and kinase-dead AKT over-expression abrogated GLP-2-induction of IGF-1 mRNA, and ISEMF from GLP-2R null mice demonstrated reductions in IGF-1 mRNA and cellular IGF-1, but not in media IGF-1, vs. wild-type ISEMF. These findings suggest a possible mechanism by which GLP-2 increases intestinal growth in-vivo.

Insulin-like growth factor-1 (IGF-1), a known secretory product of intestinal subepithelial myofibroblasts (ISEMF), is essential for the intestinotrophic effects of glucagon-like peptide-2(GLP-2). I hypothesized that GLP-2 increases the production of IGF-1 by primary murine ISEMF in culture. Immunocytochemistry showed that the ISEMF stained appropriately for α smooth muscle actin and vimentin but not for desmin. The ISEMF also expressed GLP-2 receptor and IGF-1 mRNA transcripts. ISEMF treated with GLP-2 revealed a maximal increase in IGF-1 mRNA transcript levels at 10-8 M GLP-2 and 2hr. Interestingly, immunoblotting revealed an increase in P-AKT/T-AKT with GLP-2, but no changes in cAMP, P-ERK/T-ERK or calcium were detected. PI3K inhibition and kinase-dead AKT over-expression abrogated GLP-2-induction of IGF-1 mRNA, and ISEMF from GLP-2R null mice demonstrated reductions in IGF-1 mRNA and cellular IGF-1, but not in media IGF-1, vs. wild-type ISEMF. These findings suggest a possible mechanism by which GLP-2 increases intestinal growth in-vivo.

No description available.

La production endogène de glucose est une fonction cruciale au maintien de l'homéostasie glucidique dont les 2 dernières étapes sont la production de glucose par la glucose-6-phosphatase (G6Pase) et la sortie du glucose hors de la cellule par le transporteur facilité GLUT2. Les mécanismes dépendants de mouvements membranaires régulant ces deux étapes ont été étudiés. La régulation de la G6Pase par l'AMPc dépend de mouvements membranaires. Cependant les mécanismes moléculaires de cette régulation restaient à caractériser. Nous avons étudié l'hypothèse d'une phosphorylation directe des sous-unités de la G6Pase par la PKA. La PKA est capable d'induire l'activité G6Pase. Cependant, aucune phosphorylation des sous-unités G6Pase n'a pu être mise en évidence par phosphorylation in vitro, mutations dirigées de sites potentiels de phosphorylation ou analyse par spectrométrie de masse. En absence de Glut2, le glucose produit de novo sort des hépatocytes par une voie dépendante de mouvements membranaires, dont le mécanisme moléculaire n'est pas caractérisé. Cette voie vésiculaire n'est pas impliquée dans la sortie du glucose glycogénolytique. À l'inverse, 50% du glucose néoglucogénique sort des hépatocytes par une voie vésiculaire, probablement dépendante de la cavéoline-1. Par microscopie confocale à fluorescence, nous avons montré que la G6Pase se déplace dans la cellule vers la membrane plasmique et co-localise avec une partie de la cavéoline1 cellulaire. Les vésicules composées de cavéoline-1 et contenant la G6Pase pourrait donc constituer un lien entre le réticulum endoplasmique, lieu de production du glucose et la membrane plasmique, lieu de libération du glucose

<p>Akrilamid&nbsp; je toksična hemijska supst anca koja je već dugi niz godina prisutna u životnoj sredini,&nbsp; jer se kao važan monomer koristi u različite industrijske i laboratorijske svrhe. U poslednjih petnaest godina, akrilamid je postao posebno zanimljiv za &scaron;ire naučne krugove jer&nbsp; se pokazalo da&nbsp; se&nbsp; nalazi&nbsp; i u&nbsp; hrani&nbsp; biljnog porekla, posebno hrani bogatoj skrobom, koja se priprema pečenjem ili prženjem na temperaturama vi&scaron;im od 120&deg;C.&nbsp; Do sada ustanovljeni negativni zdravstveni efekti akrilamida su veoma raznovrsni i mogu biti rezultat delovanja samog&nbsp; akrilamida ili delovanja njegovog metabolita glicidamida koji nastaje&nbsp; in vivo&nbsp; kada se jedan deo molekula akrilamida metaboli&scaron;e oksigenacijom dvostruke veze pomoću enzima citohrom P450 2E1 (CYP2E1). Akrilamid je supstanca koja ima dokazan negativan efekat&nbsp; na organske sisteme kod ljudi i životinja, i koja je svrstana u moguće humane karcinogene. Negativan efekat akrilamida na egzokrini pankreas je poznat, ali o mogućim efektima akrilamida na endokrini pankreas se i dalje veoma malo zna. Ima puno dokaza koji&nbsp; ukazuju na to da akrilamid ima citotoksični efekat koji se&nbsp; manifestuje kroz uticaj na redoks-status ćelija i dovodi do promena u vrednostima biomarkera oksidativnog i nitrozativnog stresa, kao i u aktivnosti antioksidativnih enzima. Pankreas&nbsp; je&nbsp; jedan od ciljnih&nbsp; organa za delovanje akrilamida te je&nbsp; glavni predmet istraživanja&nbsp; doktorske teze&nbsp; bio proučavanje potencijalnog efekta akrilamida na endokrini pankreas pacova.&nbsp; Ispitivanje je vr&scaron;eno na 3&nbsp; eksperimentalne grupe&nbsp; juvenilnih&nbsp; mužjaka pacova soja Wistar,&nbsp; od kojih je&nbsp; jedna grupa bila kontrolna, dok su dve bile tretirane&nbsp; sa akrilamidom u dozama od 25 mg/kg tm i 50 mg/kg tm,&nbsp; 5 dana nedeljno,&nbsp; tokom 3 nedelje. Po isteku tretmana,&nbsp; nakon dekapitacije, kompletno tkivo pankreasa&nbsp; je&nbsp; fiksirano u 10% rastvoru formalina&nbsp; tokom&nbsp; 24&nbsp; h i obrađeno prema&nbsp; standardnoj proceduri za kalupljenje u parafinu.&nbsp; Parafinski kalupi su sečeni na serijske preseke debljine 5 &micro;m, nakon čega su bojeni&nbsp; histohemijskom i imunohistohemijskim metodama.&nbsp; Kod eksperimentalnih grupa posmatrane&nbsp; su&nbsp; histolo&scaron;ke promene na endokrinom pankreasu, sa akcentom na &alpha;-&nbsp; i &beta;-ćelije.&nbsp; Takođe, posmatrana je&nbsp; i&nbsp; ekspresija&nbsp; hormona insulina i glukagona, enzima inducibilne azot -oksi d&nbsp; sintetaze (iNOS) i&nbsp; CYP2E1,&nbsp; kao&nbsp; i ekspresija&nbsp;&nbsp; antioksidativnih enzima&nbsp; katalaza&nbsp; (CAT) i superoksid dismut aza 1 i 2&nbsp; (SOD1 i SOD2)&nbsp; u ćelijama Langerhansovih ostrvaca. Potencijalna promena u funkcionalnosti &beta;-ćelija je ispitana i kroz analizu nivoa glukoze u serumu pacova tretiranih sa akrilamidom.<br />Budući da &beta;-ćelije čine 80% ćelija koje grade Langerhansova ostrvca pankreasa,&nbsp; pored in vivo&nbsp; eksperimenata, ispitana&nbsp; je&nbsp; i toksičnost akrilamida na&nbsp; Rin-5F ćelijsku liniju insulinoma &beta;-ćelija pacova u in vitro uslovima. Glavni cilj in vitro&nbsp; istraživanja je bio&nbsp; da se&nbsp; ispita&nbsp; uticaj&nbsp; rastućih&nbsp; koncentracija akrilamida na preživljavanje tretiranih&nbsp; Rin-5F&nbsp; ćelija, ali i efekat IC₅₀&nbsp; koncentracije ove supstance primenjene&nbsp; tokom&nbsp; različitih vremenskih intervala&nbsp; (0,5, 1, 3, 6, 12 i 24 h)&nbsp; na pojavu oksidativnog i nitrozativnog stresa. Redoks-status Rin-5F ćelija tretiranih&nbsp; sa akrilamidom je ispitan preko analize prisustva biomarkera oksidativnog i nitrozativnog stresa, akrivnosti CAT i ukupne SOD, kao i promene u ekspresiji gena za CAT, SOD1, SOD2&nbsp;&nbsp; i iNOS.&nbsp; Pored toga, analiziran je i efekat istog tretmana na&nbsp; ekspresiju gena za insulin, CYP2E1, Bax i Bcl-2. U okviru teze je pokazano da akrilamid ne dovodi do&nbsp; značajnih promena u histolo&scaron;koj građi, dijametru i broju Langerhansovih ostrvaca&nbsp; kod&nbsp; tretiranih životinja.&nbsp; Primena stereolo&scaron;kih metoda&nbsp; je&nbsp; ukazala&nbsp; na mikrostrukturne promene na&nbsp; endokrinom pankreasu na nivou &alpha;-&nbsp; i &beta;-ćelija. U ovoj tezi je po prvi put pokazano da tretman akrilamidom negativno utiče na broj i povr&scaron;inu &beta;-ćelija pankreasa.&nbsp; U tezi je, takođe,&nbsp; pokazan&nbsp; značajan dozno-zavisni pad u prisustvu insulina u &beta;-ćelijama&nbsp;&nbsp; pankreasa. Uprkos&nbsp; tome, kod&nbsp; akrilamidom tretiranih&nbsp; životinja&nbsp; nije konstatovana&nbsp; promena&nbsp; u&nbsp; koncentraciji serumske glukoze.&nbsp; U&nbsp; ovoj tezi je pokazano da tretman akrilamidom dovodi do&nbsp;&nbsp; statistički značajnog porasta&nbsp; u broju &alpha;-ćelija&nbsp; kod životinja koje su primale nižu dozu tretmana, dok se&nbsp; broj &alpha;-ćelija&nbsp; kod životinja koje su primale vi&scaron;u dozu tretmana&nbsp; ne razlikuje značajno od kontrole.&nbsp; Tretman akrilamidom je doveo do značajnog&nbsp; porasta u količini&nbsp;&nbsp; prisutnog glukagona&nbsp; u &alpha;-ćelijama pankreasa.<br />Tretman akrilamidom nije doveo do značajne promene u ekspresiji CAT, SOD1 i SOD2 u ćelijama Langerhansovih ostrvaca.&nbsp; Kod&nbsp; tretiranih životinja&nbsp; do&scaron;lo do značajnog dozno-zavisnog porasta&nbsp; u ekspresiji&nbsp; enzima iNOS,&nbsp; dok je ekspresija&nbsp; CYP2E1 značajno dozno-zavisno opala&nbsp; nakon tretmana. U&nbsp; tezi je pokazano da tretman akrilamidom negativno utiče na vijabilnost Rin-5F ćelija, i utvrđeno je da IC50&nbsp; koncentracija akrilamida za Rin-5F ćelije iznosi 10 mM.&nbsp; Rezultati teze pokazuju da tretman akrilamidom u IC₅₀&nbsp; koncentraciji u Rin-5F ćelijskoj liniji značajno povećava nivo malondialdehida (MDA) nakon tretmana u trajanju od 1, 12 i 24 h.&nbsp; Isti tretman&nbsp; značajno smanjuje nivo redukovanog GSH nakon tretmana od 1, 3, 6, 12 i<br />24 h, kao i nivo slobodnih&nbsp; &ndash;SH grupa nakon tretmana od 3 i 6 h. Tretman akrilamidom u IC_50&nbsp; koncentraciji signifikantno pojačava aktivnost CAT nakon tretmana od 1 h, dok tretman u trajanju od 12 h značajno smanjuje aktivnost ovog enzima. Ovaj tretman smanjuje aktivnost SOD nakon 1, 12 i 24 h, dok&nbsp; tretman u trajanju od 6 h značajno pojačava aktivnost enzima SOD.&nbsp; U tezi je, takođe, pokazan i veoma značajan porast&nbsp; u nivou prisutnih nitrita,&nbsp; koji&nbsp; je direktno proporcionalan&nbsp; sa nivoom azot-oksida i nivoom akivnosti enzima iNOS.&nbsp; Ovaj&nbsp; nalaz ukazuje na potencijalnu pojavu nitrozati vnog stresa u akrilamidom-tretiranim Rin-5F ćelijama.&nbsp; U&nbsp; tezi je po prvi put pokazano da tretman&nbsp; akrilamidom dovodi do&nbsp; značajnih&nbsp; varijacija&nbsp; u transkripciji gena za iNOS, SOD1, SOD2,&nbsp; CAT,&nbsp; CYP2E1,&nbsp; Bax i Bcl-2 u tretiranim Rin-5F ćelijama, dok isti tretman ne dovodi do&nbsp; promene nivoa&nbsp; transkripcije gena za insulin.&nbsp; Tretman akrilamidom u koncentraciji od 10<br />mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK<br />gena za iNOS u svim tačkama tretmana, do porasta&nbsp; nivoa&nbsp; iRNK za SOD1 i SOD2 nakon tretmana od 12 i 24 h, kao i do porasta&nbsp; količine&nbsp; iRNK za CAT nakon tretmana od 3 h.&nbsp; U&nbsp; tezi je pokazano&nbsp; i&nbsp; da akrilamid&nbsp; izaziva&nbsp; promene&nbsp; u sintezi&nbsp; iRNK&nbsp; za enzim&nbsp; CYP2E1&nbsp; koji je&nbsp; posebno značajan u kontekstu detoksikacije ove toksične supstance.&nbsp; Porast u transkripciji gena za&nbsp; CYP2E1&nbsp; je uočen&nbsp; nakon tretmana u trajanju od 0,5 i 1 h, dok je&nbsp; do smanjenja transkripcije&nbsp; do&scaron;lo&nbsp; nakon tretmana od 12&nbsp; i 24&nbsp; h.&nbsp; Tretman akrilamidom u koncentraciji od&nbsp; 10 mM tokom rastućih vremenskih perioda dovodi do porasta u relativnoj količini iRNK&nbsp; gena za Bax u svim tačkama tretmana, i do porasta u transkripciji gena za Bcl-2 nakon tretmana od 0,5, 1 i 3 h.<br />Sumirajući&nbsp; sve&nbsp; rezultate&nbsp; ove teze,&nbsp; moze se zaključiti&nbsp; da je endokrini pankreas&nbsp; jedno od&nbsp; ciljnih tkiva, na koje akrilamid ostvaruje vi&scaron;estruki negativni uticaj.</p> / <p>Acrylamide is a toxic chemical used as an important monomer for various industrial and laboratory purposes, which makes it highly present in the environment. In the last fifteen years, acrylamide has become especially interesting for wider scientific circles when it was found in staple foodstuff rich in starch, prepared at temperatures higher than 120&deg;C. The established negative health effects of acrylamide are very diverse and can be the result of the acrylamide action itself or the action of its metabolite glycidamide that occurs in vivo, when acrylamide molecule is metabolized via oxygenation of the double bond by the cytochrome P450 2E1 (CYP2E1). Acrylamide is a substance with a proven adverse effect on humans and animals, and it is classified as a possible human carcinogen. The negative effect of acrylamide on the exocrine pancreas has already been recognized, but the possible effects of acrylamide&nbsp; on endocrine pancreas are still mostly undetermined. There is a significant amount of evidence to suggest that acrylamide exerts a cytotoxic effect which manifests through the changes in level of oxidative and nitrosative stress biomarkers, as well as in the activity of antioxidant enzymes. Since, pancreas is one of the target organs for acrylamide, the main subject of doctoral thesis was to investigate the potential effect of acrylamide on the rat endocrine pancreas. The investigation was conducted on 3 experimental groups of juvenile male Wistar rats, of which one group was the control group, while two groups were treated with acrylamide at doses of 25 mg/kg bw and 50 mg/kg bw, 5 days a week, during 3 weeks. After termination of the treatment, decapitation was performed, and the complete pancreatic tissue was fixed in a 10% formalin solution for 24 h and treated according to the standard paraffin embedding procedure. Paraffin molds were cut into 5 &mu;m thick serial sections, after which they were stained with histochemical and immunohistochemical methods. Histological changes ofthe endocrine pancreas, with the emphasis on &alpha;- and &beta;-cells, were examined in three experimental groups of rats. In addition, the expression of insulin and glucagon hormone, the inducible nitric oxide synthase (iNOS) and CYP2E1 enzymes, and the expression of antioxidative enzymes catalase (CAT) and superoxide dismutases 1 and 2&nbsp; (SOD1 and SOD2) in the islets of Langerhans were also investigated. A potential change in the functionality of &beta;-cells was also examined by analyzing glucose level in the serum of rats treated with acrylamide. In pancreatic islets of Langerhans the majority of cells (&gt;80%) are &beta;-cells. Therefore, in addition to in vivo experiments, the toxicity of acrylamide was examined in vitro on rat insulinoma Rin-5F cell line.The main goal of in vitro research was to investigate the impact of increasing acrylamide concentrations on the viability of treated Rin-5F cells, and also to examine whether IC50 concentration of this substance, applied at different intervals of time (0.5, 1, 3, 6, 12 and 24 h), induce oxidative and nitrosative stress. Redox-status of Rin-5F cells treated with acrylamide was examined by analyzing oxidative and nitrosative stress biomarkers, CAT and total SOD activity, as well as changes in the expression of the CAT, SOD1, SOD2 and iNOS. In addition, the effect of the same treatment on the transcription of the insulin, CYP2E1, Bax and Bcl-2 gene was analyzed.The results of the thesis showed that acrylamide treatment does not lead to significant changes in the histological structure, diameter and number of islets of Langerhans of treated animals. Application of stereological methods indicated microstructural changes of &alpha;- and &beta;-cells ofendocrine pancreas. It has been shown for the first time that treatment with acrylamide negatively affects the number and surface area of pancreatic &beta;-cells. In addition, a significant dose-dependent decline in the amount of insulin in pancreatic &beta;-cells was also demonstrated. However, no change in serum glucose level was observed in treated animals. Acrylamide treatment led to a statistically significant increase in the number of &alpha;-cells in animals receiving a lower dose of treatment, while the number of &alpha;-cells in animals receiving a higher dose of treatment did not differ significantly from the control. Treatment with acrylamide led to a significant increase in the amount of the glucagon in &alpha;-cells. Treatment with acrylamide did not cause a significant change in the expression of CAT, SOD1 and SOD2 in islets of Langerhans. However, there was a significant dosedependent increase in the&nbsp; expression of iNOS enzyme, whereas expression of CYP2E1 significantly decreased in dose-dependent manner in treated animals. Results of the thesis showed that acrylamide exerts a negative effect on the viability of Rin-5F cell line. It has been established that the IC50 concentration of acrylamide for the Rin-5F cell line is 10 mM. The results of the thesis indicate that treatment of Rin-5F cell line with IC50 concentration of acrylamide for 1, 12, and 24 h significantly increased the level of malondialdehyde (MDA). Exposure to acrylamide for 1, 3, 6, 12 and 24 h significantly decreased the level of reduced GSH, while the level of free -SH groups was reduced after 3 and 6 h of acrylamide treatments. Treatment with IC50 concentration of acrylamide significantly enhanced CAT activity after 1 h of acrylamide exposure, while 12 h exposure significantly reduced the activity of this enzyme. Application of acrylamide reduced SOD activity after 1, 12, and 24 h exposure, while 6 h exposure significantly increased the activity of SOD enzymes. Results of the thesis also showed a very significant increase of the nitrite level, which is directly proportional to the level of nitrogen oxide (NO) and the level of the iNOS activity. This finding points to the potential occurrence of nitrosative stress in acrylamide-treated Rin-5F cells. It has been shown for the first time that acrylamide treatment leads to significant variations in transcription of iNOS, SOD1, SOD2, CAT, CYP2E1, Bax and Bcl-2 genes in treated Rin-5F cells, while the same treatment does not affect transcription of the insulin gene. Treatment with acrylamide at a concentration of 10 mM for increasing periods of time leads to an increase in the relative amount of the iNOS gene iRNA at all treatment points. Twelve and and 24 h of acrylamide exposure increased the transcription of the SOD1 and SOD2 genes. Transcription of CAT gene was increased after 3 h&nbsp; ofacrylamide exposure. Furthermore, it has been shown that acrylamide treatment leads to variations in the mRNA synthesis of CYP2E1 gene, which is particularly significant in the context of detoxification of this toxic substance. An increase in the transcription ofthe CYP2E1&nbsp; gene was observed after 0.5 and 1 h of acrylamide exposure, while the reduction of&nbsp; transcription occurred after 12 and 24 h of acrylamide exposure. The treatment with 10 mM acrylamide has led to an increase of the transcription of the Bax gene at all treatment points, and also to an increase of transcription of the Bcl-2 gene after of 0.5, 1, and 3 h of acrylamide exposure. Summarizing all the results of this thesis, it can be concluded that the endocrine pancreas&nbsp; is one of the target tissues of acrylamide, to which this substance exerts a multiple adverse effects.</p>

<p>Therapeutic peptides have become a clinically and commercially important drug class providing novel treatment options in variety of disease areas. Today, more than 80 peptide drugs are marketed worldwide and hundreds more are in development. However, the development of peptide drugs can be hindered by their tendency to self-associate to form fibrils, an impurity that can affect potency and increase the potential for adverse immune responses in patients. Fibrillation of therapeutic peptides can present significant quality concerns and poses challenges for manufacturing and storage. From a pharmaceutical development perspective, early detection of instabilities can inform the development of mitigation strategies to minimize the risk of product failure and avoid costly delays in clinical development. A fundamental understanding of the mechanisms of fibrillation is critical for the rational design of fibrillation-resistant peptide drugs and formulations.</p> <p>The objective of this dissertation was to develop structurally modified fibrillation-resistant peptides based on a mechanistic understanding of the fibrillation process. The therapeutic peptides studied were human calcitonin (hCT), a glucagon/GLP-1 analog, and human insulin B-chain (INSB). Pulsed hydrogen-deuterium exchange mass spectrometry (HDX-MS) and other biophysical methods were used to provide mechanistic understanding of the intermolecular interactions and structural transitions during peptide fibrillation. Coupled with proteolytic digestion, pulsed HDX-MS of fibrillating peptides enabled identification of the residues involved in the early interactions leading to fibrillation based on their differential deuterium exchange rates. The high-resolution residue level information was used to make site-specific modifications to hCT, with phosphorylation in the central region resulting in complete inhibition of fibrillation for the phospho-Thr-13 hCT analog under the stress conditions employed. Reversible ‘prodrug’ modifications such as phosphorylation can aid the rational design of fibrillation-resistant therapeutic peptides. Furthermore, the effects of structural modifications on peptide fibrillation were evaluated by reducing the Cys1-Cys7 disulfide bond in hCT, and by C-terminal amidation or substitution with a helix-stabilizing residue (α-aminoisobutyric acid, Aib) in the glucagon/GLP-1 analog peptide. Finally, studies of insulin B-chain probed fibrillation mechanisms of this therapeutically important peptide, contributing to our understanding of the mechanisms of insulin fibrillation with the broad goal of developing fibrillation-resistant, rapid-acting, monomeric insulin analogs. Overall, the results demonstrate that small structural changes can have significant effects on peptide fibrillation, that pulsed HDX-MS can be used to probe these effects, and that an understanding of these effects can inform the rational development of fibrillation-resistant peptide drugs. </p>

Indiana University-Purdue University Indianapolis (IUPUI) / Glucagon-like peptide 1 (GLP-1) receptor agonists are a class of incretin based therapeutics which aid in blood glucose management in Type II diabetes mellitus (T2DM). Recent studies have demonstrated direct cardiovascular benefits conferred by these agents including protection in ischemia and heart failure. Despite these observations, human clinical trials fail to support improvements in cardiovascular outcomes independent of glucose lowering effects in the T2DM populations. Prior data from our lab demonstrate that obesity impairs GLP-1 associated increases in myocardial glucose uptake. However, the reasons for this impairment/resistance to cardiac effects of GLP-1 in the setting of obesity remain ill defined. This investigation tested the hypothesis that underlying differences in the cardiac proteome and microRNA (miR) transcriptome could contribute to distinct cardiac responses to ischemia and activation of GLP-1 signaling in the setting of obesity. To identify whether obesity modulated cardiac functional responses to GLP 1 related drugs, we first examined the effects of obesity on cardiac function, miR transcriptome, and proteome in response to short duration ischemia-reperfusion (I/R). We observed divergent physiologic responses (e.g. increased diastolic volume and systolic pressure in lean, decreased diastolic volumes in obese) to regional I/R in obese vs lean hearts that were associated with significant molecular changes as detected by protein mass spectrometry and miR microarray. Molecular changes were related to myocardial calcium handling (SERCA2a, histidine-rich Ca2+ binding protein), myocardial structure and function (titin), and miRs relating to cardiac metabolism, hypertrophy, and cell death, including miR-15, miR-30, miR-199a, miR-214. Importantly, these effects were modified differently by GLP-1 agonism in lean vs obese swine. Additional studies investigated the functional effects of 30 days of treatment with the GLP-1 analogue liraglutide on a model of slowly-developing, unrelieved coronary ischemia. Liraglutide failed to reduce infarct size or collagen deposition. However, analysis of left ventricular pressure-volume relationships support that liraglutide improved diastolic relaxation/filling, load-dependent indices of cardiac function, and cardiac efficiency in response to sympathetic stimulation in obese swine. Taken together, these findings support that miR and proteomic differences underlie distinct changes in functional cardiac responses to I/R and pharmacologic activation of GLP-1 signaling in the setting of obesity.

<p> Obesity is a growing concern as 42.3% of people in the U.S were considered obese in the years 2017- 2018. Little is known about the genetic components that contribute to weight gain. In humans, the hormone glucagon is a major contributor to the body’s energy demand as it helps break down lipids. Therefore, learning more about this pathway could enable a range of therapeutics. In fact, studies have shown that glucagon treatments have helped patients with both weight loss and appetite suppression. In this project, we analyzed candidate genes that modify the glucagon pathway in <em>Drosophila melanogaster.</em> We reduced the expression of the fly version of the glucagon receptor (AKHR) in our model. This induces fat retention in the L3 larvae, which mimics obesity in humans. We then crossed our model to the DGRP and looked for natural variation in fat content using a density assay. The density assay examines the relative fat levels of the larvae by slowly increasing the amount of sucrose in water. This enables us to observe whether we have lean larvae which float later or fat larvae which float early on. We used the variation in floating concentration to identify candidate modifier genes through GWA or genome-wide association study. We crossed our <em>AKHR</em> RNAi model to RNAi for various candidate modifier genes that may enhance or suppress fat retention. We screened these candidates initially with the same density assay used in the original study. This resulted in four candidate genes that significantly impacted the density of the larvae: <em>THADA</em>, <em>AmyD</em>, <em>GluRIIC</em>, and <em>CG9826</em>. We further characterized these candidates using biochemical assays to analyze stored metabolites such as triglycerides, glucose, glycogen, and protein. These have been further analyzed under control, high sugar, and high fat conditions to see if the larvae are resistant to environmental changes. <em>CG9826</em> showed significant increase in stored fats across all environments. <em>THADA</em> RNAi showed an increase in fat in the high fat environment. Overexpression of <em>THADA</em> showed a decrease in fat storage in the high fat environment. Our goal is to advance our understanding of the glucagon signaling pathway, obesity, and lipid metabolism. We are also hopeful to provide candidate genes that can be regarded as future therapeutic targets. </p>

[ES] La regulación de los niveles de glucosa en el cuerpo humano es el resultado de la secreción coordinada de hormonas. La Diabetes Tipo 1 (DT1) es una enfermedad crónica que provoca la destrucción de las células responsables de la producción de insulina, uno de los principales agentes en la regulación de glucosa. Por tanto, las personas con DT1 dependen de la administración exógena de insulina. No obstante, la gestión de la terapia no es sencilla y está sujeta a una gran variabilidad. Los sistemas de Páncreas Artificial se diseñaron con el objetivo de simplificar la gestión de la enfermedad, administrando insulina de manera automática a través de una bomba de insulina, en base a la lógica de un algoritmo de control que emplea información de un monitor continuo de glucosa. Sin embargo, la acción de la insulina es unidireccional (disminuye el valor de la glucosa), y a veces resulta insuficiente para mantener unos niveles seguros de glucosa en sangre. Por eso, en ocasiones se administran otras hormonas, con efectos opuestos (como el glucagón), o complementarios (como la pramlintida) a la insulina. Para que los sistemas automáticos se beneficien de estas acciones de control, es necesario estudiar y conocer sus dinámicas para poder simular su comportamiento, diseñar controladores que los tengan en cuenta y realizar experimentos in silico previos a utilizar los sistemas en pacientes. El uso del glucagón ya cuenta con una larga trayectoria y ha sido utilizado en varios sistemas automáticos. Sin embargo, existe mucha heterogeneidad en las formulaciones de modelos del efecto del glucagón, sobre todo en relación con su interacción con la insulina, y es necesario profundizar en el desarrollo de modelos que reflejen mejor la fisiología subyacente. Por otra parte, los modelos de pramlintida apenas se han estudiado. El objetivo principal de esta tesis es contribuir a mejorar simuladores para validar sistemas de páncreas artificial. En concreto, se realiza un análisis detallado del estado del arte para conocer las propuestas de modelos fisiológicos en la literatura, para luego centrarse en la descripción del efecto de glucagón en la producción endógena de glucosa y la farmacocinética y farmacodinámica de la pramlintida. El trabajo incluye la propuesta de nuevos modelos para glucagón y pramlintida basados en la fisiología, validados con datos clínicos individuales en el caso del glucagón y con datos poblacionales de la literatura en el caso de la pramlintida, mejorando en ambos casos los resultados previamente existentes. / [CA] La regulació dels nivells de glucosa en el cos humà és el resultat de la secreció coordinada d'hormones. La Diabetis Tipus 1 (DT1) és una malaltia crònica que provoca la destrucció de les cèl·lules responsables de la producció d'insulina, un dels principals agents en la regulació de glucosa. Per tant, les persones amb DT1 depenen de l'administració exògena d'insulina. No obstant això, la gestió de la teràpia no és senzilla i està subjecta a una gran variabilitat. Els sistemes de Pàncrees Artificial es van dissenyar amb l'objectiu de simplificar la gestió de la malaltia, administrant insulina de manera automàtica a través d'una bomba d'insulina, en funció de la lògica d'un algorisme de control que empra informació d'un monitor continu de glucosa. No obstant això, l'acció de la insulina és unidireccional (disminueix el valor de la glucosa), i de vegades resulta insuficient per a mantindre uns nivells segurs de glucosa en sang. Per això, ocasionalment s'administren altres hormones, amb efectes oposats (com el glucagó), o complementaris (com la pramlintida) a la insulina. Perquè els sistemes automàtics es beneficien d'aquestes accions de control, és necessari estudiar i conéixer les seues dinàmiques per a poder simular el seu comportament, dissenyar controladors que els tinguen en compte i realitzar experiments in silico previs a utilitzar els sistemes en pacients. L'ús del glucagó ja compta amb una llarga trajectòria i ha sigut utilitzat en diversos sistemes automàtics. No obstant això, existeix molta heterogeneïtat en les formulacions de models de l'efecte del glucagó, sobretot en relació amb la seua interacció amb la insulina, i és necessari aprofundir en el desenvolupament de models que reflectisquen millor la fisiologia subjacent. D'altra banda, els models de pramlintida a penes s'han estudiat. L'objectiu principal d'aquesta tesi és contribuir a millorar simuladors per a validar sistemes de pàncrees artificial. En concret, es fa una anàlisi detallada de l'estat de l'art per a conéixer les propostes de models fisiològics en la literatura, per a després centrar-se en la descripció de l'efecte de glucagó en la producció endògena de glucosa i la farmacocinètica i farmacodinàmica de la pramlintida. El treball inclou la proposta de nous models per a glucagó i pramlintida basats en la fisiologia, validats amb dades clíniques individuals en el cas del glucagó i amb dades poblacionals de la literatura en el cas de la pramlintida, millorant en tots dos casos els resultats prèviament existents. / [EN] Glucose regulation in the human body results from the coordinated secretion of hormones. Type 1 Diabetes (T1D) is a chronic disease that destroys insulin-producing cells, one of the main agents in the glucose regulation process. Consequently, people with T1D depend on exogenous insulin administration. However, therapy management is not an easy task, and it faces great variability. Artificial Pancreas systems were designed to ease the disease management, administering insulin automatically through an insulin pump based on the logic of a control algorithm that reads information from a continuous glucose monitor. Nevertheless, insulin action is uni-directional (lowering glucose values), and sometimes, it is insufficient to maintain safe plasma glucose levels. That is why, occasionally, other hormones are also administered, with opposite (like glucagon) or complementary effects (like pramlintide) to insulin. For automatic systems to benefit from these control actions, it is necessary to study and know their dynamics to simulate their behavior, design aware controllers, and carry out in silico experiments before using the system with patients. Glucagon use in T1D has a long trajectory; and has been used in automatic systems. However, there exists a wide heterogeneity in the definitions of glucagon effect, especially related to its interaction with insulin, and it is necessary to develop models that are more physiologically accurate. On the other hand, pramlintide models have barely been studied. This thesis' main objective is to improve T1D simulators to validate artificial pancreas systems. Specifically, a detailed analysis of the state of the art is carried out to know the physiological model proposals in the literature. Then, the focus moves to describing the glucagon effect on endogenous glucose production and the pharmacokinetics and pharmacodynamics of pramlintide. This work includes the proposal of new physiology-based models for glucagon and pramlintide. The glucagon model was validated with individual clinical data, and the pramlintide model was validated with populational data. Both proposals improved previously existing results. / This work was supported by grant FPU17/03404, grant EST19/00740, and project PID2019-107722RB-C21, funded by MCIN/AEI/10.13039/501100011033. / Furió Novejarque, C. (2024). Contributions to Glucagon and Pramlintide Pharmacokinetics and Pharmacodynamics Modeling for Multi-Hormone Artificial Pancreas Systems [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202872