Institute of Physiology

ARRS J3-9289

The role of cyclic adenosine monophosphate in normal beta cell physiology and during development of type 2 diabetes mellitus

Vloga cikličnega adenozin monofosfata v normalni fiziologiji celic beta in med razvojem sladkorne bolezni tipa 2

Abstract

In beta cells, glucose stimulates insulin secretion by way of intracellular metabolism, which yields ATP and closes ATP-dependent potassium channels. This, in turn, leads to depolarization, opening of voltage-dependent calcium channels, and calcium influx. The resulting increase in intracellular calcium is the secondary message triggering exocytosis. The therapeutically important sulfonylureas bind directly to ATP-dependent potassium channels and depolarize the cell and trigger insulin secretion in a largely glucose-independent manner. In contrast, the incretin hormones or therapeutic agents that mimic their action act on beta cells by binding to extracellular G-protein coupled receptors that stimulate adenylyl cyclase and thus increase intracellular cAMP. This second messenger acts via two distinct intracellular pathways, either PKA or Epac2A, to influence different steps of the triggering cascade, but seems to be unable to trigger exocytosis in the absence of glucose- or sulfonylurea-initiated triggering signal. Lately, evidence has been presented that sulfonylureas directly modulate Epac2A activity, and that incretins are also able to produce the triggering increase in calcium concentration, suggesting that there are many intersections of signaling pathways activated by glucose, sulfonylureas, and incretins. In addition to its intracellular effects on stimulus-secretion coupling, cAMP might influence intercellular spreading of glucose- or sulfonylurea-induced depolarization and calcium waves. This intercellular functional coupling normally serves to align beta cell glucose responsiveness at the level of all beta cells coupled in an islet of Langerhans, but is also target of diabetogenic insults during development of insulin resistance and type 2 diabetes. However, how exactly cAMP influences intra- and intercellular signal conduction and what is its role for whole body glucose tolerance in normal conditions and during development of type 2 diabetes, remains poorly understood and this might at least partly account for the present lack of long-term therapeutic success of sulfonylureas and our inability to better stratify patients to different treatment groups depending on the duration of disease or knowledge of specific beta cell defects. More specifically, there is gap in our knowledge on the differential roles of PKA and Epac2A in the above processes and a better understanding of how their mode of action could lead to development of novel therapeutic agents. By studying glucose-, sulfonylurea- and incretin-induced stimulus-secretion coupling from most proximal (membrane depolarization) to most distal steps (exocytosis), intercellular coupling, and whole animal glucose tolerance in control and Epac2 knock-out mice, fed either a control diet or a western diet to induce insulin resistance and beta cell adaptation and failure, within the framework of this project we aim to achieve the following four main objectives:

To clarify the role of Epac2A and PKA in mediating the effects of cAMP on various steps of stimulus-secretion coupling.
To clarify the role of Epac2A and PKA in mediating the effects of cAMP on intercellular functional coupling.
To explore the role of Epac2A and PKA in mediating the effects of not only glucose but also various sulfonylureas, especially their influence upon changes in membrane potential and calcium oscillations, the sensitivity of the exocytotic machinery and intercellular functional coupling between beta cells.
To help mechanistically explain the role of Epac2A and PKA in beta-cell adaptation and dysfunction during development of western diet-induced mouse model of type 2 diabetes mellitus at the multicellular level of islets of Langerhans, as well as their effect on whole animal glucose tolerance in both normal and insulin resistant mice.

References:

PODOBNIK, Boris, KOROŠAK, Dean, SKELIN, Maša, STOŽER, Andraž, DOLENŠEK, Jurij, RUPNIK, Marjan, IVANOV, Plamen Ch., HOLME, P., JUSUP, Marko. β-cells operate collectively to help maintain glucose homeostasis. Biophysical journal, ISSN 1542-0086, 17 str. https://www.cell.com/biophysj/fulltext/S0006-3495(20)30308-8#%20, doi: 10.1016/j.bpj.2020.04.005. [COBISS.SI-ID 512974648]

GRUBELNIK, Vladimir, MARKOVIČ, Rene, LIPOVŠEK DELAKORDA, Saška, LEITINGER, Gerd, GOSAK, Marko, DOLENŠEK, Jurij, VALLADOLID-ACEBES, Ismael, BERGGREN, Per-Olof, STOŽER, Andraž, PERC, Matjaž, MARHL, Marko. Modelling of dysregulated glucagon secretion in type 2 diabetes by considering mitochondrial alterations in pancreatic α-cells. Royal Society Open Science, ISSN 2054-5703, 2020, vol. 7, iss. 1, str. 1-17, doi: 10.1098/rsos.191171. [COBISS.SI-ID 25073672]

STOŽER, Andraž, MARKOVIČ, Rene, DOLENŠEK, Jurij, PERC, Matjaž, MARHL, Marko, RUPNIK, Marjan, GOSAK, Marko. Heterogeneity and delayed activation as hallmarks of self-organization and criticality in excitable tissue. Frontiers in physiology, ISSN 1664-042X, 2019, vol. 10, str. 1-19, ilustr., doi: 10.3389/fphys.2019.00869. [COBISS.SI-ID 512903480]

STOŽER, Andraž, HOJS, Radovan, DOLENŠEK, Jurij. Beta cell functional adaptation and dysfunction in insulin resistance and the role of chronic kidney disease. Nephron journals, ISSN 2235-3186, 2019, vol. 143, no. 1, str. 33-37, ilustr. https://www.karger.com/Article/FullText/495665, doi: 10.1159/000495665. [COBISS.SI-ID 512876344]

GÁL, Eleonóra, DOLENŠEK, Jurij, STOŽER, Andraž, POHOREC, Viljem, ÉBERT, Attila, VENGLOVECZ, Viktória. A novel in situ approach to studying pancreatic ducts in mice. Frontiers in physiology, ISSN 1664-042X, July 2019, vol. 10, str. 1-9, ilustr. https://www.frontiersin.org/articles/10.3389/fphys.2019.00938/full, doi: 10.3389/fphys.2019.00938. [COBISS.SI-ID 512907576]

DOLENŠEK, Jurij, SKELIN KLEMEN, Maša, GOSAK, Marko, KRIŽANČIĆ BOMBEK, Lidija, POHOREC, Viljem, SLAK RUPNIK, Marjan, STOŽER, Andraž. Glucose-dependent activation, activity, and deactivation of beta cell networks in acute mouse pancreas tissue slices. BioRxiv. doi: https://doi.org/10.1101/2020.03.11.986893

Povzetek

V celicah beta glukoza stimulira izločanje inzulina preko znotrajceličnega metabolizma, ki da ATP in zapre od ATP odvisne kalijeve kanale. To vodi v depolarizacijo in odprtje od napetosti odvisnih kalcijevih kanalov in vdor kalcijevih ionov v celico. Posledično povečanje znotrajcelične koncentracije kalcijevih ionov deluje kot sekundarni sporočevalec in sprožitelj eksocitoze. Terapevtsko pomembne sulfonilsečnine se neposredno vežejo na od ATP odvisne kalijeve kanale, depolarizirajo celico in sprožijo izločanje inzulina na način, ki je pretežno glukozno neodvisen. Po drugi strani pa inkretinski hormoni oz. terapevtski agensi, ki posnemajo njihovo delovanje, vplivajo na celice beta tako, da vežejo zunajcelične receptorje sklopljene z beljakovino G, ki stimulirajo delovanje adenilil ciklaze in povečajo znotrajcelično koncentracijo cAMP. Ta sekundarni sporočevalec vrši svoj vpliv preko dveh znotrajceličnih poti, bodisi PKA bodisi Epac2A, in preko teh vpliva na različne korake v sprožilni kaskadi, vendar, kot kaže, ni sposoben sprožiti eksocitoze v odsotnosti od glukoze ali s sulfonilsečninami povzročenega začetnega sprožilnega signal. V zadnjem času se pojavljajo dokazi, da sulfonilsečnine neposredno vplivajo na aktivnost Epac2A in da so inkretini sposobni sprožiti povečanje koncentracije kalcijevih ionov, kar kaže na številna križanja signalnih poti, ki jih aktivirajo glukoza, sulfonilsečnine in inkretini. cAMP ima morda poleg znotrajceličnega učinka na sklopitev med stimulusom in sekrecijo vpliv tudi na širjenje depolarizacije in kalcijevih valov, ki so sproženi z glukozo ali s sulfonilsečninami. Ta znotrajcelična funkcionalna sklopitev normalno služi uskladitvi odzivnosti na glukozo celic beta na nivoju vseh celic, ki so sklopljene v Langerhansovem otočku, prav tako pa je tudi tarčno mesto diabetogenih učinkov v razvoju inzulinske rezistence in sladkorne bolezni tipa 2. Kljub temu pa je slabo razumljen način vpliva cAMP na znotraj- in medcelično prevajanje signalov ter njegova vloga v vzdrževanju glukozne tolerance v normalnih pogojih in med razvojem sladkorne bolezni tipa 2. To pomanjkanje razumevanja lahko vsaj delno razloži sedanje pomanjkanje dolgoročnega uspeha terapevtskih učinkov sulfonilsečnin in sposobnosti razvrstitve bolnikov v različne skupine zdravljenja glede na trajanje bolezni oz. znanje o specifičnih poškodbah celic beta. Natančneje, obstaja praznina v našem znanju o različnih vlogah PKA in Epac2A v zgoraj omenjenem procesu in boljšem razumevanju o tem, kako lahko mehanizmi delovanja PKA in Epac2A vodijo v razvoj novejših terapevtskih agensov. Z raziskovanjem sklopitve med stimulusom in sekrecijo, ki jo povzročijo glukoza, sulfonilsečnine in inkretini od najbolj zgodnjih (depolarizacija membrane) do najbolj poznih (eksocitoza) korakov, medcelične sklopitve in glukozne tolerance na nivoju cele živali v kontrolni skupini miši in miših z izbitim genom Epac2A, ki so hranjene bodisi s kontrolno dieto bodisi z zahodno dieto, ki povzroči inzulinsko rezistenco, adaptacijo in odpoved celic beta, želimo v okviru tega projekta doseči naslednje:

Razjasniti vlogo Epac2A in PKA v uravnavanju učinkov cAMP na različnih nivojih sklopitve med stimulusom in sekrecijo.
Razjasniti vlogo Epac2A in PKA v uravnavanju učinkov cAMP na nivoju medcelične funkcionalne sklopitve.
Raziskati vlogo Epac2A in PKA v uravnavanju učinkov ne samo glukoze, temveč tudi različnih sulfonilsečnin, še posebej njihov vpliv na spremembe v membranskem potencialu in kalcijevih oscilacijah, občutljivost eksocitotskega aparata in funkcionalno medcelično sklopitev celic beta.
Pripomoči k mehanistični razlagi vloge Epac2A in PKA v adaptaciji in disfunkciji celic beta tekom razvoja z zahodno hrano povzročene sladkorne bolezni tipa 2 v mišjih modelih na večceličnem nivoju Langerhansovih otočkov, kakor tudi učinkov na glukozno toleranco celotne živali v normalnih in inzulinsko rezistentnih miših.

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