Maf and Mitf transcription factors regulate pancreatic endocrine cell differentiation and function

Abstract

Diabetes - the imbalance in glucose homeostasis is partially caused by loss or dysfunction of pancreatic insulin producing β cells or development of insulin resistance. In Type 1 diabetes β cells are destroyed in the process of an autoimmune attack, whereas in Type 2 diabetes, islets produce insufficient amounts of insulin or the insulin cannot be used adequately. So far, the most promising therapy to treat Type 1 diabetes is pancreatic islet transplantation. However, this method cannot be used in a larger scale because of the severe scarcity of donors. Therefore, there is a great and urgent need to develop new methods and therapies. One of the potential sources of obtaining functional insulin cells are human embryonic stem cells (ESC) or inducible pluripotent stem cells (iPS). Hence, differentiating stem cells into functional β-cells requires a detailed understanding of pancreas development with the focus on molecular programs underlying β-cells formation and function. Pancreas development and differentiation of insulin and glucagon secreting cells is a complex process controlled by a variety of transcription factors. Two of these factors, MafA and MafB, have been shown to play a major role in the regulation of several genes critical to pancreas development and endocrine cell formation. MafB is essential for both α- and β-cell differentiation, as mice lacking MafB have fewer insulin+ and glucagon+ cells during pancreas development. In contrast, in mouse embryos lacking MafA, the development of β-cells is normal, but adult animals develop diabetes. In adult animals these two transcription factors have a distinct expression pattern, with MafB being expressed in α-cells, whereas MafA is exclusively found in β-cells. We have performed gene expression profiling of wild type and MafA/B mutant pancreata to identify genes important for β-cell maturation and function. In these microarray studies several known (insulin, glucagon, Glut2, PC2) but also novel genes were shown to be differentially expressed in MafB and MafA/MafB compound mutant embryos. Gene ontology analysis revealed that the differentially expressed genes were mainly associated with mature β-cell function. Our findings show that Neuronatin (Nnat), islet-specific zinc transporter (Slc30a8), islet-specific glucose-6-phosphatase catalytic subunit-2 protein (G6pc2) and Microphthalmia associated transcription factor (Mitf) are downregulated in embryonic and adult mutant pancreata. In contrast, the mRNA level of Retinol Binding Protein-4 (Rbp4) was upregulated in mutant tissue. Given the differences in spatio-temporal expression pattern of MafA and MafB in developing and adult pancreas, we propose that these two factors regulate islet β-cell formation and maturation in a unique and sequential manner.

In MafA deficient models studied so far pancreas development is unaffected, most likely due to compensatory functions of MafB. Therefore, to be able to study the actual role of MafA in β-cell function we developed a β-cell-specific deletion of MafA. Our MafAΔβcell mutant animals lack expression of MafA and MafB in adult β-cells. In contrast to other MafA mutants, our animals have normal islet architecture, β-cells mass, β- to α-cell ratio, and MafB expression is restricted to α-cells. Thus we have created a system suitable for studying the true contribution of MafA to adult β-cell function.

Our gene expression experiments have shown that in MafA and MafB mutant embryonic pancreata levels of Mitf expression are reduced comparing to levels found in wild type animals. We also show that Mitf loss of function mutation alters functionality of islet β-cell. In response to an intraperitoneal glucose challenge, but also during non-fasted conditions, Mitf mutant mice have lower blood glucose levels than wild type animals. Mutant islets secrete more insulin upon exposure to high glucose concentrations and Mitf mutant animals have higher circulating insulin levels in fasted conditions. Additionally, Mitf directly regulates the expression of genes regulating blood glucose levels and β-cell formation, which is significantly higher in Mitf mutant than in wild type animals. Thereby, we demonstrate that Mitf is an important factor in modulation of β-cell function.

Abstract (Swedish)

Popular Abstract in Swedish

Patienter med diabetes kan inte kontrollera blodsockernivåerna på grund av att de saknar insulinproducerande celler eller har icke fungerande insulinproducerande celler. Det finns olika typer av diabetes där typ 1 diabetes och typ 2 diabetes är de vanligaste formerna. Den mest framgångsrika behandling mot typ 1 diabetes är transplantation av insulinproducerande celler, så kallade öar, från donatorer. Dock finns det inte tillräckligt med donatorer. Ett annat alternativ är att framställa insulinproducerande celler från bland annat mänskliga embryonala stamceller. För att lyckas med detta krävs det att vi vet hur bukspottkörteln normalt bildas i däggdjur. Bukspottkörteln är det organ där de insulinproducerande cellerna finns. Bukspottkörteln har bland annat till uppgift att producera hormoner (insulin och glukagon) som kontrollerar blodsockernivåerna. Denna process styrs av olika faktorer såsom MafA och MafB. MafB är viktigt för bildandet av både insulinpoducerande och glukagonrpoducerande celler. Möss som saknar MafB har färre insulin- och glukagonproducerande celler men de insulin celler som finns kvar har MafA. Möss som saknar MafA är normala under fosterstadiet men utvecklar så småningom diabetes. Jag har jämfört olika gener i vanliga möss och i möss som saknar Maf faktorer för att se vilka gener som är viktiga för insulincellers mognad och funktion. Vi såg att många kända faktorer var reglerade såsom insulin, glukagon, Glut2 och PC2 men också icke kända gener involverade i funktionen av insulinceller identifierades. I de möss som saknade Maf faktorer var följande gener nedreglerade; Neuronatin (Nnat), islet-specific zinc transporter (Slc30a8), islet-specific glucose-6-phosphatase catalytic subunit-2 protein ( G6pc2) och microphthalmia associated transcription factor (Mitf) medans retinol binding protein-4 (Rbp4) var uppreglerad. Jag har visat att det finns lägre nivåer av Mitf i bukspottkörteln från möss som saknar MafA och MafB. Jag har också visat att Mitf produceras i bukspottkörteln under fosterstadiet och i öarna i vanliga möss. Möss som saknar Mitf har icke fungerande insulinproducerande celler. Detta såg jag både när mössen injicerades med sockerlösning och när djuren fastades. När öarna ifrån dessa djur utsätts för socker utsöndrar de mer insulin och dessa möss har även högre nivåer av insulin i blodet när de är fastande. De faktorer som styr regleringen av blodsockernivåer i blodet och insulincellernas funktion finns i högre nivåer i möss som saknar Mitf än i vanliga möss. Jag har också visat att Mitf direkt reglerar dessa gener som styr både blodsocker nivåerna och insulincellernas funktion.

• author: Mazur, Magdalena
• supervisor: Isabella Artner; Henrik Semb
• opponent: Grapin-Botton, Anne, The Danish Stem Cell Center (DanStem) University of Copenhagen
• organization: Stem Cell Center; EXODIAB: Excellence of Diabetes Research in Sweden; StemTherapy: National Initiative on Stem Cells for Regenerative Therapy
• publishing date: 2013
• type: Thesis
• publication status: published
• subject: Cell and Molecular Biology
• keywords: pancreas development, beta cells, insulin, MafA, MafB, Mitf, diabetes
• in: Lund University Faculty of Medicine Doctoral Dissertation Series
• volume: 2013:81
• pages: 144 pages
• defense location: Segerfalsalen, BMC A10, Sölvegatan 19, Lund
• defense date: 2013-09-05 09:00:00
• ISSN: 1652-8220
• ISBN: 978-91-87449-53-6
• language: English
• LU publication?: yes
• id: 510ea156-a3ab-4d38-83fa-316f879c4e1e (old id 3972394)
• date added to LUP: 2016-04-01 15:01:40
• date last changed: 2019-05-22 04:36:12