Research

Diabetes mellitus is a disease of major significance to human world health, with an increasing prevalence (Steinbrook 2006) and a considerable macroeconomic burden in both developed and developing countries (Yach et al. 2006). A key element in the development of diabetes is the reduced insulin secretion from the pancreatic beta cells (Fajans et al. 2001). To improve current, insufficient therapeutic strategies, there is an urgent need to identify the underlying signaling pathways and the regulatory mechanisms in human pancreatic beta cells, which are much less understood than rodent beta cells.

MODY families

mody

Patients from families with monogenic diabetes (MODY, maturity-onset diabetes of the young) provide an excellent opportunity to study molecular events at protein level both before and after the onset of the clinical manifest disease. MODY is caused by pancreatic beta cell dysfunction due to a single germ line mutation in genes involved in transcriptional regulation in pancreatic beta cells. More than 90 % of MODY mutation carriers ultimately develop diabetes; non-diabetic family members are therefore assumed to be in a pre-diabetic state (Fajans et al. 2001). The advantage of studying MODY patient-derived samples lies in the monofactorial/monogenic origin of the disease, and the absence of the immune attack (unlike type 1 diabetes) and insulin resistance (unlike type 2 diabetes) allowing an unambiguous characterization of regulatory mechanisms. Secondly, the heredity transmission of the disease allows the use of healthy family members as controls, as such reducing the risk of genetic background interference. To date, 11 MODY genes have been described including the transcription factor MODY forms (hepatocyte nuclear factor, HNF): MODY1 (HNF4A gene), MODY3 (HNF1A), MODY4 (PDX1), MODY5 (HNF1B), MODY6 (NEUROD1), MODY7 (KLF11), and MODY9 (PAX4), and the non-transcription factor MODY forms MODY2 (GCK), MODY8 (CEL), MODY10 (INS) and MODY11 (BLK) (Haldorsen, Ræder et al. 2012). Recently, we discovered and characterized the MODY8 form with both diabetes and pancreatic exocrine dysfunction caused by a mutation in different locations of a gene encoding the enzyme Carboxyl-ester lipase (CEL-MODY, (Ræder et al. 2006; Ræder et al. 2007), possibly leading to protein misfolding (Johansson et al. 2011). It is assumed that the breakdown of the underlying transcription factor networks affects pancreatic organogenesis, differentiation, and beta cell growth and function for the transcription factor-related MODY forms (Maestro, Cardalda et al. 2007). However, how mutation of the underlying genes lead to the devastating effect on beta cell signaling and regulation is mostly unknown. Animal models, at least for CEL-MODY, have not been useful for the characterization of human pancreatic beta cell function in a patho-physiological context (Vesterhus, Ræder et al. 2010; Ræder, Vesterhus et al. 2013). Like CEL-MODY, several of the other MODY forms were also found to be associated with pancreatic exocrine dysfunction (Vesterhus, Ræder et al. 2008) and pancreatic hypoplasia (Haldorsen, Vesterhus et al. 2008; Vesterhus, Haldorsen et al. 2008) suggesting coupled mechanisms during the early embryonic development of pancreatic structures, which affect both endocrine and exocrine differentiation. The efficacy of sulfonylurea drugs to treat both some MODY forms (Pearson et al. 2003) and type 2 diabetes (Morsink et al. 2013) exemplifies that at least some underlying molecular mechanisms are shared between different diabetes forms, and a treatment strategy for MODY may be valid for diabetes patients in general.

Targeted proteomic quantification

We have developed selected reaction monitoring (SRM) assays based on the most differentially increased proteins detected among the CEL-MODY patients (Ræder et al., 2014) and compared their performance as biomarkers in secretin-stimulated duodenal juice from 8 nondiabetic, 8 diabetic CEL-MODY patients, and 21 unrelated controls (Bjorlykke et al., 2015).

Patients with carboxyl-ester lipase-maturity-onset diabetes of the young (CEL-MODY) display distinct disease stages toward the development of monogenic diabetes and exocrine pancreatic disease. The finding of differentially increased proteins, some related to MAPK signaling, in a discovery proteomics study of secretin-stimulated duodenal juice in three CEL-MODY patients, prompted us to monitor their abundance in an extensive number of CEL-MODY subjects at different disease stages and controls using targeted proteomics.

Patients with carboxyl-ester lipase-maturity-onset diabetes of the young (CEL-MODY) display distinct disease stages toward the development of monogenic diabetes and exocrine pancreatic disease. The finding of differentially increased proteins, some related to MAPK signaling, in a discovery proteomics study of secretin-stimulated duodenal juice in three CEL-MODY patients, prompted us to monitor their abundance in an extensive number of CEL-MODY subjects at different disease stages and controls using targeted proteomics (Bjorlykke et al., 2015).

Induced pluripotent stem cells (iPSCs) and their potential for diabetes therapy

The Nobel Prize-awarded technique to generate induced pluripotent stem cells (iPSCs) from differentiated cells, such as skin cells, bears a tremendous potential for modeling of disease (Buganim et al. 2013). A number of methodological challenges are related to the completed direction of iPSCs into beta cell differentiation (Mfopou et al. 2010; Hua et al. 2013). However, in vivo, transplanted murine pancreatic progenitor cells may be differentiated into functional insulin-secreting cells (Rezania et al. 2012) and recently, protocols for in vitro maturation of human iPSCs into mature functional pancreatic beta cells were also published (Pagliuca et al. 2014; Rezania et al. 2014). Some of the challenges with these targetted differentiation protocols include the reported high batch variability for the generated bona fide beta cells, and the subsequent potential for malignant transformation, in particular of the non- insulin producing beta-cells within the batches. Hence, there is still a need for further optimization of the existing protocols to improve the percentage of insulin-secreting bona fide beta cells.

Our Aims:

1. Demonstrating the feasibility of personalized iPSC-based cell models of diabetes for the study of new disease mechanisms.
2. The finding of patient-specific regulatory mechanisms (miRNA signatures and signatures of the phosphoproteome, the SUMOylated proteome and the ubiquitylated proteome) with time series analyses during targetted differentiation of iPSC towards pancreatic beta cells where the corresponding molecular signatures can be used diagnostically.

As a secondary objective: the identification of drugs that restore the insulin- secretory function by targeting the identified patient-specific regulatory mechanisms in order to tailor specific therapy.

Collaborators:

Frode Berven – Institute of Biomedicine, UiB

Laurence Bindoff – Dept. of Clinical Medicine, UiB

Kamal Mustafa – Dept. of Clinical Dentistry, UiB

Inge Jonassen – Computational Biology Unit, UiB

Tor Paaske Utheim – Dept. of Medical Biochemistry, Oslo University Hospital, UiO

Rohit Kulkarni – Joslin Diabetes Center of Harvard Medical School, USA

Steve Gygi – Harvard Medical School, USA

Bridget Wagner – Broad Institute of MIT, USA

Adrian Teo – Nanyang Technological University, Singapore

Paul Gadue – University of Pennsylvania, USA

Ludovic Vallier – Wellcome Trust Medical Research Council of Cambridge Stem Cell Institute, UK

Pedro Herrera – Dept. of Genetic Medicine and Development
, University of Geneva, Switzerland

Alfred Vertegaal – Leiden University Medical Center, Netherlands

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