Biomarker Discovery in Diabetes mellitus

(PIs: Prof Jochen Prehn and Dr Maria Byrne)

Objective 1: microRNAs as novel biomarkers

HNF1A-MODY which accounts for 60 % of all MODY is a monogenic form of diabetes caused by mutations in the transcription factor 1 (tcf-1)/hepatocyte nuclear factor1a (hnf1a) gene. MODY accounts for 1-2 % of all diabetes cases however the true prevalence of MODY is not known for most populations. Recently the minimum prevalence in theUK was estimated to be 108 cases per million population, which implies that the vast majority of MODY patients inIreland remain misdiagnosed or undiagnosed. Identification of HNF1A-MODY patients allows prompt and optimal treatment (sulphonylurea sensitivity), screening of family members and prevention of diabetic complications.

Previous reports on mRNA expression profiling in HNF1A-MODY models have identified numerous pathways to be dysregulated by loss-of-function mutations of HNF1A. The human genome however also expresses several non-coding RNAs, including microRNAs (miRNAs). miRNAs are short, 19-25 nucleotide RNA molecules, which suppress gene expression by binding to the 3′ untranslated region (3′UTR) of target mRNAs, leading to repression of protein production. More than 500 miRNAs in humans have been identified so far which have been estimated to regulate approximately 5000 genes or 30% of all human proteins. Because miRNAs can be secreted from cells into their environment, they represent important new targets for biomarker development. Because of their unique role in the regulation of protein translation, they also represent new therapeutic targets, and may provide novel information in relation to disease pathogenesis.

Having previously described transcriptome alterations in HNF1A-MODY, we have identified several miRNAs that are dysregulated in HNF1A-MODY. Of note, we found several miRNAs to be potently upregulated in the sera of HNF1A-MODY patients compared to the MODY-negative controls, with some miRNAs >300-fold induction. These preliminary proof-of-concept data highlight an emerging role of microRNAs as novel biomarker in HNF1A-MODY. The aim of this research is to characterise and clinically validate miRNAs as novel biomarkers for HNF1A-MODY and type 2 diabetes. We also aim to characterise whether miRNAs are involved in the regulation of beta-cell physiology, and hence may represent a new therapeutic target.

Objective 2: Biomarkers for beta cell destruction and preservation

Type 2 diabetes is affecting up to 20 % of adults in many developed countries. It is estimated that the incidence of diabetes will dramatically increase over the next decades. Type 2 diabetes was the seventh leading cause of death in theUSin 2006 with diabetic patients having a 2- to 4-fold increased risk of myocardial infarction or stroke. Despite over 170,000,000 people being affected worldwide the causes of diabetes remain largely unknown.

The pathways that control insulin secretion and regulate pancreatic b-cell mass are crucial in the development of diabetes mellitus. Despite current treatments, blood glucose levels tend to increase slowly over time, and death from myocardial infarction is not reduced by current therapy. It is now well established from post mortem studies that beta cell mass declines with time in patients with type 2 diabetes. However, there is now also a significant body of evidence demonstrating that beta cell mass in type 2 and type 1 diabetes is not static but in a constant process of renewal and destruction. These findings provide hope for new treatment paradigms that aim at increasing functional beta cell mass and ameliorating or reversing the process of beta cell destruction. New therapeutic approaches that may target the preservation of beta cell mass include glitazones, DPP-IV inhibitors, and GLP-1 analogues, all of which are clinically widely used. Islet cell transplantation and stem cell therapy represent alternative approaches for a preservation or recovery of beta cell mass. However there is very limited clinical evidence as to whether these treatment paradigms actually preserve or increase beta cell mass in patients. Moreover, there is very little clinical data about the time courses and rates of beta cell destruction in patients and their correlation to disease progression and HbA1C levels, or how individual diabetes patients respond to specific treatment regimens. This is largely due to a significant lack of clinically validated and easily accessible biomarkers for beta cell destruction or beta cell mass.

As b-cell mass cannot be evaluated in humans due to the unavailability of pancreatic biopsies, and unsuccessful and expensive imaging strategies to date, we currently use functional tests of insulin secretion as an indirect markers of beta-cell mass in humans. In previous research, we have demonstrated that PSP/reg is selectively produced in insulin-secreting beta cells during the process of beta cell destruction. We have also provided important preliminary clinical evidence that serum levels of PSP/reg may be a suitables clinical indicator of increased beta cell destructionr, suggesting that the detection of serum PSP/reg levels may be developed into a clinically reliable and easily accessible biomarker for beta cell destruction and its inhibition. The overall aim of this research project is therefore to determine whether serum PSP/reg is a clinical indicator of beta cell destruction and preservation of beta cell mass, and to develop a minimally invasive, inexpensive, serum diagnostic test that can be performed in routine clinical laboratories which will aid in evaluating beta cell destruction at diagnosis, during the course of disease, and in response to treatment.

The development of a serum biomarker for beta cell destruction and regeneration will be important clinically to correlate with pathogenesis, to monitor disease progression over time and to monitor the effects of diet, exercise and therapeutic intervention on beta cell mass. Such information would enable physicians to not only focus on glucose control but also on modulating the natural progression of the disease via targeting beta cell mass.

References and further reading:

Bacon S, Kyithar MP, Schmid J, Rizvi SR, Bonner C, Graf R, Prehn JH, Byrne MM. Serum levels of pancreatic stone protein (PSP)/reg1A as an indicator of beta-cell apoptosis suggest an increased apoptosis rate in hepatocyte nuclear factor 1 alpha (HNF1A-MODY) carriers from the third decade of life onward. BMC Endocr Disord. 2012 Jul 18;12(1):13.

Farrelly AM, Kilbride SM, Bonner C, Prehn JH, Byrne MM. Rapamycin protects against dominant negative-HNF1A-induced apoptosis in INS-1 cells. Apoptosis. 2011 Nov;16(11):1128-37.

Bonner C, Farrelly AM, Concannon CG, Dussmann H, Baquié M, Virard I, Wobser H, Kögel D, Wollheim CB, Rupnik M, Byrne MM, König HG, Prehn JH. Bone morphogenetic protein 3 controls insulin gene expression and is down-regulated in INS-1 cells inducibly expressing a hepatocyte nuclear factor 1A-maturity-onset diabetes of the young mutation. J Biol Chem. 2011 Jul 22;286(29):25719-28.

Kilbride SM, Farrelly AM, Bonner C, Ward MW, Nyhan KC, Concannon CG, Wollheim CB, Byrne MM, Prehn JH. AMP-activated protein kinase mediates apoptosis in response to bioenergetic stress through activation of the pro-apoptotic Bcl-2 homology domain-3-only protein BMF. J Biol Chem. 2010 Nov 12;285(46):36199-206.

Bonner C, Bacon S, Concannon CG, Rizvi SR, Baquié M, Farrelly AM, Kilbride SM, Dussmann H, Ward MW, Boulanger CM, Wollheim CB, Graf R, Byrne MM, Prehn JH. INS-1 cells undergoing caspase-dependent apoptosis enhance the regenerative capacity of neighboring cells. Diabetes. 2010 Nov;59(11):2799-808.

Wobser H, Düssmann H, Kögel D, Wang H, Reimertz C, Wollheim CB, Byrne MM, Prehn JH. Dominant-negative suppression of HNF-1 alpha results in mitochondrial dysfunction, INS-1 cell apoptosis, and increased sensitivity to ceramide-, but not to high glucose-induced cell death. J Biol Chem. 2002 Feb 22;277(8):6413-21.

Farrelly AM, Wobser H, Bonner C, Anguissola S, Rehm M, Concannon CG, Prehn JH, Byrne MM. Early loss of mammalian target of rapamycin complex 1 (mTORC1) signalling and reduction in cell size during dominant-negative suppression of hepatic nuclear factor 1-alpha (HNF1A) function in INS-1 insulinoma cells. Diabetologia. 2009 Jan;52(1):136-44.

Wobser H, Bonner C, Nolan JJ, Byrne MM, Prehn JH. Downregulation of protein kinase B/Akt-1 mediates INS-1 insulinoma cell apoptosis induced by dominant-negative suppression of hepatocyte nuclear factor-1alpha function. Diabetologia. 2006 Mar;49(3):519-26.