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

Maturity-onset diabetes of the young (MODY), is a monogenic form of diabetes characterized by autosomal dominant mode of inheritance including a three-generation family history of diabetes, age at diagnosis of diabetes of 25 years or less in at least one family member and reduced glucose-stimulated insulin secretion [1]. MODY can result from mutations in at least six different genes [2]. The two most common forms are HNF1A-MODY and glucokinase (GCK)-MODY in all populations studied accounting for approximately 70% of all cases [3].

Insulin secretory defects have been observed in carriers of HNF1A-MODY and GCK-MODY with and without diabetes [4, 5]. HNF1A encodes a transcription factor important for pancreatic development, beta cell differentiation and function. HNF1A-MODY subjects with pre-diabetes have defective glucose-induced beta cell insulin secretion indicative of reduced beta cell mass [4]. Homozygous hnf-1a/tcf-1 knockout mice likewise have a reduced beta cell mass [6]. HNF1A-MODY is associated with a severe and progressive clinical course with up to 50% of patients requiring insulin. The decline in functional beta cell mass may be attributed to increased beta cell apoptosis [7‐9]. Mutations in HNF1A are usually detected later in life when they are incidentally discovered through a screening programme or if subjects become symptomatic. Diabetes usually becomes manifest when additional superimposed environmental factors supervene such as a physiological decrease in insulin sensitivity with puberty and pregnancy. The decline in beta cell mass therefore, is progressive but gradual. The possible contribution of apoptosis to this decrease in beta cell mass however, has not been investigated due to a lack of accessible, specific biomarkers for beta cell apoptosis.

In contrast to HNF1A-MODY carriers, patients with GCK-MODY do not show a comparably progressive decrease in beta cell mass. Glucokinase is a key regulatory enzyme in the pancreatic beta cell catalyzing the conversion of glucose to glucose-6-phosphate, the first step in glycogen storage and glycolysis. GCK-MODY is often subclinical but can be detected at any stage of life [10, 11]. GCK-MODY carriers have mild fasting and post-prandial hyperglycaemia from birth, lack of progression, and absence of insulin requirement and vascular complications [10‐12]. Patients with GCK mutations, in contrast to HNF1A-MODY rarely require any pharmacological intervention and the majority are managed with diet alone.

One of the challenges in evaluating the contribution of beta cell apoptosis in human disease development is the lack of appropriate tools to evaluate the extent of beta cell apoptosis in patients other than post mortem studies [13]. Another complicating factor is that the actual process of apoptosis execution is very rapid and occurs within minutes [14], and apoptosing cells can rapidly detach from the extracellular matrix, and are subsequently phagocytised [15], thereby limiting the chance of detecting apoptosis by in-vivo imaging techniques. An alternative strategy therefore, is to detect signals that are generated directly or indirectly from apoptosing beta cells. Previous studies from our laboratory have demonstrated that beta cells undergoing apoptosis induce the expression of the PSP/reg gene in neighbouring beta cells. This paracrine gene induction occurs in a caspase-dependent manner, and is mediated by the shedding of microparticles from apoptosing cells. PSP/reg1A protein is subsequently secreted from neighbouring cells, and provides a regenerative cue to the microenvironment. We also demonstrated elevated serum levels of PSP/reg1A, the human homologue of PSP/reg, in human subjects with HNF1A-MODY and in subjects with type 1 diabetes mellitus, suggesting that the endocrine pancreas is the primary source of this signal [16].

In the present study, we sought to clinically validate this finding with a larger group and to investigate whether elevated PSP/reg1A levels in HNF1A-MODY correlated with a clinical phenotype and progression of disease. We also investigated whether PSP/reg1A was upregulated in GCK-MODY.

The Irish HNF1A-MODY cohort investigated in this study has been recently characterized [22]. This study reported on a mutation identification rate of 30.5% among Irish adults clinically selected for HNF1A-MODY from attendees at the diabetes clinic. In the present study, we report that PSP/reg1A levels are significantly elevated in human subjects with HNF1A-MODY when compared to controls. Higher levels of circulating PSP/reg1A in HNF1A-MODY were associated with increased age suggesting that the rate of beta cell apoptosis increases during disease progression and within the third life decade. PSP/reg1A did not correlate with HbA_1c nor did it correlate with the common clinical infectious/inflammatory markers. Patients with type 1 diabetes mellitus also showed elevated PSP/reg1A levels independent of age or disease onset. Together these data suggest that PSP/reg1A may be a clinical indicator of beta cell apoptosis.

We have previously provided biological evidence in insulinoma cell lines and transgenic mice models of HNF1A-MODY, that beta cells undergoing apoptosis induce the expression of the PSP/reg gene in neighbouring beta cells [16]. The induction of PSP/reg during apoptosis was inhibited in cells treated with caspase inhibitors. Paraffin embedded pancreatic sections from 5 month old diabetic mice expressing HNF1A-MODY in beta cells also demonstrated elevated expression of PSP/reg throughout the islets compared with wild-type mice, with PSP/reg positive cells positioned in the vicinity of cells displaying apoptotic nuclear morphology. These earlier findings demonstrated that PSP/reg1A gene expression was induced during apoptosis in in vitro and animal models of diabetes. One of the core findings of the present study is that PSP/reg1A serum levels may be used as a non-invasive marker to evaluate the extent of beta cell apoptosis during disease progression in human subjects.

The development and implementation of non-invasive techniques for the quantitative measurement of beta cell apoptosis would help in the early diagnosis of beta cell dysfunction in pre-clinical phases of diabetes. Furthermore, it would enable evaluation of emerging therapeutic approaches which focus on preservation of beta cell mass through stimulation of anti-apoptotic signalling. Currently, only indirect methods that evaluate total beta cell mass through secretory responses of islet cells are available, such as arginine-induced insulin secretion [23, 24]. These rely on measuring insulin secretion following different metabolic stimuli. Direct visualization of either native or transplanted pancreatic islets was unsuccessful due to their small size, the little difference in physical characteristics from the surrounding tissue, and their relatively low number dispersed over a large area of the pancreas or the liver as the most common site of transplantation [25]. However, modern diagnostic equipment may provide very high sensitivity by positron emission tomography (PET) and single photon emission computed tomography (SPECT), spatial resolution by magnetic resonance imaging (MRI), or both (by PET/CT) [26]. Despite these advances in imaging devices, the main problem is the lack of a specific structural or molecular marker to enable differentiation between scattered islets or single beta cells, and surrounding tissue. Moreover, these techniques require dedicated imaging centres and are costly.

A previously reported paper hypothesized as to whether serum reg protein levels could be representative of the regenerative process at the beta cell level during the early phases of type 1 diabetes mellitus in humans [27]. We have found that PSP/reg1A levels are higher in type 1 diabetes mellitus but independent of disease onset or age. This finding should be considered in the context of recent investigations suggesting that beta cell regeneration may occur even in patients with long standing type 1 diabetes mellitus [28‐31]. Our study indicates that this regeneration may be accompanied by continuing beta cell apoptosis, even in patients with long standing type 1 diabetes mellitus. Therefore, therapeutic interventions that boost beta cell survival could be a valuable therapeutic approach even in long standing type 1 diabetes mellitus.

In contrast to type 1 diabetes mellitus patients who have a rapid disease onset, HNF1A-MODY carriers represent an interesting and important study group for the study of beta cell apoptosis during disease progression as carriers will ultimately develop diabetes [1]. As demonstrated in HNF1A-MODY subjects’ beta cell apoptosis appeared to correlate with age, but not with HbA_1c suggesting that aging is an important risk factor, and that young islets have an increased resistance towards mutant HNF1A-induced apoptosis. Although patients with a GCK-MODY mutation did not have significantly elevated levels of serum PSP/reg1A when compared to controls, we cannot draw any conclusions in relation to this group due to the limited numbers available. There was also no statistically significant difference between serum PSP/reg1A levels of GCK-MODY and HNF1A-MODY or type 1 diabetes mellitus patients.

The pancreatic acinar cells are considered to be the most important source of PSP/reg under normal conditions however in pathological conditions other cells/tissues may contribute to elevated levels of the protein. PSP has been shown to be elevated in liver/pancreatic disease and in chronic renal failure [32]. When originally discovered, PSP/reg was proposed to be a marker of pancreatic injury in pancreatitis [33, 34] however; subsequent studies have failed to demonstrate elevated levels even after severe pancreatitis [35]. In this study the subjects had normal liver and renal function. Furthermore, PSP/reg1A levels did not correlate with infection/inflammation markers such as white cell count.

As a result of the growing interest in beta cell preservation as a potential cure for diabetes, a number of different treatments aimed at protecting the beta cell and maintaining beta cell mass have been evaluated. Insulinotropic agents such as repaglinide or GLP-1 have been shown to be cytoprotective in-vitro and in animal models of diabetes [36, 37]. From a clinical aspect, beta cell protection may also be induced by reducing the peripheral insulin demand by either improving sensitivity e.g. through physical exercise, pharmacologically using metformin/glitazones, or by lowering blood glucose through the administration of exogenous insulin [38‐40]. Our study suggests that PSP/reg1A serum levels may be useful as a biomarker for such potentially cytoprotective treatment paradigms.

PSP/reg1A may be developed as a serum marker to detect alterations in the rate of beta cell apoptosis during disease or in response to treatment. Furthermore, our data suggests that beta cell apoptosis contributes to the pathophysiology of HNF1A-MODY in patients 25 years and over.