Haptoglobin as an early serum biomarker of virus-induced autoimmune type 1 diabetes in biobreeding diabetes resistant and LEW1.WR1 rats

Abstract

Proteomic profiling of serum is a powerful technique to identify differentially expressed proteins that can serve as biomarkers predictive of disease onset. In this study, we utilized two-dimensional (2D) gel analysis followed by matrix-assisted-laser desorption/ionization time-of-flight mass spectrometry analysis to identify putative serum biomarkers for autoimmune type 1 diabetes (T1D) in biobreeding diabetes resistant (BBDR) rats induced to express the disease. Treatment with toll-like receptor 3 ligand, polyinosinic:polycytidilic acid (pIC), plus infection with Kilham rat virus (KRV), a rat parvovirus, results in nearly 100% of young BBDR rats becoming diabetic within 11-21 d. Sera collected from prediabetic rats at early time points following treatment with pIC + KRV were analyzed by 2D gel electrophoresis and compared with sera from control rats treated with phosphate-buffered saline, pIC alone or pIC + H1, a non-diabetogenic parvovirus. None of the latter three control treatments precipitates T1D. 2D gel analysis revealed that haptoglobin, an acute phase and hemoglobin scavenger protein, was differentially expressed in the sera of rats treated with pIC + KRV relative to control groups. These results were confirmed by Western blot and enzyme-linked immunosorbent assay studies, which further validated haptoglobin levels as being differentially increased in the sera of pIC + KRV-treated rats relative to controls during the first week following infection. Early elevations in serum haptoglobin were also observed in LEW1.WR1 rats that became diabetic following infection with rat cytomegalovirus. The identification and validation of haptoglobin as a putative serum biomarker for autoimmune T1D in rats now affords us the opportunity to test the validity of this protein as a biomarker for human T1D, particularly in those situations where viral infection is believed to precede the onset of disease.

Figure 1

Western blot analysis of undepleted sera from rats treated with PBS alone (1), pIC alone (2), pIC + H-1 (3) or pIC + KRV (4). Protein concentration was assessed by the Bradford assay and equal protein was loaded in each well. Rats were tail bled (a) or cardiac bled (b) at indicated time points. Purified rat haptoglobin was used as a positive control; molecular weight of precursor prohaptoglobin is 47 kD and cleaved, activated haptoglobin is 37 kD (β chain) and 10 kD (α chain). In (a), serum was collected from a single rat in each group for the time points indicated; shown are data from 3 rats per group. In (b), each lane represents serum from a single rat.

Figure 2

Serum haptoglobin levels determined by ELISA in BBDR rats treated with PBS alone, pIC alone, pIC + H-1 and pIC + KRV. Serum samples were obtained from rats that were (a) tail bled or (b) cardiac bled at indicated time points, and were run in duplicate against a standard curve of purified rat haptoglobin. *P < 0.05 between pIC + KRV and pIC + H-1 groups.

Figure 3

Native gel of sera from BBDR rats treated with PBS alone, pIC alone or pIC+KRV on indicated days. Protein concentration was quantified using the Bradford assay and equivalent concentrations of proteins were loaded into each well. Protein bands (indicated by arrow) that differed between groups were excised from individual lanes using a spot cutter; a molecular weight marker is shown in lane 1. Gel plugs from each lane were tryptically digested and peptides identified by MALDI-TOF/MS/MS.

Figure 4

Diabetes incidence and serum haptoglobin levels in BBDR rats treated with KRV alone. (a) Kaplan Meier survival curve depicts the percent of rats exhibiting diabetes free survival; n = 24 KRV treated rats, 10 developed diabetes. (b) Rats were tail bled on the indicated days and serum haptoglobin levels determined by ELISA. For any given time point, there were no significant differences in serum haptoglobin levels between KRV treated rats that became diabetic and those that remained diabetes-free.

Figure 5

Diabetes incidence and serum haptoglobin levels in LEW1.WR1 rats treated with RCMV alone. (a) Kaplan Meier survival curve depicts the percent of rats exhibiting diabetes free survival; n = 11 RCMV treated rats, 3 developed diabetes. (b) Rats were tail bled on the indicated days and serum haptoglobin levels were determined by ELISA; n = 3 for each group (3 of 8 serum samples from RCMV treated non-diabetic rats were analyzed). Serum haptoglobin levels were significantly higher (*P < 0.05) in diabetic RCMV treated rats compared to untreated rats.