Effects of hepatocyte nuclear factor-1A and -4A on pancreatic stone protein/regenerating protein and C-reactive protein gene expression: implications for maturity-onset diabetes of the young

Abstract

Background: There is a significant clinical overlap between patients with hepatocyte nuclear factor (HNF)-1A and HNF4A maturity-onset diabetes of the young (MODY), two forms of monogenic diabetes. HNF1A and HNF4A are transcription factors that control common and partly overlapping sets of target genes. We have previously shown that elevated serum pancreatic stone protein / regenerating protein A (PSP/reg1A) levels can be detected in subjects with HNF1A-MODY. In this study, we investigated whether PSP/reg is differentially regulated by HNF1A and HNF4A.

Methods: Quantitative real-time PCR (qPCR) and Western blotting were used to validate gene and protein expression in cellular models of HNF1A- and HNF4A-MODY. Serum PSP/reg1A levels and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY subjects. The two groups were matched for age, body mass index, diabetes duration, blood pressure, lipid profile and aspirin and statin use.

Results: Inducible repression of HNF1A and HNF4A function in INS-1 cells suggested that PSP/reg induction required HNF4A, but not HNF1A. In contrast, crp gene expression was significantly reduced by repression of HNF1A, but not HNF4A function. PSP/reg levels were significantly lower in HNF4A subjects when compared to HNF1A subjects [9.25 (7.85-12.85) ng/ml vs. 12.5 (10.61-17.87) ng/ml, U-test P = 0.025]. hsCRP levels were significantly lower in HNF1A-MODY [0.22 (0.17-0.35) mg/L] compared to HNF4A-MODY group [0.81 (0.38-1.41) mg/L, U-test P = 0.002], Parallel measurements of serum PSP/reg1A and hsCRP levels were able to discriminate HNF1A- and HNF4A-MODY subjects.

Conclusion: Our study demonstrates that two distinct target genes, PSP/reg and crp, are differentially regulated by HNF1A and HNF4A, and provides clinical proof-of-concept that serum PSP/reg1A and hsCRP levels may distinguish HNF1A-MODY from HNF4A-MODY subjects.

Figure 1

Serum levels of PSP/reg1A in HNF4A-MODY (▲) versus HNF1A-MODY (○). Solid lines/box plot showing median and interquartile range and dotted line showing mean. Median of PSP/reg1A in HNF1A-MODY is 9.25 ng/ml (IQR = 7.85-12.85 ng/ml, n = 9) while median in HNF1A-MODY is 12.50 ng/ml (IQR = 10.61-17.87 ng/ml, n = 31). The distributions differ significantly (*): Mann–Whitney U-test p = 0.025.

Figure 2

Effect of suppression of Hnf1a and Hnf4a function on Glut2 and insulin expression in INS-1 cells. DN-HNF1A and DN-HNF4A in INS-1 cells were induced with doxycycline for 0 to 48 h. Glut2 mRNA expression (A) and insulin mRNA expression (B) were determined using real-time qPCR. Experiments were carried out in triplicate three times, and normalized to β-actin. Data are represented as mean ± SEM. *p < 0.05.

Figure 3

A Quantification of PSP/reg gene expression following inhibition of HNF1A and HNF4A function in INS-1 cells. INS-1 cells were treated with 500 ng /ml doxycycline from 0 to 48 h to induce DN-HNF1A and DN-HNF4A respectively. mRNA expression of PSP/reg was examined using real-time qPCR relative to β-actin. Expression levels were normalized to control cells and data represent means ± SEM from n = 3 cultures.* p < 0.05 difference from non-induced controls. Experiments were repeated 3 times with similar results (A, B) Whole cell lysates were analysed by Western blotting on 15% SDS-PAGE. Membranes were probed with a polyclonal antibody recognizing PSP/reg. Tubulin served as a loading control. (C, D) Quantification of PSP/reg protein levels by densitometry in cells treated with doxycycline as indicated. Western blots were analysed as described in the Materials and methods section and normalised to control. Data shown represent mean ± SEM from three independent experiments. *indicates p < 0.05 compared with untreated controls (E, F).

Figure 4

Suppression of HNF1A, but not HNF4A function leads to a loss of crp expression. DN-HNF1A and DN-HNF4A expression was induced in INS-1 cells by treatment with 500 ng/ml of doxycycline for 24 and 48 h as indicated. Cells were harvested and total RNA isolated. (A-B)crp mRNA expression was determined using quantitative real-time PCR following normalization to β-actin. Data are represented as means ± SEM from n = 3 cultures. The experiment was repeated 3 times with similar results.* p < 0.05 indicates the difference from non-induced controls. (C-D) Whole cell lysates were analysed by Western blotting on 15% SDS-PAGE. Membranes were probed with an anti-CRP polyclonal antibody. Antibodies raised against β-actin served as a loading control. (E-F) Quantification of CRP protein levels by densitometry in cells treated with doxycycline as indicated. Western blots were analysed as described in the Materials and methods section. Data shown represent mean ± SEM from three independent experiments. *indicates p < 0.05 compared with untreated controls.

Figure 5

Serum levels of hsCRP in HNF4A-MODY (▲) versus HNF1A-MODY (○). Solid lines/box plot showing median and interquartile range and dotted line showing mean. (A) Serum levels excluding two subjects with extreme hsCRP levels. Median of hsCRP in HNF4A-MODY is 0.81 mg/l (IQR = 0.38-1.41 mg/l, n = 9), while median in the. HNF1A-MODY (IQR = 0.17-0.35 mg/l, n = 31) is 0.22 mg/l. The distributions differ significantly (*): Mann–Whitney U-test p = 0.002. (B) Including all hsCRP levels into analysis, increases IQR in HNF1A-MODY (median = 0.22 mg/l, IQR = 0.17-0.38 mg/l, n = 33). There is a significant difference in medians between HNF1A- and HNF4A-MODY (*): Mann–Whitney U-test p = 0.008.