Modulation of Nuclear Receptor 4A1 Expression Improves Insulin Secretion in a Mouse Model of Chronic Pancreatitis.

OBJECTIVES: Diabetes secondary to chronic pancreatitis (CP) presents clinical challenges due to lack of understanding on factor(s) triggering insulin secretory defects. Therefore, we aimed to delineate the molecular mechanism of ß-cell dysfunction in CP. MATERIALS AND METHODS: Transcriptomic analysis was conducted to identify endocrine-specific receptor expression in mice and human CP on microarray. The identified receptor (NR4A1) was overexpressed in MIN6 cells using PEI linear transfection. RNA-Seq analysis of NR4A1-overexpressed (OE) MIN6 cells on NovaSeq6000 identified aberrant metabolic pathways. Upstream trigger for NR4A1OE was studied by InBio Discover and cytokine exposure, whereas downstream effect was examined by Fura2 AM-based fluorimetric and imaging studies. Mice with CP were treated with IFN-γ-neutralizing monoclonal antibodies to assess NR4A1 expression and insulin secretion. RESULTS: Increased expression of NR4A1 associated with decreased insulin secretion in islets (humans: controls 9 ± 0.2, CP 3.7 ± 0.2, mice: controls 8.5 ± 0.2, CP 2.1 ± 0.1 µg/L). NR4A1OE in MIN6 cells (13.2 ± 0.1) showed reduction in insulin secretion (13 ± 5 to 0.2 ± 0.1 µg/mg protein per minute, P = 0.001) and downregulation of calcium and cAMP signaling pathways. IFN-γ was identified as upstream signal for NR4A1OE in MIN6. Mice treated with IFN-γ-neutralizing antibodies showed decreased NR4A1 expression 3.4 ± 0.11-fold ( P = 0.03), showed improved insulin secretion (4.4 ± 0.2-fold, P = 0.01), and associated with increased Ca 2+ levels (2.39 ± 0.06-fold, P = 0.009). CONCLUSIONS: Modulating NR4A1 expression can be a promising therapeutic strategy to improve insulin secretion in CP.

Interferon-γ Decreases Nuclear Localization of Pdx-1 and Triggers ß-Cell Dysfunction in Chronic Pancreatitis.

Interferon-gamma (IFN-γ) is shown to play a major role in ß-cell dysfunction in chronic pancreatitis (CP). However, the underlying mechanisms are to be elucidated. The present study was conducted to determine the role of IFN-γ subverting insulin gene expression in CP. Pancreatic tissues from control (n=15) and CP patients (n=30) were analyzed for nuclear localization of pancreatic and duodenal homeobox transcription factor (Pdx-1) after ascertaining their diabetic status. By immunofluorescence and western blot analysis, the influence of IFN-γ, anti-inflammatory cytokine (interleukin-10), and anti-IFN-γ agent epigallocatechin-3-gallate (EGCG) on nuclear localization of Pdx-1was examined in the islets isolated from resected normal pancreatic tissue. Nuclear localization of Pdx-1 was 20.25±2.19 in the islets of diabetic CP patients and 31.44±2.09 in nondiabetic CP patients as compared with controls (60.45±5.11) and the corresponding distribution of Pdx-1 protein in the nuclear compartment was also decreased. Exposure of normal islets to IFN-γ revealed decreased nuclear localization of Pdx-1. Pretreatment with polyphenolic compound EGCG restored the nuclear localization of Pdx-1. These results suggest that IFN-γ induced ß-cell dysfunction is mediated through decreased nuclear localization of Pdx-1.

Reduced expression of PDX-1 is associated with decreased beta cell function in chronic pancreatitis.

OBJECTIVES: The present study was conducted to monitor the expression of pancreas and duodenal homeobox gene (PDX-1) for assessing beta-cell function in islets from patients with chronic pancreatitis (CP). METHODS: Islets isolated from the pancreata of 40 surgical patients categorized as control group, patients with mild CP, and patients with advanced CP were assessed for their yield, size, and glucose-stimulated insulin secretion. Expressions of genes coding for PDX-1, insulin, and glucagon were simultaneously monitored by reverse transcription polymerase chain reaction and confirmed by immunohistochemistry. RESULTS: In comparison with the control group (2673 +/- 592 islet equivalents [IEq]/g), islet yield did not differ much in the patients with mild CP (2344 +/- 738 IEq/g) but was significantly reduced (P < 0.0001) in the patients with advanced CP (731 +/- 167 IEq/g). Although the marginal decrease in islet size observed in the patients with mild CP was not significantly different from that observed in the control group, there was a 58% decrease observed in the patients with advanced CP that was also accompanied by a significant reduction in beta-cell mass (P < 0.05). The expression of insulin and PDX-1 genes, but not of glucagon, was significantly reduced in the patients with advanced CP as confirmed by immunohistochemistry. Islets obtained from the patients with advanced CP retained 53% glucose-stimulated insulin secretion function in comparison with those of the control group. CONCLUSION: The results indicate that beta-cell dysfunction during progression of CP correlates with the decrease in PDX-1 gene expression.