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 insulin secretory defects and associated metabolic alterations. Owing to lack of molecular understanding, no pharmacotherapies to treat insulin secretory defects have been approved to date. We aimed to delineate the molecular mechanism of ß-cell dysfunction in CP. 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 on NovaSeq 6000 of NR4A1 overexpressed (OE) MIN6 cells was performed to identify aberrant metabolic pathways. Upstream trigger for NR4A1OE was studied by InBio Discover and cytokine exposure. Downstream effect of NR4A1OE was examined by Fura2 AM based fluorometric and imaging studies of intracellular calcium. 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/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), improved insulin secretion (4.4 ± 0.2-fold, p = 0.01), associated with increased Ca2+ 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.