Effects of a novel curcumin derivative on insulin synthesis and secretion in streptozotocin-treated rat pancreatic islets in vitro

Mohammed Talaat Abdel Aziz, Mohammed Farid El-Asmar, Ameen Mahmoud Rezq, Mohammed Abdel Aziz Wassef, Hanan Fouad, Nagwa Kamal Roshdy, Hanan Hosni Ahmed, Laila Ahmed Rashed, Dina Sabry, Fatma Mohammed Taha, Amira Hassouna, Mohammed Talaat Abdel Aziz, Mohammed Farid El-Asmar, Ameen Mahmoud Rezq, Mohammed Abdel Aziz Wassef, Hanan Fouad, Nagwa Kamal Roshdy, Hanan Hosni Ahmed, Laila Ahmed Rashed, Dina Sabry, Fatma Mohammed Taha, Amira Hassouna

Abstract

Background: Hyperglycemia induces activation of the c-Jun N-terminal kinase (JNK) pathway, which suppresses insulin gene expression and reduces DNA binding of pancreatic and duodenal homeobox factor (PDX)-1. This study aims to investigate the effects of a novel curcumin derivative (NCD) on JNK signaling pathway on insulin synthesis and secretion in streptozotocin (STZ)-treated rat pancreatic islets in vitro.

Methods: Isolated rat pancreatic islets were divided into five groups: untreated control group; group treated with NCD (10 μM); group exposed to STZ (5 mM); group treated with NCD (10 μM) and then exposed to STZ (5 mM); and group exposed to STZ (5 mM) and then treated with NCD (10 μM). The pancreatic islets from all groups were used for DNA fragmentation assays and quantitative assessments of the JNK, Pdx1, glucose transporter-2 (GLUT2), heme oxygenase (HO)-1, transcription factor 7-like 2 (TCF7L2), and glucagon-like peptide (GLP)-1 gene expression levels. The intracellular calcium, zinc, and the phosphorylated and total JNK protein levels were assessed. The insulin (secreted/total) and C-peptide levels were examined in islet culture medium.

Results: NCD protected pancreatic islets against STZ-induced DNA damage, improved total insulin (P = 0.001), secreted insulin (P = 0.001), and C-peptide levels (P = 0.001), normalized mRNA expressions of insulin, Pdx1, and GLUT2 (P = 0.0001), and significantly elevated calcium and zinc levels (P = 0.0001). All effects were significant when islets were treated with NCD before STZ (P = 0.05). JNK gene overexpression and JNK protein levels induced by STZ were significantly inhibited after NCD treatment of islets ( P = 0.0001). NCD-treated islets showed significantly elevated gene expressions of HO-1, TCF7L2, and GLP-1 (P = 0.0001), and these upregulated gene expressions were more significantly elevated with NCD treatment before STZ than after STZ (P = 0.05).

Conclusions: NCD improved insulin synthesis and secretion in vitro in isolated pancreatic islets treated with STZ through inhibition of the JNK pathway, up-regulation of the gene expressions of HO-1, TCF7L2, and GLP-1 and enhancing effects on calcium and zinc levels.

Figures

Figure 1
Figure 1
Agarose gel electrophoresis of DNA isolated from cultured pancreatic β-cells. Lane M: DNA markers (100 bp); lanes 1–3: STZ-treated group; lanes 4–5: STZ then NCD-treated group; lanes 6–7: NCD then STZ-treated group; lanes 8–9: control NCD-treated group; lane 10: control group.
Figure 2
Figure 2
Secreted insulin levels (pg/mL) after 1 h of NCD treatment. Insulin secretion was measured at 5.5 and at 16.5 mM glucose. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets; ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.
Figure 3
Figure 3
Total insulin contents (pg/mg protein) after 1 h of NCD treatment. Insulin secretion was measured at 16.5 mM glucose. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus NCD-treated group.
Figure 4
Figure 4
Ratios between secreted insulin and total insulin after 1 h of NCD treatment. Insulin secretion was measured at 16.5 mM glucose. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus NCD-treated group.
Figure 5
Figure 5
C-peptide levels (ng/mL) after 1 h of NCD treatment. C-peptide levels were measured at 5.5 and at 16.5 mM glucose. The results are presented as means ± SD. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets; ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.
Figure 6
Figure 6
Real-time PCR of insulin gene expression levels in Ct values relative to a housekeeping gene. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets; ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.
Figure 7
Figure 7
Real-time PCR of PDX-1, GLUT2, and JNK gene expressions in Ct values relative to a housekeeping gene. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets, ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.
Figure 8
Figure 8
Real-time PCR of TCF7L2, GLP-1, and HO-1 gene expressions in Ct values relative to a housekeeping gene. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets; ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.
Figure 9
Figure 9
Phospho-JNK and total JNK levels expressed as relative fluorescence units (RFUs). *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets; ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.
Figure 10
Figure 10
Ratios of phospho-JNK/total JNK expressed as normalized RFUs determined by dividing the phospho-JNK fluorescence at 600 nm by the total JNK fluorescence at 450 nm in each well. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets; ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.
Figure 11
Figure 11
Zinc levels (μg/dL) in islet groups. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets; ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.
Figure 12
Figure 12
Calcium levels (mg/dL) levels in islet groups. *Significant difference between all islet groups versus control islets; #significant difference between all islet groups versus STZ-treated islets; ##significant difference between islets treated with NCD before STZ versus islets treated with NCD after STZ.

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