Diabetes Metab J.  2011 Feb;35(1):65-71. 10.4093/dmj.2011.35.1.65.

Decreased Expression and Induced Nucleocytoplasmic Translocation of Pancreatic and Duodenal Homeobox 1 in INS-1 Cells Exposed to High Glucose and Palmitate

• Ryu GR
• Yoo JM
• Lee E
• Ko SH
• Ahn YB
• Song KH

• KMID: 2281494
• DOI: http://doi.org/10.4093/dmj.2011.35.1.65

Abstract

BACKGROUND
Type 2 diabetes mellitus (T2DM) is often accompanied by increased levels of circulating fatty acid. Elevations in fatty acids and glucose for prolonged periods of time have been suggested to cause progressive dysfunction or apoptosis of pancreatic beta cells in T2DM. However, the precise mechanism of this adverse effect is not well understood.
METHODS
INS-1 rat-derived insulin-secreting cells were exposed to 30 mM glucose and 0.25 mM palmitate for 48 hours.
RESULTS
The production of reactive oxygen species increased significantly. Pancreatic and duodenal homeobox 1 (Pdx1) expression was down-regulated, as assessed by reverse transcription-polymerase chain reaction and Western blot analyses. The promoter activities of insulin and Pdx1 were also diminished. Of note, there was nucleocytoplasmic translocation of Pdx1, which was partially prevented by treatment with an antioxidant, N-acetyl-L-cysteine.
CONCLUSION
Our data suggest that prolonged exposure of beta cells to elevated levels of glucose and palmitate negatively affects Pdx1 expression via oxidative stress.

Keyword

Fatty acid; High glucose; Insulin; Oxidative stress; Palmitate; Pancreatic beta cell; Pdx1

MeSH Terms

Apoptosis
Blotting, Western
Diabetes Mellitus, Type 2
Fatty Acids
Genes, Homeobox
Glucose
Insulin
Insulin-Secreting Cells
Oxidative Stress
Reactive Oxygen Species
Fatty Acids
Glucose
Insulin
Reactive Oxygen Species

Figure

• 

  Fig. 1 The expression levels of insulin and pancreatic and duodenal homeobox 1 (Pdx1) after treatment with 30 mM glucose and 0.25 mM palmitate (A) and H2O2 (B). The expressions of insulin, Pdx1 and cyclophillin (as an internal control, Cyc) mRNAs were measured using reverse transcription-polymerase chain reaction (RT-PCR). The PCR products were resolved on a 1% agarose gel and were stained with ethidium bromide. The result shown is representative of three independent experiments. LG, 5.6 mM glucose; HG, 30 mM glucose; Pal, 0.25 mM palmitate. aP < 0.05 vs. LG. bP < 0.05 vs. 0.

• 

  Fig. 2 Reactive oxygen species (ROS) generation in INS-1 cells treated with 30 mM glucose and 0.25 mM palmitate. ROS were measured as described in Materials and Methods. LG, 5.6 mM glucose; HG, 30 mM glucose; Pal, 0.25 mM palmitate; NAC, N-acetyl-L-cysteine. aP < 0.05 vs. LG, bP < 0.05 vs. HG+Pal.

• 

  Fig. 3 Promoter activity assay for insulin and pancreatic and duodenal homeobox 1 (Pdx1) in INS-1 cells treated with 30 mM glucose and 0.25 mM palmitate. The promoter activities of insulin and Pdx1 were measured as described in Materials and Methods. RFU, rate fluorescence unit; LG, 5.6 mM glucose; HG, 30 mM glucose; Pal, 0.25 mM palmitate; NAC, N-acetyl-L-cysteine. aP < 0.05 vs. LG, bP < 0.05 vs. HG+Pal.

• 

  Fig. 4 Pancreatic and duodenal homeobox 1 (Pdx1) protein expression and nucleo-cytoplasmic translocation of Pdx1 in INS-1 cells after treatment with 30 mM glucose and 0.25 mM palmitate. (A, B) Total proteins and nuclear proteins of Pdx1 were analyzed using Western blotting. (C) The staining of Pdx1 was performed as described in Materials and Methods. The cells were stained with anti-GFP antibody (1:100, green) to detect Pdx1 and with DAPI (blue) to detect nuclei. LG, 5.6 mM glucose; HG, 30 mM glucose; Pal, 0.25 mM palmitate; NAC, N-acetyl-L-cysteine.

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