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Kwon, Kim, Park, Ko, Cha, Son, and Yoon: AICAR Reversed the Glucolipotoxicity Induced β-cell Dysfunction through Suppression of PPAR-γ-coactivator-1 (PGC-1) Overexpression
Original Article
The Journal of Korean Diabetes Association

The Journal of Korean Diabetes Association 2007; 31(4): 310-318.

Published online: 31 July 2007

DOI: https://doi.org/10.4093/jkda.2007.31.4.310

AICAR Reversed the Glucolipotoxicity Induced β-cell Dysfunction through Suppression of PPAR-γ-coactivator-1 (PGC-1) Overexpression

Hyuk-Sang Kwon1, Ji-Won Kim2, Heon-Seok Park2, Seung-Hyun Ko1, Bong-Yun Cha1, Ho-Young Son1, Kun-Ho Yoon1, 2

1Department of Internal Medicine, The Catholic University of Korea, Korea.

2Catholic Research Institutes of Medical Science, The Catholic University of Korea, Korea.

Received 2 October 2006       Accepted 3 July 2007

Copyright © 2007 Korean Diabetes Association

Abstract

Background

Glucolipotoxicity plays an important role in the progression of type 2 diabetes mellitus via inducing insulin secretory dysfunction. Expression of insulin gene in pancreatic beta cell might be regulated by AMP-activated protein kinase (AMPK), which is recognized as a key molecule of energy metabolism. We studied the effects of AMPK on glucolipotoxicity-induced β-cell dysfunction by suppression of PPAR-γ-coactivator-1 (PGC-1) in vitro and in vivo.

Method

Glucolipotoxicity was induced by 33.3 mM glucose and 0.6 mM (palmitate and oleate) for 3 days in isolated rat islets. Messenger RNA (mRNA) expressions of β-cell specific gene like insulin, BETA2/NeuroD and PGC-1 induced by glucolipotoxic condition and their changes with 5-aminoimidazole-4-carboxy-amide-1-D-ribofuranoside (AICAR) treatment were investigated using RT-PCR. We also examined glucose stimulated insulin secretion in same conditions. Furthermore, SD rats were submitted to a 90% partial pancreatectomy (Px) and randomized into two groups; Ad-GFP-infected Px rats (n = 3) and Ad-siPGC-1-infected Px rats (n = 3). Then, the Px rats were infected with Ad-GFP or Ad-siPGC-1 (1 × 109 pfu) via celiac artery. After 12 days of viral infection, we measured body weight and performed the intraperitoneal glucose tolerance test (IP-GTT).

Results

Glucolipotoxicity resulted in blunting of glucose-stimulated insulin secretion, which was recovered by the AICAR treatment in vitro. Suppression in their expressions of insulin and BETA2/NeuroD gene by glucolipotoxic condition were improved with AICAR treatment. However, PGC-1α expression was gradually increased by glucolipotoxicity, and suppressed by AICAR treatment. Overexpression of PGC-1 using an adenoviral vector in freshly isolated rat islets suppressed insulin gene expression. We also confirmed the function of PGC-1 using an Ad-siPGC-1 in vivo. Direct infection of Ad-siPGC-1 in 90% pancreatectomized rats significantly improved glucose tolerance and increased body weight.

Conclusion

AMPK could protect against glucolipotoxicity induced β-cell dysfunction and the suppression of PGC-1 gene expression might involved in the insulin regulatory mechanism by AMPK.

Keywords: AICAR (5-aminoimidazole-4-carboxy-amide-1-D-ribofuranoside), AMP-activated protein Kinase (AMPK), Pancreatectomy, PPAR-γ-coactivator-1 (PGC-1)

Figures and Tables

Fig. 1
Change of insulin secretion by glucolipotoxicity and chronic AICAR treatment. Islets were isolated and subsequently cultured at the indicated glucolipotoxicity with or without AICAR treatment, prior to assay of insulin secretion. After incubation with each condition for 3 days, Cultured islets were washed in KRB washing buffer and incubated in KRB buffer containing 5.5 mM glucose for 1 h. And then, islets were stimulated for 1 h in KRB buffer containing 17.7 mM glucose. Results are expressed as means S.D. for five independent experiments (*P < 0.05).
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Fig. 2
Change of insulin gene expression by glucolipotoxicity and chronic AICAR treatment. A, islets were incubated with or without 0.6 mM FFA (a mixture of palmitate and oleate) in the presence of a high concentration of glucose (33.3 mM) for 3 days. Induction of glucolipotoxicity was confirmed by measuring insulin mRNA level using RT-PCR; B, The bar graphs show quantification of the results of PCR of rat islet extracts. Insulin gene expression was suppressed in a time-dependent manner and was notably increased by AICAR treatment at 3 day. Results are expressed as means S.D. for five independent experiments (*P < 0.05).
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Fig. 3
Expressions of beta-cell specific gene by glucolipotoxicity and chronic AICAR treatment. A, rat islets were cultured in suspension for three days in 5 mM or 33.3 mM glucose and 0.6 mM FFA or in the presence of 400 µM AICAR. Insulin, BETA2 and PGC-1 were estimated by semi-quantitative RT-PCR; B, The bar graphs show quantification of the results of PCR of rat islet extracts. Expression of glucose-induced insulin and BETA2 genes were repressed by glucolipotoxicity but rescued by AICAR treatment. However, PGC-1 The mRNA levels of these genes were normalized to GAPDH mRNA, and the RT-PCR data from the three independent experiments were summarized as means standard errors relative to those of untreated islets (*P < 0.05, **P < 0.01).
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Fig. 4
Adenovirus-mediated expression of PGC-1 reduces expression of the insulin gene. A, After infection with the indicated adenovirus at 100 MOI and culture for 24 h at 11 mM glucose, isolated rat islets were infected with PGC-1-expressing adenovirus. After infection with the indicated adenovirus 100 MOI for 2 h, islets were cultured at low or high glucose levels with or without FFA, and/or AICAR treatment for 72 h. Overexpression of PGC-1 induced by the glucolipotoxic condition was clearly suppressed by AICAR co-treatment and the expression of the insulin gene was normalized. In contrast, overexpression of PGC-1 induced by the adenoviral vector was not suppressed by AICAR treatment. Results are expressed as means S.D. for five independent experiments (*P < 0.05).
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Fig. 5
Body weight and blood glucose change during the 12-day study period. Hyperglycemic Px rats were infected with a celiac artery of Ad-GFP or Ad-siPGC-1 (1 × 109 pfu/300 µL, n = 3) for 12 days. The morning nonfasting glucose values of the Ad-siPGC-1 group were significantly lower than those of the Ad-GFP group 6 days after Ad-siPGC-1 infection. A, Body weight did not differ between Ad-GFP and Ad-siPGC-1-infected Px rats; B, IPGTT was performed using a 20% glucose solution and a dose rate of 2 g/kg. The mean AUCg was elevated in the AICAR-treated Px rats. Data are expressed as means S.D., n = 6 (*P < 0.05). (*P < 0.05, **P < 0.01); C and D, PGC-1 was expressed in islets of pancreas tissue of Ad-GFP-infected (C) Px rat but PGC-1 was remarkably decreased in islet of Ad-siPGC-1-infected Px rat (D). (400X).
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Table 1
Primer sequences for RT-PCR or real-time PCR amplification
jkda-31-310-i001

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