J Korean Diabetes Assoc.  2007 Jul;31(4):302-309. 10.4093/jkda.2007.31.4.302.

gamma-glutamylcysteine Synthetase (gamma-GCS) mRNA Expression in INS-1 Cells and Patients with Type 2 Diabetes Mellitus

• Kim JH
• Lee CH
• Moon JS
• Yoon JS
• Won KC
• Lee HW

• KMID: 2222517
• DOI: http://doi.org/10.4093/jkda.2007.31.4.302

Abstract

BACKGROUND: Hyperglycemia is a well-recognized pathogenic factor of long term complications in diabetes mellitus and hyperglycemia also generates reactive oxygen species (ROS) in beta cells when ROS accumulate in excess for prolonged periods of time, they cause chronic oxidative stress and adverse effects. Unfortunately, the islet contacts low capacity of endogenous antioxidant effects. But, gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for glutathione synthesis, is well represented in islets.
METHODS
This study is to evaluate the changes in the activity of gamma-GCS, glutathione in beta-cells exposed to high glucose, in pancreatic tissue of OLETF (Otsuka Long Evans Tokushima Fatty) and LETO (Long-Evans Tokushima Otsuka) rats, in leukocytes from patients with Korean type 2 DM (T2DM) and to disclose the effects of high blood glucose on this impairment in patients with T2DM. We divided our patients into 3 groups by HbA1c (controls: n = 20, well controls diabetes: n=24, poorly controlled diabetes: n = 36).
RESULTS
We observed that decreased glutathione level, gamma-GCS expression, glucose-stimulated (GSIS) and increased intracellular peroxide level in the INS-1 cells exposed to 30 mM glucose condition. Also decreased glutathione level at erythrocytes, gamma-GCS expression at leukocytes and increased oxidized LDL, MDA (malondialdehyde) level at plasma from patients with T2DM compared to controls (esp, poorly controlled patients).
CONCLUSION
These results suggest that insufficient antioxidant defenses by the glutathione pathway may be one of the factors responsible for development of complications in T2DM.

Keyword

gamma-glutamylcysteine synthetase (gamma-GCS) mRNA; glutathione (GSH); INS-1 Cells; Insulin secretion; Type 2 Diabetes Mellitus

MeSH Terms

Animals
Antioxidants
Blood Glucose
Diabetes Mellitus
Diabetes Mellitus, Type 2*
Erythrocytes
Glucose
Glutathione
Humans
Hyperglycemia
Leukocytes
Ligases*
Oxidative Stress
Plasma
Rats
Reactive Oxygen Species
RNA, Messenger*
Antioxidants
Blood Glucose
Glucose
Glutathione
Ligases
RNA, Messenger
Reactive Oxygen Species

Figure

• 

  Fig. 1 The effects of high glucose on the intracellular peroxide level, PDX-1, MafA, insulin mRNA and glucose stimulated insulin secretion (GSIS) in the INS-1 cells. A, INS-1 cells incubated for 72 hours at 30 mM glucose increased levels of intracellular peroxides compared with the 5.6 mM glucose (P < 0.05). And, INS-1 cells incubated at 30 mM glucose decreased GSIS compared with the 5.6 mM glucose. *P < 0.05 vs. 5.6 mM glucose; B, INS-1 cells incubated for 72 hours at 30 mM glucose decreased PDX-1, MafA, insulin mRNA compared with the 5.6 mM glucose. *P < 0.05; C, INS-1 cells incubated for 72 hours at 30 mM glucose decreased the expression of γ-GCS mRNA and GSH level compared with the 5.6 mM glucose in INS-1 cell (P < 0.05). *P < 0.05 vs. 30 mM glucose. Data are mean ± SE from 3 separate experiments.

• 

  Fig. 2 GSH level at pancreatic tissues in OLETF (n = 5) and LETO rats (n = 5). GSH level at pancreatic tissues in OLETF rats was significantly decreased compared with LETO rats. *P < 0.05 vs. LETO rats.

• 

  Fig. 3 Expression of γ-GCS mRNA in leukocytes and GSH level in erythrocytes from patients with T2DM. Expression of γ-GCS mRNA in leukocytes and GSH level in erythrocytes from patients with T2DM (n = 60) were significantly decreased compared with controls (n = 20). *P < 0.05 vs. Diabetics.

• 

  Fig. 4 GSH, ox LDL and MDA of the subjects (controls: n = 20, well controlled diabetics: n = 24, poorly controlled diabetics: n=36). GSH was significantly decreased and ox LDL and MDA levels were significantly increased compared with controls. *P < 0.05 vs. Controls.

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