Effect Of Thioctyl7linolenic Acid On The Activation Of Stressactivated Protein Kinases

Mitogen-activated protein (MAP) kinases are proline-directed serine/threonine kinases that are activated by dual phosphorylation in response to a wide variety of extracellular stimuli. Three distinct groups of MAP kinases have been identified in mammalian cells; extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38MAP kinase (p38) (see Refs. 97 and 98 for review). MAP kinase activation is achieved through kinase cascades, which serve as information relays connecting cell surface receptors to specific transcription factors and other regulatory proteins, thus allowing extracellular signals to regulate the expression of specific genes (99).

Recently, among this large family of MAP kinases, a family of stress-activated protein kinases (SAPKs), including JNK and p38, have been delineated and characterized (see Ref. 100 for review). JNK exits in three forms in mammalian cells: JNK1, JNK2, and JNK3 of 46, 54, and 56 kDa molecular weight, respectively. JNK has been found to be activated by a variety of different stimuli, including inflammatory cytokines such as interleukin-1 and tumor necrosis factor a, y-irradiation, ultraviolet irradiation, and oxidative stress (101). Once activated, JNK phosphorylates a number of different substrates, including c-jun and ATF2. Both of these transcription factors can form part of the AP-1 transcription factor complex, which is known to regulate the transcription of many different genes (reviewed in Ref. 98). Because oxidative stress, as part of the dysmetabolism of diabetes mellitus, seemed an excellent candidate for activation of SAPKs, we included measurement of activation of these molecules in the present study.

The expression of the subtypes of JNK was investigated using antibodies raised against either the native protein (JNK-FL) of the phosphorylated (activated) form (pJNK) in the DRG from 8-week diabetic rats and diabetic rats treated with 2.5% GLAaaTA. Western blots were analyzed by densitometry and levels of protein compared by normalizing to controls.

Eight weeks of diabetes resulted in a significant increase in the level of the p54 form of activated JNK (p < 0.05 compared with controls). This increase in activation of p54 JNK as measured by Western blotting with phospho-specific antibodies was reversed by 2.5% GLA/VXTA (Fig. 3) Total levels of the full-length JNK protein were unchanged by diabetes or GLA TA treatment.

Levels of the phosphorylated transcription factors, ATF2, and c-jun were investigated using antibodies specific to the phosphorylated forms of these transcription factors and were also unchanged by diabetes or GLAaaTA treatment.



Diabetic + GLAAATA

Native JNK


Figure 3 Western blots from lumbar DRG exposed to antibodies against a nonphos-phorylated epitope of JNK (JNK-FL) or a phosphorylated epitope (pJNK). Bar chart shows diabetes-induced increase in p54/p56 JNK phosphorylation (p < 0.05 controls) can be reversed by GLA'TA. There were no changes in the levels of the native protein, JNK-FL.

The reduction in the activation of JNK by GLA^TA may reflect a reduction in neuronal stress. TA treatment is known to increase levels of GSH in diabetic sciatic nerve (19), and GSH is a critical regulator for the induction of SAPKs, including JNK (102). However, it is not yet known whether the conjugate has similar effects on glutathione metabolism, but this warrants closer investigation.

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