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Characterization of insulin-stimulated protein kinase cascade in normal and diabetic rats and the effects of vanadium

University of British Columbia

Multiple protein kinases acting in a sequential manner have been identified to constitute a kinase cascade that mediates growth factor or mitogen-activated signal transduction. The cascade is triggered at the cell membrane by the activation of receptor tyrosine kinases, which pass on the signal in the order of p21 [ras superscript] → p74 [raf superscript] → or mitogen-activated protein (MAP) kinase kinase kinase (MKKK) → MAP kinase kinase (MKK) → MAP kinase or extracellular-signal regulated kinase (ERK) → S6 kinase → S6 protein phosphorylation. Insulin also activates such a cascade in cultured cells or isolated tissues, but very few studies have investigated the kinase cascade in an intact animal model. In this study, MAP kinase, S6 kinase, p74 [raf1 superscript] and casein kinase-2 (CK-2) were characterized by chromatographic and immunoblotting techniques using both control and diabetic rats. The effects of insulin-mimetic vanadium compounds on these kinases were also examined. Intravenous injection of insulin activated the previously identified 42-kDa (p42 [erk2 superscript]) and 44-kDa (p44 [erk1 superscript]) MAP kinases as well as other potential novel MAP kinases in rat skeletal muscle and spleen. p42 [erk2 superscript] appears to represent a major portion of the insulin-stimulated MAP kinase activity. Also activated in rat skeletal muscle following insulin injection were S6 kinases and p74 [raf1 superscript]. A 100-kDa S6 kinase was tentatively identified as a member of the previously identified 90-kDa ribosomal S6 kinase (p90 [rsk superscript]) family, while another novel S6 kinase activity was also identified and purified to near homogeneity as a potentially novel 32-kDa S6 kinase. This kinase appears to play a major role in the insulin-stimulated activation of S6 kinase activity. CK-2 was not significantly activated by insulin in skeletal muscle, but was activated significantly in rat spleen. These results suggest that the kinase cascade may be physiologically significant and that the intact rat may serve as a useful model in characterizing these kinases. In streptozotocin (STZ)-induced diabetic rats, basal MAP kinase and S6 kinase activity were elevated in 2-month diabetic rats, but significantly reduced in 6-month diabetic rats. Furthermore, activation of MAP kinase and S6 kinase was defective in the 2-month diabetic rats, indicating a disruption of the kinase cascade that mediates the action of insulin during diabetes. The molecular mechanism of these changes requires further investigation. When the diabetic rats were treated with vanadyl sulphate or an organic compound of vanadium, bis-maltolato-oxo-vanadium (IV) (BMOV), the changes in MAP kinase and S6 kinase did not occur, suggesting that the insulin-like effects of vanadium may involve the effects of vanadium on the kinases that are critical components in the mitogen-activated kinase cascade.

Thesis/Dissertation

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2008-07-30

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http://hdl.handle.net/2429/1212

Pharmaceutical Sciences

University of British Columbia

UBCV

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