AMP-activated protein kinase (AMPK) is activated within the cell in response to multiple stresses that increase the intracellular AMP:ATP ratio. Here we show that incubation of muscle cells with the thiazolidinedione, rosiglitazone, leads to a dramatic increase in this ratio with the concomitant activation of AMPK. This finding raises the possibility that a number of the beneficial effects of the thiazolidinediones could be mediated via activation of AMPK. Furthermore, we show that in addition to the classical activation pathway, AMPK can also be stimulated without changing the levels of adenine nucleotides. In muscle cells, both hyperosmotic stress and the anti-diabetic agent, metformin, activate AMPK in the absence of any increase in the AMP:ATP ratio. However, although activation is no longer dependent on this ratio, it still involves increased phosphorylation of threonine 172 within the catalytic (␣) subunit. AMPK stimulation in response to hyperosmotic stress does not appear to involve phosphatidylinositol 3-phosphate kinase, protein kinase C, mitogen-activated protein (MAP) kinase kinase, or p38 MAP kinase ␣ or ␤. Our results demonstrate that AMPK can be activated by at least two distinct signaling mechanisms and suggest that it may play a wider role in the cellular stress response than was previously understood.

The AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that plays a major role in energy sensing. AMPK is activated pharmacologically by 5-amino-4-imidazolecarboxamide (AICA) riboside monophosphate (ZMP), which mimics the e¡ects of AMP on the AMPK cascade. Here we show that uptake of AICA riboside into cells, mediated by the adenosine transport system, is blocked by a number of protein kinase inhibitors. Under these conditions, ZMP does not accumulate to su⁄cient levels to stimulate AMPK. Our results demonstrate that careful interpretation is required when using AICA riboside in conjunction with protein kinase inhibitors to investigate the physiological role of AMPK. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.