The aim of this work was to study the e¡ect of a sustained activation of AMP-activated protein kinase (AMPK) on liver cell survival. AMPK activation was achieved by incubating FTO2B cells with AICA-riboside, which is transformed into ZMP, an AMP analogue, or by adenoviral transfection of hepatocytes with a constitutively active form of AMPK. Prolonged AMPK activation triggered apoptosis and activated c-Jun N-terminal kinase (JNK) and caspase-3. Experiments with iodotubercidin, dicoumarol and z-VAD-fmk, which inhibited AMPK, JNK and caspase activation, respectively, supported the notion that prolonged AMPK activation in liver cells induces apoptosis through an activation pathway that involves JNK and caspase-3. ß

Aims/hypothesis. Prolonged exposure of beta cells to low glucose concentrations triggers their apoptosis and is known to activate AMP-activated protein kinase (AMPK) in beta cell lines. We examined whether prolonged activation of AMPK can trigger apoptosis in rodent beta cells. Methods. Primary beta cells were FACS-purified from rats, and from wild-type and AMPK(alpha2)-deficient mice. AMPK activation in beta cells was induced by the adenosine analog AICA-riboside and detected by immunoblotting using a phosphospecific antibody. Apoptosis of rodent beta cells was monitored by FACS analysis of beta cell DNA content, by direct counting of apoptotic cells using fluorescence microscopy, or by measurement of their caspase-3 activity.Results. Dose-dependent and time-dependent apoptosis of the cells, concommittant with an activation of caspase-3, were suppressed by the caspase inhibitors zVAD-fmk and zDEVD-fmk. Apoptosis induction by AICA-riboside was also prevented by adding the MAPK-inhibitor SB203580 which blocked the AICAriboside-induced phosphorylation of AMPK. Beta cells isolated from AMPK-(alpha2)-deficient mice were resistant against AICA-riboside induced apoptosis. Conclusion/interpretation. Sustained activation of AMPK by AICA-riboside can trigger a caspasedependent apoptosis of pancreatic beta cells. [Diabetologia (2003) 46:250-254] Keywords AICA-riboside, AICAR, AMP-activated protein kinase, AMPK, knockout, apoptosis, signalling, beta cell, glucose, diabetes. nine nucleotides that express and signal the metabolic state of the cells. The rate of glucose oxidation influences cellular ATP, ADP and AMP concentrations and hence their regulatory role on beta cell functions. During glucose deprivation, the AMP over ATP ratio is expected to increase and thus to activate the AMP-activated protein kinase (AMPK). AMPK is a serinethreonine protein kinase that is composed of a catalytic subunit (α) and two regulatory subunits (β and γ) [2]. The control of its activity involves allosteric activation by AMP, as well as phosphorylation by AMPKkinase, the upstream kinase [2]. In several cell types, this enzyme has been described as a sensor for the energy state of the cells and as a metabolic master switch [2,3]. In beta cell lines, the AMPK activity is increased by culture at low glucose concentrations [4,5]. 5-aminoimidazole-4-carboxamide (AICA)-riboside Prolonged exposure of pancreatic beta cells to low glucose concentrations results in apoptosis of the cells [1]. The underlying mechanism is not known but might be initiated by altered concentrations of the ade-

The discovery of the AMP-activated protein kinase (AMPK) more than a decade ago has shed much light on the cellular response to stresses characterized by a fall in the concentration of ATP and an increase in the AMP/ATP ratio. All conditions known to increase this ratio activate AMPK, whose major role is to act as an emergency signal to conserve ATP. It does so by inhibiting anabolic processes and by activating pathways producing ATP. In recent years, our laboratory has discovered new targets of AMPK. The purpose of this short review is to summarize our contribution to this field. Control of AMP-activated protein kinase (AMPK) activityAMPK is a well-conserved eukaryotic heterotrimeric protein kinase that senses nutritional and environmental stresses and acts as a metabolic master switch (for a review, see [1]). The control of AMPK activity is complex and involves allosteric stimulation by AMP as well as phosphorylation by AMPK kinase. AMPK activation requires phosphorylation of Thr-172 in the activation loop of its catalytic α subunit, by an upstream AMPK kinase sensitive to AMP [2]. Moreover, AMPK is allosterically activated by a decreased phosphocreatine/creatine ratio, thus reinforcing the activitydependence of AMPK on the energy state of the cell [3]. In normoxic cells, the very low AMP/ATP ratio (about 0.05) keeps AMPK mainly in the inactive form. In contrast, any energy inbalance increases the AMP/ATP ratio and activates AMPK. This is the case when oxygen supply is limited or when energy demand exceeds supply. An adenosine analogue, 5-amino-4-imidazolecarboxamide riboside (AICAR), is commonly used to activate AMPK in certain cells after its conversion into ZMP (AICAR monophosphate), which is an AMP analogue. This does not occur in all cell types, probably because of a limited transport or phosphorylation of AICAR [4][5][6][7]. In addition, caution should be exerted because AMP, and conceivably ZMP, interact with enzymes such as glycogen phosphorylase, 6-phosphofructo-1-kinase (PFK-1) and fructose-1,6-bisphosphatase.In normoxic heart, insulin decreases the basal activity of AMPK [8,9]. We found that AMPK activation by ischaemia was also inhibited in hearts treated with insulin [10]. This effect of insulin was wortmannin-sensititive, presumably involving the phosphoinositide 3-kinase pathway, and decreased the phosphorylation state of Thr-172 in AMPK.

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The effect of cell swelling on the expression of the K K2-macroglobulin (K K2M) gene was studied in hepatocytes in culture. Hypoosmolarity induced an increase (3-fold increase) in the level of K K2M mRNA through a corresponding stimulation of the rate of transcription of the K K2M gene. The addition of raffinose (100 mM) corrected the effect of hypoosmolarity at both mRNA and transcriptional level, demonstrating that cell swelling per se was responsible for the observed effect on the expression of the K K2M gene. Moreover, the effect of cell swelling was additive to that of interleukin 6, a major mediator of the acute-phase response.z 1998 Federation of European Biochemical Societies.

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In this paper, we demonstrated that in cultured rat hepatocytes cell swelling induced the activation of STAT1 and STAT3 proteins without any effect on STAT4, STAT5 and STAT6 proteins. Cell swelling induced an activation of STAT proteins through an increase in the phosphorylation of the tyrosine residue also phosphorylated by interleukin-6, but without any activation of JAK kinases. The signaling pathway by which cell swelling activated STAT1 and STAT3 is discussed.z 1999 Federation of European Biochemical Societies.

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