AMPK:a cellular energy sensor primarily regulated by AMP

AMPK (AMP-activated protein kinase) is a cellular energy sensor that monitors the ratio of AMP/ATP, and possibly also ADP/ATP, inside cells. Once activated by falling cellular energy levels, it acts to restore energy homoeostasis by switching on catabolic pathways that generate ATP, while switching off anabolic pathways and other processes consuming ATP. AMPK is switched on by increases in AMP via three mechanisms, all of which are antagonized by ATP: (i) promotion of phosphorylation of Thr172 by upstream activating kinases; (ii) inhibition of dephosphorylation of Thr172 by phosphatases; and (iii) allosteric activation of the phosphorylated kinase. Recently, it has been proposed that the first two mechanisms are also triggered by ADP, which might be the physiological signal rather than AMP, and that the third mechanism may not be physiologically significant. We have re-evaluated these questions, and found that only mechanism (ii) is mimicked by ADP, and that ADP is also less potent than AMP, which we still believe to be the primary signal. We have also provided evidence that mechanism (iii), i.e. allosteric activation by AMP, is a quantitatively significant mechanism in intact cells.</jats:p

AMPK Causes Cell Cycle Arrest in LKB1-deficient Cells via Activation of CAMKK2

The AMP-activated protein kinase (AMPK) is activated by phosphorylation at Thr172, either by the tumor suppressor kinase LKB1 or by an alternate pathway involving the Ca(2+)/calmodulin-dependent kinase, CAMKK2. Increases in AMP:ATP and ADP:ATP ratios, signifying energy deficit, promote allosteric activation and net Thr172 phosphorylation mediated by LKB1, so that the LKB1-AMPK pathway acts as an energy sensor. Many tumor cells carry loss-of-function mutations in the STK11 gene encoding LKB1, but LKB1 re-expression in these cells causes cell cycle arrest. Therefore, it was investigated as to whether arrest by LKB1 is caused by activation of AMPK or of one of the AMPK-related kinases, which are also dependent on LKB1 but are not activated by CAMKK2. In three LKB1-null tumor cell lines, treatment with the Ca(2+) ionophore A23187 caused a G1-arrest that correlated with AMPK activation and Thr172 phosphorylation. In G361 cells, expression of a truncated, CAMKK2 mutant also caused G1-arrest similar to that caused by expression of LKB1, while expression of a dominant negative AMPK mutant, or a double knockout of both AMPK-α subunits, also prevented the cell cycle arrest caused by A23187. These mechanistic findings confirm that AMPK activation triggers cell cycle arrest, and also suggest that the rapid proliferation of LKB1-null tumor cells is due to lack of the restraining influence of AMPK. However, cell cycle arrest can be restored by re-expressing LKB1 or a constitutively active CAMKK2, or by pharmacological agents that increase intracellular Ca(2+) and thus activate endogenous CAMKK2. IMPLICATIONS: Evidence here reveals that the rapid growth and proliferation of cancer cells lacking the tumor suppressor LKB1 is due to reduced activity of AMPK, and suggests a therapeutic approach by which this block might be circumvented

Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser(487) in the ‘ST loop’ (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr(172). Surprisingly, the equivalent site on AMPK-α2, Ser(491), is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr(172) phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca(2+) ionophore A23187, effects we show to be dependent on Akt activation and Ser(487) phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr(172) phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)–AMPK pathway, which would otherwise inhibit cell growth and proliferation

The Na+/Glucose Cotransporter Inhibitor Canagliflozin Activates AMPK by Inhibiting Mitochondrial Function and Increasing Cellular AMP Levels

Canagliflozin, dapagliflozin and empagliflozin, all recently approved for treatment of Type 2 diabetes, were derived from the natural product phlorizin. They reduce hyperglycemia by inhibiting glucose re-uptake by SGLT2 in the kidney, without affecting intestinal glucose uptake by SGLT1. We now report that canagliflozin also activates AMP-activated protein kinase (AMPK), an effect also seen with phloretin (the aglycone breakdown product of phlorizin), but not to any significant extent with dapagliflozin, empagliflozin or phlorizin. AMPK activation occurred at canagliflozin concentrations measured in human plasma in clinical trials, and was caused by inhibition of Complex I of the respiratory chain, leading to increases in cellular AMP or ADP. Although canagliflozin also inhibited cellular glucose uptake independently of SGLT2, this did not account for AMPK activation. Canagliflozin also inhibited lipid synthesis, an effect that was absent in AMPK knockout cells and that required phosphorylation of ACC1 and/or ACC2 at the AMPK sites. Oral administration of canagliflozin activated AMPK in mouse liver, although not in muscle, adipose tissue or spleen. As phosphorylation of acetyl-CoA carboxylase by AMPK is known to lower liver lipid content, these data suggest a potential additional benefit of canagliflozin therapy compared to other SGLT2 inhibitors

AMP Is a True Physiological Regulator of AMP-Activated Protein Kinase by Both Allosteric Activation and Enhancing Net Phosphorylation

SummaryWhile allosteric activation of AMPK is triggered only by AMP, binding of both ADP and AMP has been reported to promote phosphorylation and inhibit dephosphorylation at Thr172. Because cellular concentrations of ADP and ATP are higher than AMP, it has been proposed that ADP is the physiological signal that promotes phosphorylation and that allosteric activation is not significant in vivo. However, we report that: AMP is 10-fold more potent than ADP in inhibiting Thr172 dephosphorylation; only AMP enhances LKB1-induced Thr172 phosphorylation; and AMP can cause >10-fold allosteric activation even at concentrations 1–2 orders of magnitude lower than ATP. We also provide evidence that allosteric activation by AMP can cause increased phosphorylation of acetyl-CoA carboxylase in intact cells under conditions in which there is no change in Thr172 phosphorylation. Thus, AMP is a true physiological regulator of AMPK, and allosteric regulation is an important component of the overall activation mechanism

INO80 Chromatin Remodeling Coordinates Metabolic Homeostasis with Cell Division

Adaptive survival requires the coordination of nutrient availability with expenditure of cellular resources. For example, in nutrient-limited environments, 50% of all S. cerevisiae genes synchronize and exhibit periodic bursts of expression in coordination with respiration and cell division in the yeast metabolic cycle (YMC). Despite the importance of metabolic and proliferative synchrony, the majority of YMC regulators are currently unknown. Here, we demonstrate that the INO80 chromatin-remodeling complex is required to coordinate respiration and cell division with periodic gene expression. Specifically, INO80 mutants have severe defects in oxygen consumption and promiscuous cell division that is no longer coupled with metabolic status. In mutant cells, chromatin accessibility of periodic genes, including TORC1-responsive genes, is relatively static, concomitant with severely attenuated gene expression. Collectively, these results reveal that the INO80 complex mediates metabolic signaling to chromatin to restrict proliferation to metabolically optimal states