Paradoxical activation of AMPK by glucose drives selective EP300 activity in colorectal cancer.

Coordination of gene expression with nutrient availability supports proliferation and homeostasis and is shaped by protein acetylation. Yet how physiological/pathological signals link acetylation to specific gene expression programs and whether such responses are cell-type-specific is unclear. AMP-activated protein kinase (AMPK) is a key energy sensor, activated by glucose limitation to resolve nutrient supply-demand imbalances, critical for diabetes and cancer. Unexpectedly, we show here that, in gastrointestinal cancer cells, glucose activates AMPK to selectively induce EP300, but not CREB-binding protein (CBP). Consequently, EP300 is redirected away from nuclear receptors that promote differentiation towards ß-catenin, a driver of proliferation and colorectal tumorigenesis. Importantly, blocking glycogen synthesis permits reactive oxygen species (ROS) accumulation and AMPK activation in response to glucose in previously nonresponsive cells. Notably, glycogen content and activity of the ROS/AMPK/EP300/ß-catenin axis are opposite in healthy versus tumor sections. Glycogen content reduction from healthy to tumor tissue may explain AMPK switching from tumor suppressor to activator during tumor evolution.

AMPK Activation Promotes Tight Junction Assembly in Intestinal Epithelial Caco-2 Cells.

The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.

Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis.

Dysfunctions in the intestinal barrier, associated with an altered paracellular pathway, are commonly observed in inflammatory bowel disease (IBD). The AMP-activated protein kinase (AMPK), principally known as a cellular energy sensor, has also been shown to play a key role in the stabilization and assembly of tight junctions. Here, we aimed to investigate the contribution of intestinal epithelial AMPK to the initiation, progression and resolution of acute colitis. We also tested the hypothesis that protection mediated by metformin administration on intestinal epithelium damage required AMPK activation. A dextran sodium sulfate (DSS)-induced colitis model was used to assess disease progression in WT and intestinal epithelial cell (IEC)-specific AMPK KO mice. Barrier integrity was analyzed by measuring paracellular permeability following dextran-4kDa gavage and pro-inflammatory cytokines and tight junction protein expression. The deletion of intestinal epithelial AMPK delayed intestinal injury repair after DSS exposure and was associated with a slower re-epithelization of the intestinal mucosa coupled with severe ulceration and inflammation, and altered barrier function. Following intestinal injury, IEC AMPK KO mice displayed a lower goblet cell counts with concomitant decreased Muc2 gene expression, unveiling an impaired restitution of goblet cells and contribution to wound healing process. Metformin administration during the recovery phase attenuated the severity of DSS-induced colitis through improvement in intestinal repair capacity in both WT and IEC AMPK KO mice. Taken together, these findings demonstrate a critical role for IEC-expressed AMPK in regulating mucosal repair and epithelial regenerative capacity following acute colonic injury. Our studies further underscore the therapeutic potential of metformin to support repair of the injured intestinal epithelium, but this effect is conferred independently of intestinal epithelial AMPK.

Deletion of intestinal epithelial AMP-activated protein kinase alters distal colon permeability but not glucose homeostasis.

OBJECTIVE: The intestinal epithelial barrier (IEB) restricts the passage of microbes and potentially harmful substances from the lumen through the paracellular space, and rupture of its integrity is associated with a variety of gastrointestinal disorders and extra-digestive diseases. Increased IEB permeability has been linked to disruption of metabolic homeostasis leading to obesity and type 2 diabetes. Interestingly, recent studies have uncovered compelling evidence that the AMP-activated protein kinase (AMPK) signaling pathway plays an important role in maintaining epithelial cell barrier function. However, our understanding of the function of intestinal AMPK in regulating IEB and glucose homeostasis remains sparse. METHODS: We generated mice lacking the two α1 and α2 AMPK catalytic subunits specifically in intestinal epithelial cells (IEC AMPK KO) and determined the physiological consequences of intestinal-specific deletion of AMPK in response to high-fat diet (HFD)-induced obesity. We combined histological, functional, and integrative analyses to ascertain the effects of gut AMPK loss on intestinal permeability in vivo and ex vivo and on the development of obesity and metabolic dysfunction. We also determined the impact of intestinal AMPK deletion in an inducible mouse model (i-IEC AMPK KO) by measuring IEB function, glucose homeostasis, and the composition of gut microbiota via fecal 16S rRNA sequencing. RESULTS: While there were no differences in in vivo intestinal permeability in WT and IEC AMPK KO mice, ex vivo transcellular and paracellular permeability measured in Ussing chambers was significantly increased in the distal colon of IEC AMPK KO mice. This was associated with a reduction in pSer425 GIV phosphorylation, a marker of leaky gut barrier. However, the expression of tight junction proteins in intestinal epithelial cells and pro-inflammatory cytokines in the lamina propria were not different between genotypes. Although the HFD-fed AMPK KO mice displayed suppression of the stress polarity signaling pathway and a concomitant increase in colon permeability, loss of intestinal AMPK did not exacerbate body weight gain or adiposity. Deletion of AMPK was also not sufficient to alter glucose homeostasis or the acute glucose-lowering action of metformin in control diet (CD)- or HFD-fed mice. CD-fed i-IEC AMPK KO mice also presented higher permeability in the distal colon under homeostatic conditions but, surprisingly, this was not detected upon HFD feeding. Alteration in epithelial barrier function in the i-IEC AMPK KO mice was associated with a shift in the gut microbiota composition with higher levels of Clostridiales and Desulfovibrionales. CONCLUSIONS: Altogether, our results revealed a significant role of intestinal AMPK in maintaining IEB integrity in the distal colon but not in regulating glucose homeostasis. Our data also highlight the complex interaction between gut microbiota and host AMPK.

Promise and challenges for direct small molecule AMPK activators.

AMP-activated protein kinase (AMPK) is an evolutionary conserved and ubiquitously expressed serine/threonine kinase playing a central role in the coordination of energy homeostasis. Based on the beneficial outcomes of its activation on metabolism, AMPK has emerged as an attractive target for the treatment of metabolic diseases. Identification of novel downstream targets of AMPK beyond the regulation of energy metabolism has renewed considerable attention in exploiting AMPK signaling for novel therapeutic targeting strategies including treatment of cancer and inflammatory diseases. The complexity of AMPK system with tissue- and species-specific expression of multiple isoform combination regulated by various inputs, post-traductional modifications and subcellular locations presents unique challenges for drug discovery. Here, we review the most recent advances in the understanding of the mechanism(s) of action of direct small molecule AMPK activators and the potential therapeutic opportunities.

Knockdown of Human AMPK Using the CRISPR/Cas9 Genome-Editing System.

AMP-activated protein kinase (AMPK) is a critical energy sensor, regulating signaling networks involved in pathology including metabolic diseases and cancer. This increasingly recognized role of AMPK has prompted tremendous research efforts to develop new pharmacological AMPK activators. To precisely study the role of AMPK, and the specificity and activity of AMPK activators in cellular models, genetic AMPK inactivating tools are required. We report here methods for genetic inactivation of AMPK α1/α2 catalytic subunits in human cell lines by the CRISPR/Cas9 technology, a recent breakthrough technique for genome editing.