We study mechanisms controlling of cell function, particularly in relation to diseases involving metabolic disorders, such as type 2 diabetes and cancer. We also study metabolic adaptations in animals that can withstand stresses, including low temperature, anoxia, freezing and dessication, with medical implications for organ transplantation. Lastly, we house the core mass spectrometry facility on the Brussels campus of UCL devoted to protein analysis.

Metformin is the most prescribed drug used worldwide for the treatment of type 2 diabetes, acting mainly on the liver to decrease glucose output. Metformin's action can partly be explained by activation of an enzyme, called a protein kinase, in this case the AMP-activated protein kinase (AMPK). AMPK catalyzes the phosphorylation of other proteins, notably key enzymes of metabolic pathways, switching them on or off. In general, AMPK activation in tissues and cells switches on energy producing pathways, such as glycolysis and fatty acid oxidation, while at the same time switching off energy consuming processes, such as protein synthesis. In this way, AMPK restores the energy balance of cells in response to ATP-depletion, such as occurs during anoxia or vigorous exercise in muscle. Thus, its main role is as a sensor of cellular and whole body energy homeostasis. Interestingly, individuals taking metformin have a reduced incidence of certain cancers, and there is growing evidence that AMPK activation could correct metabolic dysfunction in disease states. Therefore, pharmaceutical firms are actively searching for new drugs that activate AMPK for the treatment of type 2 diabetes and cancer.

We have made important contributions to the AMPK field by finding new targets in the control of glucose uptake, glycogen synthesis, glycolysis and protein synthesis. The search for AMPK targets is still our main line of research, which we are pursuing using state-of-the-art mass spectrometry techniques. We are also testing new direct AMPK activators and exploring alternative ways to activate AMPK, particularly in muscle and liver. AMPK is activated during muscle contraction and regular exercise is recommended to patients as a treatment for type 2 diabetes.

Research topics:

Our group focuses on the control of cell function by reversible phosphorylation of proteins. Our main interests are the AMP-activated protein kinase (AMPK), insulin signaling via protein kinase B (PKB) and the role of fructose 2,6-bisphosphate (Fru-2,6-P2) in cell proliferation and cancer. We also investigate protein covalent modification and differential protein expression by electrospray ionization (ESI) mass spectrometry (MS) techniques.

1. AMPK: (link to relevant page) We are currently testing the effects of a potent small-molecule AMPK activator in muscle and liver and exploring new ways to activate AMPK by targeting AMP-metabolizing enzymes. We are also trying to understand the precise mechanism by which AMPK activation inhibits protein synthesis by promoting eukaryotic elongation factor-2 phosphorylation. 

2. Insulin signalling: (link to relevant page) We re-investigated which insulin-stimulated protein kinase is responsible for activating 6-phosphofructo-2-kinase (PFK-2) to stimulate glycolysis in heart. We also studied insulin-stimulated glucose metabolism in incubated skeletal muscle and the role of PKB using a novel inhibitor, MK-2206. We are currently evaluating the role of protein kinase PKB in the stimulation of lipogenesis by insulin in white adipose tissue.

3. Role of Fru-2,6-P2 in the control of cell proliferation: (link to relevant page) We are studying the potential role of Fru-2,6-P2 in coupling glycolysis to cell proliferation in cancer cell models and thymocytes stimulated by mitogens.

 

 

4. Mass spectrometry: (link to relevant page) We are developing mass spectrometry (MS)-based phosphoproteomics strategies and we routinely use MS for phosphorylation site identification and the quantification of protein expression (gel-free proteomics).

 

Photo Labs1 n° 2

 

1. Control by AMPK.

AMPK is a serine/threonine protein kinase involved in the control of cellular energy homeostasis. It stimulates ATP-producing pathways and inhibits energy consuming processes (Fig. 1). We contributed to this field by finding new AMPK targets. We demonstrated that the activation of PFK-2, the enzyme responsible for Fru-2,6-P2 synthesis, by AMPK participates in the stimulation of heart glycolysis by ischaemia (Pasteur Effect). We also showed that AMPK activation is associated with protein synthesis inhibition in anoxic rat hepatocytes and in ischaemic rat hearts. Protein synthesis inhibition in response to AMPK activation can partly be explained by increased eEF2 (eukaryotic elongation factor-2) phosphorylation leading to its inactivation, but this is not a direct effect. Regulation of the upstream eEF2 kinase (eEF2K) is complex involving phosphorylation-induced activation/inactivation by kinases from various signalling pathways. Using partially purified AMPK, it was reported that AMPK phosphorylates and activates eEF2K. However, we now find that eEF2K is probably not a direct AMPK substrate, and the precise mechanism by which AMPK activation leads to a rise in eEF2 phosphorylation is under re-investigation. We are also studying the role of eEF2K in the biology of cancer.

We tested effects of an AMPK activator that was provided to us by AstraZeneca but developed by Merck (ex229 from patent application WO2010036613), comparing chemical activation with contraction and those of and the Abbott A769662 compound, in incubated rat epitrochlearis muscles. Ex229 dose-dependently increased AMPK activity of α1-, α2-, β1- and β2-containing complexes with significant increases in AMPK activity seen at a concentration of 5 mM. At higher doses, AMPK activation was similar to that observed after contraction and importantly led to an ~2-fold increase in glucose uptake. In AMPK α1-/α2-subunit catalytic subunit double knockout myotubes incubated with ex229, the increases in glucose uptake and ACC phosphorylation seen in control cells were completely abolished, suggesting that the effects of the compound were AMPK-dependent. When muscle glycogen levels were reduced by ~50% after starvation, ex229-induced AMPK activation and glucose uptake were amplified in a wortmannin-independent manner and in myotubes, fatty acid oxidation was increased. In summary, ex229 efficiently activated skeletal muscle AMPK and elicited metabolic effects in muscle appropriate for treating type 2 diabetes by stimulating glucose uptake and increasing fatty acid oxidation. We are now investigating the effects of the compound on hepatic glucose production. 

 

Figure 1: Conditions leading to AMPK activation in higher eukaryotes and some of its consequences.

 

 

We found that the phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is a new AMPK target participating in contraction-stimulated glucose uptake in skeletal muscle (see Fig. 2). We took advantage of a potent, selective PIKfyve inhibitor, YM201636, to show that PIKfyve lipid kinase activity was implicated in contraction-stimulated glucose uptake in skeletal muscle. Furthermore, siRNA knockdown of PIKfyve in C2C12 myotubes confirmed its importance in the stimulation of glucose uptake as a consequence of AMPK activation. AMPK was shown to phosphorylate PIKfyve on Ser307 in vitro as identified by mass spectrometry, but without affecting its lipid kinase activity. However, AMPK activation in cells led to the recruitment of GFP-tagged PIKfyve to "ring-like" structures seen by confocal microscopy, some of which were early endosomes. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK-activation. AMPK-induced phosphorylation of PIKfyve at Ser307 could favour its translocation to endosomes for PtdIns(3,5)P2 production, which facilitates GLUT4 translocation.

 

Figure 2: Schematic representation of GLUT4 translocation to the plasma membrane. The mechanism involves not only Akt substrate of 160 kDa (also called Tbc1d4) and Tbc1d1 phosphorylation by PKB and AMPK, but also PIKfyve phosphorylation on a common site (Ser307) in response to insulin or contraction, respectively. GSV, GLUT4 storage vesicle; LKB1, liver kinase B1, CaMKKb, Ca2+/calmodulin-dependent protein kinase-b. 

 

 

Lastly, we studied the role of AMPK in animals that undergo metabolic rate suppression to adapt to severe energy stress. AMPK does not appear to play a major role in deep torpor in squirrels, but could participate in metabolic rate depression during freeze tolerance in frogs, anoxia in turtles and freeze tolerance/freeze avoidance in insects. We are now using 14-3-3 pull-down and metal oxide affinity capture (MoAC) phosphoproteomics techniques to find new targets implicated in the adaptation to squirrel hibernation.

We collaborated with the pharmaceutical company AstraZeneca (Mölndal, Sweden) to investigate whether overexpression of AMP-metabolizing enzymes in cells would modulate oligomycin-induced AMPK activation. HEK293T cells were transiently transfected with increasing amounts of plasmid vectors to obtain a graded increase in overexpression of AMP-deaminase (AMPD)-1, AMPD2 and soluble 5’-nucleotidase IA (cN-IA), (see Fig. 3), for measurements of AMPK activation and total intracellular adenine nucleotide levels induced by oligomycin treatment. Overexpression of AMPD1 and AMPD2 only slightly decreased AMP levels and oligomycin-induced AMPK activation. Increased overexpression of cN-IA, on the other hand, led to reductions in the oligomycin-induced increases in AMP and ADP concentrations concomitant with a decrease in AMPK activation. From the calculated control coefficients, we conclude that in this model cN-IA exerts a large proportion of control over intracellular AMP, with little sharing of control by AMPDs. Also, in resting muscle, AMPD flux was very low, as judged by measurements of intracellular purine nucleotide concentrations. However, AMPD flux increased during contraction when ATP reserves become depleted. In rat skeletal muscle incubated with small-molecule AMP-competitive AMPD inhibitors and in muscles from mice bearing a whole body deletion of AMPD1, AMPK activation by electrical stimulation was potentiated, as was with the rise in AMP. However, the enhanced AMPK activity during contraction in Ampd1 KO mice muscles was much less than expected from the substantial increase in AMP and the stimulation of glucose uptake by electrical stimulation was unaffected. Our results support the idea that AMPK activation is controlled by factors other than changes in adenine nucleotides. We also conclude that the principle of indirect AMPK activation via inhibition of AMPD is not a viable approach to treat metabolic disease. The main role of AMPD in muscle and other tissues is likely to protect against the loss of adenine nucleotides during energy stress conditions. However, the compound AMPD inhibitors that have been developed would be useful tools for enhancing AMPK activation in muscle and other cells and tissues during ATP-depletion.

We are now studying changes in adenine nucleotide levels and AMPK activation in contracting muscles and phenformin-treated hepatocytes from whole body soluble 5'-nucleotidase cN-IA (Nt5c1a) and cN-II (Nt5c2) knockout mice.  Small molecule inhibition of the soluble 5'-nucleotidases might be a strategy for achieving AMPK activation for the treatment of type 2 diabetes.

 

 

Figure 3: Scheme showing AMP-metabolizing pathways. The enzymes implicated are indicated in italics. ASP: aspartate; AS: adenylosuccinate; FUM: fumarate; Ado: adenosine; Ino: inosine.

 

 

2. Insulin signalling.

For many years, we studied the structure-function relationships and regulation by protein phosphorylation of the PFK-2/fructose-2,6-bisphosphatase isoenzymes. We reinvestigated the role of PKB in heart PFK-2 activation by insulin using different approaches. In rat hearts perfused with the Akti-1/2 selective PKB inhibitor, insulin-induced PFK-2 activation was abrogated. Results from PKBb-knockout mice indicated that this isoform is not required for heart PFK-2 activation by insulin. PKBa knock-down by siRNA transfection suggested that this isoform likely mediates insulin-induced heart PFK-2 activation. Using Akti-1/2 and the next generation MK-2206 PKB inhibitor, we found that PKB played an important role in the insulin-induced stimulation of glucose transport, glycogen synthesis and protein synthesis in incubated rat epitrochlearis muscle. In rat epididymal adipocytes, we are now studying the role of PKB in the stimulation of lipogenesis by insulin using Akti-1/2 and the MK-2206 inhibitor. Although incubation with the inhibitors blocked insulin-stimulated lipogenesis, the changes in phosphorylation states of some of the key enzymes was unaffected. Therefore, athough PKB activation is important, other pathways are involved in insulin-stimulated lipogenesis in white adipose tissue.

 

3. Role of Fru-2,6-P2 in the control of cell proliferation.

Many years ago, concentrations of Fru-2,6-P2 were found to be unusually elevated in cancer cells and much higher than needed to stimulate glycolysis, suggesting that Fru-2,6-P2 might have other roles. Indeed in cancer cells and proliferating rat thymocytes, which express the PFKFB3 isoenzyme, incubation with "3PO", a small-molecule inhibitor of this enzyme, decreased Fru-2,6-P2 levels, lactate production and proliferation measured by 3H-thymidine incorporation, without having cytotoxic effects. The mechanism by which Fru-2,6-P2 could control cell proliferation is being investigated. 

 

4. Mass spectrometry.

The development of mass spectrometry (MS) facilities within our laboratory, and for our Institute and University, has been an asset (link to “Massprot”: http://www.uclouvain.be/en-proteomics.html). Since the acquisition of an electrospray mass spectrometer in 1997, the application of mass spectrometry techniques to protein identification, identification of sites of covalent modification and quantification of changes in protein expression has led to over 50 joint publications. In our own research, it enabled us to identify new phosphorylation sites in the AMPK complex and to demonstrate that in heart, insulin antagonized AMPK activation during ischaemia via PKB-induced phosphorylation of the AMPK catalytic a-subunits at Ser495/491.

 

The 2D-LC-LTQ ESI-MS system used in our laboratory

 

 

We are collaborating with the group of J.-F. Collet on the development of new proteomics strategies to study sulfenic acid formation in bacteria in vivo. A new concept is rapidly emerging, in which sulfenylation of specific cysteine residues modulates signal transduction pathways by altering the activity and function of cellular proteins in an analagous way to phosphorylation/dephosphorylation cycles. However, the regulation of protein function by sulfenic acid formation has only been clearly and unambiguously shown for a few proteins. Therefore, we are using MS to clearly establish the role of reversible sulfenic acid formation in living cells and to identify new proteins and pathways regulated by sulfenylation.

We study differential protein expression by label-free multidimensional LC-MS. One application is the screening of proteins and neuropeptides from cerebro-spinal fluid of patients with neurodegenerative diseases to discover biomarkers. This research was undertaken within the framework of the DIANE consortium on neurodegenerative diseases. These diseases include Alzheimer’s disease, Parkinson’s disease and multiple sclerosis.

Lastly, we use phosphoproteomics strategies to identify new targets downstream of signalling pathways. We are developping innovative approaches based on two parallel workflows: 1) the use of 14-3-3 proteins to pull-down phosphoproteins from cell extracts 2) the use of a combination of strong cation exchange and electrostatic repulsion hydrophilic liquid chromatography (ERLIC) followed by MoAC to enrich and concentrate phosphopeptides.

Complete list on PubMed
Mark Rider
Institut de Duve
Avenue Hippocrate 75 - B1.74.02
B-1200 Bruxelles
Tel:
+32 2 764 75 29
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