198 results found
Banskota S, Wang H, Kwon YH, et al., 2021, Salicylates Ameliorate Intestinal Inflammation by Activating Macrophage AMPK, INFLAMMATORY BOWEL DISEASES, Vol: 27, Pages: 914-926, ISSN: 1078-0998
Jørgensen NO, Kjøbsted R, Larsen MR, et al., 2021, Direct small molecule ADaM-site AMPK activators reveal an AMPKγ3-independent mechanism for blood glucose lowering., Mol Metab
OBJECTIVE: Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK, that bind to the Allosteric Drug and Metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. The aim of this study was to illuminate the mechanism by which the ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e. ADaM-site binding small molecules and the prodrug AICAR. METHODS: The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2-as well as whole-body AMPKγ3-deficient mouse models. In vitro complex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by Western Blotting in muscle lysate. To investigate the translatability of our studies, we treated mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle. RESULTS: Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR, increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2β2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2β2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscle. In vivo PF739 treatment lowered blood glucose leve
Boyle J, Seneviratne A, Cave L, et al., 2021, Metformin directly suppresses atherosclerosis in normoglycemic mice via haematopoietic Adenosine Monophosphate-Activated Protein Kinase (AMPK), Cardiovascular Research, Vol: 117, Pages: 1295-1308, ISSN: 0008-6363
AimsAtherosclerotic vascular disease has an inflammatory pathogenesis. Heme from intraplaque hemorrhage may drive a protective and pro-resolving macrophage M2-like phenotype, Mhem, via AMPK and ATF1. The anti-diabetic drug metformin may also activate AMPK-dependent signalling.HypothesisMetformin systematically induces atheroprotective genes in macrophages via AMPK and ATF1, and thereby suppresses atherogenesis.Methods and ResultsNormoglycemic Ldlr-/- hyperlipidemic mice were treated with oral metformin, which profoundly suppressed atherosclerotic lesion development (p < 5x10−11). Bone marrow transplantation from AMPK-deficient mice demonstrated that metformin-related atheroprotection required haematopoietic AMPK (ANOVA, p < 0.03). Metformin at a clinically relevant concentration (10μM) evoked AMPK-dependent and ATF1-dependent increases in Hmox1, Nr1h2 (Lxrb), Abca1, Apoe, Igf1 and Pdgf, increases in several M2-markers and decreases in Nos2, in murine bone marrow macrophages. Similar effects were seen in human blood-derived macrophages, in which metformin induced protective genes and M2-like genes, suppressible by si-ATF1-mediated knockdown. Microarray analysis comparing metformin with heme in human macrophages indicated that the transcriptomic effects of metformin were related to those of heme, but not identical. Metformin induced lesional macrophage expression of p-AMPK, p-ATF1 and downstream M2-like protective effects.ConclusionMetformin activates a conserved AMPK-ATF1-M2-like pathway in mouse and human macrophages, and results in highly suppressed atherogenesis in hyperlipidemic mice via haematopoietic AMPK.Translational perspectiveThe work shows that oral antidiabetic drug metformin may suppress atherosclerotic lesion development via hematopoietic AMPK at clinically relevant concentrations, rather than via a hypoglycemic effect. Activating Transcription Factor 1 (ATF1) may mediate induction of key atheroprotective genes
Bonnard C, Navaratnam N, Ghosh K, et al., 2020, A loss-of-function NUAK2 mutation in humans causes anencephaly due to impaired Hippo-YAP signaling, JOURNAL OF EXPERIMENTAL MEDICINE, Vol: 217, ISSN: 0022-1007
Seneviratne A, Han Y, Wong E, et al., 2020, Hematoma resolution in vivo is directed by Activating Transcription Factor 1, Circulation Research, Vol: 127, Pages: 928-944, ISSN: 0009-7330
Rationale: The efficient resolution of tissue hemorrhage is an important homeostatic function. In human macrophages in vitro, heme activates an adenosine monophosphate activated protein kinase / activating transcription factor 1 (AMPK/ATF1) pathway that directs Mhem macrophages through coregulation of heme oxygenase 1 (HMOX1, HO-1) and lipid homeostasis genes.Objective: We asked whether this pathway had an in vivo role in mice.Methods and Results: Perifemoral hematomas were used as a model of hematoma resolution. In mouse bone marrow derived macrophages (mBMM), heme induced HO-1, lipid regulatory genes including LXR, the growth factor IGF1, and the splenic red pulp macrophage gene Spic. This response was lost in mBMM from mice deficient in AMPK (Prkab1-/-) or ATF1 (Atf1-/-). In vivo, femoral hematomas resolved completely between day 8 and day 9 in littermate control mice (n=12), but were still present at day 9 in mice deficient in either AMPK (Prkab1-/-) or ATF1 (Atf1-/-) (n=6 each). Residual hematomas were accompanied by increased macrophage infiltration, inflammatory activation and oxidative stress. We also found that fluorescent lipids and a fluorescent iron-analog were trafficked to lipid-laden and iron-laden macrophages respectively. Moreover erythrocyte iron and lipid abnormally colocalized in the same macrophages in Atf1-/- mice. Therefore, iron-lipid separation was Atf1-dependent.Conclusions: Taken together, these data demonstrate that both AMPK and ATF1 are required for normal hematoma resolution.
Spengler K, Zibrova D, Woods A, et al., 2020, Protein kinase A negatively regulates VEGF-induced AMPK activation by phosphorylating CaMKK2 at serine 495, BIOCHEMICAL JOURNAL, Vol: 477, Pages: 3453-3469, ISSN: 0264-6021
Muñoz-Clares RA, González-Segura L, Juárez-Díaz JA, et al., 2020, Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics., Biochem J, Vol: 477, Pages: 2095-2114
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Munoz-Clares RA, Gonzalez-Segura L, Andres Juarez-Diaz J, et al., 2020, Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C-4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics, BIOCHEMICAL JOURNAL, Vol: 477, Pages: 2095-2114, ISSN: 0264-6021
Garcia E, Guo W, Kumar S, et al., 2020, FLIM, FRET and high content analysis, Symposium on Multiphoton Microscopy in the Biomedical Sciences XX held at SPIE BiOS Conference, Publisher: SPIE-INT SOC OPTICAL ENGINEERING, ISSN: 0277-786X
Boyle J, Seneviratne A, Han Y, et al., 2019, VERTEBRATE HEMATOMA RESOLUTION IS DIRECTED BY ACTIVATING TRANSCRIPTION FACTOR 1 (ATF1) AND ADENOSINE-MONOPHOSPHATE-ACTIVATED-PROTEIN-KINASE (AMPK), 87th Congress of the European-Atherosclerosis-Society (EAS), Publisher: ELSEVIER IRELAND LTD, Pages: E246-E246, ISSN: 0021-9150
Boyle J, Seneviratne A, Tsao A, et al., 2019, SMARCA4 REDIRECTS BINDING OF MACROPHAGE ACTIVATING TRANSCRIPTION FACTOR 1 (ATF1) FROM GENES FOR INFLAMMATION RESOLUTION TO GENES FOR ERYTHROCYTE RESOLUTION, 87th Congress of the European-Atherosclerosis-Society (EAS), Publisher: ELSEVIER IRELAND LTD, Pages: E78-E78, ISSN: 0021-9150
Mcglone ER, Siebert M, Minnion J, et al., 2019, SLEEVE GASTRECTOMY IS ASSOCIATED WITH WEIGHT LOSS-INDEPENDENT IMPROVEMENT IN HEPATIC STEATOSIS Basic science and research in bariatric surgery, 24th World Congress of the International-Federation-for-the-Surgery-of-Obesity-and-Metabolic-Disorders (IFSO) / 21st SECO Congress, Publisher: SPRINGER, Pages: 479-479, ISSN: 0960-8923
Boyle J, Seneviratne A, Hyde G, et al., 2019, METFORMIN DIRECTLY SUPPRESSES ATHEROSCLEROSIS IN NORMOGLYCEMIC MICE VIA HAEMATOPOIETIC ADENOSINE MONOPHOSPHATE-ACTIVATED PROTEIN KINASE (AMPK), 87th Congress of the European-Atherosclerosis-Society (EAS), Publisher: ELSEVIER IRELAND LTD, Pages: E45-E46, ISSN: 0021-9150
Carling D, 2019, AMPK hierarchy: A matter of space and time, Cell Research, Vol: 29, Pages: 425-426, ISSN: 1001-0602
AMP-activated protein kinase (AMPK) is a key sensor of energy balance in eukaryotic cells, responding to low energy status by switching off anabolic pathways and upregulating catabolic processes. Zong and colleagues now show that different intensities of stimulation trigger activation of specific subcellular pools of AMPK, resulting in phosphorylation of different downstream targets.
Steinberg GR, Carling D, 2019, AMP-activated protein kinase: the current landscape for drug development, Nature Reviews Drug Discovery, Vol: 18, Pages: 527-551, ISSN: 1474-1776
Since the discovery of AMP-activated protein kinase (AMPK) as a central regulator of energy homeostasis, many exciting insights into its structure, regulation and physiological roles have been revealed. While exercise, caloric restriction, metformin and many natural products increase AMPK activity and exert a multitude of health benefits, developing direct activators of AMPK to elicit beneficial effects has been challenging. However, in recent years, direct AMPK activators have been identified and tested in preclinical models, and a small number have entered clinical trials. Despite these advances, which disease(s) represent the best indications for therapeutic AMPK activation and the long-term safety of such approaches remain to be established.
Pollard AE, Martins L, Muckett PJ, et al., 2019, AMPK activation protects against diet induced obesity through Ucp1-independent thermogenesis in subcutaneous white adipose tissue, Nature Metabolism, Vol: 1, Pages: 340-349, ISSN: 2522-5812
Obesity results from a chronic imbalance between energy intake and energy output but remains difficult to prevent or treat in humans. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an important regulator of energy homeostasis1,2,3 and is a molecular target of drugs used for the treatment of metabolic diseases, including obesity4,5. Here we show that mice expressing a gain-of-function AMPK mutant6 display a change in morphology of subcutaneous white adipocytes that is reminiscent of browning. However, despite a dramatic increase in mitochondrial content, Ucp1 expression is undetectable in these adipocytes. In response to a high-fat diet (HFD), expression of skeletal muscle–associated genes is induced in subcutaneous white adipocytes from the gain-of-function AMPK mutant mice. Chronic genetic AMPK activation results in protection against diet-induced obesity due to an increase in whole-body energy expenditure, most probably because of a substantial increase in the oxygen consumption rate of white adipose tissue. These results suggest that AMPK activation enriches, or leads to the emergence of, a population of subcutaneous white adipocytes that produce heat via Ucp1-independent uncoupling of adenosine triphosphate (ATP) production on a HFD. Our findings indicate that AMPK activation specifically in adipose tissue may have therapeutic potential for the treatment of obesity.
Penfold L, Woods A, Muckett P, et al., 2018, CAMKK2 promotes prostate cancer independently of AMPK via increased lipogenesis, Cancer Research, Vol: 78, Pages: 6747-6761, ISSN: 1538-7445
New targets are required for treating prostate cancer, particularly castrate-resistant disease. Previous studies reported that calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) expression is increased in human prostate cancer. Here, we show that Camkk2 deletion or pharmacologic inhibition protects against prostate cancer development in a preclinical mouse model that lacks expression of prostate-specific Pten. In contrast, deletion of AMP-activated protein kinase (Ampk) β1 resulted in earlier onset of adenocarcinoma development. These findings suggest for the first time that Camkk2 and Ampk have opposing effects in prostate cancer progression. Loss of CAMKK2 in vivo or in human prostate cancer cells reduced the expression of two key lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase. This reduction was mediated via a posttranscriptional mechanism, potentially involving a decrease in protein translation. Moreover, either deletion of CAMKK2 or activation of AMPK reduced cell growth in human prostate cancer cells by inhibiting de novo lipogenesis. Activation of AMPK in a panel of human prostate cancer cells inhibited cell proliferation, migration, and invasion as well as androgen-receptor signaling. These findings demonstrate that CAMKK2 and AMPK have opposing effects on lipogenesis, providing a potential mechanism for their contrasting effects on prostate cancer progression in vivo. They also suggest that inhibition of CAMKK2 combined with activation of AMPK would offer an efficacious therapeutic strategy in treatment of prostate cancer.
Hinchy EC, Gruszczyk AV, Willows R, et al., 2018, Mitochondria-derived ROS activate AMP-activated protein kinase (AMPK) indirectly, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 293, Pages: 17208-17217, ISSN: 0021-9258
Mitochondrial reactive oxygen species (ROS) production is a tightly regulated redox signal that transmits information from the organelle to the cell. Other mitochondrial signals, such as ATP, are sensed by enzymes, including the key metabolic sensor and regulator, AMP-activated protein kinase (AMPK). AMPK responds to the cellular ATP/AMP and ATP/ADP ratios by matching mitochondrial ATP production to demand. Previous reports proposed that AMPK activity also responds to ROS, by ROS acting on redox-sensitive cysteine residues (Cys-299/Cys-304) on the AMPK α subunit. This suggests an appealing model in which mitochondria fine-tune AMPK activity by both adenine nucleotide–dependent mechanisms and by redox signals. Here we assessed whether physiological levels of ROS directly alter AMPK activity. To this end we added exogenous hydrogen peroxide (H2O2) to cells and utilized the mitochondria-targeted redox cycler MitoParaquat to generate ROS within mitochondria without disrupting oxidative phosphorylation. Mitochondrial and cytosolic thiol oxidation was assessed by measuring peroxiredoxin dimerization and by redox-sensitive fluorescent proteins. Replacing the putative redox-active cysteine residues on AMPK α1 with alanines did not alter the response of AMPK to H2O2. In parallel with measurements of AMPK activity, we measured the cell ATP/ADP ratio. This allowed us to separate the effects on AMPK activity due to ROS production from those caused by changes in this ratio. We conclude that AMPK activity in response to redox changes is not due to direct action on AMPK itself, but is a secondary consequence of redox effects on other processes, such as mitochondrial ATP production.
Thomas EC, Hook SC, Gray A, et al., 2018, Isoform-specific AMPK association with TBC1D1 is reduced by a mutation associated with severe obesity, Biochemical Journal, Vol: 475, Pages: 2969-2983, ISSN: 1470-8728
AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energyhomeostasis which achieves this through the phosphorylation of a myriad of downstreamtargets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucoseuptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser237 in TBC1D1 is a target for phosphorylation by AMPK, an event which maybe important in regulating glucose uptake. Here, we show AMPK heterotrimers containingthe α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosinebinding (PTB) domains of TBC1D1 and is increased by two different pharmacologicalactivators of AMPK (AICAR and A769962). The interaction enhances the efficiency bywhich AMPK phosphorylates TBC1D1 on its key regulatory site, Ser237. Furthermore, theinteraction is reduced by a naturally occurring R125W mutation in the PTB1 domain ofTBC1D1, previously found to be associated with severe familial obesity in females, with aconcomitant reduction in Ser237 phosphorylation. Our observations provide evidence fora functional difference between AMPK α-subunits and extend the repertoire of proteinkinases that interact with substrates via stabilisation mechanisms that modify the efficacyof substrate phosphorylation.
AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αβγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca(2+) release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.
Willows R, Sanders MJ, Xiao B, et al., 2017, Phosphorylation of AMPK by upstream kinases is required for activity in mammalian cells, Biochemical Journal, Vol: 474, Pages: 3059-3073, ISSN: 1470-8728
AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism andhas attracted significant attention as a therapeutic target for treating metabolic disorders.AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172(Thr172). Binding of AMP to the γ subunit allosterically activates the kinase. Additionally,many small molecules, e.g. 991, have been identified that bind between the kinasedomain and the carbohydrate-binding module of the β subunit, stabilising their interactionand leading to activation. It was reported recently that non-phosphorylated Thr172 AMPKis activated by AMP and A769662. We present here the crystal structure of non-phosphorylatedThr172 AMPK in complex with AMP and 991. This structure reveals that theactivation loop, as well as the complex overall, is similar to the Thr172 phosphorylatedcomplex. We find that in the presence of AMP and 991 non-phosphorylated Thr172,AMPK is much less active than the Thr172 phosphorylated enzyme. In human cells, thebasal level of Thr172 phosphorylation is very low (∼1%), but is increased 10-fold by treatmentwith 2-deoxyglucose. In cells lacking the major Thr172 kinases, LKB1 and CaMKKβ,Thr172 phosphorylation is almost completely abolished, and AMPK activity is virtuallyundetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr172AMPK can induce an ordered, active-like, conformation of the activation loop explaininghow AMPK activity can be measured in vitro without Thr172 phosphorylation. However, ina cellular context, phosphorylation of Thr172 is critical for significant activation of AMPK.
Willows R, Navaratnam N, Lima A, et al., 2017, Effect of different γ subunit isoforms on the regulation of AMPK, Biochemical Journal, Vol: 474, Pages: 1741-1754, ISSN: 1470-8728
AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. AMPK activation results in a wide range of downstream responses, many of which are associated with improved metabolic outcome, making AMPK an attractive target for the treatment of metabolic diseases. AMPK is a heterotrimeric complex consisting of a catalytic subunit (α) and two regulatory subunits (β and γ). The γ-subunit harbours the nucleotide-binding sites and plays an important role in AMPK regulation in response to cellular energy levels. In mammals, there are three isoforms of the γ-subunit and these respond differently to regulation by nucleotides, but there is limited information regarding their role in activation by small molecules. Here, we determined the effect of different γ-isoforms on AMPK by a direct activator, 991. In cells, 991 led to a greater activation of γ2-containing AMPK complexes compared with either γ1 or γ3. This effect was dependent on the long N-terminal region of the γ2-isoform. We were able to rule out an effect of Ser108 phosphorylation, since mutation of Ser108 to alanine in the β2-isoform had no effect on activation of AMPK by 991 in either γ1- or γ2-complexes. The rate of dephosphorylation of Thr172 was slower for γ2- compared with γ1-complexes, both in the absence and presence of 991. Our studies show that activation of AMPK by 991 depends on the nature of the γ-isoform. This finding may have implications for the design of isoform-selective AMPK activators.
Carling D, Woods A, Williams JR, et al., 2017, Liver-specific activation of AMPK prevents steatosis on a high fructose diet, Cell Reports, Vol: 18, Pages: 3043-3051, ISSN: 2211-1247
AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. We identified a mutation in the γ1 subunit (γ1D316A) that leads to activation of AMPK. We generated mice with this mutation to study the effect of chronic liver-specific activation of AMPK in vivo. Primary hepatocytes isolated from these mice have reduced gluconeogenesis and fatty acid synthesis, but there is no effect on fatty acid oxidation compared to cells from wild-type mice. Liver-specific activation of AMPK decreases lipogenesis in vivo and completely protects against hepatic steatosis when mice are fed a high-fructose diet. Our findings demonstrate that liver-specific activation of AMPK is sufficient to protect against hepatic triglyceride accumulation, a hallmark of non-alcoholic fatty liver disease (NAFLD). These results emphasize the clinical relevance of activating AMPK in the liver to combat NAFLD and potentially other associated complications (e.g., cirrhosis and hepatocellular carcinoma).
Carling D, 2017, AMPK signalling in health and disease, Current Opinion in Cell Biology, Vol: 45, Pages: 31-37, ISSN: 1879-0410
In eukaryotic cells AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance. AMPK responds to changes in intracellular adenine nucleotide levels, being activated by an increase in AMP/ADP relative to ATP. Activation of AMPK increases the rate of catabolic (ATP-generating) pathways and decreases the rate of anabolic (ATP-utilising) pathways. In addition to its role in maintaining intracellular energy balance, AMPK regulates whole body energy metabolism. Given its key role in controlling energy homeostasis, AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases, including type 2 diabetes and, more recently, cancer. Here I review the regulation of AMPK and its potential as a target for therapeutic intervention in human disease.
Boyle JJ, Seneviratne A, Carling D, et al., 2017, Hematoma Resolution In Vivo is Mediated by AMP-Activated Kinase (AMPK) and Activating Transcription Factor 1 (ATF1) Via Coregulation of Tissue Homeostatic Genes, 2nd Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 29-29, ISSN: 1018-1172
Boyle JJ, Seneviratne A, Haskard DO, et al., 2017, AMP-Activated Protein Kinase-Mediated Chromatin Remodelling Redirects Activating Transcription Factor 1 From Cyclic-AMP Response Genes to Heme-Response Genes, 2nd Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 29-29, ISSN: 1018-1172
Boyle JJ, Seneviratne AA, Haskard DO, et al., 2017, Oral Metformin Profoundly Suppresses Atherosclerotic Lesion Development In Vivo Independently of Glucose Lowering in a Mild Hyperlipidemic Model Via AMPK, 2nd Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 8-9, ISSN: 1018-1172
Maioli V, Chennell G, Sparks H, et al., 2016, Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates, Scientific Reports, Vol: 6, ISSN: 2045-2322
Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format.
Chennell G, Willows RJW, Warren SC, et al., 2016, Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM, Sensors, Vol: 16, ISSN: 1424-8239
We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation.
Siggs OM, Stockenhuber A, Deobagkar-Lele M, et al., 2016, Mutation of Fnip1 is associated with B-cell deficiency, cardiomyopathy, and elevated AMPK activity, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E3706-E3715, ISSN: 1091-6490
Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt–Hogg–Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1. Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the γ2 subunit of AMPK. Concordantly, γ2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51–like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.
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