Imperial College London

ProfessorDavidCarling

Faculty of MedicineInstitute of Clinical Sciences

Professor of Biochemistry
 
 
 
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Contact

 

+44 (0)20 3313 4313david.carling

 
 
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Location

 

3rd Fl CRBHammersmith HospitalHammersmith Campus

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Summary

 

Publications

Publication Type
Year
to

196 results found

Bonnard C, Navaratnam N, Ghosh K, Chan PW, Tan TT, Pomp O, Ng AYJ, Tohari S, Changede R, Carling D, Venkatesh B, Altunoglu U, Kayserili H, Reversade Bet al., 2020, A loss-of-function NUAK2 mutation in humans causes anencephaly due to impaired Hippo-YAP signaling., J Exp Med, Vol: 217

Failure of neural tube closure during embryonic development can result in anencephaly, one of the most common birth defects in humans. A family with recurrent anencephalic fetuses was investigated to understand its etiology and pathogenesis. Exome sequencing revealed a recessive germline 21-bp in-frame deletion in NUAK2 segregating with the disease. In vitro kinase assays demonstrated that the 7-amino acid truncation in NUAK2, a serine/threonine kinase, completely abrogated its catalytic activity. Patient-derived disease models including neural progenitor cells and cerebral organoids showed that loss of NUAK2 activity led to decreased Hippo signaling via cytoplasmic YAP retention. In neural tube-like structures, endogenous NUAK2 colocalized apically with the actomyosin network, which was disrupted in patient cells, causing impaired nucleokinesis and apical constriction. Our results establish NUAK2 as an indispensable kinase for brain development in humans and suggest that a NUAK2-Hippo signaling axis regulates cytoskeletal processes that govern cell shape during neural tube closure.

Journal article

Spengler K, Zibrova D, Woods A, Langendorf CG, Scott JW, Carling D, Heller Ret al., 2020, Protein kinase A negatively regulates VEGF-induced AMPK activation by phosphorylating CaMKK2 at serine 495., Biochem J, Vol: 477, Pages: 3453-3469

Activation of AMP-activated protein kinase (AMPK) in endothelial cells by vascular endothelial growth factor (VEGF) via the Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) represents a pro-angiogenic pathway, whose regulation and function is incompletely understood. This study investigates whether the VEGF/AMPK pathway is regulated by cAMP-mediated signalling. We show that cAMP elevation in endothelial cells by forskolin, an activator of the adenylate cyclase, and/or 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases, triggers protein kinase A (PKA)-mediated phosphorylation of CaMKK2 (serine residues S495, S511) and AMPK (S487). Phosphorylation of CaMKK2 by PKA led to an inhibition of its activity as measured in CaMKK2 immunoprecipitates of forskolin/IBMX-treated cells. This inhibition was linked to phosphorylation of S495, since it was not seen in cells expressing a non-phosphorylatable CaMKK2 S495C mutant. Phosphorylation of S511 alone in these cells was not able to inhibit CaMKK2 activity. Moreover, phosphorylation of AMPK at S487 was not sufficient to inhibit VEGF-induced AMPK activation in cells, in which PKA-mediated CaMKK2 inhibition was prevented by expression of the CaMKK2 S495C mutant. cAMP elevation in endothelial cells reduced basal and VEGF-induced acetyl-CoA carboxylase (ACC) phosphorylation at S79 even if AMPK was not inhibited. Together, this study reveals a novel regulatory mechanism of VEGF-induced AMPK activation by cAMP/PKA, which may explain, in part, inhibitory effects of PKA on angiogenic sprouting and play a role in balancing pro- and anti-angiogenic mechanisms in order to ensure functional angiogenesis.

Journal article

Boyle J, Seneviratne A, Cave L, Hyde G, Moestrup SK, Carling D, Mason JC, Haskard DOet al., 2020, Metformin directly suppresses atherosclerosis in normoglycemic mice via haematopoietic Adenosine Monophosphate-Activated Protein Kinase (AMPK), Cardiovascular Research, 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

Journal article

Seneviratne A, Han Y, Wong E, Walter E, Jiang L, Cave L, Long NJ, Carling D, Mason JC, Haskard DO, Boyle Jet al., 2020, Hematoma resolution in vivo is directed by Activating Transcription Factor 1 (ATF1), Circulation Research, 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.

Journal article

Muñoz-Clares RA, González-Segura L, Juárez-Díaz JA, Mújica-Jiménez Cet 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.

Journal article

Munoz-Clares RA, Gonzalez-Segura L, Andres Juarez-Diaz J, Mujica-Jimenez Cet 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

Journal article

Garcia E, Guo W, Kumar S, Gorlitz F, Sparks H, Alexandrov Y, Munro I, Kelly DJ, Warren S, Chennell G, Sardini A, Carling D, Thorpe P, Dunsby C, French PMWet 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

Conference paper

Boyle J, Seneviratne A, Han Y, Jiang L, Walter E, Cave L, Shaikh A, Long NJ, Carling D, Mason JC, Haskard DOet 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

Conference paper

Boyle J, Seneviratne A, Tsao A, Shaikh A, Carling D, Haskard DO, Justin MC, Cave Let 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

Conference paper

Mcglone ER, Siebert M, Minnion J, Owen B, Goldin R, Li J, Carling D, Bado A, Bloom S, Le Gall M, Tan Tet 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

Conference paper

Boyle J, Seneviratne A, Hyde G, Moestrup SK, Carling D, Mason JC, Haskard DOet 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

Conference paper

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.

Journal article

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.

Journal article

Pollard AE, Martins L, Muckett PJ, Khadayate S, Bornot A, Clausen M, Admyre T, Bjursell M, Fiadeiro R, Wilson L, Whilding C, Kotiadis VN, Duchen MR, Sutton D, Penfold L, Sardini A, Bohlooly-Y M, Smith DM, Read JA, Snowden MA, Woods A, Carling Det 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.

Journal article

Penfold L, Woods A, Muckett P, Nikitin A, Kent T, Zhang S, Graham R, Pollard A, Carling Det 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.

Journal article

Hinchy EC, Gruszczyk AV, Willows R, Navaratnam N, Hall AR, Bates G, Bright TP, Krieg T, Carling D, Murphy MPet 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.

Journal article

Thomas EC, Hook SC, Gray A, Chadt A, Carling D, Al-Hasani H, Heesom KJ, Hardie DG, Tavare JMet 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.

Journal article

Yavari A, Bellahcene M, Bucchi A, Sirenko S, Pinter K, Herring N, Jung JJ, Tarasov KV, Sharpe EJ, Wolfien M, Czibik G, Steeples V, Ghaffari S, Nguyen C, Stockenhuber A, Clair JRS, Rimmbach C, Okamoto Y, Yang D, Wang M, Ziman BD, Moen JM, Riordon DR, Ramirez C, Paina M, Lee J, Zhang J, Ahmet I, Matt MG, Tarasova YS, Baban D, Sahgal N, Lockstone H, Puliyadi R, de Bono J, Siggs OM, Gomes J, Muskett H, Maguire ML, Beglov Y, Kelly M, Dos Santos PPN, Bright NJ, Woods A, Gehmlich K, Isackson H, Douglas G, Ferguson DJP, Schneider JE, Tinker A, Wolkenhauer O, Channon KM, Cornall RJ, Sternick EB, Paterson DJ, Redwood CS, Carling D, Proenza C, David R, Baruscotti M, DiFrancesco D, Lakatta EG, Watkins H, Ashrafian Het al., 2017, Mammalian γ2 AMPK regulates intrinsic heart rate., Nature Communications, Vol: 8, ISSN: 2041-1723

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.

Journal article

Willows R, Sanders MJ, Xiao B, Patel BR, Martin SR, Wilson JR, Hubbard J, Gamblin SJ, Carling Det 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.

Journal article

Willows R, Navaratnam N, Lima A, Read J, Carling Det 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.

Journal article

Carling D, Woods A, Williams JR, Muckett PJ, Mayer FV, Liljevald M, Bohlooly-Y Met 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).

Journal article

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.

Journal article

Boyle JJ, Seneviratne A, Carling D, Haskard DOet 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

Conference paper

Boyle JJ, Seneviratne A, Haskard DO, Carling Det 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

Conference paper

Boyle JJ, Seneviratne AA, Haskard DO, Carling D, Hyde Get 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

Conference paper

Maioli V, Chennell G, Sparks H, Lana T, Kumar S, Carling D, Sardini A, Dunsby CWet 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.

Journal article

Chennell G, Willows RJW, Warren SC, Carling D, French PMW, Dunsby C, Sardini Aet 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.

Journal article

Siggs OM, Stockenhuber A, Deobagkar-Lele M, Bull KR, Crockford TL, Kingston BL, Crawford G, Anzilotti C, Steeples V, Ghaffari S, Czibik G, Bellahcene M, Watkins H, Ashrafian H, Davies B, Woods A, Carling D, Yavari A, Beutler B, Cornall RJet 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.

Journal article

Trousil S, Kaliszczak M, Schug Z, Nguyen Q, Tomasi G, Favicchio R, Brickute D, Fortt R, Twyman FJ, Carroll L, Kalusa A, Navaratnam N, Adejumo T, Carling D, Gottlieb E, Aboagye EOet al., 2016, The novel choline kinase inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth., Oncotarget, Vol: 7, Pages: 37103-37120, ISSN: 1949-2553

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2-2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids.

Journal article

Maioli V, Gorlitz F, Warren S, Kumar S, French PMW, Chennell G, Sardini A, Carling D, Alwes F, Dunsby CWet al., 2016, Three-dimensional fluorescence imaging by stage-scanning oblique plane microscopy, Conference on Three-Dimensional and Multidimensional Microscopy - Image Acquisition and Processing XXIII, Publisher: SPIE, ISSN: 0277-786X

Conference paper

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