Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator.

The AMP-activated protein kinase (AMPK) αßγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2ß2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2ß2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating ß2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2ß2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2ß1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2ß2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.

AMPK mediates energetic stress-induced liver GDF15.

Growth differentiating factor-15 (GDF15) is an emerging target for the treatment of obesity and metabolic disease partly due to its ability to suppress food intake. GDF15 expression and secretion are thought to be regulated by a cellular integrated stress response, which involves endoplasmic reticulum (ER) stress. AMPK is another cellular stress sensor, but the relationship between AMPK, ER stress, and GDF15 has not been assessed in vivo. Wildtype (WT), AMPK ß1 deficient (AMPKß1-/- ), and CHOP-/- mice were treated with three distinct AMPK activators; AICAR, which is converted to ZMP mimicking the effects of AMP on the AMPKγ isoform, R419, which indirectly activates AMPK through inhibition of mitochondrial respiration, or A769662, a direct AMPK activator which binds the AMPKß1 isoform ADaM site causing allosteric activation. Following treatments, liver Gdf15, markers of ER-stress, AMPK activity, adenine nucleotides, circulating GDF15, and food intake were assessed. AICAR and R419 caused ER and energetic stress, increased GDF15 expression and secretion, and suppressed food intake. Direct activation of AMPK ß1 containing complexes by A769662 increased hepatic Gdf15 expression, circulating GDF15, and suppressed food intake, independent of ER stress. The effects of AICAR, R419, and A769662 on GDF15 were attenuated in AMPKß1-/- mice. AICAR and A769662 increased GDF15 to a similar extent in WT and CHOP-/- mice. Herein, we provide evidence that AMPK plays a role in mediating the induction of GDF15 under conditions of energetic stress in mouse liver in vivo.

AMPK activation by SC4 inhibits noradrenaline-induced lipolysis and insulin-stimulated lipogenesis in white adipose tissue.

The effects of small-molecule AMP-activated protein kinase (AMPK) activators in rat epididymal adipocytes were compared. SC4 was the most effective and submaximal doses of SC4 and 5-amino-4-imidazolecarboxamide (AICA) riboside were combined to study the effects of AMPK activation in white adipose tissue (WAT). Incubation of rat adipocytes with SC4 + AICA riboside inhibited noradrenaline-induced lipolysis and decreased hormone-sensitive lipase (HSL) Ser563 phosphorylation, without affecting HSL Ser565 phosphorylation. Preincubation of fat pads from wild-type (WT) mice with SC4 + AICA riboside inhibited insulin-stimulated lipogenesis from glucose or acetate and these effects were lost in AMPKα1 knockout (KO) mice, indicating AMPKα1 dependency. Moreover, in fat pads from acetyl-CoA carboxylase (ACC)1/2 S79A/S212A double knockin versus WT mice, the effect of SC4 + AICA riboside to inhibit insulin-stimulated lipogenesis from acetate was lost, pinpointing ACC as the main AMPK target. Treatment with SC4 + AICA riboside decreased insulin-stimulated glucose uptake, an effect that was still observed in fat pads from AMPKα1 KO versus WT mice, suggesting the effect was partly AMPKα1-independent. SC4 + AICA riboside treatment had no effect on the insulin-induced increase in palmitate esterification nor on sn-glycerol-3-phosphate-O-acyltransferase activity. Therefore in WAT, AMPK activation inhibits noradrenaline-induced lipolysis and suppresses insulin-stimulated lipogenesis primarily by inactivating ACC and by inhibiting glucose uptake.

CaMKK2 is inactivated by cAMP-PKA signaling and 14-3-3 adaptor proteins.

The calcium-calmodulin-dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495 We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.

Effects of PKB/Akt inhibitors on insulin-stimulated lipogenesis and phosphorylation state of lipogenic enzymes in white adipose tissue.

We investigated acute effects of two allosteric protein kinase B (PKB) inhibitors, MK-2206 and Akti-1/2, on insulin-stimulated lipogenesis in rat epididymal adipocytes incubated with fructose as carbohydrate substrate. In parallel, the phosphorylation state of lipogenic enzymes in adipocytes and incubated epididymal fat pads was monitored by immunoblotting. Preincubation of rat epididymal adipocytes with PKB inhibitors dose-dependently inhibited the following: insulin-stimulated lipogenesis, increased PKB Ser473 phosphorylation, increased PKB activity and decreased acetyl-CoA carboxylase (ACC) Ser79 phosphorylation. In contrast, the effect of insulin to decrease the phosphorylation of pyruvate dehydrogenase (PDH) at Ser293 and Ser300 was not abolished by PKB inhibition. Insulin treatment also induced ATP-citrate lyase (ACL) Ser454 phosphorylation, but this effect was less sensitive to PKB inhibitors than ACC dephosphorylation by insulin. In incubated rat epididymal fat pads, Akti-1/2 treatment reversed insulin-induced ACC dephosphorylation, while ACL phosphorylation by insulin was maintained. ACL and ACC purified from white adipose tissue were poor substrates for PKBα in vitro. However, effects of wortmannin and torin, along with Akti-1/2 and MK-2206, on recognized PKB target phosphorylation by insulin were similar to their effects on insulin-induced ACL phosphorylation, suggesting that PKB could be the physiological kinase for ACL phosphorylation by insulin. In incubated epididymal fat pads from wild-type versus ACC1/2 S79A/S212A knockin mice, effects of insulin to increase lipogenesis from radioactive fructose or from radioactive acetate were reduced but not abolished. Together, the results support a key role for PKB in mediating insulin-stimulated lipogenesis by decreasing ACC phosphorylation, but not by decreasing PDH phosphorylation.

Mice with Whole-Body Disruption of AMPK-Glycogen Binding Have Increased Adiposity, Reduced Fat Oxidation and Altered Tissue Glycogen Dynamics.

The AMP-activated protein kinase (AMPK), a central regulator of cellular energy balance and metabolism, binds glycogen via its ß subunit. However, the physiological effects of disrupting AMPK-glycogen interactions remain incompletely understood. To chronically disrupt AMPK-glycogen binding, AMPK ß double knock-in (DKI) mice were generated with mutations in residues critical for glycogen binding in both the ß1 (W100A) and ß2 (W98A) subunit isoforms. We examined the effects of this DKI mutation on whole-body substrate utilization, glucose homeostasis, and tissue glycogen dynamics. Body composition, metabolic caging, glucose and insulin tolerance, serum hormone and lipid profiles, and tissue glycogen and protein content were analyzed in chow-fed male DKI and age-matched wild-type (WT) mice. DKI mice displayed increased whole-body fat mass and glucose intolerance associated with reduced fat oxidation relative to WT. DKI mice had reduced liver glycogen content in the fed state concomitant with increased utilization and no repletion of skeletal muscle glycogen in response to fasting and refeeding, respectively, despite similar glycogen-associated protein content relative to WT. DKI liver and skeletal muscle displayed reductions in AMPK protein content versus WT. These findings identify phenotypic effects of the AMPK DKI mutation on whole-body metabolism and tissue AMPK content and glycogen dynamics.

Post-Translational Modifications of the Energy Guardian AMP-Activated Protein Kinase.

Physical exercise elicits physiological metabolic perturbations such as energetic and oxidative stress; however, a diverse range of cellular processes are stimulated in response to combat these challenges and maintain cellular energy homeostasis. AMP-activated protein kinase (AMPK) is a highly conserved enzyme that acts as a metabolic fuel sensor and is central to this adaptive response to exercise. The complexity of AMPK's role in modulating a range of cellular signalling cascades is well documented, yet aside from its well-characterised regulation by activation loop phosphorylation, AMPK is further subject to a multitude of additional regulatory stimuli. Therefore, in this review we comprehensively outline current knowledge around the post-translational modifications of AMPK, including novel phosphorylation sites, as well as underappreciated roles for ubiquitination, sumoylation, acetylation, methylation and oxidation. We provide insight into the physiological ramifications of these AMPK modifications, which not only affect its activity, but also subcellular localisation, nutrient interactions and protein stability. Lastly, we highlight the current knowledge gaps in this area of AMPK research and provide perspectives on how the field can apply greater rigour to the characterisation of novel AMPK regulatory modifications.

Relationships between Mitochondrial Function, AMPK, and TORC1 Signaling in Lymphoblasts with Premutation Alleles of the FMR1 Gene.

The X-linked FMR1 gene contains a non-coding trinucleotide repeat in its 5' region that, in normal, healthy individuals contains 20-44 copies. Large expansions of this region (>200 copies) cause fragile X syndrome (FXS), but expansions of 55-199 copies (referred to as premutation alleles) predispose carriers to a neurodegenerative disease called fragile X-associated tremor/ataxia syndrome (FXTAS). The cytopathological mechanisms underlying FXTAS are poorly understood, but abnormalities in mitochondrial function are believed to play a role. We previously reported that lymphoblastoid cell lines (LCLs, or lymphoblasts) of premutation carriers have elevated mitochondrial respiratory activities. In the carriers, especially those not clinically affected with FXTAS, AMP-activated protein kinase (AMPK) activity was shown to be elevated. In the FXTAS patients, however, it was negatively correlated with brain white matter lesions, suggesting a protective role in the molecular mechanisms. Here, we report an enlarged and extended study of mitochondrial function and associated cellular stress-signaling pathways in lymphoblasts isolated from male and female premutation carriers, regardless of their clinical status, and healthy controls. The results confirmed the elevation of AMPK and mitochondrial respiratory activities and reduction in reactive O2 species (ROS) levels in premutation cells and revealed for the first time that target of rapamycin complex I (TORC1) activities are reduced. Extensive correlation, multiple regression, and principal components analysis revealed the best fitting statistical explanations of these changes in terms of the other variables measured. These suggested which variables might be the most "proximal" regulators of the others in the extensive network of known causal interactions amongst the measured parameters of mitochondrial function and cellular stress signaling. In the resulting model, the premutation alleles activate AMPK and inhibit both TORC1 and ROS production, the reduced TORC1 activity contributes to activation of AMPK and of nonmitochondrial metabolism, and the higher AMPK activity results in elevated catabolic metabolism, mitochondrial respiration, and ATP steady state levels. In addition, the results suggest a separate CGG repeat number-dependent elevation of TORC1 activity that is insufficient to overcome the inhibition of TORC1 in premutation cells but may presage the previously reported activation of TORC1 in FXS cells.

AMPK-ACC signaling modulates platelet phospholipids and potentiates thrombus formation.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.

AMPK ß1 activation suppresses antipsychotic-induced hyperglycemia in mice.

Olanzapine (OLZ) is a second-generation antipsychotic that is used to treat schizophrenia but also causes acute hyperglycemia. This study aimed to determine if the ablation of AMPK ß1-containing complexes potentiates acute OLZ-induced metabolic dysfunction and if the activation of AMPK ß1 suppresses these effects. Female AMPK ß1-/- or wild-type (WT) control mice were treated with OLZ, and changes in blood glucose, serum and liver metabolites, whole-body fuel oxidation, and pyruvate-induced increases in blood glucose were measured. Additionally, WT mice were cotreated with OLZ and A769662, a specific AMPK ß1 activator, and we determined if cotreatment protected against acute, OLZ-induced metabolic dysfunction. OLZ-induced increases in blood glucose were exacerbated in AMPK ß1-/- mice compared with WT mice, and this was paralleled by greater OLZ-induced increases in markers of liver glucose production, such as pyruvate tolerance, serum glucagon, and glucagon responsiveness. Cotreatment with A769662 attenuated OLZ-induced increases in blood glucose, serum nonesterified fatty acid, and glycerol. Furthermore, this effect was absent in AMPK ß1-/- mice, consistent with A769662's specificity for the AMPK ß1 subunit. Reductions in AMPK activity potentiate the effects of acute OLZ treatment on blood glucose, whereas specifically targeting AMPK ß1-containing complexes is sufficient to protect against OLZ-induced hyperglycemia.-Shamshoum, H., Medak, K. D., Townsend, L. K., Ashworth, K. E., Bush, N. D., Hahn, M. K., Kemp, B. E., Wright, D. C. AMPK ß1 activation suppresses antipsychotic-induced hyperglycemia in mice.

ATP synthase inhibitory factor 1 (IF1), a novel myokine, regulates glucose metabolism by AMPK and Akt dual pathways.

ATPase inhibitory factor 1 (IF1) is an ATP synthase-interacting protein that suppresses the hydrolysis activity of ATP synthase. In this study, we observed that the expression of IF1 was up-regulated in response to electrical pulse stimulation of skeletal muscle cells and in exercized mice and healthy men. IF1 stimulates glucose uptake via AMPK in skeletal muscle cells and primary cultured myoblasts. Reactive oxygen species and Rac family small GTPase 1 (Rac1) function in the upstream and downstream of AMPK, respectively, in IF1-mediated glucose uptake. In diabetic animal models, the administration of recombinant IF1 improved glucose tolerance and down-regulated blood glucose level. In addition, IF1 inhibits ATP hydrolysis by ß-F1-ATPase in plasma membrane, thereby increasing extracellular ATP and activating the protein kinase B (Akt) pathway, ultimately leading to glucose uptake. Thus, we suggest that IF1 is a novel myokine and propose a mechanism by which AMPK and Akt contribute independently to IF1-mediated improvement of glucose tolerance impairment. These results demonstrate the importance of IF1 as a potential antidiabetic agent.-Lee, H. J., Moon, J., Chung, I., Chung, J. H., Park, C., Lee, J. O., Han, J. A., Kang, M. J., Yoo, E. H., Kwak, S.-Y., Jo, G., Park, W., Park, J., Kim, K. M., Lim, S., Ngoei, K. R. W., Ling, N. X. Y., Oakhill, J. S., Galic, S., Murray-Segal, L., Kemp, B. E., Mantzoros, C. S., Krauss, R. M., Shin, M.-J., Kim, H. S. ATP synthase inhibitory factor 1 (IF1), a novel myokine, regulates glucose metabolism by AMPK and Akt dual pathways.

Functional analysis of an R311C variant of Ca2+ -calmodulin-dependent protein kinase kinase-2 (CaMKK2) found as a de novo mutation in a patient with bipolar disorder.

OBJECTIVES: Loss-of-function mutations in the gene encoding the calcium-calmodulin (Ca2+ -CaM)-dependent protein kinase kinase-2 (CaMKK2) enzyme are linked to bipolar disorder. Recently, a de novo arginine to cysteine (R311C) mutation in CaMKK2 was identified from a whole exome sequencing study of bipolar patients and their unaffected parents. The aim of the present study was to determine the functional consequences of the R311C mutation on CaMKK2 activity and regulation by Ca2+ -CaM. METHODS: The effects of the R311C mutation on CaMKK2 activity and Ca2+ -CaM activation were examined using a radiolabeled adenosine triphosphate (ATP) kinase assay. We performed immunoblot analysis to determine whether the R311C mutation impacts threonine-85 (T85) autophosphorylation, an activating phosphorylation site on CaMKK2 that has also been implicated in bipolar disorder. We also expressed the R311C mutant in CaMKK2 knockout HAP1 cells and used immunoblot analysis and an MTS reduction assay to study its effects on Ca2+ -dependent downstream signaling and cell viability, respectively. RESULTS: The R311C mutation maps to the conserved HRD motif within the catalytic loop of CaMKK2 and caused a marked reduction in kinase activity and Ca2+ -CaM activation. The R311C mutation virtually abolished T85 autophosphorylation in response to Ca2+ -CaM and exerted a dominant-negative effect in cells as it impaired the ability of wild-type CaMKK2 to initiate downstream signaling and maintain cell viability. CONCLUSIONS: The highly disruptive, loss-of-function impact of the de novo R311C mutation in human CaMKK2 provides a compelling functional rationale for being considered a potential rare monogenic cause of bipolar disorder.

Foam Cell Induction Activates AMPK But Uncouples Its Regulation of Autophagy and Lysosomal Homeostasis.

The dysregulation of macrophage lipid metabolism drives atherosclerosis. AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics and plays essential roles regulating macrophage lipid dynamics. Here, we investigated the consequences of atherogenic lipoprotein-induced foam cell formation on downstream immunometabolic signaling in primary mouse macrophages. A variety of atherogenic low-density lipoproteins (acetylated, oxidized, and aggregated forms) activated AMPK signaling in a manner that was in part due to CD36 and calcium-related signaling. In quiescent macrophages, basal AMPK signaling was crucial for maintaining markers of lysosomal homeostasis as well as levels of key components in the lysosomal expression and regulation network. Moreover, AMPK activation resulted in targeted upregulation of members of this network via transcription factor EB. However, in lipid-induced macrophage foam cells, neither basal AMPK signaling nor its activation affected lysosomal-associated programs. These results suggest that while the sum of AMPK signaling in cultured macrophages may be anti-atherogenic, atherosclerotic input dampens the regulatory capacity of AMPK signaling.

AMP-activated protein kinase selectively inhibited by the type II inhibitor SBI-0206965.

Inhibition of the metabolic regulator AMP-activated protein kinase (AMPK) is increasingly being investigated for its therapeutic potential in diseases where AMPK hyperactivity results in poor prognoses, as in established cancers and neurodegeneration. However, AMPK-inhibitory tool compounds are largely limited to compound C, which has a poor selectivity profile. Here we identify the pyrimidine derivative SBI-0206965 as a direct AMPK inhibitor. SBI-0206965 inhibits AMPK with 40-fold greater potency and markedly lower kinase promiscuity than compound C and inhibits cellular AMPK signaling. Biochemical characterization reveals that SBI-0206965 is a mixed-type inhibitor. A co-crystal structure of the AMPK kinase domain/SBI-0206965 complex shows that the drug occupies a pocket that partially overlaps the ATP active site in a type IIb inhibitor manner. SBI-0206965 has utility as a tool compound for investigating physiological roles for AMPK and provides fresh impetus to small-molecule AMPK inhibitor therapeutic development.

Absence of the ß1 subunit of AMP-activated protein kinase reduces myofibroblast infiltration of the kidneys in early diabetes.

Activation of the heterotrimeric energy-sensing kinase AMP-activated protein kinase (AMPK) has been reported to improve experimental diabetic kidney disease. We examined the effect of type 1 diabetes in wild-type (WT) mice and mice lacking the ß1 subunit of AMPK (AMPK ß1-/- mice), which have reduced AMPK activity in kidneys and other organs. Diabetes was induced using streptozotocin (STZ) and the animals followed up for 4 weeks. Hyperglycaemia was more severe in diabetic AMPK ß1-/- mice, despite the absence of any difference in serum levels of insulin, adiponectin and leptin. There was no change in AMPK activity in the kidneys of diabetic WT mice by AMPK activity assay, or phosphorylation of either the αT172 activation site on the α catalytic subunit of AMPK or the AMPK-specific phosphosite S79 on acetyl CoA carboxylase 1 (ACC1). Phosphorylation of the inhibitory αS485 site on the α subunit of AMPK was significantly increased in the WT diabetic mice compared to non-diabetic controls. Despite increased plasma glucose levels in the diabetic AMPK ß1-/- mice, there were fewer myofibroblasts in the kidneys compared to diabetic WT mice, as evidenced by reduced α-smooth muscle actin (α-SMA) protein by Western blot, mRNA by qRT-PCR and fewer α-SMA-positive cells by immunohistochemical staining. Albuminuria was also reduced in the AMPK ß1-/- mice. In contrast to previous studies, therefore, myofibroblasts were reduced in the kidneys of AMPK ß1-/- diabetic mice compared to diabetic WT mice, despite increased circulating glucose, suggesting that AMPK can worsen renal fibrosis in type 1 diabetes.

Phosphorylation of Acetyl-CoA Carboxylase by AMPK Reduces Renal Fibrosis and Is Essential for the Anti-Fibrotic Effect of Metformin.

BACKGROUND: Expression of genes regulating fatty acid metabolism is reduced in tubular epithelial cells from kidneys with tubulointerstitial fibrosis (TIF), thus decreasing the energy produced by fatty acid oxidation (FAO). Acetyl-CoA carboxylase (ACC), a target for the energy-sensing AMP-activating protein kinase (AMPK), is the major controller of the rate of FAO within cells. Metformin has a well described antifibrotic effect, and increases phosphorylation of ACC by AMPK, thereby increasing FAO. METHODS: We evaluated phosphorylation of ACC in cell and mouse nephropathy models, as well as the effects of metformin administration in mice with and without mutations that reduce ACC phosphorylation. RESULTS: Reduced phosphorylation of ACC on the AMPK site Ser79 occurred in both tubular epithelial cells treated with folate to mimic cellular injury and in wild-type (WT) mice after induction of the folic acid nephropathy model. When this effect was exaggerated in mice with knock-in (KI) Ser to Ala mutations of the phosphorylation sites in ACC, lipid accumulation and fibrosis increased significantly compared with WT. The effect of ACC phosphorylation on fibrosis was confirmed in the unilateral ureteric obstruction model, which showed significantly increased lipid accumulation and fibrosis in the KI mice. Metformin use was associated with significantly reduced fibrosis and lipid accumulation in WT mice. In contrast, in the KI mice, the drug was associated with worsened fibrosis. CONCLUSIONS: These data indicate that reduced phosphorylation of ACC after renal injury contributes to the development of TIF, and that phosphorylation of ACC is required for metformin's antifibrotic action in the kidney.

Ghrelin-AMPK Signaling Mediates the Neuroprotective Effects of Calorie Restriction in Parkinson's Disease.

Calorie restriction (CR) is neuroprotective in Parkinson's disease (PD) although the mechanisms are unknown. In this study we hypothesized that elevated ghrelin, a gut hormone with neuroprotective properties, during CR prevents neurodegeneration in an 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. CR attenuated the MPTP-induced loss of substantia nigra (SN) dopamine neurons and striatal dopamine turnover in ghrelin WT but not KO mice, demonstrating that ghrelin mediates CR's neuroprotective effect. CR elevated phosphorylated AMPK and ACC levels in the striatum of WT but not KO mice suggesting that AMPK is a target for ghrelin-induced neuroprotection. Indeed, exogenous ghrelin significantly increased pAMPK in the SN. Genetic deletion of AMPKß1 and 2 subunits only in dopamine neurons prevented ghrelin-induced AMPK phosphorylation and neuroprotection. Hence, ghrelin signaling through AMPK in SN dopamine neurons mediates CR's neuroprotective effects. We consider targeting AMPK in dopamine neurons may recapitulate neuroprotective effects of CR without requiring dietary intervention.

SnRK1 from Arabidopsis thaliana is an atypical AMPK.

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic ß and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, ß(3) and ßγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique ßγ subunit and one of three non-catalytic ß subunits: ß(1), ß(2) or the plant-specific ß(3) isoform. Thus, the AtSnRK1 heterotrimers contain the atypical ßγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the ß(1), ß(2) and ßγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.

AMPK-dependent inhibitory phosphorylation of ACC is not essential for maintaining myocardial fatty acid oxidation.

RATIONALE: The energy sensor AMP-activated protein kinases (AMPK) is thought to play an important role in regulating myocardial fatty acid oxidation (FAO) via its phosphorylation and inactivation of acetyl coenzyme A carboxylase (ACC). However, studies supporting this have not directly assessed whether the maintenance of FAO rates and subsequent cardiac function requires AMPK-dependent inhibitory phosphorylation of ACC. OBJECTIVE: To determine whether preventing AMPK-mediated inactivation of ACC influences myocardial FAO or function. METHODS AND RESULTS: A double knock-in (DKI) mouse (ACC-DKI) model was generated in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser221 (Ser212 mouse) on ACC2 were mutated to prevent AMPK-dependent inhibitory phosphorylation of ACC. Hearts from ACC-DKI mice displayed a complete loss of ACC phosphorylation at the AMPK phosphorylation sites. Despite the inability of AMPK to regulate ACC activity, hearts from ACC-DKI mice displayed normal basal AMPK activation and cardiac function at both standard and elevated workloads. In agreement with the inability of AMPK in hearts from ACC-DKI mice to phosphorylate and inhibit ACC, there was a significant increase in cardiac malonyl-CoA content compared with wild-type mice. However, cardiac FAO rates were comparable between wild-type and ACC-DKI mice at baseline, during elevated workloads, and after a more stressful condition of myocardial ischemia that is known to robustly activate AMPK. CONCLUSIONS: Our findings show AMPK-dependent inactivation of ACC is not essential for the control of myocardial FAO and subsequent cardiac function during a variety of conditions involving AMPK-independent and AMPK-dependent metabolic adaptations.