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AMP-activated protein kinase (AMPK) is a key regulator of metabolism and a recognised target for the treatment of metabolic diseases such as Type 2 diabetes (T2D). Here, we review how mass spectrometry (MS) can be used to study short-term control by AMPK via protein phosphorylation and long-term control due to changes in protein expression. We discuss how MS can quantify AMPK subunit levels in tissues from different species. We propose hydrogen-deuterium exchange (HDX)-MS to investigate molecular mechanisms of AMPK activation and thermoproteomic profiling (TPP) to assess off-target effects of pharmacological AMPK activators/inhibitors. Lastly, because large MS data sets are generated, we consider different approaches that can be used for their interpretation., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Insulin resistance in obesity and type 2 diabetes has been shown to be associated with decreased de novo fatty acid (FA) synthesis in adipose tissue. It is known that insulin can acutely stimulate FA synthesis in adipocytes; however, the mechanisms underlying this effect are unclear. The rate-limiting step in FA synthesis is catalyzed by acetyl-CoA carboxylase (ACC), known to be regulated through inhibitory phosphorylation at S79 by the AMP-activated protein kinase (AMPK). Previous results from our laboratory showed an inhibition of AMPK activity by insulin, which was accompanied by PKB-dependent phosphorylation of AMPK at S485. However, whether the S485 phosphorylation is required for insulin-induced inhibition of AMPK or other mechanisms underlie the reduced kinase activity is not known. To investigate this, primary rat adipocytes were transduced with a recombinant adenovirus encoding AMPK-WT or a nonphosphorylatable AMPK S485A mutant. AMPK activity measurements by Western blot analysis and in vitro kinase assay revealed that WT and S485A AMPK were inhibited to a similar degree by insulin, indicating that AMPK S485 phosphorylation is not required for insulin-induced AMPK inhibition. Further analysis suggested an involvement of decreased AMP-to-ATP ratios in the insulin-induced inhibition of AMPK activity, whereas a possible contribution of phosphodiesterases was excluded. Furthermore, we show that insulin-induced AMPK S485 phosphorylation also occurs in human adipocytes, suggesting it to be of an importance yet to be revealed. Altogether, this study increases our understanding of how insulin regulates AMPK activity, and with that, FA synthesis, in adipose tissue.

NLRP3 inflammasome plays a key role in Western diet-induced systemic inflammation and was recently shown to mediate long-lasting trained immunity in myeloid cells. Saturated fatty acids (SFAs) are sterile triggers able to induce the assembly of the NLRP3 inflammasome in macrophages, leading to IL-1β secretion while unsaturated ones (UFAs) prevent SFAs-mediated NLRP3 activation. Unlike previous studies using LPS-primed bone marrow derived macrophages, we do not see any ROS or IRE-1α involvement in SFAs-mediated NLRP3 activation in human monocytes-derived macrophages. Rather we show that SFAs need to enter the cells and to be activated into acyl-CoA to lead to NLRP3 activation in human macrophages. However, their β-oxidation is dispensable. Instead, they are channeled towards phospholipids but redirected towards lipid droplets containing triacylglycerol in the presence of UFAs. Lipidomic analyses and Laurdan fluorescence experiments demonstrate that SFAs induce a dramatic saturation of phosphatidylcholine (PC) correlated with a loss of membrane fluidity, both events inhibited by UFAs. The silencing of CCTα, the key enzyme in PC synthesis, prevents SFA-mediated NLRP3 activation, demonstrating the essential role of the de novo PC synthesis. This SFA-induced membrane remodeling promotes a disruption of the plasma membrane Na, K-ATPase, instigating a K + efflux essential and sufficient for NLRP3 activation. This work opens novel therapeutic avenues to interfere with Western diet-associated diseases such as those targeting the glycerolipid pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

AMP-activated protein kinase (AMPK) plays a key role in energy homeostasis and is activated in response to contraction-induced ATP depletion in skeletal muscle via a rise in intracellular AMP/ADP concentrations. AMP can be deaminated by AMP-deaminase (AMPD) to IMP, which is hydrolyzed to inosine by cytosolic 5'-nucleotidase II (NT5C2). AMP can also be hydrolyzed to adenosine by cytosolic 5'-nucleotidase 1A (NT5C1A). Previous gene silencing and overexpression studies indicated control of AMPK activation by NT5C enzymes. In the present study using gene knockout mouse models, we investigated the effects of NT5C1A and NT5C2 deletion on intracellular adenine nucleotide levels and AMPK activation in electrically stimulated skeletal muscles. Surprisingly, NT5C enzyme knockout did not lead to enhanced AMP or ADP concentrations in response to contraction, with no potentiation of increases in AMPK activity in extensor digitorum longus (EDL) and soleus mouse muscles. Moreover, dual blockade of AMP metabolism in EDL using an AMPD inhibitor combined with NT5C1A deletion did not enhance rises in AMP and ADP or increased AMPK activation by electrical stimulation. The results on muscles from the NT5C knockout mice contradict previous findings where AMP levels and AMPK activity were shown to be modulated by NT5C enzymes., (Copyright © 2017 the American Physiological Society.)

AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α-subunit (α1 and α2) and regulatory β (β1 and β2)- and γ (γ1, γ2, γ3)-subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently described potent AMPK activator called 991, in combination with the commonly used activator 5-aminoimidazole-4-carboxamide riboside or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that the γ3-subunit is exclusively expressed in skeletal muscle and has been implicated in contraction-induced glucose transport, we measured the activity of AMPKγ3 as well as ubiquitously expressed γ1-containing complexes. We initially validated the specificity of the antibodies for the assessment of isoform-specific AMPK activity using AMPK-deficient mouse models. We observed that a low dose of 991 (5 μM) stimulated a modest or negligible activity of both γ1- and γ3-containing AMPK complexes. Strikingly, dual treatment with 991 and 5-aminoimidazole-4-carboxamide riboside or 991 and contraction profoundly enhanced AMPKγ1/γ3 complex activation and glucose transport compared with any of the single treatments. The study demonstrates the utility of a dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types, including skeletal muscle., (Copyright © 2016 the American Physiological Society.)

Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal α-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

AMPK (AMP-activated protein kinase) is an attractive therapeutic drug target for treating metabolic disorders. We studied the effects of an AMPK activator developed by Merck (ex229 from patent application WO2010036613), comparing chemical activation with contraction in intact incubated skeletal muscles. We also compared effects of ex229 with those of the Abbott A769662 compound and AICAR (5-amino-4-imidazolecarboxamide riboside). In rat epitrochlearis muscle, ex229 dose-dependently increased AMPK activity of α1-, α2-, β1- and β2-containing complexes with significant increases in AMPK activity seen at a concentration of 50 μM. At a concentration of 100 μM, AMPK activation was similar to that observed after contraction and importantly led to an ~2-fold increase in glucose uptake. In AMPK α1-/α2-catalytic subunit double-knockout myotubes incubated with ex229, the increases in glucose uptake and ACC (acetyl-CoA carboxylase) phosphorylation seen in control cells were completely abolished, suggesting that the effects of the compound were AMPK-dependent. When muscle glycogen levels were reduced by ~50% after starvation, ex229-induced AMPK activation and glucose uptake were amplified in a wortmannin-independent manner. In L6 myotubes incubated with ex229, fatty acid oxidation was increased. Furthermore, in mouse EDL (extensor digitorum longus) and soleus muscles, ex229 increased both AMPK activity and glucose uptake at least 2-fold. In summary, ex229 efficiently activated skeletal muscle AMPK and elicited metabolic effects in muscle appropriate for treating Type 2 diabetes by stimulating glucose uptake and increasing fatty acid oxidation.

PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.

Muscle protein synthesis rates decrease during contraction/exercise, but rapidly increase post-exercise. Previous studies mainly focused on signaling pathways that control protein synthesis during post-exercise recovery, such as mTOR and its downstream targets S6K1 and 4E-BP1. In this study, we investigated the effect of high-frequency electrical stimulation on the phosphorylation state of signaling components controlling protein synthesis in rat skeletal muscle. Electrical stimulation increased S6K1 Thr389 phosphorylation, which was unaffected by Torin1, a selective mTOR inhibitor, suggesting that S6K1 phosphorylation by contraction was mTOR-independent. Phosphorylation of eIF4B Ser422 was also increased during electrical stimulation, which was abrogated by inhibition of MEK/ERK/RSK1 activation. Moreover, although phosphorylation of conventional mTOR sites in 4E-BP1 decreased during contraction, mTOR-independent phosphorylation was also apparent, which was associated with the release of 4E-BP1 from eIF4E. The results indicate mTOR-independent phosphorylation of S6K1 and 4E-BP1 and suggest MEK/ERK/RSK1-dependent phosphorylation of eIF4B during skeletal muscle contraction. These phosphorylation events would keep the translation initiation machinery "primed" in an active state so that protein synthesis could quickly resume post-exercise., (Copyright © 2013 Elsevier Inc. All rights reserved.)

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose 2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is involved in cell proliferation owing to its role in carbohydrate metabolism. In the present study we analysed the mechanism of regulation of PFKFB3 as an immediate early gene controlled by stress stimuli that activates the p38/MK2 [MAPK (mitogen-activated protein kinase)-activated protein kinase 2] pathway. We report that exposure of HeLa and T98G cells to different stress stimuli (NaCl, H2O2, UV radiation and anisomycin) leads to a rapid increase (15-30 min) in PFKFB3 mRNA levels. The use of specific inhibitors in combination with MK2-deficient cells implicate control by the protein kinase MK2. Transient transfection of HeLa cells with deleted gene promoter constructs allowed us to identify an SRE (serum-response element) to which SRF (serum-response factor) binds and thus transactivates PFKFB3 gene transcription. Direct binding of phospho-SRF to the SRE sequence (-918 nt) was confirmed by ChIP (chromatin immunoprecipiation) assays. Moreover, PFKFB3 isoenzyme phosphorylation at Ser461 by MK2 increases PFK-2 activity. Taken together, the results of the present study suggest a multimodal mechanism of stress stimuli affecting PFKFB3 transcriptional regulation and kinase activation by protein phosphorylation, resulting in an increase in Fru-2,6-P2 concentration and stimulation of glycolysis in cancer cells.

Eukaryotic elongation factor 2 (eEF-2) and mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K) signaling pathways control protein synthesis and are inhibited during myocardial ischemia. Intracellular acidosis and AMP-activated protein kinase (AMPK) activation, both occurring during ischemia, have been proposed to participate in this inhibition. We evaluated the contribution of AMPKα2, the main cardiac AMPK catalytic subunit isoform, in eEF2 and mTOR-p70S6K regulation using AMPKα2 KO mice. Hearts were perfused ex vivo with or without insulin, and then submitted or not to ischemia. Insulin pre-incubation was necessary to activate mTOR-p70S6K and evaluate their subsequent inhibition by ischemia. Ischemia decreased insulin-induced mTOR-p70S6K phosphorylation in WT and AMPKα2 KO mice to a similar extent. This AMPKα2-independent p70S6K inhibition correlated well with the inhibition of PKB/Akt, located upstream of mTOR-p70S6K and can be mimicked in cardiomyocytes by decreasing pH. By contrast, ischemia-induced inhibitory phosphorylation of eEF-2 was drastically reduced in AMPKα2 KO mice. Interestingly, AMPKα2 also played a role under normoxia. Its deletion increased the insulin-induced p70S6K stimulation. This p70S6K over-stimulation was associated with a decrease in inhibitory phosphorylation of Raptor, an mTOR partner identified as an AMPK target. In conclusion, AMPKα2 controls cardiac p70S6K under normoxia and regulates eEF-2 but not the mTOR-p70S6K pathway during ischemia. This challenges the accepted notion that mTOR-p70S6K is inhibited by myocardial ischemia mainly via an AMPK-dependent mechanism., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This study was undertaken to investigate whether the mechanism of increased Na(+)-K(+)-2Cl(-) (NKCC1) cotransporter activity by osmotic shrinkage involved AMP-activated protein kinase (AMPK) activation. AMPK was found to phosphorylate a recombinant GST-dogfish (1-260) NKCC1 fragment at Ser38 and Ser214, corresponding to Ser77 and Ser242 in human NKCC1, respectively. Incubation of human erythrocytes with 20 microM A769662 AMPK activator increased Ser242 NKCC1 phosphorylation but did not stimulate (86)Rb(+) uptake. Under hypertonic conditions in human red blood cells (RBCs) incubated with 0.3 M sucrose, NKCC1 activity increased as measured by bumetanide-sensitive (86)Rb(+) uptake and AMPK was activated. However, there was no effect of AMPKalpha1 deletion in mouse RBCs on the increased rate of (86)Rb(+) uptake induced by hyperosmolarity. AMPK activation by osmotic shrinkage of mouse RBCs was abrogated by 10 microM STO-609 CaMKKbeta inhibitor, but incubation with STO-609 did not affect the increase in (86)Rb(+) uptake induced by hyperosmolarity. Osmotic shrinkage of human and mouse RBCs led to activation loop phosphorylation of the STE20/SPS1-related proline/alanine-rich kinase (SPAK) at Thr233, which was accompanied by phosphorylation of NKCC1 at Thr203/207/212, one of which (Thr207) is responsible for cotransporter activation. Therefore, phosphorylation-induced activation of NKCC1 by osmotic shrinkage does not involve AMPK and is likely to be due to SPAK activation.

Long-term survival of oxygen deprivation by animals with well-developed anoxia tolerance depends on multiple biochemical adaptations including strong metabolic rate depression. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the suppression of protein synthesis that occurs when turtles experience anoxic conditions. AMPK activity and the phosphorylation state of ribosomal translation factors were measured in liver, heart, red muscle and white muscle of red-eared slider turtles (Trachemys scripta elegans) subjected to 20 h of anoxic submergence. AMPK activity increased twofold in white muscle of anoxic turtles compared with aerobic controls but remained unchanged in liver and red muscle, whereas in heart AMPK activity decreased by 40%. Immunoblotting with phospho-specific antibodies revealed that eukaryotic elongation factor-2 phosphorylation at the inactivating Thr56 site increased six- and eightfold in red and white muscles from anoxic animals, respectively, but was unchanged in liver and heart. The phosphorylation state of the activating Thr389 site of p70 ribosomal protein S6 kinase was reduced under anoxia in red muscle and heart but was unaffected in liver and white muscle. Exposure to anoxia decreased 40S ribosomal protein S6 phosphorylation in heart and promoted eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) dephosphorylation in red muscle, but surprisingly increased 4E-BP1 phosphorylation in white muscle. The changes in phosphorylation state of translation factors suggest that organ-specific patterns of signalling and response are involved in achieving the anoxia-induced suppression of protein synthesis in turtles.

The kinetics of myosin regulatory light chain (MLC) phosphorylation by recombinant AMP-activated protein kinase (AMPK) were compared with commercial AMPK from rat liver and smooth muscle myosin light chain kinase (smMLCK). With identical amounts of activity units, initial rates of phosphorylation of MLC were at least 100-fold less with recombinant AMPK compared to smMLCK, whereas with rat liver AMPK significant phosphorylation was seen. In Madin-Darby Canine Kidney cells, AMPK activation led to an increase in MLC phosphorylation, which was decreased by a Rho kinase inhibitor without affecting AMPK activation. Therefore, MLC phosphorylation during energy deprivation does not result from direct phosphorylation by AMPK.

Class IIa histone deacetylases (HDACs) act as key transcriptional regulators in several important developmental programs. Their activities are controlled via phosphorylation-dependent nucleocytoplasmic shuttling. Phosphorylation of conserved serine residues triggers association with 14-3-3 proteins and cytoplasmic relocalization of class IIa HDACs, which leads to the derepression of their target genes. Although a lot of effort has been made toward the identification of the inactivating kinases that phosphorylate class IIa HDAC 14-3-3 motifs, the existence of an antagonistic protein phosphatase remains elusive. Here we identify PP2A as a phosphatase responsible for dephosphorylating the 14-3-3 binding sites in class IIa HDACs. Interestingly, dephosphorylation of class IIa HDACs by PP2A is prevented by competitive association of 14-3-3 proteins. Using both okadaic acid treatment and RNA interference, we demonstrate that PP2A constitutively dephosphorylates the class IIa member HDAC7 to control its biological functions as a regulator of T cell apoptosis and endothelial cell functions. This study unravels a dynamic interplay among 14-3-3s, protein kinases, and PP2A and provides a model for the regulation of class IIa HDACs.

In rat epitrochlearis skeletal muscle, contraction inhibited the basal and insulin-stimulated rates of protein synthesis by 75 and 70%, respectively, while increasing adenosine monophosphate-activated protein kinase (AMPK) activity. Insulin, on the other hand, stimulated protein synthesis (by 30%) and increased p70 ribosomal protein S6 kinase (p70S6K) Thr389, 40S ribosomal protein S6 (rpS6) Ser235/236, rpS6 Ser240/244 and eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) Thr37/46 phosphorylation over basal values. Electrical stimulation had no effect on mammalian target of rapamycin complex 1 (mTORC1) signalling, as reflected by the lack of reduction in basal levels of p70S6K, rpS6 Ser235/236, rpS6 Ser240/244 and 4E-BP1 phosphorylation, but did antagonize mTORC1 signalling after stimulation of the pathway by insulin. Eukaryotic elongation factor-2 (eEF2) Thr56 phosphorylation increased rapidly on electrical stimulation reaching a maximum at 1 min, whereas AMPK Thr172 phosphorylation slowly increased to reach threefold after 30 min. Eukaryotic elongation factor-2 kinase (eEF2K) was not activated after 30 min of contraction when AMPK was activated. This could not be explained by the expression of a tissue-specific isoform of eEF2K in skeletal muscle lacking the Ser398 AMPK phosphorylation site. Therefore, in this skeletal muscle system, the contraction-induced inhibition of protein synthesis could not be attributed to a reduction in mTORC1 signalling but could be due to an increase in eEF2 phosphorylation independent of AMPK activation.

Survival in the frozen state depends on biochemical adaptations that deal with multiple stresses on cells including long-term ischaemia and tissue dehydration. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the metabolic re-sculpting that occurs during freezing. AMPK activity and the phosphorylation state of translation factors were measured in liver and skeletal muscle of wood frogs (Rana sylvatica) subjected to anoxia, dehydration, freezing, and thawing after freezing. AMPK activity was increased 2-fold in livers of frozen frogs compared with the controls whereas in skeletal muscle, AMPK activity increased 2.5-, 4.5- and 3-fold in dehydrated, frozen and frozen/thawed animals, respectively. Immunoblotting with phospho-specific antibodies revealed an increase in the phosphorylation state of eukaryotic elongation factor-2 at the inactivating Thr56 site in livers from frozen frogs and in skeletal muscles of anoxic frogs. No change in phosphorylation state of eukaryotic initiation factor-2alpha at the inactivating Ser51 site was seen in the tissues under any of the stress conditions. Surprisingly, ribosomal protein S6 phosphorylation was increased 2-fold in livers from frozen frogs and 10-fold in skeletal muscle from frozen/thawed animals. However, no change in translation capacity was detected in cell-free translation assays with skeletal muscle extracts under any of the experimental conditions. The changes in phosphorylation state of translation factors are discussed in relation to the control of protein synthesis and stress-induced AMPK activation.

Previous studies showed that insulin antagonizes AMP-activated protein kinase activation by ischemia and that protein kinase B might be implicated. Here we investigated whether the direct phosphorylation of AMP-activated protein kinase by protein kinase B might participate in this effect. Protein kinase B phosphorylated recombinant bacterially expressed AMP-activated protein kinase heterotrimers at Ser(485) of the alpha1-subunits. In perfused rat hearts, phosphorylation of the alpha1/alpha2 AMP-activated protein kinase subunits on Ser(485)/Ser(491) was increased by insulin and insulin pretreatment decreased the phosphorylation of the alpha-subunits at Thr(172) in a subsequent ischemic episode. It is proposed that the effect of insulin to antagonize AMP-activated protein kinase activation involves a hierarchical mechanism whereby Ser(485)/Ser(491) phosphorylation by protein kinase B reduces subsequent phosphorylation of Thr(172) by LKB1 and the resulting activation of AMP-activated protein kinase.

Fructose 2,6-bisphosphate is a potent allosteric activator of trypanosomatid pyruvate kinase and thus represents an important regulator of energy metabolism in these protozoan parasites. A 6-phosphofructo-2-kinase, responsible for the synthesis of this regulator, was highly purified from the bloodstream form of Trypanosoma brucei and kinetically characterized. By searching trypanosomatid genome databases, four genes encoding proteins homologous to the mammalian bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) were found for both T. brucei and the related parasite Leishmania major and four pairs in Trypanosoma cruzi. These genes were predicted to each encode a protein in which, at most, only a single domain would be active. Two of the T. brucei proteins showed most conservation in the PFK-2 domain, although one of them was predicted to be inactive due to substitution of residues responsible for ligating the catalytically essential divalent metal cation; the two other proteins were most conserved in the FBPase-2 domain. The two PFK-2-like proteins were expressed in Escherichia coli. Indeed, the first displayed PFK-2 activity with similar kinetic properties to that of the enzyme purified from T. brucei, whereas no activity was found for the second. Interestingly, several of the predicted trypanosomatid PFK-2/FBPase-2 proteins have long N-terminal extensions. The N-terminal domains of the two polypeptides with most similarity to mammalian PFK-2s contain a series of tandem repeat ankyrin motifs. In other proteins such motifs are known to mediate protein-protein interactions. Phylogenetic analysis suggests that the four different PFK-2/FBPase-2 isoenzymes found in Trypanosoma and Leishmania evolved from a single ancestral bifunctional enzyme within the trypanosomatid lineage. A possible explanation for the evolution of multiple monofunctional enzymes and for the presence of the ankyrin-motif repeats in the PFK-2 isoenzymes is presented.

Familial juvenile hyperuricemic nephropathy (FJHN [MIM 162000]) is an autosomal-dominant disorder characterized by abnormal tubular handling of urate and late development of chronic interstitial nephritis leading to progressive renal failure. A locus for FJHN was previously identified on chromosome 16p12 close to the MCKD2 locus, which is responsible for a variety of autosomal-dominant medullary cystic kidney disease (MCKD2). UMOD, the gene encoding the Tamm-Horsfall/uromodulin protein, maps within the FJHN/MCKD2 critical region. Mutations in UMOD were recently reported in nine families with FJHN/MCKD2 disease. A mutation in UMOD has been identified in 11 FJHN families (10 missense and one in-frame deletion)-10 of which are novel-clustering in the highly conserved exon 4. The consequences of UMOD mutations on uromodulin expression were investigated in urine samples and renal biopsies from nine patients in four families. There was a markedly increased expression of uromodulin in a cluster of tubule profiles, suggesting an accumulation of the protein in tubular cells. Consistent with this observation, urinary excretion of wild-type uromodulin was significantly decreased. The latter findings were not observed in patients with FJHN without UMOD mutations. In conclusion, this study points to a mutation clustering in exon 4 of UMOD as a major genetic defect in FJHN. Mutations in UMOD may critically affect the function of uromodulin, resulting in abnormal accumulation within tubular cells and reduced urinary excretion.

The stimulation of heart glycolysis by insulin and ischemia involves the recruitment of the glucose transporter GLUT4 to the plasma membrane and the activation of 6-phosphofructo-2-kinase (PFK-2), which in turn increases the concentration of fructose 2,6-bisphosphate, a well-known stimulator of glycolysis. This review focuses on the mechanisms responsible for PFK-2 activation by insulin and ischemia in heart. Heart PFK-2 is phosphorylated by various protein kinases, including protein kinase B (PKB), thought to mediate most, if not all, short-term effects of insulin, and the AMP-activated protein kinase (AMPK), known to be activated under anaerobic conditions. We found that PKB is not required for PFK-2 activation by insulin and we partially purified an insulin-sensitive PFK-2 kinase, that differs from PKB and from other insulin-stimulated protein kinases. We also demonstrated that AMPK mediates PFK-2 activation by ischemia. Finally, our study of the interaction between the signaling pathways of insulin and ischemia revealed opposite effects on signaling. Intracellular acidosis induced by ischemia inhibited insulin signaling, whereas insulin pretreatment antagonized AMPK activation by ischemia.