Salicylate, a plant product, has been in medicinal use since ancient times. More recently it has been replaced by synthetic derivatives such as aspirin and salsalate, both rapidly broken down to salicylate in vivo. At concentrations reached in plasma following administration of salsalate, or aspirin at high doses, salicylate activates adenosine monophosphate-activated protein kinase (AMPK), a central regulator of cell growth and metabolism. Salicylate binds at the same site as the synthetic activator, A-769662, to cause allosteric activation and inhibition of dephosphorylation of the activating phosphorylation site, Thr172. In AMPK knockout mice, effects of salicylate to increase fat utilization and lower plasma fatty acids in vivo were lost. Our results suggest that AMPK activation could explain some beneficial effects of salsalate and aspirin in humans.The medicinal effects of willow bark have been known since the time of Hippocrates. The active component is salicylate, a hormone produced by plants in response to pathogen infection (1). For medicinal use it was largely replaced by aspirin (acetyl salicylate), which is rapidly broken down to salicylate in vivo (2, 3). Salicylate can also be administered as salsalate, which shows promise for treatment of insulin resistance and type 2 diabetes (4, 5). Aspirin and salicylate inhibit cyclo-oxygenases and hence prostanoid biosynthesis (6), as well as the protein kinase IKKβ in the NF-κB pathway (7). However, some effects of these drugs are still observed in mice deficient in these pathways (8).Adenosine monophosphate-activated protein kinase (AMPK) is a cellular energy sensor conserved throughout eukaryotes. This heterotrimeric enzyme is composed of catalytic α Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts subunits and regulatory β and γ subunits (9, 10). Once activated in response to metabolic stress, AMPK phosphorylates targets that switch off adenosine triphosphate (ATP) consuming processes, while switching on catabolic pathways that generate ATP. AMPK is activated >100-fold by phosphorylation at Thr172 in the α subunit by the tumour suppressor protein kinase, LKB1, or the Ca 2+ -dependent kinase, CaMKKβ (9, 10). Binding of AMP or adenosine diphosphate (ADP) to the γ subunit triggers a conformational change that promotes phosphorylation and inhibits dephosphorylation (11-15), causing a switch to the active form. Binding of AMP (but not ADP) to a second site (15) causes further allosteric activation, leading to >1,000-fold activation overall (16). Most drugs or xenobiotics that activate AMPK work by inhibiting mitochondrial ATP synthesis and increasing the concentration of AMP and ADP (17). However, a synthetic activator, A-769662 (18), which also causes allosteric activation and inhibits Thr172 dephosphorylation, binds directly to AMPK at distinct site(s) (19-21).Salicylate, but not aspirin, activated AMPK when applied to HEK-293 cells, with its effects being significant at 1 mM and above ( Fig. 1A; it appears that the estera...

Abstract-The predominant cardiac Ca 2ϩ /calmodulin-dependent protein kinase (CaMK) is CaMKII␦. Here we acutely overexpress CaMKII␦ C using adenovirus-mediated gene transfer in adult rabbit ventricular myocytes. This circumvents confounding adaptive effects in CaMKII␦ C transgenic mice. CaMKII␦ C protein expression and activation state (autophosphorylation) were increased 5-to 6-fold. Basal twitch contraction amplitude and kinetics (1 Hz) were not changed in CaMKII␦ C versus LacZ expressing myocytes. However, the contraction-frequency relationship was more negative, frequency-dependent acceleration of relaxation was enhanced ( 0.5Hz / 3Hz ϭ2.14Ϯ0.10 versus 1.87Ϯ0.10), and peak Ca 2ϩ current (I Ca ) was increased by 31% (Ϫ7.1Ϯ0.

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by -linked β--acetylglucosamine (-GlcNAc). In the present study, we tested the hypothesis that AMPK controls -GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced -GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

Type 2 diabetes is characterized by a progressive resistance of peripheral tissues to insulin. Recent data have established the lipid phosphatase SH2 domain-containing inositol phosphatase 2 (SHIP2) as a critical negative regulator of insulin signal transduction. Mutations in the SHIP2 gene are associated with type 2 diabetes. Here, we used hyperglycemic and hyperinsulinemic KKA y mice to gain insight into the signaling events and metabolic changes triggered by SHIP2 inhibition in vivo. Liver-specific expression of a dominant-negative SHIP2 mutant in KKA y mice increased basal and insulin-stimulated Akt phosphorylation. Protein levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase were significantly reduced, and consequently the liver produced less glucose through gluconeogenesis. Furthermore, SHIP2 inhibition improved hepatic glycogen metabolism by modulating the phosphorylation states of glycogen phosphorylase and glycogen synthase, which ultimately increased hepatic glycogen content. Enhanced glucokinase and reduced pyruvate dehydrogenase kinase 4 expression, together with increased plasma triglycerides, indicate improved glycolysis. As a consequence of the insulin-mimetic effects on glycogen metabolism, gluconeogenesis, and glycolysis, the liverspecific inhibition of SHIP2 improved glucose tolerance and markedly reduced prandial blood glucose levels in KKA y mice. These results support the attractiveness of a specific inhibition of SHIP2 for the prevention and/or treatment of type 2 diabetes. Diabetes 56:2235-2241, 2007 A key event in insulin signal transduction is the increase in phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P 3 ], which recruits and activates downstream effectors at the plasma membrane (1). The lipid phosphatase SH2 domain-containing inositol phosphatase 2 (SHIP2) is expressed in insulin target tissues and dephosphorylates PI(3,4,5)P 3 at the D5 position (2,3). Activating mutations in the SHIP2 gene contribute to the genetic susceptibility to type 2 diabetes in humans (4). Mice lacking the SHIP2 gene (Inppl1 Ϫ/Ϫ mice) are viable and have normal glucose and insulin levels with normal insulin and glucose tolerances. However, Inppl1Ϫ/Ϫ mice are protected from diet-induced obesity, hyperglycemia, and insulin resistance (5). The function of SHIP2 in differentiated L6 myotubes and 3T3-L1 adipocytes was extensively studied by inhibition of the endogenous SHIP2 via overexpression of a dominantnegative SHIP2 mutant (dnSHIP2). SHIP2 inhibition increases phosphorylation of Akt (also known as protein kinase B) and glycogen synthesis in both cell lines (6,7). The liver-specific overexpression of dnSHIP2 in db/db mice results in dramatically decreased fasting glucose levels due to the reduced expression of the gluconeogenic genes glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) (8). However, the strong reduction in fasting blood glucose concentration confounds the interpretation of an improved glucose tolerance test in this model. Furthermore, an effect o...

Abstract-The effect of the 12-kDa isoform of FK-506 -binding protein (FKBP)12.0 on cardiac excitationcontraction coupling was studied in adult rabbit ventricular myocytes after transfection with a recombinant adenovirus coding for human FKBP12.0 (Ad-FKBP12.0). Western blots confirmed overexpression (by 2.6Ϯ0.4 fold, nϭ5). FKBP12.0 association with rabbit cardiac ryanodine receptor (RyR2) was not detected by immunoprecipitation. However, glutathione S-transferase pull-down experiments indicated FKBP12.0 -RyR2 binding to proteins isolated from human and rabbit but not dog myocardium. (FKBP), which binds to the RyR2 with a maximum stoichiometry of 4 FKBP:1 RyR2. 1 Two members of the FKBP family are expressed within mammalian ventricular cardiomyocytes: FKBP12.0 (12.0 kDa) and FKBP12.6 (12.6 kDa). The cytosolic concentration of FKBP12.0 is almost 10-fold higher than FKBP12.6. 1,2 In some species (eg, canine), the affinity of FKBP12.0 for RyR2 is Ϸ500 times lower than for FKBP12.6, hence binding is negligible under physiological conditions. 1 In others (including rabbit and human), the affinity of FKBP12.0 for RyR2 is only Ϸ7 times lower than for FKBP12.6 1 ; therefore, it is conceivable that FKBP12.0 -RyR2 interaction occurs in these species under physiological conditions. 1 The consequences of FKBP12.0 binding on RyR2 function are unclear; currently, no pharmacological intervention can differentiate between the effects of FKBP12.0 and -12.6. FKBP12.0-null mice showed RyR2 dysfunction 3 either resulting from the absence of RyR2-FKBP12.0 binding or as an indirect consequence of the accompanying developmental defects. FKBP12.6-null mice lack developmental defects, supporting the view that the FKBP12.0 isoform is indispensable for normal cardiac development. In the current study, adenoviral-mediated transfection of the human FKBP12.0 gene was used to overexpress FKBP12.0 in isolated rabbit ventricular cardiomyocytes over 48 hours. The choice of rabbit heart tissue was based on the similarities to human heart in terms of relative affinities of FKBP12.0 and FKBP12.6 for RyR2. Materials and Methods Recombinant Adenovirus Vector ConstructionFull-length cDNA of the human FKBP12.0 gene was cloned by polymerase chain reaction (PCR) from human heart muscle-specific cDNA samples by the use of PCR primers that span the entire coding region of FKBP12.0 cDNA. This sequence was inserted downstream from a cytomegalovirus promoter into vector pACCMV ⅐ pLpA, and recombination with vector pJM17 was performed in HEK293 cells. The production, purification, and titration of adenovirus containing the FKBP12.0 gene (Ad-FKBP12.0) were performed according to standard procedures. 4 Previous studies have used an adenovirus containing the human FKBP12.6 gene. 2,5 At 100 multiplicities of infection, this Ad-FKBP12.6 vector caused an overexpression of FKBP12.6 of Ϸ6-fold normal values after 48 hours of incubation. Ventricular Cardiomyocyte Isolation and TransfectionNew Zealand White rabbits (2 to 2.5 kg) were euthanized by administration of ...

In Type 2 diabetes, increased glycogenolysis contributes to the hyperglycaemic state, therefore the inhibition of GP (glycogen phosphorylase), a key glycogenolytic enzyme, is one of the possibilities to lower plasma glucose levels. Following this strategy, a number of GPis (GP inhibitors) have been described. However, certain critical issues are associated with their mode of action, e.g. an impairment of muscle function. The interaction between GP and the liver glycogen targeting subunit (termed G(L)) of PP1 (protein phosphatase 1) has emerged as a new potential anti-diabetic target, as the disruption of this interaction should increase glycogen synthesis, potentially providing an alternative approach to counteract the enhanced glycogenolysis without inhibiting GP activity. We identified an inhibitor of the G(L)-GP interaction (termed G(L)-GPi) and characterized its mechanism of action in comparison with direct GPis. In primary rat hepatocytes, at elevated glucose levels, the G(L)-GPi increased glycogen synthesis similarly to direct GPis. Direct GPis significantly reduced the cellular GP activity, caused a dephosphorylation of the enzyme and decreased the amounts of GP in the glycogen-enriched fraction; the G(L)-GPi did not influence any of these parameters. Both mechanisms increased glycogen accumulation at elevated glucose levels. However, at low glucose levels, only direct GPis led to increased glycogen amounts, whereas the G(L)-GPi allowed the mobilization of glycogen because it did not block the activity of GP. Due to this characteristic, G(L)-GPi in comparison with GPis could offer an advantageous risk/benefit profile circumventing the potential downsides of a complete prevention of glycogen breakdown while retaining glucose-lowering efficacy, suggesting that inhibition of the G(L)-GP interaction may provide an attractive novel approach for rebalancing the disturbed glycogen metabolism in diabetic patients.

Vascular aging is based on the development of endothelial dysfunction, which is thought to be promoted by senescent cells accumulating in aged tissues and is possibly affected by their environment via inflammatory mediators and oxidative stress. Senescence appears to be closely interlinked with changes in cell metabolism. Here, we describe an upregulation of both glycolytic and oxidative glucose metabolism in replicative senescent endothelial cells compared to young endothelial cells by employing metabolic profiling and glucose flux measurements and by analyzing the expression of key metabolic enzymes. Senescent cells exhibit higher glycolytic activity and lactate production together with an enhanced expression of lactate dehydrogenase A as well as increases in tricarboxylic acid cycle activity and mitochondrial respiration. The latter is likely due to the reduced expression of pyruvate dehydrogenase kinases (PDHKs) in senescent cells, which may lead to increased activity of the pyruvate dehydrogenase complex. Cellular and mitochondrial ATP production were elevated despite signs of mitochondrial dysfunction, such as an increased production of reactive oxygen species and extended mitochondrial mass. A shift from glycolytic to oxidative glucose metabolism induced by pharmacological inhibition of PDHKs in young endothelial cells resulted in premature senescence, suggesting that alterations in cellular glucose metabolism may act as a driving force for senescence in endothelial cells.

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