AMPK inhibits liver gluconeogenesis: fact or fiction?

Is there a role for AMPK in the control of hepatic gluconeogenesis and could targeting AMPK in liver be a viable strategy for treating type 2 diabetes? These are frequently asked questions this review tries to answer. After describing properties of AMPK and different small-molecule AMPK activators, we briefly review the various mechanisms for controlling hepatic glucose production, mainly via gluconeogenesis. The different experimental and genetic models that have been used to draw conclusions about the role of AMPK in the control of liver gluconeogenesis are critically discussed. The effects of several anti-diabetic drugs, particularly metformin, on hepatic gluconeogenesis are also considered. We conclude that the main effect of AMPK activation pertinent to the control of hepatic gluconeogenesis is to antagonize glucagon signalling in the short-term and, in the long-term, to improve insulin sensitivity by reducing hepatic lipid content.

Inhibition of basal and glucagon-induced hepatic glucose production by 991 and other pharmacological AMPK activators.

Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKß1-/- mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKß1-/- mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia.

New insight in understanding the contribution of SGLT1 in cardiac glucose uptake: evidence for a truncated form in mice and humans.

Although sodium glucose cotransporter 1 (SGLT1) has been identified as one of the major SGLT isoforms expressed in the heart, its exact role remains elusive. Evidence using phlorizin, the most common inhibitor of SGLTs, has suggested its role in glucose transport. However, phlorizin could also affect classical facilitated diffusion via glucose transporters (GLUTs), bringing into question the relevance of SGLT1 in overall cardiac glucose uptake. Accordingly, we assessed the contribution of SGLT1 in cardiac glucose uptake using the SGLT1 knockout mouse model, which lacks exon 1. Glucose uptake was similar in cardiomyocytes isolated from SGLT1-knockout (Δex1KO) and control littermate (WT) mice either under basal state, insulin, or hyperglycemia. Similarly, in vivo basal and insulin-stimulated cardiac glucose transport measured by micro-PET scan technology did not differ between WT and Δex1KO mice. Micromolar concentrations of phlorizin had no impact on glucose uptake in either isolated WT or Δex1KO-derived cardiomyocytes. However, higher concentrations (1 mM) completely inhibited insulin-stimulated glucose transport without affecting insulin signaling nor GLUT4 translocation independently from cardiomyocyte genotype. Interestingly, we discovered that mouse and human hearts expressed a shorter slc5a1 transcript, leading to SGLT1 protein lacking transmembrane domains and residues involved in glucose and sodium bindings. In conclusion, cardiac SGLT1 does not contribute to overall glucose uptake, probably due to the expression of slc5a1 transcript variant. The inhibitory effect of phlorizin on cardiac glucose uptake is SGLT1-independent and can be explained by GLUT transporter inhibition. These data open new perspectives in understanding the role of SGLT1 in the heart.NEW & NOTEWORTHY Ever since the discovery of its expression in the heart, SGLT1 has been considered as similar as the intestine and a potential contributor to cardiac glucose transport. For the first time, we have demonstrated that a slc5a1 transcript variant is present in the heart that has no significant impact on cardiac glucose handling.

Modeling of the Electromechanical Behavior of Barium Titanate Under Mechanical Stress and Comparison With Experimental Measurements.

Barium titanate (BaTiO3) is increasingly studied to replace lead-based piezoelectric materials, such as those which belong to the lead zirconate titanate (PZT) family, due to lead toxicity. In many applications, such as Tonpilz transducers, piezoelectric materials undergo mechanical stress simulation of which is important to control and predict electroacoustic effects. Thus, this article deals with a fully tensorial model that allows to simulate the behaviors of electrical displacements and elastic strains under mechanical stress. Simulated curves are compared with experimental ones obtained for BaTiO3 samples. It can be verified that the hysteretic curves of strains are well predicted for unpoled samples as well as for poled ones. The order of values and global behavior of the theoretical electrical displacement are also verified, even if a less precise agreement is observed. The optimized values of the physical parameters, such as d33 , are discussed, and improvements both of the model and the optimization procedure are finally proposed in order to better predict the mechanical behavior of BaTiO3.

Electromechanical Behavior of a Doped Barium Titanate Piezoceramic Under Mechanical Stress: Modeling and Comparison With Experimental Measurements.

Barium titanate (BaTiO3) is being studied extensively to replace lead-based piezoelectric materials, such as the lead zirconate titanate (PZT) family, due to lead toxicity. As a result, researchers are turning to materials such as BaTiO3 and seek to improve their properties with the use of dopants. In many applications such as Tonpilz transducers, piezoelectric materials undergo mechanical stress which is important to control and predict their electro-acoustic performance. Thus, this study deals with a fully tensorial model that allows us to simulate the behaviors of electrical displacements and elastic strains under mechanical stress. The simulated curves are compared with the experimental ones obtained for a doped BaTiO3 composition and the hysteretic curves of strains are in good agreement both for the unpoled and poled samples. The values and global behavior of the theoretical electrical displacement are also found to be in fair agreement, though some discrepancies are observed. The optimized values of the physical parameters, such as d33 , are discussed and improvements both of the model and the optimization procedure are finally proposed to better predict the mechanical behavior of the doped BaTiO3 piezoceramics.

Internal Electric Field in Co-Doped BaTiO3 With Co2+/3+, Nb5+, Li+, and F-: Impact on Functional Properties and Charge Compensation With Niobium and Fluorine Ions.

Dense barium titanate (BaTiO3) ceramics ( [Formula: see text]) with a microscale grain size are obtained at 800 °C-1100 °C by a solid-state ceramic process. BaTiO3 (BT) doped with Co2+/3+ leads to a significant improvement in the properties ( pC/N). Soft and hard characteristics of the piezoceramics are observed depending on the dopant ions. The Co/Li acceptor dopants lead to hard piezoceramics and aging phenomena. Aged BT:Co, Li exhibits double loops and a distorted hysteresis cycle for nonpoled and poled ceramics, respectively. Ceramics poled by the increasing field process at room temperature and the field cooling process present different poled and aged states, which are dependent on the thermal history and poling process. The distorted hysteresis loops for BT:Co, Li indicate an increased internal bias field with aging time. Insertion of donor dopants, such as Nb5+ ions, significantly reduces the internal field. These behaviors are related to the presence of defect dipoles ( [Formula: see text]"- [Formula: see text] due to the insertion of acceptor dopants in the B-sites following the oxygen vacancies to equilibrate charge compensation. BT:Co sintered with LiF leads to a quasi-symmetric hysteresis loop, indicating that F- may insert into an oxygen site and counteract the formation of oxygen vacancies. Dielectric drift of BT:Co, Li shows resilience to an ac electric field, which is related to the increased internal field. BT doped with 0.75 mol% Co2+/3+ and 1 mol% Li2CO3 presents hard piezoelectric behavior with a Rayleigh coefficient α = 2.53 10-7 m/V and the capability to handle high electrical stress of up to 400 [Formula: see text]/mm.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake.

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with [13C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation reverses leucine's action, suggesting acetylation involvement in this phenomenon.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/leucine-metabolism-inhibits-cardiac-glucose-uptake/.

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes.

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P2) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~2-fold and ~15-fold, respectively) and rates of cell proliferation (~40-fold) and protein synthesis (10-fold) after 68h of incubation compared with resting cells. After 54h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/ß in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P2 in coupling glycolysis to cell proliferation and protein synthesis in this model.

Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart.

Hyperglycemia (HG) stimulates the production of reactive oxygen species in the heart through activation of NADPH oxidase 2 (NOX2). This production is independent of glucose metabolism but requires sodium/glucose cotransporters (SGLT). Seven SGLT isoforms (SGLT1 to 6 and sodium-myoinositol cotransporter-1, SMIT1) are known, although their expression and function in the heart remain elusive. We investigated these 7 isoforms and found that only SGLT1 and SMIT1 were expressed in mouse, rat and human hearts. In cardiomyocytes, galactose (transported through SGLT1) did not activate NOX2. Accordingly, SGLT1 deficiency did not prevent HG-induced NOX2 activation, ruling it out in the cellular response to HG. In contrast, myo-inositol (transported through SMIT1) reproduced the toxic effects of HG. SMIT1 overexpression exacerbated glucotoxicity and sensitized cardiomyocytes to HG, whereas its deletion prevented HG-induced NOX2 activation. In conclusion, our results show that heart SMIT1 senses HG and triggers NOX2 activation. This could participate in the redox signaling in hyperglycemic heart and contribute to the pathophysiology of diabetic cardiomyopathy.

Effects of pharmacological AMP deaminase inhibition and Ampd1 deletion on nucleotide levels and AMPK activation in contracting skeletal muscle.

AMP-activated protein kinase (AMPK) plays a central role in regulating metabolism and energy homeostasis. It achieves its function by sensing fluctuations in the AMP:ATP ratio. AMP deaminase (AMPD) converts AMP into IMP, and the AMPD1 isoenzyme is expressed in skeletal muscles. Here, effects of pharmacological inhibition and genetic deletion of AMPD were examined in contracting skeletal muscles. Pharmacological AMPD inhibition potentiated rises in AMP, AMP:ATP ratio, AMPK Thr172, and acetyl-CoA carboxylase (ACC) Ser218 phosphorylation induced by electrical stimulation, without affecting glucose transport. In incubated extensor digitorum longus and soleus muscles from Ampd1 knockout mice, increases in AMP levels and AMP:ATP ratio by electrical stimulation were potentiated considerably compared with muscles from wild-type mice, whereas enhanced AMPK activation was moderate and only observed in soleus, suggesting control by factors other than changes in adenine nucleotides. AMPD inhibitors could be useful tools for enhancing AMPK activation in cells and tissues during ATP-depletion.

AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes.

Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). α2-AMPK, the major isoform expressed in cardiomyocytes (but not α1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either α-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of α2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in α2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-ß2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces α2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity.

Controlled growth of 1D and 2D ZnO nanostructures on 4H-SiC using Au catalyst.

A perfect control of nanostructure growth is a prerequisite for the development of electronic and optoelectronic device/systems. In this article, we demonstrate the growth of various ZnO-derived nanostructures, including well-ordered arrays of high aspect ratio single crystalline nanowires with preferred growth direction along the [0001] axis, nanowalls, and hybrid nanowire-nanowall structures. The growths of the various ZnO nanostructures have been carried out on SiC substrates in a horizontal furnace, using Au thin film as catalyst. From experimental observations, we have ascribed the growth mechanisms of the different ZnO nanostructures to be a combination of catalytic-assisted and non-catalytic-assisted vapor-liquid-solid (VLS) processes. We have also found that the different ZnO nanoarchitectures' material evolution is governed by a Zn cluster drift effects on the SiC surface mainly driven by growth temperature. Au thin film thickness, growth time, and temperature are the parameters to optimize in order to obtain the different ZnO nanoarchitectures.

Theoretical and experimental study of the electroacoustic behavior of lithium niobate under an initial mechanical stress.

Recently, a second-order formalism of piezoelectric structures under an external mechanical stress was developed. Because the yield strength of lithium niobate was unknown, this study was not able to describe and evaluate realistic benefits of a prestress load on electromechanical properties. Therefore, in this study, experimental determination of the yield strength of lithium niobate sample is performed and shows that this limit is close to 110 MPa. Then, the nonlinearities and evolutions of electroacoustic parameters of this piezoelectric material under mechanical stress are numerically studied. By varying the initial prestress, as well as azimuthal and elevation angles, the cut planes in which a prestress induces significant benefits on velocities and coupling coefficient are identified. Finally, approximate relations describing changes between electroacoustic parameters defined in the two coordinate systems of the study are determined.

Insulin resistance protects the heart from fuel overload in dysregulated metabolic states.

Reversing impaired insulin sensitivity has been suggested as treatment for heart failure. However, recent clinical evidence suggests the opposite. Here we present a line of reasoning in support of the hypothesis that insulin resistance protects the heart from the consequences of fuel overload in the dysregulated metabolic state of obesity and diabetes. We discuss pathways of myocardial fuel toxicity, as well as several layers of defense against fuel overload. Our reassessment of the literature suggests that in the heart, insulin-sensitizing agents result in an elimination of some of the defenses, leading to cytotoxic damage. In contrast, a normalization of fuel supply should either prevent or reverse the process. Taken together, we offer a new perspective on insulin resistance of the heart.

Differential regulation of eEF2 and p70S6K by AMPKalpha2 in heart.

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.

Non-planar pad-printed thick-film focused high-frequency ultrasonic transducers for imaging and therapeutic applications.

Pad-printed thick-film transducers have been shown to be an interesting alternative to lapped bulk piezoceramics, because the film is deposited with the required thickness, size, and geometry, thus avoiding any subsequent machining to achieve geometrical focusing. Their electromechanical properties are close to those of bulk ceramics with similar composition despite having a higher porosity. In this paper, padprinted high-frequency transducers based on a low-loss piezoceramic composition are designed and fabricated. High-porosity ceramic cylinders with a spherical top surface are used as the backing substrate. The transducers are characterized in view of imaging applications and their imaging capabilities are evaluated with phantoms containing spherical inclusions and in different biological tissues. In addition, the transducers are evaluated for their capability to produce high-acoustic intensities at frequencies around 20 MHz. High-intensity measurements, obtained with a calibrated hydrophone, show that transducer performance is promising for applications that would require the same device to be used for imaging and for therapy. Nevertheless, the transducer design can be improved, and simulation studies are performed to find a better compromise between low-power and high-power performance. The size, geometry, and constitutive materials of optimized configurations are proposed and their feasibility is discussed.

Overexpression of AMP-metabolizing enzymes controls adenine nucleotide levels and AMPK activation in HEK293T cells.

We investigated whether overexpression of AMP-metabolizing enzymes in intact cells would modulate oligomycin-induced AMPK activation. Human embryonic kidney (HEK) 293T cells were transiently transfected with increasing amounts of plasmid vectors to obtain a graded increase in overexpression of AMP-deaminase (AMPD) 1, AMPD2, and soluble 5'-nucleotidase IA (cN-IA) for measurements of AMPK activation and total intracellular adenine nucleotide levels induced by oligomycin treatment. Overexpression of AMPD1 and AMPD2 slightly decreased AMP levels and oligomycin-induced AMPK activation. Increased overexpression of cN-IA led to reductions in the oligomycin-induced increases in AMP and ADP concentrations by ∼70 and 50%, respectively, concomitant with a 50% decrease in AMPK activation. The results support the view that a rise in ADP as well as AMP is important for activation of AMPK, which can thus be regulated by the adenylate energy charge. The control coefficient of cN-IA on AMP was 0.3-0.7, whereas the values for AMPD1 and AMPD2 were <0.1, suggesting that in this model cN-IA exerts a large proportion of control over intracellular AMP. Therefore, small molecule inhibition of cN-IA could be a strategy for AMPK activation.

AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase.

Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.

NADPH oxidase activation by hyperglycaemia in cardiomyocytes is independent of glucose metabolism but requires SGLT1.

AIMS: Exposure to high glucose (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase in cardiomyocytes, but the underlying mechanism remains elusive. In this study, we have dissected the link between glucose transport and metabolism and NADPH oxidase activation under hyperglycaemic conditions. METHODS AND RESULTS: Primary cultures of adult rat cardiomyocytes were exposed to HG concentration (HG, 21 mM) and compared with the normal glucose level (LG, 5 mM). HG exposure activated Rac1GTP and induced p47phox translocation to the plasma membrane, resulting in NADPH oxidase (NOX2) activation, increased ROS production, insulin resistance, and eventually cell death. Comparison of the level of O-linked N-acetylglucosamine (O-GlcNAc) residues in LG- and HG-treated cells did not reveal any significant difference. Inhibition of the pentose phosphate pathway (PPP) by 6-aminonicotinamide counteracted ROS production in response to HG but did not prevent Rac-1 upregulation and p47phox translocation leading to NOX2 activation. Modulation of glucose uptake barely affected oxidative stress and toxicity induced by HG. More interestingly, non-metabolizable glucose analogues (i.e. 3-O-methyl-D-glucopyranoside and α-methyl-D-glucopyranoside) reproduced the toxic effect of HG. Inhibition of the sodium/glucose cotransporter SGLT1 by phlorizin counteracted HG-induced NOX2 activation and ROS production. CONCLUSION: Increased glucose metabolism by itself does not trigger NADPH oxidase activation, although PPP is required to provide NOX2 with NADPH and to produce ROS. NOX2 activation results from glucose transport through SGLT1, suggesting that an extracellular metabolic signal transduces into an intracellular ionic signal.