AICAR improves depression-like behaviors and is associated with hippocampal AMPK activation and modulation of neurogenesis and neuroinflammation in a microbiota disruption model.

AMP-activated protein kinase (AMPK), an evolutionarily conserved serine-threonine kinase that senses cellular energy status, is activated by stress and neurohumoral stimuli. We investigated the mechanisms by which adrenergic signaling alters AMPK activation in vivo. Brown adipose tissue (BAT) is highly enriched in sympathetic innervation, which is critical for regulation of energy homeostasis. We performed unilateral denervation of BAT in wild type (WT) mice to abolish neural input. Six days post-denervation, UCP-1 protein levels and AMPK α2 protein and activity were reduced by 45%. In β(1,2,3)-adrenergic receptor knock-out mice, unilateral denervation led to a 25-45% decrease in AMPK activity, protein expression, and Thr(172) phosphorylation. In contrast, acute α- or β-adrenergic blockade in WT mice resulted in increased AMPK α Thr(172) phosphorylation and AMPK α1 and α2 activity in BAT. But short term blockade of α-adrenergic signaling in β(1,2,3)-adrenergic receptor knock-out mice resulted in decreased AMPK activity in BAT, which strongly correlated with enhanced phosphorylation of AMPK on Ser(485/491), a site associated with inhibition of AMPK activity. Both PKA and AKT inhibitors attenuated AMPK Ser(485/491) phosphorylation resulting from α-adrenergic blockade and prevented decreases in AMPK activity. In vitro mechanistic studies in BAT explants showed that the effects of α-adrenergic blockade appeared to be secondary to inhibition of oxygen consumption. In conclusion, adrenergic pathways regulate AMPK activity in vivo acutely via alterations in Thr(172) phosphorylation and chronically through changes in the α catalytic subunit protein levels. Furthermore, AMPK α Ser(485/491) phosphorylation may be a novel mechanism to inhibit AMPK activity in vivo and alter its biological effects.

The AMP-activated protein kinase (AMPK) is activated by a fall in the ATP:AMP ratio within the cell in response to metabolic stresses. Once activated, it phosphorylates and inhibits key enzymes in energy-consuming biosynthetic pathways, thereby conserving cellular ATP. The creatine kinase-phosphocreatine system plays a key role in the control of ATP levels in tissues that have a high and rapidly fluctuating energy requirement. In this study, we provide direct evidence that these two energy-regulating systems are linked in skeletal muscle. We show that the AMPK inhibits creatine kinase by phosphorylation in vitro and in differentiated muscle cells. AMPK is itself regulated by a novel mechanism involving phosphocreatine, creatine and pH. Our findings provide an explanation for the high expression, yet apparently low activity, of AMPK in skeletal muscle, and reveal a potential mechanism for the co-ordinated regulation of energy metabolism in this tissue. Previous evidence suggests that AMPK activates fatty acid oxidation, which provides a source of ATP, following continued muscle contraction. The novel regulation of AMPK described here provides a mechanism by which energy supply can meet energy demand following the utilization of the immediate energy reserve provided by the creatine kinase-phosphocreatine system.

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Despite its importance in terms of energy homeostasis, the role of AMP-activated protein kinase in adipose tissue remains controversial. Initial studies have described an anti-lipolytic role for AMP-activated protein kinase, whereas more recent studies have suggested the converse. Thus we have addressed the role of AMP-activated protein kinase in adipose tissue by modulating AMP-activated protein kinase activity in primary rodent adipocytes using pharmacological activators or by adenoviral expression of dominant negative or constitutively active forms of the kinase. We then studied the effects of AMP-activated protein kinase activity modulation on lipolytic mechanisms. Finally, we analyzed the consequences of a genetic deletion of AMP-activated protein kinase in mouse adipocytes. AMP-activated protein kinase activity in adipocytes is represented mainly by the alpha(1) isoform and is induced by all of the stimuli that increase cAMP in adipocytes, including fasting. When AMP-activated protein kinase activity is increased by 5-aminoimidazole-4-carboxamide-riboside, phenformin, or by the expression of a constitutively active form, isoproterenol-induced lipolysis is strongly reduced. Conversely, when AMP-activated protein kinase activity is decreased either by a dominant negative form or in AMP-activated protein kinase alpha(1) knock-out mice, lipolysis is increased. We present data suggesting that AMP-activated protein kinase acts on hormone-sensitive lipase by blocking its translocation to the lipid droplet. We conclude that, in mature adipocytes, AMP-activated protein kinase activation has a clear anti-lipolytic effect.

Endocannabinoids and ghrelin are potent appetite stimulators and are known to interact at a hypothalamic level. However, both also have important peripheral actions, including beneficial effects on the ischemic heart and increasing adipose tissue deposition, while ghrelin has direct effects on carbohydrate metabolism. The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that functions as a fuel sensor to regulate energy balance at both cellular and whole body levels, and it may mediate the action of anti-diabetic drugs such as metformin and peroxisome proliferator-activated receptor gamma agonists. Here we show that both cannabinoids and ghrelin stimulate AMPK activity in the hypothalamus and the heart, while inhibiting AMPK in liver and adipose tissue. These novel effects of cannabinoids on AMPK provide a mechanism for a number of their known actions, such as the reduction in infarct size in the myocardium, an increase in adipose tissue, and stimulation of appetite. The beneficial effects of ghrelin on heart function, including reduction of myocyte apoptosis, and its effects on lipogenesis and carbohydrate metabolism, can also be explained by its ability to activate AMPK. Our data demonstrate that AMPK not only links the orexigenic effects of endocannabinoids and ghrelin in the hypothalamus but also their effects on the metabolism of peripheral tissues.

The myocardium possesses intrinsic protective mechanisms against ischemia and reperfusion injury (I/R). 5′AMP-activated protein kinase (AMPK) is known as regulator of cellular energy status and is reduced during diabetes mellitus. Recently, AMPK is also implicated in ischemic preconditioning leading to cardioprotection against I/R. We hypothesize that AMPK is involved in anesthetic-induced cardioprotection and that this AMPK activation is evoked by production of reactive oxygen species (ROS). Isolated Langendorff-perfused rat hearts were subjected to 35 minutes of global ischemia followed by 120 minutes of reperfusion. Hearts were divided into 2 groups: a Control group and a Sevo group receiving three times 5-minute episodes of 2.5 vol% sevoflurane before I/R. AMPK phosphorylation was determined with Western Blot analysis. Cardioprotection was assessed as recovery of left ventricular pressure after I/R and as infarct size using triphenyltetrazolium chloride staining. In the Control group, I/R resulted in a twofold increase in phosphorylation levels of AMPK (Control 1.0 ± 0.1 vs. Control-I/R: 2.3 ± 0.1 a.u., n = 4, p < 0.05). Sevoflurane preconditioning did not immediately, prior to ischemia, affect the AMPK phosphorylation (Sevo 0.9 ± 0.2 vs. Control 1.0 ± 0.2, n = 6), but doubled the increase in AMPK phosphorylation to control after ischemia (Sevo-I: 2.0 ± 0.5 (vs. Control-I), n = 6, p < 0.05), as well as after I/R (Sevo-I/R: 2.1 ± 0.3 vs. (Control-I/R), n = 6, p < 0.05). The AMPK-inhibitor compound C (1 and 10 μM) reduced the increase in AMPK phosphorylation and abolished the cardioprotection derived from functional recovery and infarct size. In addition, the ROS-scavenger n-(2-mercaptopropionyl)-glycine (MPG, 1mM) also reduced the sevoflurane-mediated increase in AMPK phosphorylation and completely prevented cardioprotection. These results demonstrate for the first time a direct link between AMPK activation and the production of ROS in cardioprotection. We conclude that anesthetic-induced AMPK activation protects the heart against I/R and relies on production of ROS, which might be especially important in the context of impaired cardioprotection in the diabetic myocardium.

We have studied the mechanism of A-769662, a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators, it directly activates native rat AMPK by mimicking both effects of AMP, i.e. allosteric activation and inhibition of dephosphorylation. We found that it has no effect on the isolated alpha subunit kinase domain, with or without the associated autoinhibitory domain, or on interaction of glycogen with the beta subunit glycogen-binding domain. Although it mimics actions of AMP, it has no effect on binding of AMP to the isolated Bateman domains of the gamma subunit. The addition of A-769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC), effects that are completely abolished in AMPK-alpha1(-/-)alpha2(-/-) cells but not in TAK1(-/-) mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in isolated mouse skeletal muscle lacking LKB1, a major upstream kinase for AMPK in this tissue. However, in HeLa cells, which lack LKB1 but express the alternate upstream kinase calmodulin-dependent protein kinase kinase-beta, phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells, the effects of A-769662 are independent of the upstream kinase utilized. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK.

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.

LKB1 is an upstream activating kinase for the AMP-activated protein kinase (AMPK) and at least 12 other AMPK-related kinases. LKB1 therefore acts as a master kinase regulating the activity of a wide range of downstream kinases, which themselves have diverse physiological roles. Here we identify a second form of LKB1 generated by alternative splicing of the LKB1 gene. The two LKB1 proteins have different C-terminal sequences generating a 50-kDa form (termed LKB1L) and a 48-kDa form (LKB1S). LKB1L is widely expressed in mouse tissues, whereas LKB1S has a restricted tissue distribution with predominant expression in the testis. LKB1S, like LKB1L, forms a complex with MO25 and STRAD, and phosphorylates and activates AMPK both in vitro and in intact cells. A phosphorylation site (serine 431 in mouse) and a farnesylation site (cysteine 433 in mouse) within LKB1L are not conserved in LKB1S raising the possibility that these sites might be involved in differential regulation and/or localization of the two forms of LKB1. However, we show that phosphorylation of serine 431 has no effect on LKB1L activity and that both LKB1L and LKB1S have similar patterns of subcellular localization. These results indicate that the physiological significance of the different forms of LKB1 is not related directly to differences in the C-terminal sequences but may be due to their differential patterns of tissue distribution.

Caloric restriction has emerged as an effective strategy for lengthening lifespan in a variety of species.1 In mammals, one mechanism for this phenomenon may be the prevention of detrimental age-related alterations in cellular function1 and presumably subsequent improvement in organ function. The effects of caloric restriction on the heart, at least in rats and mice, involve a number of changes in gene expression that are beneficial to the aged cardiomyocyte2 and/or protect the heart from ischemic injury.3 Although it is likely that all of the beneficial mediators of caloric restriction have not been identified, a number of proteins in the mammalian sirtuin family may play key roles in the regulation of health and longevity.4 In addition, recent evidence has suggested that alterations in whole-body energy metabolism contribute to the beneficial effects of caloric restriction.5 Indeed, caloric restriction in mammals leads to loss of adipose tissue and dramatically alters the action of this endocrine organ.6 As such, caloric restriction contributes to changes in adipose tissue–derived hormone (adipokine) secretion, which can govern whole-body metabolism.7 Furthermore, studies using isolated cardiac myocytes suggest that these adipokines may exert direct end-organ effects that are independent from alterations in whole-body metabolism.8–10 One adipokine that is significantly increased during caloric restriction is adiponectin.11 Previous work has shown that adiponectin exerts a host of protective effects on the cardiovascular system12 and as such may be an essential component mediating the effects of caloric restriction. Article p 2809 The focus on adiponectin in the cardiovascular system has been due largely to the fact that in humans, circulating adiponectin levels are negatively correlated with increased body mass index.13 Because increased body mass index is associated with a number of obesity-linked disorders, including cardiovascular disease, the reduction in serum …

Glucose-Based Regulation of miR-451/AMPK Signaling Depends on the OCT1 Transcription Factor

The activation of AMP-activated protein kinase (AMPK) has a beneficial effect on hyperglycaemia. The aim of this study was to examine whether an azuki bean (Vigna angularis) extract (ABE) stimulates the AMPK or insulin signalling pathways in a liver cell line in response to hyperglycaemia, as well as in a diabetic rat liver. HepG2 cells were incubated with 5 or 20 mmol L(-1) glucose and then treated with ABE. Streptozotocin-induced diabetic rats received 0, 10, or 40 mg kg(-1) ABE orally. Blood chemistry and phosphorylation of AMPK and Akt (a serine/threonine kinase) in the livers were examined. There was a significant increase in the levels of AMPK and Akt phosphorylation in ABE-treated HepG2 cells. AMPK phosphorylation increased significantly in glucose-stimulated HepG2 cells that were treated with ABE. In the 40 mg kg(-1) ABE-treated diabetic rats, the glucose levels were lower than in the control. Phosphorylation of AMPK in ABE-untreated diabetic rat livers decreased significantly. Conversely, ABE treatment increased the phosphorylation of AMPK and Akt in the diabetic rat liver. ABE treatment upregulated AMPK phosphorylation in HepG2 cells, and upregulated AMPK and Akt phosphorylation in the diabetic rat liver. These data suggest that ABE can potentially improve glucose intolerance.

transient intracellular calcium oscillations were the first signaling mediator described to regulate contraction-stimulated glucose transport in skeletal muscle ([6][1]). Thirty years later, the discoveries, that 5′-AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-β-

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.