Uncoupling AMPK from autophagy: a foe that hinders the beneficial effects of metformin treatment on metabolic syndrome-associated atherosclerosis? Focus on "glucose and palmitate uncouple AMPK from autophagy in human aortic endothelial cells".

Dysregulated autophagy and decreased AMP-activated protein kinase (AMPK) activity are each associated with atherogenesis. Atherogenesis is preceded by high circulating concentrations of glucose and fatty acids, yet the mechanism by which these nutrients regulate autophagy in human aortic endothelial cells (HAECs) is not known. Furthermore, whereas AMPK is recognized as an activator of autophagy in cells with few nutrients, its effects on autophagy in nutrient-rich HAECs has not been investigated. We maintained and passaged primary HAECs in media containing 25 mM glucose and incubated them subsequently with 0.4 mM palmitate. These conditions impaired basal autophagy and rendered HAECs more susceptible to apoptosis and adhesion of monocytes, outcomes attenuated by the autophagy activator rapamycin. Glucose and palmitate diminished AMPK activity and phosphorylation of the uncoordinated-51-like kinase 1 (ULK1) at Ser555, an autophagy-activating site targeted by AMPK. 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR)-mediated activation of AMPK phosphorylated acetyl-CoA carboxylase, but treatment with AICAR or other AMPK activators (A769662, phenformin) did not restore ULK1 phosphorylation or autophagosome formation. To determine whether palmitate-induced ceramide accumulation contributed to this finding, we overexpressed a ceramide-metabolizing enzyme, acid ceramidase. The increase in acid ceramidase expression ameliorated the effects of excess nutrients on ULK1 phosphorylation, without altering the effects of the AMPK activators. Thus, unlike low nutrient conditions, AMPK becomes uncoupled from autophagy in HAECs in a nutrient-rich environment, such as that found in patients with increased cardiovascular risk. These findings suggest that combinations of AMPK-independent and AMPK-dependent therapies may be more effective alternatives than either therapy alone for treating nutrient-induced cellular dysfunction.

AMP-activated protein kinase (AMPK) inactivation in chronic kidney disease (CKD) leads to energy status deterioration in the kidney, constituting the vicious cycle of CKD exacerbation. Unc-51-like kinase 1 (ULK1) is considered a downstream molecule of AMPK; however, it was recently reported that the activity of AMPK could be regulated by ULK1 conversely. We demonstrated that AMPK and ULK1 activities were decreased in the kidneys of CKD mice. However, whether and how ULK1 is involved in the underlying mechanism of CKD exacerbation remains unknown. In this study, we investigated the ULK1 involvement in CKD, using ULK1 knockout mice. The CKD model of Ulk1-/- mice exhibited significantly exacerbated renal function and worsening renal fibrosis. In the kidneys of the CKD model of Ulk1-/- mice, reduced AMPK and its downstream β-oxidation could be observed, leading to an energy deficit of increased AMP/ATP ratio. In addition, AMPK signaling in the kidney was reduced in control Ulk1-/- mice with normal renal function compared to control wild-type mice, suggesting that ULK1 deficiency suppressed AMPK activity in the kidney. This study is the first to present ULK1 as a novel therapeutic target for CKD treatment, which regulates AMPK activity in the kidney.

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

Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser(555) in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser(774) and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr(180) that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.

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.

Regulation of autophagy in human muscle in many aspects differs from the majority of previous reports based on studies in cell systems and rodent muscle. An acute bout of exercise and insulin stimulation reduce human muscle autophagosome content. An acute bout of exercise regulates autophagy by a local contraction-induced mechanism. Exercise training increases the capacity for formation of autophagosomes in human muscle. AMPK activation during exercise seems insufficient to regulate autophagosome content in muscle, while mTORC1 signalling via ULK1 probably mediates the autophagy-inhibiting effect of insulin. Studies in rodent muscle suggest that autophagy is regulated by acute exercise, exercise training and insulin stimulation. However, little is known about the regulation of autophagy in human skeletal muscle. Here we investigate the autophagic response to acute one-legged exercise, one-legged exercise training and subsequent insulin stimulation in exercised and non-exercised human muscle. Acute one-legged exercise decreased (P<0.01) lipidation of microtubule-associated protein 1A/1B-light chain 3 (LC3) (∼ 50%) and the LC3-II/LC3-I ratio (∼ 60%) indicating that content of autophagosomes decreases with exercise in human muscle. The decrease in LC3-II/LC3-I ratio did not correlate with activation of 5'AMP activated protein kinase (AMPK) trimer complexes in human muscle. Consistently, pharmacological AMPK activation with 5-aminoimidazole-4-carboxamide riboside (AICAR) in mouse muscle did not affect the LC3-II/LC3-I ratio. Four hours after exercise, insulin further reduced (P<0.01) the LC3-II/LC3-I ratio (∼ 80%) in muscle of the exercised and non-exercised leg in humans. This coincided with increased Ser-757 phosphorylation of Unc51 like kinase 1 (ULK1), which is suggested as a mammalian target of rapamycin complex 1 (mTORC1) target. Accordingly, inhibition of mTOR signalling in mouse muscle prevented the ability of insulin to reduce the LC3-II/LC3-I ratio. In response to 3 weeks of one-legged exercise training, the LC3-II/LC3-I ratio decreased (P<0.05) in both trained and untrained muscle and this change was largely driven by an increase in LC3-I content. Taken together, acute exercise and insulin stimulation reduce muscle autophagosome content, while exercise training may increase the capacity for formation of autophagosomes in muscle. Moreover, AMPK activation during exercise may not be sufficient to regulate autophagy in muscle, while mTORC1 signalling via ULK1 probably mediates the autophagy-inhibiting effect of insulin.

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.

Skeletal muscle ischemia-reperfusion (I/R) injury is a critical clinical condition. AMP-activated protein kinase (AMPK) during IR injury remains unclear. A hindlimb I/R model was established in Sprague-Dawley rats using vascular occlusion. AMPK activator Metformin and AMPK inhibitor Compound C were applied to investigate the effects of AMPK on hindlimb I/R injury. Differentiated C2C12 myotubes were subjected to hypoxia/reoxygenation to mimic I/R injury in vitro. Histopathology, apoptosis, and angiogenesis were observed by Hematoxylin-Eosin, TUNEL staining, and immunohistochemistry. Western blotting and Enzyme-Linked Immunosorbent Assay were performed to assess autophagy and ferroptosis-related indicators. Co-immunoprecipitation validated AMPK-ULK1 interactions. Furthermore, Unc-51-like kinase 1 (ULK1) was inhibited to explore its effects in vivo and in vitro. AMPK activation via Metformin reduced I/R-induced muscle damage, apoptosis, and endothelial dysfunction, while Compound C exacerbated these effects. Metformin upregulated p-AMPK and suppressed ferroptosis in vivo. In C2C12 cells, AMPK overexpression attenuated hypoxia/reoxygenation-induced ferroptosis (increased GPX4 and SLC7A11) and activated autophagy (increased LC3-II/LC3-I and decreased p62). Mechanistically, AMPK phosphorylated ULK1 at Ser555, promoting ATG7/ATG5-dependent autophagy. ULK1 inhibition suppressed autophagy and abolished AMPK's protective effects, exacerbating muscle injury and ferroptosis. AMPK activation protects against skeletal muscle I/R injury by enhancing ULK1/ATG-mediated autophagy and inhibiting ferroptosis.

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

Morin promotes autophagy in human PC3 prostate cancer cells by modulating AMPK/mTOR/ULK1 signaling pathway

Here we examine recent evidence suggesting that many drugs and diet supplements (DS), experimental AMP-activated protein kinase (AMPK) agonists as well as energy-depleting stress, lead to decreases in anabolism, growth or proliferation, and potency of cultured oocytes, embryos, and stem cells in an AMPK-dependent manner. Surprising data for DS and drugs that have some activity as AMPK agonists in in vitro experiments show possible toxicity. This needs to be balanced against a preponderance of evidence in vivo that these drugs and DS are beneficial for reproduction. We here discuss and analyze data that leads to two possible conclusions: First, although DS and drugs that have some of their therapeutic mechanisms mediated by AMPK activity associated with low ATP levels, some of the associated health problems in vivo and in vitro fertilization/assisted reproductive technologies (IVF/ART) may be better-treated by increasing ATP production using CoQ10 (Ben-Meir et al., Aging Cell 14:887-895, 2015). This enables high developmental trajectories simultaneous with solving stress by energy-requiring responses. In IVF/ART, it is ultimately best to maintain handling and culture of gametes and embryos in the quietest state with low metabolic activity (Leese et al., Mol Hum Reprod 14:667-672, 2008; Leese, Bioessays 24 (9):845-849, 2002) using back-to-nature or simplex algorithms to identify optima (Biggers, Reprod Biomed Online 4 Suppl 1:30-38, 2002). Stress markers, such as checkpoint proteins like TRP53 (aka p53) (Ganeshan et al., Exp Cell Res 358:227-233, 2017); Ganeshan et al., Biol Reprod 83:958-964, 2010) and a small set of kinases from the protein kinome that mediate enzymatic stress responses, can also be used to define optima. But, some gametes or embryos may have been stressed in vivo prior to IVF/ART or IVF/ART optimized for one outcome may be suboptimal for another. Increasing nutrition or adding CoQ10 to increase ATP production (Yang et al., Stem Cell Rev 13:454-464, 2017), managing stress enzyme levels with inhibitors (Xie et al., Mol Hum Reprod 12:217-224, 2006), or adding growth factors such as GM-CSF (Robertson et al., J Reprod Immunol 125:80-88, 2018); Chin et al., Hum Reprod 24:2997-3009, 2009) may increase survival and health of cultured embryos during different stress exposure contexts (Puscheck et al., Adv Exp Med Biol 843:77-128, 2015). We define "stress" as negative stimuli which decrease normal magnitude and speed of development, and these can be stress hormones, reactive oxygen species, inflammatory cytokines, or physical stimuli such as hypoxia. AMPK is normally activated by high AMP, commensurate with low ATP, but it was recently shown that if glucose is present inside the cell, AMPK activation by low ATP/high AMP is suppressed (Zhang et al., Nature 548:112-116, 2017). As we discuss in more detail below, this may also lead to greater AMPK agonist toxicity observed in two-cell embryos that do not import glucose. Stress in embryos and stem cells increases AMPK in large stimulation indexes but also direness indexes; the fastest AMPK activation occurs when stem cells are shifted from optimal oxygen to lower or high levels (Yang et al., J Reprod Dev 63:87-94, 2017). CoQ10 use may be better than risking AMPK-dependent metabolic and developmental toxicity when ATP is depleted and AMPK activated. Second, the use of AMPK agonists, DS, and drugs may best be rationalized when insulin resistance or obesity leads to aberrant hyperglycemia and hypertriglyceridemia, and obesity that negatively affect fertility. Under these conditions, beneficial effects of AMPK on increasing triglyceride and fatty acid and glucose uptake are important, as long as AMPK agonist exposures are not too high or do not occur during developmental windows of sensitivity. During these windows of sensitivity suppression of anabolism, proliferation, and stemness/potency due to AMPK activity, or overexposure may stunt or kill embryos or cause deleterious epigenetic changes.

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.

Myotonic Dystrophy type I (DM1) is the most prevalent adult form of muscular dystrophy and is characterized by myotonia, skeletal muscle weakness and wasting. The disease pathogenesis is characterized by a CTG microsatellite repeat expansion, leading to the dysregulation of pre-messenger RNA splicing of numerous muscle genes. There is currently no cure for this disorder, but recent evidence indicate that exercise is a safe and modestly effective therapy. AMP-activated protein kinase (AMPK) is critical to the acute responses and chronic adaptations to physical activity at the cellular and molecular level in the healthy condition, as well as in various disease states. However, the AMPK-signaling response to exercise in DM1 remains largely unknown. Therefore, the purpose of this study is to examine whether a single bout of exercise 1) activates AMPK and its downstream signalling network in DM1 skeletal muscle, and 2) modulates the DM1 molecular signature. Wild-type (WT) and HSA-LR (DM1) mice ran on a motor-driven treadmill until the inability to continue exercise was objectively determined, and the molecular response to physical activity at various timepoints post-exercise was examined using Western blotting, immunofluorescence microscopy, and qPCR techniques. WT mice ran for 937.6 ± 193.8 meters, which was significantly greater than DM1 mice at 573.3 ± 181.4meters. In the skeletal muscle of WT mice, AMPK activation status was significantly augmented immediately after exercise. This coincided with an increase (p < 0.05) in peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) transcript levels, as well as significant elevations in unc-51-like kinase 1 (ULK1) and calcium/calmodulin-dependent protein kinase type 2β (CAMKIIβ) activation. In DM1 mice, acute exercise also significantly stimulated AMPK, however, PGC-1α, ULK1 and CAMKIIβ were unaffected. In resting muscle, several markers related to autophagy downstream of AMPK, for example ULK1 activation status, p62 protein level, and the lipidated form of microtubule-associated protein 1-light chain 3, were significantly elevated in DM1 mice compared to their WT counterparts. These normalized after exercise. Acute physical activity did not impact either the prevalence of toxic myonuclear foci formed by the CTG microsatellite repeat or the proportion of mis-spliced muscle-specific chloride channel in DM1 mice. Collectively, these results indicate that while exercise-induced AMPK phosphorylation was preserved in the skeletal muscle of DM1 mice, markers of upstream and downstream AMPK signalling were attenuated in response to acute physical activity. Nonetheless, our data also suggest that exercise-evoked AMPK activation normalizes perturbations in the autophagy pathway observed in DM1 muscle. Thus, skeletal muscle AMPK stimulation after a single bout of exercise is very likely necessary but insufficient to elicit beneficial structural and functional adaptations in DM1.