Adrenergic regulation of AMP-activated protein kinase in brown adipose tissue in vivo

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.     

PKA phosphorylates and inactivates AMPKα to promote efficient lipolysis

The mobilization of metabolic energy from adipocytes depends on a tightly regulated balance between hydrolysis and resynthesis of triacylglycerides (TAGs). Hydrolysis is stimulated by β‐adrenergic signalling to PKA that mediates phosphorylation of lipolytic enzymes, including hormone‐sensitive lipase (HSL). TAG resynthesis is associated with high‐energy consumption, which when inordinate, leads to increased AMPK activity that acts to restrain hydrolysis of TAGs by inhibiting PKA‐mediated activation of HSL. Here, we report that in primary mouse adipocytes, PKA associates with and phosphorylates AMPKα1 at Ser‐173 to impede threonine (Thr‐172) phosphorylation and thus activation of AMPKα1 by LKB1 in response to lipolytic signals. Activation of AMPKα1 by LKB1 is also blocked by PKA‐mediated phosphorylation of AMPKα1 in vitro. Functional analysis of an AMPKα1 species carrying a non‐phosphorylatable mutation at Ser‐173 revealed a critical function of this phosphorylation for efficient release of free fatty acids and glycerol in response to PKA‐activating signals. These results suggest a new mechanism of negative regulation of AMPK activity by PKA that is important for converting a lipolytic signal into an effective lipolytic response.     

Intracellular signals mediating the food intake-suppressive effects of hindbrain glucagon-like peptide-1 receptor activation

Glucagon-like peptide-1 receptor (GLP-1R) activation within the nucleus tractus solitarius (NTS) suppresses food intake and body weight (BW), but the intracellular signals mediating these effects are unknown. Here, hindbrain (fourth i.c.v.) GLP-1R activation by Exendin-4 (Ex-4) increased PKA and MAPK activity and decreased phosphorylation of AMPK in NTS. PKA and MAPK signaling contribute to food intake and BW suppression by Ex-4, as inhibitors RpcAMP and U0126 (fourth i.c.v.), respectively, attenuated Ex-4's effects. Hindbrain GLP-1R activation inhibited feeding by reducing meal number, not meal size. This effect was attenuated with stimulation of AMPK activity by AICAR (fourth i.c.v.). The PKA, MAPK, and AMPK signaling responses by Ex-4 were present in immortalized GLP-1R-expressing neurons (GT1-7). In conclusion, hindbrain GLP-1R activation suppresses food intake and BW through coordinated PKA-mediated suppression of AMPK and activation of MAPK. Pharmacotherapies targeting these signaling pathways, which mediate intake-suppressive effects of CNS GLP-1R activation, may prove efficacious in treating obesity.     

p70S6 kinase phosphorylates AMPK on serine 491 to mediate leptin's effect on food intake

The PI3K-AKT, mTOR-p70S6 kinase and AMPK pathways play distinct and critical roles in metabolic regulation. Each pathway is necessary for leptin's anorexigenic effects in the hypothalamus. Here we show that these pathways converge in an integrated phosphorylation cascade to mediate leptin action in the hypothalamus. We identify serine(491) on α2AMPK as the site of convergence and show that p70S6 kinase forms a complex with α2AMPK, resulting in phosphorylation on serine(491). Blocking α2AMPK-serine(491) phosphorylation increases hypothalamic AMPK activity, food intake, and body weight. Serine(491) phosphorylation is necessary for leptin's effects on hypothalamic α2AMPK activity, neuropeptide expression, food intake, and body weight. These results identify an inhibitory AMPK kinase, p70S6 kinase, and demonstrate that AMPK is a substrate for mTOR-p70S6 kinase. This discovery has broad biologic implications since mTOR-p70S6 kinase and AMPK have multiple, fundamental and generally opposing cellular effects that regulate metabolism, cell growth, and development.     

Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogated ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activated PKCzeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.     

Differential AMPK phosphorylation by glucagon and metformin regulates insulin signaling in human hepatic cells

Insulin and glucagon signaling in the liver are major contributors to glucose homeostasis. Patients with Type 1 and Type 2 diabetes have impaired glycemic control due, in part, to dysregulation of the opposing actions of these hormones. While hyperglucagonemia is a common feature in diabetes, its precise role in insulin resistance is not well understood. Recently, metformin, an AMPK activator, was shown to regulate hepatic glucose output via inhibition of glucagon-induced cAMP/PKA signaling; however, the mechanism for how PKA inhibition leads to AMPK activation in human hepatic cells is not known. Here we show that glucagon impairs insulin-mediated AKT phosphorylation in human hepatic cell line Huh7. This impairment of AKT activation by glucagon is due to PKA-mediated inhibition of AMPK via increased inhibitory phosphorylation of AMPK^Ser173 and reduced activating phosphorylation of AMPK^Thr172. In contrast, metformin decreases PKA activity, leading to decreased pAMPK^Ser173 and increased pAMPK^Thr172. These data support a novel mechanism involving PKA-dependent AMPK phosphorylation that provides new insight into how glucagon and metformin modulate hepatic insulin resistance.     

α1-肾上腺素受体激活大鼠心脏腺苷酸活化蛋白激酶

为了验证心脏腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）是否为肾上腺素受体（adrenergic receptor,AR）的下游信号分子,本实验在大鼠心室肌源细胞和大鼠心脏中观察了α-AR对AMPK的激活作用,利用Western blot检测了AMPK-α总蛋白表达量及其172位苏氨酸磷酸化水平。雄性Sprague-Dawley大鼠皮下植入去甲肾上腺素（norepinephrine,NE）,苯肾上腺素（phenylephrine,PE）或者溶剂载体[0．01％（W／V）维生素C]的缓释微泵（osmotic minipump）。NE或PE以每小时0．2 mg／kg的速率持续输注,7 d后用AMPK-α抗体免疫沉淀处理样本并测定AMPK的活性。结果显示,在细胞水平,NE引起的AMPK磷酸化水平增高具有时间依赖和剂量依赖特点, NE处理细胞10 min后AMPK磷酸化水平达到最高峰;NE引起的这种效应对β-AR的拮抗剂普萘洛尔（propranolol）不敏感,但是可以被α1-AR拮抗剂哌唑嗪（prazosin）所阻断。结果提示,α1-AR介导AMPK的磷酸化,但β-AR无此作用。AR激动剂持续灌注7 d后,AMPK的活性在NE（7．4倍）和PE（6．0倍）灌注组较对照组显著增高（P〈0．05,H=6）。PE持续灌注组大鼠与对照组相比无明显的心肌肥厚和组织纤维化变化。本文证明α1-AR激动剂可以增强AMPK的活性,揭示了心脏中α1-AR激动在调控AMPK活性方面的重要作用。深入了解α1-AR介导的AMPK激活可能在心衰治疗中具有重要的临床意义。     

The active form of the metabolic sensor AMP-activated protein kinase α (AMPKα) directly binds the mitotic apparatus and travels from centrosomes to the spindle midzone during mitosis and cytokinesis

The metabolic rheostat AMP-activated protein kinase (AMPK) is unexpectedly required for proper cell division and faithful chromosomal segregation during mitosis. Although it is conceptually attractive to assume that AMPK-interpreted microenvironmental bioenergetics may strictly engage cell’s energy status, cell grow, and cell division to avoid that energy stresses trigger cell death, the ultimate framework of AMPK activity towards chromosomal and cytoskeletal mitotic regulation is a question that remains unanswered. We herein reveal that the active form of the α-catalytic AMPK subunit (P-AMPKαThr172) -but not its total form (AMPKα)- transiently associates with several mitotic structures including centrosomes, spindle poles, the central spindle midzone and the midbody throughout all of the mitotic stages and cytokinesis in human cancer-derived epithelial cells. At prophase, P-AMPKαThr172 associates with the two asters of microtubules that begin to nucleate from mature centrosomes. The overlapping localization of P-AMPKαThr172 with the mitotic centrosomal Aurora-A kinase is also apparent on the microtubules near the spindle poles in metaphase and in early anaphase. This Aurora A-like centrosomal localization of P-AMPKαThr172 cannot be detected following chromatid separation following anaphase-telophase transition. Rather, toward the end of anaphase and in telophase P-AMPKαThr172 reactivity exhibited a similar but not identical localization to that occupied by the bona fide chromosomal passenger proteins INCENCP and Aurora-B. This localization of P-AMPKαThr172 at the central spindle and midbody persisted during the furrowing process and, at the completion of telophase, a prominent staining of P-AMPKαThr172 as doublet was apparent on either side of the midbody within the intercellular cytokinetic bridge. An identical mitotic geography of P-AMPKαThr172 was observed in cancer cells lacking the AMPK kinase LKB1, in non-cancerous human epithelial cells, and in mouse fibroblasts. The active form of AMPKα bound to the mitotic apparatus may physically tether the bioenergetic state of a cell to the four-dimensional regulation of the chromosomal and cytoskeletal mitotic events, thus suggesting a putative cytokinetic suppressor function. In this newly discovered scenario, we suggest a primordial mitotic role for the α catalytic AMPK subunit in the eukaryotic evolutionary process as it may ensure, at the cell level, an exquisite coordination between sensing of energy resources and the fundamental biological process of genome division.     

Triiodothyronine (T3) stimulates food intake via enhanced hypothalamic AMP-activated kinase activity

Thyroid hormone regulates food intake. We previously reported that rats with triiodothyronine (T3)-induced thyrotoxicosis display hyperphagia associated with suppressed circulating leptin levels, increased hypothalamic neuropeptide Y (NPY) mRNA and decreased hypothalamic pro-opiomelanocortin (POMC) mRNA. AMP-activated kinase (AMPK) is a serine/threonine protein kinase that is activated when cellular energy is depleted. We hypothesized that T3 causes an increase in hypothalamic AMPK activity, which in turn contributes to the development of T3-induced hyperphagia. Rats that were given s.c. injections of T3 (4.5 nmol/kg) had increased food intake 2 h later without alterations in NPY and POMC mRNA levels, but with increased hypothalamic phosphorylated AMPK (169%) and phosphorylated acetyl-CoA carboxylase (194%). To determine the more chronic effects of T3, rats were given 6 daily s.c. injection of T3 or the vehicle. Food intake was significantly increased. Multiple T3 injections increased hypothalamic phosphorylated AMPK (278%) and phosphorylated acetyl-CoA carboxylase (335%) compared to the controls. Intracerebroventricular administration of compound C, an AMPK inhibitor, blocked the food intake induced by a single or multiple injections of T3. Taken together, these results suggest that enhanced hypothalamic AMPK phosphorylation contributes to T3-induced hyperphagia. Hypothalamic AMPK plays an important role in the regulation of food intake and body weight.     

Covalent activation of heart AMP-activated protein kinase in response to physiological concentrations of long-chain fatty acids.

Rat hearts were perfused for 1 h with 5 mm glucose with or without palmitate or oleate at concentrations characteristic of the fasting state. The inclusion of fatty acids resulted in increased activities of the alpha-1 or the alpha-2 isoforms of AMP-activated protein kinase (AMPK), increased phosphorylation of acetyl-CoA carboxylase and a decrease in the tissue content of malonyl-CoA. Activation of AMPK was not accompanied by any changes in the tissue contents of ATP, ADP, AMP, phosphocreatine or creatine. Palmitate increased phosphorylation of Thr172 within AMPK alpha-subunits and the activation by palmitate of both AMPK isoforms was abolished by protein phosphatase 2C leading to the conclusion that exposure to fatty acid caused activation of an AMPK kinase or inhibition of an AMPK phosphatase. In vivo, 24 h of starvation also increased heart AMPK activity and Thr172 phosphorylation of AMPK alpha-subunits. Perfusion with insulin decreased both alpha-1 and alpha-2 AMPK activities and increased malonyl-CoA content. Palmitate prevented both of these effects. Perfusion with epinephrine decreased malonyl-CoA content without an effect on AMPK activity but prevented the activation of AMPK by palmitate. The concept is discussed that activation of AMPK by an unknown fatty acid-driven signalling process provides a mechanism for a 'feed-forward' activation of fatty acid oxidation.     

AMP-activated protein kinase kinase: detection with recombinant AMPK alpha1 subunit

The AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase important for the responses to metabolic stress. It consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, and is regulated both by the allosteric action of AMP and by phosphorylation of the alpha and beta subunits catalyzed by AMPKK(s) and autophosphorylation. The Thr172 site on the alpha subunit has been previously characterized as an activating phosphorylation site. Using bacterially expressed AMPK alpha1 subunit proteins, we have explored the role of Thr172-directed AMPKKs in alpha subunit regulation. Recombinant alpha1 subunit proteins, representing the N-terminus, have been expressed as maltose binding protein (MBP) 6x His fusion proteins and purified to homogeneity by Ni(2+) chromatography. Both wild-type alpha1(1-312) and alpha1(1-312)T172D are inactive when expressed in bacteria, but the former can be fully phosphorylated (1 mol/mol) on Thr172 and activated by a surrogate AMPKK, CaMKKbeta. The corresponding AMPKalpha1(1-392), an alpha construct containing its autoinhibitory sequence, can be similarly phosphorylated, but it remains inactive. In an insulinoma cell line, either low glucose or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) treatment leads to activation and T172 phosphorylation of endogenous AMPK. Under the same conditions of cell incubation, we have identified an AMPKK activity that both phosphorylates and activates the recombinant alpha1(1-312), but this Thr172-directed AMPKK activity is unaltered by low glucose or AICAR, indicating that it is constitutively active.     

LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells

LKB1 is a 50 kDa serine/threonine kinase that phosphorylates and activates the catalytic subunit of AMPK at its T-loop residue Thr 172. We prepared adenoviruses expressing the constitutive active (wild-type) form (CA) or dominant negative (kinase inactive, D194A mutant) form (DN) of LKB1 and overexpressed these proteins in cultured myotubes (C2C12 cells) and rat hepatoma cells (FAO cells). When analyzed by immunoblotting with the antibody against Thr172-phosphorylated AMPK, the phosphorylation of AMPK was increased (2.5-fold) and decreased (0.4-fold) in cells expressing CA and DN LKB1, respectively, as compared with Lac-Z expressing control cells. Immunoprecipitation experiments, using isoform-specific antibody, revealed these alterations of AMPK phosphorylation to be attributable to altered phosphorylation of AMPK alpha2, but not alpha1 catalytic subunits, strongly suggesting the alpha2 catalytic subunit to be the major substrate for LKB1 in mammalian cells. In addition, adiponectin or AICAR-stimulated AMPK phosphorylation was inhibited by overexpression of DN LKB1, while phenformin-stimulated phosphorylation was unaffected. These results may explain the difference in AMPK activation mechanisms between AMP and phenformin, and also indicate that AMPK phosphorylation by LKB1 is involved in AMP-stimulated AMPK activation. As a downstream target for AMPK, AICAR-induced glucose uptake and ACCbeta phosphorylation were found to be significantly reduced in DN LKB1 expressing C2C12 cells. The expression of key enzymes for gluconeogenesis, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was also dependent on LKB1 activities in FAO cells. These results demonstrate that LKB1 is a crucial regulator of AMPK activation in muscle and liver cells and, therefore, that LKB1 activity is potentially of importance to our understanding of glucose and lipid metabolism.     

Glucagon regulates ACC activity in adipocytes through the CAMKKβ/AMPK pathway

Glucagon is important for regulating lipid metabolism in part through its inhibition of fatty acid synthesis in adipocytes. Acetyl-CoA carboxylase 1 (ACC1) is the rate-limiting enzyme for fatty acid synthesis. Glucagon has been proposed to activate cAMP-dependent protein kinase A (PKA), which phosphorylates ACC1 to attenuate the lipogenic activity of ACC1. Because AMP-activated protein kinase (AMPK) also inhibits fatty acid synthesis by phosphorylation of ACC1, we examined the involvement of AMPK and its upstream kinase in the glucagon-elicited signaling in adipocytes in vitro and in vivo. LC-MS-MS analysis suggested that ACC1 was phosphorylated only at Ser(79), an AMPK-specific site, in glucagon-treated adipocytes. Pharmacological inhibitors and siRNA knockdown of AMPK or PKA in adipocytes demonstrate that glucagon regulates ACC1 and ACC2 activity through AMPK but not PKA. By using Ca(2+)/calmodulin-dependent protein kinase kinase-β knockout (CaMKKβ(-/-)) mice and cultured adipocytes, we further show that glucagon activates the CaMKKβ/AMPK/ACC cascade. Additionally, fasting increases the phosphorylation of AMPK and ACC in CaMKKβ(+/+) but not CaMKKβ(-/-) mice. These results indicate that CaMKKβ/AMPK signaling is an important molecular component in regulating lipid metabolism in adipocytes responding to glucagon and could be a therapeutic target for the dysregulation of energy storage.     

Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis

Bacterially expressed heterotrimeric (alpha1, beta1, and gamma1) wild-type, catalytically inactive, and constitutively active forms of AMP-activated protein kinase (AMPK) were used to study phosphorylation by an upstream AMPK kinase preparation. Here, we report the identification of two new phosphorylation sites in the alpha-subunit, viz. Thr258 and Ser485 (Ser491 in the alpha2-subunit) by mass spectrometry, in addition to the previously characterized Thr172 site. Also, autophosphorylation sites in the beta1-subunit were identified as Ser96, Ser101, and Ser108. Mutagenesis of Thr172, Thr258, and Ser485 to acidic residues to mimic phosphorylation in the recombinant proteins indicated that Thr172 was involved in AMPK activation, whereas Thr258 and Ser485 were not. Transfection of the non-phosphorylatable S485A and T258A mutants in CCL13 cells subjected to stresses known to activate AMPK either by increasing the AMP:ATP ratio (slow lysis) or without changing adenine nucleotide concentrations (hyperosmolarity) resulted in no significant differences in AMPK activation. All three sites within the alpha-subunit were phosphorylated in vivo, as seen in AMPK immunoprecipitated from anoxic rat liver. In transfected CCL13 cells, the level of Ser485 phosphorylation did not change upon AMPK activation. The newly identified phosphorylation sites could play a subtle role in the regulation of AMPK, e.g. in subcellular localization or substrate recognition.     

Regulation of AMP-activated protein kinase by cAMP in adipocytes: Roles for phosphodiesterases,protein kinase B,protein kinase A,Epac and lipolysis

AMP-activated protein kinase (AMPK) is an important regulator of cellular energy status. In adipocytes, stimuli that increase intracellular cyclic AMP (cAMP) have also been shown to increase the activity of AMPK. The precise molecular mechanisms responsible for cAMP-induced AMPK activation are not clear. Phosphodiesterase 3B (PDE3B) is a critical regulator of cAMP signaling in adipocytes. Here we investigated the roles of PDE3B, PDE4, protein kinase B (PKB) and the exchange protein activated by cAMP 1 (Epac1), as well as lipolysis, in the regulation of AMPK in primary rat adipocytes. We demonstrate that the increase in phosphorylation of AMPK at T172 induced by the adrenergic agonist isoproterenol can be diminished by co-incubation with insulin. The diminishing effect of insulin on AMPK activation was reversed upon treatment with the PDE3B specific inhibitor OPC3911 but not with the PDE4 inhibitor Rolipram. Adenovirus-mediated overexpression of PDE3B and constitutively active PKB both resulted in greatly reduced isoproterenol-induced phosphorylation of AMPK at T172. Co-incubation of adipocytes with isoproterenol and the PKA inhibitor H89 resulted in a total ablation of lipolysis and a reduction in AMPK phosphorylation/activation. Stimulation of adipocytes with the Epac1 agonist 8-pCPT-2′O-Me-cAMP led to increased phosphorylation of AMPK at T172. The general lipase inhibitor Orlistat decreased isoproterenol-induced phosphorylation of AMPK at T172. This decrease corresponded to a reduction of lipolysis from adipocytes. Taken together, these data suggest that PDE3B and PDE4 regulate cAMP pools that affect the activation/phosphorylation state of AMPK and that the effects of cyclic AMP on AMPK involve Epac1, PKA and lipolysis.     

The elusive short gene--an ensemble method for recognition for prokaryotic genome

Glucagon-like peptide-1 (GLP-1) and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important to elucidate a mechanism to maintain energy balance. In the present study, coadministration of subthreshold GLP-1 and leptin dramatically reduced feeding in rats. Although coadministration of GLP-1 with leptin did not enhance leptin signal transduction in the hypothalamus, it significantly decreased phosphorylation of AMP-activated protein kinase (AMPK). In addition, coadministration of GLP-1 with leptin significantly increased proopiomelanocortin (POMC) mRNA levels. Considering that α-melanocortin stimulating hormone (α-MSH) is derived from POMC and functions through the melanocortin-4-receptor (MC4-R) as a key molecule involved in feeding reduction, the interaction of GLP-1 and leptin on feeding reduction may be mediated through the α-MSH/MC4-R system. As expected, the interaction of GLP-1 and leptin was abolished by intracerebroventricular preadministration of the MC4-R antagonists agouti-related peptide and SHU9119. Taken together, GLP-1 and leptin cooperatively reduce feeding at least in part via inhibition of AMPK following binding of α-MSH to MC4-R.     

Activation of AMP-activated protein kinase is involved in vincristine-induced cell apoptosis in B16 melanoma cell

The molecular basis for induction of apoptosis in melanoma cells by vincristine remains unknown. Here we tested the potential involvement of AMP-activated protein kinase (AMPK) in this process. We found for the first time that vincristine induces AMPK activation (AMPKα, Thr 172) and Acetyl-CoA carboxylase (ACC, Ser 79) (a downstream molecular target of AMPK) phosphorylation in cultured melanoma cells in vitro. Reactive oxygen species (ROS) dependent LKB1 activation serves as the upstream signal for AMPK activation. AMPK inhibitor (compound C) or AMPKα siRNA knockdown inhibits vincristine induced B16 melanoma cell apoptosis, while AMPK activator 5-aminoimidazole-4-carboxamide-1-β-riboside (AICAR) enhances it. AMPK activation is involved in vincristine induced p53 phosphorylation and stabilization, the latter is known to mediate melanoma cell apoptosis. Further, activation of AMPK by vincristine inhibits mTOR Complex 1 (mTORC1) in B16 melanoma cells, which serves as another important mechanism to induce melanoma cell apoptosis. Our study provides new insights into understanding the cellular and molecular mechanisms of vincristine induced cancer cell death/apoptosis. We suggest that combining AMPK activator AICAR with vincristine may have potential to be used as a new therapeutic intervention against melanoma.